The main challenge of advertising is to catch consumers’ attention and evoke in them positive attitudes to consequently achieve product preference and higher purchase intentions. In modern advertising, visual metaphors are widely used due to their effects such as improving advertising recall, enhancing persuasiveness, and generating consumers’ positive attitudes. Previous research has pointed out the existence of an “inverted U-curve” that describes a positive relationship between the conceptual complexity of metaphors and consumers’ positive reactions to them, which ends where complexity outweighs comprehension. Despite the dominance of visual metaphors in modern advertising, academic research on this topic has been relatively sparse. The inverted U-curve pattern has been validated regarding ad appreciation, ad liking, and purchase intention by using declarative methods. However, at present, there is no evidence of consumers’ neurophysiological responses to visual metaphors included in advertising. Given this gap, the aim of this research is to assess consumer neurophysiological responses to print advertisements that include visual metaphors, using neuroscience-based techniques. Forty-three participants (22W–21M) were exposed to 28 stimuli according to three levels of visual complexity, while their reactions were recorded with an electroencephalogram (EEG), eye tracking (ET), and galvanic skin response (GSR). The results indicated that, regardless of metaphor type, ads with metaphors evoke more positive reactions than non-metaphor ads. EEG results revealed a positive relationship between cognitive load and conceptual complexity that is not mediated by comprehension. This suggests that the cognitive load index could be a suitable indicator of complexity, as it reflects the amount of cognitive resources needed to process stimuli. ET results showed significant differences in the time dedicated to exploring the ads; however, comprehension doesn’t mediate this relationship. Moreover, no cognitive load was detected from GSR. ET and GSR results suggest that neither methodology is a suitable measure of cognitive load in the case of visual metaphors. Instead, it seems that they are more related to the attention and/or emotion devoted to the stimuli. Our empirical analysis reveals the importance of using neurophysiological measures to analyze the appropriate use of visual metaphors and to find out how to maximize their impact on advertising effectiveness. Marketing scholars and practitioners are continuously facing the challenge to find out how to enhance advertising effectiveness. The decrease of traditional advertising media such as TV and newspapers, the rise of new ones like mobiles or videos, and the growth of interactive and targeted advertising represent a huge limitation to print advertising, characterized by a static image. Given this limitation, graphic print advertising must focus on seeking the most optimal design to catch consumers’ attention and evoke positive attitudes, in order to trigger a higher preference for the products and, consequently, higher purchase intentions.Advertisers and academics have analyzed the key factors that influence effective print advertisement such as element location , advertiImages themselves can be extremely complex, as they are “capable of representing concepts, abstractions, actions, metaphors and modifiers” , p. 253.A rhetorical figure is an artful deviation relative to audience expectation , which cAmong the visual rhetoric figures, metaphors are the most commonly used because, according to the theory, they can formulate, sustain, or modify the attention, perceptions, attitudes, or behaviors of their audiences ; they alScholar research defines metaphors as comparisons between two things that are originally different in nature but have something in common , and wheConsumer studies have concluded that advertisements with complex layouts evoke positive attitudes , 2014, hAs mentioned, previous studies have consistently reported that advertisements with complex layouts result in audiences’ more positive attitudes than advertisements based on stand-alone images , 2014. OIt seems that decoding the message increases the subject’s sense of pleasure and decreases the sense of tension, leading to the enhancement of the subject’s attitude toward the ad (Aad)  and to iAccording to The pleasure evoked by complex visual images used in advertising has been studied from different perspectives. Previous studies suggest that if metaphors demand too much or too little cognitive processing effort, consumers may opt out, and appreciation will decrease; thus, advertisement appreciation follows the pattern of the aforementioned inverted U-curve . In the Despite of the valuable findings of the aforementioned research, empirical evidence for the inverted U-curve is still relatively scarce, and its validity has not yet been proven regarding Aad, a concept extensively examined that reveals consumers’ precise perceptions and impressions toward advertisement designs . NeitherHypothesis 1:The effects of metaphors on (a) Aad, (b) purchase intention, and (c) preference follow the inverted U-curve pattern according to which there is a positive relationship between complexity and positive feelings until a tipping point is reached where complexity exceeds comprehension.According to In cognitive psychological terms, elaboration “indicates the amount, complexity, or range of cognitive activity occasioned by a stimulus” . p 39. W;Jeong, 2008;Chang and Yen, 2013), where it is stated that more complex visual figures lead to more cognitive elaboration. Such higher elaboration is a consequence of comprehension efforts, and it manifests as an enhanced memory of the ad , and especially those based on the recording of event-related brain potentials (ERPs)–an EEG methodology that offers great insights into processing mechanisms with millisecond precision –suggest In the same line, Pileliene and Grigaliunaite (2016) analyzed the allocation of attentional resources to process advertising with complex layouts , through the use of P300, a component that provides information about the neural activity of cognitive operations . The obtMoreover, regarding temporal and spatial studies of the brain, the EEG study developed by On the other hand, studies using eye tracking (ET) revealed that the time devoted to exploring the stimuli could be indicative of the cognitive processes involved in comprehending the metaphors, as experimental studies showed that extra time is needed to comprehend more complex metaphors . BesidesFinally, some authors have suggested that galvanic skin response (GSR) seems to be a suitable tool for measuring cognitive activity and have pointed out the correlation between GSR features and cognitive functions and more specific cognitive workload .Based on the preceding discussion, we can identify a positive relationship between complexity and elaboration that can be represented as a higher cognitive load as metaphors increase their complexity. According to literature, the cognitive load can be measured with EEG, analyzing oscillations of alpha–theta bands; with ET, analyzing the time devoted to exploring the visual metaphor; and by analyzing GSR features. Hence, it is hypothesized:Hypothesis 2:To the extent that a visual metaphor increases its difficulty, the subject will have a higher cognitive load. This situation will be reflected in (a) a longer time to explore the advertisement, (b) a higher index of EEG cognitive load, and (c) a higher index on GSR activation.Forty-three undergraduate students  voluntarily participated in the study in June 2018. The mean age was 23.3 years with a standard deviation of 2.8 years. The participants were recruited using convenience sampling. All participants were right-handed, healthy people with normal or corrected-to-normal vision and were free of any hearing problems. All participants provided signed consent before participating and received monetary compensation at the end of the session.The EEG is a measurement of the whole sphere of brainwave activity emerging in various cortical areas, which helps to understand the way the brain responds to various stimuli. EEG is a non-invasive instrument that provides information from areas underneath the cortex and, combined with other instruments, may provide very accurate results on a subject’s response to a marketing stimulus .Cerebral activity was recorded using the Bitbrain Versatile EEG with 16 channels at a sampling rate of 256 Hz, while impedances were kept below 5 kΩ. For the experiment, we used 12 electrodes placed by following the International 10–20 system.This biometric technique is based on the relationship between human eye movements, visual attention, and information acquisition, with the latter two both being closely related to higher-order cognitive processes . ET has Consumers’ behavior is measured with an ET technique by recording either the number of fixations or dwell time of the eyes during an individual or group exposure to external stimuli. The specific ET device used in the present study was a Tobii X2-30 Eye-Tracker Compact Edition, a screen-based eye tracker capturing gaze data at 60 Hz.The GSR is defined as a change in the electro-physiological properties of the skin due to sweat gland function. GSR provides an indication of changes in the human sympathetic nervous system (SNS)  and is wNeurophysiological measurements comprise cognitive load, time in AOI and arousal. These measures are described following and the instruments used to measure them are related in The EEG technique can be used to obtain many different psychological metrics such as cognitive workload. Studies on the area have found that EEG power in the theta and alpha frequency range is related to cognitive performance . In factBased on previous studies , in the The ET technique has been used as a direct measure of attention by analyzing the number of fixations on specific areas, the viewing time, or the time that users take to reach each area  and alsoTaking into account that there is evidence regarding “a sufficiently close connection between time spent fixating on display items and the amount of cognitive processing” , p. 1237To get the time in AOI, we previously defined the AOIs on each image by selecting the area of the metaphor. In order to avoid the bias derived from having a different number of images according to the type of metaphor, we defined as “area of interest” the same zone (same size and shape) for all four conditions in each set.Previous studies have found that the GSR signal represents a suitable measure for detecting emotional responses but also for differentiating between stress and cognitive load . BesidesA computer-based questionnaire was also applied, through an Internet platform, to obtain declarative Aad, purchase intention, preference, and perceived complexity see .(1)Aad was measured adopting (2)Purchase intention was measured as in previous studies , on a se(3)Participants ranked their preference from 1 (the most preferred stimulus) to 4 (the least preferred stimulus).