Ginsenoside Rg3 Protects Pancreatic β-cells By Enhancing Autophagolysosomal Function

In the past thirty years,the number of type 2 diabetes patients in the world has tripled,and Asia is the main area of the rapid epidemic of type 2 diabetes(T2DM)all over the world,and my country China is the epicenter of type 2 diabetes.Unhealthy eating habits and sedentary lifestyle are also important driving factors for the current global prevalence of type 2 diabetes.Type 2 diabetes patients are often accompanied by abnormal blood glucose and lipid metabolism.Long-term high levels of free fatty acids(FFA)can cause lipotoxic damage to pancreatic β-cells,induce compensatory disorders of insulin secretion in pancreatic β-cells,and ultimately reduce the function of pancreatic β-cells.Towards apoptosis.Therefore,the research on the mechanism of lipotoxic damage of pancreatic β-cells and the development of therapeutic drugs based on this is of great significance for the prevention and treatment of diabetes.Lipid toxicity refers to the harmful effects of high concentrations of lipids and lipid derivatives on cells.Accumulated lipids and their derivatives induce a series of organelles such as endoplasmic reticulum stress,mitochondrial damage,and blocked autophagy lysosome clearance.Injury and inflammatory response,thereby triggering cell apoptosis.Studies have shown that pancreatic β-cells play a role in lipotoxicity.In order to remove excessive free fatty acids,the cells feedback increase the level of autophagy,but blindly promote the occurrence of autophagy,while the corresponding lysosome function is not enhanced simultaneously.Will cause autophagolysosomal-dependent clearance to be blocked.As a result,autophagosomes accumulate,pathological changes that cannot be degraded,and eventually go to apoptosis.Therefore,based on the increase of autophagosomes,improving the function of lysosomes,balancing the relationship between autophagy and lysosomes,and maintaining the flow of autophagolysosomal is the key to solving metabolic disorders.Studies have confirmed that high levels of free fatty acids can also inhibit the expression and activity of the lysosomal membrane protein LAMP2,which results in the obstruction of the fusion of autophagosomes and lysosomes,and reducing the formation of autophagolysosomal.At the same time,lipotoxicity can also cause the activity of autophagy-lysosomal cathepsin B to decrease,which leads to the obstruction of autophagy-lysosome-dependent clearance,which ultimately leads to pancreatic β-function failure and induces apoptosis.Lysosomal key nuclear transcription factor EB(TFEB)is a key regulator of lysosomal function.After dephosphorylation,it enters the nucleus and regulates the expression of autophagy-lysosomal function-related proteins,which is closely related to lysosomal biogenesis.TFEB is regulated by mTOR.mTOR complex 1(mTORC1)prevents nuclear translocation after phosphorylation of TFEB.When mTORC1 is inhibited,TFEB undergoes dephosphorylation and enters the nucleus to regulate downstream target genes.A large number of studies have shown that the activation of the energy metabolism regulation switch AMPK can inhibit the activity of mTORC1,suggesting that drugs acting on AMPK activation may inhibit mTOR and promote TFEB nuclear translocation,thereby up-regulating autophagylysosome fusion and the expression of lysosomal enzyme-related genes.Ginseng,a traditional herbal medicine,has been extensively studied in Southeast Asian countries.Ginsenosides are the main active components in ginseng.Among them,Ginsenoside Rg3(Ginsenoside Rg3),as one of the biologically active steroid saponins group,has shown a variety of pharmacological activities.Research reports have pointed out that in obese rat models with insulin resistance,ginsenoside Rg3 can activate AMPK in the liver to improve insulin resistance.In obese insulin-resistant rat skeletal muscle,Rg3 is also phosphorylated by AMPK.Plays the role of hypoglycemia and weight loss.Interestingly,in the mouse liver sepsis model,Rg3 activates the AMPK/mTOR pathway to promote autophagy and improve liver cell function.However,this therapeutic effect is in the lysosomal function