College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
Phytomedicine. 2019 Feb;53:263-273. doi: 10.1016/j.phymed.2018.09.023. Epub 2018 Sep 3.
BACKGROUND: Sedum sarmentosum, which is recorded in Chinese Pharmacopoeia, has been applied clinically to treat liver and gallbladder diseases. PURPOSE: This study aimed to explore the hepatoprotective effect of S. sarmentosum less polar extract (SSE) against ANIT-induced liver injury in rats, and the protective activity and mechanism of one major constituent isolated from this extract on D-GalN-induced human hepatic QSG7701 cell damage. METHODS: Rats were divided into groups and then administrated intragastrically with SSE at doses of 100, 200 and 400 mg/kg for 7 days. They were modeled in the experiments with ANIT (70 mg/kg) to induce liver injury after the sixth day administration. The levels of serum biochemical markers ALT, AST, ALP, GGT/γ-GT, DBiL, TBiL, ALB, TP, and bile flow rate, as well as the histopathology of the liver tissue were used as indices of liver damage and measured. The inflammatory response and oxidative stress were thought to be key contributors to ANIT-induced liver injury in rats. Therefore, the inflammatory mediators (TNF-α, IFN-γ, IL-4) and oxidative stress (ROS, SOD, GSH-PX) were measured in the serum and liver homogenates, respectively. Next, phytochemical research was performed to produce the main component, and the isolated compound was evaluated for its hepatoprotective activity against QSG7701 cell injured by D-GalN through the measurement of cell viabilities, ALT, AST, IL-1β, TNF-α, IL-6, ROS, GSH-PX and SOD productions. Furthermore, the protein expression of the Nrf2 and NF-κB pathways were analyzed by western blotting. RESULTS: SSE had an obvious effect on the decreases of ALT, AST, ALP, GGT/γ-GT, DBiL and TBiL levels, the increases of ALB and TP levels in serum, and the ANIT-induced deceleration in bile flow for liver injury. Meanwhile, SSE pretreatment alleviated ANIT-induced liver pathological injuries exhibited by HE stain of the liver. Moreover, SSE significantly suppressed levels of pro-inflammatory cytokines TNF-α and IFN-γ, and elevated level of anti-inflammatory cytokine IL-4 in serum. SSE also attenuated oxidative stress by reducing ROS level and by enhancing antioxidative enzymes (SOD and GSH-PX) activities after ANIT administration in liver tissue. Further, the major compound shown in HPLC was isolated from SSE. Its structure was identified by the spectroscopic data analysis and comparison with literature values. The principal constituent had potent protective effect on D-GalN-induced QSG7701 cells damage in a dose dependent manner with survival rates of 58.2% and 69.5% at 10 μM and 20 μM, respectively. Its cytoprotective effect was associated with the reduction of ALT, AST, IL-1β, TNF-α, IL-6 and ROS levels, and the elevation of GSH-PX and SOD productions in QSG7701 cells induced by D-GalN. Western blotting showed that this compound enhanced the expression of Nrf2, HO1, NQO1 and GCLC, and inhibited D-GalN-induced IκBα and NF-κB p65 phosphorylation. CONCLUSIONS: Current study showed that SSE treatment exerted a protective effect on ANIT-induced liver injury. The main compound δ-amyrone isolated from the extract was characterized as the effective component with hepatoprotective activity by promoting Nrf2 antioxidant defense and suppressing NF-κB inflammatory response.
背景：贯叶连翘在中国药典中有记载，已被临床用于治疗肝胆疾病。 目的：本研究旨在探讨贯叶连翘非极性提取物（SSE）对 ANIT 诱导的大鼠肝损伤的保护作用，以及从该提取物中分离得到的一种主要成分对 D-GalN 诱导的人肝 QSG7701 细胞损伤的保护活性和机制。 方法：将大鼠分为几组，然后分别灌胃 SSE 剂量为 100、200 和 400mg/kg，连续 7 天。在第六天给药后，用 ANIT（70mg/kg）对它们进行建模，以诱导肝损伤。血清生化标志物 ALT、AST、ALP、GGT/γ-GT、DBiL、TBiL、ALB、TP 和胆汁流速以及肝组织病理学作为肝损伤的指标进行测量。炎症反应和氧化应激被认为是 ANIT 诱导大鼠肝损伤的关键因素。因此，在血清和肝匀浆中分别测量了炎症介质（TNF-α、IFN-γ、IL-4）和氧化应激（ROS、SOD、GSH-PX）。接下来，进行植物化学研究以产生主要成分，并通过测量 D-GalN 诱导的 QSG7701 细胞活力、ALT、AST、IL-1β、TNF-α、IL-6、ROS、GSH-PX 和 SOD 产生来评估分离得到的化合物对 D-GalN 诱导的 QSG7701 细胞损伤的保护活性。此外，通过 Western blot 分析了 Nrf2 和 NF-κB 通路的蛋白表达。 结果：SSE 对 ALT、AST、ALP、GGT/γ-GT、DBiL 和 TBiL 水平的降低、血清中 ALB 和 TP 水平的升高以及 ANIT 诱导的胆汁流速减慢有明显的作用，从而减轻肝损伤。同时，SSE 预处理减轻了 ANIT 诱导的肝病理损伤，通过肝组织的 HE 染色显示。此外，SSE 显著抑制了 TNF-α和 IFN-γ等促炎细胞因子的水平，并升高了抗炎细胞因子 IL-4 的水平。SSE 还通过降低 ROS 水平和增强抗氧化酶（SOD 和 GSH-PX）在肝组织中的活性，减轻了 ANIT 给药后的氧化应激。此外，HPLC 中显示的主要化合物从 SSE 中分离出来。通过光谱数据分析和与文献值的比较，确定了其结构。主要成分具有很强的保护作用，对 D-GalN 诱导的 QSG7701 细胞损伤具有剂量依赖性，在 10μM 和 20μM 时的存活率分别为 58.2%和 69.5%。其细胞保护作用与降低 ALT、AST、IL-1β、TNF-α、IL-6 和 ROS 水平以及升高 D-GalN 诱导的 QSG7701 细胞中的 GSH-PX 和 SOD 产生有关。Western blot 显示，该化合物增强了 Nrf2、HO1、NQO1 和 GCLC 的表达，并抑制了 D-GalN 诱导的 IκBα和 NF-κB p65 磷酸化。 结论：目前的研究表明，SSE 治疗对 ANIT 诱导的肝损伤有保护作用。从提取物中分离得到的主要成分δ-桉叶醇被鉴定为具有保护活性的有效成分，通过促进 Nrf2 抗氧化防御和抑制 NF-κB 炎症反应。
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