Regulatory Role And Mechanisms Of Acid-sensing Ion Channel 1a In The Process Of Liver Fibrosis Under Hyperglycemia

Diabetes mellitus (DM) is a complex, multisystem disease characterized by hyperglycemia and tissue acidification. Hepatic fibrosis is the wound healing process that occurs in response to many causes of chronic injury. It is characterized by the accumulation of extracellular matrix (ECM) following liver injury. Hepatic stellate cells (HSCs) are the main liver cells that produce matrix such as a-SMA and Collagen I. For fibrogenesis to occur, quiescent HSCs must be activated, and transform to proliferating myofibroblast-like cells. This event was regarded as the pivotal point of the progression of liver fibrosis.Recent studies have found that hyperglycemia play an important role in the activation and proliferation of HSC and the formation of collagen. Other studies have shown that hyperglycemia is a stimulative factor in the increasing mortality of liver disease. The standardized mortality ratios of diabetics with liver disease even higher than these with cardiovascular disease. Therefore, many researchers have focus on the effect of hyperglycemia on the lesion of liver fibrosis, and the mechanism of activation and proliferation of HSC stimulated by hyperglycemia.Acid-sensing ion channels (ASICs) are cation channels activated by protons (H+). When the concentration of H+ fall, the channels opening and conduct Ca2+ and Na+, thus cause a series of physiological pathology. In the process of liver fibrosis, the changes of ASIC1a bring about HSC activation and proliferation and thus promote the development of liver fibrosis. However, there still lack of reports about the effect of high glucose on the change of ASIC1a in HSC. In order to explore the effect of ASIC1a in the process of liver fibrosis under high glucose and the potential mechanisms, we used STZ and CC14 to established diabetes combining with hepatic fibrosis rat model, high glucose and PDGF co-culture HSC-T6 to mimic diabetes combining with hepatic fibrosis model in vitro. The main contents are summarized as follows:1. Effect of diabetes on the liver demage of hepatic fibrosisTo detect the effect of diabetes on the liver demage of hepatic fibrosis, STZ and CC14 were used to establish diabetes rat model, liver fibrosis rat model and diabetes combining with hepatic fibrosis rat model. Histopathological changes of experimental rats’ liver were observed by H&E staining, and the variations of liver fibrosis related factors α-SMA and Collagen I were detected by immunohistochemistry, Western Blot and Q-PCR. The results showed that rats from diabetes group, liver fibrosis group and diabetes combining with hepatic fibrosis group were all observed with liver damage at different level compared to control group, and tissue injury of diabetes combining with hepatic fibrosis group was most serious. However, the expression of α-SMA and Collagen I in the three model groups were significantly increased compared to the control group, which was most obvious in the diabetes combining with hepatic fibrosis rats, and also remained statistically significant with diabetes group and liver fibrosis group.These suggests that diabetes aggravate the pathogenesis of hepatic fibrosis.2. The expression of ASIC1a in rat liver tissues of liver fibrosis with diabetesTo detect the expression of ASIC la in rat liver tissues of hepatic fibrosis with diabetes, immunohistochemistry, Western blot and Q-PCR were used to detect the expression of ASIC1a. Increased expression were found in the liver tissues of diabetes group, hepatic fibrosis group and diabetes combining with hepatic fibrosis double models group compared to control group. However, the expression of ASIC1a was highest in double models group, and even showed significant statistical difference with both diabetes group and hepatic fibrosis group. All these suggest that ASIC1a may involve in the lesion of liver fibrosis with diabetes.3. The expression of Notch1/Hes-1 in rat liver tissues of liver fibrosis with diabetesWestern blot was used to detect the expression of Notchl and Hes-1 in the liver tissues of each group rats. The result showed that both the expression of Notchl and Hes-1 were significantly increased in diabetes group, hepatic fibrosis group and diabetes combining with hepatic fibrosis double models group, and the double models group showed the highest expression of