缝隙连接在脑缺血后处理中的作用及可能机制
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摘要
脑缺血性疾病的病死率仅次于心血管病和癌症，位列第三。脑缺血性疾病的治疗原则是尽早恢复血液再灌注，但恢复血液再灌注可能导致缺血/再灌注损伤。缺血/再灌注损伤的机制主要涉及到氧化应激、自由基损伤、兴奋性氨基酸毒性、炎症损伤、钙超载及细胞凋亡等机制。寻找有效的抗缺血/再灌注损伤的措施，一直是研究的热点。目前实验研究主要集中于缺血/再灌注损伤内在保护机制的探讨和抗脑缺血/再灌注损伤有效药物的发现。
对缺血/再灌注损伤内在保护机制的研究结果发现，缺血预适应及缺血后处理对缺血/再灌注损伤具有保护作用。后处理在实际应用方面具有先天优势，因而倍受关注。研究发现，抑制缝隙连接功能对脑缺血/再灌注损伤具有保护作用，表明缝隙连接可能参与脑缺血/再灌注损伤，而缝隙连接是否参与脑缺血后处理的保护作用及其可能机制还未见文献报道。
研究目的：
研究大鼠局灶性脑缺血后处理中缝隙连接的作用及其可能机制。
研究方法：
1．采用大鼠大脑中动脉栓塞法（middle cerebral artery occlusion, MCAO)制备大鼠局灶性脑缺血/再灌注损伤的动物模型（缺血2h/再灌注24h）。成年雄性SD大鼠随机分为5组，即假手术组，缺血/再灌注组，缺血后处理组，缺血/再灌注+甘珀酸（缝隙连接抑制剂）组，缺血后处理+甘珀酸组。缝隙连接阻断剂甘珀酸按2mg/ml/kg的剂量于再灌注前1分钟尾静脉注射给药，其它组分别注射相应剂量的生理盐水。研究中，对大鼠的神经功能障碍情况进行评分，计算脑梗死体积百分比，进行脑组织病理检查； Western blotting法检测大鼠皮层组织Cx43蛋白、Src激酶及凋亡相关蛋白Bax、Bcl-2的表达。
2．利用原代培养的星形胶质细胞，制备缺氧/复氧损伤细胞模型(缺氧8h/再灌注24h)。研究中，将细胞分为正常对照组、缺氧/复氧组、缺氧后处理组、缺氧后处理+维甲酸（缝隙连接增强剂，终浓度为10μM）组、缺氧后处理+油酸酰胺（缝隙连接抑制剂，终浓度为25μM）组。MTT法检测各组细胞的存活率；Hoechst33258染色和AnnexinV/PI双染流式细胞术检测细胞凋亡情况；细胞接种荧光示踪法测定培养细胞间的GJ功能；细胞免疫荧光法检测细胞膜Cx43蛋白表达； Westernblotting法检测星形胶质细胞Cx43蛋白、Src激酶蛋白、Bax蛋白、Bcl-2蛋白的表达。
研究结果：
1.制备大鼠脑缺血/再灌注损伤模型，观察脑缺血后处理的保护作用结果显示，与假手术组相比，缺血/再灌注组大鼠出现明显的神经功能障碍，出现细胞坏死、核固缩，甚至细胞融解消失及血管充血等脑组织病理学改变以及脑组织梗死；与缺血/再灌注组相比，缺血后处理可明显降低脑缺血/再灌注损伤引起的大鼠神经功能障碍的评分，减轻细胞坏死、核固缩及血管充血等病理学改变，降低脑梗死体积百分比。结果提示，大鼠局灶性脑缺血/再灌注损伤动物模型制备是成功的，脑缺血后处理对脑缺血/再灌注损伤具有保护作用。Western blotting检测Cx43总蛋白的表达结果发现，与假手术组相比，缺血再灌组Cx43总蛋白的表达明显增加（P <0.05）；与缺血/再灌注组相比，缺血后处理组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05），结果提示，缺血后处理对脑缺血再灌注损伤的保护作用可能与缝隙连接的抑制有关。
为探讨缝隙连接在脑缺血后处理中的作用，研究中使用了缝隙连接抑制剂甘珀酸。结果发现，与缺血/再灌注组相比，缺血/再灌注+甘珀酸组大鼠神经功能障碍评分及脑梗死百分率明显降低（P <0.05），脑组织病理学改变明显减轻；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠神经功能障碍评分及脑梗死百分率明显降低（P <0.05），脑组织病理学改变得以明显改善。Cx43总蛋白表达的检测结果发现，与缺血/再灌注组相比，缺血/再灌注+甘珀酸组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05）；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05）。结果说明，缝隙连接抑制剂可增强脑缺血后处理的保护作用。
为进一步明确缺血后处理抑制缝隙连接功产生保护作用的机理，进一步检测了Src激酶的表达水平及凋亡相关蛋白Bax/Bcl-2的表达情况。
大鼠脑组织Src激酶表达情况的检测结果显示，与假手术组相比，缺血/再灌注组大鼠脑组织Src激酶表达水平明显升高（P <0.01）；与缺血/再灌注组相比，缺血后处理组大鼠脑组织Src激酶的表达水平明显降低（P <0.01）；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠脑组织Src激酶的表达出现明显增减弱（P <0.01）。凋亡相关蛋白Bcl-2及Bax表达的检测结果显示，与假手术组相比，缺血再灌组Bax与Bcl-2的比值升高（P <0.01）；与缺血/再灌注组相比，缺血后处理组其比值明显降低（P <0.01）。
2．制备星形胶质细胞缺氧/复氧损伤模型，观察缺氧后处理对缺氧/复氧损伤保护作用的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞存活率明显降低（P <0.01）而细胞凋亡率则明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞存活率明增加（P <0.01）而细胞凋亡率则明显降低（P <0.01）结果说明，星形胶质细胞缺氧/复氧损伤模型制备是成功的，且缺氧后处理对星形胶质细胞缺氧/复氧损伤具有保护作用。
