基于PI3K/AKT/mTOR信号通路探讨野黄芩苷联合虎杖苷抗慢性支气管炎的作用机制

Effects of scutellarin combined with polydatin on chronic bronchitis via PI3K/AKT/mTOR signaling pathway

目的:探讨野黄芩苷联合虎杖苷对脂多糖(LPS)联合烟熏法诱导的大鼠肺组织和支气管损伤的影响及其机制。方法:雄性SD大鼠分为对照组、模型组、黄芩茎叶-虎杖水煎液(5 g/kg)组、野黄芩苷(40 mg/kg)组、虎杖苷(60 mg/kg)组、野黄芩苷+虎杖苷(20 mg/kg+30 mg/kg、40 mg/kg+60 mg/kg和80 mg/kg+120 mg/kg)组和桂龙咳喘宁片(1 g/kg)组,每组10只。建立慢性支气管炎模型。ELISA法测定大鼠支气管肺泡灌洗液(BALF)和血清中白细胞介素1β(IL-1β)、IL-6、IL-17A、IL-10和肿瘤坏死因子(TNF-α)水平;通过试剂盒检测肺组织中超氧化物歧化酶(SOD)和丙二醛(MDA)水平;测定BALF中蛋白、白细胞及中性粒细胞含量;测定右肺组织湿/干(W/D)质量比值;HE染色观察肺组织和支气管病理变化,并对肺组织损伤评分;RT-qPCR检测磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(PKB/AKT)和哺乳动物雷帕霉素靶蛋白(mTOR)的mRNA表达;Western blot检测p-PI3K、PI3K、p-AKT、AKT、p-mTOR和mTOR的蛋白水平。结果:与对照组相比,模型组大鼠支气管和血清中IL-1β、IL-6、IL-17A、TNF-α和MDA水平显著升高(P<0.01),IL-10和SOD水平显著降低(P<0.01),右肺组织W/D质量比值显著升高(P<0.01),BALF中蛋白含量、白细胞和中性粒细胞水平均显著升高(P<0.01),p-PI3K、p-AKT和p-mTOR蛋白水平显著升高(P<0.01);与模型组相比,野黄芩苷+虎杖苷(20 mg/kg+30 mg/kg、40 mg/kg+60 mg/kg和80 mg/kg+120 mg/kg)组IL-1β、IL-6、IL-17A、TNF-α和MDA水平均显著降低(P<0.01或P<0.05),IL-10和SOD水平显著升高(P<0.01),右肺组织W/D质量比值显著下降(P<0.01或P<0.05),BALF中蛋白含量、白细胞和中性粒细胞水平均显著降低(P<0.01或P<0.05),肺组织和支气管炎症反应损伤病变明显减轻,p-PI3K、p-AKT和p-mTOR蛋白水平显著降低(P<0.01)。结论:野黄芩苷联合虎杖苷可减轻LPS联合烟熏法诱导的大鼠肺组织和支气管损伤,其机制可能与抑制PI3K/AKT/mTOR信号通路的活化,降低氧化应激反应,抑制炎症因子IL-17A、TNF-α、IL-1β和IL-6有关。

AIM:To investigate the effect of scutellarin combined with polydatin on pulmonary and bronchial injury induced by lipopolysaccharide(LPS)combined with smoking in rats and its mechanism.METHODS:Male SD rats were randomly divided into control group,model group,Huangqin Jingye-Huzhang decoction(5 g/kg)group,scutel‐larin(40 mg/kg)group,polydatin(60 mg/kg)group,scutellarin+polydatin(20 mg/kg+30 mg/kg,40 mg/kg+60 mg/kg and 80 mg/kg+120 mg/kg)groups,and Guilong Kechuanning tablet(1 g/kg)group,with 10 rats each group.Chronic bronchitis model was established in 90 rats.The levels of interleukin-1β(IL-1β),IL-6,IL-17A,IL-10 and tumor necro‐sis factor-α(TNF-α)in bronchoalveolar lavage fluid(BALF)and serum were measured by ELISA,and the levels of su‐peroxide dismutase(SOD)and malondialdehyde(MDA)in lung tissues were detected using kits.Protein content and cell counts in BALF were determined.Wet/dry(W/D)mass ratio of the right lung was estimated,and HE staining was used to observe the pathological changes of pulmonary and bronchial tissues and score the lung tissue injury.The mRNA expres‐sion of phosphatidylinositol 3-kinase(PI3K),protein kinase B(PKB/AKT)and mammalian target of rapamycin(mTOR)was detected by RT-qPCR.The protein levels of p-PI3K,PI3K,p-AKT,AKT,p-mTOR and mTOR were detected by Western blot.RESULTS:Compared with control group,the levels of IL-1β,IL-6,protein,leukocytes and neutrophils in BALF,the levels of IL-17A and TNF-αin serum,the content of MDA in lung tissues and the W/D mass ratio of the right lung in model group were significantly increased(P<0.01),while the content of IL-10 in serum and the activity of SOD in lung tissue were significantly decreased(P<0.01).The protein levels of p-PI3K,p-AKT and p-mTOR were signif‐icantly up-regulated(P<0.01).Compared with model group,the levels of IL-1β,IL-6,protein,white blood cells and neutrophils in BALF,the levels of IL-17A and TNF-αin serum,the content of MDA in lung tissues and the W/D mass ra‐tio of the right lung in scutellarin+polydatin(20 mg/kg+30 mg/kg,40 mg/kg+60 mg/kg and 80 mg/kg+120 mg/kg)groups were significantly lower than those in model group(P<0.01 or P<0.05),while the content of IL-10 in serum and the activi‐ty of SOD in lung tissue were significantly increased(P<0.01).The pulmonary and bronchial inflammatory response was alleviated,and the injury was obviously attenuated.The protein levels of p-PI3K,p-AKT and p-mTOR were significantly down-regulated(P<0.01).CONCLUSION:Scutellarin combined with polydatin protects pulmonary and bronchial tis‐sues against the injury induced by LPS combined with smoking in rats.The mechanism may be related to inhibiting the ac‐tivation of PI3K/AKT/mTOR pathway,reducing oxidative stress and inhibiting inflammatory factors such as IL-17A,TNF-α,IL-1βand IL-6.