# The Preventive Effect ofLactobacillus plantarum ZS62 on DSS-Induced IBD by Regulating Oxidative Stress and the Immune Response

Authors: Yanni Pan; Yujing Ning; Jing Hu; Zhiying Wang; Xiufeng Chen; Xin Zhao
Journal: Oxidative Medicine and Cellular Longevity
 (2021)

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## Abstract

In this study, we used DSS to establish an IBD mouse model to study the preventive effect ofLactobacillus plantarum (L. plantarum) ZS62 on IBD in the context of oxidative stress and the immune response. We assessed the mitigating effect of this strain on IBD mice by examining the length of and histopathological changes in the colon, determining the serum antioxidant index and the levels of inflammatory cytokines, as well as the mRNA and protein expression levels of relevant genes. The study results showed that L. plantarum ZS62 could inhibit colonic atrophy in IBD mice, reduce the degree of colonic damage, downregulate the serum levels of MDA, MPO, IL-1β, IL-6, IL-12, TNF-α, and IFN-γ and the relative mRNA and protein expression of IL-1β, IL-12, TNF-α, COX-2, iNOS, and NF-κB p65 in mouse colon tissues, and upregulate the serum levels of CAT, T-SOD, and IL-10 and the relative mRNA and protein expression of Cu/Zn SOD, Mn SOD, GSH-Px, CAT, IL-10, and IκB-α in colon tissues. In summary, L. plantarum ZS62 exhibited a good preventive effect on DSS-induced IBD by regulating oxidative stress and the immune response.


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## Body

## 1. Introduction

Lactic acid bacteria are widely regarded as a probiotic, which is a class of gram-positive bacteria that can ferment carbohydrates to produce lactic acid [1]. Lactobacillus plantarum belongs to the genus Lactobacillus, which is the largest genus of lactic acid bacteria [2]. A number of in vitro and in vivo studies have shown that L. plantarum has many physiological functions that are beneficial to human health, such as anticancer, antitumor, and antiatherosclerotic by exerting antioxidant and immune-modulatory effects [3–6]. Research has shown that oral L. plantarum 299v supplementation improves vascular endothelial function and reduces systemic inflammation in humans with stable coronary artery disease, and circulating gut-derived metabolites likely account for these improvements [7]. The probiotic L. plantarum KU15149 derived from Korean kimchi was found to have antioxidant and anti-inflammatory effects [8]. Since L. plantarum has many beneficial physiological effects on the human body, the functionality of L. plantarum has become a research hotspot in the food and medicine fields in recent years. Therefore, to further explore the function of L. plantarum, this study used the strain L. plantarum ZS62, which was isolated and purified from traditionally fermented yak yogurt from the homes of Baleksu Kaisk grassland herders in Zhaosu County, Ili Kazak Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China. According to previous research reports in our laboratory, L. plantarum ZS62 has a protective effect on the stomach, and the stomach and intestine are often considered to be two inseparable organs that ensure the normal function of the gut [9, 10]. Therefore, we speculate that L. plantarum ZS62 may have the effect of protecting the intestine. Consequently, relevant experiments on the preventive effects of L. plantarum ZS62 on inflammatory bowel disease were conducted.Inflammatory bowel disease (IBD) is a group of chronic and recurrent inflammatory conditions in the bowel. Ulcerative colitis (UC) and Crohn’s disease (CD) are the two major phenotypes [11]. At present, IBD has been found to be a global disease, and reducing the prevalence and morbidity is essential to reduce the global burden of IBD [12]. A number of studies have pointed out that the colitis model induced by 3~5% dextran sodium sulfate (DSS) exhibits similar to the clinical symptoms of human IBD and has good reproducibility [13–15]. The possible mechanism by which DSS induces IBD is by destroying the intestinal mucosal barrier, inhibiting the proliferation of intestinal epithelial cells, and causing an imbalance in the intestinal flora, thereby generating an immune response [16]. The existing treatment methods for IBD include medication and surgery. Among them, medication mesalazine (5-aminosalicylic acid) and sulfasalazine (SSZ) are the common medications for the treatment of mild to moderate IBD. However, medications usually have side effects [17, 18]. In recent years, probiotic preparations have often been used to treat gastrointestinal diseases and can effectively alleviate the symptoms of gastrointestinal diseases. Lactobacillus bulgaricus has been reported to prevent or alleviate colon inflammation through antioxidant effects, immune regulation, etc. [19–21]. Therefore, in order to reduce the global burden of IBD, in this study, 5% DSS was selected to induce IBD, SSZ was used as a positive control for drug, and Lactobacillus bulgaricus was used as a positive control for lactic acid bacteria, which intends to use L. plantarum ZS62 to develop probiotic preparations to relieve the symptoms of IBD.Oxidative stress and the immune response play important roles in the pathological development of IBD [22]. The mechanism of some drugs that are used clinically to treat IBD is related to the elimination of excess free radicals in the body, which in turn proves that excessive oxidative free radicals are related to the development of IBD [23]. The body’s antioxidant system mainly plays a role in scavenging reactive oxygen species (ROS), including the enzymatic and nonenzymatic systems. Among them, the enzymatic system mainly refers to antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) [24]. In addition, IBD is an autoimmune deficiency disease, and patients are prone to immune dysfunction. Mucosal immunity plays an important role in innate immunity, including the epithelial barrier and mucosal immune cells [25]. Inflammatory effector T cells (Th1 cells) and anti-inflammatory regulatory T cells (Treg cells) in the lamina propria of the mucosa play important roles in adaptive immunity. Once the regulation of these two cell types is out of balance, the number of inflammatory T cells increases, the mucosal barrier is damaged, and intestinal inflammation occurs [26]. The occurrence of inflammation is accompanied by the release of cytokines, such as TNF-α, IL-6, and IL-10 [27–29]. Therefore, this study is aimed at simply clarifying the preventive effect of L. plantarum ZS62 on IBD through the mechanisms of oxidative stress and the immune response.In this study, we established a mouse IBD model by DSS to study the preventive effect ofL. plantarum ZS62 on IBD in the context of oxidative stress and the immune response. We assessed the mitigating effect of this strain on IBD mice by determining the serum antioxidant index and the levels of inflammatory cytokines, examining the length of and histopathological changes in the colon, as well as mRNA and protein expression levels of relevant genes, to clarify the mechanism by which L. plantarum ZS62 prevents IBD. Our results provide a theoretical basis and evidence for the in-depth study of lactic acid bacteria as IBD treatments and the development of new lactic acid bacteria preparations.


## 2. Materials and Methods

### 2.1. Experimental Strains

Our research team isolated and purified a new lactic acid bacteria strain from traditionally fermented yak yogurt from the homes of herders in Baleksu Kaisk grassland in Zhaosu County, Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China. The 16S rDNA sequence analysis showed that it was aL. plantarum strain, and the colony morphology of this strain was observed by gram staining. The strain was stored in the China General Microbiological Culture Collection Centre (CGMCC, Beijing, China) and named L. plantarum ZS62, and its preservation number was 18228. In addition, Lactobacillus bulgaricus was used as a positive control in this study and was purchased from the China Centre for Type Culture Collection (CCTCC, Wuhan City, Hubei Province, China), and its strain preservation number was CCTCC AB 200048.


