State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang 550004, China; School of Pharmacy, Guizhou Medical University, Guiyang 550004, China.
Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of Education) and State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550004, China.
Ecotoxicol Environ Saf. 2024 Jan 15;270:115813. doi: 10.1016/j.ecoenv.2023.115813. Epub 2023 Dec 19.
To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.
为了研究杠柳乙醇提取物(P. forrestii)对改善氟化物摄入引起的肝损伤的影响,并探讨其潜在机制。首先,通过氟化物诱导人正常肝细胞系(L-02)构建了体外氟中毒细胞模型。通过 CCK-8 试剂盒评估细胞活力。使用乳酸脱氢酶(LDH)试剂盒测量细胞上清液中的 LDH 含量,使用丙二醛(MDA)试剂盒测量细胞内 MDA 水平。随后,建立氟中毒大鼠模型,使用 LDH 试剂盒测量细胞上清液中的 LDH 含量。使用适当的试剂盒检测 MDA、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)和细胞内活性氧(ROS)含量等各种参数。此外,通过 Annexin V-FITC/PI 细胞凋亡检测试剂盒测定细胞凋亡率。通过 Western blot 分析 B 细胞淋巴瘤-2(Bcl-2)、Bcl-2 相关 X 蛋白(Bax)、Caspase-3、Cleaved Caspase-3、Caspase-9 和 Cleaved Caspase-9 的蛋白表达水平。与模型组相比,杠柳乙醇提取物 D(Fr.D)增加了细胞活力(P < 0.01)并降低了 LDH 和 MDA 水平(P < 0.01)。在氟中毒大鼠的高剂量 Fr.D 治疗组中,血清 ALT、AST、LDH 和 MDA 水平显著降低(P < 0.01)。Fr.D 给药组的大鼠原代细胞结果显示,与氟化物组相比,细胞存活率显著提高(P < 0.01)。同样,与模型组相比,Fr.D 处理的原代大鼠细胞表现出更高的细胞活力(P < 0.05)和更低的凋亡率、LDH、MDA、SOD、GSH-Px、CAT 和 ROS 水平(P < 0.05)。Western blot 分析表明,与模型组相比,Fr.D 处理组提高了 Bcl-2/Bax 蛋白表达比值,并降低了 Caspase-3 和 Caspase-9 的激活水平(P < 0.01)。结果表明,杠柳 Fr.D 中的成分可能通过对抗氧化应激和细胞凋亡来缓解氟化物引起的肝损伤。