Effect Of PI3K/AKT Upregulated ASIC1a Expression And ERS On PDGF-Induced HSC-T6 Activation And Related Mechanisms

Acid sensitive ion channel 1a?ASIC1a?is a kind of H⁺activation of cation channels formed by extracellular acidification.The opening of the channels allows influx of extracellular calcium ions and changes the steady state of intracellular calcium ions,causing a series of physiological and pathological phenomena.Some studies have shown that PI3K/Akt signaling pathway upregulates the expression and activation of ASIC1a.Endoplasmic reticulum is an important Ca²⁺storage station,which participates in the regulation of Ca²⁺signal in cytoplasm and plays an important role in the signal pathway.Ca²⁺disruption affects the function of the endoplasmic reticulum on protein synthesis and folding,including translational modifications of proteins that induce endoplasmic reticulum stress?ERS?and activate unfolded protein responses?UPR?.UPR can increase the survival rate of cells in short and light ERS,or conversely promote cell death by activating downstream apoptotic signaling molecules.There are three main UPR paths:protein kinase RNA-like endoplasmic reticulum kinase?PERK?,inositol-requiring 1??IRE1??and activating transcription factor 6?ATF6?.Hepatic fibrosis?HF?is the result of a self-wound repair response to multiple liver injuries,with a complex mechanism including self-adaptation,apoptosis and inflammatory responses.Hepatic stellate cells?HSC?are the major fibroblasts in the liver that can be stimulated by various cytokines to produce large amounts of extracellular matrix?ECM?deposits.Our previous study demonstrated thatASIC1a and ERS are both involved in the formation of hepatic fibrosis,especially the activation of HSC.However,it is unclear whether these two mechanisms exist.Activation of ASIC1a leads to influx of extracellular calcium,which leads to the expression of ERS in liver fibrosis and aggravates liver fibrosis.Therefore,based on the previous research group,we detected the expression of ASIC1a and ERS in liver tissues of patients with liver fibrosis and PDGF-stimulated HSC-T6 cells to establish an in vitro hepatic fibrosis model in this study.The mechanism of ERS in the progress of liver fibrosis and the interaction of ASIC1a and ERS in activating HSC are also researched.The main research contents are summarized as follows:1.The expression of ASIC1a in human liver fibrosisThe liver tissue of patients with bile duct stones as a normal control group,liver cancer with moderate and severe liver fibrosis in liver tissue as liver fibrosis group.With H&E observed in each patient's liver pathological changes,the results showed that liver fibrosis patients showed significant liver injury,there are significant differences in fiber level when compared with the normal control group.The expression of a-SMA,a marker of hepatic fibrosis,was higher than the control group.The expression of ASIC1a in human liver fibrosis was detected by immunohistochemistry,and the results showed that there was an expression of ASIC1a in human liver tissue,and the expression of liver fibrosis was significantly increased.The ERS marker GRP78 was detected at the same time,and the results showed that the expression of GRP78 was higher than the control group.All above suggests that both ASIC1a and ERS are expressed in human liver fibrosis.2.Expression of ASIC1a and ERS-related proteins in hepatic fibrosis rats and HSC-T6CCl₄ was used to establish hepatic fibrosis rat model,H&E staining was used to observe the pathological changes of hepatic fibrosis in rats.Immunohistochemistry was used to detect the expressions of hepatic fibrosis related factors,such as?-SMA and Collagen I.The results showed that liver fibrosis group appeared significant liver injury than the control group,?-SMA and Collagen I expression was also increased significantly compared with the control group.Western Blot was used to detect the expression of ASIC1a and ERS-related proteins in hepatic fibrosis group and PDGF-BB-stimulated HSC-T6 cells.The results showed that the expressions of ASIC1a,GRP78 and Caspase12 in rat liver tissue and HSC-T6 cells were significantly increased compared with the control group,further suggesting that both ASIC1a and ERS are involved in the development of liver fibrosis.3.Effect of ASIC1a on PDGF-BB-induced HSC-T6 activation and proliferationHSC-T6 cells were stimulated with PDGF-BB?10ng/ml?for 24 hours to establish an model of liver fibrosis in vitro,ASIC1a nonspecific blocker Amiloride?Amiloride?or ASIC1a-specific blocker Toxigenin?PcTx1?was used to block the high expression of ASIC1a stimulated by PDGF;or use specific ASIC1a-ShRNA to interfere with the expression of ASIC1a.Western Blot and q-PCR were used to detect the expression of ASIC1a,hepatic fibrosis related proteins?