Study On The Role And Mechanism Of CaM/CaMKⅡ In ASIC1a Regulating The Activation And Proliferation Of Hepatic Stellate Cells

Liver fibrosis is a kind of chronic liver disease caused by a variety of pathogenic factors acting on the liver for a long time,which is actually a process of injury and repair,as well as a large number of extracellular matrix precipitation,as well as abnormal liver tissue and function.Phenotypic activation of hepatic stellate cells plays an important role in the process of hepatic fibrosis.inhibiting the activation and proliferation of hepatic stellate cells in the damaged liver is an effective therapeutic strategy to solve and treat hepatic fibrosis.However,the mechanism leading to the activation of hepatic stellate cells is not completely clear.Acid sensitive ion channel 1a(ASIC1a)is a cation channel with permeability to Na+and Ca2+,and its mechanism is extracellular acid activation.It is widely involved in the occurrence of inflammatory diseases such as cancer,Synovial inflammation and infection,suggesting that ASIC1 a may play an important role in liver fibrosis.In our previous study,when ASIC1 a regulated the activation and proliferation of hepatic stellate cells(HSC),which was the core link in the course of hepatic fibrosis,we found that when the membrane channel protein ASIC1 a was opened,the intracellular Ca2+increased,the expression of CaM/CaMKⅡ in HSC increased,and HSC activated and proliferated.In this process,Is CaM/CaMKⅡ highly expressed in HSC of rats with hepatic fibrosis regulated by ASIC1a? Is CaM/CaMKⅡ involved in the regulation of HSC and hepatic fibrosis by ASIC1 a as an intramembrane protein? For this reason,the main research contents are summarized as follows:1.Expression of ASIC1 a in rat hepatic stellate cells induced by acid.HSC-T6 cells were stimulated with acid(pH6.0)for 24 h in vitro.Western Blot analysis showed that,compared with normal group,acid-induced HSC-T6 cells showed significantly increased expressions of ASIC1 a and fibrosis-related proteins(p<0.01),the effect of psalmooxin 1(Pc Tx-1)on the activity of HSC-T6 cells was detected by CCK8 method,and the ASIC1 a channel was specifically blocked by Pc Tx-1.The results showed that Pc Tx-1 had little effect on HSC-T6 cell viability when the concentration of Pc Tx-1 was 100 ng/m L.The expression of ASIC1 a on the cell membrane surface of HSC-T6 was detected by immunofluorescence.The results showed that extracellular acid promoted the expression of ASIC1 a,and Pc Tx-1inhibited the ASIC1 a channel and decreased its activity.It was suggested that acid induced stimulation of ASIC1 a was expressed in hepatic stellate cells.2.To verify the expression and function of CaM/CaMKⅡ in HSC of rats with hepatic fibrosis regulated by ASIC1 a.When HSC-T6 was acidified to pH6.0,the expression of ASIC1 a was increased,and the protein and m RNA levels of CaM and CaMKⅡ were also increased.At the same time,we established the models of drug blocking,overexpression and silencing of ASIC1 a.The results of Western blotting and qRT-PCR showed that after blocking and silencing ASIC1 a,the m RNA and protein expression levels of ASIC1 a,Collagen-1,α-SMA,CaM and CaMKⅡ decreased significantly compared with the model group.The results of ASIC1 a overexpression plasmid group were contrary to the above results.Confocal microscopy was used to study the effect of activation of ASIC1 a channel on the change of Ca2+ concentration.The results showed that pH6.0 could increase the inflow of Ca2+ into the cell,and the concentration of Ca2+ decreased after silencing ASIC1 a,Pc Tx-1 and Amiloride.Overexpression of ASIC1 a can promote Ca2+ to enter HSC-T6 cells.In addition,flow cytometry detected the cell cycle changes of HSC cells.The results showed that acid-induced accelerated G1 max S transformation was reduced after exposure to ASIC1a-si RNA,hindered the cycle progression and inhibited the proliferation of HSC-T6 cells,while overexpression of ASIC1 