目的 探讨蛇葡萄素(AMP)对氧糖剥夺/再灌注(OGD/R)诱导的神经元损伤的影响及其作用机制,为新生儿缺氧缺血性脑损伤的研究奠定基础.方法 分离并体外培养新生SD大鼠神经元细胞,分为5组:对照组(AMP浓度为0 μmol/L)、OGD/R组、AMP低剂量组(OGD/R处理+AMP 20 μmol/L)、AMP高剂量组(OGD/R 处理+AMP 30 μmol/L)、JAK2/STAT3 激活剂组(OGD/R 处理+AMP 30 μmol/L+Coumermy-cin A1 10 μmol/L).CCK-8法检测不同处理组细胞活力;乳酸脱氢酶(LDH)试剂盒检测培养基中LDH活性;流式细胞术检测细胞凋亡率;酶联免疫吸附试验检测白细胞介素6(IL-6)、白细胞介素10(IL-10)、肿瘤坏死因子α(TNF-α)水平;试剂盒检测活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平;Western blotting检测凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、酶切含半胱氨酸的天冬氨酸蛋白水解酶-3(C-caspase-3)、酪氨酸激酶2(JAK2)、磷酸化JAK2(p-JAK2)、信号传导及转录活化因子3(STAT3)、磷酸化STAT3(p-STAT3)表达情况.结果 与AMP浓度为0 μmol/L比较,AMP浓度为5~30 μmol/L时,细胞活力比较差异无统计学意义(P>0.05),AMP浓度为40 μmol/L细胞活力明显下降(P<0.05).与对照组比较,OGD/R组细胞活力、SOD、IL-10、Bcl-2水平明显下降,LDH活性、细胞凋亡率、ROS荧光强度、MDA、IL-6、TNF-α、Bax、C-caspase-3、p-JAK2/JAK2、p-STAT3/STAT3 水平明显升高(P<0.05).与 OGD/R 组比较,AMP低、高剂量组神经元细胞活力、SOD、IL-10、Bcl-2水平上升,LDH活性、细胞凋亡率、ROS荧光强度、MDA、IL-6、TNF-α、Bax、C-caspase-3、p-JAK2/JAK2、p-STAT3/STAT3 水平下降(P<0.05),而 JAK2/STAT3激活剂可逆转AMP对OGD/R诱导的神经元细胞损伤的保护作用.结论 AMP通过减少氧化应激和炎症反应减轻OGD/R诱导的神经元细胞损伤,其作用机制可能与抑制JAK2/STAT3信号通路磷酸化有关.
Objective To investigate the impact of ampelopsin(AMP)on oxygen glucose deprivation/reperfusion(OGD/R)induced neuronal damage and its mechanism,and to lay a foundation for the study of neonatal hypoxic-ischemic brain damage.Methods Neurons of newborn SD rats were isolated and cultured in vitro,and they were divided into 5 groups:control group(AMP 0 μmol/L),OGD/R group,low dose AMP group(OGD/R+AMP 20 μmol/L),high dose AMP group(OGD/R+AMP 30 μmol/L)and JAK2/STAT3 activator group(OGD/R+AMP 30 μmol/L+Coumermycin A1 10 μmol/L).CCK-8 method was used to de-tect the cell viability of different treatment groups,the lactate dehydrogenase(LDH)kit was used to detect the cell activity of LDH in the medium,flow cytometry was used to detect the apoptosis rate,enzyme-linked immunosorbent assay was used to detect the levels of interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor α(TNF-α),the kit was used to detect the levels of reactive oxygen species(ROS),malondial-dehyde(MDA)and superoxide dismutase(SOD),and Western blotting was used to detect the expression of apoptosis related proteins B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),enzymatic cleavage of cysteine containing aspartate protein hydrolase-3(C-caspase-3),tyrosine kinase 2(J AK2),phosphorylated JAK2(p-JAK2),signal transduction and transcription activating factor 3(STAT3)and phosphorylated STAT3(p-STAT3).Results Compared with the concentration of AMP of 0 μmol/L,the cell viability in con-centration of AMP of 5-30 μmol/L was not obvious different(P>0.05),when the concentration of AMP was 40 μmol/L,the cell viability decreased obviously(P<0.05).Compared with the control group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in OGD/R group decreased obviously,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 increased obviously(P<0.05).Compared with OGD/R group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in low and high dose AMP groups increased,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 decreased(P<0.05),and JAK2/STAT3 activator was able to reverse the protective effect of AMP on OGD/R induced neuronal.Conclusion AMP attenuates OGD/R induced neuronal by reducing oxidative stress and inflammatory response,and its mechanism may be related to inhibition of JAK2/STAT3 signal pathway phosphorylation.