| Schistosomiasis japonica is a disease that is seriously harmful to human health.By the end of 2012,there are still approximately 240000 patients who are suffered from schistosomiasis japonica in China.The life history of bilharzia is very complicated,the antigen excreted by them during every stage of development in vivo can lead to the immunopathological effects in host,in which type Th1/Th2 cell response play an important role during the infection process.Hepatic egg granuloma and subsequent hepatic fibrosis are the main pathological injuries of bilharziasis.The activation of HSCs is the key link of hepatic fibrosis.Besides the functions of lipid droplet storage and fibrosis participation that have been verified,there are also some other important functions of HSCs such as participation in the hepatic immunoreactions process.Recent research has found that HSCs shows heterogeneity and plasticity in liver which has different subgroups of various cell phenotypes and functions.Therefore,we will do ressearch into the functions of various subgroups of HSCs during the process of schistosoma infection,so as to carry on more thorough understanding to hepatic fibrosis.As a preferred antischistosomal drug,praziquantel has the characteristics of higher efficiency,lower toxicity and less side effects.Besides the insecticidal action towards worm such as fluke and tapeworm,in recent years,some researchers have proposed that praziquantel has direct pesticide effect on the host tissue and cells.The preliminary study of our lab found that,in the experimental model of mice which had been infected by schistosoma japonicum,the long-course treatment of praziquantel could inhibit the deterioration of hepatic fibrosis.Thus,we studied the effects of praziquantel on the function of various subgroups of HSCs,to further uncover the mechanism of the anti-fibrosis function of praziquantel.miRNA is a kind of highly conservative non-coding small RNA that can inhibit the translation process after transcription of target gene by the combination of its seed sequence with complementary sequence at the 3'UTR end of target gene,so as to regulate a series of physiological and pathological processes.Researches show that miRNA plays an important regulative role in the occcurance and development of hepatopathy and hepatic fibrosis caused by various factors.For instance,the expression of miRNA-454 decreases during the infection process by schistosoma japonicum,it can inhibit the activation of HSCs by complementation with smad4 3?UTR end of target gene.We analyzed the miRNA that involved in the activation process of HSCs,and its regulatory function towards various subgroups of HSCs by observing the dynamic change of HSCs miRNA during the process of infection by schistosoma japonicum.This research chose HSCs as research objective,studied the functional characteristics of HSCs in the infection process by schistosoma japonicum.First of all,we established a schistosoma japonicum infection model in mice,studied the characteristic of HSCs functional differentiation in the process of infection and its possible mechanism;Then,we established praziquantel long-course treatment model in chronic schistosoma japonicum infected mice,to observe the effects of praziquantel on the function of various subgroups of HSCs;Finally,we used the miRNA expression chip to uncover the miRNA expression profiles of HSCs in the process of infection,and found miRNA that related to regulating functional differentiation.The main results are listed as follows:1.The activated HSCs have functional plasticity during the infection process by schistosoma japonicumWe found that,the expression of MHC II and immune costimulatory molecules CD80,CD86,B7-H1 on the surface of HSCs,increased in the process of infection,suggested that the activated HSCs had the function of immune cells which enabled them to participate in the process of hepatic immune response;and the expression of?-SMA,Collal gene of HSCs also increased significantly in the process of infection,suggested that the activated HSCs also had the function of myofibroblastic-like cells and played an important role in the promotion of fibrosis.The above results suggested that,in the process of schistosoma japonicum infection,the function of the activated HSCs is plastic.2.Functional differentiation of activated HSCs can be divided into two populations as MHC II+and MHC II-HSCs by MHC II in the process of schistosoma japonicum infection.Recent studies showed that there are function difference of HSCs at different position and condition of liver,HSCs'structure and function are heterogeneous in the liver.So,we speculated that there are different subgroups of HSCs in the process of schistosoma japonicum infection because of their functional differentiation.So using magnetic cell sorting method,choosing MHC II as tagged molecule,HSCs were divided into MHC II+and MHC II-HSCs in schistosoma japonicum infected mice.There were no statistical difference between MHC II+and MHC II-HSCs in the levels of LRAT and vitamin A,which suggested that MHC II+and MHC II-HSCs both came from quiescent HSCs to participate in the storage and metabolism of vitamin A in the process of schistosoma japonicum infection.But,the expression of?-SMA in MHC II-HSCs was obviously higher than that of MHC II+HSCs,which suggested that there existed heterogeneity in the condition of two groups of cells after activated,only MHC II-HSCs had the characteristic of myofibroblastic-liked cells.To further study the function of MHC II+and MHC II-HSCs,we found that the level of CD80,CD86 and B7-H1 on the surface of MHC II+HSCs was significantly higher than of MHC II-HSCs in the process of infection,but the mRNA level of?