为明确缺氧后处理的保护作用是否与缝隙连接的功能有关，在缺氧后处理的基础上合用了缝隙连接增强剂维甲酸及缝隙连接抑制剂油酸酰胺。
检测细胞存活率及细胞凋亡情况的结果显示，与缺氧/复氧后处理组相比，缺氧后处理+维甲酸组星形胶质细胞存活率明显降低（P <0.01）而细胞凋亡率则明显升高（P <0.01），而缺氧后处理+油酸酰胺组则出现星形胶质细胞存活率明显增加（P <0.01）而细胞凋亡率则明显降低（P <0.01）。
细胞缝隙连接通讯功能检测的结果显示，与正常对照组相比，缺氧/复氧损伤组星形胶质细胞的缝隙连接通讯功能明显增强（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞缝隙连接通讯功能明显减弱（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞缝隙连接通讯功能分别出现明显增强（P <0.01）和减弱（P <0.01）。
星形胶质细胞Cx43膜蛋白免疫荧光检测的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞膜表面荧光强度增强；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞膜表面荧光强度减弱；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞膜表面荧光强度出现明显增强和减弱。
星形胶质细胞Cx43总蛋白表达检测的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Cx43总蛋白的表达明显增加（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Cx43总蛋白的表达明显降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Cx43总蛋白的表达分别出现明显增强（P <0.05）和减弱（P <0.05）。
结果说明，缺氧后处理对星形胶质细胞缺氧复氧损伤的保护作用与抑制缝隙连接的功能有关，增强缝隙连接功能可降低缺氧后处理的保护作用，而抑制缝隙连接功能则可增强缺氧后处理的保护作用。
3．为了进一步明确后处理（缺血后处理和缺氧后处理）中缝隙连接功能降低，产生保护作用的机理，进一步检测了缝隙连接蛋白磷酸化相关蛋白Src激酶的表达水平及凋亡相关蛋白Bax/Bcl-2的表达情况。
星形胶质细胞Src激酶表达情况的检测结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Src激酶表达水平明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Src激酶的表达水平明显降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Src激酶的表达出现明显增强（P <0.05）和减弱（P <0.05）。
凋亡相关蛋白Bax/Bcl-2的比值结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Bax/Bcl-2的比值明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Bax/Bcl-2的比值降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Bax/Bcl-2的比值分别出现增强（P <0.05）和减弱（P <0.05）。
研究结论：
(1)脑缺血/再灌注损伤中出现缝隙连接蛋白Cx43表达增多，提示缺血/再灌注引起的脑损伤可能与缝隙连接功能增强有关。
(2)缺血后处理或缺氧后处理对脑缺血/再灌注损伤或星形胶质细胞缺氧/复氧损伤具有保护作用，且这种保护作用可能与缝隙连接功能抑制有关。
(3)脑缺血后处理中缝隙连接功能的抑制可能与src激酶的表达降低有关。
(4)脑缺血后处理中，缝隙连接功能抑制引起细胞凋亡的减少可能与Bax/Bcl-2比值降低有关。
Cerebral ischemia is the third cause of death worldwide following cardiovasculardiseases and cancer. Reperfusion has the potential to produce ischemia/reperfusioninjury, although rapid initiation of reperfusion is the most effective treatment for thedisease. The mechanisms of ischemia-reperfusion injury include oxidative stress, freeradical damage, excitatory amino acid toxicity, inflammation, calcium overload andapoptosis. Exploring effective measure to anti-ischemia-reperfusion injury has been aresearch focus. The experimental research focused on internal protective mechanisms ofischemia-reperfusion injury and the discovery of effective drugs for cerebralischemia/reperfusion injury.
