ROCK通路抑制剂对糖氧剥夺星形胶质细胞的保护作用及机制

Protective effect and mechanism of ROCK pathway inhibitor on oxygen and glucose deprivation astrocytes

目的研究糖氧剥夺(OGD)及ROCK通路抑制对星形胶质细胞的保护作用及机制的影响。方法2022年6月—12月以大鼠CTX TNA2星形胶质细胞系为实验对象,分为对照组A组、糖氧剥夺组B组和法舒地尔组C组。B组构建糖氧剥夺(OGD)干预星形胶质细胞体外模型,C组选用ROCK通路抑制剂法舒地尔(Fasudil)干预模型细胞。分析糖氧剥夺及Fasudil对星形胶质细胞形态、焦亡通路、炎性反应及NRF2/HO-1通路mRNA表达的影响。结果与对照组相比,糖氧剥夺(OGD)干预星形胶质细胞胞体皱缩,延展性减低,细胞伪足减少,细胞体萎缩;转录组测序结果显示,焦亡通路NLRP3、Caspase-1及炎性因子IL-1β、IL-18 mRNA表达相对增加(P<0.05);NRF2、OH-1 mRNA表达明显降低(P<0.05)。与糖氧剥夺(OGD)干预组相比,法舒地尔(Fasudil)干预后星形胶质细胞延展性增强,细胞伪足增多;转录组测序结果显示,焦亡通路NLRP3、Caspase-1及炎性因子IL-1β、IL-18 mRNA表达降低(P<0.05);NRF2、OH-1 mRNA表达明显升高(P<0.05);所有数据以mean±SEM形式显示,两组比较选用Student’s t-test;多组比较选用one-way ANOVA,多组组内比较选用LSD post-hoc test;P<0.05代表有统计学意义。结论本实验证实,糖氧剥夺(OGD)能够破坏星形胶质细胞形态,抑制NRF2/HO-1信号通路表达;促进焦亡通路及炎性因子表达。法舒地尔(Fasudil)能够改善糖氧剥夺(OGD)干预后星形胶质细胞形态;逆转糖氧剥夺(OGD)干预对星形胶质细胞焦亡通路、炎性反应及NRF2/HO-1通路相关基因NLRP3、Caspase-1、IL-1β、IL-18、NRF2、OH-1 mRNA表达的影响;进而发挥保护作用。

Objective To study the protective effect and mechanism of oxygen glucose deprivation(OGD)and ROCK pathway inhibition on astrocytes.Methods From June 2022 to December 2022,CTX TNA2 astrocytes line originate from rats were selected as study subjects,and were divided into control group A,glucose-oxygen deprivation group B and Fasudil group C.In group B,OGD interfered astrocytes in vitro model was construct;Fasudil,a ROCK pathway inhibitor,was used in group C.The effects of oxygen glucose deprivation and Fasudil on astrocytes morphology,pyroptosis pathway,inflammatory response and NRF2/HO-1 pathway mRNA expression were analyzed.All data were displayed in the form of mean±SEM,and Student's t-test was used for comparison between the two groups.One-way ANOVA was used for multi-group comparison,and LSD post-hoc test was used for intra-group comparison.P<0.05 indicated statistical significance.Results Compared with the control group,oxygen glucose deprivation(OGD)interfered astrocytes appeared cell body shrinkage,reduced ductility,reduced pseudopod and cell body atrophy.Transcriptome sequencing results showed that the mRNA expressions of NLRP3,Caspase-1 and inflammatory cytokines IL-1βand IL-18 in pyroptosis pathway were increased(P<0.05).The mRNA expressions of NRF2 and OH-1 were significantly decreased(P<0.05).Compared with OGD treatment group,Fasudil treatment enhanced astrocytes malleability and increased pseudopodia.Transcriptome sequencing results showed that the mRNA expressions of NLRP3,Caspase-1 and inflammatory cytokines IL-1βand IL-18 in pyroptosis pathway were decreased(P<0.05).The mRNA expressions of NRF2 and OH-1 were significantly increased(P<0.05).Conclusions In this study,oxygen glucose deprivation(OGD)can destroy the morphology of astrocytes and inhibit the expression of NRF2/HO-1 signaling pathway,promote the expression of pyroptosis pathway and inflammatory factors.Fasudil can improve the morphology of astrocytes after OGD intervention.The mechanism may related to reverse the impact of OGD intervention on pyroptosis pathway,inflammatory response and mRNA expression of NRF2/HO-1 pathway related genes NLRP3,Caspase-1,IL-1β,IL-18,NRF2 and OH-1 in astrocytes.