ghrelin对海马神经干细胞糖氧剥夺性损伤的影响及机制

Effects of ghrelin on oxygen-glucose deprivation-induced injury of hippocampal neural stem cells

目的观察ghrelin对糖氧剥夺诱导海马神经干细胞损伤的影响,并探讨核受体辅阻遏子1(N-CoR1)/磷脂酰肌醇3-激酶(PI3K)通路在该影响中的作用。方法将细胞随机分为正常对照组、损伤模型组、ghrelin治疗组、GHRP组、LY294002组、siRNA空白对照组、N-CoR1 siRNA组。正常对照组在正常糖含量的培养基、正常氧含量混合气体的普通培养箱中培养24 h,其余各组细胞行糖氧剥夺处理,处理后培养条件同正常对照组。siRNA空白对照组、N-CoR1 siRNA组行糖氧剥夺处理前1 h,培养液更换为含等量转染溶剂Lipofectamine2000或N-CoR siRNA脂质体的培养液,持续转染至糖氧剥夺之后的24 h。ghrelin治疗组在糖氧剥夺处理结束后,将培养液更换为含ghrelin的培养基继续培养24 h。GHRP-6组、LY294002组在糖氧剥夺处理结束后,预先给予ghrelin受体拮抗剂D-Lys ^3-GHRP-6或PI3K抑制剂LY294002处理1 h,再同时给予ghrelin处理,继续培养24 h。收集各组细胞,采用CCK-8法检测海马神经干细胞增殖活力,Western blotting法检测N-CoR1、PI3K、增殖标志蛋白增殖细胞核抗原(PCNA)及凋亡标志蛋白Bax、Bcl-2。CCK-8实验中ghrelin治疗组ghrelin浓度分别为4、20、100 nmol/L,其余实验中ghrelin浓度均为100 nmol/L。结果与正常对照组比较,损伤模型组海马神经干细胞增殖活力降低,细胞PCNA表达减少、Bax/Bcl-2增大,N-CoR表达明显增多、PI3K表达减少(P均<0.01);与损伤模型组比较,ghrelin治疗组随ghrelin作用浓度增高细胞增殖活力增高(P均<0.01),细胞PCNA表达增多、Bax/Bcl-2减小,N-CoR表达减少、PI3K表达增多(P均<0.01);与ghrelin治疗组(100 nmol/L)比较,GHRP组、LY29400组神经干细胞增殖活力降低,PCNA表达减少、Bax/Bcl-2增大,N-CoR表达明显增多、PI3K表达减少(P均<0.01)。与siRNA空白对照组比较,N-CoR siRNA组神经干细胞增殖活力增高,N-CoR siRNA组PCNA表达增多、Bax/Bcl-2减小,N-CoR siRNA组N-CoR表达减少、PI3K表达增多(P均<0.01)。结论ghrelin可抑制糖氧剥夺诱导的海马神经干细胞损伤,其机制与调节N-CoR/PI3K信号通路有关。

Objective To observe the influence of ghrelin on the oxygen-glucose deprivation-induced injury of hippocampal neural stem cells(NSCs)and to explore the potential role of the nuclear receptor corepressor 1(N-CoR1)/phosphoinositide 3-kinase(PI3K)pathway in mediating the actions of ghrelin.Methods Isolated hippocampal NSCs from neonatal C57BL/6 mice were randomly divided into 7 groups:the normal control group,injury model group,ghrelin group,GHRP-6 group,LY294002 group,blank siRNA group,and N-CoR siRNA group.The cells in the normal control group were cultured under normal glucose and normal oxygen for 24 h.After 3-hour oxygen-glucose deprivation,the cells in the other groups were also cultured under normal glucose and normal oxygen for 24 h.In addition,for N-CoR siRNA group and blank siRNA group,transfection with N-CoR siRNA or blank siRNA began at 1 h before oxygen-glucose deprivation continued until 24 h after deprivation.In the ghrelin group,we changed the nutrient solution into the ghrelin-containing medium after oxygen-glucose deprivation,followed by incubation for 24 h.After oxygen-glucose deprivation,the cells in the GHRP-6 group and LY294002 group were pre-treated with D-Lys ^3-GHRP-6 or LY294002 for 1 h and then received ghrelin treatment for 24 h.After drug treatment,the cells from each group were collected and used for the measurement of NSC proliferative viability by CCK-8 assay,and the protein expression of N-CoR1,PI3K,proliferating cell nuclear antigen(PCNA),Bax,and Bcl-2 by Western blotting.In some experiments for CCK-8 assay,the NSCs in the ghrelin group were treated with different doses of ghrelin(4,20,and 100 nmol/L)to evaluate the concentration-dependent effects;and in other experiments,the concentration of ghrelin was 100 nmol/L.Results Compared with the control group,the injury model group showed decreased proliferative viability of NSCs,reduced PCNA expression,increased Bax/Bcl-2,enhanced N-CoR expression,and decreased PI3K expression(all P<0.01).In contrast,ghrelin concentration-dependently increased the proliferative viability of NSCs(P<0.01),up-regulated PCNA expression,reduced Bax/Bcl-2,down-regulated N-CoR expression,and enhanced PI3K expression(all P<0.01).Compared with the ghrelin group,GHRP group and LY29400 group exhibited decreased proliferative viability of NSCs,reduced PCNA expression,elevated Bax/Bcl-2,increased N-CoR expression,and decreased PI3K expression(all P<0.01).Compared with the blank siRNA group,N-CoR siRNA group exhibited increased proliferative viability of NSCs,elevated PCNA expression,down-regulated Bax/Bcl-2,decreased N-CoR expression,and increased PI3K expression(all P<0.01).Conclusion Ghrelin can protect hippocampal NSCs against oxygen-glucose deprivation-induced injury in vitro by regulating the N-CoR/PI3K signaling pathway.