基于RhoA/ROCK/NF-κB信号通路探究益肾活血化痰方对动脉粥样硬化小鼠炎症反应的影响

Effects of Yishen Huoxue Huatan recipe on inflammatory response in atherosclerotic mice via RhoA/ROCK/NF-κB signaling pathway

目的探讨益肾活血化痰方对动脉粥样硬化(AS)小鼠炎症反应的影响及机制。方法采用高脂饲养法制备AS模型。将小鼠分为对照组、模型组、药物组(303 mg/kg益肾活血化痰方灌胃给药)、抑制剂组(仅给予5 mg/kg的ROCK抑制剂Y-27632腹腔注射)、阳性药物组(3.03 mg/kg的阿托伐他汀灌胃给药),每组10只。油红O染色观察小鼠主动脉组织斑块形成。ELISA法检测血清炎症因子(TNF-α、IL-1β、IL-6)水平,自动生化分析仪检测血脂(TG、TC、LDL-C、HDL-C)水平,免疫组化法检测主动脉组织IL-1β表达。Western blot法检测主动脉组织RhoA、ROCK蛋白表达及NF-κB p65磷酸化水平。结果与对照组比较,模型组小鼠血清TG、TC、LDL-C、TNF-α、IL-1β、IL-6水平显著升高(P<0.05),HDL-C水平显著降低(P<0.05),主动脉斑块面积增大(P<0.05),主动脉组织IL-1β、RhoA、ROCK蛋白表达及p-NF-κB p65/NF-κB p65显著升高(P<0.05)。与模型组比较,药物组、抑制剂组、阳性药物组小鼠血清TG、TC、LDL-C、TNF-α、IL-1β、IL-6水平显著降低(P<0.05),HDL-C水平显著升高(P<0.05),主动脉斑块面积减小(P<0.05),主动脉组织IL-1β、RhoA、ROCK蛋白表达及p-NF-κB p65/NF-κB p65显著降低(P<0.05)。药物组与阳性药物组、抑制剂组各指标比较差异无统计学意义(P>0.05)。结论益肾活血化痰方可能通过抑制RhoA/ROCK/NF-κB信号通路激活,改善AS小鼠血脂代谢,减轻AS小鼠炎症反应。

Objective To investigate the effect and its mechanism of Yishen Huoxue Huatan recipe on inflammatory response in athe-rosclerotic(AS)mice.Methods The AS model was established by high-fat feeding method.The mice were divided into control group,model group,drug group(303 mg/kg Yishen Huoxue Huatan recipe by gavage),inhibitor group(only intraperitoneal injection of 5 mg/kg ROCK inhibitor Y-27632),and positive drug group(3.03 mg/kg of atorvastatin by gavage),with 10 mice in each group.Oil red O staining was applied to observe the plaque formation in mouse aortic tissue.ELISA method was applied to detect serum levels of inflammatory factors(TNF-α,IL-1β,IL-6).Automatic biochemical analyzer was applied to detect blood lipid(TG,TC,LDL-C,HDL-C)levels.Immunohistochemistry was used to detect the expression of IL-1βin aortic tissue.Western blot was used to detect the protein expression of RhoA and ROCK and the phosphorylation of NF-κB p65 in aortic tissue.Results Compared with control group,the serum levels of TG,TC,LDL-C,TNF-α,IL-1βand IL-6 in model group were significantly increased(P<0.05),the level of HDL-C was statistically decreased(P<0.05),the aortic plaque area was increased(P<0.05),and the protein expression levels of IL-1β,RhoA,ROCK and p-NF-κB p65/NF-κB p65 in aortic tissue were increased(P<0.05).Compared with model group,the serum levels of TG,TC,LDL-C,TNF-α,IL-1βand IL-6 of the mice in drug group,inhibitor group and positive drug group were decreased(P<0.05),the level of HDL-C was increased(P<0.05),the aortic plaque area was decreased(P<0.05),and the protein expression levels of IL-1β,RhoA,ROCK and p-NF-κB p65/NF-κB p65 in aortic tissue were decreased(P<0.05).There was no significant difference in the above indexes between drug group and positive drug group,and between drug group and inhibitor group(P>0.05).Conclusion Yishen Huoxue Huatan formula may improve the blood lipid metabolism and reduce the inflammation in AS mice by inhibiting the activation of RhoA/ROCK/NF-κB signaling pathway.