Molecular Mechanism Of HSCs Apoptosis Related Signaling Pathway During Hepatic Fibrosis Of Mice Infected With Schistosomiasis Japonicum Regulated By ICOSL/ICOS

Schistosomiasis is a major infectious parasitic disease mainly caused by the infection of schistosoma cercariae,and its pathogenic mechanism has been very clear,mainly because of the deposit of eggs in the liver which cause egg granuloma,resulting in liver fibrosis,secondary schistosomiasis cirrhosis.Hepatic stellate cells,as non-parenchymal cells of the liver,are the main effector cells in the process of hepatic fibrosis formation.At present,an important method for the treatment of hepatic fibrosis is to promote the apoptosis of activated HSCs,while mitochondrial dependent pathway and death receptor pathway(Fas/FasL,TNFR,Trail)are currently identified as important pathways involved in HSCs apoptosis.a lot of in-depth studies have been carried out on the pathways of hepatic stellate cell proliferation and activation,while the molecules of HSCs apoptosis and its related signaling pathways in liver fibrosis have not been elucidated.The liver fibrosis level and HSCs activation level of ICOS transgenic(ICOS-Tg)mice were severe than that of wild-type control group.The same experimental model was used to observe the expression dynamics of schistosomiasis progression HSCs apoptosis,and to explore the mechanism of co-stimulatory ICOSL/ICOS mediating apoptosis during liver fibrosis and its effect on liver fibrosis and its molecular mechanism.?.Molecular expression dynamics of primary HSCs apoptosis and related signaling pathways in chronic disease-causing mice infected with S.japonicumObjective:To investigate the apoptosis trend of primary HSCs and the expression dynamics of related signaling pathway molecules in HSCs from mice infected with S.japonicum.Methods:1.Microscopically count 5?+7-10? seropositive cercariae attached to the abdomen of the mice,infected 15 minutes to establish a disease model,collect and fixed pre-infection(0 weeks),infected acute lesion period(6 weeks,9 weeks),infected chronic period(12 weeks),late infection(16 weeks)of the liver tissue,to observe the area changes of egg granuloma in liver sections.2.Primary HSCs of before infection(0 weeks),acute lesion of infection(6 weeks,9 weeks),and chronic infection(12 weeks)were isolated by taking situ liver perfusion and density gradient centrifugation.We use fluorescence microscopy and cellular immunofluorescence to identify them.3.TUNEL was used to detect apoptosis of HSCs in liver sections.Annexin V-FITC/PI co-staining was used to detect isolated primary HSCs apoptosis.We detect the expression of molecular genes related to apoptosis in primary HSCs and to compare the dynamic changes of each gene in mice infected with S.japonicum by Real-time fluorescence quantitative PCR.Results:1.HE showed that the hepatic lobules were intact before infection(0 weeks),the hepatocytes were well-formed,no inflammatory infiltration,and no collagen fibers were deposited.After S.japonicum infection[6 weeks(7.14±0.74)× 104?m2,(P<0.000 1)],the lobule structure was destroyed,and the inflammatory cell infiltrated,collagen fiber deposited.With the increase of infection time,the granuloma area decreased slowly,and the collagen deposition gradually increased[9weeks,(5.58±0.77)×104?m2,P<0.001;12weeks,(4.78±0.45)×104?m2,P<0.01;16weeks,(4.31±0.42)×104?m2].2.Freshly isolated cells were rounded,containing lipid droplets with strong refraction.Spontaneous bluish-green fluorescence was observed with the help of 328 nm UV fluorescence microscope.After immunocytochemical staining of the isolated cells,we can see more than 90%cells with red fluorescence in the cytoplasm.3.TUNEL results showed that HSCs with apoptosis around the ovary granuloma of liver worms increased after infection and peaked after 6 weeks(19.5±2.53,P<0.0001),then slowly decreased but still at a high level[9 weeks,(10.7±0.895),P<0.01;12 weeks,(9.3±0.7895);16 weeks,(9.2± 1.041)].Flow cytometry showed that the apoptosis rate of primary HSCs,peaked after 6 weeks of infection[(45.19±0.7463)%P<0.0001]and then gradually decreased[9 weeks,(31.93±2.292)%,P<0.01;12 weeks,(13.61 ±1.407)%,P<0.01]but still higher than that before infection(5.907±0.8856)%.The real-time PCR showed that dr5 and its ligand TRAIL in the death receptor signaling pathway was highest at 6 weeks,down-regulated at 9 weeks and 12 weeks[Trail 6 weeks,(7.7±1.3,gene/GAPDH),P<0.05;9 weeks,(1.5±0.2,gene/GAPDH),P<0.05;Dr5 6 weeks,(2.29±0.09,gene/GAPDH),P<0.01;9 weeks,(2.015±0.065,gene/GAPDH);12 weeks,(1.055 ± 0.005,gene/GAPDH),P<0.01],and the expression of Fas was elevated after infection,but there was no statistical difference.FasL expression levels continued to increase after infection,peaked at 9 weeks(2.873±0.5113,gene/GAPDH),and down-regulated at 12 weeks[(1.04±0.01528,gene/GAPDH),P<0.05].The expression Bax pro-apoptotic genes in mitochondrial signaling pathway was the highest 6 weeks after infection,then the expression decreased,and the Bcl-2 showed the same trend:6 weeks,[Bax(2.247±0.3047,gene/GAPDH),P<0.05;Bcl-2(9.023±0.7823,gene/GAPDH),P<0.0001];9weeks,[Bax(1.43±0.1572),gene/GAPDH);Bcl-2(3.253±0.508,gene/GAPDH),P<0.01];12 