Ischemic preconditioning and ischemic postconditioning are internal protectionmechanisms of ischemia-reperfusion injury. Postconditioning has inherent advantages in practical applications, thus attracting much attention. Studies have shown that gapjunction inhibiors have protective effect against cerebral ischemia/reperfusion.Whether gap junction is involved in cerebral ischemic postconditioning, and thepossible mechanism is still unclear.
Objective:
To explore the role and possible mechanisms of gap junctions in cerebral ischemicpostconditioning.
Method:
1. The middle cerebral artery occlusion rat models were established, and the middlecerebral artery was occluded for2hours following with24hours reperfusion. Adultmale SD rats were randomly divided into five groups, including sham group,ischemia-reperfusion group,‘ischemic postconditioning’ group,‘ischemia-reperfusion+carbenoxolone’ group and ‘ischemic postconditioning+carbenoxolone’ group.Carbenoxolone (a gap junction inhibitor) was administered intravenously in1minutebefore the initiation of reperfusion, the other groups were injected with proportionabledose of saline. Neurological deficit scores of different group were evaluated with5-point scoring method. TTC stain method was used to calculate the percentage ofinfarct volume, and H-E stain method was used to detect the pathology of brain tissue.Western blotting was used to detect the expressionCx43protein. Src kinases andapoptosis-related protein Bax/Bcl-2were also detected by Western blotting.
2. The primary cultured astrocytes went with hypoxia-reoxygenation injury (hypoxia8h/reoxygenation24h). Astrocytes of the study were divided into normal control group, hypoxia-reoxygenation group, hypoxic postconditioning group,‘hypoxicpostconditioning+retinoic acid’ group and ‘hypoxic postconditioning+oleamide’group. MTT assay was used to measure the survival fraction of astrocytes;Hoechst33258staining and Annexin V/PI double staining following with flowcytometry were applied to detect apoptosis of astrocytes; Parachute assay was used todetect the function of gap junction of astrocytes; Cx43membrane protein was observedby cellular immune fluorescence; Western blotting was used to detect the expression ofCx43protein, Src kinase protein, Bax protein and Bcl-2protein.
Results:
1. In order to observe the protective effect of iscehmic postconditioning,ischemia-reperfusion injury model of rats was preparaed. Compared with the shamgroup, rats in ischemia-reperfusion group had significant neurological dysfunction,brain tissue pathological changes and brain tissue infarction; compared withischemia-reperfusion group, the ischemic postconditioning can significantly reduce theneurological deficit scores score, reduce histopathological changes of necrosis andpyknosis of neurons and congestion of blood vessels, and reduce the percentage of theinfarct volume of brain tissue. The results suggest that the preparation of rat model oflocal cerebral ischemia and reperfusion injury is successful, and ischemicpostconditioning has the protective effect on cerebral ischemia-reperfusion injury.
Results of Cx43total protein showed that Cx43protein expression had differencesbetween the groups. Compared with sham group, Cx43protein expression in theischemia group was significantly increased (P <0.05); Compared withischemia-reperfusion group, the expression of Cx43protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.05). The results indicate that the potective effect of cerebral ischemic postconditioning may related with theinhibition of gap junctions.
In order to realize the role of gap junctions in cerebral ischemic postconditioning, gapjunction inhibitor carbenoxolone was used in the study. Compared withischemia-reperfusion group, neurological function disability scores and the percentageof cerebral infarction of rat in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.05), and brain tissue pathological change wassignificantly reduced; Results of Cx43total protein showed that Cx43proteinexpression had differences between the groups. Compared with the ischemicpostconditioning group, Cx43protein expression of rat brain in ‘ischemicpostconditioning+carbenoxolone’ group was significantly reduced (P <0.05). Theresults suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions, and gap junction inhibitors canenhance the protective effect of cerebral ischemic postconditioning.
In order to further clarify the mechanism of the protective effect of ischemicpostconditioning by inhibiting the gap junction, expression of Src kinase protein and theratio of Bax/Bcl-2were detected.
Compared with sham group, Src kinase protein expression in the ischemia-reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of Src kinase protein of rat brains in ischemic postconditioning groupwas significantly decreased (P <0.05); Compared with ischemic postconditioning group,expression of Src kinase protein in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.01).
Compared with sham group, ratio of Bax/Bcl-2protein in the ischemia/reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of ratio of Bax/Bcl-2protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.01).
