毛酸浆果乙醇提取物抗炎作用及其机制研究

Anti-inflammatory effect and mechanism of Physalis pubescens

目的观察毛酸浆果乙醇提取物（AEFPP）的抗炎作用,并探索其作用机制。方法取二甲苯涂于小鼠耳廓制备耳廓肿胀急性炎症模型,将棉花植入大鼠皮下构建棉球肉芽肿慢性炎症模型,观察AEFPP的在体抗炎作用;采用MTT法检测质量浓度为0.25~20.00 mg/m L的AEFPP对RAW 264.7细胞的细胞毒性;质量浓度为0.625、1.250、2.500 mg/m L的AEFPP预处理3 h后,脂多糖（LPS）作用于RAW 264.7细胞制备炎症模型,ELISA法检测上清中肿瘤坏死因子（TNF-α）和白细胞介素-6（IL-6）的水平;实时定量PCR（q RT-PCR）法检测NF-κB p65 m RNA表达;western blotting法测定NF-κB p65蛋白表达。结果 0.8 g/kg AEFPP可显著抑制小鼠耳廓肿胀率;0.1、0.2、0.4 g/kg剂量组对大鼠棉球肉芽肿具显著的抑制作用,且表现出剂量依赖性;AEFPP在0~3 mg/m L范围内对细胞无明显毒性;另外,0.625、1.250、2.500 mg/m L AEFPP均可显著抑制RAW 264.7细胞上清液中的TNF-α和IL-6的水平;0.625、1.250 mg/m L AEFPP显著抑制NF-κB p65 m RNA及其蛋白的表达。结论 AEFPP具良好的抗炎活性,其作用机制可能与下调p65蛋白的表达和抑制炎症因子TNF-α和IL-6的释放有关。

Objective To investigate the anti-inflammatory effect of the alcohol extract from fruit of Physalis pubescens（AEFPP） and explore its mechanism. Methods To observe the anti-inflammatory effect of AEFPP in vivo, dimethylbenzene was daubed on mice auricle to replicate mouse ear edema acute inflammatory model, and cotton ball was sc implanted to establish rat cotton ball granuloma model. The cytotoxicity of 0.25—20.00 mg/m L AEFPP was examined with MTT method. Pretreatment with 0.625, 1.250, and 2.500 mg/m L AEFPP for 3 h, RAW 264.7 mononuclear macrophages cells were stimulated with lipopolysaccharides so as to build an inflammatory cell model. The culture supernatant of the cells was collected in order to determine tumor necrosis factor-α（TNF-α） and interleukin 6（IL-6） by ELISA. The expression of NF-κB p65 and its anti-inflammatory activity were assayed by Western blotting and real-time quantitative PCR（q RT-PCR）. Results High dose（0.8 g/kg） of AEFPP could diminish mouse ear edema, which had been induced by dimethylbenzene. Besides, all the three dosages of AEFPP（0.1, 0.2 and 0.4 g/kg） could markedly inhibit rats cotton ball granuloma. AEFPP was not toxic to the cells in the range of 0—3 mg/m L. Furthermore, the other three dosages of AEFPP（0.625, 1.250, and 2.500 mg/m L） significantly decreased the concentration of TNF-α and IL-6 in the culture supernatants. AEFPP of 0.625 and 1.25 mg/m L could markedly inhibit the expression levels of NF-κB p65 m RNA and its protein. Conclusion AEFPP has a wonderful anti-inflammatory effect. The mechanism might be related with down-regulating the expression of p65 protein and inhibiting the release of inflammatory factors, such as TNF-α and IL-6.