inhibitor chloroquine Disappeared under the intervention of ginsenoside Rg3,suggesting that the pharmacological effects of ginsenoside Rg3 are closely related to lysosomal function.As a key transcription factor of lysosome,TFEB is precisely regulated by mTOR.Therefore,we speculate that ginsenoside Rg3 activates AMPK,inhibits mTOR,and promotes TFEB nuclear translocation,thereby promoting autophagy on the one hand,enhancing lysosome function on the other hand,improving pancreatic β-cell function,and reducing apoptosis.This project uses palmitic acid incubation to induce pancreatic β-cell lipotoxic injury model,administers ginsenoside Rg3,combined with lysosomal function inhibitor chloroquine or AMPK siRNA intervention,and clarifies that ginsenoside Rg3 promotes TFEB nuclear translocation by activating AMPK/mTOR signaling pathway,Enhance autophagy lysosome function,improve pancreatic β cell function,and reduce the mechanism of apoptosis.The research contents of this thesis are as follows:1.To clarify the effect of ginsenoside Rg3 on the autophagy lysosome function of mouse pancreatic islet β cells.In this part of the experiment,palmitic acid incubation induced lipotoxicity injury model of pancreatic β-cells,and ginsenoside Rg3 was given,combined with lysosomal function inhibitor chloroquine.Autophagosomes were detected by MDC fluorescent staining;Lysotracker fluorescent staining was used to detect lysosomes;Hochest33258fluorescent staining was used to detect β cell apoptosis.The expression of autophagy proteins LC3I/II and SQSTM1/p62;the expression of β cell lysosomal function-related proteins LAMP2 and Cathepsin B;and the expression of β cell apoptosis-related proteins Bcl2,Bax and Cleaved Caspase3 were further detected by Western blot and ELISA.Compared with the blank control group,in the palmitic acid model group,MDC green fluorescent particles increased and autophagosome accumulation increased;β cell autophagy-related proteins LC3 II and SQSTM1/p62 increased significantly;Lysotracker staining results showed that red fluorescent particles decreased,and the number of lysosomes Decrease;the expression of β cell lysosomal-associated proteins LAMP2 and Cathepsin B decreased;Hochest33258 staining showed that the nucleus was shrunk,glass bead-like lesions increased,and apoptosis increased;the expression of β cell anti-apoptotic protein Bcl2 decreased significantly,and the pro-apoptotic proteins Bax and Cleaved Caspase3 Significant increase,suggesting that palmitic acid intervention makes pancreatic β-cell autophagosomes accumulate,lysosome function declines,and cell apoptosis occurs.After treatment with ginsenosides,compared with the palmitic acid model group,MDC staining showed that MDC green fluorescent particles increased and autophagosomes increased;autophagy-related protein LC3 II expression increased,but SQSTM1/p62 decreased significantly;Lysotracker staining results It showed that the number of red fluorescent particles increased and the number of lysosomes increased;the expression of lysosomal-associated proteins LAMP2 and Cathepsin B increased;Hochest33258 staining showed that the nucleus shrinkage disappeared,the bead-like lesions decreased,and the apoptosis decreased;the cell anti-apoptotic protein Bcl2 expression The expression of pro-apoptotic proteins Bax and Cleaved Caspase3 decreased significantly.After further treatment with chloroquine,compared with the ginsenoside administration group,MDC green fluorescent particles increased and autophagosome accumulation increased;autophagy-related proteins LC3 II,SQSTM1/p62 significantly increased;Lysotracker staining results showed that the red fluorescent particles decreased and dissolved The number of enzyme bodies decreased;the expression of lysosomal-related proteins LAMP2 and Cathepsin B decreased;Hochest33258 staining showed that the nucleus was shrunk,the bead-like lesions increased,and the β cell apoptosis increased;the expression of cell anti-apoptotic protein Bcl2 decreased distinctly,and the β cell apoptosis-promoting protein Bax And Cleaved Caspase3 increased significantly.The results suggest that Rg3 can protect pancreatic β cells from lipotoxic damage by improving