(4)Perceived complexity was measured as in A within-subjects research was conducted to assess consumer responses to print advertisements that include visual metaphors. Twenty-eight advertisements were developed in total, seven sets of four print advertisements for seven different product categories  and a control condition. Each set corresponded to one of the product categories  selected on the basis of a focus group performed with eight people from 18 to 35 years, in which age range were found high levels of familiarity with categories of food, house, and personal care.All images used were full-color, and product images were previously used in real printed or Internet ads. However, in order to avoid familiarization bias , althougTaking into account that metaphorical content could have multiple interpretations  a pre-teThe study was performed at the Laboratory of Neuromarketing of Complutense University of Madrid, and its total duration was 60 min, including both blocks to be described in the following paragraphs. All 43 participants were right-handed, healthy people with normal or corrected-to-normal vision and were free of any hearing problems. All participants provided signed consent before participating and received monetary compensation at the end of the session.The experimental procedure had two phases, which entailed the use of neurophysiological (Block 1) and declarative (Block 2) methods. In Block 1, after briefing the protocol to the participants, they were sat in front of the computer screen where the ET was installed and were affixed with the EEG and GSR devices for collecting their brain electrical activity and skin conductance. The screen used was 21 inches with full HD resolution .To calibrate the ET, subjects were instructed to follow the points appearing on the screen with their sight without moving their heads. Once the ET was calibrated, the researcher checked that the signal of all three devices was good and started running the experiment. It is important to highlight that in order to measure the workload, the emotional activation, and the time spent exploring each ad, we used simultaneous EEG, GSR, and ET measurements during the whole experiment. The software used to present stimuli and simultaneously record data was SensLab, developed by Bitbrain.In this block, the 43 participants were exposed to the 28 aforementioned stimuli (7 product categories × 4 complexity modification designs), presented individually and randomly. First, a fixed cross was presented in the middle of the screen, followed by the stimulus for 5 s, and then a black slide, with the word “Rest,” so participants could take a 2 s rest see .During Block 2, which entailed the declarative test phase, participants had to answer the questionnaire while they were visualizing the stimuli individually. The respondents initially answered the Aad scale for the 28 advertisements. The second scale, with the same structure, asked participants about their purchase intention. The third task was to rank the four advertisement modulations  from 1 to 4, to assess their preference. Finally, the last declarative section asked about their perceived complexity. In this second block, the exposure time depended on the subject’s response time instead of being standardized as in Block 1.All raw data coming from the neurophysiological techniques were provided by SennsLab.Raw EEG data were first filtered using a band-pass filter between 1 and 25 Hz with a four-order Butterworth filter. After that, a filtering pipeline was implemented. First, an ASR (artifact subspace reconstruction) filter was used to remove big amplitude artifacts . Then ICSkin conductance data are usually characterized by a sequence of overlapping phasic SCRs overlying a tonic component . The extThe post-processed EEG and GSR signals, next to the ET information about time in AOI were subsequently analyzed by using SPSS. The declarative information also was analyzed using that statistical software.t-test analysis was performed. The second stage was oriented to find differences in the complexity continuum. To get those results, we applied a repeated measures ANOVA. In the third stage, all the metrics (implicit and declarative) were regressed onto conceptual complexity derived from metaphorical content, with perceived complexity as a mediating variable. Finally, a Sobel test was used to statistically investigate the effect of the proposed mediator on the predictor–outcome relationship.The data analysis was performed in three stages. First, we focused on the differences between advertisements with and without metaphors. To obtain those differences, a F = 39.42, p = 0.000. More specifically, differences were found between juxtaposition (M = 6.1) and replacement  and between fusion (M = 5.9) and replacement .The first step to analyze Hypothesis 1 was to validate that the complexity continuum was well constructed. For this purpose, a repeated measures ANOVA was performed. It showed that the perceived complexity of the levels of the ads was statistically and significantly different, Note that in order to maintain the negativity of the scales to the left, subjects rated the images difficult to understand as 1 and those easy to understand as 7. t-test was conducted. Results showed that there was a significant difference in the scores for ads without metaphors  and ads including metaphors (M = 5.64 SD = 0.71); t(42) = −2.71, p = 0.010. It means that ads without metaphors were perceived as easier to understand than those with metaphorical content.To compare the perceived complexity between ads with and without metaphors, a paired-samples In order to obtain more accurate results of the self-reported measures, we performed two types of analysis: (1) a comparison between advertisements with and without metaphors and (2) a comparison between the three levels of complexity included in the experiment.t-test was conducted. Results revealed a significant difference between advertisements without metaphors  and those including metaphors ; t(42) = −9.235, p = 0.000.Regarding the analysis of Aad with and without metaphors, a paired-samples F = 75.877, p = 0.000. Regarding the level of complexity, post hoc tests using the Bonferroni correction revealed significant differences between juxtapositions (M = 4.8) and fusions (M = 5.4) and between fusions and replacement metaphors (M = 3.9). However, there were no significant differences between juxtaposition and replacement metaphors  described as an inverted U-curve in the literature, a mediation analysis was performed. Statistically, mediation is often analyzed through path analytic models with one X variable, one mediator M, and one outcome variable Y . In the a = −0.62, p = 0.000) and that perceived complexity was a significant predictor of Aad . These results support the mediational hypothesis. Ad complexity was not a significant predictor of Aad after controlling for the mediator (comprehension), c’ = 0.16, p = 0.179. The standardized indirect effect (B) was a (−0.62) × b (0.51) = −0.31 .The results of mediation analysis indicated that complexity of metaphors was a significant predictor of perceived complexity . The results revealed that comprehension/perceived complexity is a significant mediator of the relationship between advertisement complexity induced by metaphors and Aad; thus, the inverted U-curve pattern is validated, and consequently, results support Hypothesis 1a.A Sobel test was also conducted to validate the effect of the mediator, finding full mediation  and advertisements including metaphors ; t(42) = −3.395, p = 0.020.Regarding purchase intention, a paired-sample F = 41.742, p = 0.000. Post hoc tests using the Bonferroni correction revealed significant differences between all three levels of complexity: juxtapositions (M = 4.8), fusions (M = 5.4), and replacement metaphors (M = 3.9).In order to test Hypothesis 1b, a repeated measures ANOVA also determined that the different complexity levels of the advertisement produced statistically significant differences in purchase intention, a = −0.62, p = 0.000) and that perceived complexity was a significant predictor of purchase intention . Advertisement complexity induced by metaphors was no longer a significant predictor of purchase intention after controlling for the mediator , in consistence with a full mediation. The standardized indirect effect was a (−0.62) × b (0.43) = −0.27  . Thus, it can be concluded that comprehension mediated the relationship between advertisement complexity induced by metaphors and purchase intention. Consequently, the inverted U-curve pattern is validated for this construct, and the hypothesis 1b is supported, as displayed in Lastly, the Sobel test found full mediation (2(2) = 40.812, p = 0.000. A post hoc analysis with a Wilcoxon signed-rank test was conducted with a Bonferroni correction, resulting in a significance level set at p < 0.000. There were significant differences between the three levels of complexity.To test the preference hypothesis (H1c), due to the ordinal nature of the preference metric, a Friedman test was conducted as the non-parametric alternative to the one-way repeated measures ANOVA. The Friedman test revealed a statistically significant difference in preference depending on the complexity of the images visualized, χa = −0.062, p = 0.000). To investigate how conceptual complexity of metaphors could influence the preference of ads with metaphorical content, an ordinal logistic regression analysis was conducted. The conceptual complexity was found to contribute to the model . The estimated odds ratio shows an inverse relationship between complexity and preference. It suggests a decreasing probability of improving the preference level with increasing complexity level of ads .To examine the effect of comprehension of different metaphors on preference, we performed a path analysis, described in X2(1) = 16.536, p = 0.000]. The coefficient shows that when increasing the perceived complexity, there is a predicted increase of 0.659 in the log-odds of being in a higher level of preference . These results support the mediational hypothesis. The standardized indirect effect was a (−0.62) × b (0.659) = −0.4085.On the other hand, the analysis showed that comprehension was a significant predictor in the model [Z = −4.059, p = 0.000) and (2) ads not including metaphorical images and fusions . However, the analysis shows that there were no differences between ads including replacements and ads not including metaphorical images .Finally, a Wilcoxon signed-rank test was conducted to analyze the differences between ads with and without metaphorical content. Results showed statistically significant differences between (1) ads not including metaphorical images and ads including juxtapositions  is a suitable indicator of comprehension  and cognt-test was conducted to compare the time in AOI for ads including and not including metaphorical images. Results yielded a significant difference in the time in AOI between ads without metaphors  and ads including metaphors (M = 4.5 SD = 0.62); t(42) = −3.200, p = 0.003. Besides, a repeated measures ANOVA determined that the different complexity levels of the ads produced statistically significant differences in time in AOI, F = 11.609, p = 0.000. Post hoc tests using the Bonferroni correction revealed significant differences in time in AOI between juxtapositions (M = 4.1) and fusions (M = 4.8) and between fusions and replacements (M = 4.4).First, a paired-sample a = 0.62, p = 0.000), perceived complexity was not a significant predictor of time in AOI , so perceived