-SMA?Col1a1?TIMP1 on the surface of MHC II-HSCs was significantly higher than that of MHC II+HSCs,which suggested that there were MHC II+and MHC II-HSCs with phenotype and function hetemgeneity in the process of schistosoma japonicum infection.MHC II+HSCs had immunologic functions that could stimulate allogeneic T-cell response,MHC II-HSCs had myofibroblastic-like cell functions that contributed to fibrosis.3.The functional differentiation of MHC II+and MHC II-can be regulated by IFN-?in the process of schistosoma japonicum infectionWe found that,in the process of schistosoma japonicum infection,the espression level of IFN-?began to rise at the early stage of infection,which had time consistency with the expression of HSCs MHC II molecules,so the functional differentiation of HSCs were inferred to be related to the expression of IFN-?.When stimulating the HSCs in different conditions with IFN-?in vitro,IFN-?could increase the expression of MHC II and CIITA and decrease the expression of fibrogenic related genes Col1a1in all cases;and the expression level of MHC II in HSC decreased significantly when IFN-?-/-mice were infected with schistosome.These results indicated that IFN–?could induce the pheotype change in HSCs and promote the transform from MHC II-HSCs to MHC II+HSCs.The functional difference in HSCs might be regulated by IFN-?in the process of schistosoma japonicum infection.4.Praziquantel play the function of anti-fibrosis by acting on MHC II-HSCsRecently,some researchers considered that praziquantel had direct pesticide effect on the host tissue and cells.In early stage of our research,we found that the long-course treatment of praziquantel could inhibit the deterioration of hepatic fibrosis in the experimental model of mice infected with schistosoma japonicum.We observed the effect of praziquantel on different subgroups of cells by treating the mice of chronic infection?12W?with long-course treatment of praziquantel for continuous 4W.The results showed that praziquantel could inhibit the expression of MHC II molecules and CD86 on the surface of HSCs.In the early stage of our research,we found that praziquantel could inhibit the secretion of Th1 cytokines,such as IFN-?,which might lead to the function inhibiting of MHC II+HSCs.At the same time,praziquantel could also inhibit the expression of the fibrosis-related genes of MHC II–HSCs,such as?-SMA?Col1a1?TIMP1,which indicated that the anti-fibrosis function of praziquantel might be related to MHC II–HSCs.To further study the possible mechanism of the effect of praziquantel on the function of MHC II+/-HSCs,we observed the apoptosis of two groups of cells after treated with praziquantel,the results showed that there were no sign of apoptosis in two groups HSCs of MHC II+/-.All the above results suggested that praziquantel played the role of anti-fibrosis by acting on MHC II-HSCs,the mechanism of which might not be explained by promoting the apoptosis of MHC II-HSCs,but by decreasing the expression of fibrogenic related genes.5.The miRNA dynamic expression profile of HSCs during the infection and the function differentiation-related miRNAIn order to study the dynamic change of HSCs miRNA during the infection process by schistosoma japonicum and the possible regulatory function of different miRNA in different subgroups of HSCs,we carried out the testing by using the miRNA expression profile chip.The results showed that HSCs miRNA had already changed significantly in the early stage of schistosoma japonicum infection,including the upregulated of 17 miRNAs and downregulated of 22 miRNAs.By further pathway analysis of different miRNA,it showed that the high concentration of signaling pathways included regulation of actin cytoskeleton signal pathway,MAPK signal pathways and PI3K-Akt signaling pathways,etc.The miRNA that may be associated with fibrosis and immune function included miR-21a-3p?miR-21a-5p?miR-146b-5p?miR-29c-3p and miR-217-3p.By forecasting target genes,we found that miR-21a-5p could complement and combine with the 3'UTR of smad7,inhibiting smad7 translation.The results of qRT-PCR test showed that,the expression level of miR-21a-5p persistently increased during the infection process and is higher in MHCII-HSCs than in MHCII+HSCs,suggested that the miR-21a-5p might through the activation TGF-beta/Smad signaling pathways,promoting the transformation of HSCs to MHC II-HSCs in the process of infection by schistosoma japonicum.The miRNA may participate in the regulation of the functional differentiation of MHC II+/-groups of HSCs by regulating the translation level after transcription.In conclusion,we demonstrated that the activated HSCs had heterogeneity in the process of schistosoma japonicum infection,and it could be divided into two population as MHC II+and MHC II-HSCs by MHC II.The functional differentiation of HSCs might be regulated by IFN-?in the process of infection.Additionally,we found that praziquantel played the role of anti-fibrosis by acting on MHC II-HSCs,the mechanism of which might due to decrease the expression of fibrogenic related genes;at the same time,we analyzed the dynamic expression profiles of HSCs in the process of infection by using miRNA chips,and found that miRNA could regulate the progress of differentiation.These results will help us to understand the function of HSCs from a new perspective in the process of schistosoma japonicum infection,providing us a new reference in anti-fibrosis treatment. |
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