### 2.2. Determination of the Tolerance of Experimental Strains in Artificial Gastric Juice

The artificial gastric juice was prepared with 0.2% NaCl (Beijing Solarbio Biotechnology Co. Ltd., Beijing, China) and 0.35% pepsin (Beijing Solarbio Biotechnology Co. Ltd.), the pH was adjusted to 3.0 with 1 mol/L HCl after preparation, and then, the juice was filtered with a 0.22μm filter (Tullagreen, Carrigtwohill, Ireland) and set aside for later use. 5 mL of the activated strain culture solution was placed in a 10 mL centrifuge tube and centrifuged at 3000 r/min for 10 min at room temperature. The supernatant was discarded, and the bacteria were collected. The collected bacteria were washed twice with sterile normal saline and then resuspended in 5 mL of normal saline to make a bacterial suspension. The prepared experimental bacterial suspension was mixed with the artificial gastric juice at a volume ratio of 1 : 9, and after shaking, 2 mL of the mixed solution was taken and stored for later use (0 h time point). The remaining 8 mL was incubated in a water bath shaker at 37°C and 80 rpm for 3 h. The number of viable bacteria was determined at 0 h and 3 h. The samples collected at 0 h and 3 h were subjected to a series of 10-fold dilutions, and the dilutions were incubated on de Man, Rogosa, and Sharpe (MRS; Becton, Dickinson and Company, Franklin Lake, New Jersey, USA) solid media in a constant temperature incubator at 37°C for 48 h; the plate counting method was applied for feasible counts [30]. The survival rate was calculated according to
(1)Survivalrate%=3hNumberofviablebacteriaCFU/mL0hNumberofviablebacteriaCFU/mL×100.


### 2.3. Determination of the Growth Efficiency of Experimental Strains in Bile Salt

The bacterial solution that was cultured overnight after activation was collected and inoculated into MRS-THIO media (the MRS medium contained 0.2% sodium thioglycolate) containing 0.0% and 0.3% bovine bile salt at an inoculum concentration of 2%; a blank medium (uninoculated MRS-THIO medium) was used as a control; all media were cultured in a constant temperature incubator at 37°C for 24 h. A microplate reader was used to measure the absorbance (OD) values of the media with different mass concentrations at 600 nm, and the growth efficiencies of the experimental strains in different concentrations of bile salt were calculated according to Equation (2) [31]:
(2)Growthefficiency%=BilesaltmediumOD600nmBlankmediumOD600nm×100.


### 2.4. Determination of the Surface Hydrophobicity of Experimental Strains

Strains with survival rates greater than 70% in artificial gastric juice were selected for this experiment. The surface hydrophobicity of the strain was determined by BATH (carbon-hydrocarbon adhesion method); a total of 5 mL of the cultured bacterial solution was collected and centrifuged at 3 000 r/min for 10 min to collect the bacterial cells. The cells were washed twice with 5 mL of PBS (50 mmol/L, pH 6.5; Beijing Solarbio Biotechnology Co. Ltd.). PBS was used as a blank control, and the concentration of bacteria was adjusted with PBS to achieve an OD560 nm value of approximately 1.00. 4 mL of the bacterial solution with the adjusted cell concentration was placed in a 10 mL sterile centrifuge tube, 0.8 mL of xylene was added, and the tube was shaken for 30 s, paused for 10 s, shaken again for 30 s, and allowed to stand for 5-10 min for stratification. The lower aqueous phase was removed, and the OD560 nm value was determined with PBS buffer as a blank control. The hydrophobicity was calculated according to Equation (3) (A0 and A are the OD560 nm values of the bacterial solution before and after mixing with xylene, respectively) [32].
(3)Hydrophobicityrate%=A0−AA0×100.


### 2.5. Experimental Animal Model

Six-week-old C57BL/6 mice (n=50, male, bodyweight: 20±2g) were purchased from the Experimental Animal Centre of Chongqing Medical University. The mice were housed in an environment with a temperature of 25±2°C and a relative humidity of 50±5%, with 12 h light/dark cycles. The mice were allowed free access to drinking water and standard mouse food, the pads were replaced every two days, and adaptive feeding lasted for one week. To study the preventive effect of L. plantarum ZS62 on DSS-induced IBD, the mice were randomly divided into 5 groups (10 mice in each group). The entire experiment lasted for 5 weeks, and the specific experiment plan was as follows: (1) normal group: standard food and drinking water, daily gavaged with 0.1 mL/10 g (mouse bodyweight) 0.9% normal saline; (2) DSS group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and were gavaged with 0.1 mL/10 g (mouse bodyweight) 0.9% normal saline daily; at week 4, normal drinking water was replaced with 5% DSS (MP Biomedicals, Santa Ana, CA), and the other conditions remaining unchanged; (3) L. plantarum ZS62 (ZS62) group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and gavaged with 0.1 mL/10 g of L. plantarum ZS62 at a concentration of 1.0×109 CFU/mL; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remaining unchanged; (4) sulfasalazine (SSZ) group: at weeks 1, 2, 3, and 5, the mice were fed with standard food and drinking water and gavaged with 0.1 mL/10 g sulfasalazine (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) at a concentration of 500 mg/kg daily; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remain unchanged; and (5) L. bulgaricus (LB) group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and gavaged with 0.1 mL/10 g of Lactobacillus bulgaricus at a concentration of 1.0×109 CFU/mL daily; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remain unchanged. After the last dose, the mice were fasted for 16-24 h, but were allowed free access to drinking water. The mice were killed by cervical dislocation, eyeball blood was collected, the mice were dissected, and the colon was separated for later use [15].


### 2.6. Histopathological Analysis of Colon Tissues

The colon tissue was washed with normal saline, cut into pieces of approximately 0.5 cm in size, and fixed in 10% formalin solution. The colon tissue was dehydrated with an ethanol gradient, soaked in xylene and ethanol for approximately 30 min for clarification, embedded in paraffin, cut into slices of approximately 2-3μm with a microtome, and fixed on a glass slide. Haematoxylin and eosin (H&E) were used to stain the cytoplasm in different shades of pink or red [15]. Finally, the morphological changes were observed under an optical microscope (BX43; Olympus, Tokyo, Japan).


### 2.7. Serum Index Determination

The obtained mouse blood was centrifuged at 4000 rpm for 10 min at 4°C. The mice serum was then separated, collected, and stored at -80°C for later use. Appropriate biochemical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) were used to determine the levels of the oxidation indicators CAT, T-SOD, MDA, and MPO in the serum according to the procedures recommended by the manufacturer.


### 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of the inflammatory cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α, and IFN-γ in the preserved mouse serum were determined according to the manufacturer’s instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).