lysosomal function,enhancing autophagic lysosomal flow,inhibiting cell apoptosis.2.Verify that ginsenoside Rg3 activates AMPK/mTOR/TFEB pathway to enhance autophagolysosomal function.In this part of the experiment,palmitic acid incubation induced lipotoxic injury model of pancreatic β-cells,and ginsenoside Rg3 was given,combined with AMPK siRNA intervention.Autophagosomes were detected by MDC fluorescent staining;Lysotracker fluorescent staining was used to detect lysosomes;Hochest33258fluorescent staining was used to detect β cell apoptosis.The expression of β cell pathway-related proteins AMPK/p-AMPK and mTOR/p-mTOR were further detected by Western blot;the expression of β cell autophagy-related proteins LC3I/II,SQSTM1/p62;and the expression of β cell lysosomal function-related proteins LAMP2 and Cathepsin B;apoptosis Related proteins Bcl2,Bax,Cleaved Caspase3 are expressed.The β cell nuclear translocation of TFEB was detected by immunefluorescence.Compared with the control group,the expression ofβ cell pAMPK/AMPK was decreased and the expression of β cell p-mTOR/mTOR was increased in the palmitic acid model group;immunofluorescence showed decreased βcell nuclear translocation of TFEB;MDC staining showed that MDC green fluorescent particles increased,and autophagosome accumulation increased;Autophagy-related proteins LC3 II,SQSTM1/p62 significantly increased;Lysotracker staining results showed that the red fluorescent particles decreased,and the number of lysosomes decreased;the expression of lysosomal-related proteins LAMP2 and Cathepsin B decreased significantly;Hochest33258 staining showed that the β cell nucleus was shrunk and glass Bead-like lesions increased and cell apoptosis increased;the expression of β cell anti-apoptotic protein Bcl2 decreased significantly,and the pro-apoptotic proteins Bax and Cleaved Caspase3 increased significantly,suggesting that palmitic acid can induce cell apoptosis.After treatment with ginsenosides,compared with the palmitic acid model group,the expression of β cell p-AMPK/AMPK increased and the expression of p-mTOR/mTOR decreased;β cell MDC staining showed that βcell MDC green fluorescent particles increased,and autophagosomes increased;β cell autophagy-related proteins LC3 II expression increased,SQSTM1/p62 expression decreased;Lysotracker staining results showed that red fluorescent particles increased,and the number of lysosomes increased;the expression of β cell lysosomal-associated proteins LAMP2 and Cathepsin B increased significantly;Hochest33258 staining showed that the nucleus shrinkage disappeared,and the glass beads The expression ofβ cell anti-apoptotic protein Bcl2 increased,and the expression of β cell pro-apoptotic proteins Bax and Cleaved Caspase3 decreased.After combined with AMPK siRNA intervention,compared with the ginsenoside administration group,the expression of p-AMPK/AMPK decreased and the expression of p-mTOR/mTOR increased;immunofluorescence showed that TFEB nuclear translocation decreased;MDC staining showed that MDC green fluorescent particles decreased,Autophagosome formation decreased;autophagy-related protein LC3 II expression significantly decreased,SQSTM1/p62 significantly increased;Lysotracker staining results showed that the red fluorescent particles decreased,the number of lysosomes decreased;the expression of β cell lysosomal-related proteins LAMP2 and Cathepsin B decreased;Hochest33258 staining showed that the β cell nucleus shrank,glass bead-like lesions increased,and β cell apoptosis increased;the expression of β cell anti-apoptotic protein Bcl2 decreased significantly,and the β cell pro-apoptotic proteins Bax and Cleaved Caspase3 increased significantly.The results show that ginsenoside Rg3 up-regulates autophagy by activating AMPK/mTOR/TFEB signaling pathway,improves lysosome function,and protects pancreatic β cells.In summary,β cell autophagy lysosomal dysfunction plays an important role in palmitic acid-induced lipotoxic damage to pancreatic β-cells.Ginsenoside Rg3 regulates the AMPK/mTOR/TFEB pathway,enhances autophagic lysosome function,and balances autophagy.Phage-lysosome flow improves pancreatic β-cell function and inhibits cell apoptosis.