2. In order to observe the protective effect of hypoxic postconditioning, hypoxiareoxygenation injury model of astrocytes was preparaed. Compared with the controlgroup, survival rate of astrocyte in hypoxia reoxygenation was significantly decreased(P <0.01), and the apoptosis rate was significantly increased (P <0.01); Compared withhypoxia reoxygenation group, survival of astrocytes in postconditioning group wasincreased (P <0.01), and apoptosis rate was significantly decreased (P <0.01). Theresults suggest that hypoxia reoxygenation model of astrocytes was preparaedsuccessfully, and hypoxic postconditioning has protective effect on hypoxiareoxygenation injury.
In order to realize the role of gap junctions in hypoxic postconditioning, gap junctionpotentiator (retinoid acid) and inhibitor (oleamide) were used in the study.
Compared with hypoxic postconditioning group, the survival fraction of astrocytes in‘hypoxic postconditioning+retinoic acid’ group was reduced (P <0.01), and theapoptosis rate of astrocytes was significantly increased (P <0.01). The survival fractionof astrocytes in ‘hypoxic postconditoning+oleamide’ group was increased (P <0.01),and the apoptosis rate of astrocytes was significantly decreased (P <0.01).
Compared with the control group, astrocyte gap junctional intercellular communicationin hypoxia/reoxygenation injury group was significantly increased (P <0.01);Compared with hypoxia/reoxygenation group, gap junctional intercellular communication of astrocytes in hypoxic postconditioning group was decreasedsignificantly (P <0.01); Compared with the hypoxic postconditioning group, gapjunctional intercellular communication of astrocytes in ‘hypoxic postconditioning+oleamide’ group was decreased (P <0.01), which in ‘hypoxic postconditioning+retinoid acid’ group was increased (P <0.01).
Compared with normal control group, Cx43membrane protein immunofluorescence ofastrocytes in the hypoxia/reoxygenation group was increased; Compared withhypoxia/reoxygenation group, fluorescence intensity of astrocytes in hypoxicpostconditioning group was decreased; Compared with hypoxic postconditioning group,fluorescence intensity of astrocytes in ‘hypoxic postconditioning+retinoid acid’ wasincreased, and which in ‘hypoxic postconditioning+oleamide’ group was decreased.Compared with normal control group, Cx43total protein of astrocyte in the hypoxiareoxygenation group was increased (P <0.01); Compared with hypoxia reoxygenationgroup, Cx43total protein in hypoxic postconditioning group was decreased(P <0.01);
Compared with hypoxic postconditioning group, Cx43total protein in ‘hypoxicpostconditioning+retinoid acid’ group increased significantly (P <0.05), which in‘hypoxic postconditioning+oleamide’ group decreased significantly (P <0.05).
The results suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions. Enhancement of gap junctiondecrease the protective effect of the cerebral ischemic postconditioning, and inhibitionof gap junction increase the protective effect.
3. In order to further clarify the mechanism of the protective effect of ischemia andhypoxic postconditioning by reducing the gap junction function processing, expression of Src kinase protein which related to phosphorylation of connexin and the ratio of Baxand Bcl-2which related to apoptosis were detected.
Compared with normal control group, Src kinase of astrocyte in the hypoxia/reoxygenation group was increased (P <0.01); Compared with hypoxia/reoxygenationgroup, expression of Src kinase in hypoxic postconditioning group was decreased (P <0.01); Compared with hypoxic postconditioning group, Src kinase of astrocyte in‘hypoxic postconditioning+retinoid acid’ group was significantyly increased (P <0.05), and which in ‘hypoxic postconditioning+oleamide’ group was significantylydecreased (P <0.05).
Compared with normal control group, ratio of Bax/Bcl-2protein of astrocytes inhypoxia/reoxygenation group was increased (P <0.01); Compared withhypoxia/reoxygenation group, ratio of Bax/Bcl-2protein of astrocytes in hypoxicpostconditioning group was decreased (P <0.01); Compared with hypoxicpostconditioning group, ratio of Bax/Bcl-2protein of astrocytes in ‘hypoxicpostconditioning+retinoid acid’ group was increased(P <0.05), and which in ‘hypoxicpostconditioning+oleamide’ group was decreased (P <0.05).
Conclusion:
(1) The expression of Cx43protein increased during cerebral ischemia-reperfusioninjury，which suggests that cerebral ischemia/reperfusion injury may related with thefunctional enhancement of gap junctions.
(2) The protective effect of cerebral ischemic postconditioning or hypoxicpostconditiong is probably related to the inhibition of gap junctions.
(3) Gap junction inhibition in the ischemic postconditioning is probably related to the reduced src kinase expression.
(4) Decreased apoptosis in ischemic postconditioning through gap junction inhibition isprobably related to the reduced ratio of Bax/Bcl-2.
