| ObjectiveTo reveal the mechanism of XST on prevention and treatment of CHF with diuretic resistance,we use human cardiomyocytes to evaluate the level of gene, protein, pathological morphology and function to investigate the effect of aqueous extract of XST on AQPs.MethodsHuman cardiomyocytes cultured in vitro and inducted with hypoxia/reoxygenation.Different concentration of aqueous extract of XST were used to intervene human cardiomyocytes.MTT assay was examined to determine cell viability.Flow cytometry and laser confocal were used to evaluate the change of mitochondrial membrane potential.Lysotracker Red were used to assess the level of Cell autophagy.Real-time PCR, immunocytochemistry and western blot were performed to investigate the mRNA and protein expression of AQP-1,4,7.Result1.MTT assay showed that XST-200 and XST-400 group significantly increased the cell viability of hypoxia/reoxygenated induced HCM, compared to the model group(P<0.05).2.The change of mitochondrial membrane potential by flow cytometry and laser confocal showed that mitochondrial membrane potential in model group was significantly decreased, compared to the control group(.P<0.05). Mitochondrial membrane potential in the XST-400 group was significantly increased, compared to the model group(P<0.05).3.Lysotracker red staining showed that the red fluorescence intensity of the model group was lower than the control group. The red fluorescence intensity in XST-400 were significantly increased, compared to the model group(P<0.05), respectively.4.Real-time PCR analysis showed that hypoxia/reoxygenation significantly up-regulated the expression of AQP-1,4 and 7 mRNA, respectively.The level of mRNA of AQP-1 in XST-200 group significantly decreased(P<0.05),and XST treatment dose-dependently increased the expression of AQP-1 mRNA.Compared to the model group,the level of mRNA of AQP-4 and 7 in XST-400 group significantly decreased(P<0.05) respectively.5.Immunocytochemistry showed that it can be detected the expression of AQP-1,4 and 7 protein on human cardiomyocyte membrane.The number of positive cell and integral light denesity in model group were more than control group. As to AQP-1, the number of positive cell and integral light denesity in XST-200 group were significantly decreased, compared to the model group(P<0.05).As to AQP-4 and 7, the number of positive cell and integral light denesity in XST-400 group was significantly decreased respectively, compared to the model group(P<0.05).6.Western blot analysis indicated that hypoxia/reoxygenation significantly up-regulated the expression of AQP-1,4 and 7, respectively.As to AQP-1,the expression of AQP-1 protein in XST-200 was significantly down-regulated,compared to the model group(P<0.05).As to AQP-4 and 7,the level of AQP-4 and 7 protein was significantly decreased(P<0.05), respectively.Conclusion1.XST showed a protective effect that can improve the tolerance of hypoxia/reoxygenated induced human cardiomyocytes.2.XST have protective effect on hypoxia/reoxygenated induced human cardiomyocytes by stabilizing the mitochondrial membrane potential and lysosome, alleviated mitochondrial oxidative stress injure and maintain the activity of lysosomal enzymes.3.the expression of AQP-1,4 and 7 mRNA and protein were up-regulated in human cardiomyocytes induced with hypoxia/reoxygenated.4.XST regulated the expression of AQP-1,4 and 7 mRNA and protein and stable mitochondrial membrane potential and lysosome to protect human cardiomyocytes induced with hypoxia/reoxygenated.It may be one of protective mechanism.5.The expression of AQP-1,4 and 7 mRNA and protein were different when different concentration of aqueous extract of XST intervented hypoxia/reoxygenated induced with human cardiomyocytes. XST treatment dose-dependently increased the expression of AQP-1 mRNA and protein.However,the expression of AQP-4 and 7 have no relation with dosage.lt may be due to the different of structure and function,but the mechanisms remain to be investigated. |
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