Shen Yu-Li, Jiang Yi-Ping, Li Xiao-Qin, Wang Su-Juan, Ma Ming-Hua, Zhang Chun-Yan, Zhu Jian-Yong, Rahman Khalid, Zhang Li-Jun, Luan Xin, Zhang Hong
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Front Pharmacol. 2020 Jan 24;10:1596. doi: 10.3389/fphar.2019.01596. eCollection 2019.
Diabetic nephropathy (DN) is one of the main causes of renal fibrosis and is associated with high morbidity and mortality. Traditional Chinese Medicine (TCM) therapy has a long history of usage in a clinical setting and its usage is increasing. ErHuang Formula (EHF), a Chinese herbal compound, has been clinically used in treating DN for more than 30 years. However, its mechanism of action is still unknown. This study was conducted to evaluate the effect of EHF on renal fibrosis in a DN rat model and explore its underlying mechanism. The DN rat model was established by high-sugar-fat diet combined with a single intraperitoneal injection of streptozotocin (STZ), and EFH extract (4, 2, 1 g/kg d) was administered orally for 8 weeks. The biochemical parameters (blood glucose, weight, Scr, BUN, UA, U-Alb and UAE) were analyzed. The pathological changes in renal tissue were observed by histological staining with H&E and Masson. The effect of EHF on the proliferation of NRK-49F cells was examined by CCK-8 assay and the levels of several inflammation and fibrosis related cytokines (IL-6, TNF-α, TGF-β1, Collagen I/III, MMP2/9) in serum and NRK-49F cell culture supernatants were detected by enzyme-linked immunoassay (ELISA). The mRNA levels of CXCL6, CXCR1, Collagen I/III, MMP2/9 in renal tissue were also measured by quantitative RT-PCR. Furthermore, the protein expression of PCNA, Collagen I/III, MMP2/9, CXCL6, CXCR1, p-STAT3, STAT3 in renal tissue and NRK-49F cells were determined by western blot. EHF improved the abnormal biochemical parameters and ameliorated the abnormal histology and fibrosis of renal tissue in a dose-dependent manner. EHF inhibited NRK-49F proliferation and decreased the expressions of inflammation and fibrosis related factors both and . Interestingly, the levels of Collagen I/III, PCNA, MMP2/9 and p-STAT3 were positively correlated with CXCL6. The amelioration of renal fibrosis in DN by EHF is related to CXCL6/JAK/STAT3 signal pathway, which is associated with inflammation and fibrosis of the tissue. These findings may have clinical implications for the treatment of DN.
糖尿病肾病（DN）是肾纤维化的主要原因之一，与高发病率和高死亡率相关。中医治疗在临床应用中历史悠久，且其应用正在增加。二黄方（EHF）是一种中药复方，已在临床上用于治疗DN超过30年。然而，其作用机制仍不清楚。本研究旨在评估EHF对DN大鼠模型肾纤维化的影响，并探讨其潜在机制。通过高糖高脂饮食联合单次腹腔注射链脲佐菌素（STZ）建立DN大鼠模型，并口服给予EHF提取物（4、2、1 g/kg·d）8周。分析生化参数（血糖、体重、Scr、BUN、UA、U-Alb和UAE）。通过苏木精-伊红（H&E）和Masson组织学染色观察肾组织的病理变化。通过CCK-8法检测EHF对NRK-49F细胞增殖的影响，并通过酶联免疫吸附测定（ELISA）检测血清和NRK-49F细胞培养上清液中几种炎症和纤维化相关细胞因子（IL-6、TNF-α、TGF-β1、胶原蛋白I/III、MMP2/9）的水平。还通过定量逆转录-聚合酶链反应（RT-PCR）测量肾组织中CXCL6、CXCR1、胶原蛋白I/III、MMP2/9的mRNA水平。此外，通过蛋白质印迹法测定肾组织和NRK-49F细胞中PCNA、胶原蛋白I/III、MMP2/9、CXCL6、CXCR1、p-STAT3、STAT3的蛋白表达。EHF以剂量依赖的方式改善了异常的生化参数，减轻了肾组织的异常组织学和纤维化。EHF抑制NRK-49F增殖，并降低炎症和纤维化相关因子在体内和体外的表达。有趣的是，胶原蛋白I/III、PCNA、MMP2/9和p-STAT3的水平与CXCL6呈正相关。EHF改善DN肾纤维化与CXCL6/JAK/STAT3信号通路有关，该通路与组织的炎症和纤维化相关。这些发现可能对DN的治疗具有临床意义。
