OGD模型乳小鼠的星状胶质细胞内miR-31对共培养神经元氧化应激损伤的影响

Effects of miR-31 in Astrocytes of OGD Model Suckling Mice on Oxidative Stress-Induced Injury in Co-Cultured Neurons

目的探究氧-糖剥夺(OGD)模型乳小鼠的星状胶质细胞内miR-31调控PKD1基因表达,进而抑制JAK-STAT3信号通路活化介导的OGD共培养神经元炎症反应以及氧化应激损伤的保护机制.方法提取乳小鼠原代星状胶质细胞和神经元,流式细胞仪分选纯化,之后将星状胶质细胞分别转染miR-31 mimic、miR-31 inhibitor、si-PKD1或加入JAK-STAT3通路抑制剂;构建OGD模型将转染后的星状胶质细胞与神经元共培养,并分为七组:normal组,blank OGD组,NC OGD组,miR-31 mimic OGD组,miR-31 inhibitor OGD组,si-PKD1 OGD组,si-PKD1+CdCl_(2)OGD组.MTT法检测OGD共培养体系中神经元存活率;LDH漏出率测定OGD共培养体系中神经元细胞损伤;TUNEL法检测OGD共培养体系中神经元凋亡;免疫荧光法检测OGD共培养体系中氧化应激损伤相关蛋白4-HNE和8-OHdG表达;TBA法检测OGD共培养体系中MDA含量;黄嘌呤氧化酶法检测OGD共培养体系中SOD含量;ELISA检测OGD共培养体系中炎症因子;qRT-PCR检测OGD共培养体系中miR-31和PKD1的表达;Western blot检测OGD共培养体系中PKD1及JAK-STAT3通路相关蛋白表达.结果OGD模型乳小鼠星状胶质细胞miR-31调控炎症反应结果显示:与blank组和NC组相比,miR-31 mimic组炎症因子IL-1β、IL-6、TNF-α的表达量显著降低(均P<0.05),miR-31 inhibitor组炎症因子IL-1β、IL-6、TNF-α的表达量显著上升(均P<0.05).与blank组和NC组相比,miR-31 mimic组神经元存活率上升,LDH漏出率、ROS表达量显著降低(均P<0.05),miR-31 inhibitor组神经元存活率显著降低,LDH漏出率和ROS表达量显著升高(均P<0.05);OGD模型乳小鼠星状胶质细胞miR-31调控对神经元的活性的影响结果显示:与单独培养(Neuron组)相比,共培养条件下神经元(Neuron+astrocytes)的增殖及存活率、LDH表达及凋亡率差异均无统计学意义(均P>0.05);与神经元组(Neuron组)相比,神经元OGD模型组(Neuron OGD组)细胞增殖及存活率减少,培养液中LDH表达增加,细胞凋亡率增加(均P<0.05);与神经元OGD模型(Neuron OGD组)相比,神经元+星状胶质细胞OGD组(Neuron+astrocytes OGD)细胞存活率减少,培养液中LDH表达增加,细胞凋亡率增加(均P<0.05);OGD模型乳小鼠星状胶质细胞miR-31调控PKD1各组细胞中mRNA、蛋白表达水平及炎症因子表达结果显示:相比于normal组,blank OGD组的星状胶质细胞中miR-31的表达量下降(P<0.05),而PKD1表达和p-STAT3/STAT3显著上升(P<0.05),炎症因子IL-1β、IL-6、TNF-α表达量上调(P<0.05);相比于blank OGD组,miR-31 mimic OGD组星状胶质细胞中miR-31的表达量上调(P<0.05),而PKD1表达和p-STAT3/STAT3显著降低(P<0.05),炎症因子IL-1β、IL-6、TNF-α表达量下调(P<0.05);miR-31 inhibitor OGD组miR-31的表达量下降(P<0.05),而PKD1表达和p-STAT3/STAT3显著上升(均P<0.05),炎症因子IL-1β、IL-6、TNF-α表达量上调(P<0.05);si-PKD1 OGD组和si-PKD1+CdCl_(2)OGD组miR-31的表达量无显著变化(P>0.05),而PKD1表达和p-STAT3/STAT3显著降低(均P<0.05),炎症因子IL-1β、IL-6、TNF-α表达量下调(P<0.05);NC OGD组各指标差异无统计学意义(均P>0.05);OGD模型乳小鼠星状胶质细胞miR-31调控PKD1基因抑制OGD导致的神经元氧化应激损伤结果显示:相比于normal组,blank OGD组LDH和MDA表达量上升,SOD表达下降,神经元膜脂质过氧化损伤标志蛋白4-HNE和DNA氧化损伤标志蛋白8-OHdG免疫染色强度增强,神经元存活率降低,神经元出现氧化应激损伤(均P<0.05);与blank OGD组相比,miR-31 inhibitor OGD组LDH漏出量和MDA表达量上升,SOD表达下降,神经元膜脂质过氧化损伤标志蛋白4-HNE和DNA氧化损伤标志蛋白8-OHdG免疫染色强度增强,神经元存活率降低,细胞凋亡率上升,神经元氧化应激损伤加重(均P<0.05);miR-31 mimic OGD组、si-PKD1 OGD组和si-PKD1+CdCl_(2)OGD组LDH漏出量和MDA表达量下降,SOD表达上升,神经元膜脂质过氧化损伤标志蛋白4-HNE和DNA氧化损伤标志蛋白8-OHdG免疫染色强度降低,神经元存活率增加,细胞凋亡率下降,神经元氧化应激损伤减缓(均P<0.05);NC OGD组各指标差异无统计学意义(均P>0.05);采用TUNEL染色及Western blot检测OGD共培养体系中神经元凋亡结果显示:相比于normal组,blank OGD组神经元细胞凋亡率及凋亡相关蛋白cleaved caspase-3、Bax表达显著增加(均P<0.05);与blank OGD组相比,miR-31 inhibitor OGD组神经元细胞凋亡率及cleaved caspase-3、Bax表达明显上升(均P<0.05);miR-31 mimic OGD组、si-PKD1 OGD组和si-PKD1+CdCl_(2)OGD组神经元细胞凋亡率及cleaved caspase-3、Bax表达明显下调(均P<0.05);NC OGD组各cleaved caspase-3、Bax表达差异无统计学意义(均P>0.05).结论OGD模型乳小鼠星状胶质细胞内miR-31靶向下调PKD1基因,进而通过抑制JAK/STAT3信号通路降低炎症反应,从而减轻OGD神经元氧化应激损伤.