对缺血/再灌注损伤内在保护机制的研究结果发现，缺血预适应及缺血后处理对缺血/再灌注损伤具有保护作用。后处理在实际应用方面具有先天优势，因而倍受关注。研究发现，抑制缝隙连接功能对脑缺血/再灌注损伤具有保护作用，表明缝隙连接可能参与脑缺血/再灌注损伤，而缝隙连接是否参与脑缺血后处理的保护作用及其可能机制还未见文献报道。
研究目的：
研究大鼠局灶性脑缺血后处理中缝隙连接的作用及其可能机制。
研究方法：
1．采用大鼠大脑中动脉栓塞法（middle cerebral artery occlusion, MCAO)制备大鼠局灶性脑缺血/再灌注损伤的动物模型（缺血2h/再灌注24h）。成年雄性SD大鼠随机分为5组，即假手术组，缺血/再灌注组，缺血后处理组，缺血/再灌注+甘珀酸（缝隙连接抑制剂）组，缺血后处理+甘珀酸组。缝隙连接阻断剂甘珀酸按2mg/ml/kg的剂量于再灌注前1分钟尾静脉注射给药，其它组分别注射相应剂量的生理盐水。研究中，对大鼠的神经功能障碍情况进行评分，计算脑梗死体积百分比，进行脑组织病理检查； Western blotting法检测大鼠皮层组织Cx43蛋白、Src激酶及凋亡相关蛋白Bax、Bcl-2的表达。
2．利用原代培养的星形胶质细胞，制备缺氧/复氧损伤细胞模型(缺氧8h/再灌注24h)。研究中，将细胞分为正常对照组、缺氧/复氧组、缺氧后处理组、缺氧后处理+维甲酸（缝隙连接增强剂，终浓度为10μM）组、缺氧后处理+油酸酰胺（缝隙连接抑制剂，终浓度为25μM）组。MTT法检测各组细胞的存活率；Hoechst33258染色和AnnexinV/PI双染流式细胞术检测细胞凋亡情况；细胞接种荧光示踪法测定培养细胞间的GJ功能；细胞免疫荧光法检测细胞膜Cx43蛋白表达； Westernblotting法检测星形胶质细胞Cx43蛋白、Src激酶蛋白、Bax蛋白、Bcl-2蛋白的表达。
研究结果：
1.制备大鼠脑缺血/再灌注损伤模型，观察脑缺血后处理的保护作用结果显示，与假手术组相比，缺血/再灌注组大鼠出现明显的神经功能障碍，出现细胞坏死、核固缩，甚至细胞融解消失及血管充血等脑组织病理学改变以及脑组织梗死；与缺血/再灌注组相比，缺血后处理可明显降低脑缺血/再灌注损伤引起的大鼠神经功能障碍的评分，减轻细胞坏死、核固缩及血管充血等病理学改变，降低脑梗死体积百分比。结果提示，大鼠局灶性脑缺血/再灌注损伤动物模型制备是成功的，脑缺血后处理对脑缺血/再灌注损伤具有保护作用。Western blotting检测Cx43总蛋白的表达结果发现，与假手术组相比，缺血再灌组Cx43总蛋白的表达明显增加（P <0.05）；与缺血/再灌注组相比，缺血后处理组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05），结果提示，缺血后处理对脑缺血再灌注损伤的保护作用可能与缝隙连接的抑制有关。
为探讨缝隙连接在脑缺血后处理中的作用，研究中使用了缝隙连接抑制剂甘珀酸。结果发现，与缺血/再灌注组相比，缺血/再灌注+甘珀酸组大鼠神经功能障碍评分及脑梗死百分率明显降低（P <0.05），脑组织病理学改变明显减轻；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠神经功能障碍评分及脑梗死百分率明显降低（P <0.05），脑组织病理学改变得以明显改善。Cx43总蛋白表达的检测结果发现，与缺血/再灌注组相比，缺血/再灌注+甘珀酸组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05）；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠脑组织的Cx43总蛋白的表达明显降低（P <0.05）。结果说明，缝隙连接抑制剂可增强脑缺血后处理的保护作用。
为进一步明确缺血后处理抑制缝隙连接功产生保护作用的机理，进一步检测了Src激酶的表达水平及凋亡相关蛋白Bax/Bcl-2的表达情况。
大鼠脑组织Src激酶表达情况的检测结果显示，与假手术组相比，缺血/再灌注组大鼠脑组织Src激酶表达水平明显升高（P <0.01）；与缺血/再灌注组相比，缺血后处理组大鼠脑组织Src激酶的表达水平明显降低（P <0.01）；与缺血后处理组相比，缺血后处理+甘珀酸组大鼠脑组织Src激酶的表达出现明显增减弱（P <0.01）。凋亡相关蛋白Bcl-2及Bax表达的检测结果显示，与假手术组相比，缺血再灌组Bax与Bcl-2的比值升高（P <0.01）；与缺血/再灌注组相比，缺血后处理组其比值明显降低（P <0.01）。
2．制备星形胶质细胞缺氧/复氧损伤模型，观察缺氧后处理对缺氧/复氧损伤保护作用的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞存活率明显降低（P <0.01）而细胞凋亡率则明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞存活率明增加（P <0.01）而细胞凋亡率则明显降低（P <0.01）结果说明，星形胶质细胞缺氧/复氧损伤模型制备是成功的，且缺氧后处理对星形胶质细胞缺氧/复氧损伤具有保护作用。
为明确缺氧后处理的保护作用是否与缝隙连接的功能有关，在缺氧后处理的基础上合用了缝隙连接增强剂维甲酸及缝隙连接抑制剂油酸酰胺。
