健肾颗粒对5/6肾切除大鼠的初步药效学研究

Preliminary study of the mechanism underlying how Jianshen granules ameliorate renal failure in 5/6 nephrectomy model rats

慢性肾功能衰竭(CRF)是一个世界性的公共卫生问题。目前,可供选择的治疗方法有限。健肾颗粒在空军特色中心临床应用多年,疗效显著。本文探讨健肾颗粒对慢性肾衰大鼠(chronic renal failure,CRF)的治疗作用。建立5/6肾切除慢性肾衰大鼠模型,尿毒清为阳性对照。尿毒清组给予3.12 mg/g/d混悬液灌胃,健肾颗粒低、中、高剂量组分别给予0.96、1.92、3.84 mg/g/d混悬液灌胃,模型组给予等体积的生理盐水,共给药8周。治疗期间,观察大鼠的行为状态、体质量变化;测定各组大鼠血清肌酐、尿素氮、24 h尿蛋白含量;光镜下观察肾组织病理变化;蛋白免疫迹法、qRT-PCR检测肾组织中TGF-β1表达量。建立过氧化氢(H_(2)O_(2))诱导的人类肾小管上皮细胞(Human kidney cells,HKC)氧化损伤细胞模型,给予健肾颗粒,试剂盒法检测细胞存活率,流式细胞术检测线粒体膜电位(mitochondrial membrane potential,MMP)、活性氧(reactive oxygen species,ROS)和Ca2+水平,蛋白免疫迹法、RT-PCR检测细胞TGF-β1表达量。健肾颗粒能降低CRF大鼠血清肌酐、尿素氮、24 h尿蛋白。肾组织病理学检查显示健肾颗粒对肾损伤有改善作用。健肾颗粒可显著降低CRF大鼠TGF-β1(P<0.05)水平。给予健肾颗粒处理后,HKC细胞活力和MMP水平升高,ROS和Ca2+水平明显降低。TGF-β1表达量下降。健肾颗粒对CRF大鼠肾功能具有保护作用,可缓解肾衰症状。

Chronic renal failure(CRF)is a worldwide public health concern.At present,there are limited treatment options available.Jianshen granules,a traditional Chinese herbal medicine,have been used clinically to treat CRF for many years in the Air Force Medical Center of PLA and have shown significant efficacy.In the present study,the effectiveness of Jianshen granules in treating CRF was investigated.A rat model of CRF was established by 5/6 nephrectomy.Rats in the sham model groups were given distilled water,while rats in Uremic Clearance Granule(UCG)group were given a gavage of 3.12 mg/g/d premixing solution.Low,medium,and high doses of Jianshen granule(JS-L,JS-M and JS-H)was given premixing solution once daily to rats with gavage of 0.96,1.92 and 3.84 mg/g/d with continuous administration for 8 weeks.During the administration period,the physiological state of the rats was observed,and the biochemical parameters were measured and the weight changes were recorded every week.The pathology of the kidney tissues were assessed using hematoxylin and eosin(H.E.),and Masson’s staining.In addition,Western blot and quantitate real-time polymerase chain reaction(qRT-PCR)were used to detect the expression of TGF-β1 in renal tissue.Human kidney cells(HKC cells)were used to investigate the effects of Jianshen Granules on apoptosis induced by hydrogen peroxide(H_(2)O_(2)),whereas cell viability was assessed by Cell Counting Kit 8(CCK 8)assay.The level of apoptosis,the mitochondrial membrane potential(MMP),and reactive oxygen species(ROS)and Ca2+levels were measured using a flow cytometry.Western blot and qRT-PCR were used to detect the expression of TGF-β1 in HKC cell.The results revealed that Jianshen granules could reduce the levels of serum creatinine,blood urea nitrogen,and 24 h urinary protein in CRF rats.Kidney histopathological examination showed that Jianshen granules had an ameliorative effect on renal injury.HKC cells were used to investigate the effects of Jianshen granules on apoptosis induced by H_(2)O_(2).ROS and Ca2+levels were decreased,whereas cell viability and the level of MMP were increased in Jianshen groups.Besides,Jianshen granules could reduce the level of TGF-β1.Jianshen granules can protect renal function and relieve the symptoms of renal failure in CRF rats.