Long Non-coding RNA During Hepatic Fibrosis Of Mice Infected With Schistosoma Japonicum Regulated By ICOSL/ICOS Pathway

Schistosomiasis japonicum is an acute and chronic disease caused by Schistosoma japonicum.The main pathogenic mechanism of liver fibrosis is that the deposition of worm eggs leads to granuloma of liver worm eggs and the formation of secondary liver fibrosis.Due to the continuous immune response of the host to the egg antigen,the stationary phase of hepatic stellate cells(HSCs)are abnormally activated,and the HSCs begin to proliferate and transform into myofibroblasts,and express ?-smooth actin Extracellular matrix(ECM)is secreted.Excessive ECM cannot be degraded and deposited in the liver,causing liver fibrosis.In recent years,lncRNA that does not have the function of encoding proteins has been gradually recognized by people.It is abnormally expressed in many tumors and participates in the regulation of various biological processes such as cell proliferation,activation and apoptosis.Recent studies on liver fibrosis have found that lncRNA can affect the occurrence and development of liver fibrosis by regulating the activation of HSCs.At present,there are many studies on the regulation of lncRNA-p21,GAS5,MALAT1,PVT1,lncRNA-H19 in liver fibrosis.However,the mechanism of these lncRNAs in the process of Schistosoma japonicum infection in the host and liver fibrosis has not been elucidatedPrevious research by the research group showed that down-regulation of ICOS signal can alleviate liver fibrosis and HSCs activation to a certain extent.In this research project,ICOSL-KO mice and their wild-type C57BL/6J mice were used to establish a schistosomiasis,experimental animal model,investigate the effect of related lncRNA on the activation of HSCs and the formation of liver fibrosis,find new ways to regulate HSCs activation and control the occurrence and development of liver fibrosisPart ?.The isolation,identification and culture of primary HSCs in chronic pathogenic process after mice infected with Schistosomajaponicum.Objective:To extract the high-purity primary HSCs of the mice and culture them for 7 days in vitro to simulate the activated state of HSCs in mice and lay the foundation for subsequent experiments.Methods:(1)Pick 5?+7? Schistosoma japonicum cercariae to infect ICOS-KO mice and wild-type control C57/BL6J mice to establish a model of Schistosoma japonicum liver fibrosis.Using in situ liver enzyme perfusion and centrifugation by density gradient to obtain the primary HSCs in mice with different five disease stages include uninfected(0 weeks),4 weeks infected,7 weeks infected,9 weeks infected,and 12 weeks infected,among them,7 weeks is the acute infection period,and 12 weeks is the late infection period.The method of identifying the survival rate of primary HSCs is trypan blue staining.(2)The methods that are used to identify the purity of the cells is Observed and photographed under the live cell workstation,under the excitation light of 328nm,the blue-green fluorescence of HSCs spontaneously counted.(3)The method of identifying the purity of cells is to perform GFAP immunofluorescence staining on HSCs and observe and count under a fluorescence microscope.Results:(1)The primary cells'growth and activation deformation of mice which is freshly extracted during the culture process is godd and obvious.The average number of cells obtained per mouse was(1.95±0.2)x 106 cells/mice,its survival rate can reach more than 90%.(2)Under the living cell workstation,it can be observed that after freshly isolated cells are cultured overnight,they spontaneously emit blue-green fluorescence under ultraviolet excitation light of 328 nm.(3)After culturing HSCs for 3 days,perform GFAP immunofluorescence staining.Under the fluorescence microscope,red fluorescence in the cytoplasm was observed,and the purity of the cells was 90%or more by counting.Conclusion:The survival rate and purity of the primary HSCs obtained from experimental animal models of Schistosoma japonicum-infected mice are above 90%,which meets the requirements of subsequent experimentsPart II.Liver Tissue Immunopathology and Dynamic Expression of HSCs-related lncRNA in Chronic Pathogenesis after Mice Infected with Schistosoma japonicum.Objective:To detect the immunopathology and dynamic expression of related lncRNA in liver of mice after C57BL/6J mice were infected with Schistosoma japonicum cercariae In the course of chronic disease.Methods:(1)HE staining of liver tissue sections,Then observe and compare the liver granuloma area of mice with different infection stages under the microscope.(2)Isolation of primary HSCs uninfected and infected for 4 weeks,7 weeks,9 weeks,and 12 weeks.After 7 days of incubation,use the TRIzol Reagent to extract the total RNA,using the real time fluorescence quantitative PCR(Real-time PCR)to detect the expression levels of lncRNA-p21?lncRNA-H19?lncRNA-GAS5?lncRNA-MALAT1?lncRNA-PVT1 in primary HSCs of C57BL/6J mice and analyzing and comparing the dynamic expression of each gene during schistosomiasis.Results:(1)Results of HE staining:in liver tissue sections from mice which is uninfected with Schistosoma japonicum,it was observed that hepatic lobules are intact and hepatocytes are well-formed;However,in infected liver tissue sections,it was observed that the normal structure of the hepatic lobes was destroyed,and inflammatory cells infiltrated around the eggs to form granulomas,and with the occurrence of fibrosis,the area of granulomas was the largest at 7 weeks of infection with the increase of the infection cycle,at 9 weeks 12 Peripheral area shrinks and collagen deposition gradually increases.