【摘要】 目的:探讨细胞外调节蛋白激酶1/2(extracellcular regulated protein kinase 1/2,ERK1/2)信号转导通路在ghrelin上调热休克蛋白70(heat shock protein 70, HSP70)表达及抑制氧化应激诱导PC12细胞凋亡中的作用。方法:采用硝普钠(sodium nitroprusside, SNP)诱导PC12细胞构建氧化应激损伤模型;实验分为SNP损伤组(0.5 mmol/L SNP分别孵育PC12细胞6 h、12 h、18 h和24 h),ghrelin预处理组(加SNP前30 min给予100 nmol/L ghrelin),HSP70抑制剂组[ghrelin处理前60 min加入10μmol/L槲皮素(quercetin)],ERK阻断剂组(ghrelin处理前60 min加入ERK1/2抑制剂PD98059)和对照组(只加入等量的培养液);利用流式细胞术检测细胞凋亡率,通过Western blot和免疫细胞化学染色法检测蛋白表达的变化。结果:(1)与对照组比较, SNP损伤组PC12细胞的凋亡率显著增加(P<0.05);(2)与SNP损伤组比较,100 nmol/L ghrelin预处理可显著抑制SNP诱导的PC12细胞凋亡(P<0.05),且显著上调HSP70蛋白表达(P<0.05);(3)时-效分析表明,SNP损伤后18 h, ghrelin上调HSP70表达的作用最为显著。使用HSP70抑制剂quercetin可显著削弱ghrelin的抗凋亡作用(P<0.05);(4)Ghrelin预处理促进ERK1/2的磷酸化。ERK1/2抑制剂PD98059可显著削减ghrelin对HSP70表达的上调作用(P<0.05)。结论:Ghrelin可通过促进ERK1/2磷酸化上调HSP70的表达,进而抑制氧化应激诱导的PC12细胞凋亡。更多还原

【Abstract】 AIM: To study the role of ghrelin in cell protection by up-regulating heat shock protein 70(HSP70) and inhibiting apoptosis induced by oxidative stress through extracellular regulated protein kinases 1/2(ERK1/2) signaling pathway in the PC12 cells. METHODS: Sodium nitoprusside(SNP) was used to induce oxidative stress injury in the PC12 cells. The cultured PC12 cells were divided into SNP-injured group(incubated with SNP at 0.5 mmol/L for 6, 12, 18 and 24 h), ghrelin pretreatment group(ghrelin at 100 nmol/L was given 30 min before adding SNP); HSP70 inhibitor group(quercetin at 10 μmol/L was added 60 min before ghrelin treatment), ERK inhibitor group(ERK 1/2 inhibitor PD98059 was added 60 min before ghrelin treatment) and control group(added same amount of culture medium only). The apoptotic rate was detected by flow cytometry. The protein expression was determined by Western blot and immunocytochemistry. RESULTS: Compared with control group, the apoptotic rate of PC12 cells in SNP-injured group was significantly increased(P<0.05). Compared with SNP-injured group, ghrelin(100 nmol/L) pretreatment significantly inhibited SNP-induced apoptosis of PC12 cells(P<0.05), and significantly up-regulated the protein expression of HSP70(P<0.05). Time-effect analysis showed that ghrelin had the most significant effect at 18 h after SNP injury. Quercetin, an inhibitor of HSP 70, significantly reduced the anti-apoptotic effect of ghrelin(P<0.05). Ghrelin pretreatment promoted the phosphorylation of ERK1/2. ERK1/2 inhibitor PD98059 significantly inhibited the effects of ghrelin on up-regulation of HSP70 expression(P<0.05). CONCLUSION: Ghrelin upregulates the expression of HSP70 and inhibits the apoptosis in the PC12 cells induced by oxidative stress by promoting the phosphorylation of ERK1/2.更多还原