Study On The Mechanism Of Platelet Hyperactivation And Protective Effect Of Ginkgolide B In Type 2 Diabetic Rats

Part One Objective:To investigate the effect of ginkgolide B on platelet hyperactivation in diabetic rats.Method:(1)The model of T2DM rats was established by high fat diet combined with intraperitoneal injection of STZ,that is diabetes group.The model of T2 DM was established that the fasting blood glucose of rats was ≥16.7mmol/ L.The control group and the high fat group were fed with normal diet and high fat diet,respectively.Ginkgolide B treatment(GB)group received intraperitoneal injection of ginkgolide B(10mg/kg daily for 8 weeks).Body weight and fasting blood glucose were measured every 6 weeks.(2)The protein expressions of platelet active molecule CD62 P /PAC-1 and VAMP8/SNAP 23 in each group were detected by Western blot.(3)The expression levels of VAMP8 mRNA and miR-96 in platelets of rats in each group were detected by RT-PCR.(4)P-selectin content in serum was detected by P-selectin ELISA kit.The ultrastructure of platelets was observed by transmission electron microscope.Result:(1)The body weight and FBG of rats were measured every 6 weeks.Compared with the control group,the weight of rats in Diabetes group decreased significantly,and the FBG increased significantly(P < 0.01).However,there were no significant differences in blood glucose and body weight between the GB group and the Diabetes group(P > 0.05).(2)Protein expression in platelets was detected by Western blot.There were no significant differences in the expression of CD62 P,PAC-1,SNAP-23 and VAMP8 between the HF-control group and the control group(P > 0.05).Compared with the control group,the expression levels of CD62 P,PAC-1,SNAP-23 and VAMP8 in the Diabetes group were significantly increased(P < 0.05).Compared with Diabetes group,the protein expression levels of CD62 P,PAC-1,VAMP8 and SNAP 23 in GB group were significantly decreased,with statistical significance(P < 0.05).(3)There was no significant difference in the expression of VAMP8 m RNA and miR-96 between the HF-control group and the control group(P > 0.05).Compared with the control group,the m RNA expression level of VAMP8 in the Diabetes group was significantly increased(P < 0.001),and the expression level of miR-96 was significantly decreased(P < 0.001).Compared with the Diabetes group,the m RNA expression level of VAMP8 in the GB group was significantly decreased(P < 0.05),and the expression level of miR-96 was significantly increased(P < 0.01).(4)P-selectin content in serum of rats was determined by ELISA.Compared with control group,P-selectin content in serum of Diabetes group was significantly increased(P < 0.01).Compared with the Diabetes group,the level of P-selectin in serum of the GB group was significantly decreased(P < 0.05).(5)Ultrastructure of platelets in SD rats was observed by electron microscopy.The general shape of platelets in normal control group was similar to ellipse,and the surface of cell membrane was regular without indentation and pseudopod protruding.The distribution of α granules and dense granules was uniform and the number of α granules and dense granules was normal.The morphology of platelet in diabetic group was changed.The surface of the cell membrane is irregular.The number and volume of α granules increased significantly,and the granules were close to and fused with the cell membrane,indicating the increase of exocytosis.The ultrastructure of platelets in the ginkgolide B treatment group was significantly improved,and the general shape of platelets was generally normal,no depression was formed on the surface of cell membrane,and the distribution of αgranules was uniform and the volume was decreased.Conclusion:(1)T2DM rat model was successfully established,and high glucose stimulation induced increased platelet exocytosis and hyperactivation.The expression of miR-96 was decreased and the expression of VAMP8 was increased in diabetic rats.(2)Ginkgolide B treatment can up-regulate the expression of miR-96,inhibit the expression of VAMP8 m RNA and protein,and have a certain protective effect on the hyperactivated platelets of type 2 diabetic rats.Part twoObjective: To investigate the mechanism of platelet hyperactivation in type 2 diabetic ratsMethod:(1)MEG-01 cells cultured in vitro were used as research subjects.Study groups: normal control group(control): 5m M low-glucose medium culture;Hypertonic group(OC): 33 m M hypertonic medium;High glucose group(HG): Cells cultured in 33 m M high glucose medium were used to construct high glucose injury cell model;HG+ miR96 Mimic group: MEG-01 cells were incubated with 33 m M high glucose medium for 24 h and then transfected with miR-96 mimics.HG+Mimic control group: miR-96 simulated control after incubating MEG-01 cells with 33 m M high glucose medium for 24 h.(2)CCK-8 kit was used to detect the activity of MEG-01 cells.(3)The expression of platelet active molecule CD62p/PAC-1 and VAMP8 in MEG-01 cells in each group were detected by Western blot.(4)The expression levels of VAMP8 m RNA and miR-96 in MEG-01 cells were detected by RT-PCR assay.Result:(1)MEG-01 cell activity was detected by CCK8.The cell activity of Meg-01 in HG group was significantly lower than that in control group and OC group(P < 0.01),while there was no statistical difference between control group and OC group(P > 0.05).(2)Protein expression in MEG-01 cells was detected by Western blot.There were no significant differences in CD62 P,PAC-1 and VAMP8 protein expression between OC group and control group(P > 0.05).Compared with control group,the expression levels of CD62 P,PAC-1 and VAMP8 in HG group were significantly increased(P < 0.05).(3)The expression of VAMP8 m RNA and miR-96 in MEG-01 cells was detected by RT-PCR assay.There were no significant differences in VAMP8 m RNA and miR-96 expression between OC group and control group(P > 0.05).Compared with the control group,the expression level of VAMP8 m RNA in HG group was significantly increased(P < 0.01),and the expression level of miR-96 was significantly decreased(P < 0.01).(4)The effect of overexpression of miR-96 on the expression level of VAMP8 m RNA in MEG-01 cells stimulated by high glucose.Compared with the control group,the expression of miR-96 was significantly increased in the HG+miR-96 Mimic group(P < 0.001),suggesting that the MEG-01 cells had been successfully transfected with miR-96.There was no significant difference in miR-96 expression between HG+ Mimic Control group and HG group(P > 0.05).The m RNA expression of VAMP8 in HG+miR-96 Mimic group was significantly lower than that in HG group and HG+Mimic Control group(P < 0.01).There was no significant difference in VAMP8 m RNA expression between HG+Mimic Control group and HG group(P > 0.05).(5)The effect of overexpression of miR-96 on the expression of VAMP8 and CD62 P protein in MEG-01 cells stimulated by high glucose.The expression of VAMP8 and CD62 P in HG+miR-96 Mimic group was significantly lower than that in HG group and HG+ Mimic Control group(P < 0.01),and there was no significant difference in the expression of VAMP8 and CD62 P in HG+ Mimic Control group and HG group(P > 0.05).Conclusion:(1)High glucose stimulation down-regulated the expression of miR-96 and up-regulated the expression of target gene VAMP8 in MEG-01 cells.(2)Overexpression of miR-96 inhibited the activation of MEG-01 cells induced by high glucose stimulation by down-regulating the expression of VAMP8 protein.