Regulatory Effects Of RhoA Inhibitor Fasudil On Th17 Cells And Hepatic Fibrosis In Mice Infected With Schistosoma Japonicum

RhoA is an important intracellular signaling molecules belonging to Rho subfamily of small GTPases.Rho GTPases behave like“molecular switches”in the process of cellular signal transduction.Rho GTPases switch between the active GTP-bound(Rho-GTP)and inactive GDP-bound(Rho-GDP)forms.Once activated,the Rho-GTPases are capable of interacting with multiple downstream effector proteins until switching back to their inactive GDP-bound(Rho-GDP)form.In vivo,Rho GTPases control complex cellular physiological processes by cycling between the active GTP-bound form(Rho-GTP)and inactive GDP-bound form(Rho-GDP).Among over twenty members of this subfamily,three of them,RhoA,CDC42 and Rac1,have been studied a great deal,especially RhoA.RhoA of Rho GTPases is widely distributed in mammalian cells,which regulates multiple pathways and plays central roles in singling transduction.Rho GTPases are becoming the focus of research interests as therapeutic targets for intervention of various diseases.RhoA is an intracellular signal transducer of the Rho family GTPases that cycles between the active GTP-bound form(Rho-GTP)and inactive GDP-bound(Rho-GDP)forms.RhoA has been shown to modulate actin cytoskeleton organization,migration,cell adhesion,survival,and proliferation.Especially in T cells,RhoA plays roles in T cell polarization,thymic egress and thymocyte development.Asthma is a disease of chronic allergic airway inflammation,in which T cells play a fundamental role in its pathogenesis.Thus,RhoA dependent metabolic pathway can be explored as a potent therapeutic target in treating allergic airway inflammation.Studies are now suggesting that Th1/Th2could be involved in regulating hepatic fibrosis observed in chronic schistosomiasis disease.As egg deposition builds up,Th1 immune responses are dampened and a more pronounced Th2 response is observed.This process is characterized by a granulomatous reaction with subsequent fibrosis,which is dependent mainly on Th2 mediated cytokines including interleukin-4(IL-4),interleukin-5(IL-5),and interleukin-13(IL-13).RhoA deficiency impairs glycolysis in activated T cells and Th2 cells.Therefore,blockade of glycolysis also inhibits Th2 differentiation.In cytology,the basis of hepatic fibrosis is the activation of hepatic stellate cells.Studies have shown that selective blocking of the signaling pathway of Rho GTPases can inhibit the progression of fibrosis and improve the prognosis.At present,the molecular mechanisms of Th1 and Th2 cells on the regulation of hepatic fibrosis through RhoA are relatively clear,whereas Th17 cells remains elusive on this topic.In this study,we investigated the roles and underlying molecular mechanisms of RhoA on Th17 cells and hepatic fibrosis with the use of RhoA pharmacological inhibitor fasudil and experimental infection model by Schistosoma japonicum.Fasudil is a selective inhibitor of Rho-associated kinase(ROCK),one of the main downstream signaling molecules of RhoA.It has widely applied for the functional studies of RhoA.In our study,na?ve CD4~+T cells were differentiated under Th17 conditions in the presence or absence of fasudil to observe its effect on Th17 differentiation.Moreover,effects of fasudil on the apoptosis of hepatic stellate cells were also carried out.In the basis of above in vitro experiments,the effects of fasudil on the worm burden,fecundity and hepatic fibrosis in mice infected with S.japonicum were further investigated.Objective:To explore the effect of fasudil on Th17 cells and hepatic stellate cells in vitro.And the effects of fasudil on worm burden,fecundity and hepatic fibrosis in mice infected with S.japonicum.Finally,combined with its molecular mechanism,we elucidated the regulatory effects of RhoA inhibitor fasudil on Th17 cells and hepatic fibrosis in mice infected with S.japonicum.Methods:1.Purified CD4~+T cells were differentiated under Th17 conditions for 4 d and restimulated with PMA plus ionomycin for 5 h in the presence or absence of fasudil(30mM).Cells were processed for IL-17 intracellular staining.2.IL-17F and IL-21 in the culture supernatants were determined by ELISA.3.Hepatic stellate cells were cultured in vitro and observe the effects of different concentrations of fasudil(0mM?5mM?10mM?20mM?50mM)on hepatic stellate cells.4.Mice were infected with S.japonicum through their abdominal skin.At 4,5,6 and 7 wk post infection,eggs of S.japonicum were counted in the feces of infected mice.At 8 wk after infection,all the mice were sacrificed and the adult worms were collected and counted.5.A portion of liver was digested in 4%KOH for 18 hr,and the eggs were counted and expressed as numbers per g of liver.The right lobes of livers were fixed and processed for histological examination with hematoxylin and eosin and Masson trichrome staining.Other two parts of liver were used for hydroxyproline detection and qPCR analysis for Col-?,Col-? and TGF-b(16).Results:1.Fasudil suppressed Th17 differentiation and effector cytokines secretion in vitro(p<0.05).2.Fasudil was able to induce the apoptosis of hepatic stellate cells in a dose-dependent manner(p<0.05).3.At 4,5,6 and 7 wk after infection,the numbers of eggs have no statistical difference between fasudil-treated and untreated groups at each time-point (p>0.05).And the number of adult worms between fasudil-treated and untreated groups are also no statistical difference.4.Fasudil treated group showed reduced size of the granuloma compared to control Group(p<0.05).By Masson Trichrome staining,the blue fiber areas,were significantly lower in fasudil treated groups compared to control ones(p<0.05).The results of hydroxyproline were consistent with Masson trichrome staining.Furthermore,the mRNA lever of several important genes related to hepatic fibrosis,such as Col-I,Col-III and TGF-b(16)were significantly down-regulated in fasudil treated groups compared to control ones(p<0.05).Conclusions:1.Fasudil,a selective inhibitor of RhoA-ROCK pathway,suppressed Th17 differentiation and effector cytokine secretion.2.Fasudil was able to induce the apoptosis of hepatic stellate cells in a dose-dependent manner.3.In an experimental infection model with S.japonicum,we found that fasudil treatment did not significantly affect worm burden and fecundity.However,circumoval granuloma formation and hepatic fibrosis were significantly inhibited in fasudil-treated mice.Furthermore,fasudil treatment downregulated the mRNA lever of several key genes related to hepatic fibrosis,such as Col-I,Col-III and TGF-b1.4.With the use of the selective inhibitor of RhoA-ROCK pathway,this study firstly revealed the regulatory effects of fasudil on Th17 cells and hepatic fibrosis.