红毛五加多糖对大鼠肝细胞免疫损伤的保护作用及其机制

Protective effects of polysaccharide AHP-Ⅱ on hepatocyte injury in rats and its mechanism

关键词:  红毛五加多糖, 脂多糖, 肝损伤, JNK

Key words:   Acanthopanax giraldii polysaccharide, lipopolysaccharide, liver injury, JNK

摘要:

红毛五加属于五加科植物,而五加科植物大多具有调节免疫力的双向调节作用,红毛五加多糖是从中药红毛五加中提取的单一组分,目前关于红毛五加多糖的抑炎作用还少有报道。为了探究红毛五加多糖AHP-Ⅱ对LPS导致的大鼠肝细胞免疫损伤的保护作用及其作用机制,该研究将实验分为空白组、模型组(LPS,40 μg·mL^-1)和AHP-Ⅱ低、中、高剂量组(25,50,100 μg·mL^-1)五组,以LPS(40 μg·mL^-1)来制备大鼠肝细胞免疫损伤模型,先用ELISA方法检测细胞分泌TNF-α的水平及流式检测ROS的含量,来探究AHP-Ⅱ不同剂量对炎症因子的抑制作用,再用western方法检测P-JNK2的蛋白水平来进一步探究AHP-Ⅱ的抑制作用机制。结果表明:低、中、高剂量组的AHP-Ⅱ均可使肝细胞损伤后的TNF-α含量下降。同时,AHP-Ⅱ中、高剂量组均可使损伤后的肝细胞ROS的分泌量下降,且AHP-Ⅱ高剂量组抑制ROS的作用最强。在加入AHP-Ⅱ后,JNK2蛋白的磷酸化水平呈剂量依赖性降低,AHP-Ⅱ高剂量组抑制作用最强。这说明红毛五加多糖AHP-Ⅱ可以通过降低P-JNK2的蛋白含量来抑制炎症因子TNF-α及ROS的水平,以发挥免疫保护作用。

Abstract:

Acanthopanax giraldii belongs to the Araliaceae plant, and most of the Araliaceae plants have a two-way regulation effect in regulating immunity. A. giraldii polysaccharide is a single component extracted from the traditional Chinese medicine A. giraldii. The anti-inflammatory effect of polysaccharides has rarely been reported. This study aimed to explore the protective effect and mechanism of polysaccharide AHP-Ⅱ on LPS-induced hepatocyte injury in rats. This experiment was divided into five groups: blank group, model group(LPS, 40 μg·mL^-1), AHP-Ⅱ low, moderate, high-dose group(25, 50, 100 μg·mL^-1). LPS(40 μg·mL^-1)was used to prepare the rat hepatocyte immune injury model. The cytotoxic effect of AHP-Ⅱ on hepatocytes was first examined by CCK-8 method to eliminate interference, and the secreted level of TNF-α by the cells and the level of ROS were measured by ELISA and flow cytometry respectively to establish the hepatocyte immune injury model. Then the inhibitory effect of different doses of AHP-Ⅱ on inflammatory factors was explored, and the level of JNK2 protein and its phosphorylation were detected by western method to further explore the inhibitory mechanism of AHP-Ⅱ. The results showed that the experimental dose of AHP-Ⅱ had no damage to cells by CCK-8 method, which indicates that the reduction of inflammatory factors secreted by cells treated by AHP-Ⅱ is not due to its cytotoxic effect. AHP-Ⅱ groups(25, 50, 100 μg·mL^-1)decreased TNF-α content durig hepatocyte injury. At the same time, the high-dose AHP-Ⅱ group can reduce the secretion of ROS durig hepatocytes injury, and the high-dose AHP-Ⅱ group has the strongest effect in inhibiting ehe conrent of ROS. After the addition of AHP-Ⅱ, the phosphorylation level of JNK2 protein decreased in a dose-dependent manner, and the AHP-Ⅱ high-dose group had the strongest inhibitory effect. This indicate that the polysaccharide AHP-Ⅱ can play an immunoprotective role by reducing the protein content of P-JNK2 and inhibiting the levels of TNF-α and ROS. The results of this study provide a scientific reference for the hepatoprotective effect of A. giraldii, and promote the further development and utilization of the resources of A. giraldii.