The Second Clinical Medical College, Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China.
The Second Clinical Medical College, Guangdong Provincial Key Laboratory of Chinese Medicine for Prevention and Treatment of Refractory Chronic Diseases, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China.
J Ethnopharmacol. 2020 Jul 15;257:112789. doi: 10.1016/j.jep.2020.112789. Epub 2020 Mar 29.
Tripterygium wilfordii Hook F. (TwHF), a traditional Chinese herb medicine, has been widely used for clinical treatment of various rheumatic immune diseases. Tripterygium glycosides (TG) extracted from TwHF has been verified to process multiple bioactivities, including immunosuppressive, anti-inflammatory and anti-cancer effects. However, the clinical application of TG is limited due to its severe toxicity and narrow therapeutic window. For the clinical safety of TG usage, attenuation of toxicity is the key issue to be solved.
Tripterygium glycoside fraction n2 (TG-n2) is a detoxified mixture obtained from TG using a new preparation method. In our previous study, we have demonstrated that TG-n2 has a lower toxicity than TG. The aim of the present study was to screen the renal protective effect of TG-n2 in nephrotic syndrome (NS) induced by adriamycin (ADR) in rats and its effect on apoptosis, as well as the effective difference between TG-n2 and TG.
The ADR-induced NS rat model was established. Rats were intravenously injected with ADR (6 mg/kg), then treated with either TG-n2 (10 mg/kg/day) or TG (10 mg/kg/day) by oral gavage for 4 weeks. Clinical indexes in each group were determined. HE staining and electron microscopic analysis were used to evaluate renal histopathological damage. Caspase-3 activity reagent and TUNEL staining were used to estimate renal apoptosis. Protein levels of caspase-3, caspase-9, caspase-8, caspase-12, Bax, Bcl-2, p53, TNF-R1, FLIP and podocin were measured by Western Blot.
TG-n2 and TG intervention ameliorated renal function as assessed by the levels of 24-h proteinuria, Cr, BUN, TC, TG, ALB and LDL-c. TG-n2 and TG alleviated the decrease of podocin protein expression and morphological injury of podocyte as screened by Western Blot and electron microscopic analysis. Besides, renal tubular injury was reduced as inspected by light microscopic analysis. TG-n2 and TG could significantly inhibit the apoptosis and activity of caspase-3 in kidney tissues as examined by fluorescence microscopic analysis and reagent. After intervention of TG-n2 and TG, protein levels of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, p53 and TNF-R1 in renal issues were significantly decreased compared with ADR group. In contrast, protein level of Bcl-2 was elevated remarkedly.
Our data suggested that attenuated TG-n2 may have a similar protective effect with TG in ADR-induced NS in rats by inhibiting activation of apoptosis.
ETHNOPHARMACOLOGICAL 相关性：雷公藤（TwHF）是一种传统的中草药，已广泛用于治疗各种风湿免疫性疾病。从 TwHF 中提取的雷公藤苷（TG）已被证明具有多种生物活性，包括免疫抑制、抗炎和抗癌作用。然而，由于其严重的毒性和狭窄的治疗窗，TG 的临床应用受到限制。为了保证 TG 使用的临床安全性，降低毒性是需要解决的关键问题。
雷公藤苷组分 n2（TG-n2）是一种用新的制备方法从 TG 中获得的解毒混合物。在我们之前的研究中，已经证明 TG-n2 的毒性低于 TG。本研究的目的是筛选 TG-n2 在阿霉素（ADR）诱导的大鼠肾病综合征（NS）中的肾保护作用及其对细胞凋亡的影响，并比较 TG-n2 和 TG 的有效差异。
建立 ADR 诱导的 NS 大鼠模型。大鼠静脉注射 ADR（6mg/kg），然后分别用 TG-n2（10mg/kg/天）或 TG（10mg/kg/天）灌胃 4 周。测定各组临床指标。HE 染色和电镜分析评估肾组织病理学损伤。用 caspase-3 活性试剂和 TUNEL 染色评估肾细胞凋亡。用 Western Blot 测定 caspase-3、caspase-9、caspase-8、caspase-12、Bax、Bcl-2、p53、TNF-R1、FLIP 和 podocin 的蛋白水平。
TG-n2 和 TG 干预改善了 24 小时蛋白尿、Cr、BUN、TC、TG、ALB 和 LDL-c 等肾功能指标。Western Blot 和电镜分析筛选出 TG-n2 和 TG 可减轻 podocin 蛋白表达降低和足细胞形态损伤。此外，光镜分析显示肾小管损伤减轻。荧光显微镜分析和试剂检测表明，TG-n2 和 TG 可显著抑制肾组织细胞凋亡和 caspase-3 活性。与 ADR 组相比，TG-n2 和 TG 干预后肾组织中 cleaved caspase-3、cleaved caspase-8、cleaved caspase-9、Bax、p53 和 TNF-R1 的蛋白水平显著降低，而 Bcl-2 蛋白水平显著升高。
我们的数据表明，减毒的 TG-n2 通过抑制细胞凋亡的激活，可能在 ADR 诱导的 NS 大鼠中与 TG 具有相似的保护作用。
