锌原卟啉-9腹腔注射对脑出血大鼠神经功能的改善作用及其机制

Effect of intraperitoneal injection of zinc protoporphyrin-Ⅸ on neurological function in rats with intracerebral hemorrhage

目的探讨锌原卟啉-9(ZPP-Ⅸ)腹腔注射对脑出血(ICH)大鼠神经功能的影响,并探讨其可能机制。方法95只SD大鼠随机分为4组,ZPP-Ⅸ组(30只)、ICH组(30只)、DMSO组(30只)大鼠于脑立体定位仪上缓慢注射自体股动脉血50μL至基底节区,以制备ICH模型;正常组(5只)大鼠于相同部位打孔进针,不注血。ZPP-Ⅸ组在制模后30 min内腹腔注射10 mg/kg ZPP-Ⅸ,DMSO组给予10 mg/kg的35%DMSO,正常组、ICH组均给予等量生理盐水。采用Gareia18分测评法评价大鼠神经功能,Western blotting法检测脑组织Nrf2、结合型Nrf2(bindingNrf2)、HO-1、NF-κB、TNF-α蛋白,RT-PCR法检测脑组织Nrf2、HO-1、NF-κB、TNF-αmRNA,免疫荧光双标法检测脑组织HO-1、Nrf2、NF-κB、TNF-α与CD11b(小胶质细胞标记物)共染的阳性细胞数。结果与正常组比较,ICH组、ZPP-Ⅸ组、DMSO组制模后6 h、12 h、24 h、48 h、72 h、7 d神经功能评分减少(P均<0. 05);与ICH组比较,ZPP-Ⅸ组12 h、24 h、48 h、72 h、7 d的神经功能评分减少(P均<0. 05)。与正常组比较,ICH组、DMSO组、ZPP-Ⅸ组各时间点HO-1、Nrf2、NF-κB、TNF-α蛋白表达增加,binding-Nrf2蛋白水平减少(P均<0. 05);与ICH组比较,ZPP-Ⅸ组12 h、24 h、48 h、72 h、7 d HO-1蛋白表达减少,binding-Nrf2、NF-κB、TNF-α表达增加(P均<0. 05)。与正常组比较,ICH组和DMSO组HO-1、Nrf2 mRNA表达在制模后各时间点均增加,NF-κB、TNF-αmRNA表达于制模24 h、48 h、72 h、7 d增加(P均<0. 05);与ICH组比较,ZPP-Ⅸ组24 h、48 h、72 h、7 d的HO-1、Nrf2 mRNA表达减少,NF-κB、TNF-αmRNA表达在制模后各时间点均增加(P均<0. 05)。与ZPP-Ⅸ组比较,ICH组制模6 h、12 h、24 h、48 h、72h、7 d的HO-1与CD11b共表达阳性细胞增加,ICH组制模12 h、24 h、48 h、72 h、7 d Nrf2与CD11b共表达阳性细胞增加(P均<0. 05)。结论 ZPP-Ⅸ可能是通过抑制HO-1的表达,从而抑制Nrf2的解离、入核及增加下游炎症因子NF-κB、TNF-α的表达,来加剧ICH大鼠神经功能损伤的作用。

Objective To explore the influence and possibly mechanism of intraperitoneal injection of protoporphyrin zinc-Ⅸ(ZPP-Ⅸ)on the neurological function in rats with intracerebral hemorrhage(ICH).Methods Ninety-five rats were randomly divided into 4 groups:the normal group(n=5),ZPP-Ⅸgroup(n=30),ICH group(n=30),and DMSO group(n=30).In the ZPP-IX group,ICH group,and DMSO group,the rats were slowly injected with 50μL of autologous femoral artery blood to the basal ganglia on the brain stereotaxic apparatus to prepare the ICH model;the rats in the normal group were punched at the same position,without blood injection.In the ZPP-IX group,10 mg/kg ZPP-IX was intraperitoneally injected within 30 min after modeling.The rats in the DMSO group were given 10 mg/kg of 35%DMSO.The rats in the normal group and ICH group were given the same amount of normal saline.Garcia 18-test was used to evaluate the neurological function of rats.Western blotting was used to detect the protein expression of Nrf2,binding-Nrf2,HO-1,NF-κB,and TNF-α.RT-PCR was used to detect the mRNA expression of Nrf2,binding-Nrf2,HO-1,NF-κB,and TNF-α.Double immunofluoresence staining method was employed to observe the co-expression of HO-1,Nrf2,NF-κB,TNF-αand CD11b in the brain tissues.Results Compared with the normal group,the neurological function scores significant decreased in the ICH group,ZPP-Ⅸgroup,and ZPP group at 6,12,24,48,72 h and 7 d(all P<0.05).Compared with the ICH group,the neurological function scores of the ZPP-IX group decreased at 12,24,48,72 h,and 7 d(all P<0.05).The HO-1,Nrf2,NF-κB,and TNF-αlevels significantly increased at 6,12,24,48,72 h and 7 d,while the binding-Nrf2 level significantly decreased in the ICH group as compared with those of the normal group(all P<0.05).Compared with the ICH group,the expression of HO-1 protein decreased in the ZPP-IX group at 12,24,48,72 h and 7 d,and the expression of binding-Nrf2,NF-κB and TNF-αincreased(all P<0.05).Compared with the normal group,the expression of HO-1 and Nrf2 mRNA in the ICH group and DMSO group increased at each time point after modeling,and NF-κB and TNF-αmRNA expression significantly increased at 24,48,72 h and 7 d(all P<0.05).Compared with the ICH group,the expression of HO-1 and Nrf2 mRNA decreased in the ZPP-IX group at 24,48,72 h and 7 d,and the expression of NF-κB and TNF-αmRNA increased at each time point after modeling(all P<0.05).Compared with ZPP-IX group,the positive cells with HO-1 and CD11b co-expression increased in the ICH group at 6,12,24,48,72 h and 7 d,and the positive cells with co-expression of Nrf2 and CD11b increased in the ICH group at 12,24,48,72 h and 7 d(all P<0.05).Conclusion ZPP-Ⅸaggravates the neurological impairment of ICH rats by inhibiting the HO-1 expression,then inhibiting the Nrf2 dissociating and entering nucleus,and activating the NF-κB and TNF-αexpression.