complexity did not mediate the relationship between ad complexity induced by metaphors and time in AOI . These results were also supported by results of the Sobel test .As shown in M = 4.8) than ads with metaphors (M = 4.5); (2) the time that participants spent exploring an ad did not always increase when the ad increased in complexity ; and (3) perceived complexity was not a significant predictor of time in AOI.Regarding the time spent exploring the ads, obtained results provide three interesting findings: (1) participants spent more time exploring ads without metaphors  and ads including metaphors ; t(41) = −0.503, p = 0.617. However, a repeated measures ANOVA determined that cognitive load measured through EEG yielded statistically significant differences as a function of the different complexity levels of the ads, F = 3.102, p = 0.050. Post hoc tests using Bonferroni revealed differences in cognitive load between juxtaposition (M = 26.1) and replacement (M = 28.4).In order to determine the cognitive load required by ads including and not including metaphorical images, a paired-sample Regarding these results, it is interesting to remark that EEG results reflect a linear relationship between complexity and cognitive load, as shown in a = −0.61, p = 0.000), perceived complexity was not a significant predictor of cognitive load , so perceived complexity did not mediate the relationship between ad complexity induced by metaphors and cognitive load .The mediation analysis showed that comprehension did not mediate the relationship between ad complexity induced by metaphorical content and cognitive load. Although ad complexity was a significant predictor of perceived complexity (M = 27.6) than ads without metaphors (M = 27.1), (2) there is a linear relationship between complexity and cognitive load, and (3) perceived complexity is not a significant predictor of cognitive load. Since Hypothesis 2b stated that higher levels of difficulty could be reflected on a higher index of EEG cognitive load, we can state that Hypothesis 2a was supported.According to the obtained results: (1) ads with metaphors had a higher cognitive load index  and ads including metaphors ; t(38) = 0.372, p = 0.712. Nor was there any significant difference in activation as a function of the three levels of complexity, F = 0.288, p = 0.636. However, it is interesting to note that the means obtained reflect the aforementioned inverted U-curve pattern, as displayed in Again, the first step was to conduct a M = 0.06) than when they visualized ads with metaphors (M = 0.05) and (2) the participants’ arousal did not always increase when the ad increased in complexity . On the basis of the obtained results and since Hypothesis 2c predicted that higher levels of difficulty and, consequently, a higher index of cognitive load could be reflected by higher levels of arousal, we can state that Hypothesis 2c was not supported. Regarding the arousal, obtained results provide two findings: (1) participants had more arousal when they were exposed to ads without metaphors  could be suitable to measure the cognitive load derived from complex tasks or contents , 2012. TIn advertising, visual metaphors are widely used to draw individuals’ attention and entice them to buy the product . HoweverVisual metaphors are creative resources that try to provide differentiation and recognition of the brand. Our results highlight the importance of assessing the perceived complexity of ads in order to find the optimal level of complexity of the visual metaphors to achieve the desired brand awareness and advertising effectiveness .Finally, the present study provides evidence on how useful the use of neuroscientific techniques can be to have an objective measure of consumer reactions to marketing stimuli and to find new and useful insights that cannot be detected with declarative methodologies . Our resWhile this study provides theoretical and practical implications, some limitations must be acknowledged, mainly regarding the sample size, similarity of participants, and forced exposure to the stimuli.Although previous studies have demonstrated that consumer neuroscience studies with small sample sizes can produce predictive results and significant insights , the useBesides, the sample used is very similar because it is formed by students, and their age, education level, cultural context, and lifestyle are pretty similar. Since previous studies have proven significant cross-cultural variations in the interpretation of advertisements , it woulOn the other hand, the present study was conducted in a laboratory, and subjects were forced to visualize the stimuli contrary to how they behave in real life, where they devote their attention voluntarily. The degree of control exerted over potential extraneous variables determines the level of internal validity of a study . AlthougFinally, on the basis of previous findings according to which advertisement appreciation follows an inverted U-curve pattern , we tookThe datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.All authors listed contributed to the design of the experiment, data collection, data analysis, literature review and writing and reviewing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The collection of phenotypes related to livestock methane emissions is hampered by costly and time-demanding techniques. In the present research, a laser methane detector was used to measure several novel phenotypes, including mean and aggregate of methane records, and mean and number of methane peak records, considering Simmental heifers as a case study. Phenotypes showed satisfactory repeatability and reproducibility for log-transformed data. The number of emission peaks had great variability across animals and thus it is a promising candidate to discriminate between high and low emitters.4) emissions represent a worldwide problem due to their direct involvement in atmospheric warming and climate change. Ruminants are among the major players in the global scenario of CH4 emissions, and CH4 emissions are a problem for feed efficiency since enteric CH4 is eructed to the detriment of milk and meat production. The collection of CH4 phenotypes at the population level is still hampered by costly and time-demanding techniques. In the present study, a laser methane detector was used to assess repeatability and reproducibility of CH4 phenotypes, including mean and aggregate of CH4 records, slope of the linear equation modelling the aggregate function, and mean and number of CH4 peak records. Five repeated measurements were performed in a commercial farm on three Simmental heifers, and the same protocol was repeated over a period of three days. Methane emission phenotypes expressed as parts per million per linear meter (ppm × m) were not normally distributed and, thus, they were log-transformed to reach normality. Repeatability and reproducibility were calculated as the relative standard deviation of five measurements within the same day and 15 measurements across three days, respectively. All phenotypes showed higher repeatability and reproducibility for log-transformed data compared with data expressed as ppm × m. The linear equation modelling the aggregate function highlighted a very high coefficient of determination (≥0.99), which suggests that daily CH4 emissions might be derived using this approach. The number of CH4 peaks resulted as particularly diverse across animals and therefore it is a potential candidate to discriminate between high and low emitting animals. Results of this study suggest that laser methane detector is a promising tool to measure bovine CH4 emissions in field conditions.Methane (CH Anthropic activities related to the primary and secondary sectors are responsible for the majority of GHG released in the atmosphere. Methane , and sniffer-based systems [4 measurement or prediction is an emerging research field of global interest. Until now, mid-infrared spectroscopy [2 to CH4 ratio [4 production in cattle. Laser methane detector (LMD) has been proposed as an alternative instrument to directly measure CH4 emissions. The sensitivity and accuracy of LMD have been assessed in controlled conditions, by comparing data acquired through LMD with those measured through the respiration chamber  [2 = 0.64) [4 emissions in field conditions. For decades, the scientific community has focused on different strategies and approaches aiming at reducing ruminants CH systems . For thetroscopy , archaeotroscopy , sulphurH4 ratio  have beeto 0.97)  and Gree = 0.64) . However4-related phenotypes, including mean and aggregate of CH4 records, slope of the linear equation modelling the aggregate function, and mean and number of CH4 peak records. For this purpose, CH4 emissions were measured on Simmental heifers as a case study.This research question was investigated by assessing repeatability and reproducibility of different CHProcedures used in this study are excluded from the authorization of the animal welfare committee. Methane emission measurements were performed in September 2019, in a commercial dairy farm located in Padova province  on three pregnant Simmental heifers: heifer 1 , heifer 2 , and heifer 3 . Animals were housed in the same open-aerated barn. Heifers received the same diet containing wheat straw , corn silage , meadow hay , protein mix , corn meal , and mineral/vitamin mix , distributed through a total mixed ration. Protein, neutral detergent fiber, acid detergent fiber, starch, fat, and ashes content, calculated on dry matter basis, were 12.39%, 57.12%, 36.25%, 5.30%, 2.62%, and 7.32%, respectively.4 was expressed as parts per million per linear meter (ppm × m). Each measurement was performed by pointing the laser to the nostril of a single animal for 5 min, at a distance of 3 m, according to the protocol proposed by Chagunda et al. [4 emission every 0.5 s, for a total of 600 records for each measurement (5 min). Every single measurement was forwarded via Bluetooth from the laser device to a Lenovo Tab E7  tablet, equipped with an Android operating system and Gas Viewer  application, saved as .csv file, sent to a dedicated e-mail box, and downloaded in a computer workstation to allow for the local storage of data. The methanogram plot resulting from a single measurement is depicted in Methane emissions were measured through Laser Methane Mini  and CHa et al. . Each he4 emissions expressed as ppm × m is depicted in 4 emissions were loge-transformed (lnCH4) to achieve normality and homogeneity of variances. The distribution of the probability function of lnCH4 is reported in 4, n = 27,000), which resulted in 26,856 records available for subsequent analysis.The distribution of CH4 and lnCH4 records, (ii) aggregate of CH4 and lnCH4 records, (iii) slope of the linear equation modelling the aggregate function, (iv) mean of CH4 and lnCH4 peak records, calculated on the last decile of the distribution, and (v) number of CH4 and lnCH4 peak records. Repeatability of the previously mentioned phenotypes was calculated as the relative standard deviation (RSDr) of five consecutive measurements carried out within the same day and within the same animal. Similarly, reproducibility of phenotypes was calculated as the relative standard deviation (RSDR) of 15 measurements collected across three days of analyses and within the same animal, as