The Role Mechanism Of Clostridium Metabolite P-cresol Sulfate In Primary Biliary Cholangitis

Part I Expression of Clostridium metabolite p-cresol sulfate/p-cresol glucuronic acid in patients with primary biliary cholangitis and influencing factorsObjective:To study the expression levels and influencing factors of Clostridium metabolites p-cresol sulfate(PCS)/p-cresol glucuronic acid(PCG)in patients with primary biliary cholangitis(PBC).Methods:Thirty-four patients with PBC and thirty healthy controls who were matched with age and sex were selected.Metabolomics was used to detect the expression levels of PCS/PCG,aromatic amino acid(tyrosine,phenylalanine and tryptophan)in the serum of the two groups.Analyze the distribution differences of PCS in different stages and different liver functions Child classification.Analyze the correlation between PCS and alkaline phosphatase,glutamyltransferase,alanine aminotransferase and aspartate aminotransferase.Results:(1)The difference between the concentration of PCS and PCG in the serum of PBC patients and the healthy control group:The concentration of PCS in the serum of PBC patients is significantly lower than that of the healthy control group,and the difference is statistically significant;There is no statistical difference in the expression level of PCG between the PBC group with the healthy control group.(2)The difference in serum peak thresholds of aromatic amino acids between PBC patients and healthy controls:The peak thresholds of tyrosine and phenylalanine in the serum of PBC patients are significantly higher than those of healthy controls,and the difference is statistically significant.Tryptophan level was no statistical difference in two groups.(3)The difference in the concentration of PCS in different stages:the concentration of stage ? patients is lower than that of PBC stage III and ? patients,the difference is statistically significant,and the concentration levels of stage III and? have no statistical difference.(4)The difference of PCS in liver function classification:PCS has no statistical difference between the average concentration of Child-A,Child-B and Child-C patients.(5)Correlation analysis between PCS and liver function indicators:Correlation analysis between PCS and alkaline phosphatase,glutamyltransferase,alanine aminotransferase and aspartate aminotransferase,the correlation coefficient R value are all less than 0.4.Conclusion(s):1.The PCS level in the serum of PBC patients decreased,the tyrosine level and the phenylalanine level increased,and there was no significant difference between the PCG level and the tryptophan level;2.The serum distribution of PBC patients and healthy control group is mainly PCS,not PCG;3.The concentration of PCS in the serum of PBC patients in different stages is different;4.There is no difference in the concentration of PCS in the serum of PBC patients with different liver function grades;5.The concentration of PCS in the serum of PBC patients are not correlated with the levels of alkaline phosphatase,glutamyltransferase,alanine aminotransferase and aspartate aminotransferase.Part II Study on the expression and effect of clostridium metabolite p-cresol sulfate in mice with primary biliary cholangitisObjective(s):To study the role of clostridium metabolite p-cresol sulfate(PCS)in mice with primary biliary cholangitis(PBC).Methods:C57BL/6 female mice were divided into 3 groups,normal control group,PBC group,and PBC tyrosine group.The PBC mouse model was made by chemical induction.According to the results of the preliminary experiment,the PBC tyrosine group was fed with tyrosine-rich feed for 10 days after PBC was modeled,and the PBC group was fed with ordinary feed for 10 days.Normal control was fed with ordinary feed without any treatment.After the mice were treated,the liver inflammation reaction of the three groups was compared.UPLC-ESI-QTOF-MS detects the concentration of PCS in the serum and liver of the three groups of mice.CD86 labeled liver M1 Kupffer cells and CD206 labeled M2 Kupffer cells,respectively,compared the polarization of liver Kupffer cells in three groups of mice.ELASA analyzed the expression levels of IL-6,IL-10,TNF-? and TGF-? in the serum of each group of mice.The expression levels of IL-6,IL-10,TNF-? and TGF-? in the liver of each group of mice were tested by PCR and WB,respectively.Results:(1)The PBC mouse model is verified to meet the PBC diagnostic criteria;(2)The concentration of PCS in PBC mice:The concentration of PCS in the liver and serum of PBC mice is lower than that of normal control mice.The concentration of PCS in the liver and serum of mice in PBC tyrosine group was no different from that in the normal control group;(3)The effect of PCS on liver inflammation in PBC