HSP47-shRNA Interference Of Activated HSCs Associated Receptors In Schistosoma Japonicum-induced Hepatic Fibrosis In Mice

Backgroud and objective: Schistosomiasis, with an estimated200million people infected,mostly epidemic in the Middle East, eastern Asia, South America[1,2]. In China,Schistosoma japonica still represents a serious disease burden for50million people peryear[3]. As a common parasitic diseases endangering human health, one of the keyschistosome hepatic fibrosis pathogenesis and treatment research is clinical and basicresearch.Liver fibrosis, not only represents the final pathway of virtually Schistosoma chronicinflammatory injuries, also reveals the immunological tolerance induced by Schistosomajaponicum infection resulting in a long-term parasitism of immune escape. The pathologyassociated with schistosomiasis results from schistosome egg deposition and granulomatousreactions, followed by accumulation of hepatic fibrosis, obstruction of hepatic blood flow,and finally the portal hypertension. Portal hypertension (portal hypertension, PHT), acommon clinical syndrome, can cause severe life-threatening complication. Moreover, theendothelin system (ETS) plays a critical role in the course of portal hypertension. ET(Endothelin) was first reported in1988by Yanagisawa[4]et al as a novel potent vasocon-strictor peptide isolated from porcine endothelial cells. The endothelins are a family of isopeptides named ET-1, ET-2and ET-3. ET-1is the most abundant and appears to be themost clinically important form of endothelin. ET binds to two receptor subtypes, ETA andETB which mediate a range of it’s biologic effects. ETA receptors are expressed with therank order affinities of the peptide as ET-1> ET-2ET-3, while ETB receptors haveequal affinity for all ET subtypes. The relative densities of the ETA and ETB receptors varybetween tissues and can be altered by disease. The pathological roles of ET are determinedby the distribution of ET receptors. For example, expression of ET receptors varies indifferent states of PHT. The expression of ETR is regulated by many factors, such ashypoxia, hypothermia, fat protein level, and ET level. There may be a negative feedbackmechanism between ET and its receptors; AT present, ETR receptor antagonist has becomea hot topic in pharmaceutical research.Hepatic stellate cells (HSCs), which play a key role in the process of hepatic fibrosis,are an important source of cytokines and their receptors, such as ET, TGF-β, PDGF andIGF. The expression of receptors can be provoked by a range of chronic injuries to the liver,amongst which are aging, hypoxia, statement of activation. Endothelin system (ETS) hasbeen implicated in regulating the HSCs function owing to their interaction whichcontributes to aggradation of liver fibrosis, changes of intrahepatic and extrahepaticvascular resistance and the blood flow of portal vein[5,6].Heat shock protein47(HSP47) is a collagen-binding, stress-inducible proteinlocalized in the endoplasmic reticulum, as well as playing a specific role as a molecularchaperone in the folding and secreting of procollagen molecules[7]. HSP47expression wasupregulated in many experimental animal models of fibrosis. Selective blocking of HSP47may reduce collagen accumulation and delay the progression of fibrotic diseases[8,9].However, the relationship between HSP47and the progress of liver fibrosis is still in theexploratory phase.The relationship between Schistosoma japonicum hepatic fibrosis and ETR has notbeen fully understood. Still, the effect of HSP47on the activation of HSC and ETR expression has not been reported. The purpose of this study is applying HSP47-shRNA onmice with schistosomiasis liver fibrosis, and further exploring its effect on HSC receptorexpression, looking for the diagnosis and treatment of new targets.Methods: The constructed HSP47-shRNA expression plasmid was transfected into theNIH/3T3cell lines with lipofectamine transfection technique. HSP47mRNA and proteinexpression were examined by real-time PCR and Western blotting respectively to detect theefficiency of interference. The ETBR expression on NIH/3T3cells were detected by flowcytometry. In vivo study, we used HSP47-shRNA plasmid administered the same injectionsi.v. per week from9weeks to14weeks post infection with a mouse model of S.japonicum-induced hepatic fibrosis. Liver portal vein was calculated. The efficiency ofHSP47mRNA interference was detected and the protein expression was examined. flowcytometry was used to determine receptors on HSCs.Results: The interference efficacy of HSP47-shRNA on its targeted gene expression were（25.39±2.358）%and （48.71±1.391）%after24h and48h transfection, respectively,with statistical significant difference compared with that of negative control group(p<0.05). The HSP47protein expression was analyzed by Western-blotting aftertransefection, and the protein was down-regulated in accordance with mRNA.The flow cytometry results showed that expression of ETBR on NIH/3T3cells posttransfection was up-regulated to (53.433±5.243), compared with the negative controlgroup（p<0.05）. Meanwhile, TGFβreceptor, PDGF receptor were detected and there wereno significant changes.In vivo, we used HSP47-shRNA intravenous injection per week from9weeks to14weeks post infection with a mouse model of S. japonicum-induced hepatic fibrosis.HSP47-mRNA relative expression was (2.686±0.7114), showing statistical significancecompared with control group (6.001±0.4583)(p<0.01). The expression of HSP47proteinwas detected by Western-blot, which was consistent with the mRNA level. Liver portalvein was down-regulated after shRNA administered. The mean fluorescence intensity on HSCs of shRNA intervention group showed ETAR and ETBR were (4249±344.42) and(3706±193.76), compared with infected group (8538±493.71) and (5052±212.93)respectively (p<0.01).Conclusion: In this study, the shRNA interference plasmids targeting HSP47, both in vitroand in vivo experiments, showed that it could effectively interfere with the expression ofHSP47mRNA and protein. In vitro, ETBR increased on the NIH/3T3cells aftertransfection. While in vivo, ETRs were down-regulated with portal vein pressure decreased.To sum up, we conclude that there may be feedback among HSP47, HSCs and ETS, whichhas the stability on regulating hepatic fibrosis.