a significantly increased the S phase and accelerated the G1 max S transformation of HSC-T6 cells,thus aggravating the proliferation of activated HSC-T6 cells.It is suggested that acid-stimulated ASIC1 a can promote the activation of HSC and CaM/CaMKⅡ is expressed in HSC of rats with hepatic fibrosis regulated by ASIC1 a.3.Effect and mechanism of CaM/CaMKⅡ on activation and proliferation of HSCThe in vitro model of acid-induced HSC-T6 was established to observe the effect of KN93,a specific inhibitor of CaMKⅡ,on acid-induced HSC proliferation.The cells were treated with media containing different concentrations of KN93(1.25μM,2.5μM,5μM,10μM,20μM,40μM).The results showed that the proliferation rate of HSC in pH6.0 group was higher than that in normal control group.Compared with the pH6.0group without the addition of KN93,the cell proliferation decreased after the addition of KN93.And it is concentration-dependent in a certain range.It was found that KN93(10μM)could significantly inhibit the proliferation of HSC,so this concentration was chosen as the concentration of drug action in the follow-up test.Then,we established the model of drug blocking,overexpression and silencing of CaMKⅡ.The results of Westernblotting and qRT-PCR showed that after blocking and silencing CaMKⅡ,the m RNA and protein expression levels of ASIC1 a,Collagen-1,α-SMA,CaM and CaMKⅡ decreased significantly compared with the model group.The results of CaMKⅡ overexpression plasmid group were contrary to the above results.After inhibition,silencing and overexpression of CaMKⅡ,the protein and m RNA levels of downstream related proteins MMP-13,NF-κB and NFAT also changed.At the same time,we detected the expression of CaM/CaMKⅡ and α-SMA by immunofluorescence.Compared with the normal group,the expression of CaM/CaMKⅡ and α-SMA in the model group was significantly increased,while KN93 could significantly inhibit the expression of CaM/CaMKⅡ and α-SMA.In addition,flow cytometry showed that acid-induced accelerated G1 max S transformation in HSC cells was reduced after exposure to CaMKⅡ-si RNA,which hindered the cycle progression and inhibited the proliferation of HSC-T6 cells,while overexpression of CaMKⅡ could significantly increase the S phase and accelerate the G1 max S transformation of HSC-T6 cells,thus aggravating the proliferation of activated HSC-T6 cells.These results suggest that the activation and proliferation of HSC induced by acid stimulation of ASIC1 a are related to CaM/CaMKⅡ.To sum up,CaM/CaMKⅡ promotes the activation and proliferation of HSC.4.Protective effect and mechanism of drug blocking CaMKⅡ on hepatic fibrosis in ratsThe animal model of hepatic fibrosis in CCl4 rats was established.Except for the normal control group,the rats in the other groups were injected intraperitoneally with40%CCl4 0.1ml/0.1kg twice a week for 12 weeks.From the 4th week of the model,each group was injected with drugs with different concentration of KN93 every day,and the model group and the normal group were injected with the same amount of NS through tail vein.The results showed that KN93 could significantly reduce the increased serum ALT,AST;HE staining and Masson staining to show the improvement of liver fibrosis in rats,and immunohistochemistry showed that the positive cells decreased significantly after treatment.Further studies showed that KN93 could significantly inhibit the expression of ASIC1 a,fibrosis protein,CaM,CaMKⅡ and downstream related proteins in liver tissue,suggesting that KN93 can improve the regulation of HSC activation,proliferation and fibrosis by ASIC1 a.To sum up,this study found that ASICla is involved in acid-induced activation of hepatic stellate cells.Blocking ASICla has protective effect on liver fibrosis.CaM/CaMKⅡ is highly expressed in HSC of rats with hepatic fibrosis regulated by ASIC1 a,CaM/CaMKⅡ promotes the activation and proliferation of HSC,and drug blocking CaMKⅡ has a protective effect on rats with hepatic fibrosis.