### 2.9. Real-Time Quantitative PCR Assay

In this study, messenger RNA (mRNA) expression in mouse colon tissue was determined by the SYBR green method. The mouse colon tissue was cut into pieces, and the total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The RNA concentration was measured using a microspectrophotometer (Nano 300, Ao Sheng, Hangzhou, Zhejiang, China). Reverse transcription was performed with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Lithuania, Vilnius) to obtain the cDNA template. Then, amplification was performed on a StepOne Plus real-time PCR system (Thermo Fisher Scientific) with 10μL of SYBR green PCR master mix (Thermo Fisher Scientific), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of cDNA template, and 7 μL of DEPC. The conditions were as follows: predenaturation at 95°C for 3 min; then denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s for 40 cycles in total; the final dissolution curves were completed at 95°C for 30 s, 60°C for 30 s, and 95°C for 15 s. Finally, the relative expression level of each gene was calculated by the 2-ΔΔCT method, where CT was the cycle threshold, and β-actin was used as an internal reference gene [33]. Table 1 shows the primer sequence information used in this study.Table 1
Primer sequences of RT-qPCR assay.
Gene nameSequenceCu/Zn SODForward: 5′-AACCAGTTGTGTTGTCAGGAC-3′Reverse: 5′-CCACCATGTTTCTTAGAGTGAGG-3′Mn SODForward: 5′-CAGACCTGCCTTACGACTATGG-3′Reverse: 5′-CTCGGTGGCGTTGAGATTGTT-3′GSH-PxForward: 5′-GTGCAATCAGTTCGGACACCA-3′Reverse: 5′-CACCAGGTCGGACGTACTTG-3′CATForward: 5′-GGAGGCGGGAACCCAATAG-3′Reverse: 5′-GTGTGCCATCTCGTCAGTGAA-3′IL-1βForward: 5′-GAAATGCCACCTTTTGACAGTG-3′Reverse: 5′-TGGATGCTCTCATCAGGACAG-3′IL-12Forward: 5′-CCTCCACTGTGCTGGTTTTAT-3′Reverse: 5′-TCAGCAACATGCTCCAGAAG-3′TNF-αForward: 5′-CAGGCGGTGCCTATGTCTC-3′Reverse: 5′-CGATCACCCCGAAGTTCAGTAG-3′IL-10Forward: 5′-CCAAGCCTTATCGGAAATGA-3′Reverse: 5′-TTTTCACAGGGGAGAAATCG-3′COX-2Forward: 5′-TTCCAATCCATGTCAAAACCGT-3′Reverse: 5′-AGTCCGGGTACAGTCACACTT-3′iNOSForward: 5′-GTTCTCAGCCCAACAATACAAGA-3′Reverse: 5′-GTGGACGGGTCGATGTCAC-3′NF-κB p65Forward: 5′-TGCGATTCCGCTATAAATGCG-3′Reverse: 5′-ACAAGTTCATGTGGATGAGGC-3′IκB-αForward: 5′-CGAGACTTTCGAGGAAATACCC-3′Reverse: 5′-GTCTGCGTCAAGACTGCTACA-3′β-ActinForward: 5′-ATGGAGCCGGACAGAAAAGC-3′Reverse: 5′-TGGGAGGTGTCAACATCTTCTT-3′Cu/Zn SOD: cuprozinc-superoxide dismutase; Mn SOD: manganese superoxide dismutase; GSH-Px: glutathione peroxidase; CAT: catalase; IL-1β: interleukin 1β; IL-12: interleukin12; TNF-α: tumor necrosis factor-alpha; IL-10: interleukin 10; COX-2: cyclooxygenase-2; iNOS: inducible nitric oxide synthase; NF-κB p65: nuclear factor κ-light-chain-enhancer of activated B cells; IκB-α: nuclear factor of κ-light polypeptide gene enhancer in B-cell inhibitor-α.


### 2.10. Western Blot Analysis

100 mg of colon tissue was homogenized using 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) and 10μL of PMSF and then centrifuged at 12,000 rpm for 5 min at 4°C. The intermediate protein solution was collected, and protein quantification was performed with a bicinchoninic acid (BCA) kit (Beijing Solarbio Biotechnology Co. Ltd.). Each set of samples was diluted to 50 μg/mL, and then, the diluted protein was mixed with the sample buffer and heated at 95°C for 5-10 min for denaturation. The prestained protein ladder and samples were added to the sample wells of 10% SDS-PAGE gels, subjected to vertical gel electrophoresis, and then transferred to a polyvinylidene fluoride (PVDF; Thermo Fisher Scientific) membrane. The PVDF membrane was then blocked with 5% skim milk (Becton, Dickinson and Company) for 1 h. After blocking was completed, the PVDF membrane was washed with TBST (Beijing Solarbio Biotechnology Co. Ltd.) 3-5 times for 5-8 min each time at 100 rpm and then incubated with the corresponding primary antibodies overnight at 4°C and 80 rpm: Cu/Zn SOD, Mn SOD, GSH-Px, CAT, IL-1β, IL-10, IL-12, TNF-α, COX-2, iNOS, NF-κB p65, and IκB-α (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Then, the PVDF membrane was washed with TBST and incubated with the corresponding secondary antibody at 25°C at 80 rpm for 1 h. Finally, the chromogenic solution was prepared according to the SuperSignal West Pico PLUS kit (Thermo Fisher Scientific), and the membrane was observed on the Amersham imager 680 (GE Healthcare, Chicago, IL, USA) [34]. In addition, ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyse the images, and β-actin was used as the internal reference protein to calculate the relative expression of the target proteins.


### 2.11. Data Analysis

Three or more parallel experiments were performed on the serum and tissue indicators of each mice to obtain the average values. Statistical analysis of the data was performed with the IBM SPSS 22 statistical software package (SPSS Inc., IL, USA). The experimental results are expressed as themean±standarddeviation (SD). The differences between the averages of each group were evaluated by one-way ANOVA with Duncan’s multiple range test (MRT). Differences with p<0.05 were considered statistically significant.


## 2.1. Experimental Strains

Our research team isolated and purified a new lactic acid bacteria strain from traditionally fermented yak yogurt from the homes of herders in Baleksu Kaisk grassland in Zhaosu County, Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China. The 16S rDNA sequence analysis showed that it was aL. plantarum strain, and the colony morphology of this strain was observed by gram staining. The strain was stored in the China General Microbiological Culture Collection Centre (CGMCC, Beijing, China) and named L. plantarum ZS62, and its preservation number was 18228. In addition, Lactobacillus bulgaricus was used as a positive control in this study and was purchased from the China Centre for Type Culture Collection (CCTCC, Wuhan City, Hubei Province, China), and its strain preservation number was CCTCC AB 200048.


## 2.2. Determination of the Tolerance of Experimental Strains in Artificial Gastric Juice

The artificial gastric juice was prepared with 0.2% NaCl (Beijing Solarbio Biotechnology Co. Ltd., Beijing, China) and 0.35% pepsin (Beijing Solarbio Biotechnology Co. Ltd.), the pH was adjusted to 3.0 with 1 mol/L HCl after preparation, and then, the juice was filtered with a 0.22μm filter (Tullagreen, Carrigtwohill, Ireland) and set aside for later use. 5 mL of the activated strain culture solution was placed in a 10 mL centrifuge tube and centrifuged at 3000 r/min for 10 min at room temperature. The supernatant was discarded, and the bacteria were collected. The collected bacteria were washed twice with sterile normal saline and then resuspended in 5 mL of normal saline to make a bacterial suspension. The prepared experimental bacterial suspension was mixed with the artificial gastric juice at a volume ratio of 1 : 9, and after shaking, 2 mL of the mixed solution was taken and stored for later use (0 h time point). The remaining 8 mL was incubated in a water bath shaker at 37°C and 80 rpm for 3 h. The number of viable bacteria was determined at 0 h and 3 h. The samples collected at 0 h and 3 h were subjected to a series of 10-fold dilutions, and the dilutions were incubated on de Man, Rogosa, and Sharpe (MRS; Becton, Dickinson and Company, Franklin Lake, New Jersey, USA) solid media in a constant temperature incubator at 37°C for 48 h; the plate counting method was applied for feasible counts [30]. The survival rate was calculated according to
(1)Survivalrate%=3hNumberofviablebacteriaCFU/mL0hNumberofviablebacteriaCFU/mL×100.


