Experimental Study On Metformin Improving Pulmonary Fibrosis Induced By Acute Paraquat Poisoning In Mice

Objective:To investigate the protective effect of metformin on pulmonary fibrosis after paraquat(PQ)poisoning and its mechanism.Methods: Sixty healthy 12 week old SPF male C57BL/6J mice were randomly divided into blank control group(control group),paraquat poisoning model group(PQ group),metformin intervention group(PQ+MET group),AMPK agonist group(PQ+AICAR group)and AMPK inhibitor group(PQ+MET+CC group),12 animals each group.PQ poisoning model was established by one-time intraperitoneal injection of PQ solution(20mg/kg).Mice in control group were injected with the same amount of normal saline through peritoneal cavity.Two hours after intraperitoneal injection of PQ solution,each mouse in PQ+MET group,PQ+AICAR group and PQ+MET+CC group was given MET(200mg/kg)by gavage,injected AICAR solution(200mg/kg)through intraperitoneal,treated MET(200mg/kg)by gavage while CC solution(20mg/kg)with intraperitoneal injection,once a day for 21 consecutive days.On the 21 st day of the experiment,the lung tissue of mice was taken for related detection.The observed indexes of experimental animals include:(1)Behavioral change.(2)21-day survival rate.(3)The pathological changes of lung tissue.(4)The content of hydroxyproline in lung tissue.(5)MDA content and SOD activity in lung tissue.(6)The expression of EMT markers E-cadherin and α-SMA protein in lung tissue was detected by Western blot(WB).(7)WB was used to detect protein expression,including phospho-AMPK,TGF-β1 and phospho-Smad3,in lung tissue.Results:(1)The mice in PQ group and PQ+MET+CC group gradually showed shortness of breath,arch back fatigue and decreased activity after 1 to 6 hours exposure to paraquat,and began to eat less after 3 to 7 days.Compared with PQ group,at the same time,the above-mentioned manifestations of mice in PQ+MET group and PQ+AICAR group were relatively mild.(2)The 21-day survival rate of MET group was higher than that of PQ group(P<0.05).(3)The lung tissue structure of PQ group and PQ+MET+CC mice was seriously damaged,and the fibrosis changes were obvious.The degree of lung tissue structure damage and fibrosis changes in PQ+MET group and PQ+AICAR group were significantly reduced compared with PQ group.(4)The content of hydroxyproline in PQ group was higher than that in control group(P<0.05),the content of hydroxyproline in PQ+ MET group and PQ + AICAR group was lower than that in PQ group(P<0.05),the content of hydroxyproline in PQ+MET group was lower than that in PQ+AICAR group(P<0.05),and the content of hydroxyproline in PQ+MET+CC group was higher than that in PQ+MET group(P<0.05).(5)Compared with control group,the content of MDA in PQ group was significantly higher(P<0.05)and the activity of SOD was significantly lower(P<0.05).Compared with PQ group,the content of MDA in PQ+MET group and PQ+AICAR group was significantly lower(P<0.05)and the activity of SOD was significantly higher(P<0.05).Compared with PQ+MET group,the content of MDA in PQ+MET+CC group was significantly higher(P<0.05),and the activity of SOD was significantly lower(P<0.05);(6)Compared with the control group,the expression of E-cadherin in PQ group decreased(P<0.05)and the expression of α-SMA protein increased(P<0.05).Compared with PQ group,the expression of E-cadherin in PQ + MET group and PQ+AICAR group increased(P<0.05)and the expression of α-SMA decreased(P<0.05).Compared with PQ+AICAR group,the expression of E-cadherin in PQ+MET group increased(P<0.05)and there was no significant difference in the expression ofα-SMA(P > 0.05).Compared with PQ + MET group,the expression of E-cadherin decreased(P<0.05)and α-SMA increased(P<0.05)in PQ+MET+CC group.(7)Compared with the control group,the expression of Phospho-AMPK in PQ group decreased(P<0.05),and the expression of TGF-β1 and Phospho-Smad3 increased(P<0.05).Compared with PQ group,the expression of Phospho-AMPK increased in PQ+MET group and PQ+AICAR group(P<0.05),and the expression of TGF-β1 and Phospho-Smad3decreased(P<0.05).Compared with PQ + MET group,the expression of phospho-AMPK was lower(P<0.05),and the expression of TGF-β1 and phospho-Smad3 was higher(P<0.05)in PQ+MET+CC group.There was no significant difference in the expression of phospho-AMPK between PQ+AICAR group and PQ+MET group(P>0.05),however,the expression of TGF-β1 and phospho-Smad3 protein in PQ+AICAR group was higher than that in PQ+MET group(P < 0.05).Conclusion: Metformin can effectively reduce pulmonary fibrosis caused by paraquat poisoning,and its mechanism is related to the activation of AMPK and the inhibition of TGF-β1 / Smad3.