proposed by Chagunda et al. [Phenotypes considered in the present study were: (i) mean of CHa et al. , Franzoia et al. , and Niea et al. .4 emissions expressed as ppm × m, obtained in a single measurement on a single heifer. The methanogram featured a baseline signal, including the majority of records, which is likely due to environmental CH4 and to the basal eructation activity. The plot highlighted clear emission signals, as the minority of records, associated with peaks of CH4 eructation. Methane emissions averaged 105.48 and 98.26 ppm × m with standard deviation of 77.92 and 58.02 ppm × m for the pre-edited and post-edited datasets, respectively . The loge-transformation of CH4 produced a much more normal trait (lnCH4), as reported in p > 0.05), both in the pre-edited and in the post-edited datasets. Skewness and kurtosis were close to zero in the pre-edited and post-edited dataset. Logarithmic transformations were proposed also by Ali and Shook [ectively . Averagea et al. . In terma et al. . These da et al.  who measrom zero . Visual rom zero  and Shapnd Shook  and Benend Shook  to achie4 emissions was assessed through RSDr and RSDR (4 highlighted notable improvements in terms of RSDr and RSDR. Repeatability varied from 8.93% (heifer 1 in day 3) to 14.85% (heifer 1 in day 1), whereas reproducibility ranged from 11.98% (heifer 2) to 15.35% (heifer 3). Still, such repeatability and reproducibility values remain greater than values from other studies describing the precision of analytical methods carried out under controlled conditions [The precision of LMD for determining the mean of CHand RSDR . Methanenditions ,17. Overnditions , and  of the linear equation modelling the aggregate function are reported in 4 emissions showed the lowest value for heifer 3 in day 1, being equal to 153,393 ppm × m and 11,212 lnCH4. The greatest aggregate value was observed for heifer 1 in day 2 . The slopes of the linear equation mirrored the tendency of aggregate values, being lower and greater concurrently with lower and greater aggregates. Although the agreement between aggregates and slopes may support and reinforce the significance of these traits, it can be argued that the consideration of both phenotypes is redundant since their biological meaning is likely the same. Overall, the R2 of the aggregate functions of lnCH4 (0.999) was greater than the R2 of the aggregate functions of CH4 (0.989 to 0.997). Such a great accuracy suggests that the linear equation modelling the aggregate function might be used in the future to estimate long-term or daily CH4 emissions.The aggregate value of CH4 and lnCH4 emissions was assessed through RSDr and RSDR (4 emissions showed greater precision compared with the same indexes calculated for means of CH4 emissions. This translated into a relatively low RSDr, ranging from 17.58% to 20.15% for CH4, and 4.50% to 5.34% for lnCH4, and RSDR, which was always lower than 20% and 5% for CH4 and lnCH4, respectively. The average values for peaks of CH4 emissions did not vary much across different animals, which suggests that this phenotype might be not adequate to discriminate between high and low CH4 emitters. Such a low variability was somewhat expected. Peaks of CH4 emissions were defined as records belonging to the highest decile of both the datasets, which leads to a considerable decrease of variability. For this reason, the number of peaks emitted from every single animal is more informative because it is more differentiated across heifers. A similar approach was adopted by Bobbo et al. [The precision of LMD for determining peaks of CHand RSDR . Repeatao et al.  in the s4 emissions, measured through LMD using Simmental heifers as a key study. The distribution of emission events expressed as ppm × m showed a significant deviation from the normal distribution, but the logarithmic transformation of the data led to normality. Repeatability and reproducibility were much better for lnCH4 than for CH4. The coefficient of determination of the linear equation modelling the aggregate function showed high precision. Such results are promising since these equations might be used to estimate daily or long-term CH4 emissions. Peaks of CH4 emissions were rather different across animals in terms of the number of events but were homogeneous in terms of average values. For this reason, the number of peaks may be an interesting phenotype to discriminate between high and low emitting animals. Overall, results of the present study indicate that measures carried out through LMD are fairly repeatable and reproducible. Therefore, in terms of accuracy, LMD may be considered as a promising tool enabling to measure bovine CH4 emissions in field conditions at relatively low costs. Future studies will focus on the application of LMD for large-scale studies to assess sources of variation of CH4 emissions.The present research assessed the repeatability and the reproducibility of phenotypes related to CH


A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19. Betacoronavirus. In general, coronavirus is the name of viruses that belong to the family Coronaviridae. These are classified into four categories: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. Alpha- and betacoronaviruses infect mammals, gammacoronaviruses infect avian species, and deltacoronaviruses infect both mammalian and avian species  discovered in Wuhan, China, in December 2019, has caused a global disruption and COVID-19 pandemic. SARS-CoV-2 is classified as cies Li, . CoronavCoronaviruses have characteristic clove-shape spikes (“corona”) protruding from the surface. The spikes are protein complexes that virus uses to bind to a receptor (receptor-binding subunit S1) and mediate entry into host cells (a membrane-fusion subunit S2). Upon binding virus fuses with the human cell membrane, allowing the genome of the virus to enter the cell and begin replication. The receptor-binding domain of SARS-CoV-2 spikes are closely related to those of SARS-CoVs (73.8–74.9% amino acid identity) and SARS-like CoVs (75.9–76.9% amino acid identity)  have demonstrated potent and broad immunomodulatory and anti-inflammatory capacity , Middle East Respiratory Syndrome (MERS), and now, COVID-19. The severity of these infections also varies widely between age-groups and different ethnic populations. Older people are at increased risk of acquiring the infection and are likely to develop severe symptoms culminating in death.Interestingly, there appears to also be gender disparity in the number of acquired cases of COVID-19 with higher percentage of men (~60%) than women being infected, as was first reported in China . A recent study presented a deleterious effect on the cells Currently, there are 17 clinical trials investigating the therapeutic potential of MSCs in COVID-19 patients that are registered on clinicaltrials.gov website; most of these trials are either recruiting patients or have not yet started the recruitment. The vast majority of the trials are selecting patients with COVID-19 and pneumonia, and utilizing allogeneic bone-marrow or umbilical cord-derived MSCs transplanted intravenously on three different occasions . Approxi6 per kg) and 3 patients in the placebo controlled group albeit consisting of all female patients, showed significant improvement in pulmonary function and symptoms as well as substantial reduction in inflammation compared to the placebo controlled group  and follows strict regulations prior to being approved for the use in humans. The use of unauthorized and unapproved stem cell therapies not validated through stringent multicenter clinical trials should be strongly discouraged. At the same time current findings imply that there is a need for globally coordinated approach and support to conduct multicenter clinical trials to demonstrate safety and effectiveness of various types of stem cells to treat COVID-19-induced health complications. It also suggests that there is a need in biomedical research and development to establish the most effective stem cell types that are ideally suited for the treatment of aforementioned complications. These developments will also require (a) GMP compliant technologies to mass produce stem cells, and (b) testing platforms that mimic human pathophysiology  to allow high-throughput screening and rapid testing of stem cells safety and efficacy.Extracellular vesicles (EVs) are emerging as an attractive alternative to the whole cell–based therapy. EVs have several advantages compared to the whole cell therapy including lower risk of tumorigenic effects, lower susceptibility to damage by hostile disease microenvironment and possibility for long-term storage. The long-term storage is fundamental to make the treatment accessible in developing countries and it circumvents the need to have expensive GMP cell manufacturing facilities on-site. Nevertheless, production of EVs must follow the same strict guidelines that apply to stem cells and any EV-based therapy needs to be approved by the governing bodies after being tested in clinical trials to demonstrate the safety and efficacy.in vivo environments on the integrity of these cells. Stem cells can be environmentally preconditioned by certain stimuli such as hypoxia or ischemia, which can induce certain signaling pathways to improve engraftment, survival, and function of transplanted cells in harsh environments within the injured lung  will potentially be a paradigm shift. In the light of a recent study that demonstrated efficacy of inhaled stem cell-derived therapy in both The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this report, we present a detailed comparison of the lipid composition of human milk (HM) and formula milk (FM) targeting different lactation stages and infant age range. We studied HM samples collected from 26 Polish mothers from colostrum to 19 months of lactation, along with FM from seven brands available on the Polish market . Lipid extracts were analysed using liquid chromatography coupled to high-resolution mass spectrometry (LC–Q-TOF–MS). We found that the lipid composition of FM deviates significantly from the HM lipid profile in terms of qualitative and quantitative differences. FM had contrasting lipid profiles mostly across brands and accordingly to the type of fat added but not specific to the target age range. The individual differences were dominant in HM; however, differences according to the lactation stage were also observed, especially between colostrum and HM collected in other lactation stages. Biologically and nutritionally important lipids, such as long-chain polyunsaturated fatty acids (LC-PUFAs) containing lipid species, sphingomyelines or ether analogues of glycerophosphoethanoloamines were detected in HM collected in all studied lactation stages. The observed differences concerned all the major HM lipid classes and highlight the importance of the detailed compositional studies of both HM and FM. Human m infants . However infants . HM lipiThe World Health Organization (WHO) and United Nations Children’s Fund (UNICEF) recommends exclusive breastfeeding for six months after birth and continued breastfeeding along with appropriate complementary foods for up to two years of age or beyond, while human milk is a source of a wide range of different valuable components . HoweverMany studies have shown that human milk metabolite