## 2.3. Determination of the Growth Efficiency of Experimental Strains in Bile Salt

The bacterial solution that was cultured overnight after activation was collected and inoculated into MRS-THIO media (the MRS medium contained 0.2% sodium thioglycolate) containing 0.0% and 0.3% bovine bile salt at an inoculum concentration of 2%; a blank medium (uninoculated MRS-THIO medium) was used as a control; all media were cultured in a constant temperature incubator at 37°C for 24 h. A microplate reader was used to measure the absorbance (OD) values of the media with different mass concentrations at 600 nm, and the growth efficiencies of the experimental strains in different concentrations of bile salt were calculated according to Equation (2) [31]:
(2)Growthefficiency%=BilesaltmediumOD600nmBlankmediumOD600nm×100.


## 2.4. Determination of the Surface Hydrophobicity of Experimental Strains

Strains with survival rates greater than 70% in artificial gastric juice were selected for this experiment. The surface hydrophobicity of the strain was determined by BATH (carbon-hydrocarbon adhesion method); a total of 5 mL of the cultured bacterial solution was collected and centrifuged at 3 000 r/min for 10 min to collect the bacterial cells. The cells were washed twice with 5 mL of PBS (50 mmol/L, pH 6.5; Beijing Solarbio Biotechnology Co. Ltd.). PBS was used as a blank control, and the concentration of bacteria was adjusted with PBS to achieve an OD560 nm value of approximately 1.00. 4 mL of the bacterial solution with the adjusted cell concentration was placed in a 10 mL sterile centrifuge tube, 0.8 mL of xylene was added, and the tube was shaken for 30 s, paused for 10 s, shaken again for 30 s, and allowed to stand for 5-10 min for stratification. The lower aqueous phase was removed, and the OD560 nm value was determined with PBS buffer as a blank control. The hydrophobicity was calculated according to Equation (3) (A0 and A are the OD560 nm values of the bacterial solution before and after mixing with xylene, respectively) [32].
(3)Hydrophobicityrate%=A0−AA0×100.


## 2.5. Experimental Animal Model

Six-week-old C57BL/6 mice (n=50, male, bodyweight: 20±2g) were purchased from the Experimental Animal Centre of Chongqing Medical University. The mice were housed in an environment with a temperature of 25±2°C and a relative humidity of 50±5%, with 12 h light/dark cycles. The mice were allowed free access to drinking water and standard mouse food, the pads were replaced every two days, and adaptive feeding lasted for one week. To study the preventive effect of L. plantarum ZS62 on DSS-induced IBD, the mice were randomly divided into 5 groups (10 mice in each group). The entire experiment lasted for 5 weeks, and the specific experiment plan was as follows: (1) normal group: standard food and drinking water, daily gavaged with 0.1 mL/10 g (mouse bodyweight) 0.9% normal saline; (2) DSS group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and were gavaged with 0.1 mL/10 g (mouse bodyweight) 0.9% normal saline daily; at week 4, normal drinking water was replaced with 5% DSS (MP Biomedicals, Santa Ana, CA), and the other conditions remaining unchanged; (3) L. plantarum ZS62 (ZS62) group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and gavaged with 0.1 mL/10 g of L. plantarum ZS62 at a concentration of 1.0×109 CFU/mL; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remaining unchanged; (4) sulfasalazine (SSZ) group: at weeks 1, 2, 3, and 5, the mice were fed with standard food and drinking water and gavaged with 0.1 mL/10 g sulfasalazine (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) at a concentration of 500 mg/kg daily; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remain unchanged; and (5) L. bulgaricus (LB) group: at weeks 1, 2, 3, and 5, the mice were fed standard food and drinking water and gavaged with 0.1 mL/10 g of Lactobacillus bulgaricus at a concentration of 1.0×109 CFU/mL daily; at week 4, normal drinking water was replaced with 5% DSS, and the other conditions remain unchanged. After the last dose, the mice were fasted for 16-24 h, but were allowed free access to drinking water. The mice were killed by cervical dislocation, eyeball blood was collected, the mice were dissected, and the colon was separated for later use [15].


## 2.6. Histopathological Analysis of Colon Tissues

The colon tissue was washed with normal saline, cut into pieces of approximately 0.5 cm in size, and fixed in 10% formalin solution. The colon tissue was dehydrated with an ethanol gradient, soaked in xylene and ethanol for approximately 30 min for clarification, embedded in paraffin, cut into slices of approximately 2-3μm with a microtome, and fixed on a glass slide. Haematoxylin and eosin (H&E) were used to stain the cytoplasm in different shades of pink or red [15]. Finally, the morphological changes were observed under an optical microscope (BX43; Olympus, Tokyo, Japan).


## 2.7. Serum Index Determination

The obtained mouse blood was centrifuged at 4000 rpm for 10 min at 4°C. The mice serum was then separated, collected, and stored at -80°C for later use. Appropriate biochemical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) were used to determine the levels of the oxidation indicators CAT, T-SOD, MDA, and MPO in the serum according to the procedures recommended by the manufacturer.


## 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of the inflammatory cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α, and IFN-γ in the preserved mouse serum were determined according to the manufacturer’s instructions (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).


## 2.9. Real-Time Quantitative PCR Assay

In this study, messenger RNA (mRNA) expression in mouse colon tissue was determined by the SYBR green method. The mouse colon tissue was cut into pieces, and the total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The RNA concentration was measured using a microspectrophotometer (Nano 300, Ao Sheng, Hangzhou, Zhejiang, China). Reverse transcription was performed with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Baltics UAB, Lithuania, Vilnius) to obtain the cDNA template. Then, amplification was performed on a StepOne Plus real-time PCR system (Thermo Fisher Scientific) with 10μL of SYBR green PCR master mix (Thermo Fisher Scientific), 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of cDNA template, and 7 μL of DEPC. The conditions were as follows: predenaturation at 95°C for 3 min; then denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 15 s for 40 cycles in total; the final dissolution curves were completed at 95°C for 30 s, 60°C for 30 s, and 95°C for 15 s. Finally, the relative expression level of each gene was calculated by the 2-ΔΔCT method, where CT was the cycle threshold, and β-actin was used as an internal reference gene [33]. Table 1 shows the primer sequence information used in this study.Table 1
Primer sequences of RT-qPCR assay.
Gene nameSequenceCu/Zn SODForward: 5′-AACCAGTTGTGTTGTCAGGAC-3′Reverse: 5′-CCACCATGTTTCTTAGAGTGAGG-3′Mn SODForward: 5′-CAGACCTGCCTTACGACTATGG-3′Reverse: 5′-CTCGGTGGCGTTGAGATTGTT-3′GSH-PxForward: 5′-GTGCAATCAGTTCGGACACCA-3′Reverse: 5′-CACCAGGTCGGACGTACTTG-3′CATForward: 5′-GGAGGCGGGAACCCAATAG-3′Reverse: 5′-GTGTGCCATCTCGTCAGTGAA-3′IL-1βForward: 5′-GAAATGCCACCTTTTGACAGTG-3′Reverse: 5′-TGGATGCTCTCATCAGGACAG-3′IL-12Forward: 5′-CCTCCACTGTGCTGGTTTTAT-3′Reverse: 5′-TCAGCAACATGCTCCAGAAG-3′TNF-αForward: 5′-CAGGCGGTGCCTATGTCTC-3′Reverse: 5′-CGATCACCCCGAAGTTCAGTAG-3′IL-10Forward: 5′-CCAAGCCTTATCGGAAATGA-3′Reverse: 5′-TTTTCACAGGGGAGAAATCG-3′COX-2Forward: 5′-TTCCAATCCATGTCAAAACCGT-3′Reverse: 5′-AGTCCGGGTACAGTCACACTT-3′iNOSForward: 5′-GTTCTCAGCCCAACAATACAAGA-3′Reverse: 5′-GTGGACGGGTCGATGTCAC-3′NF-κB p65Forward: 5′-TGCGATTCCGCTATAAATGCG-3′Reverse: 5′-ACAAGTTCATGTGGATGAGGC-3′IκB-αForward: 5′-CGAGACTTTCGAGGAAATACCC-3′Reverse: 5′-GTCTGCGTCAAGACTGCTACA-3′β-ActinForward: 5′-ATGGAGCCGGACAGAAAAGC-3′Reverse: 5′-TGGGAGGTGTCAACATCTTCTT-3′Cu/Zn SOD: cuprozinc-superoxide dismutase; Mn SOD: manganese superoxide dismutase; GSH-Px: glutathione peroxidase; CAT: catalase; IL-1β: interleukin 1β; IL-12: interleukin12; TNF-α: tumor necrosis factor-alpha; IL-10: interleukin 10; COX-2: cyclooxygenase-2; iNOS: inducible nitric oxide synthase; NF-κB p65: nuclear factor κ-light-chain-enhancer of activated B cells; IκB-α: nuclear factor of κ-light polypeptide gene enhancer in B-cell inhibitor-α.


