华泽兰根部乙酸乙酯萃取物基于NF-κB及NLR-P3炎症小体信号通路对NIH-3T3小鼠胚胎成纤维细胞功能的影响

Effects of Ethyl Acetate Extract of Huazelan(Eupatorium chinense)Root on Function of Embryonic Fibroblasts in NIH-3T3 Mice Based on NF-κB and NLR-P3 Inflammasome Signaling Pathways

目的探讨华泽兰乙酸乙酯萃取物对小鼠胚胎成纤维细胞(NIH-3T3)功能的影响。方法从炎症因子一氧化氮(NO)及氧化应激重要因素活性氧(ROS)介入,通过核因子κB(NF-κB)及核苷酸结合寡聚化结构域样受体蛋白3(NLR-P3)炎症小体等信号通路角度阐明其作用机制。体外培养NIH-3T3细胞,分为空白对照组(Control)、不同浓度华泽兰根部乙酸乙酯萃取物组,检测不同浓度下华泽兰根部乙酸乙酯萃取物对NIH-3T3细胞Toll增殖活性(MTT法)、细胞迁移(细胞划痕实验)、分泌NO及胞内ROS生成的影响,Western blot法检测其对Toll样受体4(TLR-4)、磷脂酰肌醇3激酶(PI3K)、NLR-P3、NF-κB、髓样分化因子(Myd88)、核因子抑制蛋白α(IκBα)、白细胞介素-18(IL-18)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、凋亡相关斑点样蛋白(ASC)、环氧化酶-2(COX-2)蛋白表达的影响。结果细胞增殖与细胞划痕实验表明,华泽兰根部乙酸乙酯萃取物对于NIH-3T3细胞的增殖与划痕愈合影响具有双向性。与对照组相比在低浓度下(<6.25μg·mL^(-1)),萃取物有促进NIH-3T3细胞增殖与迁移的作用,而高浓度(>25μg·mL^(-1))反而表现出对NIH-3T3细胞的增殖与迁移抑制作用,且不同阶段呈现出相应的剂量依赖性,与对照组相比有统计学意义(P<0.05)。在萃取物浓度为6.25μg·mL^(-1)时,NO生成量相较于低浓度组差异有显著统计学意义(P<0.01),而高浓度组(>12.5μg·mL^(-1))相较对照组差异无统计学意义(P>0.05)。细胞内ROS生成实验表明,经华泽兰根部乙酸乙酯萃取物处理后ROS生成量显著高于对照组(P<0.05,P<0.01)。Western blot实验表明,与对照组相比,萃取物作用浓度为6.25μg·mL^(-1)时,细胞内NF-κB、TLR-4、Myd88、NLR-P3、ASC表达显著升高(P<0.01),IκBα表达升高(P<0.05),IL-18表达显著降低(P<0.01),Caspase-3表达降低(P<0.05)。结论华泽兰乙酸乙酯萃取物对小鼠胚胎成纤维细胞(NIH-3T3)增殖的影响具有双向性,即低浓度促进细胞增殖而高浓度抑制细胞增殖。其作用机制可能与NF-κB及NLR-P3炎症小体信号通路和细胞凋亡有关。

Objective To investigate the effect of Huazelan(Eupatorium chinense)ethyl acetate extract on the function of mouse embryonic fibroblasts(NIH-3T3).Methods From the intervention of inflammatory factor nitric oxide(NO)and Reactive Oxygen Species(ROS),an important factor of oxidative stress,and through signaling pathways such as nuclear factor kappa B(NF-κB)and NOD-like receptor protein-3(NLR-P3)inflammasome,the mechanism of action was elucidated.NIH-3T3 cells were cultured in vitro and divided into blank control group(Control)and ethyl acetate extract group of Huazelan(Eupatorium chinense)roots with different concentrations.MTT(Thiazolyl blue)assay,cell migration(cell scratch assay),NO secretion and intracellular ROS generation were used to observe the effects of Huazelan(Eupatorium chinense).Western blot assay was used to detect the protein expressions of Toll-like receptor 4(TLR-4),phosphatidylinositol 3-kinase(PI3K),NLR-P3,NF-κB,myeloiddifferentiationfactor-88(Myd88),inhibitorαof NF-κB(IκBα),interleukin-18(IL-18),cysteinyl aspartate specific proteinase-1(Caspase-1),apoptosis-associated speck-like protein containing CARD(ASC)and cyclooxygenase-2(COX-2).Results The cell proliferation and cell scratch experiments showed that the ethyl acetate extract of Huazelan(Eupatorium chinense)root had bidirectional effects on the proliferation and scratch healing of NIH-3T3 cells.The extract can promote the proliferation and migration of NIH-3T3 cells at low concentration(<6.25μg·mL^(-1)).On the contrary,high concentration(>25μg·mL^(-1))exhibited inhibitory effects on the proliferation and migration of NIH-3T3 cells,and showed a corresponding dose-dependent effect at different stages.It was statistically significant compared with the control group(P<0.05).When the extract concentration was 6.25μg·mL^(-1),the NO production was significantly different from that in the low-concentration group(P<0.01),but the NO production of the high-concentration group(>12.5μg·mL^(-1))had no significant difference compared with that of the control group(P>0.05).The intracellular ROS generation experiment showed that the amount of ROS generated by the ethyl acetate extract of Huazelan(Eupatorium chinense)roots was significantly higher than that of the control group(P<0.05,P<0.01).Western blot experiments showed that compared with those of the control group,when the concentration of the extract was 6.25μg·mL^(-1),the expressions of NF-κB,TLR-4,Myd88,NLR-P3 and ASC in cells were significantly increased(P<0.01),the expression of IκBαwas increased(P<0.05),the expression of IL-18 was significantly decreased(P<0.01)and the expression of Caspase-3 was decreased(P<0.05).Conclusion The effect of Huazelan(Eupatorium chinense)ethyl acetate extract on the proliferation of mouse embryonic fibroblasts(NIH-3T3)is bidirectional,that is,low concentration promotes cell proliferation and high concentration inhibits cell proliferation.Its mechanism of action may be related to the NF-κB and NLR-P3 inflammasome signaling pathways and apoptosis.