Effect Of Autophagy Induced By ASIC1a On HSC-T6 Activation And Relative Mechanisms Research

Diabetes mellitus(DM)is a complex multiple systems metabolic disease characterized mainly by high blood sugar,along with the acidification of organization,can lead to a variety of organ damage and dysfunction.Hepatic fibrosis(HF)is the wound healing response to various chronic liver injury,the essence of which is the accumulation of extracellular matrix(ECM).Hepatic stellate cells(HSCs)are the main cells which generate ECM components in the liver,which must be activated,and transform into myofibroblast-like cells.Research has shown that high blood sugar in patients with chronic hepatitis C is an independent synergy factor of fibrosis progression,with higher pro-fibrosis effect.Hyperglycemia is an important factor to promote liver diseases development,the morbidity of diabetes complicated with hepatic fibrosis rises gradually in recent years.Further study of the influence of hyperglycemia on the pathogenesis and progress of hepatic fibrosis,the proliferation and activation of HSC,are of great significance,which be focused by scientists at home and abroad.Acid sensitive ion channel 1a(ASIC1a)is a kind of cation channel protein complex which can be activated by extracellular H+,open channel has permeability to Na+,Ca2+.Activated ASIC1 a can lead to the inflow of extracellular calcium.Research has shown that intracellular calcium ion flowed in through a series of signaling pathways can lead to the occurrence of autophagy.Autophagy plays an important role in the process of HSC activation.In the case of liver injury,the quiet HSC can increase lipid metabolism by raising autophagy to increases the generation of energy to activate the HSC and lead to the occurrence of hepatic fibrosis.Preliminary study of our research group found that high blood glucose may induce liver damage,promote the HSC proliferation and activation to aggravate hepatic fibrosis,with increased expression of ASIC1 a in animal and cell model.Further study found that ASIC1 a is involved in the HSC proliferation and activation induced by the high glucose with PDGF and led to aggravated hepatic fibrosis.However,the specific mechanism is still not very clear.In the process of the HSC proliferation and activation induced by high glucose and PDGF,whether autophagy induced by ASIC1 a upregulates to promote the HSC proliferation and activation to aggravate hepatic fibrosis? Related research has not been reported.Therefore,based on the preliminary study,with high sugar and PDGF trained the HSC in vitro to establish diabetes combined hepatic fibrosis double model to explore the effect of autophagy induced by ASIC1 a in the process of hepatic fibrosis under high glucose and relevant mechanism.The main research content is summarized as follows:1.The expression of autophagy related protein in rat liver tissues of liver fibrosis with diabetesOn the animal models established by streptozotocin and carbon tetrachloride through the early stage of our team,the experimental rat liver pathological changes was observed by H&E staining and Masson staining,the results found that diabetic rats,hepatic fibrosis rats liver tissue appeared significant liver damage,and hepatic fibrosis with diabetes group is most serious.Western Blot detected autophagy related proteins LC3 II and Beclin1,hepatic fibrosis associated protein alpha SMA and Collagen I,it was found that diabetic rats,liver fibrosis rats and hepatic fibrosis with diabetes rats liver tissue LC3 II and Beclin1,alpha SMA and Collagen I express were enhanced via control group,the difference is statistically significant,and the expression of LC3 II and Beclin1,alpha SMA and Collagen I of hepatic fibrosis with diabetes rats is the strongest,the difference is statistically significant via liver fibrosis group or hepatic fibrosis group.The results showed that autophagy may participate in diabetic aggravates hepatic fibrosis process.2.The change of ASIC1 a and autophagy on the HSC-T6 stumulated by high glucose and PDGFIn order to detect the change of ASIC1 a and autophagy in hepatic fibrosis cell model,we used high glucose(6000 mg/L)and PDGF(10ng/ml)to stimulate HSC-T6 to establish diabetes with hepatic fibrosis cell