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