composition differs significantly from the composition of milk formula due to the dynamic nature of HM and unique chemical structure of HM lipids ,14,15,16Lipidomics is now widely used in the comprehensive analysis of the lipidomes of various biological samples and is a valuable tool to analyse human milk ,28,29,30Besides these studies, data concerning lipid composition differences between HM and FM, especially regarding advanced lactation (HM after 1 year postpartum) and corresponding growing-up formula, are remarkably scarce. Filling this knowledge gap is necessary to further determine the impact of these components on infant health, growth, and development, as well as to provide suitable nutrition for all infants.In this study, we performed comparative LC–MS-based lipidomic analysis of the HM and FM samples to evaluate the differences between HM at different lactation stages, including HM samples at advanced lactation (>12 months postpartum) and the corresponding FM. The lipid fingerprints were compared and analysed using statistical and/or chemometric methods in terms of differences in the lipid composition: (i) within the HM samples collected in various stages of lactation; (ii) within the FM samples with various age range targets and fat sources; (iii) between the HM and FM samples.To our best knowledge, this study is the first report to comprehensively describe differences in HM and FM lipidomes regarding the lactation stage, including longitudinal changes in HM samples collected after 1 year postpartum.LC–MS-grade methanol, 2-propanol, and HPLC-grade chloroform were purchased from Merck . Chemicals—ammonium formate (99.9% purity), formic acid and ammonium hydroxide solution (28%)—were purchased from Sigma-Aldrich . All aqueous solutions were prepared with ultra-pure water produced by an HLP5 system . The lipid standards 1-pentadecanoyl-2-oleoyl(d7)-sn-glycero-3-phosphoethanolamine (15:0-18:1-d7-PE) and 1,3-dipentadecanoyl-2-oleyol(d7)-glycerol (15:0-18:1-d7-15:0 TG) were purchased from Sigma-Aldrich .n = 11); samples collected between 0 and 6 months (n = 10); samples collected between 6 and 12 months (n = 8); samples collected after 12 months (n = 16). Colostrum samples were obtained by a qualified midwife at the Obstetric Clinic, University Clinical Centre of the Medical University of Gdańsk. All volunteering mothers were given oral and written instructions for the standardised collection of milk samples. Written informed consent was obtained from each participant. The milk samples were obtained by the full expression of one breast using an electronic breast pump in the morning and/or in the evening. After collection, 10 mL of HM was transferred to the polypropylene laboratory tube and kept frozen at −20 °C until delivery to the laboratory. Samples were stored at −80 °C until analysis for a maximum of three months.Human milk samples were donated by 26 healthy women living in Pomeranian Voivodship, Poland, who had delivered healthy full-term neonates and had met the criteria of inclusion to the study , follow-on formula (age range target 6–12 months of life), and the growing-up formula milk  of seven brands were included in the study. The detailed characteristics of all analysed samples are presented in Research ethics approval was obtained from the Human Research Ethics Committee of the Medical University of Gdańsk, Poland .Sample preparation was performed using a previously developed extraction method based on the LLE and SPE techniques . The HM v/v) and vigorous mixing for 10 s. After that, 310 μL chloroform and 310 μL deionised water were added. Next, the samples were mixed for another 30 s and centrifuged for 10 min at 4300 rpm to separate the aqueous and organic phases. The upper phase was discarded and the lower phase was transferred to a new borosilicate glass tube by a Pasteur pipette. Next, 20 μL of the obtained extract was diluted by adding 980 μL methanol to further dissolve the SPE extract.A total of 225 μL of the milk sample was transferred to a borosilicate-glass tube with a PTFE cap, followed by the addition of 950 μL of a chloroform/methanol mixture  15:0-18:1-d7-PE were added. The contents were mixed for 30 s at 2000 rpm, followed by centrifugation for 10 min at 10,000 rpm. After that, 900 μL of the supernatant was loaded onto a HybridSPE-Phospholipid  cartridge , washed in sequence with 2 × 1 mL methanol and 2 × 1 mL 2-propanol. Finally, the phospholipids were eluted with 2 mL 5% ammonia in methanol. The extract was evaporated under a nitrogen stream at 35 °C. Next, 90 μL of the diluted LLE extract was used to dissolve the dried phospholipid extract. Additionally, 10 μL internal standard (2) 15:0-18:1-d7-15:0 TG was added to the final extract, which was then transferred to a 1.5-mL chromatographic vial and subsequently analysed by LC–Q-TOF–MS.v/v)) and component B (2-propanol). The mobile phase was pumped with a flow rate of 0.3 mL/min. The gradient elution program was initiated with 20% component B, which was ramped to 40% from 0 to 20 min, then from 40% to 60% from 20 to 40 min and finally from 60% to 100% from 40 to 45 min. The column was then equilibrated with the starting conditions for 10 min. The total run time was 55 min, and the injection volume was set to 0.5 μL. The data were collected in the positive ion mode using the SCAN acquisition mode in a range from 100 to 1700 m/z in the high-resolution mode (4 GHz). MS analysis was carried out using the following parameters: capillary voltage, 3500 V; fragmentation voltage, 120 V; nebulising gas, 35 psig; drying gas temperature, 300 °C. MS/MS analysis was performed using identical chromatographic and ion source conditions. The collision energy was set to the following values: 35 V and 80 V. The two most abundant peaks were selected for fragmentation and excluded for the next 0.3 min. The MS/MS spectra were acquired in the m/z range of 50–1700. Lipid extracts were injected randomly using one Quality Control (QC) sample  injected every five real samples for the LC–MS stability control. The LC–MS batch started with the extraction blank and the five subsequent QC samples to equilibrate the chromatographic column. The lipid extracts were kept in the autosampler at 10 °C during the batch run.Lipid analysis was conducted using an LC–Q-TOF–MS system—an Agilent 1290 LC system equipped with a binary pump, an online degasser, an autosampler and a thermostated column compartment coupled to a 6540 Q-TOF–MS with a dual electrospray ionisation (ESI) source . Lipid extracts were injected into a reversed-phase column Poroshell 120 EC-C8, 2.1 × 150 mm, 1.9 μm particle size  with a 0.2-μm in-line filter. The column was maintained at 60 °C. The mobile phase comprised component A  and (2) manual interpretation of the obtained MS/MS spectra of milk samples. Identification resulted in the determination of the lipid class, number of carbon atoms, and number of unsaturated bonds in fatty acid residues, as well as the presence of ether bonds instead of ester bonds in the lipid structure . The fatty acid composition was evaluated based on MS/MS spectra interpretation. Lipid species with ether-linked substituents were not differentiated regarding ether and vinyl ether bonds in position sn-1. The position of fatty acyl substituents and the position of double bonds were not evaluated. The fragmentation lipid patterns for TGs, DGs, PEs, PCs and SMs were previously published [m/z 184.0726 for confirmation of the SM and PC identity; neutral loss of 141.02 Da for the confirmation of the PE identity; neutral loss of 185.01 Da for the confirmation of PS identity; m/z 264.27 for confirmation of the C18 sphingoid base backbone.Lipid identification was carried out using the two-step procedure: (1) a custom HM database automated search based on an accurately measured ublished . The fra+; isotope model, common organic ; charge state range, 1–2; MFE score, ≥70. Next, the peak areas of the identified lipids  filtering on peak height, 1000 counts. In both data pre-treatment approaches, the .cef files were exported and imported to Mass Profiler Professional 15.1 software  for data alignment and filtration. Missing values were exported as missing. The alignment parameters were set as follows: alignment slope = 0.0%; alignment intercept = 0.4 min; mass tolerance slope = 20.0 ppm; intercept = 2.0 mDa. Filtration was based on frequency (the MFs remained in the dataset if they were present in 80% of the samples in at least one specified group) and the QC %RSD . The MFs that were present in the extraction blank, with the average peak volume higher than 10% of the average peak volume in the real samples, were removed. The statistical analyses and fold change calculation  were conducted using Mass Profiler Professional 15.1 software  The parameters in the statistical tests were as follows: p ≤ 0.01; Multiple Testing Correction: Benjamini–Hochberg; p-value computation—asymptotic; missing values were excluded from the fold change and p-value calculations ; corrected p-value cut-off, 0.01; Post-hoc test: Tukey HSD(for ANOVA). Statistical tests and fold change calculations were conducted using the average peak area of samples within defined group of samples. The Metaboanalyst 4.0 web tool [The Molecular Feature Extraction (MFE) algorithm, implemented in the Agilent MassHunter Workstation Profinder 10.0 , was used to extract the total molecular features (MFs) from the raw LC–MS data using the following parameters: ion threshold, >1000 counts; ion type, Hweb tool  was usedp < 0.01)The analytical procedure adapted from our previous studies  allowed First, we studied the detailed composition of human milk according to the lactation stage. We analysed 90 lipid extracts of 45 samples of human milk collected from 26 women. The highest number of molecular features (MFs) was detected in the colostrum samples , as shown in The lipidome coverage of both colostrum and mature (HM) comprised lipid species belonging to the classes of triacylglycerols (TGs), diacylglycerols (DGs), monoacylglycerols (MGs), sphingomyelines (SMs), glycerophosphoethanoloamines (PEs), glycerophosphocholines (PCs), glycerophosphoinositoles (PIs) and glycerophosphoserines (PSs). We also detected ether analogues of PE, PC, and TG containing one ether-linked fatty acid in its structure apart from the acyl-linked FA. The percentage relative amount of many lipid species within all detected lipid classes was diversified between individual mothers and lactation stage, with the highest difference between colostrum and other lactation stages HM, as shown in PCA was used to visualise the differences in the lipid pattern among the collected HM samples. The results of the unsupervised analysis are shown in n = 215) were statistically significantly changed (p < 0.01) with a fold change (fc) ≥2 among colostrum and mature HM samples, with the peak area of 76 lipid species higher in mature milk and that of 40 lipid species higher in colostrum. The detailed list of the lipid components that were statistically different between colostrum and mature milk is shown in To further study the difference between the colostrum samples and mature milk samples, we performed statistical tests using the dataset containing only the identified lipids. Fifty-five percent of the lipid compounds included in the test  was higher in mature HM (mainly