藏荆芥乙醇提取物抗炎作用及机制研究

The Effect and Mechanism of Ethanol Extract of Nepeta Angustifoia C.Y.Wu on Inflammation

目的研究藏荆芥乙醇提取物对脂多糖(LPS)诱导的体内外炎症模型的作用及其作用机制。方法将小鼠随机分为空白对照组、脂多糖模型组、阳性药地塞米松(0.000 5 g·kg^(-1))组及藏荆芥乙醇提取物低、中、高剂量组(0.500、1.000、2.000 g·kg^(-1)),每组10只。通过腹腔注射脂多糖(0.001 g·kg^(-1))建立小鼠急性腹膜炎模型,采用酶联免疫法(ELISA)检测各组小鼠腹腔灌洗液中炎症因子水平。另外,通过脂多糖诱导小鼠巨噬细胞RAW264.7建立细胞炎症模型,将RAW264.7细胞分为空白对照组、脂多糖模型组及不同浓度藏荆芥乙醇提取物组(25、50、100、200、400μg·mL^(-1)),检测细胞存活率以筛选给药浓度;ELISA法检测藏荆芥乙醇提取物对促炎因子一氧化氮(NO)、肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-6、IL-1β的抑制作用;蛋白电泳法检测促炎介质诱导型一氧化氮合酶(iNOS)和环氧化酶-2(COX-2)的表达水平,并采用蛋白电泳法和免疫荧光法检测细胞中NF-κB通路相关蛋白(p65、p-IκBα、IκBα)的表达水平。结果体内实验中,与脂多糖模型组比,藏荆芥乙醇提取物中、高剂量组均不同程度降低了小鼠腹腔中TNF-α、IL-6、IL-1β的表达水平(P<0.05,P<0.01)。在体外实验中,根据实验结果,与空白对照组比,400μg·mL^(-1)的藏荆芥乙醇提取物表现出明显的细胞毒性(P<0.01),而25~200μg·mL^(-1)的藏荆芥乙醇提取物对细胞存活率无明显影响(P>0.05),用于进一步实验;与脂多糖模型组比,藏荆芥组NO、TNF-α、IL-6、IL-1β水平明显降低(P<0.05,P<0.01)。蛋白电泳结果显示,与脂多糖模型组比,不同浓度(150、200μg·mL^(-1))的藏荆芥组明显抑制p65、p-IκBα的表达,逆转了IκBα的表达下降,且差异有统计学意义(P<0.05,P<0.01)。免疫荧光结果显示,与脂多糖模型组比,藏荆芥乙醇提取物明显抑制了细胞核中的p65的表达。结论藏荆芥提取物具有明显的抗炎活性,其作用机制可能通过抑制NF-κB通路活化而改善体内、外炎症。

Objective To study the effect and mechanism of ethanol extract of Nepeta Angustifoia C.Y.Wu(NA)on lipopolysaccharide(LPS)-induced inflammation in vivo and in vitro.Methods KM mice were randomly divided into the control group,LPS group and positive drug group(dexamethasone,0.000 5 g·kg^(-1)),NA low,medium and high dose groups(0.500,1.000,2.000 g · kg^(-1)),10 mice in each group.Mice model of acute peritonitis was established by intraperitoneal injection of LPS(0.001 g·kg^(-1)),and enzyme linked immunosorbent assay(ELISA)was used to detect the level of inflammatory cytokines in the peritoneal lavage solution of mice.In vitro,LPS was used to induce mouse macrophages RAW 264.7 to establish a cell inflammation model.RAW 264.7 cells were divided into the control group,LPS group and difference concentration of NA groups(25,50,100,200,400 μg·mL^(-1)).MTT assay was used to detect the cell viability and screen the concentration of drug administration,and the effect of NA on pro-inflammatory cytokines such as nitric oxide(NO),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β) were measured by ELISA.The protein expression level of inducible nitric oxid synthase(iNOS)and cyclooxygenase-2(COX-2)also were measured by Western Blotting.The expression levels of NF-κB signaling pathway related proteins(p65,p-IκBα and IκBα)were detected by western blotting and immunofluorescence.Results In vivo,the levels of TNF-α,IL-6 and IL-1β in mice peritoneal of NA medium and high dose groups were significantly decreased when compared with LPS group in a dose-independent manner(P<0.05,P<0.01).In vitro,MTT results showed that 25~200 μg·mL^(-1) of NA has no significant effect on the cell viability(P>0.05),while 400 μg·mL^(-1) of NA exhibited signficant cytotoxicity compared with the control group.Thus,the concentration of NA from 25 μg·mL^(-1) to 200 μg·mL^(-1) was used for further experiment.Compared with LPS group,the levels of NO,TNF-α,IL-6 and IL-1β in NA group were significantly decreased in a dose-independent manner(P<0.05,P<0.01).Additionally,Western Blotting results showed that the protein expression levels of iNOS and COX-2 in LPS group were significantly increased compared of that in the control group,while NA treatment doseindependently decreased the protein expression of iNOS and COX-2(P<0.05,P<0.01).Western Blotting results showed that different concentration of NA(150,200 μg·mL^(-1))suppressed the phosphorylation of p65 and p-IκBα,and restored the expression of IκBα compared of that in LPS group with statistical difference(P<0.05,P<0.01).Immunofluorescence results showed that NA treatment significantly inhibited the p65 translocation into the nucleus compared of that in LPS group.Conclusion NA may improve inflammation in vivo and in vitro by inhibiting the activation of NF-κB signaling pathway,thereby exerts an anti-inflammatory effect.