| Objective: In this study,mice with type 2 diabetes mellitus were given an 8-week combined aerobic and resistance exercise(combined exercise)to explore the effects of combined exercise on the proliferation,adhesion,migration and angiogenesis of mouse myelo-derived endothelial progenitor cells(EPCs),as well as PI3 K,AKT and mi RNA-378a-5p.Moreover,PI3K/AKT pathway inhibitor was used to act on EPCs to further explore the regulatory role of PI3K/AKT signaling pathway in function of EPCs and mi RNA-378a-5p,and to further study the mechanism of regulation of combined exercise on the functions of EPCs proliferation,adhesion,migration and angiogenesis through the PI3K/AKT signaling pathway via mi RNA-378a-5p.Methods:(1)Male db/db mice with the background of C57BL/ks.db were used as the mouse model of type 2 diabetes,with the age of 8 weeks.And the random blood glucose of all the mice exceeding 300 mg/dl,purchased from Nanjing university--Nanjing institute of biological medicine [license no.: SYXK(su)2016-0012].According to the random number table method,the mice were randomly divided into the combined aerobic-resistance exercise group and the control group,with20 mice in each group.The control group had no exercise training.The intervention mode of mice in the combined aerobic-resistance exercise group was carried out in combination with aerobic and resistance exercise,and the exercise training was carried out six days a week,that is,the resistance exercise was carried out on Monday,Wednesday and Friday respectively,while the aerobic exercise was carried out on Tuesday,Thursday and Saturday.During the intervention,mice in both groups were kept in the normal feeding mode.(2)Mouse bone marrow endothelial progenitor cells were isolated and cultured in vitro after 8 weeks of combined cardio-resistance exercise intervention.The culture bottle was coated with fibronectin(FN-protein)in advance,and the monocytes obtained by density gradient method were transferred to the culture bottle for culture.The expression of cell surface markers was detected by immunofluorescence staining.(3)The identified EPCs were divided into groups,in which the isolated and cultured cells of the control group were randomly divided into the control group and the control + inhibitor group,and the isolated and cultured cells of the combined aerobic and resistance exercise group were randomly divided into the combined exercise group and the combined exercise + inhibitor group.The PI3K/AKT signaling pathway of EPCs was blocked by the PI3K/AKT pathway inhibitor LY294002.The proliferation,migration,adhesion and angiogenesis of EPCs in each group were observed,and the expressions of PI3 K,AKT and mi RNA-378a-5p were detected by real-time PCR and Western Blot.Results:(1)Cell identification results: the positive rate of EPCs surface antigen identification was 91.2± 3.6% for CD31,95.3± 2.6% for CD34,94.8± 3.2% for CD133,93.1±4.2% for CD144,and 95.8±2.5% for VEGFR2.The identification results showed that the cultured cells were EPCs.(2)The proliferation,migration,adhesion and angiogenesis of bone marrow EPCs in type 2 diabetic mice were significantly enhanced after 8 weeks of combined cardio-resistance exercise training,with statistically significant differences compared with the control group(P<0.05).After blocking the PI3K/AKT signaling pathway with the inhibitor LY294002,the proliferation,migration,adhesion and angiogenesis of EPCs in the combined exercise +inhibitor group decreased significantly compared with the combined exercise group(P<0.05).(3)Compared with the control group,the expression levels of EPCs PI3 K 、AKT and mi RNA-378a-5p m RNA in the combined exercise group were increased,and the difference was statistically significant(P<0.05).After blocking the PI3K/AKT signaling pathway with LY294002,the m RNA expression levels of PI3K、AKT and mi RNA-378a-5p of EPCs in the combined exercise + inhibitor group were lower than those in the combined exercise group,and the difference was statistically significant(P<0.05).(4)After 8 weeks of exercise intervention,Phospho-PI3Kp85 and PhosphoAKT protein expression levels of EPCs in the combined exercise group were significantly increased,and the difference was statistically significant compared with the control group(P<0.05).After blocking the PI3K/AKT signaling pathway with the inhibitor LY294002,Phospho-PI3Kp85 and Phospho-AKT protein expression levels of EPCs in the combined exercise + inhibitor group were lower than those in the combined exercise group,with statistically significant differences(P<0.05).Conclusions:(1)Combined with aerobic resistance exercise at 8 weeks promoted the proliferation,migration,adhesion and angiogenesis of bone marrow EPCs in type2 diabetic mice,and increased the expression levels of Phospho-PI3Kp85,Phospho-AKT and mi RNA-378a-5p.(2)The addition of PI3K/AKT pathway inhibitor can block the PI3K/AKT signaling pathway,reduce the expression levels of Phospho-PI3Kp85,PhosphoAKT and mi RNA-378a-5p in EPCs,and weaken the effect of combined aerobic resistance exercise on the proliferation,migration,adhesion and angiogenesis of EPCs.(3)Combined aerobic-resistance exercise may through the activation of PI3K/AKT signaling pathway,up-regulate the mi RNA-378a-5p levels,and strengthen the function of EPCs in mice with type 2 diabetes,which further for joint aerobic-resistance exercise EPCs proliferation,migration,adhesion and promote angiogenesis function,provide theoretical basis for prevention and treatment of patients with type 2 diabetes vascular complications. |
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