MiR-181b-5p Promotes Shistosomiasis-induced Liver Fibrosis By Targeting Smad7

Schistosomiasis is a common parasite disease in clinic,which is only less than the hazard degrees of malaria.There are more than 230 million people infected with schistosomiasis in the world including 76 countries,and at least 200 thousand people dead for schistosomiasis every year.The main pathological change of schistosomiasis is schistosoma eggs-induced liver fibrosis characterized by excessive extracellular matrix(ECM)deposition,which can markedly influence physiological functions of liver,even causes liver failure.Hepatic stellate cells(HSCs)is the derived cells of ECM.The main biological control measures for schistosomiasis is praziquantel.However,praziquantel has not shown benefit in improving of liver fibrosis,and the drug resistance and chemical toxicity of praziquantel also limit its application.Therefore,it is very important to develop new an effective strategy in prevention and treatment of schistosomiasis-induced liver fibrosis.MicroRNAs(miRNA)are highly conservative endogenous non-coding RNAs with 20-25 nucleotides,those miRNAs exert negative post-transcription regulation by complementary combining to the 3'untranslated region(3'UTR)zone of target genes'mRNA.Previous studies have shown that miRNAs play important regulatory roles in liver diseases and hepatic fibrosis,that give evidence miRNA can be a therapeutic target for liver fibrosis.MiR-181b-5p is an important miRNA which is significantly increased in schistosomiasis,and it also exerts regulatory effect on activating hepatic stellate cells in cell experiment.In the present study,we consider miR-181b-5p as a potential target for liver fibrosis treatment,and try to develop a possible anti-hepatic fibrosis strategy through deeply studying the mechanism of miR-181b-5p on schistosomiasis-induced liver fibrosis.Furthermore,providing theoretical basis and experimental data for the prevention and treatment of schistosomiasis and liver fibrosis.Objective:In the present study,we explored the mechanism of miR-181b-5p on schistosomiasis-induced liver fibrosis from animal and cellular level,respectively.Firstly,we verified the dynamic expression changes of miR-181b-5p in the progression of schistosomiasis in mice model.Then,the regulatory effects and its potential mechanisms of miR-181b-5p in schistosomiasis-induced hepatic fibrosis was further studied by interfering expression of miR-181b-5p.Furthermore,providing experimental basis for the basic research and clinical intervention strategy of schistosomiasis based on miR-181b-5p.Methods:Female BALB/C mice were purchased from Wuhan University Animal Laboratory Center,aged 6-8 weeks and weighing 20-22 grams.Modelling method:To establish a schistosomiasis infection model,mice were infected with 20-30 S.japonicum cercariae through a clear and bald abdominal skin.To verify the expression change of miR-181b-5p in schistosoma,mice were randomly divided into two groups:(1)Control group:mice without any treatment;(2)Model group:mice were infected with 15-20 S.japonicum cercariae.All the mice were sacrificed at 6,8,10 weeks post infection.To examine the role of miR-181b-5p in schistosomiasis-related hepatic fibrosis in vivo,mice were first infected with S.japonicum cercariae.Next,they were injected with a rAAV8-miR-181b-5p-sponge vector for sustained inhibiting miR-181b-5p expression through tail vein,a control vector,or PBS at day 10 post-infection as negative control or blank control,all the mice were sacrificed at 8 weeks post infection.To observe the expression change of miR-181 b-5p in vitro,LX2 cells were activated by TGF-?1.Then,a miR-181b-5p mimic or inhibitor was transfected into LX2 cells for up-regulating or down-regulating miR-181b-5p expression,in order to explore the mechanism of miR-181b-5p on the activation of hepatic stellate cells and the occurrence of liver fibrosis.The data were analyzed by student-T test and One-Way ANOVA,P<0.05 indicated the difference was statistically significant.Results:(1)The expression of miR-181b-5p were increased gradually with the prolongation of infection,and Smad7 expression were decreased gradually with the extension of infection;(2)Down-regulation of miR-181b-5p significantly reduced ECM deposits and liver granulation by HE and Masson staining,decreasing the expression of liver fibrosis genes COL1A1 by RT-PCR and Western Blot,reducing fibrosis-related cytokine TGF-?1 by ELISA,and increasing Smad7 protein expression by RT-PCR and Western Blot in the liver of schistosomiasis infected mice;(3)The expression of miR-181b-5p was elevated in TGF-?1(5ng/ml,10 ng/ml,15ng/ml)stimulated LX2 cells,and the administration dose with the highest fold change was 10 ng/ml.And expression of miR-181b-5p was increased at different time post 10ng/ml TGF-?1 administration,while 24 hours post TGF-?1 administration the level of miR-181b-5p expression increased to peak;(4)Down-regulating of miR-181b-5p significantly decreased COL1A1 expression in TGF-?1-stimulated LX2.Conclusion:(1)The expression of miR-181b-5p was gradually increased with the progression of Schistosoma japonicum infection,while the expression of Smad7 was gradually decreased;(2)Down-regulation of miR-181b-5p significantly alleviate Schistosoma japonicum-induced hepatic granuloma and liver fibrosis;(3)MiR-181b-5p aggravate the process of schistosomiasis induced liver fibrosis by targeting Smad7,while inhibition of miR-181b-5p alleviate liver fibrosis induced by schistosomiasis through up-regulating expression of Smad7.