## 2.10. Western Blot Analysis

100 mg of colon tissue was homogenized using 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) and 10μL of PMSF and then centrifuged at 12,000 rpm for 5 min at 4°C. The intermediate protein solution was collected, and protein quantification was performed with a bicinchoninic acid (BCA) kit (Beijing Solarbio Biotechnology Co. Ltd.). Each set of samples was diluted to 50 μg/mL, and then, the diluted protein was mixed with the sample buffer and heated at 95°C for 5-10 min for denaturation. The prestained protein ladder and samples were added to the sample wells of 10% SDS-PAGE gels, subjected to vertical gel electrophoresis, and then transferred to a polyvinylidene fluoride (PVDF; Thermo Fisher Scientific) membrane. The PVDF membrane was then blocked with 5% skim milk (Becton, Dickinson and Company) for 1 h. After blocking was completed, the PVDF membrane was washed with TBST (Beijing Solarbio Biotechnology Co. Ltd.) 3-5 times for 5-8 min each time at 100 rpm and then incubated with the corresponding primary antibodies overnight at 4°C and 80 rpm: Cu/Zn SOD, Mn SOD, GSH-Px, CAT, IL-1β, IL-10, IL-12, TNF-α, COX-2, iNOS, NF-κB p65, and IκB-α (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Then, the PVDF membrane was washed with TBST and incubated with the corresponding secondary antibody at 25°C at 80 rpm for 1 h. Finally, the chromogenic solution was prepared according to the SuperSignal West Pico PLUS kit (Thermo Fisher Scientific), and the membrane was observed on the Amersham imager 680 (GE Healthcare, Chicago, IL, USA) [34]. In addition, ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyse the images, and β-actin was used as the internal reference protein to calculate the relative expression of the target proteins.


## 2.11. Data Analysis

Three or more parallel experiments were performed on the serum and tissue indicators of each mice to obtain the average values. Statistical analysis of the data was performed with the IBM SPSS 22 statistical software package (SPSS Inc., IL, USA). The experimental results are expressed as themean±standarddeviation (SD). The differences between the averages of each group were evaluated by one-way ANOVA with Duncan’s multiple range test (MRT). Differences with p<0.05 were considered statistically significant.


## 3. Results

### 3.1. Morphological Characteristics of the Strain

As shown in Figure1(a), in MRS agar medium, the experimental strain bacteria were opaque, white round colonies with a moist and smooth surface. Figure 1(b) shows that the experimental strain was gram-positive. There was no spore production, and the bacteria were short rod-shaped. In addition, the 16S rRNA gene sequence was determined and compared with the genome of the known standard strain Lactobacillus plantarum strain (Accession number: KM350169.1/KJ026622.1) in the GenBank database, and the similarity was 99.80%. These results indicate that this strain is Lactobacillus plantarum.Figure 1
Morphological characteristics of experimental lactic acid bacteriumLactobacillus plantarum ZS62.
(a)(b)


### 3.2. In Vitro Resistance Test of Experimental Strains

To determine whether the experimental strain could survive and colonize the gastrointestinal tract, artificial gastric juice and a 0.3% bile salt test were used to screen the strain to ensure that the probiotics could perform their probiotic functions in the human body. The pH value of human gastric juice is generally maintained at approximately 3.0, and the bile salt concentration is usually between 0.03 and 0.3%. The residence time of food in this environment is relatively short, usually 1-3 h [35]. To evaluate the in vitro probiotic properties of L. plantarum ZS62, we measured the survival rate at pH 3.0 and growth efficiency in 0.3% bile salt. The results showed that the survival rate of L. plantarum ZS62 in artificial gastric juice at pH 3.0 was 89.48%, and the growth efficiency in 0.3% bile salt was 11.2%. The surface hydrophobicity was 10.92%. Based on the positive in vitro resistance and surface hydrophobicity of L. plantarum ZS62, the strain was preliminarily believed that it has the potential to survive in the gastrointestinal tract, and its functional properties were further evaluated through animal experiments.


### 3.3. Colon Lengths of the Experimental Mice

To evaluate the effect of DSS-induced IBD on the length of the mouse colon, we measured the colon length of all mice (Figure2). The colon length in the normal group was 7.75±0.16 cm, and the colon length in the DSS group was 5.68±0.17 cm, which was significantly (p<0.05) different. In addition, the colon lengths in the SSZ, ZS62, and LB groups were 6.18±0.34, 6.23±0.50, and 6.26±0.31 cm, respectively, and the colons were significantly (p<0.05) longer than those in the DSS group. In addition, the average colon length in the ZS62 group was longer than that in the SSZ group. These results suggest that L. plantarum ZS62 effectively inhibits atrophy of the mouse colon, indicating to a certain extent that L. plantarum ZS62 could prevent DSS-induced IBD.Figure 2
Colon length (cm) of experimental mice. (a) Average length of experimental mouse colon. (b) Photo of mouse colon length. Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62:Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS. a–cMean values with different letters in the same bars are significantly different (p<0.05) according to Duncan’s new multiple range test (MRT).
(a)(b)