model according to the early stage of our group.Western Blot was used to detected the expression of ASIC1 a,autophagy related proteins LC3 II and Beclin1,liver fibrosis associated protein alpha SMA and Collagen I,ptf LC-3 plasmid transfection and MDC staining were used to observe the change of cell autophagy.Results showed that high glucose and PDGF can enhance the expression of ASIC1 a,alpha SMA and Collagen I,along with the increase of the autophagy,and the expression of high glucose and PDGF group is was the strongest via high glucose group or PDGF group,the difference is statistically significant3.Effect of autophagy blockers 3-MA to HSC-T6 proliferation and activation cultured with high glucose and PDGFTo observe the effects of autophagy on HSC activation and proliferation,3-MA was given to block HSC autophagy under high glucose and PDGF stimulation,Western Blot was used to detect the expression of autophagy related proteins LC3 II and Beclin1,liver fibrosis related protein alpha SMA and Collagen I.The results showed that the 3-MA reduced the expression of LC3 II and Beclin1,as well as alpha SMA and Collagen I via high glucose group and PDGF group,the difference is statistically significant.4.Effect of autophagy blockers 3-MA to HSC-T6 cell cycle cultured with high glucose and PDGFTo further observe the effect of autophagy on HSC proliferation and activation,3-MA was used to block HSC autophagy under high glucose and PDGF stimulation,flow cytometry was used to detect the change of each group cell cycle.The results showed that 3-MA can block the HSC from G0 into G1 phase transformation and inhibited HSC proliferation via high glucose group and PDGF group,the difference is statistically significant.5.Effect of Amiloride to HSC-T6 autophagy cultured with high glucose and PDGF stimulationTo observe whether ASIC1 a affect HSC autophagy under high glucose and PDGF stimulation,ASIC1 a nonspecific blocker Amiloride was used to block ASIC1 a under high glucose and PDGF stimulation,Western Blot was used to detect the expression of ASIC1 a,autophagy related proteins LC3 II and Beclin1,liver fibrosis related protein alpha SMA and Collagen I,ptf LC-3 plasmid transfection and MDC staining were used to observe the change of each group cell autophagy.Results showed that Amiloride can reduce the expression of ASIC1 a on HSC under high glucose and PDGF stimulation,as well as autophagy related proteins LC3 II and Beclin1,liver fibrosis associated protein alpha SMA and Collagen I,ptf LC-3 plasmid transfection and MDC staining results also showed that Amiloride can reduce the HSC autophagy.6.Effect of ASIC1 a Sh RNA to HSC-T6 autophagy cultured with high glucose and PDGFIn order to further observe the effect of ASIC1 a to HSC-T6 autophagy,ASIC1 a Sh RNA was transfected to HSC-T6 under high glucose and PDGF stimulation.Western Blot was used to detected the expression of ASIC1 a,autophagy related proteins LC3 II and Beclin1,fibrosis alpha SMA and Collagen protein.Results showed that the ASIC1 a protein expression is reduced,as well as LC3 II and Beclin1,alpha SMA and Collagen I also is reduced via high glucose group and PDGF group,the difference is statistically significant.7.The change of Ca MKK?/ERK pathway protein under high glucose and PDGF stimulation.To observe the possible pathways that ASIC1 a induce autophagy under high glucose and PDGF stimulation,Western Blot was used to detect the expression of Ca MKK? protein and ERK phosphorylation level on HSC under high glucose and PDGF stimulation.Results showed that Ca MKK? expression and ERK phosphorylation level were enhanced via high glucose group or PDGF group,the difference is statistically significant.8.Effect of Amiloride to Ca MKK?/ERK pataway of HSC-T6 cultured with high glucose and PDGFIn order to further observe the effect of Amiloride to Ca MKK?/ERK pataway of HSC-T6 cultured with high glucose and PDGF,Amiloride was used to block ASIC1 a under high glucose and PDGF stimulation,Western Blot was used to detected the expression of Ca MKK? and ERK phosphorylation level on HSC.Results showed that Ca MKK? expression and ERK phosphorylation level were reduced by Amiloride on HSC via high glucose group and PDGF group,the difference is statistically significant.