检测细胞存活率及细胞凋亡情况的结果显示，与缺氧/复氧后处理组相比，缺氧后处理+维甲酸组星形胶质细胞存活率明显降低（P <0.01）而细胞凋亡率则明显升高（P <0.01），而缺氧后处理+油酸酰胺组则出现星形胶质细胞存活率明显增加（P <0.01）而细胞凋亡率则明显降低（P <0.01）。
细胞缝隙连接通讯功能检测的结果显示，与正常对照组相比，缺氧/复氧损伤组星形胶质细胞的缝隙连接通讯功能明显增强（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞缝隙连接通讯功能明显减弱（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞缝隙连接通讯功能分别出现明显增强（P <0.01）和减弱（P <0.01）。
星形胶质细胞Cx43膜蛋白免疫荧光检测的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞膜表面荧光强度增强；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞膜表面荧光强度减弱；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞膜表面荧光强度出现明显增强和减弱。
星形胶质细胞Cx43总蛋白表达检测的结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Cx43总蛋白的表达明显增加（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Cx43总蛋白的表达明显降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Cx43总蛋白的表达分别出现明显增强（P <0.05）和减弱（P <0.05）。
结果说明，缺氧后处理对星形胶质细胞缺氧复氧损伤的保护作用与抑制缝隙连接的功能有关，增强缝隙连接功能可降低缺氧后处理的保护作用，而抑制缝隙连接功能则可增强缺氧后处理的保护作用。
3．为了进一步明确后处理（缺血后处理和缺氧后处理）中缝隙连接功能降低，产生保护作用的机理，进一步检测了缝隙连接蛋白磷酸化相关蛋白Src激酶的表达水平及凋亡相关蛋白Bax/Bcl-2的表达情况。
星形胶质细胞Src激酶表达情况的检测结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Src激酶表达水平明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Src激酶的表达水平明显降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Src激酶的表达出现明显增强（P <0.05）和减弱（P <0.05）。
凋亡相关蛋白Bax/Bcl-2的比值结果显示，与正常对照组相比，缺氧/复氧组星形胶质细胞Bax/Bcl-2的比值明显升高（P <0.01）；与缺氧/复氧组相比，缺氧后处理组星形胶质细胞Bax/Bcl-2的比值降低（P <0.01）；与缺氧后处理组相比，缺氧后处理+维甲酸组和缺氧后处理+油酸酰胺组星形胶质细胞Bax/Bcl-2的比值分别出现增强（P <0.05）和减弱（P <0.05）。
研究结论：
(1)脑缺血/再灌注损伤中出现缝隙连接蛋白Cx43表达增多，提示缺血/再灌注引起的脑损伤可能与缝隙连接功能增强有关。
(2)缺血后处理或缺氧后处理对脑缺血/再灌注损伤或星形胶质细胞缺氧/复氧损伤具有保护作用，且这种保护作用可能与缝隙连接功能抑制有关。
(3)脑缺血后处理中缝隙连接功能的抑制可能与src激酶的表达降低有关。
(4)脑缺血后处理中，缝隙连接功能抑制引起细胞凋亡的减少可能与Bax/Bcl-2比值降低有关。
Cerebral ischemia is the third cause of death worldwide following cardiovasculardiseases and cancer. Reperfusion has the potential to produce ischemia/reperfusioninjury, although rapid initiation of reperfusion is the most effective treatment for thedisease. The mechanisms of ischemia-reperfusion injury include oxidative stress, freeradical damage, excitatory amino acid toxicity, inflammation, calcium overload andapoptosis. Exploring effective measure to anti-ischemia-reperfusion injury has been aresearch focus. The experimental research focused on internal protective mechanisms ofischemia-reperfusion injury and the discovery of effective drugs for cerebralischemia/reperfusion injury.
