二甲双胍激活AMP活化蛋白激酶通路对急性百草枯中毒致肺纤维化的保护作用

Protective effect of metformin on pulmonary fibrosis caused by paraquat through activating AMP-activated protein kinase pathway

目的观察二甲双胍(MET)是否通过激活AMP活化蛋白激酶(AMPK)来抑制转化生长因子-β_(1)(TGF-β_(1))/Smad3信号通路,从而减轻百草枯(PQ)中毒所致小鼠肺纤维化。方法按随机数字表法将雄性C57BL/6J小鼠分为对照组(Control组)、PQ中毒模型组(PQ组)、MET干预组(PQ+MET组)、AMPK激动剂组(PQ+AICAR组)和AMPK抑制剂组(PQ+MET+CC组)。经腹膜腔一次性注射20 mg/kg PQ溶液1 mL建立PQ中毒小鼠模型;Control组注射等量生理盐水。制模后2 h,PQ+MET组灌胃200 mg/kg MET溶液2 mL,PQ+AICAR组经腹膜腔注射200 mg/kg腺苷类似物AICAR溶液2 mL,PQ+MET+CC组灌胃200 mg/kg MET溶液2 mL后经腹膜腔注射20 mg/kg复合物C(CC)溶液1 mL,Control组和PQ组灌胃生理盐水2 mL;均每日1次,连续干预21 d。每组取10只小鼠用于计算21 d存活率;剩余小鼠于制模后21 d麻醉取肺组织,苏木素-伊红(HE)染色和Masson染色后于光镜下观察肺组织病理学改变,并对肺纤维化程度进行Ashcroft评分;检测肺组织羟脯氨酸含量以及丙二醛(MDA)和超氧化物歧化酶(SOD)等氧化应激指标;采用蛋白质免疫印迹试验(Western blotting)检测肺组织E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、磷酸化AMPK(p-AMPK)、TGF-β_(1)、磷酸化Smad3(p-Smad3)蛋白表达。结果与Control组比较,PQ组小鼠21 d存活率明显降低;肺组织出现明显纤维化改变,Ashcroft评分显著升高;肺组织羟脯氨酸、MDA含量以及α-SMA、TGF-β_(1)、p-Smad3蛋白表达均明显升高,SOD活性以及E-cadherin、p-AMPK蛋白表达均明显降低。与PQ组比较,PQ+MET组和PQ+AICAR组小鼠21 d存活率明显提高(70%、60%比20%,均P<0.05);肺组织纤维化程度明显减轻,Ashcroft评分显著降低(分:1.50±0.55、2.00±0.63比6.67±0.52,均P<0.05);肺组织羟脯氨酸、MDA含量以及α-SMA、TGF-β_(1)、p-Smad3蛋白表达均明显降低〔羟脯氨酸(mg/L):2.03±0.11、3.00±0.85比4.92±0.65,MDA(kU/g):2.06±1.48、2.10±1.80比4.06±1.33,α-SMA/GAPDH:0.23±0.06、0.16±0.06比1.00±0.09,TGF-β_(1)/GAPDH:0.28±0.03、0.53±0.05比0.92±0.06,p-Smad3/GAPDH:0.52±0.04、0.69±0.06比1.11±0.10,均P<0.05〕,SOD活性以及E-cadherin、p-AMPK蛋白表达均明显升高〔SOD(μmol/g):39.76±1.35、33.03±1.28比20.08±1.79,E-cadherin/GAPDH:0.91±0.08、0.72±0.08比0.26±0.04,p-AMPK/GAPDH:0.62±0.04、0.60±0.01比0.20±0.04,均P<0.05〕。而加入AMPK抑制剂CC后,MET的这些保护作用受到抑制。结论MET能有效减轻PQ中毒小鼠肺纤维化程度,其作用机制可能与激活AMPK并抑制TGF-β_(1)/Smad3信号通路相关,而这一效应可以被AMPK抑制剂CC所抑制。

