基因沉默诱导型环氧化酶协同高压氧对脑出血大鼠神经功能修复及细胞凋亡、自噬的影响

Effects of gene silencing Cyclooxygenase-2 cooperated with hyperbaric oxygen on neurological repair and apoptosis, autophagy in cerebral hemorrhage rats

目的探讨基因沉默诱导型环氧化酶(cyclooxygenase-2,COX-2)联合高压氧(hyperbaric oxygen,HBO)对脑出血大鼠神经细胞水肿、凋亡、自噬及神经功能修复影响。方法SPF级成年雄性SD大鼠(n=96),立体定向下于尾状核注射凝血酶Ⅶ构建脑出血模型。采用随机数字法分为4组(n=24/组):对照组、HBO组、COX-2 RNAi组及联合组。构建靶向沉默COX-2基因表达的siRNA质粒。大鼠分组治疗后,每组再随机抽取4只大鼠用于每类项目检测。术后第1、7、14天采用神经功能缺损评分(modified neurological severity score,mNSS评分)评估。术后第7天,干/湿比重法检测脑组织含水量;Evans法检测血脑屏障通透性;Annexin V法及TUNEL法检测神经细胞凋亡率;RT-PCR法检测脑组织COX-2的mRNA表达水平;免疫蛋白印迹法(western blot,WB)检测自噬相关蛋白Beclin-1、COX-2、水通道蛋白4(aquaporin 4,AQP-4)、B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia,Bcl-2)、半胱天冬酶-3(Caspase-3)、缺氧诱导因子-1α(hypoxia inducible factor-1,HIF-1α)、基质金属蛋白-2/9(matrix metalloproteinase-2/9,MMP-2/9)表达水平。采用SPSS软件对组间多样本行单因素方差分析,进一步两两比较采用LSD-t检验。结果SD大鼠脑出血模型、质粒构建均获成功。术后第7、14天,与对照组比较,COX-2 RNAi、HBO、联合组mNss评分均明显降低(均P<0.01),以联合组效果最优(P<0.01)。术后第7天,与对照组比较,各治疗组脑组织含水量、血脑屏障通透性均明显降低(均P<0.05),以联合组降低最明显(P<0.01)。神经细胞凋亡率各治疗组与对照组相比均有所下降(均P<0.05),以联合组下降最明显(P<0.01)。COX-2 mRNA表达水平各治疗组均较对照组明显降低(均P<0.01),以联合组沉默COX-2表达最明显(P<0.05)。WB结果显示,与对照组比较,各治疗组均能降低Beclin-1、COX-2、AQP-4、Caspase-3、HIF-1α、MMP-2/9蛋白表达(均P<0.05),而增加Bcl-2表达(均P<0.01),其中以联合组趋势最明显(P<0.01)。结论基因沉默COX-2协同高压氧可有效修复脑出血大鼠神经功能,其机制可能与降低血脑屏障通透性,减轻脑水肿,抑制神经细胞凋亡、自噬有关。

Objective To investigate the effects of gene silencing inducible cyclooxygenase-2(COX-2)combined with hyperbaric oxygen(HBO)on neuronal cell edema,apoptosis,autophagy and neural functional recovery in rats with intracerebral hemorrhage.Methods SPF-grade adult male SD rats(n=96)were used to establish a cerebral hemorrhage model through stereotactic injection of thrombin VII into the caudate nucleus.They were randomized(random number)into 4 groups(n=24/group):control group,hyperbaric oxygen(HBO)group,COX-2 RNAi group and combined group(COX-2 RNAi+HBO).The siRNA plasmid targeting silencing COX-2 gene expression was constructed.After group treatment,the four rats were randomly selected from each group for testing in each category.Postoperative day 1,7,and 14 were assessed using the modified neurological severity score(mNSS)for evaluating neurofunctional deficits.On the 7th day,the water content of the brain tissue was measured using the dry/wet weight method.The blood-brain barrier permeability was assessed using the Evans method.Annexin V and TUNEL assays were employed to assess the apoptotic rate of neural cells.The mRNA expression level of COX-2 in brain tissue was determined using the RT-PCR method.The protein expression levels of Beclin-1,COX-2,aquaporin 4(AQP-4),B cell lymphoma/lewkmia-2(Bcl-2),caspase-3,hypoxia-inducible factor-1α(HIF-1α)and matrix metalloprotein-2/9(MMP-2/9)were detected by Western blot(WB).SPSS software was used for data analysis.One-way ANOVA was used for inter group comparisons and LSD-t test was used for further pairwise comparison.Results The SD rat intracerebral hemorrhage model and plasmid construction were successfully achieved.The mNSS scores were significantly decreased in COX-2 RNAi,HBO and combined groups compared with control group on the 7th day and 14th day(all P<0.01),especially in combined group(P<0.01).The contents of Evans blue and the water content of brain tissue of all treatment groups were significantly lower than those in control group(all P<0.05),especially in combined group(P<0.01).The apoptotic rate of neural cells decreased in all treatment groups compared with the control group(all P<0.05),and the combined group decreased the most(P<0.01).The mRNA expression levels of COX-2 were significantly decreased in all treatment groups compared with the control group(all P<0.01),and combined group silenced COX-2 expression most obviously(P<0.05).The results of WB showed that the protein expression levels of Beclin-1,COX-2,AQP-4,Caspase-3,HIF-1α,MMP-2/9 were significantly lower than control group(all P<0.05),while the expression of Bcl-2 was increased in all treatment groups(all P<0.01).Among them,the combined group exhibited the most pronounced trend(P<0.01).Conclusions Gene silencing of COX-2 in combination with hyperbaric oxygen therapy can effectively restore neurological function in rats with cerebral hemorrhage.The mechanism may be associated with reduced blood-brain barrier permeability,alleviated brain edema,and inhibition of neuronal apoptosis and autophagy.