Roles Of TRPC6-Mediated SOCE In Hepatic Fibrosis And The Underlying Mechanism

Background:Liver fibrosis is a common outcome of chronic liver injury that can eventually lead to cirrhosis,which is a worldwide public problem.There is no clear treatment for cirrhosis and liver transplantation is the only option for advanced liver disease.The main characteristics of hepatic fibrosis are excessive Extracellular Matrix（ECM）accumulation and hepatic lobule structure disorder.Hepatic Stellate Cell（HSC）is the main producer of ECM and plays an important role in the formation of Hepatic fibrosis.HSCS are stationary in a healthy liver.Liver fibrosis is a dynamic process involving the interaction of different types of cells in the liver tissue.Resting hepatic stellate cells transform into fibroblast-like cells that express-smooth muscle actin（α-SMA）,triggering a chronic process of hepatic fibrosis.Toxins,viruses,oxidative stress,necrosis,apoptosis and growth factors are all involved in the activation of hepatic stellate cells.In activated stellate cells,multiple signal transduction pathways are activated,constituting the pathogenesis of hepatic fibrosis.These signaling molecules involved in HSCS activation and transformation provide potential therapeutic targets for the prevention of liver fibrosis and cirrhosis.Ca²⁺play a vital role in the physiological functions of the human body,including myocardial contraction,vascular muscle contraction,bone mineralization,fertilization,synaptic transmission,blood coagulation and neuron function.Store-operated Ca²⁺Entry（SOCE）is also called voluminous Ca²⁺flow（CCE）,which is the main mechanism of calcium ion flow in non-excited cells.TRPC is a non-selective calcium channel,which is involved in the regulation of a variety of physiological functions and pathological processes.The TRPC family of channels are calcium channels that are expressed on the plasma membranes of many tissues,including the heart.TRPC channels are expressed in almost all tissues and cell types and play an important role in regulating various cell functions.There was also a correlation between TRPC channel protein expression levels and disease symptoms.TRPC channels also play an important role in some systemic diseases because they can act as targets for stimulants,inflammatory products and foreign body toxins.Currently,people are working on the relationship between the function of TRPC channel and human diseases.Objective:The purpose of this study was to use TGF-β1 to treat LX-2 cells,establish an in vitro fibrosis model,and explore the role of TRPC6-mediated SOCE in LX-2 cells activation.The effect of TRPC6 gene knockout on hepatic fibrosis and its mechanism were discussed through the classical CBDL model.Methods and results:1.In order to verify the existence of SOCE in human hepatic stellate cell line LX-2,we used Tg to treat LX-2,and then detected the change of calcium concentration in LX-2 cells induced by Tg.In order to confirm whether the use of TGF-β1 treatment of LX-2 can activate the intracellular SOCE,this experiment used TGF-β1 to pretreatment for LX-2 for 24 h to detect the changes of intracellular Ca²⁺and the expression of TRPC6.The results showed that LX-2 cells can product significant intracellular flow of Ca²⁺under the stimulation of Tg.With the application of SOCE inhibitors SAR,Gd³⁺,2-APB,Ca²⁺influx was significantly decreased.The results also showed that SOCE was activated after LX-2 was treated with TGF-β1,TRPC6 expression was significantly up-regulated.2.The expression of fibrosis-related proteinα-SMA and pathway protein PI3K/AKT/P70S6K were detected.The results showed that the expression ofα-SMA,p-AKT and p-P70S6K were also significantly increased.After the use of TRPC6inhibitor SAR,the expression ofα-SMA,p-AKT and p-P70S6K were decreased.3.In order to investigate the effect of TRPC6 gene knockout on hepatic fibrosis,the classic CBDL surgery was used in this study to establish the fibrosis model in vivo,and the protein expression ofα-SMA and pathway protein PI3K/AKT/P70S6K were detected.The results showed that after CBDL operation,the expression ofα-SMA protein was significantly increased,HE staining and Masson staining in liver tissue showed that damage was serious,the obvious collagen fiber formation and p-AKT and p-P70S6K protein were also significantly increased.TRPC6 gene knockout reduced liver tissue injury and reduced collagen fiber decreased significantly,and the expression ofα-SMA,p-AKT,p-P70S6K were decreased.Conclusion:1.Typical SOCE is existed in LX-2 cells and can be inhibited by SOCE inhibitors SAR,Gd³⁺and 2-APB.TGF-β1 can activate SOCE in LX-2.The expression of TRPC6 in LX-2 was increased after treated by TGF-β1.And the SOCE activated by TGF-1 in LX-2 cells was inhibited by SAR.2.TGF-β1 can enhance the expression ofα-SMA in LX-2,and TRPC6inhibition may inhibit the activation of hepatic stellate cells by inhibiting the PI3K/AKT/P70S6K pathway.3.TRPC6 gene knockout may inhibit the formation of hepatic fibrosis by inhibiting the PI3K/AKT/P70S6K pathway.