摘要
目的:探讨肾康注射液治疗慢性肾衰竭(chronic renal failure,CRF)的效果,及其对肾脏内质网应激和氧化应激的影响.方法:从50只SD大鼠中随机选取10只作为对照组,其余大鼠灌胃腺嘌呤(200 mg·kg-1)复制CRF模型.将造模成功的大鼠随机分为模型组、肾康注射液低剂量组(2 g·kg-1)、肾康注射液高剂量组(4 g·kg-1)及N-乙酰半胱氨酸(N-acetylcys-teine,NAC 组,100 mg·kg-1),腹腔注射相应药物进行治疗.HE染色观察肾脏病理形态,免疫组织化学法和Western blot法检测肾脏磷酸化蛋白激酶 R 样内质网激酶(phosphorylated protein kinase R-like endoplasmic reticulum kinase,p-PERK)、磷酸化真核起始因子-2α(phosphorylated eukaryotic initiation factor-2α,p-eIF2ct)、活化转录因子-4(activating transcription factor-4,ATF-4)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白(Collagen Ⅰ)表达水平.以叔丁基过氧化氢(tert-butyl hydroperoxide,TBHP)处理大鼠肾系膜细胞R5200,复制氧化应激损伤模型.将R5200细胞分为空白组、TBHP组(150 μmol·L-1 TBHP)、TBHP+NAC 组(150 μmol·L-1 TBHP+5 mmol·L-1 NAC)、TBHP+SK 组(150 μmol·L-1TBHP+50 mg·L-1肾康注射液含药血清),孵育24 h.采用DCFH-DA荧光探针检测细胞活性氧(reactive oxygen species,ROS)水平,JC-1染色检测线粒体膜电位(mitochondrial membrane potential,MMP)水平,免疫荧光法检测ATF-4表达水平,Western blot法检测p-PERK、p-eIF2α、ATF-4表达水平.结果:HE染色显示,对照组大鼠肾小管未见明显异常.模型组大鼠出现大量肾小管扩张,上皮细胞水样变性,肾小管内可见尿酸盐结晶和坏死细胞.药物组大鼠肾脏可见少量肾小管扩张、尿酸盐结晶及上皮细胞水样变性.免疫组化显示,与对照组比较,模型组大鼠肾脏p-PERK、p-eIF2α、ATF-4、α-SMA、Collagen Ⅰ表达水平增加(P＜0.01).与模型组比较,药物组大鼠肾脏p-PERK、p-eIF2α、ATF-4、α-SMA、Collagen Ⅰ表达水平减少(P＜0.01).Western blot显示,与对照组比较,模型组大鼠肾脏p-PERK、p-eIF2α表达水平增加(P＜0.01).与模型组比较,药物组大鼠肾脏p-PERK、p-eIF2α表达水平减少(P＜0.05).与空白组比较,TBHP组细胞ROS水平升高、MMP水平降低(P＜0.01).与 TBHP 组比较,TBHP+NAC 组、TBHP+SK 组细胞 ROS 水平降低、MMP 水平升高(P＜0.01).Western blot 显示,与空白组比较,TBHP组细胞p-PERK、p-eIF2α、ATF-4表达水平增加(P＜0.01).与TBHP组比较,TBHP+NAC组、TBHP+SK组细胞p-PERK、p-eIF2α、ATF-4表达水平减少(P＜0.05).结论:肾康注射液可有效缓解大鼠CRF,其机制可能与抑制肾脏内质网应激和氧化应激反应有关.
Abstract
Objective:To investigate the effect of Shenkang Injection in treatment of chronic renal failure(CRF)and its effect on renal endoplasmic reticulum stress and oxidative stress.Methods:Ten rats were randomly selected from 50 SD rats as the control group,and the rest were replicated the CRF model by gavage of adenine(200 mg·kg-1).The successful rats were randomly divided into the model group,the low-dose(2 g·kg-1),the high-dose(4 g·kg-1)and the N-acetylcysteine(NAC,100 mg·kg-1)groups,and were treated with intraperitoneal injections of the corresponding drugs.The pathological morphology of the kidneys was observed with HE stai-ning,and the renal phosphorylates were detected with immunohistochemistry and Western blotting.The pathological morphology of kid-ney was observed with HE staining,and immunohistochemistry and Western blot were used to detect phosphorylated protein kinase R-like endoplasmic reticulum kinase(p-PERK),phosphorylated eukaryotic initiation factor-2alpha(p-ERK),phosphorylated eu-karyotic initiation factor-2alpha(p-ERK),and phosphorylated eukaryotic initiation factor-2alpha(p-ERK).eukaryotic initiation factor-2α(p-eIF2α),activating transcription factor 4(ATF-4),α-smooth muscle actin(α-SMA),and collagen I expression levels.Rat renal mesangial cells R5200 were treated with tert-butyl hydroperoxide(TBHP)to replicate the oxidative stress injury model.R5200 cells were divided into blank group,TBHP group(150 μmol·L-1 TBHP),TBHP+NAC group(150 μmol·L-1 TBHP+5 mmol·L-1 NAC),and TBHP+SK group(150 μmol·L-1 TBHP+50 mg·L-1 nephrokine injection-containing serum)and incu-bated for 24 h.The cells were treated with tert-butyl hydroperoxide(TBHP),and the model of oxidative stress injury was reproduced.DCFH-DA fluorescent probe was used to detect the level of reactive oxygen species(ROS),JC-1 staining was used to detect the lev-el of mitochondrial membrane potential(MMP),immunofluorescence was used to detect the level of ATF-4,and the expression levels of p-PERK,p-eIF2α and ATF-4 were detected with immunofluorescence and Western blot.Results:with HE staining,it was showed that there was no obvious abnormalities in the renal tubules of rats in the control group.Rats in the model group showed many renal tu-bular dilatation,hydropic degeneration of epithelial cells,and urate crystals and necrotic cells were seen in the renal tubules.The kid-neys of rats in the drug group showed a small amount of renal tubular dilatation,urate crystals and hydropic degeneration of epithelial cells.Immunohistochemistry showed that the expression levels of p-PERK,p-eIF2α,ATF-4,α-SMA,and Collagen I were in-creased in the kidneys of the rats in the model group compared with the control group(P＜0.01).Compared with the model group,the expression levels of p-PERK,p-eIF2α,ATF-4,α-SMA,Collagen I in the kidney of rats in the drug group were decreased(P＜0.01).With Western blot,it is showed that the expression levels of p-PERK,p-eIF2α in the kidney of rats in the model group were increased compared with the control group(P＜0.01).Compared with the model group,the expression levels of p-PERK and p-eIF2α in the kidneys of rats in the drug group were decreased(P＜0.05).Compared with that of the blank group,the cellular ROS level was increased and MMP level was decreased in TBHP group(P＜0.01).Compared with that of the TBHP group,cellular ROS level was decreased and MMP level was increased in TBHP+NAC group and TBHP+SK group(P＜0.01).With Western blot,it was showed that cellular p-PERK,p-eIF2α,ATF-4 expression level was increased in TBHP group compared with blank group(P＜0.01).Compared with that of the TBHP group,cellular p-PERK,p-eIF2α,ATF-4 expression levels decreased in TBHP+NAC group and TBHP+SK group(P＜0.05).Conclusion:Shenkang Injection can effectively alleviate CRF in rats,and its mechanism may be related to the inhibition of renal endoplasmic reticulum stress and oxidative stress response.
维普
万方数据