Ischemic preconditioning and ischemic postconditioning are internal protectionmechanisms of ischemia-reperfusion injury. Postconditioning has inherent advantages in practical applications, thus attracting much attention. Studies have shown that gapjunction inhibiors have protective effect against cerebral ischemia/reperfusion.Whether gap junction is involved in cerebral ischemic postconditioning, and thepossible mechanism is still unclear.
Objective:
To explore the role and possible mechanisms of gap junctions in cerebral ischemicpostconditioning.
Method:
1. The middle cerebral artery occlusion rat models were established, and the middlecerebral artery was occluded for2hours following with24hours reperfusion. Adultmale SD rats were randomly divided into five groups, including sham group,ischemia-reperfusion group,‘ischemic postconditioning’ group,‘ischemia-reperfusion+carbenoxolone’ group and ‘ischemic postconditioning+carbenoxolone’ group.Carbenoxolone (a gap junction inhibitor) was administered intravenously in1minutebefore the initiation of reperfusion, the other groups were injected with proportionabledose of saline. Neurological deficit scores of different group were evaluated with5-point scoring method. TTC stain method was used to calculate the percentage ofinfarct volume, and H-E stain method was used to detect the pathology of brain tissue.Western blotting was used to detect the expressionCx43protein. Src kinases andapoptosis-related protein Bax/Bcl-2were also detected by Western blotting.
2. The primary cultured astrocytes went with hypoxia-reoxygenation injury (hypoxia8h/reoxygenation24h). Astrocytes of the study were divided into normal control group, hypoxia-reoxygenation group, hypoxic postconditioning group,‘hypoxicpostconditioning+retinoic acid’ group and ‘hypoxic postconditioning+oleamide’group. MTT assay was used to measure the survival fraction of astrocytes;Hoechst33258staining and Annexin V/PI double staining following with flowcytometry were applied to detect apoptosis of astrocytes; Parachute assay was used todetect the function of gap junction of astrocytes; Cx43membrane protein was observedby cellular immune fluorescence; Western blotting was used to detect the expression ofCx43protein, Src kinase protein, Bax protein and Bcl-2protein.
Results:
1. In order to observe the protective effect of iscehmic postconditioning,ischemia-reperfusion injury model of rats was preparaed. Compared with the shamgroup, rats in ischemia-reperfusion group had significant neurological dysfunction,brain tissue pathological changes and brain tissue infarction; compared withischemia-reperfusion group, the ischemic postconditioning can significantly reduce theneurological deficit scores score, reduce histopathological changes of necrosis andpyknosis of neurons and congestion of blood vessels, and reduce the percentage of theinfarct volume of brain tissue. The results suggest that the preparation of rat model oflocal cerebral ischemia and reperfusion injury is successful, and ischemicpostconditioning has the protective effect on cerebral ischemia-reperfusion injury.
Results of Cx43total protein showed that Cx43protein expression had differencesbetween the groups. Compared with sham group, Cx43protein expression in theischemia group was significantly increased (P <0.05); Compared withischemia-reperfusion group, the expression of Cx43protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.05). The results indicate that the potective effect of cerebral ischemic postconditioning may related with theinhibition of gap junctions.
In order to realize the role of gap junctions in cerebral ischemic postconditioning, gapjunction inhibitor carbenoxolone was used in the study. Compared withischemia-reperfusion group, neurological function disability scores and the percentageof cerebral infarction of rat in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.05), and brain tissue pathological change wassignificantly reduced; Results of Cx43total protein showed that Cx43proteinexpression had differences between the groups. Compared with the ischemicpostconditioning group, Cx43protein expression of rat brain in ‘ischemicpostconditioning+carbenoxolone’ group was significantly reduced (P <0.05). Theresults suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions, and gap junction inhibitors canenhance the protective effect of cerebral ischemic postconditioning.
In order to further clarify the mechanism of the protective effect of ischemicpostconditioning by inhibiting the gap junction, expression of Src kinase protein and theratio of Bax/Bcl-2were detected.
Compared with sham group, Src kinase protein expression in the ischemia-reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of Src kinase protein of rat brains in ischemic postconditioning groupwas significantly decreased (P <0.05); Compared with ischemic postconditioning group,expression of Src kinase protein in ‘ischemia-reperfusion+carbenoxolone’ group wassignificantly decreased (P <0.01).
Compared with sham group, ratio of Bax/Bcl-2protein in the ischemia/reperfusiongroup was significantly increased (P <0.01); Compared with ischemia-reperfusiongroup, expression of ratio of Bax/Bcl-2protein of rat brains in ischemicpostconditioning group was significantly decreased (P <0.01).
