Praziquantel Ameliorates Liver Fibrosis By Inhibiting Hepatic Stellate Cell Activation Via TGF-beta/Smad Signaling

Schistosomiasis belongs to the zoonotic parasitic diseases that threaten human health and hinder socio-economic development in the world.Schistosomiasis caused by Schistosoma japonicum is mainly epidemic in China.Although much productive work have been down to control this diesae,there are still about 110,000 of patients infected with Schistosoma japonicum by the end of 2014.Further more,they are getting worse and probably develop into advanced schistosomiasis.The major pathology in schistosomiasis is the formation of egg granulomas in the liver which can result in secondary fibrosis.Post-transmission schistosomiasis refers to patients that there have been no parasite lived in the body after treatment;however,liver fibrosis is still sustainable development and eventually deteriorate into cirrhosis or liver cancer.Therefore,it is a noticeable issue of schistosomiasis prevention at present in managing post-transmission schistosomiasis with anti-fibrosis treatment,to prevent the continued development of liver disease.Praziquantel(PZQ)has the features of efficient,safe and few side effects as a drug of anti-schistosomiasis.In addition to the helminthicide activity for flukes,tapeworms and other worms,recent studies showed that PZQ could also affect directly on tissues and cells of the host.Previous studies in our laboratory demonstrated that the prolonged treatment with PZQ can inhibit the development of liver fibrosis in mice infected with Schistosoma japonicum.However,the exact mechanism is still not clear.Here,we focused on the impact of PZQ on the TGF-?/Smad signaling pathway,to further reveal the mechanism of PZQ in antifibrotic function.To avoid the impact of helminthicide activity of PZQ itself against the liver fibrosis induced by Schistosoma japonicum infection,we established mouse model of hepatic fibrosis induced by carbon tetrachloride.Then,we detected the role of PZQ on the related indicators of fibrotic liver tissue as well as TGF-?/Smad signaling pathway related factors by cytology and molecular biology techniques.Further more,we also detected the effects of PZQ on LX-2 cells stimulated by exogenous TGF-?1 in apoptosis,cell cycle,fibrosis index and TGF-?/Smad signaling pathway.At last,we used the technique of siRNA interference to further confirmed the antifibrotic role of PZQ on TGF-?/Smad signaling pathway.The main results obtained in this study are as follows:1.PZQ treatment significantly improved the liver pathology of CCL4-induced hepatic fibrosis in miceThe mouse model of hepatic fibrosis in mice is built by using intraperitoneal injection of 25%CCL4 twice a week for 4 weeks.Normal group,model group and PZQ group(300mg/kg×12h)are established,respectively.The results showed that the ALT,AST in model group is higher than that in normal group,and can be significantly reduced after 4 weeks treatment of PZQ.HE staining of liver tissue showed that normal liver tissues presented integrated lobule mesh fiber structure,with clear boundaries and no infiltration of inflammatory cells,as well as no fibroplasias and hepatic sinusoid/portal area dysplasia.But in model group,the liver tissues were loss of normal lobular structure with neutrophils,lymphocytes and other inflammatory cell infiltration.The fibrous tissues surrounding lobular hyperplasia is partly formed pseudolobules.In the group of PZQ treatment,the liver tissues showed almost normal liver structure,with low degree fibrosis,well hepatic cords,a few scattered hepatocellular vacuolation,lobular lesions and no inflammatory cell infiltration in periportal area.These results indicated that PZQ treatment for 4 weeks could definitely improve liver function and histological damages in CCL4-treated mice.2.PZQ treatment reduced CCL4 induced hepatic fibrosis in miceIn order to evaluated the anti-fibrotic effects of PZQ on liver tissues,we analysed the Collal and a-SMA expressions by immunohistochemistry.In normal group,Col11?1 and ?-SMA were rarely expressed in the periportal tissues.In contrast,in model group,their expressions were significantly increased.However,in PZQ treatment group,the Collal and a-SMA expressions were obviously less than that in model group,even to be normal.Then,we further confirmed,by using western blot,that the Collal and a-SMA protein levels in the HSCs isolated from PZQ-treated mice were much lower in comparison with that of model group.These results clearly showed that PZQ treatment of 4 weeks could ameliorated CCL4-induced hepatic fibrosis of mice by reducing Col1?1 and ?-SMA expressions of HSCs.3.PZQ treatment suppresses TGF-?/Smad signaling pathway in CCL4-induced liver fibrosis of miceTo further investigate the anti-hepatic fibrosis mechanism of PZQ,we detected levels of phosphorylated-Smad2/3,Smad2/3 and Smad7 proteins in liver tissues and HSCs,respectively,by western blot method.It was obvious that in the PZQ treatment group,the expression of phosphorylated-Smad2/3 was decreased,but the expression of Smad7 was increased compared with the model group.These results suggested that treatment of PZQ for 4 weeks could strongly suppress TGF-?/Smad signaling pathway in CCL4-induced liver fibrosis of mice.4.PZQ inhibited the activation of LX-2 and TGF-?/Smad signaling pathway in vitroTo further observe the effect of PZQ on TGF-?1-induced LX-2 activation and TGF-?/Smad signaling pathway,we detected the gene and protein expression levels of Collal,a-SMA,Smad2/3,Smad4 and Smad7 by using qRT-PCR and western blot,respectively.The results presented that PZQ could reduce Smad3,but increase Smad7,gene expression in vitro.Furthermore,the levels of total Smad2/3 and phosphorylated Smad2/3 were detected.The result showed clearly that praziquantel could down-regulate phosphorylation of Smad2/3 in vitro.aken together,the results suggested that PZQ may directly inhibit activation of HSCs and TGF-?/Smad signaling pathway in vitro.5.Smad7 as a main target for PZQ to inhibit pro-fibrogenic function of HSCsIn order to further elucidate the target of PZQ involved in TGF-?/Smad signaling-mediated fibrosis,the Smad7 gene expression in LX-2 cells was knockdown by using siRNA technique and the Collal expression of cells with or without PZQ stimulation was detected.The results showed that the Collal expression of cells was no longer decreased under PZQ stimulation in comparison to that of NC-siRNA interference control.Our results suggested that PZQ inhibits TGF-?/Smad pathway possiblily by increasing the expression of Smad7,thereby inhibiting the function of HSCs.In summary,this study demonstrated that PZQ could block TGF-?/Smad signaling pathway to inhibit the activition of HSCs and ECM formation,probably through promoting Smad7 expression.It reveales a new mechanism of PZQ on anti-hepatic fibrosis and expands our understanding on the pharmacological effects of PZQ in treatment of schistosomiasis and even other diseases with hepatic fibrosis.