| Chronic diseases Diabetes mellitus(DM)is a group of metabolic disease syndromes characterized by an increase in chronic blood glucose levels.Its main pathogenesis is related to the elevation of blood glucose caused by insulin secretion and/or impaired action.In the early stages of the disease,the target is the degree of organ sensitivity leads to its relative lack of insulin;at the late stage of its onset,insulin content is absolutely insufficient due to damage of islet cells.The key to the treatment of this disease lies in promoting insulin secretion and inhibiting islet cell apoptosis;therefore,many DM therapeutic drugs are used to treat pancreatic islet ?cells and restore their normal biological functions.AWRK6(SWVGKHGKKFGLKKHKKH)is a novel bioactive peptide isolated from the skin of the Rana chensinensis in our laboratory.It can not only promote the secretion of insulin in Min6 cells but also inhibit the apoptosis of the corresponding cells.In the previous relevant research,these characteristics have been correspondingly verified,this study further explored the mechanism of the corresponding function.In order to explore the molecular mechanism of insulin secretion by AWRK6,Min6 cells were treated with fluorescently labeled AWRK6 and the location of the effect was observed.The ESR method was used to detect the insulin secretion of GLP-1R-silenced Min6 cells to analyze the insulin secretion.Role of receptors;Min6cells were treated with Epac2 inhibitors and PKA inhibitors,respectively,then treated with AWRK6 and detected insulin secretion,to explore the possible signal pathway of AWRK6 to promote insulin secretion.In order to explore the function of AWRK6 in inhibiting cell apoptosis and its molecular mechanism,the survival rate of Min6 cells was detected by flow cytometry,and the contents of phosphate and cleaved caspase-9 and Caspase-3 were detected by Western Blotting.The activity of Caspase-12,Caspase-9,and Caspase-3 was detected by Elisa,and then the phosphorylation of Akt was detected by Western Blotting,and the survival rate and Caspase-9 activity of AWRK6-treated Min6 cells were detected by Akt inhibitor.The results showed that green fluorescence was located on the cell membrane surface after AWRK6 with fluorescent labeling and Min6 cells acted;AWRK6 couldsignificantly promote insulin secretion in Min6 cells(P <0.01),but it was significantly inhibited by Ex-9(P <0.01).After GLP-1R was silenced,AWRK6 could not promote insulin secretion in Min6 cells.After treatment with Epac inhibitor and PKA inhibitor,the insulinotropic function of AWRK6 was significantly inhibited(P <0.01 and P<0.05,respectively).AWRK6 significantly inhibited the apoptosis of Min6 cells induced by endoplasmic reticulum stress(P<0.01);it significantly increased the phosphorylation levels of Caspase-9 and Caspase-3(P<0.01)and significantly reduced the lysis type content(P< 0.01);AWRK6 can significantly reduce the activity of Caspase-12,Caspase-9 and Caspase-3(P<0.01).In addition,AWRK6 significantly promoted the phosphorylation of Akt(P<0.01);after Akt inhibitor treatment,AWRK6's function of inhibiting apoptosis was significantly inhibited(P<0.01),and the function of inhibiting caspase activity was also significantly reduced(P<0.01).According to the experimental results,the following experimental conclusions can be obtained:1.AWRK6 may function as a GLP-1 receptor agonist to play its role in promoting insulin secretion,and the relevant molecular mechanism may be through the GLP-1R/cAMP/Epac signaling pathway.2.AWRK6 can ameliorate apoptosis induced by endoplasmic reticulum stress by inhibiting the activity of the Caspase family.Its related molecular mechanism is to phosphorylate Caspase-9 through Akt,thereby inhibiting its biological activity and further inhibiting cell apoptosis. |
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