### 3.4. Histopathological Evaluation of the Mouse Colon

Figure3 shows the histological morphology of mouse colon tissue after haematoxylin and eosin staining. Under normal conditions, the mucosal epithelial cells were intact, the crypts were normal, the glands were neatly arranged, and there were no ulcers. However, after DSS treatment, severe erosion of the colonic mucosa was observed, almost all crypts were destroyed, goblet cells were sharply decreased, inflammatory cell infiltration appeared in the lamina propria, glands were disordered, and severe ulcers were observed. In the morphological analysis of the colon tissues of mice in the SSZ, ZS62, and LB groups, we found that although the colonic mucosa had erosions, the number of goblet cells decreased, and a small amount of ulcers were observed, the degree of damage was significantly (p<0.05) lower than that of the DSS group. In addition, obvious erosion was not observed in the colonic mucosa of the ZS62 group, the crypts were relatively complete, the glands were arranged neatly, the goblet cells were more complete, and the morphology of the colon tissues was similar to that of the normal group. The histological results indicated that L. plantarum ZS62 effectively reduced the histopathological damage to the colon in DSS-induced mice.Figure 3
Histopathological observation of colon tissues. Magnification 100x. Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62:Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


### 3.5. The Levels of the Oxidation Indicators T-SOD, CAT, MDA, and MPO in Mouse Serum

Table2 shows the levels of the oxidation indicators T-SOD, CAT, MDA, and MPO in mouse serum. In mouse serum, the levels of T-SOD and CAT in the normal group were the highest, while the levels of MDA and MPO were the lowest. The DSS group showed the opposite trend. The levels of T-SOD and CAT in the serum of mice in the SSZ, ZS62, and LB groups were significantly (p<0.05) higher than those in the SSD group, while the levels of MDA and MPO were significantly (p<0.05) lower than those in the DSS group. Among them, the levels of the oxidation indicators T-SOD, MDA, and MPO in the serum of mice in the ZS62 group were the closest to those in the normal group. Based on these results, it could be concluded that L. plantarum ZS62 enhanced the antioxidant capacity of mice, thereby improving the preventive effect on DSS-induced IBD.Table 2
T-SOD, CAT, MDA, and MPO levels in the serum of mice.
GroupT-SOD (U/mL)CAT (U/mL)MDA (nmol/mL)MPO (U/L)Normal96.77±1.97a543.20±29.55a3.26±0.05d680.12±27.78dDSS61.86±4.14d166.82±7.57d12.09±1.55a949.14±3.39aSSZ87.13±0.55bc276.42±37.64b6.46±0.19bc732.98±25.67cZS6291.83±0.75b225.27±5.19bc4.73±0.62cd693.10±27.09cdLB85.81±0.75c177.89±25.04cd8.06±0.52b825.96±6.77ba–dMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


### 3.6. The Levels of Inflammatory Cytokines in Mouse Serum

The levels of inflammatory cytokines in mouse serum are shown in Figure4. The data show that in the serum of mice in the DSS group, the levels of the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-12, and IL-6 were the highest, while the anti-inflammatory cytokine IL-10 was the lowest. In the serum of mice administered SSZ, ZS62, and LB, the levels of TNF-α, IFN-γ, IL-1β, IL-12, and IL-6 decreased, while the level of IL-10 increased. In addition, the expression of proinflammatory cytokine levels in the serum of mice of the ZS62 group was closer to that of the normal group. These results show that L. plantarum ZS62 significantly (p<0.05) reduced the release of proinflammatory cytokines in the serum of mice induced by DSS and increased the release of anti-inflammatory cytokines.Figure 4
Concentrations of cytokines IL-6, IL-10, IL-12, TNF-α, and IFN-γ. (a–e) Mean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


### 3.7. The mRNA and Protein Expression Levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in Mouse Colon Tissue

As shown in Figure5, the relative mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in mouse colon tissue of the DSS group were the lowest. In contrast, the relative expression levels of the normal group were the highest. In addition, compared with those of the DSS group, the mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in the mouse colon tissues of the SSZ, ZS62, and LB groups were all high. Moreover, the expression levels in the ZS62 and SSZ groups were closer to the normal group than the LB group, and there were significant differences. These data indicated that L. plantarum ZS62 could enhance the antioxidant capacity of mice, reduce free radical damage caused by DSS, and protect the antioxidant balance in the body.Figure 5
The mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in mouse colon tissue.a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


### 3.8. The mRNA and Protein Expression Levels of IL-1β, IL-12, TNF-α, and IL-10 in Mouse Colon Tissue

Figure6 shows that in the colon tissue of mice in the normal group, the relative mRNA and protein expression levels of IL-1β, IL-12, and TNF-α were the lowest, and the relative mRNA and protein expression levels of IL-10 were the highest. The DSS group showed the opposite trend. The expression levels in the ZS62 group were significantly (p<0.05) lower than those in the DSS group but were slightly higher than those in the normal group. These results suggest that L. plantarum ZS62 could effectively balance proinflammatory and anti-inflammatory reactions and played a beneficial role in immune regulation.Figure 6
The mRNA and protein expression levels of IL-1β, IL-12, TNF-α, and IL-10 in mouse colon tissue. a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


### 3.9. The mRNA and Protein Expression Levels of COX-2, iNOS, NF-κB p65, and IκB-α in Mouse Colon Tissue

Compared with those of the DSS group, the mRNA and protein expression levels of COX-2, iNOS, and NF-κB p65 were high in the normal group, SSZ group, ZS62 group, and LB group, while the mRNA and protein expression levels of IκB-α were low (Figure 7). Among them, the relative mRNA and protein expression levels of iNOS and NF-κB p65 were lowest in the normal group, followed by the ZS62 group, and then the SSZ and LB groups. The relative mRNA and protein expression levels of IκB-α were highest in the normal group and the closest to those in the normal group in the ZS62 group. According to these results, L. plantarum ZS62 could reduce the inflammatory response caused by DSS by regulating the immune response.Figure 7
The mRNA and protein expression levels of COX-2, iNOS, NF-κB p65, and IκB-α in mouse colon tissue. a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.1. Morphological Characteristics of the Strain

As shown in Figure1(a), in MRS agar medium, the experimental strain bacteria were opaque, white round colonies with a moist and smooth surface. Figure 1(b) shows that the experimental strain was gram-positive. There was no spore production, and the bacteria were short rod-shaped. In addition, the 16S rRNA gene sequence was determined and compared with the genome of the known standard strain Lactobacillus plantarum strain (Accession number: KM350169.1/KJ026622.1) in the GenBank database, and the similarity was 99.80%. These results indicate that this strain is Lactobacillus plantarum.Figure 1
Morphological characteristics of experimental lactic acid bacteriumLactobacillus plantarum ZS62.
(a)(b)


## 3.2. In Vitro Resistance Test of Experimental Strains

To determine whether the experimental strain could survive and colonize the gastrointestinal tract, artificial gastric juice and a 0.3% bile salt test were used to screen the strain to ensure that the probiotics could perform their probiotic functions in the human body. The pH value of human gastric juice is generally maintained at approximately 3.0, and the bile salt concentration is usually between 0.03 and 0.3%. The residence time of food in this environment is relatively short, usually 1-3 h [35]. To evaluate the in vitro probiotic properties of L. plantarum ZS62, we measured the survival rate at pH 3.0 and growth efficiency in 0.3% bile salt. The results showed that the survival rate of L. plantarum ZS62 in artificial gastric juice at pH 3.0 was 89.48%, and the growth efficiency in 0.3% bile salt was 11.2%. The surface hydrophobicity was 10.92%. Based on the positive in vitro resistance and surface hydrophobicity of L. plantarum ZS62, the strain was preliminarily believed that it has the potential to survive in the gastrointestinal tract, and its functional properties were further evaluated through animal experiments.