-SMA and Collagen I.As a result,it was found that after blocking or silencing ASIC1a,the expression of ASIC1a was decreased,and the expression of?-SMA and Collagen I increased by PDGF stimulation was also significantly decreased.It is suggested that the inhibition of the expression of ASICla can block the activation enhancement of HSC-T6 stimulated by PDGF.4.Effect of ASIC1a on ERS induced by PDGF-BB in HSC-T6The ASIC1a-specific inhibitors PcTx1 and ASIC1a-ShRNA were used to down-regulate the expression of ASIC1a.Western Blot detection of ERS marker protein GRP78,Caspase12 and downstream pathway IRE1-XBP1 changes.After PcTx1/ASIC1a-ShRNA downregulated the expression of ASIC1a,the expression of ASIC1a was decreased,and the expression of ERS marker proteins GRP78,Caspase12and downstream pathway IRE1-XBP1 were significantly decreased.The results of q-PCR were consistent with Western blot results.It is suggested that the progress of ASIC1a in regulating hepatic fibrosis may be related to ERS.5.Effect of PI3K/AKT pathway on ASIC1a expressionTo investigate how ASIC1a is involved in regulating the development of liver fibrosis,LY294002 was used as an inhibitor of PI3K/AKT,and the optimal concentration of LY294002 was detected by MTT and Western Blot?the results were 15 mol/L?.After the inhibition of PI3K/AKT pathway by LY294002,the expression of ASIC1a was detected by Western Blot.It was found that after using LY294002,the expression of p-akt decreased,and the high expression of ASIC1a with PDGF stimulation was also significantly blocked.It suggested that LY294002 can block the activation of ASIC1a.6.PI3K/AKT pathway regulates ASIC1a membrane transportTo further investigate the regulatory effect of PI3K/AKT pathway on ASIC1a membrane transport,the membrane transport of ASIC1a was detected by cellular immunofluorescence.The results showed that ASIC1a was activated by PDGF and transported from the nuclear membrane to the surface of cell membrane,and the fluorescence intensity also increased obviously.However,the transport of ASIC1a was blocked by PcTx1,an inhibitor of ASIC1a.At the same time,it was also found that PDGF-induced ASIC1a membrane transport was also significantly inhibited after inhibiting the PI3K/AKT pathway.It is suggested that the PI3K/AKT pathway upregulates the membrane transport process of ASIC1a.7.Effect of PI3K/AKT pathway on intracellular calcium ion flow induced by ASIC1aThe effect of PI3K/AKT in regulating the function of ASIC1a was further studied by using laser confocal technology to detect the changes of Ca²⁺concentration in HSC-T6cells.The Ca²⁺channel was blocked by a voltage-gated calcium channel blocker?10 M nimodipine?in the experiment.From the results of confocal image and concentration trajectory,it can be seen that compared with the control group,Ca²⁺fluorescence intensity of HSC was significantly enhanced under PDGF stimulation,and the concentration increased significantly.PcTx1 can significantly reduce the intracellular calcium ions induced by PDGF after the specific blocking of ASIC1a.In the case of PI3K/AKT inhibitor?LY294002?,the results are similar to PcTx1,suggesting that PDGF activates ASIC1a through the PI3K/AKT pathway and induces intracellular calcium ion flow.8.The effect of ERS on the activation and proliferation of HSC-T6 induced by PDGF-BBTo explore how ERS participated in regulating the development of hepatic fibrosis,4-PBA was used as the inhibitor of ERS,MTT and Western Blot were used to detect the optimal concentration of 4-PBA?the result was 1 mol/L?.After using 4-PBA,Western Blot results showed that the expression of ERS marker protein GRP78,Caspase12 and ERS downstream IREI-XBP1 was decreased.At the same time detection of liver fibrosis associated protein?-SMA and Collagen I,it was found that 4-PBA can block induced by PDGF of high expression of?-SMA and Collagen I.It is suggested that ERS may participate in the regulation of activation and proliferation of HSC through IREI-XBP1 pathway.9.Effect of ERS on ASIC1a expression in HSC-T6 induced by PDGF-BBTo further explore the relationship between ERS and ASIC1a,Western Blot detected ASIC1a expression after 4-PBA inhibition of ERS and found that 4-PBA can inhibit PDGF-induced ASIC1a expression;simultaneous detection of ERS on p-AKT expression,the results found that 4-PBA can significantly block the expression of p-AKT.The expression of p-AKT was detected by Western Blot using ASIC1a-specific inhibitor PcTx1.The results showed that PcTx1 can inhibit the expression of p-AKT induced by PDGF.The results suggest that ERS may up-regulate the activity of ASIC1a through the regulation of PI3K/AKT pathway.