mice:immunohistochemical results showed that PBC mice had obvious inflammation and PBC tyrosine mice was partially alleviated;(4)Kupffer cell changes:In PBC mice,M1 Kupffer cells are mainly polarized by CD86;in PBC tyrosine group,M2 Kupffer cells are mainly polarized by CD206;(5)The effect of PCS on the distribution of cytokines in PBC mice:ELASA,PCR and WB results all showed that the levels of IL-6,TNF-? and TGF-? in the PBC group were higher than those in the normal group.The level of IL-10 was lower than that of normal mice.The levels of IL-6,TNF-? and TGF-? in PBC mice fed with tyrosine decreased,and the level of IL-10 increased.Conclusion(s):1.PBC mice were successfully modeled;2.PBC mice supplemented with tyrosine food would increase the levels of PCS in plasma and liver;3.PCS derived from tyrosine could partially alleviate liver inflammation in PBC mice;4.Tyrosine-derived PCS can reverse the polarization direction of Kupffer cells in the liver of PBC mice;5.Tyrosine-derived PCS can reduce the level of inflammatory factors in the liver of PBC mice and increase the level of anti-inflammatory factors.Part ? Study on the effect and mechanism of clostridium metabolite p-cresol sulfate on bile duct epithelial cells and Kupffer cellsObjective(s):To study the effect of clostridium metabolite p-cresol sulfate on bile duct epithelial cells and Kupffer cells.Methods:Different concentrations of p-cresol sulfate(PCS)were selected to stimulate the bile duct epithelial cells of SD rats to observe the cell morphology,CCK-8 to detect cell viability,flow cytometry to detect cell cycle,and EdU to detect the proliferation of bile duct epithelial cells.After adding 200 ?g/ml PCS to the bile duct epithelial cells,qRT-PCR detects NLRP3,IL-1,IFN-?,TNF-?,BCL-2,Caspase-1,Caspase-9,GSDMD,NOX2,P22,TGF-?1 and TIMP-1.The fluorescence intensity was used to measure the reactive oxygen species,WB detects the expression of NOX2 and P22.After lentivirus transfection,the expression level of NOX2 and P22 were tested again by WB.PCS was co-cultured with SD rat Kupffer cells,the polarization direction was detected by flow cytometry,the expression of IL-1?,IL-6,TNF-?,CCL3,IL-10,Arg-1 was detected by qRT-PCR,and the expression of IL-1?,IL-6,TNF-?,CCL3,IL-10,Arg-1 was detected by WB.After damaging the bile duct epithelial cells with LPS,they were co-cultured with Kupffer cells acted by PCS.After the co-cultivation,the expression levels of TNF-?,IL-8,MCP-1,IL-1?,CX3CL1,and IL-10 were detected by qRT-PCR,and the expression levels of TNF-?,IL-8,MCP-1,IL-1?,CX3CL1 and IL-10 was detected by WB.Supernatants of each group were collected,expression levels of TNF-?,IL-8,MCP-1,IL-1?,IL-10 were tested by ELAS A.Results:(1)The effect of PCS on normal bile duct epithelial cells:?Different concentrations of PCS have an effect on the morphology of bile duct epithelial cells.The higher the concentration,the more obvious the changes in cell morphology;?The results of CCK8 show that PCS inhibits the viability of bile duct epithelial cells at a dose dependence;?qRT-PCR results showed that the levels of NLRP3,IL-1,IFN-?,TNF-?,BCL-2,Caspase-1,Caspase-9,GSDMD did not change significantly after stimulating by PCS with 200?g/ml,but NOX2,P22,TGF-?1 and TIMP-1 levels increased;?PCS with 200?g/ml can produce the strongest fluorescence intensity;?PCS with 200?g/ml express the highest protein level NOX2 and P22,the expression levels of NOX2 and P22 decreased after lentivirus transfection;(2)The effect of PCS on normal Kupffer cells:?Flow cytometry results show that PCS can reverse the polarization direction of Kupffer cells;?qRT-PCR results show that in Kupffer cells,the expression levels of IL-1?,IL-6,TNF-?,and CCL3 decreased,and IL-10 and Arg-1 increased;?WB results showed that in Kupffer cells the protein level of IL-1?,IL-6,TNF-? and CCL3 decreased,IL-10 and Arg-1 increased;(3)The effect of PCS on the bile duct epithelial cells damaged by LPS through Kupffer cells:?The results of qRT-PCR and WB showed that PCS can activate Kupffer cells reduce the expression levels of TNF-?,IL-8,MCP-1,IL-1?,and CX3CL1 in LPS-injured bile duct cells,and increase the expression level of IL-10;?ELASA detects the supernatant after co-culture,and the results show Kupffer cells activated by PCS can reduce the expression levels of TNF-?,IL-8,MCP-1,and IL-1? in the supernatant of LPS-damaged bile duct cells,and increase the expression level of IL-10.Conclusion(s):1.PCS can damage normal bile duct epithelial cells to cause oxidative stress and express fibrotic factors;2.PCS can reverse the polarization of Kupffer cells to M2;3.PCS protects the injured bile duct epithelial cells by reversing the polarization direction of Kupffer cells and reduces the inflammatory damage of PBC.