medium-chain triacylglycerols (MCTGs) and long-chain triacylglycerols (LCTGs) with a low level (1–3 double bonds) of unsaturation (HM 0–6 months and HM >12 months)) than in colostrum. However, the content of some TGs was significantly higher in colostrum samples than in mature HM, corresponding to TGs containing long-chain polyunsaturated fatty acids (LC-PUFAs)  or TG 58:5, which contains a 22:4-18:0-18:1 fatty acyl composition (fc = 3.5)). Interestingly, the value of a fold change of many TG species was lower between colostrum and HM 6-12 months than the value of a fold change between colostrum and HM 0-6 months and HM >12 months .p < 0.01, fc = 3.5), TG-O-52:1  and TG-O 50:1 —which were all upregulated in colostrum samples.The colostrum samples also had a higher content of TGs containing fatty acyl substituents with more than 20 carbon atoms, such as TG 62:3 and TG 62:4, than mature milk (average fc >20). This also concerned alkyldiacylglycerols (captioned here as TG-O)—TG-O-52:2  and PE-O 38:5 (fc = 2.0), which contain LC-PUFAs eicosatetraenoic acid (C20:4) in their structure, PC-O 34:1 (fc = 1.8) and more PC34:0 (fc = 2.2), PC32:0 (fc = 2.8) and PC30:0 (fc = 2.7) containing saturated fatty acids compared with mature HM.We observed an increased content of PI38:4 (fc = 4.0) and PS36:1 (fc= 3.1) in colostrum compared with mature milk and some species of SM . However, the content of many other lipid species of these classes was higher in mature milk. Further exploration of HM lipid composition dynamics by visualising the variance in HM specific lipids, according to the four lactation stages on whiskers and box plots, showed that the lipid composition pattern is even more ambiguous, as shown in Crypthecodinium cohnii or Mortierella alpine). It was not always possible to determine whether FM contained isolated DHA or ARA from oils or contained them in the bound form in other lipid molecules, such as TGs. One of the formulas (FM.4.1.MFGM) was claimed to be enriched in milk fat globule membranes. The detailed characteristics of milk formulas and contained fat sources are presented in Next, we investigated the lipid composition of the milk formulas available on the Polish market. We studied infant formulas from seven brands, including starting formulas (0–6 months), follow-on formulas (6–12 months), and growing-up formulas . The lipid fraction of infant formulas was based on various sources of fat, including caprine whole milk, vegetable oils, fish oil, and microorganism lipids  between the FMs classified into two groups (soy-lecithin supplemented FM and caprine milk FM), as indicated by PCA, are presented in Generally, we did not observe grouping of the lipid fingerprints based on the age range target; however, the lipid fingerprints of the growing-up formulas of brands one, two and six  form a subgroup within the group of soy-lecithin-based FM, likely associated with the absence of coconut oil compared with the starting and follow-on formulas from the same brand. The lipid fingerprints of one of the studied FMs  were positioned on the PCA score far from two groups of FM samples and correspond to FM enriched in milk fat globule membranes. Some of the statistically significant differences (Detailed insight into the variance in the lipid composition among the studied FM samples with respect to different brands and specific age targets was obtained by heatmaps presented in p < 0.01) higher content of many lipid species belonging to the MCTs and LCTs with a low unsaturation level  and with an odd total number of carbon atoms in FAs compared with FMs not supplemented with caprine milk . However, soy lecithin-supplemented FMs contained a higher level of some LC-PUFA TGs than caprine-based FMs .The differences were observed in the content (measured by peak area fold change) of many medium-chain triacylglycerols (MCTGs) and long-chain TGs (LCTGs). FM with caprine whole milk  contained a significantly  were richer in phospholipids than FMs not containing this ingredient.For SM species, no specific trend of differences was observed, the content of some SM molecules was higher and others were lower in the FM caprine whole milk-based samples than in soy lecithin-supplemented FM.The observed results show that the lipid composition is generally brand-specific and depends strongly on the used fat source.Next, we compared the lipid composition of the HM and FM samples to explore the differences between them. For the statistical testing of differences at various lactation stages between HM and FM, all FM samples dedicated to the particular age target range were considered as one group without differentiation based on the fat source. We considered colostrum and HM samples collected in the period of 0–6 months separately to evaluate in detail the differences between HM samples obtained in this lactation stage and starting FM, because infants who cannot be breastfed directly after birth with colostrum are fed with FM dedicated for infants in age range 0–6 months.p < 0.01) between HM and FM samples is presented in The short-list of lipids significantly different  differences included MCTGs whose content was higher in formula milk than in HM, especially saturated TGs with the number of carbon atoms in fatty acid substituents lower than 40. Additionally, HM samples contained a higher content of TGs containing LC-PUFAs, such as DHA C22:6 , DPA C22:5 (in TG58:7) and ALA C18:3 (in TG50:4), compared with FM. These differences were the highest between colostrum and FM dedicated for infants aged 0–6 months but also between HM collected after 12 months postpartum and the corresponding FM  or TG 56:6 (fc = 53), TG58:7 (22:5-18:1-18:1) and TG58:7 (22:6-18:0/18:1)). In further lactation stages, the largest difference between HM and FM was observed in the content of TGs containing both MCFAs and LCFAs in its structure ). Moreover, the difference was observed in the fatty acid substituents of TG 50:2 between HM and FM samples, as shown in Statistically significant (p < 0.01) between the studied groups of FM and HM samples concerning the lactation stages. In particular, a higher peak area fold change was observed between samples of growing-up FM and HM samples collected after 1 year . Considering differences between colostrum and FM, the greatest differences corresponded to SMs with an LCFA residue attached to the d18:1 sphingoid base, such as SMd38:1 (fc = 40), SMd44:2 (detected only in HM), and SMd42:2 (fc = 24).Different lipid patterns were also observed for SMs. In HM, the most abundant SM was SMd42:2 (15–34%), while it comprised, on average, only 2–4% of the total SMs in FMs. SMd39:1 comprising 11–15% of the SM fraction in soy-supplemented FM was detected in HM samples in a much lower amount (0.05–0.69%). These lipid species were also statistically significantly different .A diversified lipid pattern was also observed for PCs. In HM samples, the percentage relative amounts of PC34:1 and PC32:0 were the highest in colostrum and were decreased in further stages of lactation along with an increase in percentage relative amounts of PC36:2 and PC34:2. In FM samples, percentage contribution of these components was almost equal in all FMs concerning the age range target. Regarding these lipids separately, the content of some PCs was statistically significantly higher in FM than in HM; however, that of the others was lower .The FM samples contained lipid species not detected in HM, such as TG66:18 or PI34. On the other hand, HM contained lipids that FM lacks or has at very low levels, such as PE-O36:5 and PE-O38-5, ether analogues of TGs, some LC-PUFAs TGs, SMs and PIs.Human milk is considered an ideal source of nutrients and bioactive compounds for a new-born; however, it remains a high-value liquid in further stages of lactation . AccordiSchizochytrium sp.) and predicting the FM lipid composition is not possible based on the list of ingredients. This raises a question about the similarity of the lipid composition of FM, its bioavailability, fate in the digestive system, biological functions compared with HM, and how it affects infants. Evaluation of the impact of feeding type on an infant’s growth and development should be performed using knowledge about the chemical composition of the food. Therefore, the knowledge gap regarding the molecular composition of HM and FM should be fulfilled. This should also concern lipid composition because the biological roles of these lipid components are of great importance for infant proper growth and development [FMs are designed to mimic human milk composition; therefore, various sources of lipids are added to FM to mimic the chemical composition and nutritional function of HM, such as vegetable , fish and microorganisms oils, but also whole caprine milk. The detailed lipid compositions of some of these oils are not known in the literature  of various lipids within specific classes of lipids.Analysis of the lipid composition of the HM samples collected at various stages of lactation showed the most significant difference between colostrum samples and the remaining HM samples. Samples collected in periods from the first to the 19th month of lactation showed no clear pattern according to child age as designated by FM dedication, namely 0–6 months, 6–12 months and >12 months.The individual differences were dominant among analysed samples. Based on statistical tests, specific lipid species within the lipid class undergo different trends during lactation ; however, we observed a contrasting trend for other molecules. Additionally, the individual HM samples were characterised by a higher concentration of specific compounds than the other samples. It is not without meaning when we consider the individual nutritional requirements of growing and developing infants. The intra-population variance of the HM samples can be determined by many factors, including maternal diet. The influence of maternal diet on the HM fatty acid composition has been very well documented ,39,40,41Especially alarming is the distinct lipid composition of FM from colostrum and mature milk obtained during advanced lactation (after 1 year postpartum) or lack of some lipid compounds in FM. This condition can possibly lead to the loss of intake of biologically important lipids by infants when fed with starting formula milk and at an age after 12 months when only a small number of children are still breastfed. Our results also proved that, at the advanced lactation (after 1 year postpartum), lactation milk is still rich in lipid species. This includes easily digested and accessible sources of energy—triacylglycerols—and also lipid species containing LC-PUFAs, such as arachidonic acid , linolenic acid , docosahexaenoic acid , docosapentaenoic acid , eicosatetraenoic acid  and adrenic acid (C22:4). LC-PUFAs are fatty acids known for their great nutritional value because they are important components of the brain and retina; for example, the concentration of DHA in an infant’s brain increases until at least 2 years of age ,43.On average, the most abundant TGs in the collected HM samples were as follows: TG52:2 (18:1-16:0-18:1), TG 52:3 (18:1-16:0-18:2), TG 50:2 (18:1-16:0-16:1) and TG 54:3 (18:1-18:1-18:1). It is a slightly different composition than that reported in other studies of the composition of TGs in HM ,26,44,45In contrast to HM, differences in the