(2)After C57BL/6J mice infected with Schistosoma japonicum,The lncRNA-PVT1 and lncRNA-GAS5 were expressed in HSCs With the occurrence and development of liver fibrosis.The expression of lncRNA begins to rise after infecting for 4 weeks,it reaches the peak after infecting for 7 weeks(PVT1,5.184±0.7346,gene/GAPDH,P<0.01;GAS5,5.849±0.2907,gene/GAPDH,P<0.0001).However the expression continues to decline after infecting for 9 weeks and 12 weeks.The expression of lncRNA-H19 begins to rise after infecting for 4 weeks,But it is gradually decreasing after infecting for 7 weeks,9 weeks,12 weeks.The expression of lncRNA-p21 start to decrease significantly after infecting for 4 weeks,it was starting to rise after 7 weeks,after 12 weeks the expression continues to decrease until less than none infect.The expression of lncRNA-MALAT1 begins to rise after infecting for 4 weeks,it reaches the peak after infecting for 7 weeks(6.666 ± 0.08177,gene/GAPDH,P<0.0001).It slightly decreased and still maintained at a high level after 9 weeks,it was significant decrease after 12 weeks.Conclusion:During the pathogenesis of Schistosoma japonicum,HSCs are continuously activated.The expression of lncRNA-P21 and lncRNA-H19 in HSCs is low.The expression of lncRNA-P21 decreases with the increase of liver fibrosis.The molecular expression of lncRNA-H19 decreased gradually with the increase of fibrosis at 7 weeks after infection.The expression levels of lncRNA-PVT1,lncRNA-GAS5,and lncRNA-MALAT1 are relatively high,the changes are more significant,and they are more closely related to the degree of liver fibrosis,which increases with the increase in the degree of fibrosis.The expression of the above lncRNA is down-regulated to affect the progress of liver fibrosis.Part ?.The effects of co-stimulatory signals ICOSL/ICOS on the immunopathological formation and HSCs-related lncRNA dynamic expression in mice infected with Schistosoma japonicumObjective:To investigate the down-regulation of co-stimulation signals ICOSL/ICOS,and to analyze and compare the liver pathological changes of ICOSL-KO mice and wild-type control C57BL/6J mice and their effects on the expression of related lncRNA.Methods:(1)The HE staining method was used to treat liver samples from ICOSL-KO mice and wild-type C57BL/6J mice for five infection cycles.(2)Collect primary HSCs from five infection cycles.After 7 days of in vitro culture,extract the total RNA of the cells through TRIzol Reagent and reverse transcribe them into cDNA.Using real time fluorescence quantitative PCR(Real-time PCR)to detecct the expression levels of lncRNA-p21,lncRNA-H19,lncRNA-GAS5,lncRNA-MALAT1.and lncRNA-PVT1 in the primary HSCs of ICOS-KO mice and C57BL/6J mice,and analyzing and comparing their expression changesResults:The expression of lncRNA-P21 and lncRNA-GAS5 in primary HSCs of ICOS-KO mice was higher than that of wild-type C57BL/6J mice in the same period.(lncRNA-P21,9w,2.071±0.1717 vs 0.9556 ± 0.07206,P<0.0001,gene/GAPDH)(lncRNA-GAS5,7w,14.08 ± 1.626 vs 5.849 ± 0.2907,P<0.0001,gene/GAPDH).Howerver the expression of lncRNA-PVT1(7w,3.721±0.2404 vs 5.184± 0.7346,P<0.05,gene/GAPDH)?lncRNA-H19(9w,1.09±0.139 vs 1.524±0.1206,gene/GAPDH)?lncRNA-MALAT1(9w,1.71 ± 0.4134 vs 6.235 ± 0.1149,P<0.0001,gene/GAPDH)is lower than the C57BL/6J mice in the same period.Conclusions:In fibrotic diseases caused by Schistosoma japonicum,the liver fibrosis in ICOS-KO mice is reduced,the down-regulation of the ICOSL/ICOS signal reduces the activation of HSCs,and the expression of lncRNA is also affected,which plays an anti-fibrotic effect The expressions of lncRNA-P21 and lncRNA-GAS5 were up-regulated,and the expressions of lncRNA-PVT1,lncRNA-H19,and lncRNA-MALAT1,which play a role in promoting fibrosis,were inhibited.It is suggested that in the course of liver fibrosis caused by Schistosoma japonicum,ICOS signaling has a regulatory effect on the expression of lncRNA,which can affect the occurrence and development of liver fibrosis.Part ?.The activation and proliferation of HSCs effected by interference of five kinds of lncRNA expression in vitroObjective:The effection of down-regulation of lncRNA on HSCs activation was detected,after interfering with the expression of lncrna.Methods:Transfect siRNA after JS-1 cells cultured stably,and The expression of a-SMA and type I collagen in HSCs was detected by real-time fluorescence quantitative PCR.Results:The expression of ?-SAM and type I collagen in HSCs improved by interference of lncRNA-P21?lncRNA-GAS5.Moreover,the interferece of lncRNA-PVT1?lncRNA-H19?lncRNA-MALAT1 can reduce the expression of ?-SAM and type I collagen in HSCs.Conclusion:The results showed that lncRNA-P21 and lncRNA-GAS5 could inhibit the activation of HSCs,while lncRNA-PVT1,lncRNA-H19 and lncRNA-MALAT1 could promote the activation of HSCs.LinRNA is expected to be a potential target for regulating HSCs activation and controlling liver fibrosis.