| Object:To investigate the histone acetylation level of ROR-γt gene in biliary atresia(BA)patients and mouse models.To explore the molecular mechanism of TNFα-TNFRs signaling pathway regulating the histone acetylation level of ROR-γt gene;and to investigate the role of TNFα-TNFRs signaling pathway regulating the acetylation level of ROR-γt gene in the progression of BA by injecting TNFR1 and TNFR2 antibodies in animal intervention experiments.Methods:From October 2016 to December 2018,59 patients were diagnosed with BA in the department of pediatric surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,and their liver tissues were collected during the hepatojejunostomy(Kasai)surgery.Liver tissues of 42 patients with choledochal cyst were collected as control group.Newborn Balb/c mice were injected with Rhesus Rotavirus(RRV)to establish an animal model of biliary atresia.Newborn mice were randomly divided into 4 groups:(1)RRV group: the RRV group was established by intraperitoneal injection of 20 L 1×105 pfu RRV within 12 hours after birth;(2)Control group: newborn mice were intraperitoneally injected with the same amount of normal saline within 12 hours after birth as the control group;(3)TNFR1 intervention group: newborn mice were intraperitoneally injected with 20 L 1×105 pfu RRV within 12 hours after birth,followed by TNFR1 antibody(10 μg/mouse)injection every day until 7 days after birth;(4)TNFR2 intervention group: newborn mice were intraperitoneally injected with 20 L 1×105 pfu RRV within 12 hours after birth,followed by daily injection of TNFR2 antibody(10 μg/mouse)until 7 days after birth;(5)TNFα intervention group: newborn mice were intraperitoneally injected with 20 L 1×105 pfu RRV within 12 hours after birth,followed by daily injection of TNFα antibody(10 μg/mouse)until 7 days after birth.Part 1: The expression levels of ROR-γt in the two groups were determined by RT-q PCR and western blot test;and the histone acetylation levels of ROR-γt promoter region in the two groups were detected by chromatin immunoprecipitation(Ch IP)assay.The expression levels of ROR-γt were detected by RT-q PCR and immunohistochemistry on day 3,day 7 and day 14,respectively.The histone acetylation levels of the ROR-γt gene promoter region in each group were compared by Ch IP assay;the number and proportion of Th17 cells in the each groups were determined by flow cytometry,and the expression levels of Th17-related cytokines were detected by ELISA.Part 2: The liver tissues of BA patients and the control group,as well as the BA mouse models group and the control group on day 3,7 and 14 were taken.The expression levels of TNFα,TNFR1 and TNFR2 were detected by ELISA,RT-q PCR and immunohistochemistry;Expression levels of p38,JNK,STAT3 and c-rel,p65 in liver tissues of each group were determined and compared.Part 3: Weight change,survival rate,biliary atresia phenotype and liver histopathology were observed in RRV,Control,TNFR1,TNFR2 and TNFα intervention groups.Taken the liver tissues of the five groups on day 3,7 and 14,using RT-q PCR and western blot test to explore the ROR-γt,p38,JNK,STAT3 and c-rel,p65 expression levels;the histone acetylation levels of ROR-γt promoter region in the each groups were detected by Ch IP assay;The number and proportion of Th17 cells in the each groups were determined by flow cytometry.Results: Part 1: Compared with the control group,the acetylation level of ROR-γt gene promoter was significantly increased(p<0.001)and the expressions of ROR-γt m RNA and protein were significantly up-regulated in BA(p<0.01);the Th17 cells showed increased differentiation and IL-17 A secretion(p<0.001).Moreover,in BA mouse model,the acetylation level and protein expression level of the ROR-t gene promoter increased with time and reached the peak on the 7th day,which was consistent with the up-regulation trend of Th17 cell proliferation and related cytokines.Part 2: The expression of TNFα in BA patients was not significantly increased,however the immunohistochemical staining showed that the expression of TNFR1 and TNFR2 were increased when compared with the control group,and TNFR2 was significantly up-regulated(p<0.01);a large number of inflammatory cells were locally infiltrated in the hepatic portal area.Compared with the control group,the TNFα levels were significantly increased in the RRV group at all time points(p<0.05)and the expression of TNFR1 and TNFR2 was also increased,with a higher expression level of TNFR2 p<0.01).The expressions of p38,JNK,STAT3 and c-rel in the liver tissues of the BA children and mice RRV group were significantly higher than those of the control group(p<0.01).Part 3: After the intervention of TNFR1,TNFR2 and TNFαantibody injection,we found that the BA phenotype in mice was reduced,the survival rate was improved and the liver tissue pathologic changes were alleviated.Moreover,the TNFR2 and TNFα intervention group had a more effective therapeutic effect compared with the TNFR1 intervention group.In TNFR2 and TNFα intervention group,the expression of p38,JNK,STAT3,c-Rel and p65 were decreased(p<0.01)and the histone acetylation level of ROR-γt gene promoter region was significantly down-regulated when compared with RRV group(p<0.001).While in the TNFR1 intervention group,the expression of p38,JNK,STAT3 and c-rel,p65 and the acetylation level of ROR-γt genes were not significantly different from RRV group.Conclusions: 1.In the liver tissues of BA patients and mouse models,the histone acetylation level of ROR-γt gene promoter region was significantly increased and the expression of ROR-γt was increased,promoting the proliferation and differentiation of Th17 cells and the IL-17 A secretion;2.The expression of TNFα and TNFRs were increased in the liver tissues of BA patients and mouse models,leading to up-regulated the downstream NF-κB and MAPK-STAT3 signaling pathway and promoting the histone acetylation level of ROR-γt gene;3.In the BA mouse models,the NF-κB and MAPK-STAT3 were down-regulated after blocking the TNFα-TNFR2 signaling pathway using the anti-TNFR2.The acetylation level of ROR-γt gene promoter,the differentiation of Th17 cells and the incidence and phenotype of BA were significantly decreased,with a higher survival rate. |
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