weeks,[Bax(1.31 ±0.07371,gene/GAPDH);Bcl-2(1.553±0.1713,gene/GAPDH),P<0.05].Conclusion:The apoptosis of HSCs peaked in the acute phase of infection and began to decrease in the chronic phase.The expression of molecules associated with activation and proliferation of HSCs showed a parallel change,mitochondrial apoptosis signaling pathway is more closely related to the apoptosis of HSCs.?.Effects of ICOSL/ICOS on apoptosis of HSCs and molecular dynamic expression of related signaling pathways from mice infected with S.japonicumObjective:To investigate the up-regulation of co-stimulation signal ICOSL/ICOS,the degree of liver fibrosis caused by S.japonicum infection and the regulation of apoptosis of HSCs.Methods:1.Using HE staining,ICOS-Tg mice with pre-infection(0 weeks),acute lesion(6 weeks,9 weeks),chronic infection(12 weeks),and advanced infection(16 weeks)of liver tissue pathological changes in mice with WT mice were observed.2.Using TUNEL to detect apoptosis of HSCs in liver sections of ICOS-Tg mice and WT mice.Primary apoptosis in ICOS-Tg mice and WT mice isolated HSCs were detected by Annexin V-FITC/PI.We use Real-time PCR to detect the expression levels of molecular genes related to apoptosis signaling pathways in primary HSCs of ICOS-Tg mice and WT mice,and to find the difference between them.Results:1.The results of HE staining showed that the area of egg granuloma of ICOS-Tg mice was higher than that of WT mice[6 weeks,(10.44±1.30)×104?m2 vs(7.14±0.74)× 104?m2,P<0.5],and the degree of fibrosis was more serious.2.TUNEL results showed that ICOS-Tg mice had higher apoptosis of HSCs around egg granuloma of Sjaponicum than that of WT type[6 weeks,(3 8±3.486)vs(19.5±2.535),P<0.0001;9 weeks,(15.6±1.024)vs(10.7±0.895),P<0.05].The apoptosis rate of ICOS-Tg primary HSCs detected by flow cytometry was higher than that of WT in the same period[6 weeks,(57.76±3.707)%vs(45.19±0.7463)%;9 weeks,(44.43±2.328)%vs(31.93±2.292)%,P<0.01].The real-time fluorescence quantitative PCR results showed that the overall change trend of ICOS-Tg and the apoptosis-related genes in WT mice after infection with S.japonicum was consistent,but the ICOS-Tg was higher than the level of WT gene expression in the same period:[Trail:6 weeks,(24.59±0.34)vs(7.7± 1.3),P<0.0001;9 weeks,(7.775±0.565)vs(1.5±0.2),P<0.0001];[dr5:0 week,(2.275±0.025)vs(1±0),P<0.01;6 weeks,(4.7±0.47)vs(2.29±0.09),P<0.0001;9 weeks,(4.725±0.165)vs(2.015±0.065),P<0.0001;12 weeks,(2.355± 0.125)vs(1.055±0.005),P<0.01];[Fas:12 weeks,(3.03 ± 0.46)vs(1.735±0.323),P<0.05];[FasL:6 weeks,(3.543±0.5368)vs(1.593±0.3185),P<0.01];[Bax:9 weeks,(2.463±0.006667)vs(1.43±0.1572),P<0.01];[Bcl-2:6 weeks,(6.06±0.4499)vs(9.023±0.7823),P<0.001];[Caspase-3:6 weeks,(2.923±0.1868)vs(1.627±0.07688),P<0.0001;12 weeks,(1.65 ± 0.1501)vs(1.163 ±0.06333),P<0.05].Conclusion:The up-regulation of ICOSL/ICOS signal increased the area of egg granuloma than that of WT mice.Upregulation of co-stimulation signaling ICOSL/ICOS also increased the apoptosis of HSCs,and the expression of apoptosis-related signaling pathway molecules was also higher than that of WT in the same period.After up regulation of ICOSL/ICOS,HSCs apoptosis and HSCs activation increased compared with WT.The possible mechanism is that the increase of HSCs activation number is much higher than the increase of apoptosis number,and finally the degree of fibrosis is more serious.Although the apoptosis of HSCs and the activation of HSCs decreased in the later stage of infection,the difference between them still existed,which made the degree of liver fibrosis worse.?.Preliminary study on the molecular mechanism of co-stimulation signal ICOSL/ICOS regulation HSCs apoptosis in mice with S.japonicumObjective:To investigate the molecular mechanism of co-stimulation signal ICOSL/ICOS regulating the apoptosis of HSCs in the course of mice infected with S.japonicum.Methods:Using TGF-? and IL-13 recombinant protein to stimulate HSCs cell line JS1,using Annexin V-FITC/PI co-staining flow cytometry to detect apoptosis before and after stimulation,using Giemsa staining to observe the morphology of typical apoptotic cells.Results:Flow cytometry showed that after stimulated JS1,the apoptosis of TGF-?group was more significant than control group[5ng/ml:control(13.38±0.135)vs TGF-?(20.02±1.4),P<0.5;10ng/ml:control(13.38±0.135)vs TGF-?(21.65±2.235),P<0.5].However,after stimulated by IL-13,the apoptosis rate increased but was not statistically significant.Giemsa staining showed that the normal nuclei were dark blue,the apoptotic nuclei were deeply stained.and the cytoplasm was concentrated,and the apoptotic bodies were visible.Conclusion:Previous studies showed that up-regulation of co-stimulation of ICOSL/ICOS signal over expression of Th2 cytokine TGF-? which can promote apoptosis may be the reason why the apoptosis rate of ICOS-Tg mice is higher than that of WT mice.The results showed that ICOSL/ICOS signal could affect HSCs apoptosis in regulating the progression of liver fibrosis in mice,and its molecular mechanism might be to up-regulate Th2 cytokines TGF-? expressed by costimulatory signal overexpression.