Study Of The Role And Mechanism Of Xin-Ji-Er-Kang On Nrf2/HO-1 Signaling Pathway In Kidney Injury

Background:Xin-Ji-Er-Kang?XJEK?is a Chinese herbal formula recorded and established as an effective preparation in the treatment of coronary heart disease,myocarditis and cardiovascular remodeling.This herb contains fourteen types of herbs,such as Panax ginseng C.A.Mey,Astragalus mongholicus Bunge,Polygonatum odoratum?Mill.?Druce,Ophiopogon japonicus?Thunb.?Ker Gawl.Cardiovascular diseases,such as coronary heart disease and myocardial infarction?MI?are currently considered as the leading causes of death and disability.In terms of health,heart and kidney both contribute to the wellbeing of the entire body.However,This interdependence of the two organs may lead to a vicious circle.Objectives:The aim of the present study is based on the nuclear factor erythroid2-related factor?Nrf2?/heme oxygenase-1?HO-1?pathway to investigate the protective effects of Xin-Ji-Er-Kang?XJEK?on kidney injury and renal oxidative stress.This experiment is divided into two parts.The first part is to explore the anti-oxidative stress ability of XJEK on renal injury in myocardial infarction rats.The second part explores that XJEK inhibits Ang II-induced oxidative stress in HK-2 cells and its mechanism is based on the Nrf2/HO-1 pathway.Methods:A total of 138 Sprague-Dawley rats were used in the present study.The control group was designated as 0 wk?n=8?.A total of 3 phases?2,4,6 wk?of administration were used in the sham-operated groups?sham,n=10?,MI groups?MI,n=10?,MI+XJEK groups?XJEK,n=10?and MI+fosinopril groups?fosinopril,n=10?.Additional 10 rats were used to evaluate the infarct area.At 2,4 or 6 wk post-MI,the hemodynamic parameters were monitored,the rats were sacrificed,then blood,heart and renal tissues were collected for furtherly analysis.Kidney weight/body weight?KW/BW?,cardiac function and renal morphological changes were evaluated.Commercial kits were used to determine malondialdehyde?MDA?concentration,total antioxidant capacity?T-AOC?,and superoxide dismutase?SOD?activity to assess plasma and renal tissue Oxidative stress levels.Enzyme-linked immunosorbent assay?ELISA?was used to detect 8-hydroxydeoxyguanosine?8-OHd G?and angiotensin II?Ang II?type 1 receptor in kidney tissue.Body?angiotensin II type 1 receptor,AT₁R?,NADPH oxidase-4?NADPH Oxidase-4,NOX-4?and aldosterone content,and the changes of Ang II content in the kidney and plasma were detected.Western blot?WB?method was used to detect nuclear translocation of Nrf2 and expression of HO-1,AT₁R and NOX-4 in renal cortex.In vitro,HK-2 cells were used.The first part of the experiment was the select of drug concentration and treatment time.Ang?induced pre-experimental group experiments.The cells were randomly divided into:1.blank control group 2.Ang?Induction group with different concentrations at the same treatment times 3.Ang?induction group with the same concentrations at different treatment times.CCK8 assay was used to detect cell viability;reactive oxygen species?ROS?detection kit was used to detect intracellular ROS level;western blot was used to detect cell Nrf2 nuclear translocation and HO-1 protein expression.The second part of the protective effect of XJEK on Ang?-induced HK-2 cell injury.According to the results of the first part,the cells were randomly divided into the following 6 groups:control group,Ang?group,XJEK group with different concentrations?Ang?+0.2mg/ml XJEK,Ang?+0.4mg/ml XJEK,Ang?+0.8 mg/ml XJEK,Ang II+1.6 mg/ml XJEK?.CCK8 experiment was used to detect cell viability;reactive active oxygen detection kit was used to detect intracellular active oxygen level;western blot was used to detect cell Nrf2 nuclear translocation and HO-1 protein expression.Results:Infarct areas and area-at-risk were measured by Evans blue and TTC staining and infarct area was normalized for area-at-risk?IA/AAR?.Statistical analysis indicated that the percentage of IA/AAR was 66.2±4.0%.Compared with the sham operation group,the hemodynamic parameters of the rats in the MI group increased significantly at the end of 2 and 4 wk of the experiment,and their values decreased significantly after 6 wk treatment.Compared with the MI group,XJEK administration can improve cardiac function and significantly inhibit abnormal changes in hemodynamic parameters.In addition,XJEK can significantly improve renal function by reducing KW/BW,reducing serum urea nitrogen?BUN?and serum cystatin C?Cys-C?content.XJEK administration for 2,4,and 6 wk can continuously reduce the levels of Ang II in the kidney tissues and plasam of myocardial infarction rats,reduce the levels of AT₁R and NOX4 in renal tissue,thereby inhibiting the activation of RAAS system.XJEK inhibits the oxidative stress?OS?caused by MI by increasing the activity of SOD and T-AOC,reducing the content of MDA and 8-OHd G.XJEK treatment for 2 wk potentiated Nrf2 nuclear translocation and HO-1 expression and slightly reduced nuclear Nrf2 and HO-1 at 4 wk post-MI compared with that of the MI groups,indicating the attenuation of the renal oxidative stress condition.In vitro,part I,compared with the blank control group,the damage effect of Ang?on HK-2 cells was concentration and time-dependent.With the increase of time,cell viability decreased and intracellular ROS content increased.Nrf2 nuclear translocation and expression of HO-1 protein increased gradually at 6 h,12 h,24 h,and 48 h of HK-2 cells treated with Ang?,and their values decreased significantly at the end of 72h of Ang?treatment.Part II,the protective effect of XJEK on Ang?induced HK-2cell injury.Compared with the Ang?group,XJEK can increase the viability of HK-2cells in a dose-dependent manner,reduce intracellular ROS content,and reduce intracellular Nrf2 nuclear translocation and HO-1 protein expression.Conclusion:These results demonstrated that progressive nephropathy in MI rats was associated with intrarenal activation of the renin-angiotensin-aldosterone system.Concomitantly,this process was associated with oxidative stress and impaired Nrf2activation.The improvement in the severity of nephropathy by XJEK in this model may be associated with the reversal of these abnormalities.The results of in vitro experiments demonstrated that the injury of HK-2 cells induced by Ang?was time and concentration dependent.The effect of XJEK on Ang?-induced HK-2 cell injury may be related to the Nrf2/HO-1 signaling pathway by reducing cell oxidative damage.