Objective To explore the protective mechanism of miR-31 regulating PKD1 gene expression and inhibiting the activation of JAK-STAT3 signaling pathway in oxygen-glucose deprivation(OGD)cocultured neuronal inflammatory response and oxidative stress injury in suckling mice astrocytes of OGD model.Methods Primary astrocytes and neurons of milk mice were extracted and purified by flow cytometry.After that,astrocytes were transfected miR-31 mimic,miR-31 inhibitor,si-PKD1 or added to JAK-STAT3 pathway inhibitor respectively.The OGD model was constructed and the transfected astrocytes were co-cultured with neurons and divided into seven groups:normal group,blank OGD group,NC OGD group,miR-31 mimic OGD group,miR-31 inhibitor group,si-PKD1 OGD group,and si-PKD1+CdCl_(2)OGD group.MTT was used to detect the survival rate of neurons in OGD co-culture system.The leakage rate of LDH was used to determine the damage of neurons in OGD co-culture system.TUNEL assay was used to detect neuronal apoptosis in OGD coculture system.Immunofluorescence was used to detect the expression of oxidative stress injury-related proteins 4-HNE and 8-OHdG in OGD co-culture system.MDA content in OGD co-culture system was detected by TBA method.SOD content in OGD co-culture system was detected by xanthine oxidase method.ELISA was used to detect inflammatory factors in OGD co-culture system.The expressions of miR-31 and PKD1 in OGD coculture system were detected by qRT-PCR.Western blot was used to detect PKD1 and JAK-STAT3 pathway related proteins in OGD co-culture system.Results The inflammatory response of miR-31 in astrocytes of OGD model of suckling mice showed that,compared with the blank and NC groups,the expressions of inflammatory factors IL-1β,IL-6 and TNF-αin the miR-31 mimic group were significantly decreased(all P<0.05),but in the miR-31 inhibitor group,their expression increased significantly(all P<0.05).Compared with the blank and NC groups,the neuronal survival rate,LDH leakage rate and ROS expression level of the miR-31 mimic group were significantly decreased(all P<0.05),but in the miR-31 inhibitor group,the survival rate of neurons was significantly decreased,and LDH leakage rate and ROS expression were significantly increased(all P<0.05);Effects of miR-31 regulation on neurons in OGD model:results showed that the expression of the inflammatory factors such as IL-1β,IL-6,TNF-α,LDH leakage rate,and ROS level were reduced,and the neuronal viability was enhanced due to the elevation of miR-31(all P<0.05).Downregulation of miR-31 contributed to elevated levels of IL-1β,IL-6,TNF-α,LDH leakage rate,and ROS level,but reduced the neuronal viability(all P<0.05);miR-31 regulates PKD1 in OGD model,the expression levels of mRNA,protein and inflammatory factors in each group were shown as follows:compared with the normal group,the expression of miR-31 in astrocytes of the blank OGD group decreased(P<0.05);while the expression of PKD1 and p-STAT3/STAT3 were significantly increased(P<0.05),inflammatory factors IL-1β,IL-6,TNF-αwere