| Background:Neurovascular unit is consisted of neurons, glial cells and vascular endothelial cells, which emphasizes its importance to maintain the function of brain. Gap junctions can transfer information among cells, interchange metabolin, play an important role in neuronal excitability and regulation of homeostasis. Connexin 43(Cx43) is one of the most important menbers in connexin family, which is expressed highest in nervous system. The reason of cerebral infarction is that occlusion of cerebral blood flow interrupt the energy supply, destroy brain tissues and lead to vascular dysfunction. In recent years, some study has reported that Cx43 has been found in the mitochondria of astrocyte and also in the rat heart. However, the function of it durning ischemic-reperfusion(IR) is still unkown. As the significant role of mitochondria in IR inury, we prefer to explore the role of Cx43 in mitochondria and provide a novel target in clinical treatment. Object:To observe the regulation of the quantity and quality of mitochondrial Cx43 under cerebral IR injury, analyze the mechanism and evaluate the function of SA.. Methods:In vitro, based on the oxygen glucose deprivation(OGD) model, cultured astrocytes were divided into six groups: Sham group, OGD group, inhibitor of gap junctionCarbenoxolone(CBX) group, mito KATP channel agonist-Diazoxide(DZX) group, DZX+ mito KATP channel antagonist- 5-hydroxydecanoic acid(5-HD) group, and Salvianolic acid(SA) group. At first, the relative survival rate of astrocyte was detected in each group after OGD injury. Besides, cell apoptosis was observed by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining under electron microscope and the ultrastructure of mitochondria by transmission electron microscope(TEM). Furthermore, mitochondrial membrane potential(MMP) was tested via Rhodamine 123(Rh123) staining by flow cytometry. At last, we observed the distribution and quantity of Cx43 by immunofluorescence.In vivo, Wistar rats of 250~280 g, were divided into eight goups: Sham group, IR group, CBX group, DZX group, 5-HD + DZX group, protein kinase C(PKC) inhibitor-Ro-31-8425 + DZX group, PKC agonist-Phorbol-12-myristate-13-acetate(PMA) + 5-HD group and SA group. After the middle cerebral artery occlusion(MCAO) injury, brain infarction area was detected by TTC staining and the ethology was scored by Longa’s standard. Besides, cell apoptosis was observed by TUNEL staining under electron microscope. To evaluated the morphology and function of mitochondria, we used TEM to observe and determined superoxide dismutase(SOD) activity and malondialdehyde(MDA) content. Futhermore, expression of Cx43, p- Cx43, PKC and p-PKC were detected by western blot. Results:1、Morphology of tissue and cell: compared to the model group, cerebral infarction area in CBX, DZX and SA groups were obviously reduced and the rate of cell apoptosis was down-regulated(P<0.01). Meanwhile, 5-HD reduced the protection of DZX in neurovascular unit.2、Neurology deficit score: compared to sham group, the score in other groups were higher. After MCAO injury, limb was weaken on the oppsite of the ischemic brain and scores had no significant differences among groups(P>0.05).3、The morphology and function of mitochondria: the mitochondial morphology exerted different levels of damage in model groupand intervention groups compared to the sham group(P<0.05). Model group and DZX+5-HD group were evidently damaged. The damage in mitochondial morphology was mainly as mitochondrial swelling, spine fracture and transparent of substrate; similarly, decreases in SOD activity and increases in MDA of IR group were obseverd compared to the sham group(P<0.05), while the intervetion group such as CBX, DZX, PMA and SA promoted the SOD activity and reduced MDA to some degree(P<0.05), exerting protective effect on mitochondrial function.4、Distribution and quantity of Cx43: the total Cx43 protein expression in astrocytes decreased in OGD group compared to sham group, while the intracellular Cx43 protein expression increased and co-locolization of Cx43 and mitochondria were observed by immunofluorescence; the Cx43 expression and p-Cx43 levels in brain reduced significantly compared to sham group(P<0.01), similarly, p-Cx43/Cx43 ratio was also decreased compared to the sham(P<0.01), but the abnormalities were evidently ameliorated by interference of CBX, DZX, PMA and SA(P<0.05). Conclusion:Mitochondrial Cx43 could protect neurovascular unit from acute cerebral IR injury via PKC activation induced by mito KATP channel agonist, and it is also involved in the protective effects of SA. |
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