健肾颗粒对5/6肾切除大鼠的初步药效学研究

doi:10.5246/jcps.2023.04.027

摘要/Abstract

摘要：

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(H₂O₂)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。

关键词: 健肾颗粒, 5/6肾切除, 慢性肾衰竭, TGF-β1

Abstract:

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 (H₂O₂), 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 H₂O₂. 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.

Key words: Jianshen granules, 5/6 nephrectomy, Chronic renal failure, TGF-β1

Supporting:

邱海莹, 张梦迪, 贾海燕, 周梦露, 卢洪杰, 吴燕. 健肾颗粒对5/6肾切除大鼠的初步药效学研究[J]. 中国药学(英文版), 2023, 32(4): 302-316.

Haiying Qiu, Mengdi Zhang, Haiyan Jia, Menglu Zhou, Hongjie Lu, Yan Wu. Preliminary study of the mechanism underlying how Jianshen granules ameliorate renal failure in 5/6 nephrectomy model rats[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(4): 302-316.

图/表 10

Figure 1. Scr and BUN levels of the rats in sham and model groups prior to administration of the medicine. Each value is expressed as mean ± S.E.M (n = 6). *P < 0.05 compared with the model group. Scr, serum creatinine; BUN, blood urea nitrogen.

Figure 2. Effect of Jianshen granules on body weight in the different experimental groups. Each value is expressed as mean ± S.E.M (n = 8).

Figure 3. Effects of Jianshen granules on rats subjected to 5/6 Nx. The levels of the following biochemical parameters were investigated: (A) BUN; (B) Scr; (C) SOD; (D) Upr. Each value is expressed as mean ± S.E.M (n = 6). aP < 0.01, bP < 0.05, compared with Sham group; cP < 0.01, dP < 0.05, compared with model group; eP < 0.01, fP < 0.05, compared with UCG group. 5/6 Nx, 5/6 nephrectomy; BUN, blood urea nitrogen; SCr, serum creatinine; SOD, superoxide dismutase; UPr, urine protein.

Figure 4. Histopathological changes in renal tissue obtained from 5/6 nephrectomized rats. Shown are the results from (A) H&E staining; (B) Masson's staining (magnification, ×200). The black arrows represent tubular lesions, such as tubulointerstitial infiltration by inflammatory cells, tubular dilatation, tubular atrophy, and tubulointerstitial fibrosis; (C) Scores of renal lesion tissue. (a) Sham group; (b) Model group; (c) J–L group; (d) J–M group; (e) J–H group; (f) UCG group.

Figure 5. Effect of Jianshen granules on the concentration of TGF-β1 in the kidney tissues. (A) Western blotting analysis of TGF-β1; (B) mRNA expression of TGF-β1. Each value is expressed as mean ± S.E.M (n = 6). *P < 0.05; **P < 0.01 compared with the model group. TGF-β1, transforming growth factor-β1. The Figure of 6A was cropped. aP < 0.01, bP < 0.05, compared with Sham group; cP < 0.01, dP < 0.05, compared with model group; eP < 0.01, fP < 0.05, compared with UCG group.

Figure 6. Effects of Jianshen granules on the viability of HKC cells. Values are presented as mean ± S.E.M (n = 3). cP < 0.01, dP < 0.05, compared with H2O2 group; eP < 0.01, fP < 0.05, compared with UCG group.

Figure 7. Effects of Jianshen granules on H2O2-induced oxidative stress in HKC cells. Shown are the results of experiments that investigated (A) the extent of apoptosis; (B) level of ROS; (C) level of Ca2+; and (D) level of MMP. ROS, reactive oxygen species; MMP, mitochondrial membrane potential. aP < 0.01, bP < 0.05, compared with Control group; cP < 0.01, dP < 0.05, compared with H2O2 group.

Figure 8. Effect of Jianshen granules on the concentration of TGF-β1 in the H2O2-induced apoptosis of HKC cells. The results were investigated by (A) Western blotting analysis of TGF-β1 and (B) mRNA expression of TGF-β1. Each value was expressed as mean ± S.E.M (n = 3). aP < 0.01, bP < 0.05, compared with Control group; cP < 0.01, dP < 0.05, compared with H2O2 group.

Table 1. Primers of renal tissue.

Table 2. Primers of HKC cells.

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