up-regulated(P<0.05);compared with the blank OGD group,the expression levels of miR-31 were up-regulated in astrocytes of miR-31 mimic OGD group(P<0.05);while the expression of PKD1 and p-STAT3/STAT3 were significantly decreased(P<0.05),inflammatory factors IL-1β,IL-6,TNF-αwere down-regulated(P<0.05);the miR-31 expression in miR-31 inhibitor OGD group decreased(P<0.05);while the expression of PKD1 and p-STAT3/STAT3 were significantly increased(all P<0.05),inflammatory factors IL-1β,IL-6,TNF-αwere up-regulated(P<0.05).There was no significant change in miR-31 expression in the si-PKD1 OGD group and the si-PKD1+CdCl_(2)OGD group(P<0.05),while the expression of PKD1 and p-STAT3/STAT3 were significantly decreased(all P<0.05),inflammatory factors IL-1β,IL-6,TNF-αwere down-regulated(P<0.05).There was no significant difference in each indicator in NC OGD group(P<0.05).miR-31 inhibited OGD-induced oxidative stress-induced neuronal injury by targeting PKD1,the results showed:compared with the normal group,LDH and MDA expression increased,SOD expression decreased,immunostaining intensity of 4-HNE and 8-OHdG enhanced,survival rate of neurons decreased,and oxidative stress injury occurred in neurons(all P<0.05).Compared with the blank OGD group,LDH leakage and MDA expression in miR-31 inhibitor OGD group increased,SOD expression decreased,immunostaining intensity of 4-HNE and 8-OHdG enhanced,survival rate of neurons decreased,apoptosis rate increased,and oxidative stress injury of neurons increased(all P<0.05).The levels of LDH leakage and MDA expression were decreased,SOD expression was increased,immunostaining intensity of 4-HNE and 8-OHdG were decreased,neuronal survival rate was increased,apoptosis rate was decreased,and oxidative stress injury of neurons was reduced in groups of miR-31 mimic OGD group,si-PKD1 OGD group and si-PKD1+CdCl_(2)OGD group(all P<0.05).There was no significant difference in each indicator in NC OGD group(all P<0.05).The results of apoptosis in OGD coculture system detected by TUNEL staining and Western blot showed that:compared with the normal group,the apoptosis rate of neurons and the expression of cleaved caspase-3 and Bax significantly increased in the blank OGD group(all P<0.05);compared with blank OGD group,the neuronal apoptosis rate and cleaved caspase-3 and Bax expressions in miR-31 inhibitor OGD group were significantly increased(all P<0.05).Apoptosis rate,cleaved caspase-3 and Bax expression were significantly down-regulated in the miR-31 mimic OGD group,si-PKD1 OGD group and si-PKD1+CdCl_(2)OGD group(all P<0.05).There was no significant difference in expression of cleaved caspase-3 and Bax in NC OGD group(all P<0.05).Conclusion miR-31 downregulate PKD1 expression in astrocytes of OGD model,reduces the inflammatory responses by suppressing the activation of JAK-STAT3 pathway,thereby reducing oxidative stress injury of OGD neurons.