目的 探讨紫花牡荆素(Casticin,CAS)通过调节环磷酸腺苷/蛋白激酶A信号通路(Cyclic a-denosine monophosphate/protein kinase A,cAMP/PKA)对氧糖剥夺/复糖复氧(Oxygen glucose deprivation/recovery,OGD/R)诱导的神经元损伤的影响.方法 通过体外培养海马神经元(HT-22),进行OGD/R诱导处理建立神经元损伤模型,使用2~32 μmol/L的紫花牡荆素处理细胞,筛选最佳实验水平;将细胞分为对照组(Control组)、模型组(OGD/R组)、紫花牡荆素低、中、高水平组(CAS-L组、CAS-M组、CAS-H组)、紫花牡荆素+cAMP/PKA信号通路抑制剂组(CAS+H-89组);采用细胞计数试剂-8(Cell counting kit-8,CCK-8)法检测细胞的增殖活力,倒置显微镜观察细胞形态,Hoechst染色及流式细胞术观察并计算细胞凋亡,酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)法检测过氧化氢酶(Catalase,CAT)、超氧化物歧化酶(Superoxide dismutase,SOD)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)以及cAMP水平,Western blot检测天冬氨酸特异性半胱氨酸蛋白酶-3(Aspartate-specific cysteine proteinase-3,Caspase-3)、PKA、磷酸化 PKA(phosphorylated PKA,p-PKA)表达水平.结果 与 Control 组比较,OGD/R组HT-22细胞细胞核皱缩,细胞崩解,细胞存活率、CAT及SOD,cAMP,p-PKA表达水平显著降低,细胞凋亡率、TNF-α及IL-6,Caspase-3表达水平显著升高(P<0.05);与OGD/R组比较,CAS-L组、CAS-M组、CAS-H组HT-22细胞损伤减轻,细胞存活率、CAT及SOD、cAMP、p-PKA表达水平显著升高,细胞凋亡率、TNF-α及IL-6,Caspase-3表达水平显著降低,且呈水平依赖性(P<0.05);与CAS-H组比较,CAS+H-89组HT-22细胞损伤加重,细胞存活率、CAT及SOD,cAMP,p-PKA表达水平显著降低,细胞凋亡率、TNF-α及IL-6,Caspase-3表达水平显著升高(P<0.05).结论 紫花牡荆素可以通过激活cAMP/PKA信号通路来减轻OGD/R诱导的神经元损伤.
Objective To investigate the impact of casticin(CAS)on neuronal damage induced by oxy-gen glucose deprivation/recovery(OGD/R)by regulating the cyclic adenosine monophosphate/protein kinase A signaling pathway(cAM P/PKA).Methods Rat hippocampal neurons(HT-22)were subjected to OGD/R treatment in vitro to establish a neuronal injury model.And then the cells were treated with 2~32 μmol/L purple peony extract and screened for the optimal experimental concentration.Cells were divided into control group,model group(OGD/R group),low-,medium-,and high-concentration CAS groups(CAS-L group,CAS-M group,CAS-H group),and CAS+cAMP/PKA signaling pathway inhibitor group(CAS+H-89 group).Cell counting kit-8(CCK-8)was applied to detect the proliferation activity of cells.Inverted micro-scope was used to observe cell morphology.Hoechst staining and flow cytometry were applied to detect cell ap-optosis.Enzyme-linked immunosorbent assay(ELISA)was applied to measure the levels of catalase(CAT),superoxide dismutase(SOD),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and cAMP.Western blotting was applied to detect the expression of caspase-3,PKA,and p-PKA.Results Compared with the control group,HT-22 cells in the OGD/R group showed nuclear crumpling,cell disintegration,significantly lower cell survival,CAT and SOD,cAMP and p-PKA expression levels,and significantly higher apoptosis,TNF-α and IL-6,and caspase-3 expression levels(P<0.05);compared with the OGD/R group,the cells in the CAS-L group was reduced in CAS-L group,CAS-M group and CAS-H group was reduced;the cell surviv-al rate and CAT,SOD,cAMP and p-PKA expression levels were significantly increased;and the apoptosis rate and TNF-α,IL-6,and caspase-3 expression levels were significantly decreased in a level-dependent manner(P<0.05).Compared with those in the CAS-H group,the cell survival rate was significantly increased in the CAS+H-89 group,and the expression levels of TNF-α,IL-6 and caspase-3 were significantly increased(P<0.05).Compared to the CAS-H group,the damage to HT-22 cells in the CAS+H-89 group worsened,the cell survival rate,CAT and SOD levels,cAMP level,and p-PKA expression were obviously reduced,and the apoptosis rate,TNF-α and IL-6 levels,and caspase-3 expression were obviously increased(P<0.05).Conclusion Casticin can improve OGD/R-induced neuronal damage by activating the cAMP/PKA signaling pathway.