ASIC1a Restrained Cell Pyrotosis In Acid-induced Activation Of Rat Hepatic Stellate Cells

Hepatic fibrosis is considered to be a pathological process of diffuse damage to liver cells caused by various acute and chronic pathogenic factors,during which excessive accumulation of extracellular matrix?ECM?is a prominent feature.The activation of resting hepatic stellate cells?HSC?is the most important step in the process of liver fibrosis.Excessive secretion of?-SMA is the main source of ECM.Acid-sensing ion channels?ASICs?are a type of ion channels activated by extracellular cations.When extracellular acidification occurs,acid-sensitive ion channels are opened,and at the same time,the permeability to Na⁺and Ca²⁺is increased,thereby causes physiological and pathological changes in cells.The previous experimental results of the research team showed that the high expression of ASIC1a exists in hepatic stellate cells of rat liver fibrosis,and participates in the activation process of hepatic stellate cells,accelerating the development of liver fibrosis.Studies have also shown that ASIC1a is closely related to the pyroptosis of articular chondrocytes.After the ASIC1a channel is opened,pyroptosis of the articular chondrocytes of rats will worsen the process of rheumatoid arthritis.Pyroptosis is a hot topic that scholars at home and abroad pay close attention to,and it is also closely related to liver fibrosis.So does ASIC1a participate in the process of activated hepatic stellate cells?To this end,this experiment uses extracellular acids to activate HSC-T6,and explores the most likely mechanism of ASIC1a's effect on pyroptosis.The research content of this topic includes the following 4 parts:1. Inhibition,silencing and overexpression of ASIC1a in hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmids were used to investigate the effects of ASIC1a on intracellular proliferation and matrix metabolism of HSC-T6 cells.The effect of pretreatment of ASIC1a blocker Pc Tx-1 on the proliferation of HSC-T6 cells induced by acid was observed by MTT method.Compared with the p H 6.0 group,the growth of HSC-T6 cells was significantly inhibited.To study the effect of HSC-T6 function,it was found that compared with the p H 7.4 group,the expression of?-SMA and Collagen-I increased in HSC-T6 cells treated with acid to p H 6.0,and?-SMA after Pc Tx-1 and ASIC1a-si RNA intervention The expression of Collagen-I decreased.After intervention with the ASIC1a overexpression plasmid,the above-mentioned indicators of?-SMA and Collagen-I increased.Studies suggest that ASIC1a is involved in the function and metabolism of HSCs.When stimulated by acid,ASIC1a activates and channels are opened,which in turn regulates HSC-T6 cell growth and collagen secretion.2. The effects of inhibition,silencing and overexpression of ASIC1a on Ca²⁺influx in hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmid were used to investigate the effect of ASIC1a on intracellular Ca²⁺concentration in HSC-T6 cells.Laser confocal microscopy and Ca²⁺imaging studies of changes in Ca²⁺in HSC-T6 cells found that p H 6.0 significantly promoted Ca²⁺influx into HSC-T6cells.The degree of influx was reduced after Pc Tx-1 and ASIC1a-si RNA intervention.Overexpression of ASIC1a was used,after plasmid treatment,Ca²⁺influx increased significantly.Studies suggest that when acid stimulated,ASIC1a activation and channel opening increase Ca²⁺influx in HSC-T6 cells.3. The effects of inhibition,silencing and overexpression of ASIC1a on pyroptosising of activated hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established,and ASIC1a-specific blocker Pc Tx-1,transfected with ASIC1a-si RNA and ASIC1a overexpression plasmid were used to investigate the effect of ASIC1a on activated HSC-T6 pyroptosis.The results show that ASIC1a activation can affect the expression of HSC-T6 pyroptosis related indicators ASC,caspase-1,NLRP3 and GSDMD.After treatment with Pc Tx-1and ASIC1a-si RNA,compared with the model group,the expression of pyroptosis death indicators increased,and the expression of the pyroptosis death indicators decreased after the intervention of ASIC1a overexpression plasmid.Moreover,the LDH results showed that after inhibiting ASIC1a,the LDH content in the supernatant was higher than that in the acidified group.In addition,Elisa results also showed that the levels of IL-1?and IL-18 in the culture supernatant increased after ASIC1a was inhibited or silenced.After transfection ASIC1a overexpression pretreatment,the above-mentioned indexes related to pyroptosis were contrary to the results described above.Studies suggest that ASIC1a inhibits extracellular acid-induced pyroptosising of activated hepatic stellate cells.4. Effect of Ca2+on ASIC1a-mediated focal death of hepatic stellate cellsAn in vitro model of acid-induced HSC-T6 was established and pre-treated with Ca²⁺-free medium to investigate the effects of Ca²⁺on activated HSC-T6 pyroptosis.The results showed that compared with the model group,the expression of pyroptosis-related indicators increased after pretreatment with Ca²⁺-free medium.Studies suggest that Ca²⁺inhibits extracellular acid-induced pyroptosising of activated hepatic stellate cells.To sum up,the results of this study show that:in rat hepatic stellate cells,acid induction activates them.In activated hepatic stellate cells,activation of ASIC1a inhibits its pyroptosising,and its possible mechanism is through the regulation of Ca²⁺and inflammatory bodies.