2. In order to observe the protective effect of hypoxic postconditioning, hypoxiareoxygenation injury model of astrocytes was preparaed. Compared with the controlgroup, survival rate of astrocyte in hypoxia reoxygenation was significantly decreased(P <0.01), and the apoptosis rate was significantly increased (P <0.01); Compared withhypoxia reoxygenation group, survival of astrocytes in postconditioning group wasincreased (P <0.01), and apoptosis rate was significantly decreased (P <0.01). Theresults suggest that hypoxia reoxygenation model of astrocytes was preparaedsuccessfully, and hypoxic postconditioning has protective effect on hypoxiareoxygenation injury.
In order to realize the role of gap junctions in hypoxic postconditioning, gap junctionpotentiator (retinoid acid) and inhibitor (oleamide) were used in the study.
Compared with hypoxic postconditioning group, the survival fraction of astrocytes in‘hypoxic postconditioning+retinoic acid’ group was reduced (P <0.01), and theapoptosis rate of astrocytes was significantly increased (P <0.01). The survival fractionof astrocytes in ‘hypoxic postconditoning+oleamide’ group was increased (P <0.01),and the apoptosis rate of astrocytes was significantly decreased (P <0.01).
Compared with the control group, astrocyte gap junctional intercellular communicationin hypoxia/reoxygenation injury group was significantly increased (P <0.01);Compared with hypoxia/reoxygenation group, gap junctional intercellular communication of astrocytes in hypoxic postconditioning group was decreasedsignificantly (P <0.01); Compared with the hypoxic postconditioning group, gapjunctional intercellular communication of astrocytes in ‘hypoxic postconditioning+oleamide’ group was decreased (P <0.01), which in ‘hypoxic postconditioning+retinoid acid’ group was increased (P <0.01).
Compared with normal control group, Cx43membrane protein immunofluorescence ofastrocytes in the hypoxia/reoxygenation group was increased; Compared withhypoxia/reoxygenation group, fluorescence intensity of astrocytes in hypoxicpostconditioning group was decreased; Compared with hypoxic postconditioning group,fluorescence intensity of astrocytes in ‘hypoxic postconditioning+retinoid acid’ wasincreased, and which in ‘hypoxic postconditioning+oleamide’ group was decreased.Compared with normal control group, Cx43total protein of astrocyte in the hypoxiareoxygenation group was increased (P <0.01); Compared with hypoxia reoxygenationgroup, Cx43total protein in hypoxic postconditioning group was decreased(P <0.01);
Compared with hypoxic postconditioning group, Cx43total protein in ‘hypoxicpostconditioning+retinoid acid’ group increased significantly (P <0.05), which in‘hypoxic postconditioning+oleamide’ group decreased significantly (P <0.05).
The results suggest that the protective effect of postconditioning in cerebral ischemia isrelated to the functional inhibition of gap junctions. Enhancement of gap junctiondecrease the protective effect of the cerebral ischemic postconditioning, and inhibitionof gap junction increase the protective effect.
3. In order to further clarify the mechanism of the protective effect of ischemia andhypoxic postconditioning by reducing the gap junction function processing, expression of Src kinase protein which related to phosphorylation of connexin and the ratio of Baxand Bcl-2which related to apoptosis were detected.
Compared with normal control group, Src kinase of astrocyte in the hypoxia/reoxygenation group was increased (P <0.01); Compared with hypoxia/reoxygenationgroup, expression of Src kinase in hypoxic postconditioning group was decreased (P <0.01); Compared with hypoxic postconditioning group, Src kinase of astrocyte in‘hypoxic postconditioning+retinoid acid’ group was significantyly increased (P <0.05), and which in ‘hypoxic postconditioning+oleamide’ group was significantylydecreased (P <0.05).
Compared with normal control group, ratio of Bax/Bcl-2protein of astrocytes inhypoxia/reoxygenation group was increased (P <0.01); Compared withhypoxia/reoxygenation group, ratio of Bax/Bcl-2protein of astrocytes in hypoxicpostconditioning group was decreased (P <0.01); Compared with hypoxicpostconditioning group, ratio of Bax/Bcl-2protein of astrocytes in ‘hypoxicpostconditioning+retinoid acid’ group was increased(P <0.05), and which in ‘hypoxicpostconditioning+oleamide’ group was decreased (P <0.05).
Conclusion:
(1) The expression of Cx43protein increased during cerebral ischemia-reperfusioninjury，which suggests that cerebral ischemia/reperfusion injury may related with thefunctional enhancement of gap junctions.
(2) The protective effect of cerebral ischemic postconditioning or hypoxicpostconditiong is probably related to the inhibition of gap junctions.
(3) Gap junction inhibition in the ischemic postconditioning is probably related to the reduced src kinase expression.
(4) Decreased apoptosis in ischemic postconditioning through gap junction inhibition isprobably related to the reduced ratio of Bax/Bcl-2.
引文
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