The Expression, Function And Mechanism Of Acid-sensing Ion Channels1a In Hepatic Stellate Cells Of Liver Fibrosis

Acid-sensing ion channels (ASICs) are a family of ligand-gated cation channels, which belong to the degenerin/epithelial Na+channel (DEG/ENaC) superfamily. They are Na+-selective and Ca2+-permeant, and are transiently activated by extracellular acid. To date,7subtypes of ASICs have been identified in mammals:ASICla, ASIClb, ASIClb2, ASIC2a, ASIC2b, ASIC3, and ASIC4. In recent years, ASICs were been paid more attention because of its various biological function. ASICs not only mediate pain, sensation and neurotransmitter in the nervous system, but ASICla has been demonstrated to be relevant to the physiology and pathology of non-neuronal tissues such as inflammation, tumor, ischemic injury and gastrointestinal diseases, which were often accompanied with pH value decreased, tissue acidosis, Na+or Ca2+influx. Our previous studies demonstrated that ASICla, ASIC2a and ASIC3are expressed in rat articular cartilage. ASICs are expressed at high leval in articular cartilage of adjuvant arthritis rats. Amiloride, ASICs nonspecific blocker, and aspirin have protective effect on AA rats by inhibiting the expression of ASICs. Furthermore we demonstrated ASICs are as key component of extracellular pH sensors in the cartilage. ASICs were activated by low pH, mediate Ca2+influx into chondrocytes, lead to extracellular matrix imbalance and cell injury. Liver fibrosis is recognized as a common type of liver disease with significant morbidity, and a middle stage between chronic liver disease and cirrhosis. Liver inflammation induced by hepatitis viral infections and alcohol toxicity are considered as major reasons for liver fibrosis. The activation of HSCs could be initiated by the stimulation of cytokines, oxidative stress in liver inflammation. The activated HSCs then increase the production of ECM(such as a-SMA and type I collagen) and different cytokines, including transforming growth factor-β1(TGF-β1), which further stimulates the activation of HSCs and the production of ECM. This pathological process is similar to arthritis, such as tissue acidsis of inflammation, the imbalance of ECM metabolism. Therefore, the aim of this study was to evaluate the expression and role of ASICs in liver fibrosis.1. Expression of ASIC la in liver tissues and HSCFirstly, we confirmed that ASIC1a mRNA and protein is expressed in liver tissures by RT-PCR and western blot, and expression of ASIC la increases liver of CCl4-induced rat. Secondly, we investigated the distribution of ASIC la in liver tissures by double immunofluorescence technique. The results showed that the distribution is consistent with ASIC1a and a-SMA, ASIC1a is expressed in HSC. Thirdly, we performed RT-PCR and western blot to evaluate the expression of ASIC1a in primary HSC and HSC-T6cells, we detected the expression of ASIC la at mRNA and protein levels in primary HSC and HSC-T6cells, respectively. And the expression of ASIC la in HSC of CCl4-induced rats and acid-induced HSC-T6cells was increased. ASIC1a specific blocker PcTX1could suppress ASIC1a in acid-induced HSC-T6cells. These findings suggest that ASIC1a may play an important role in liver fibrosis.2. Protective effects of ASIC1a blocker Amiloride on carbon tetrachloride-induced liver fibrosis in ratsLiver fibrosis model was established by subcutaneous injection (sc) of50%CCl4twice a week lasting for12weeks. Amiloride (10,5and2.5mg/kg) was treated gastrogavage (ig) daily since the5th week. The results showed that amiloride (10and5mg/kg) treatment for 8weeks significantly reduced the elevated liver index (liver weight/body weight) and spleen index (spleen weight/body weight), elevated levels of serum transaminases (alanine aminotransferase and aspartate aminotransferase), hyaluronic acid (HA), LN, type Ⅲ procollagen (PCⅢ), interleukin-1(IL-1), interleukin-6(IL-6). In addition, amiloride markedly improved the morphologic changes of hepatic fibrosis induced by CCl4. Moreover, amiloride (10and5mg/kg) treatment suppressed the ASIC1a, α-SMA, TGF-β1, NFκB, collagen Ⅰ expression. These evidence indicated that amiloride is able to ameliorate liver injury and prevent rats from CCl4-induced liver fibrosis by inhibiting expression of ASIC1a, mediating the function of HSC.3. ASIC1a mediate Ca2+influx, proliferation and matrix metabolism of acid-induced HSC-T6cellsTo further evaluate the role of ASIC1a in regulating of HSC, we established acid-induced HSC-T6cells model with different dose of amiloride, PcTX1treatment, investigated its role of Ca2+influx, proliferation and matrix metabolism of acid-induced HSC-T6cells. We recorded intracellular calcium ([Ca2+]i) in HSCs by the laser scanning confocal microscopy technique. In cultured HSC-T6cells, extracellular pH6.0increased intracellular calcium in the presence of extracellular Ca2+. Amiloride and PcTX1significantly reduced this increase of Ca2+. Then we used MTT to evaluate effect of ASIC1a on proliferation of acid-induced HSC-T6cells, amiloride and PcTX1significantly inhibited proliferation. Furthermore, we evaluated effects of ASIC1a on HSCs activation and matrix metabolism by RT-PCR, western blot and Immunocytochemical staining. With acid induced HSC-T6cells, expression of α-SMA, TGF-β1and collagen I were significantly increased, amiloride and PcTX1significantly inhibited these increases. Amiloride, PcTX1significantly inhibited the increases of TIMP-1, elevated the level of inhibitor MMP-13expression. These findings suggest that ASIC1a mediate Ca2+influx, proliferation and matrix metabolism of acid-induced HSC-T6cells. 4. The role of MAPK pathways in Acid-sensing ion channel la regulates matrix metabolism of acid-induced HSC-T6cellsOur research has shown that extracellular pH plays an important role in matrix synthesis and metabolism in HSC-T6by activated ASICla. However, little is kown about the underlying mechanism of ASICla on matrix synthesis and metabolism in HSC-T6. The present study was undertaken to investigate the role of MAPK pathways in Acid-sensing ion channel la regulates matrix metabolism of acid-induced HSC-T6cells. With acid induced HSC-T6cells, the expression of p38MAPK and ERK was increased rapidly, Amiloride, PcTX1, PD98059(ERK1/2inhibitor) and SB203580(p38MAPK inhibitor) inhibited these increases. PD98059, SB203580significantly inhibited collagen I, TIMP-1, elevated the level of MMP-13. Our data indicate that ASICla inhibit matrix synthesis and metabolism of acid-induced HSC-T6cells, which might contribute to via Ca2+-mediated activation of p38MAPK and ERK pathway.Conclusion:ASIC1a is expressed in rat liver, HSC. The expression of ASIC1a is increased in fibrotic liver, HSC. Inhibition of ASIC1a could protect liver fibrosis. ASIC1a mediate function of HSC by calcium influx. ERK and p38MAPK participate in function of HSC with ASIC1a activated.