Expression Of AQP9, MCTs After Cerebral Ischemia And Their Possible Roles On Lactic Acid Transportation

Part I The expression of AQP9 and MCTs after acute cerebral ischemiaObjectiveTo investigate the expression of AQP9 and MCTs after acute cerebral ischemia, so as to explore the possible role of AQP9 and MCTs in cerebral ischemia.MethodsAdult healthy male Sprague-Dawley rats were randomly divided into two groups, the ischemic group and the sham operation control group. pMCAO(Permanent middle cerebral artery occlusion) models were established by occluding unilateral middle cerebral artery (MCA) of the rats with the suture method. The operation procedure of the control group was as same as the ischemic group but without occluding unilateral MCA. The brains of the animals were taken out at interval times of 1 h、3h、6h、12h and 24h, respectively. Subsequently, TTC staining to evaluate infarct volume, Brain water content (BWC) was measured by using wet-dry weighing method. The expression of AQPs in the edema brain tissue was detected with immunofluorence (IFC) and western blotting(WB), respectively.ResultsCompared with the sham operation group, the cerebral infarction and BWC was significantly increased, and gradually increased with the extension of ischemic time in the ischemic group. IFC and WB results showed that, the expression of AQP9 and MCTs after cerebral ischemia were stronger increased, and gradually increased along with the ischemic time extension. The expression of AQP9 was correlated with BWC after pMCAO (P＜0.05).ConclusionsThe results showed that AQP9 expression correlated strongly with brain oedema, suggesting that AQP9 contributes to oedema formation after pMCAO. Upregulation of AQP9 and MCTs expression after pMCAO in both astrocytes and neurons suggested that AQP9 may be collaborate with MCTs and involved in mediating lactate shuttle between these two cell types.PartⅡ The expression and function of AQP9 and MCTs in astrocytes after hypoxia-ischemiaObjectiveTo investigate the expression of AQP9 and MCTs in astrocytes after hypoxia-ischemia, so as to explore the role of AQP9 and MCTs in hypoxia-ischemia cell injury.MethodsPrimary cortical astrocytes were obtained from the brains of new-born(0 to 2 days) rats. Immunofluorescent staining of GFAP was used to identify astrocytes. One part of astrocytes was randomly divided into two groups, control group and hypoxia-ischemia group. The hypoxia-ischemia group was intervened for 1 h,3 h,6 h respectively. The others were randomly divided into control group, low-dose group(4-CIN:150umol/L), moderate-dose group(4-CIN:425umol/L), and high-dose group(4-CIT: 900umol/L) for drug intervention. Subsequently, HE staining was used to observe the morphology of each groups, LD kit was performed to detect lactic acid of culture medium, immunofluorescence and western blotting were used to investigate the expression of AQP9 and MCTs.ResultsCompared with control group, it was observed that morphological changes, nucleus enlargement and partially coagulation necrosis in hypoxia-ischemia group. LD consentration was increased, which was positive with hypoxia-ischemia time (P＜0.05). The expression of AQP9 and MCT1,4 in hypoxia-ischemia group were higher than that in control group detected by immunofluoresence and western blotting (P＜0.05). The concentration of LD in drug intervention groups was increased. At the same time, AQP9 protein was increased in the low-, moderate-and high-dose groups (P<0.05), while MCT1,4 protein were decreased compared with control group..ConclusionsExtracellular lactate concentration has correlative variation trend with expression of AQP9 and MCTs in astrocytes following hypoxia-ischemia treatment, suggested that deregulation of intercellular lactic acid transport is probable relative to the impairment of astrocytes after hypoxia-ischemia. When MCTs were suppressed, extracellular lactic acid was increased while expression of AQP9 was upregulated, suggesting that both MCTs and AQP9 may participate in the transport of lactic acid in astrocytes after hypoxia-ischemia treatment.PartⅢ Expression of AQP9 and monocarboxylate transporter 1,4 in U87 glioma cells with hypoxia-ischemiaObjectiveTo investigate the expression patterns of AQP9 and monocarboxylate transporter 1,4 in U87 malignant glioma cells with hypoxia-ischemia, so as to explore the possible effect of AQP9 and MCT1,4 on lactic acid transporting in human glioma cells.MethodsU87 cells were routinely cultured and randomly divided into control group and hypoxia-ischemia group. Hypoxia-ischemia group was treated for 1,3 and 6 hours. Cell survival rate was tested by MTT. The expressions of AQP9 and MCT1,4 in hypoxia-ischemia group and control group were detected by immunofluorescence and western blotting.ResultsCompared with control group, morphology of U87 was changed after hypoxia-ischemia, survival rate of cells were decreased and negatively related with hypoxia-ischemia time(P<0.05). Immunofluorescence and western blotting showed that AQP9 and MCT1,4 were increased in the hypoxia-ischemia group compared with control group and increasing with the extension of hypoxia-ischemia time(P<0.05).ConclusionsThe expressions of AQP9 and MCT1,4 were increased in U87 glioma cells with hypoxia-ischemia compared to the control group, suggesting AQP9 and MCT1,4 may be involved in lactate transport of malignant glioma microenvironment, and related to its proliferation and survival.