lipid composition within FM samples were generally brand-specific and linked to a contained fat source. The analysed FM samples contained a distinct TG pattern compared with human milk. All of the studied FMs contained a higher content of MCTGs and SCTGs than HM. However, milk formula with the addition of whole caprine milk was characterised by a higher content of these compounds than FM with other sources of fat. MCTGs and SCTGs are added to formula milk to directly provide energy because of the lower level of lipase in new-born babies . These csn-2 position and unsaturated fatty acids primarily at the sn-1,3 positions in the TG structure enhancing the fat and calcium absorption [Crypthecodinium cohnii and detected in FM, described as rich in DHA. The composition of LC-PUFAs in TGs and the level of specific TGS in FM are distinct from that observed in HM; therefore, the bioavailability and digestibility, as well as the nutritional properties of TGs contained in FM should be investigated.Another important aspect of HM and FM compositional differences is the positional distribution of fatty acids in triacylglycerols, palmitic acid in the sorption . This unsorption . We coulsorption ,50. One We also detected ether analogues of TGs in HM samples (TG-O). Their content was statistically significantly higher in the colostrum samples than in the mature HM samples. Importantly, these compounds were not detected in many milk formula samples; if they were, their content was statistically significantly lower than that in HM. Information about these components in the literature is scarce. Recently, it was shown by Yu et al. that alkylglycerol-type (AKG-type) ether lipids are specific lipid signals of breast milk that are essential for healthy adipose tissue development  and therWe also observed that formula milk is also poor in the ether analogues of glycerophosphoethanolamines. In many formulas, the concentration level was below the detection limit. In other formulas, the content was significantly lower than that in HM. The PE-Os detected in HM contain LC-PUFAs in the structure, especially 20:4. This is in accordance with previous studies by Moukarzel et al. showing that human milk plasmalogens are highly enriched in long-chain PUFAs . We did sn-1 and sn-3 position) and phospholipids (LC-PUFA in the sn2 position) raises speculation of the possible dissimilar role of LC-PUFAs in these categories of lipids [Our study showed that HM also contains a higher concentration of other glycerophospholipids containing LC-PUFAs than FM, which contains a higher content of saturated phospholipids than HM. Although these LC-PUFAs are provided to the child by FM in the TG structure, the TGs and phospholipids might not be equivalent to dietary sources of PUFAs with different metabolic FA handling. In particular, it was shown that the brain accretion of AA and DHA was more effective for dietary phospholipids containing AA and DHA than for TGs . Moreovef lipids .Notably, we observed that phospholipids containing LC-PUFAs are present in the HM of all lactation stages. Interestingly, the content of many lipids known to be involved in the brain and neurological system development, such as PC and SMs, were higher in mature milk obtained during the advanced stage of lactation (after 1 year postpartum) than at earlier stages of lactation. For these compounds, the difference in the content between HM and FM was higher after the age of 1 year because the content of these compounds in FM was almost at the same level considering various age targets. The higher content of SMs in HM in the advanced lactation stage may be associated with the decreased number of those feeding with HM after introducing the complimentary food and providing the sufficient amount of these lipid compounds with HM.Differences in the content of phospholipids—not only GPs but also SMs—in FM and HM may refer to the differences in neurocognitive development between formula-fed and breastfed infants . ExogenoIn this study, we employed a lipidomic approach to comprehensively and semi-quantitatively compare the lipid composition of HM at various lactation stages and FM with different age range targets. The results of our study clearly showed that human milk and formula milk vary within all milk lipid classes. Although HM lipid composition varies individually between mothers and in the stage of lactation, FM lipid content differs concerning the fat source and brand used. Human milk lipid components change over the course of lactation. However, no trend can be indicated because this process is lipid species-specific. We also observed a higher content of lipids related to neurodevelopment in samples collected after 1 year postpartum than at the earlier lactation stages. We did not observe similar changes in formula milk samples. HM contains different contributions of lipid species within specific classes of lipids than FM and contains a higher content of many biologically important lipids than FM, such as ether analogues of glycerophosphoethanolamines. Detailed knowledge about changes in the lipid composition of HM and FM, including a long-term perspective, is required to investigate the impact of lipid components on child health and development and to move from standardised nutritional protocols to tailored, individualised nutrition in infants .


Nonalcoholic fatty liver disease (NAFLD) with pathogenesis ranging from nonalcoholic fatty liver (NAFL) to the advanced form of nonalcoholic steatohepatitis (NASH) affects about 25% of the global population. NAFLD is a chronic liver disease associated with obesity, type 2 diabetes, and metabolic syndrome, which is the most increasing factor that causes hepatocellular carcinoma (HCC). Although advanced progress has been made in exploring the pathogenesis of NAFLD and penitential therapeutic targets, no therapeutic agent has been approved by Food and Drug Administration (FDA) in the United States. Gut microbiota-derived components and metabolites play pivotal roles in shaping intrahepatic immunity during the progression of NAFLD or NASH. With the advance of techniques, such as single-cell RNA sequencing (scRNA-seq), each subtype of immune cells in the liver has been studied to explore their roles in the pathogenesis of NAFLD. In addition, new molecules involved in gut microbiota-mediated effects on NAFLD are found. Based on these findings, we first summarized the interaction of diet-gut microbiota-derived metabolites and activation of intrahepatic immunity during NAFLD development and progression. Treatment options by targeting gut microbiota and important molecular signaling pathways are then discussed. Finally, undergoing clinical trials are selected to present the potential application of treatments against NAFLD or NASH. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting about 25% of the global population . The preFactors such as lipotoxicity and inflammation can drive NAFLD progression to NASH and ending stage of liver disease . Gut micLactobacillus and Bifidobacterium spp., resulting in amelioration of adipose tissue inflammation in obesity and NAFLD [Diet plays an important role in modulating gut microbiota and metabolic pathways in the development of NAFLD ,11. Consnd NAFLD ,14. In aChange of intestinal or hepatic metabolites impacts intrahepatic immune cell profiles, as well as the expression of proinflammatory cytokines and chemokines in the fatty liver. Innate immunity plays an essential role in NAFLD or NASH pathogenesis. For example, the frequency of macrophages was increased in the NASH liver in amylin liver NASH (AMLN) diet-fed mice compared to that in standard chow diet-fed mice . Our resHowever, the role of diet and gut microbiota interaction-derived metabolites in modulating intrahepatic immune response remains to be explored. A better understanding of the underlying molecular mechanism is helpful to find a new therapeutic target for NAFLD or potential diagnostic marker. For this purpose, a search was conducted in PubMed, Web of Science, Google Scholar, and Embase with the keywords including NAFLD or NASH, metabolite, gut microbiota, signaling pathway, and immune response in the last five years. The originally retrieved publications were independently reviewed by two authors. The inclusion criteria were (1) the study contained at least three keywords, (2) either animal or human studies. Excluding criteria included (1) studies were abstracts or unpublished studies, (2) studies with similar findings from another study. All the rest studies were carefully reviewed by the authors, and representative findings in the last five years were selected. Few supporting studies prior to this period were added to explain the underlying mechanism.In this review, we first summarize the latest findings of metabolites that are implicated in the development of NAFLD, as well as the progression to NASH. Then, we investigate the underlying cellular and molecular mechanisms of these metabolites in hepatic immunity in animal models to study NAFLD or NASH or clinical samples. Finally, we summarize the currently ongoing clinical trials to evaluate potential therapeutic reagents by targeting key molecules or proteins for NAFLD and NASH treatment.LPS, a major component of Gram-negative bacterial cell membrane, plays a pivotal in the pathogenesis of mouse and human NAFLD via Toll-like receptor 4 (TLR4) signaling pathway . In addiPlasma amino acids (AAs), such as glutamate and valine, are shown to increase in NAFLD patients with or without obesity compared to non-NAFLD controls . Hoyles Bile acids (BAs) play important roles in NAFLD pathogenesis by modulating hepatic lipid and glucose metabolism, consisting of primary and secondary BAs . PrimaryFirmicutes [Choline can be metabolized by the gut microbiota to trimethylamine (TMA), which is absorbed in the liver and further converted to trimethylamine N-oxide (TMAO) by flavin-containing monooxygenase 3 (FMO3) . In addirmicutes ,33,34,35rmicutes .Klebsiella pneumoniae) to mice can increase ethanol production, increase liver injury, and impair mitochondrial function in mice, indicating a causative factor for NAFLD [Excessive consumption of alcohol causes alcohol fatty liver disease (AFLD). Endogenous ethanol produced by gut microbiota can impair mitochondrial function and promotes NAFLD development . Gavage or NAFLD . Fastingor NAFLD . Furtheror NAFLD . Thus, eDietary fibers (DF) consist of carbohydrate polymers resistant to digestive enzymes in the small intestine, which can be digested by bacteria in the large intestine . DF can Bifidobacterium and Lactobacillus genera compared to placebo or low-fiber consumption, which is associated with a high concentration of butyrate in feces [Fermentation of DF can impact the diversity of gut microbiota. For example, a meta-analysis revealed that DF intervention can increase the abundance of in feces . Consumpin feces .SCFAs, consisting of acetate, propionate, and butyrate, are produced by gut microbiota from dietary fibers and starch. They play important roles in energy metabolism, tissue homeostasis, and immune regulation. Here, we discuss their roles in the pathogenesis of NAFLD.Ruminococcus flavefaciens and portal acetic acid concentration, resulting in a reduction in hepatic fat accumulation [Oral administration of branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, significantly