Restoring The Platelet MiR-223 By Calpain Inhibition Alleviates The Neointimal Hyperplasia In Diabetes

ObjectivePlatelet hyperactivity is the hallmark of diabetes mellitus(DM),and platelet activation plays a crucial role in diabetic vascular complications.Recent studies have shown that upon activation,platelet-derived mi RNA are incorporated into vascular smooth muscle cells(VSMC),regulating the phenotypic switch of VSMC.Under diabetes,mi RNA deficiency in platelets fails to regulate the VSMC phenotypic switch.Therefore,manipulation of platelet-derived mi RNA expression may provide a therapeutic option for diabetic vascular complications.Platelet-derived mi RNA is formed by cleavage of endoribonuclease Dicer in its precursor cell-megakaryocyte.Calpain is an important enzyme involved in the regulation of the expression of the Dicer in megakaryocytes.It is unclear whether an effective calpain inhibitor can restore platelet mi RNA expression to alleviate the vascular complications of diabetes.Our research is to explore the effect of calpain inhibitor(Calpeptin)on the expression of mi RNA in diabetic platelets and clarifies the mechanism of mi R-223 in inhibition neointimal hyperplasia in mice models with diabetic and femoral artery injury.It will help to clarify the mechanism of platelet mi RNA in the process of diabetic vascular complications and provide new ideas for clinical drug treatment of diabetic vascular complications.MethodPart Ⅰ: Activated platelets(APs)from healthy subject(HS)and DM patients were cocultured with human VSMC.(1)Western blot to detect the differentiation and dedifferentiation markers of VSMC;(2)CCK-8,Ed U detect the proliferation rate of VSMC to observe the effect of platelets on the proliferation of VSMC;Part Ⅱ:(1)Bioinformatics detection and Dual-Luciferase Reporter assay system to detect the target of mi R-223 on insulin-like growth factor-1 receptor(IGF-1R).(2)Western blot,immunofluorescence to detect the targeting effect of mi R-223 on IGF-1R and the expression of downstream targets after transfection of mi R-223mimic;(3)VSMC was transfected with IGF-1R lentivirus,and then the proliferation rate of VSMC was detected by CCK-8 and Ed U;(3)Proteomic Phosphokinase Array analysis to search the proteins with changes in phosphorylation in VSMC co-cultured with activated platelets.Part Ⅲ: Use streptozotocin-induced diabetic mouse(STZ-DM)combine with the model of femoral artery injury,and use Agomir-223 and Calpain inhibitor calpeptin for treatment.(1)The recovery effect of Calpeptin on platelet mi R-223 was detected by q PCR,and the effect of platelet mi R-223 recovered by Calpeptin on the proliferation rate of VSMC was tested by Ed U experiment;(2)Observe vascular proliferation by HE staining,and IGF-1R expression by immunofluorescence to explore the role of calpeptin in vascular intimal hyperplasia in diabetes.ResultPart Ⅰ:(1)VSMC co-cultured with activated platelets of HS have reduced dedifferentiation markers and increased differentiation markers,while VSMC co-cultured with activated platelets of DM has no change trend.VSMC transfected with mi R-223 mimic have the same trend as VSMC co-cultured with HS platelets;(2)The experimental results of CCK-8 and Ed U show that platelets of HS can inhibit the proliferation of VSMC;meanwhile,transfection of mi R-223 mimic can also inhibit the proliferation of smooth muscle cells.Part Ⅱ:(1)Bioinformatics analysis Dual-Luciferase Reporter assay system identified a binding site for mi R-223 in the 3’-UTR of IGF-1R and mi R-223 mimic significantly inhibit the luciferase activity;(2)The results of Western blot and immunofluorescence show that mi R-223 can target IGF-1R and inhibit the expression of IGF-1R and the phosphorylation level of IGF-1R downstream pathway proteins is reduced after transfection of mi R-223 mimic;(3)The results of Proteomic Phosphokinase Array analysis and Western blot revealed that HS-APS and mi R-223 can increase the phosphorylation of AMPKα and activates downstream cell proliferation-inhibition Signal pathway.Part Ⅲ:(1)After Calpeptin treatment,the expression of mi R-223 in diabetic platelets was significantly increased,and the treated diabetic platelets could inhibit the proliferation of VSMC;(2)HE staining and immunofluorescence results show that Calpeptin treatment reduced the vascular intimal hyperplasia in diabetic mice after the femoral artery injury and the expression of IGF-1R were reduced in the femoral artery injury.Conclusion(1)miR-223 in platelets can inhibit the proliferation of VSMC,while mi R-223 deficiency in diabetic platelet failed to suppress VSMC Proliferation.(2)Platelet-derived mi R-223 inhibits VSMC proliferation by regulating the mi R-223 / IGF-1R / AMPK signaling pathway;(3)Inhibition of calpain by calpeptin can restore the mi R-223 in diabetic platelets and reduces the vascular intimal hyperplasia in diabetic mice.