Objective To observe whether metformin(MET)inhibits transforming growth factor-β_(1)(TGF-β_(1))/Smad3 signaling pathway by activating adenosine activated protein kinase(AMPK),so as to alleviate the pulmonary fibrosis caused by paraquat(PQ)poisoning in mice.Methods Male C57BL/6J mice were randomly divided into the Control group,PQ poisoning model group(PQ group),MET intervention group(PQ+MET group),AMPK agonist group(PQ+AICAR group),and AMPK inhibitor group(PQ+MET+CC group),according to a random number table method.A mouse model of PQ poisoning was established by one-time peritoneal injection of 1 mL PQ solution(20 mg/kg).The Control group was injected with the same volume of normal saline.After 2 hours of modeling,the PQ+MET group was given 2 mL of 200 mg/kg MET solution by gavage,the PQ+AICAR group was given 2 mL of 200 mg/kg AICAR solution by intraperitoneal injection,the PQ+MET+CC group was given 2 mL of 200 mg/kg MET solution by gavage and then 1 mL complex C(CC)solution(20 mg/kg)was intraperitoneally injected,the Control group and PQ group were given 2 mL of normal saline by gavage.The intervention was given once a day for 21 consecutive days.The 21-day survival rate of ten mice in each group was calculated,and the lung tissues of remaining mice were collected at 21 days after modeling.The pathological changes of lung tissues were observed under light microscope after hematoxylin-eosin(HE)staining and Masson staining,and the degree of pulmonary fibrosis was evaluated by Ashcroft score.The content of hydroxyproline in lung tissue and oxidative stress indicators such as malondialdehyde(MDA)and superoxide dismutase(SOD)were detected.The protein expressions of E-cadherin,α-smooth muscle actin(α-SMA),phosphorylated AMPK(p-AMPK),TGF-β_(1)and phosphorylated Smad3(p-Smad3)in lung tissue were detected by Western blotting.Results Compared with the Control group,the 21 days survival rate was significantly reduced,lung fibrosis and Ashcroft score were significantly increased in PQ group.In addition,the content of hydroxyproline,MDA and the protein expressions ofα-SMA,TGF-β_(1)and p-Smad3 in lung tissue were significantly increased,while the activity of SOD and the protein expressions of E-cadherin and p-AMPK were significantly decreased in PQ group.Compared with the PQ group,the 21 days survival rates of mice were significantly improved in the PQ+MET group and PQ+AICAR group(70%,60%vs.20%,both P<0.05).The degree of pulmonary fibrosis and the Ashcroft score were significantly reduced(1.50±0.55,2.00±0.63 vs.6.67±0.52,both P<0.05).The content of hydroxyproline and MDA in lung tissue,as well asα-SMA,TGF-β_(1)and p-Smad3 protein expressions were significantly reduced[hydroxyproline(mg/L):2.03±0.11,3.00±0.85 vs.4.92±0.65,MDA(kU/g):2.06±1.48,2.10±1.80 vs.4.06±1.33,α-SMA/GAPDH:0.23±0.06,0.16±0.06 vs.1.00±0.09,TGF-β_(1)/GAPDH:0.28±0.03,0.53±0.05 vs.0.92±0.06 p-Smad3/GAPDH:0.52±0.04,0.69±0.06 vs.1.11±0.10,all P<0.05],SOD activity and the protein expressions of E-cadherin and p-AMPK were significantly increased[SOD(μmol/g):39.76±1.35,33.03±1.28 vs.20.08±1.79,E-cadherin/GAPDH:0.91±0.08,0.72±0.08 vs.0.26±0.04,p-AMPK/GAPDH:0.62±0.04,0.60±0.01 vs.0.20±0.04,all P<0.05].However,these protective effects of MET were inhibited by the addition of AMPK inhibitor CC solution.Conclusion MET can effectively alleviate the degree of pulmonary fibrosis in mice poisoned with PQ,and its mechanism may be related to the activation of AMPK and inhibition of TGF-β_(1)/Smad3 signaling pathway,which can be inhibited by AMPK inhibitor CC.