## 3.3. Colon Lengths of the Experimental Mice

To evaluate the effect of DSS-induced IBD on the length of the mouse colon, we measured the colon length of all mice (Figure2). The colon length in the normal group was 7.75±0.16 cm, and the colon length in the DSS group was 5.68±0.17 cm, which was significantly (p<0.05) different. In addition, the colon lengths in the SSZ, ZS62, and LB groups were 6.18±0.34, 6.23±0.50, and 6.26±0.31 cm, respectively, and the colons were significantly (p<0.05) longer than those in the DSS group. In addition, the average colon length in the ZS62 group was longer than that in the SSZ group. These results suggest that L. plantarum ZS62 effectively inhibits atrophy of the mouse colon, indicating to a certain extent that L. plantarum ZS62 could prevent DSS-induced IBD.Figure 2
Colon length (cm) of experimental mice. (a) Average length of experimental mouse colon. (b) Photo of mouse colon length. Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62:Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS. a–cMean values with different letters in the same bars are significantly different (p<0.05) according to Duncan’s new multiple range test (MRT).
(a)(b)


## 3.4. Histopathological Evaluation of the Mouse Colon

Figure3 shows the histological morphology of mouse colon tissue after haematoxylin and eosin staining. Under normal conditions, the mucosal epithelial cells were intact, the crypts were normal, the glands were neatly arranged, and there were no ulcers. However, after DSS treatment, severe erosion of the colonic mucosa was observed, almost all crypts were destroyed, goblet cells were sharply decreased, inflammatory cell infiltration appeared in the lamina propria, glands were disordered, and severe ulcers were observed. In the morphological analysis of the colon tissues of mice in the SSZ, ZS62, and LB groups, we found that although the colonic mucosa had erosions, the number of goblet cells decreased, and a small amount of ulcers were observed, the degree of damage was significantly (p<0.05) lower than that of the DSS group. In addition, obvious erosion was not observed in the colonic mucosa of the ZS62 group, the crypts were relatively complete, the glands were arranged neatly, the goblet cells were more complete, and the morphology of the colon tissues was similar to that of the normal group. The histological results indicated that L. plantarum ZS62 effectively reduced the histopathological damage to the colon in DSS-induced mice.Figure 3
Histopathological observation of colon tissues. Magnification 100x. Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62:Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.5. The Levels of the Oxidation Indicators T-SOD, CAT, MDA, and MPO in Mouse Serum

Table2 shows the levels of the oxidation indicators T-SOD, CAT, MDA, and MPO in mouse serum. In mouse serum, the levels of T-SOD and CAT in the normal group were the highest, while the levels of MDA and MPO were the lowest. The DSS group showed the opposite trend. The levels of T-SOD and CAT in the serum of mice in the SSZ, ZS62, and LB groups were significantly (p<0.05) higher than those in the SSD group, while the levels of MDA and MPO were significantly (p<0.05) lower than those in the DSS group. Among them, the levels of the oxidation indicators T-SOD, MDA, and MPO in the serum of mice in the ZS62 group were the closest to those in the normal group. Based on these results, it could be concluded that L. plantarum ZS62 enhanced the antioxidant capacity of mice, thereby improving the preventive effect on DSS-induced IBD.Table 2
T-SOD, CAT, MDA, and MPO levels in the serum of mice.
GroupT-SOD (U/mL)CAT (U/mL)MDA (nmol/mL)MPO (U/L)Normal96.77±1.97a543.20±29.55a3.26±0.05d680.12±27.78dDSS61.86±4.14d166.82±7.57d12.09±1.55a949.14±3.39aSSZ87.13±0.55bc276.42±37.64b6.46±0.19bc732.98±25.67cZS6291.83±0.75b225.27±5.19bc4.73±0.62cd693.10±27.09cdLB85.81±0.75c177.89±25.04cd8.06±0.52b825.96±6.77ba–dMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.6. The Levels of Inflammatory Cytokines in Mouse Serum

The levels of inflammatory cytokines in mouse serum are shown in Figure4. The data show that in the serum of mice in the DSS group, the levels of the proinflammatory cytokines TNF-α, IFN-γ, IL-1β, IL-12, and IL-6 were the highest, while the anti-inflammatory cytokine IL-10 was the lowest. In the serum of mice administered SSZ, ZS62, and LB, the levels of TNF-α, IFN-γ, IL-1β, IL-12, and IL-6 decreased, while the level of IL-10 increased. In addition, the expression of proinflammatory cytokine levels in the serum of mice of the ZS62 group was closer to that of the normal group. These results show that L. plantarum ZS62 significantly (p<0.05) reduced the release of proinflammatory cytokines in the serum of mice induced by DSS and increased the release of anti-inflammatory cytokines.Figure 4
Concentrations of cytokines IL-6, IL-10, IL-12, TNF-α, and IFN-γ. (a–e) Mean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.7. The mRNA and Protein Expression Levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in Mouse Colon Tissue

As shown in Figure5, the relative mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in mouse colon tissue of the DSS group were the lowest. In contrast, the relative expression levels of the normal group were the highest. In addition, compared with those of the DSS group, the mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in the mouse colon tissues of the SSZ, ZS62, and LB groups were all high. Moreover, the expression levels in the ZS62 and SSZ groups were closer to the normal group than the LB group, and there were significant differences. These data indicated that L. plantarum ZS62 could enhance the antioxidant capacity of mice, reduce free radical damage caused by DSS, and protect the antioxidant balance in the body.Figure 5
The mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in mouse colon tissue.a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.8. The mRNA and Protein Expression Levels of IL-1β, IL-12, TNF-α, and IL-10 in Mouse Colon Tissue

Figure6 shows that in the colon tissue of mice in the normal group, the relative mRNA and protein expression levels of IL-1β, IL-12, and TNF-α were the lowest, and the relative mRNA and protein expression levels of IL-10 were the highest. The DSS group showed the opposite trend. The expression levels in the ZS62 group were significantly (p<0.05) lower than those in the DSS group but were slightly higher than those in the normal group. These results suggest that L. plantarum ZS62 could effectively balance proinflammatory and anti-inflammatory reactions and played a beneficial role in immune regulation.Figure 6
The mRNA and protein expression levels of IL-1β, IL-12, TNF-α, and IL-10 in mouse colon tissue. a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 3.9. The mRNA and Protein Expression Levels of COX-2, iNOS, NF-κB p65, and IκB-α in Mouse Colon Tissue

Compared with those of the DSS group, the mRNA and protein expression levels of COX-2, iNOS, and NF-κB p65 were high in the normal group, SSZ group, ZS62 group, and LB group, while the mRNA and protein expression levels of IκB-α were low (Figure 7). Among them, the relative mRNA and protein expression levels of iNOS and NF-κB p65 were lowest in the normal group, followed by the ZS62 group, and then the SSZ and LB groups. The relative mRNA and protein expression levels of IκB-α were highest in the normal group and the closest to those in the normal group in the ZS62 group. According to these results, L. plantarum ZS62 could reduce the inflammatory response caused by DSS by regulating the immune response.Figure 7
The mRNA and protein expression levels of COX-2, iNOS, NF-κB p65, and IκB-α in mouse colon tissue. a–eMean values with different letters in the same column differ significantly (p<0.05) by Duncan’s multiple range test. Values presented are the means±standarddeviation (N=10/group). Normal: mice fed a standard chow diet plus drinking water; DSS: mice fed the standard chow diet plus drinking water with 5% dextran sulfate sodium; SSZ: sulfasalazine (500 mg/kg of BW) plus 5% DSS; ZS62: Lactobacillus plantarum ZS62 (1.0×109 CFU/mL) plus 5% DSS; LB: Lactobacillus bulgaricus (1.0×109 CFU/mL) plus 5% DSS.