ASIC la, even with statistical difference compared to both diabetic group and hepatic fibrosis group.4. Effect of high glucose on PDGF-BB induced HSCs activation and proliferationTo verify the effect of high glucose on HSC-T6 in the process of liver fibrosis, HSC-T6 were pretreated with high glucose and then stimulated by PDGF-BB in vitro, which could mimic the hyperglycemia in liver fibrosis. We detected the expression of α-SMA and Collagen I by Western Blot. The results showed that the expression of a-SMA and Collagen I were all increased both in HG group and PDGF-BB group. More importantly, with the same process of stimulated by PDGF-BB, the expression of a-SMA and Collagen I were higher in the HG group than the LG group, suggesting that high glucose not only induced HSC-T6 activation and proliferation, but also promotes PDGF-BB induced HSC-T6 activation and proliferation, which further illustrate that high glucose aggravate the pathogenesis of hepatic fibrosis.5. Expression of ASIC1a in PDGF-BB induced HSC-T6 under high ambient glucoseTo detect the expression of ASIC1a in PDGF-BB induced HSC-T6 under high ambient glucose, we used Western blot to detect the expression of ASIC1a in HSC-T6. We found that both HG and PDGF-BB could increase the expression of ASIC1a. While, with the same process of stimulation by PDGF-BB, the expression of ASIC1a is higher in the HG group than the LG group, indicating that ASIC1a may involve in the pathogenesis of hepatic fibrosis under high ambient glucose.6. Effect of ASIC1a on the activation and proliferation of HSC-T6 treated with high glucoseUsing non-specific blocker Amiloride or specific blocker PcTx1 blocked ASIC1a in HG cultured HSC-T6; specific ASIC1a-ShRNA was transfected by LipofectamineTM 2000 to down-regulated ASIC1a in HSC-T6 cultured with HG. Western blot was used to detect the expression of α-SMA and Collagen I. The results showed that down-regulated ASICla, the expression of α-SMA and Collagen I also decreased in HSC-T6 treated with high glucose, indicating that inhibition of ASIC1a depress HSC-T6 activation and proliferation under high ambient glucose.7. Effect of ASIC1a expression on the activation and proliferation of HSC-T6 stimulated with PDGF-BB under high ambient glucoseIn order to further explore that whether ASIC1a contribute to the lesion of liver fibrosis under high ambient glucose, HSC-T6 were pretreated with Amiloride or PcTx1, or transfected specific ASIC1a-ShRNA by Lipofectamine TM2000, and then stimulated with high glucose and PDGF-BB successively. The change of proliferation of HSC-T6 was measured by MTT assay, and Western blot was used to detect the expression of α-SMA and Collagen I. The results showed that down-regulated ASIC1a in PDGF-BB induced HSC-T6 under high glucose significantly inhibited cell proliferation, and decreased the expression of α-SMA and Collagen I, indicating that inhibition of ASIC1a depress the promote effect of high glucose on the activation and proliferation of HSC-T6 stimulated by PDGF-BB.8. Effect of ASIC1a on the activation of Notchl/Hes-1 pathway in HSC-T6 cultured with high glucoseUsing Amiloride or PcTxl blocked ASICla in HG cultured HSC-T6; specific ASIC1a-ShRNA was transfected by LipofectamineTM2000 to down-regulated ASIC1a in HSC-T6 cultured with HG. Western blot was used to detect the expression of Notchl and Hes-1. The results showed that down-regulated ASICla, the expression of Notchl and Hes-1 also decreased in HSC-T6 treated with high glucose.9. Effect of ASICla on the activation of Notchl/Hes-1 pathway in PDGF-BB induced HSC-T6 under high glucoseTo further explore the. role of Notchl/Hes-1 pathway in the pathogenesis of diabetes combining with hepatic fibrosis and research the relation between ASIC1a and Notch1/Hes-1 pathway, we used Amiloride or PcTx1 to block ASIC1a in PDGF-BB induced HSC-T6 under high glucose, or down-regulate ASIC1a with specific ASICla-ShRNA in PDGF-BB induced HSC-T6 under high glucose. The expression of Notchl and Hes-1 were detected by Western blot. The results showed that down-regulated ASIC1a obviously decreased the expression of Notchl and Hes-1 in PDGF-BB induced HSC-T6 under high glucose, which manifest that the effect of ASIC1a on the pathogenesis of diabetes combining with hepatic fibrosis may relate to Notchl/Hes-1 pathway, ASICla inhibition may suppress the activation and proliferation of HSC-T6 by blocking Notchl/Hes-1 pathway.