increased the abundance of gut mulation . In addimulation .A randomized controlled trial study showed that dietary supplementation with inulin that is mainly metabolized into acetate in the colon increased intrahepatocellular lipid. In contrast, dietary supplementation of inulin-propionate ester, which is designed to deliver propionate to the colon and to attenuate the acetate-mediated increase in intrahepatocellular lipid .Akkermansia muciniphila and butyrate and sugar expenditure in the distal intestine [Supplementation with grape polyphenols reduced Western diet (WD)-induced adiposity and hepatic steatosis in mice by increasing the abundance of ntestine .Overall, the dietary metabolites or metabolites derived from gut microbiota impact the progression of NAFLD and NASH .The intrahepatic immune response plays an essential role in the progression of NAFLD/NASH. Gut microbiota-derived metabolites and components circulating in the portal vein system can enter the liver to modulate intrahepatic immunity to impact NAFLD. This process is involved in a complicated communication among different liver non-parenchymal cells, including macrophages, monocytes, T cells, B cells, neutrophils, and HSCs . Herein,The composition of liver macrophages was altered in mice fed a high-fat high-sucrose diet (60% fat and 10% sucrose), with a decrease in liver resident macrophage Kupffer cells (KCs) and an increase in monocyte-derived macrophages (MdMs) detected by single-cell RNA sequencing (scRNA-seq) . A subsebrightNK cells decreased in intrahepatic lymphocytes in NAFLD patients, while CD56dimNK cells increased compared to that in healthy controls, indicating the complex roles of each subtype of NK cells in NAFLD [+ NK cells can inhibit the progression of NASH and liver fibrosis via suppressing the expression of profibrogenic genes as well as M2 polarization (anti-inflammatory phenotype) of liver macrophages [The number of natural killer (NK) cells was increased in a methionine- and choline-deficient diet (MCD)-induced mouse NASH liver via C-X-C motif chemokine ligand (CXCL)10/chemokine receptor (CXCR)3 signaling . These iin NAFLD . Naturalrophages . TherefoClostridium spp. induced secondary bile species (sBAs) activated liver sinusoidal endothelial cells (LSECs) to produce the chemokine CXCL16 to attract accumulation of hepatic CXCR6+NKT cells [Activation of invariant natural killer T (iNKT) cell subsets was shown in choline-deficient L-amino acid-defined HFD (CDAHFD)-induced murine NASH, accompanying the accumulation of plasmacytoid dendritic cells (pDCs) . In addiKT cells . CD1d-deKT cells . OverallNeutrophils are one of the first response cells that are recruited to the injury site to participate in the inflammatory response and tissue repair. Neutrophil depletion treated with antibody 1A8 (200 μg/mouse per week for four times) can reduce body weight gain and attenuate liver lipid accumulation with activation of lipid β-oxidation in HFD-fed mice compared to mice treated with isotype control . Neutrop+ T cells play different roles in NAFLD pathogenesis. Fatty acid composition  can modulate the frequency of CD4+ T cell profiles in PBMCs of NAFLD patients, with an increase in CD25+CD45+CD4+ T cells and a decrease in PD1+CD4+ T cells [Different subtypes of CD4 T cells .+ T helper 17 (Th17) cells and concomitant expression of IL-17a, interferon (IFN)-γ, and TNF-α, resulting in NAFLD progression [+CD4+ T cells were significantly increased in the liver during NAFL to NASH progression [Obesity increased the accumulation of inflammatory hepatic CXCR3gression . Cellulagression . The ratgression . IL-17+Cgression . The incgression .Hepatic infiltration of Tregs was increased in CD62L-deficient mice, which was associated with less hepatic lipid accumulation, reduced liver fibrosis, and improved insulin resistance . However+ T cells were increased in obese patients with NASH, which was associated with the expression of α-SMA, a marker of HSC activation [+ T cells reduced hepatic macrophages and α-SMA expression in obesity or hyperlipidemia-induced NASH mice, but not in lean mice [+ T cells in mice with NASH [+ T cell accumulation and activation with the expression of proinflammatory cytokines, but not CD4+ T cells and NK cells. Ex vivo studies revealed that microbiota-derived extracts in NAFLD-HCC patients compared to that can induce an immunosuppressive phenotype in human PBMCs, characterized by a suppression of CD8+ T cells and expansion of Tregs [+ T cell activation and suppresses its cytotoxicity to tumor cells by inducing immune tolerance.Liver CD8tivation . Depletiean mice . RNA-seqith NASH . Perforiof Tregs . NAFLD pFecal microbiota transplantation (FMT) of gut microbiota from human NAFLD patients into recipient mice can accelerate NASH progression via inducing accumulation and activation of liver B cells . ScRNA-s+ T cells can trigger apoptosis of activated HSCs via Fas /FasL-mediated signaling [Activation of HSCs, the major cells that contribute to liver fibrosis, is mediated by the activation of intrahepatic immunity during NASH. For example, proinflammatory cytokines such as TNF-α, transforming growth factor (TGF)-β1, and IL-1β expressed by intrahepatic macrophages can activate HSCs to promote the progression of liver fibrosis and NASH . In contignaling . TherefoThe recruitment of immune cells into the fatty liver plays a critical role in the pathogenesis of NAFLD/NASH. Chemokines and their receptors are the key factors involved in the recruiting process. For example, CCL2/CCR2 and CXCL9/10/CXCR3 signaling pathways are involved in the migration of myeloid cells and T cells ,76,77,78+CD4+ T cells was positively associated with hepatic steatosis, inflammation, and fibrosis via its ligand mucosal addressin cell adhesion molecule 1 in a Western diet (WD)-fed mice [Hepatic accumulation of integrins α4β7fed mice . Anotherfed mice .A soluble form L-selectin/CD62L was dramatically increased in the liver in patients with NASH. CD62L-deficient mice showed dampened NASH features compared to wild-type mice, including less hepatic lipid accumulation, reduced liver fibrosis, and improved insulin resistance . HepaticInfiltration of hepatic infiltration of macrophages in HFD-induced NAFLD mice was associated with an increase in hepatic Runx2 expression . Both inMany treatment agents have been tested in preclinical animal studies for the treatment of NAFLD or NASH with promising effects, including modulation of gut microbiota, FXR modulators, targeting chemokines and their receptors, anti-inflammatory or antioxidant agents, and modulation of fibroblast growth factors (FGFs) and microRNAs (miRNAs).A prospective cohort in Japan showed that the prevalence of NAFLD and NASH was 82.4% and 77.5%, respectively, in morbidly obese patients . BS treaClostridium difficile infection (CDI) induced by gut dysbiosis [Christensenellaceae and Lactobacillus and intestinal tight junction protein ZO-1, and reduced hepatic lipid accumulation, proinflammatory cytokines, and NAFLD activity score (NAS) in HFD-fed mice [FMT has been tested as a therapeutic strategy to prevent and treat different diseases associated with gut microbiota dysbiosis. FMT is a procedure to transfer healthy donor stool into the gastrointestinal tract of the patient in order to restore the balance of gut microbiota. For example, FMT is an effective and safe treatment for the recurrence and reduction in severe ysbiosis ,88. The ysbiosis , the indfed mice . In addifed mice . Still, Lactobacillus acidophilus (ATCC B3208), Bifidobacterium lactis (DSMZ 32269), Bifidobacterium bifidum (ATCC SD6576), Lactobacillus rhamnosus (DSMZ 21690) for 12 weeks showed reduced liver injury and a higher percentage of normal liver sonography in compared to placebo treatment [Bacteroides uniformis (CBA7346), a strain isolated from the healthy human gut, can ameliorate liver injury, inflammation, and lipid accumulation in NAFLD mice induced by feeding an HFD via improving insulin resistance and regulating de novo lipogenesis-related proteins, such as fatty acid synthase (FAS) and peroxisome proliferator-activated receptor-gamma (PPARγ) [Treatment with probiotics significantly ameliorated HFD-induced NAFLD in rats by decreasing the abundance of pathogenic bacteria and upregulating the bile acid receptor FXR/FGF15 signaling pathway . A meta-reatment . Adminis (PPARγ) .Clifford et al. showed that FXR activation both in mice and humans can specifically decrease the levels of monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in the liver . FXR agoChemokines and their receptors, such as CCL25 and CCR9, play important roles in the hepatic infiltration of macrophages and other immune cells in NAFLD/NASH ,98. Ther+ macrophages and expression of inflammatory cytokines IL-1β and TNF-α [Treatment with aldafermin, an engineered analog of FGF19, markedly reduced serum BAs, specifically hydrophobic BAs, such as DCA, lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA), and glycocholic acid (GCA) in NASH patients . In addind TNF-α .Natural polyphenols such as resveratrol with anti-inflammatory and antioxidant properties show potential efficiency against NAFLD . PolypheAdministration of hydro-alcoholic extract of spinach reduced the expression of proinflammatory cytokine TNF-α and enhanced the expression of PPAR-γ in the livers of NAFLD rats at prevention and treatment phases . DietaryMicroRNAs (miRNAs) play important roles in regulating cell apoptosis, migration, and lipid metabolism during the development of NAFLD , which m+/hiM2c-macrophages to NAFLD mice increased serum level of high-density lipoprotein (HDL) and decreased total NAFLD pathological score via reducing liver inflammation, cell death, and fibrosis [The above-discussed treatment options are summarized in a figure . In addifibrosis .Many treatment agents have been tested in clinical trials for the treatment of NAFLD or NASH with promising effects, including chemokine receptor antagonist , FXR agonist , modulation of FGF , PPAR agonist , diet intervention , anti-inflammatory or antioxidant agents , and modulation of gut microbiota . Representative trials were selected with clinical results in the last 5 years before 11 October 2021 .NAFLD is the most chronic liver disease in the global population, and its incidence increases with the prevalence of obesity and T2D. Currently, NAFLD is the most increasing factor to induce primary liver cancer, HCC. However, there are no currently available FDA-approved treatments for NAFLD. Gut microbiota-derived metabolites and components play pivotal roles in the development and progression of NAFLD. Preclinical studies and clinical trials have been processed to evaluate potential treatment options for NAFLD and NASH, including synbiotics, omega-3, CCR2/5 antagonists, FXR agonists, and so on. A combined treatment such as combined medical treatment and physical activity could reduce the treatment time and improve the outcome. Although preclinical animal studies show the effects of pre-/probiotics and FMT, more clinical trials are waiting to verify the efficacy of balancing gut microbiota profile in patients with NALFD/NASH. In the future, meta-omics, including metabolomics with bioinformatic analysis, should be applied to search for early diagnostic markers and therapeutic targets for NAFLD and NASH.