| Objective:To observe the renal protective effects of oxymatrine (OMT) for ischemia/reperfusion induced renal injury in vivo and vitro.Methods:Part 1:Cytotoxicity test:HK-2 cells were treated with different concentrations of OMT (0.1 mg/ml,0.5mg/ml, 1.0mg/ml,2mg/ml,4mg/ml and 8mg/ml). Detect the cell vitality with CCK-8 at 24h and 48h after the treatment, to find the suitable concentration for the efficacy experiment. Establishing cell model of RIRI:Culturing cells with sugar and serum-free DMEM in hypoxic box for 2h simulated ischemia, returning to normal incubator for 3h simulated reperfusion. Efficacy experiments to find the optimal concentration. The cells were divided into four groups:Control, OMT, IR and OMT+IR. OMT and OMT+IR groups were treated with OMT for 24h; OMT+IR and IR groups were made to RIRI model. The cell vitality and percentage of apoptotic cells were tested to assess the cytoprotective effects of OMT in RIRI. Test the expression of PERK/ATF4/CHOP signaling pathway related mRNA by qPCR.Part 2:32 ICR male mice were randomly divided into 4 groups:Sham, OMT, IR and OMT+IR. IR and OMT+IR group were induced to renal ischemia/reperfusion injury by clipping the left kidney vessel for 50 minutes; All groups’right kidney were cut off. OMT and OMT+IR groups were treated with OMT (120mg/kg, i. p. qd) just after the reperfusion and 4 days before the surgery. While, the Sham and IR groups were treated with NS. Blood serum and kidney tissue were collected 24 hours after reperfusion. Serum creatinine (Cr) and blood urea nitrogen (BUN) were detected to evaluate the renal function. Superoxide dismutase (SOD) and Malondialdehyde (MDA) were tested to evaluate oxidative stress in kidney. The scores of histopathology and apoptosis were used to evaluate the renal injury. Test the expression of PERK/ATF4/CHOP signaling pathway related mRNA by qPCR.Results:Part 1:Cytotoxicity test:1mg/ml and lower concentration of OMT treating HK-2 cells for 48h, showed no significant decrease in cell viability, comparing with the control group(P<0.001). Drug efficacy experiment:the model group (including IR group, OMT+IR group) cell activity was significantly lower than the control group (P<0.01), and it was higher in OMT+IR group than in IR group (P<0.05). Model group showed obvious apoptosis and necrosis under the microscope. Flow cytometry analysis showed that the apoptotic cells of model group were significantly increased comparing with the control group (P<0.001), and the percentage of apoptotic cells decreased in OMT+IR group comparing with IR group (P<0.05). Comparing to the control group, the expression of related mRNA in HK-2 cell were increased in model group (P<0.01). The rise in OMT+IR group was lower than in IR group (P<0.05). Part 2:The level of BUN、Cr and SOD activity all increased (P<0.05) while the level of MDA decreased significantly (P<0.05) in OMT+IR group comparing with the IR group. The scores of histopathology and apoptosis in OMT+IR group were lower than in IR group (P<0.01). Comparing to the sham group, the expression of related mRNA in kidney tissues were increased in model group (P<0.01). The rise in OMT+IR group was lower than in IR group (P<0.05).Conclusion:1. OMT has cytoprotective effects on hypoxia/reoxygenation injury in vitro, by improving the cell vitality and reducing the number of apoptotic cells.2. OMT has some protecting effects on RIRI in mice by against oxidative stress and inhibiting cell apoptosis.3. RIRI can increase the expression of related mRNA of PERK/ATF4/CHOP signaling pathway in vitro and vivo. OMT can decrease the rise. OMT may inhibit apoptosis, by affecting PERK/ATF4/CHOP signaling pathway, to reduce RIRI. |
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