多黏菌素B导致大鼠肾毒性的机制研究OA

Objective To investigate the mechanism of polymyxin B(PMB)-induced kidney injury in rats.Methods ① Cell experiment:Renal interstitial cell line normal rat kidney cell(NRK)-52E in logarithmic growth phase was divided into blank control group,different doses of PMB groups[low-dose(200 μmol/L),medium-dose(500 μmol/L),high-dose(1 000 μmol/L)group],PMB+overexpression negative control(PMB+oe-NC group),and PMB+overexpression-nuclear factor erythroid 2-related factor 2(Nrf2)group(PMB+oe-Nrf2 group)according to different treatments.The blank control group received no drug or transfection intervention;PMB groups were treated with corresponding concentrations of PMB for 48 h;PMB+oe-NC group and PMB+oe-Nrf2 group were transfected with negative control vector and Nrf2 overexpression vector for 24 hours,respectively,followed by intervention with 1 000 μmol/L PMB for additional 48 hours.Three replicate wells were set in each group,and the independent experiments were repeated 3 times.Cell counting kit-8(CCK-8)was used to detect cell proliferation activity,cell apoptosis was detected by flow cytometry,and 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe method was used to detect intracellular reactive oxygen species(ROS)levels;Western blotting was used to detect the protein expression levels of Nrf2,small ubiquitin-like modifier 1(SUMO1);co-immunoprecipitation was used to detect the protein expression level of SUMOylated Nrf2.② Animal experiment:forty male Sprague-Dawley(SD)rats were randomly divided into normal control group(intraperitoneal injection of 0.9%normal saline),PMB low-dose group(intraperitoneal injection of 9.3 mg/kg PMB,twice daily),PMB medium-dose group(intraperitoneal injection of 14.0 mg/kg PMB,twice daily),and PMB high-dose group(intraperitoneal injection of 18.7 mg/kg PMB,twice daily),with 10 rats in each group.Enzyme-linked immunosorbent assay(ELISA)was used to determine blood urea nitrogen(BUN)and serum creatinine(SCr)levels,and renal function index was calculated;hematoxylin-eosin(HE)staining was used to observe the pathological damage;Western blotting was used to detect the protein expression levels of Nrf2 and SUMO1 in rat kidney tissues.Results ① Cell experiment:compared with the blank control group,PMB inhibited NRK-52E cell viability,induced cell apoptosis,and increased ROS levels,and decreased the protein expression levels of Nrf2,SUMO1 and SUMO1-modified Nrf2 in a dose-dependent manner.The changes were more significant in the PMB high-dose group compared to the blank control group[cell viability(A value):0.35±0.03 vs.0.98±0.09,cell apoptosis rate(%):51.87±7.59 vs.8.52±1.08,ROS activity(fluorescence intensity):52.35±6.84 vs.17.37±2.14,Nrf2 protein expression(Nrf2/β-actin):0.32±0.04 vs.0.84±0.07,SUMO1 protein expression(SUMO1/β-actin):0.54±0.06 vs.1.33±0.12;SUMO1-modified Nrf2 protein expression(SUMO1-Nrf2/β-actin):0.93±0.11 vs.2.35±0.34;all P<0.05].Compared with the PMB+oe-NC group,the PMB+oe-Nrf2 group presented increased cell viability,decreased apoptosis rate and ROS activity[cell proliferation(A value):0.68±0.09 vs.0.34±0.07,apoptosis rate(%):18.34±5.22 vs.37.75±6.53,ROS activity(fluorescence intensity):38.44±6.31 vs.50.79±8.25;all P<0.05].② Animal experiment:compared with the normal control group,PMB significantly elevated BUN,SCr levels,and renal function index,and decreased the protein expression levels of Nrf2 and SUMO1 in rats.The changes were more significant in the PMB high-dose group compared to the control group[BUN(mmol/L):13.24±1.25 vs.7.89±1.02,SCr(μmol/L):797.37±121.11 vs.464.98±86.63,renal function index(g/100 g):1.87±0.15 vs.1.45±0.08,Nrf2 protein expression(Nrf2/β-actin):0.31±0.04 vs.0.83±0.06,SUMO1 protein expression(SUMO1/β-actin):0.59±0.06 vs.1.24±0.16;all P<0.05].HE staining results showed that PMB induced obvious pathological injury of rat kidney tissue.Conclusion PMB aggravates nephrotoxicity in rats by inhibiting SUMO1-modified Nrf2.