TRB3沉默对氧糖剥夺/再灌注诱导的大鼠脊髓星形胶质细胞损伤的影响

Effect of TRB3 silencing on spinal astrocytes injury induced by the oxygen-glucose deprivation/reoxygenation in rats

目的探讨Tribbles同源蛋白3(TRB3)对氧糖剥夺/再灌注(OGD/R)诱导的大鼠脊髓星形胶质细胞损伤的影响。方法分离培养新生SD大鼠的脊髓星形胶质细胞,设置对照组(不做任何处理)、OGD/R组(进行OGD/R处理),采用Real-time PCR和Western blotting分别检测TRB3 mRNA和蛋白的表达;设置对照组(不做任何处理)、Scramble组(感染阴性对照慢病毒)与shTRB3组(感染TRB3 shRNA慢病毒),采用Real-time PCR和Western blotting分别检测TRB3 mRNA和蛋白的表达,确定TRB3 shRNA慢病毒干扰效率;设置对照组(正常培养)、OGD/R组(进行OGD/R处理)、shTRB3组(感染TRB3 shRNA慢病毒)、Scramble组(感染阴性对照慢病毒)、OGD/R+shTRB3组(感染TRB3 shRNA慢病毒后,进行OGD/R处理)与OGD/R+Scramble组(感染阴性对照慢病毒后,进行OGD/R处理),采用流式细胞术检测细胞凋亡情况,CCK-8法检测细胞活力,乳酸脱氢酶(LDH)活性检测试剂盒测定LDH漏出率,硫代巴比妥酸法测定丙二醛(MDA)含量,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)活性,ELISA法检测肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)水平,Western blotting检测核因子κB(NF-κB)p65蛋白表达水平。使用NF-κB p65激动剂白桦脂酸(BA)20μmol/L处理OGD/R组和OGD/R+shTRB3组细胞,采用流式细胞术检测细胞凋亡情况。结果Real-time PCR和Western blotting检测结果显示,OGD/R处理后,脊髓星形胶质细胞中TRB3 mRNA(2.15±0.12 vs.1.00±0.05)和蛋白(2.10±0.16 vs.1.00±0.08)表达水平明显升高(P<0.05);与对照组比较,shTRB3组星形胶质细胞中TRB3 mRNA(0.30±0.07 vs.1.00±0.10)和蛋白(0.30±0.04 vs.1.00±0.06)表达水平明显降低(P<0.05),表明TRB3 shRNA慢病毒感染成功。与对照组比较,OGD/R组、OGD/R+Scramble组星形胶质细胞活性、SOD活性及细胞核NF-κB p65蛋白表达水平明显降低,LDH漏出率、MDA含量、TNF-α水平、IL-1β水平及细胞凋亡率明显增高(P<0.05);与OGD/R组和OGD/R+Scramble组比较,OGD/R+shTRB3组星形胶质细胞活性、SOD活性及细胞核NF-κB p65蛋白表达水平明显增高,LDH漏出率、MDA含量、TNF-α水平、IL-1β水平及细胞凋亡率明显降低(P<0.05)。与OGD/R组比较,BA处理能明显提高细胞凋亡率(36.15%±0.87%vs.24.70%±1.05%,P<0.05);与OGD/R+shTRB3组比较,BA处理能逆转TRB3 shRNA慢病毒感染对OGD/R诱导的细胞凋亡的抑制作用(22.00%±1.04%vs.13.91%±1.20%,P<0.05)。结论TRB3沉默可能通过阻断NF-κB通路而减轻OGD/R诱导的大鼠脊髓星形胶质细胞损伤。

Objective To explore the effect of tribbles homologue 3(TRB3)on spinal astrocytes injury induced by the oxygen-glucose deprivation/reoxygenation(OGD/R)in rats.Methods The spinal astrocytes of newborn SD rats were isolated and cultured.Cells were divided into control group(without treatment)and OGD/R group(cells were exposed to OGD/R).The mRNA and protein expression levels of TRB3 were detected by Real-time PCR and Western blotting.Cells were divided into control group,Scramble group(infected with scramble virus)and shTRB3 group(infected with TRB3 shRNA lentivirus),and the mRNA and protein expression levels of TRB3 were detected by Real-time PCR and Western blotting to confirmed knockdown efficiency.Cells were divided into control group,OGD/R group,Scramble group,shTRB3 group,OGD/R+Scramble group(cells were infected with scramble virus,and then treated with OGD/R)and OGD/R+shTRB3 group(cells were infected with TRB3 shRNA lentivirus,and then treated with OGD/R).The cell apoptosis,cell viability and lactic dehydrogenase(LDH)leakage rate were detected by flow cytometry,CCK-8 assay,and LDH detection kit,respectively.Malondialdehyde(MDA)content was detected by the TBA method and the activity of superoxide dismutase(SOD)was tested by the xanthine oxidase method.The concentrations of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were determined by ELISA.The protein expression of nuclear factor kappa-B(NF-κB)p65 was measured by Western blotting.Cells in OGD/R and OGD/R+shTRB3 were also treated with NF-κB p65 agonist betulinic acid(BA,20μmol/L),and cell apoptosis was detected by flow cytometry.Results Real-time PCR and Western blotting results showed that the mRNA(2.15±0.12 vs.1.00±0.05)and protein(2.10±0.16 vs.1.00±0.08)expression levels of TRB3 in spinal astrocytes were upregulated after OGD/R(P<0.05).Compared with control group,TRB3 shRNA lentivirus infection significantly decreased mRNA(0.30±0.07 vs.1.00±0.10)and protein(0.30±0.04 vs.1.00±0.06)expression levels of TRB3(P<0.05).Compared with control group,OGD/R group and OGD/R+Scramble group showed significantly decreased cell viability,SOD activity and nuclear NF-κB p65 level,and increased LDH,MDA,TNF-α,IL-1βlevels and cell apoptosis rate(P<0.05),indicated that TRB3 shRNA lentivirus infection is successful.Compared with OGD/R group and OGD/R+Scramble group,TRB3 silencing significantly inhibited OGD/R induced decreased cell viability,SOD activity and nuclear NF-κB p65 level(P<0.05).And the increase of LDH,MDA,TNF-α,IL-1βand apoptosis induced by OGD/R was significantly inhibited by TRB3 silencing(P<0.05).Compared with OGD/R group,NF-κB p65 activator BA treatment could significantly increase cell apoptosis rate(36.15%±0.87%vs.24.70%±1.05%,P<0.05),and it also could reverse the effect of TRB3 silencing on OGD/R induced cell apoptosis rate(22.00%±1.04%vs.13.91%±1.20%,P<0.05).Conclusion TRB3 silencing inhibits OGD/R-induced rat spinal astrocyte injury,which may be mediated by blocking the NF-κB pathway.