蝉棒束胞C1菌丝体多糖抑制MAPK/NF-κB信号通路缓解LPS诱导的RAW264.7细胞炎症反应

The Anti-inflammatory Effects of Polysaccharide fromIsaria Cidada C1 Mycelial through the MAPK/NF-κB Signaling Pathways in RAW264.7 cell induced by LPS

摘要:

本文研究蝉棒束孢(Isaria cidada)C1菌丝体多糖ISP-80对脂多糖(LPS)诱导的小鼠单核巨噬细胞 RAW264.7炎症模型的抗炎效果,并探讨其作用机制。采用热水浸提法提取获得菌丝体多糖。以CCK-8法测定ISP-80 对 RAW264.7细胞的毒性作用,采用ELISA试剂盒测定TNF-α、IL-1β、IL-6细胞因子分泌量,采用Western Blot法检测 MAPK/NF-κB 信号通路中相关蛋白的含量。结果表明,ISP-80含有3种多糖组分,由葡萄糖、甘露糖和半乳糖3种单糖组成,摩尔比为6:27:8。与空白对照组相比,添加ISP-80多糖在0.01~1.0 mg/mL浓度范围内,未显示细胞毒性并能促进细胞增殖(P<0.0001)。与模型组相比,添加0.1~0.5 mg/mL 的ISP-80可明显下调 LPS 诱导的RAW264.7细胞释放炎症因子 TNF-α、IL-1β(P<0.001)和IL-6 (P<0.05),且可抑制细胞分化,恢复细胞形态。ISP-80的抗炎作用是通过MAPK和NF-κB途径介导的, ISP-80通过阻断p38、ERK和JNK的磷酸化来抑制MAPK途径,并且通过抑制IκB-α的磷酸化和降解以及阻断NF-κB p65的磷酸化来抑制NF-κB通路。因此,ISP-80可作为预防和治疗炎症性疾病的天然和安全的抗炎剂。

Abstract:

The objective of the current study is to examine the anti-inflammatory impact and mechanism of Isaria cidada C1 mycelial polysaccharide (ISP-80) in the inflammation model of lipopolysaccharide (LPS)-induced mouse monocyte-macrophage RAW264.7 cells. The polysaccharide (ISP-80) was obtained through hot water extraction from the Isaria cidada C1 mycelium. The toxic effects of ISP-80 on RAW264.7 cells were evaluated using CCK-8, and the impact of ISP-80 on the secretion of TNF-α, IL-1β, and IL-6 cytokines was measured using an ELISA kit. Furthermore, the mechanism of ISP-80 was determined using western blotting to detect the relevant protein content in the MAPK/NF-κB pathway. The findings indicated that ISP-80 was composed of three polysaccharide fractions, each containing Glc, Man, and Gal monosaccharides in a molar ratio of 6:27:8. The data demonstrated that ISP-80 polysaccharide displays no cytotoxic effects on RAW264.7 cells and promotes cell growth within the concentration range of 0.01~1.0 mg/mL (P<0.0001) when compared to the control group. Compared to the model group (LPS), the inclusion of 0.1-0.5 mg/mL of ISP-80 significantly reduced the expression of the inflammatory cytokines TNF-α, IL-1β (P<0.001) and IL-6 in RAW264.7 cells (P<0.05), while inhibiting cell differentiation and restoring cell morphology. The anti-inflammatory action of ISP-80 operated via the MAPK and NF-κB pathways. ISP-80 blocked the phosphorylation of p38, ERK, and JNK, inhibiting the MAPK pathway. Simultaneously, it inhibited the phosphorylation and degradation of IκB-α and blocked the phosphorylation of NF-κB p65, suppressing the NF-κB pathway. Therefore, ISP-80 has the potential to act as a natural and safe anti-inflammatory agent for the prevention and treatment of inflammatory diseases.