## 4. Discussion

As an important type of probiotics, lactic acid bacteria can enhance the body’s immunity and prevent the occurrence of some gastrointestinal diseases [35]. Most studies indicate that the oxidative damage to intestinal cells leads to mucosal damage closely related to the occurrence of IBD [24, 36]. Additionally, the infection of intestinal epithelial cells release harmful substances, which increase the permeability of the intestinal epithelium; pathogenic bacteria can penetrate the damaged mucosal barrier to trigger a series of immune responses and cause macrophages and other cells to produce a large number of proinflammatory cytokines, causing inflammation [37]. L. plantarum ZS62 strengthened the mucosal barrier, inhibited oxidative damage and the exposure of inflammatory signals, stimulated the immune system to adjust the unbalanced immune response, and inhibited host mucosal damage, thereby alleviating DSS-induced oxidative damage and the inflammatory response.DSS-induced IBD shortens the colon in mice. Colon length is one of the indicators used to characterize the degree of inflammation in DSS-induced colitis [38]. Simultaneously, it is an effective means to observe colon tissue damage through histopathological changes. In this study, we confirmed that DSS induction could lead to a decrease in the length and serious changes in the pathological morphology of the colon of the mice and includes inflammatory cell infiltration, intestinal cell damage, and goblet cell reductions, while L. plantarum ZS62 could effectively reduce the degree of colon shortening and inflammatory lesions in the colon.Oxidative stress plays an important role in IBD-related tissue damage [39]. Probiotics can participate in the regulation of oxidative stress and stimulate the expression of CAT, SOD, GSH-Px, and other genes [40, 41]. When the body is in a state of oxidative stress, the production of free radicals exceeds the ability to scavenge free radicals in the body, leading to the destruction of tissue structure and cell apoptosis [42]. L. plantarum ZS62 uses the antioxidant enzyme CAT that decomposes H2O2 in the body and at the same time promotes the production of GSH-Px and catalyzes the decomposition of H2O2, thereby eliminating the peroxidative stress products, preventing ROS-mediated cell damage and inhibiting oxidative stress [36, 43]. SOD plays a vital role in the oxidative balance in the body and is not only an important indicator of antioxidant capacity but also an important component of the enzymatic protection system in the process of intracellular antilipid peroxidation. L. plantarum ZS62 can scavenge free radicals by increasing the levels of T-SOD, Cu/Zn SOD, and Mn SOD in the body [44, 45]. As the final metabolite of lipid peroxidation reaction, MDA is inhibited by L. plantarum ZS62, which reflects that L. plantarum ZS62 maintains low levels of oxygen free radicals, lipid oxidation, and cell damage in the body [46]. In addition, MPO is a haem peroxidase that is present in neutrophils and is one of the signs of oxidative stress. After the mice were stimulated by DSS, MPO was released to the extracellular, and L. plantarum ZS62 alleviates the damage to cells, proteins, DNA, and fats caused by hypochlorous acid and tyrosine free radicals catalyzed by MPO [47]. In general, under the action of DSS, the levels of CAT and T-SOD in the serum of mice decreased, and the levels of MDA and MPO increased. The relative mRNA and protein expression levels of Cu/Zn SOD, Mn SOD, GSH-Px, and CAT in mouse colon tissues were reduced, indicating the presence of oxidative stress, while the opposite trend in mice administered with L. plantarum ZS62 indicates that L. plantarum ZS62 can prevent DSS-induced IBD by regulating oxidative stress.Probiotics can also, through immune regulation, improve the intestinal barrier, regulate the inflammatory process, promote the immune response, and maintain intestinal health [3, 48]. The process of inflammation must be accompanied by the release of a variety of cytokines. IL-1β is a proinflammatory factor that stimulates inflammatory cells to enter the intestine through autocrine and paracrine mechanisms, damages intestinal tissues, and causes inflammation [49]. DSS leads to the upregulation of IL-1β levels in mice, and the expression of IL-6 is positively regulated by IL-1β. At the same time, innate immune cells release inflammatory mediators IL-10 and IL-12 to affect the adaptive immune system [50, 51]. Among them, IL-10, as an effective anti-inflammatory cytokine, plays an important role in maintaining intestinal homeostasis, and research reports indicate that it has a certain protective effect on intestinal epithelial cells [52], while IL-6 and IL-12 play a proinflammatory role in immune regulation, and IL-12 can induce IFN-γ to enhance immune-mediated cell damage [53, 54].IFN-γ is a proinflammatory factor secreted by Th1 cells. It not only increases the sensitivity of endothelial cells to TNF-α but also stimulates the accumulation of NF-κB in the nucleus and promotes cell damage [55]. TNF-α is a key inflammatory factor that plays a positive feedback role to regulate the activation of NF-κB. TNF-α is not only the activator of NF-κB but also the product of its activation [56]. L. plantarum ZS62 inhibits the level of IL-12 in the body, thereby alleviating the negative stimulation of TNF-α and NF-κB caused by IFN-γ to the body. NF-κB is a vital transcriptional activator in the body that can bind with target proteins to interfere with the expression of genes, thereby affecting cell growth, differentiation, inflammation, and immune responses [57]. When NF-κB binds with the NF-κB inhibitor protein IκB, the cell is in a resting state; when the cell is stimulated, such as receiving activation of TNF-α, IκB (including IκB-α, IκB-β, and IκB-γ) is phosphorylated or ubiquitinated and then degraded by the IκB kinase IKK. The nuclear localization sequence of NF-κB is exposed, and the protein translocates into the nucleus, where it binds with the upstream regulatory sequence of its target gene, and initiates the transcription and expression of inflammatory mediators including iNOS, IL-6, TNF-α, and COX-2 [58, 59]. On the other hand, COX-2 and iNOS are almost not expressed in normal tissues. When DSS causes inflammation in the body, a large amount of COX-2 and NO expression and synthesis exacerbate inflammation. NO is a highly active oxidant, which will promote the high expression of iNOS; at the same time, NO is also an activator of COX-2 [60, 61]. This study showed that L. plantarum ZS62 could regulate IL-1β, IL-12, TNF-α, IL-10, COX-2, iNOS, NF-κB p65, and IκB-α expression in the colon tissues of mice with enteritis to inhibit colitis. This indicates that L. plantarum ZS62 improves the mouse intestinal barrier and regulates the inflammatory response through the way of immune response, thereby exerting an inhibitory effect on DSS-induced IBD.


## 5. Conclusions

Based on the preliminary confirmation of this study thatL. plantarum ZS62 exhibited beneficial in vitro resistance, we further confirmed through in vivo animal experiments that L. plantarum ZS62 could prevent DSS-induced IBD in mice by regulating oxidative stress and the immune response. In addition, this study is helpful for future IBD-related research and prevention, as well as the development of probiotic preparations. In general, L. plantarum ZS62 is a high-quality raw material that can be used as a functional food and probiotic preparation, but it needs further clinical data to support it.



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*Source: 9416794-2021-10-27.xml*