Effect Of Tacrolimus On TGF-β1/Smads Signaling Pathway In Paraquat Induced Pulmonary Fibrosis

Section One:Effect of tacrolimus on TGF-β1/Smads signaling pathway in paraquat exposed rat alveolar type II epithelial cellsBackground and aims Paraquat is a bipyridine herbicide,which is widely used in agricultural production because of its harmless to the environment,paraquat poisoning cases also increased,the lung damage of poisoning patients is the most obvious.At present,the cause of pulmonary interstitial fibrosis is not clear.The type Ⅰ and type Ⅱ epithelial cells of the lung and Clara cells of the trachea contain polyamine transport systems.The system has been found to promote paraquat transport to the lungs.TGF-β1/Smads signaling pathway is associated with pulmonary fibrosis.At present,the treatment of paraquat poisoning is mainly hormone,antioxidant,immunosuppressant and hemoperfusion.Cyclophosphamide is an immunosuppressive agent for the treatment of paraquat poisoning,but its side effects are great.Tacrolimus(FK506)is a new macrolide immunosuppressant,which is widely used in organ transplantation and the treatment of autoimmune diseases.Tacrolimus has been used in the treatment of renal disease,but the experience in the treatment of pulmonary fibrosis is still insufficient.In this study,tacrolimus was used to interfere with paraquat induced pulmonary fibrosis in rat alveolar epithelial type Ⅱ cells(RLE-6TN cells)and to explore the mechanism of tacrolimus inhibiting pulmonary fibrosis by detecting the effect of tacrolimus on inflammatory factors of TGF-β1/Smads signaling pathway,so as to provide evidence for the treatment of paraquat induced pulmonary fibrosis.Methods The experiment was divided into control group,paraquat group(PQ),tacrolimus group,TβRⅠ/Ⅱ dual inhibitor(LY2109761)1h,LY2109761 4h,LY2109761 8h,LY2109761 16h groups,each group was provided with three multiple pores.MTT method was used to detect the activity of alveolar type Ⅱ epithelial cells in rats with different concentrations of paraquat,tacrolimus+paraquat and LY2109761+paraquat.The concentrations of TGF-β1,Smad3,Smad7 and CTGF were detected by ELISA.The expressions of TGF-β1,Smad3 and Smad7 were detected by immunofluorescence.The mRNA levels of TGF-β1,Smad3,Smad7 and CTGF were detected by real-time PCR.Results ①When PQ concentration was 200 nmol/l,the inhibition rate of RLE-6TN cells was 26.05±2.99%.At the concentration of 10 ng/ml,tacrolimus had the most obvious inhibitory effect on paraquat induced injury of alveolar type Ⅱ epithelial cells,with the inhibition rate of 18.40±3.49%.The inhibition rate of LY2109761 at 5μmol/l was 26.56±4.49%.② Compared with the control group,the concentration of TGF-β1 in the supernatant of RLE-6TN cells treated with 200 nmol/1 paraquat was significantly increased(P<0.05).However,the concentration of TGF-β1 in tacrolimus group was significantly lower than that in paraquat group.Compared with paraquat group,the level of TGF-β1 decreased gradually after incubation with 5μmol/l LY2109761 for 1h,4h,8h and 16 h(P<0.05).The concentration of Smad3 in paraquat group was significantly higher than that in control cells(P<0.05).After tacrolimus treatment,Smad3 level was significantly lower than that in paraquat group.In addition,compared with paraquat group,the concentration of Smad3 in RLE-6TN cells decreased after treated with LY2109761 for 4h,8h and 16h(P<0.05)Compared with the control group and paraquat group,the concentration of Smad7 increased significantly after tacrolimus treatment.Compared with paraquat group,the levels of Smad7 in LY2109761 8h and 16h groups were significantly increased(P<0.05).The concentration of CTGF in paraquat group was significantly higher than that in control group.However,compared with paraquat group,the level of CTGF in tacrolimus pretreated cells was significantly lower(P<0.05).In line with tacrolimus treatment,CTGF levels in LY2109761 group decreased gradually with time compared with paraquat group.③ TGF-β1,Smad3 and Smad7 were expressed in cytoplasm and nucleus,and the expression was mainly in cytoplasm,The expression levels of TGF-β1 and Smad3 in paraquat group were higher than those in control group(P<0.05).Compared with the cells exposed to paraquat alone,tacrolimus pretreatment decreased the expression of TGF-β1 and Smad3,and increased the expression of Smad7(P<0.05).LY2109761 could down regulate the expression of TGF-β1 and Smad3(P<0.05).Compared with paraquat alone,LY2109761 increased the expression level of Smad7 after 4 h incubation(P<0.05).④ Compared with the control group,the expression levels of TGF-β1 and Smad3 in RLE-6TN cells exposed to paraquat were significantly increased(P<0.05).However,compared with paraquat group,the mRNA expression levels of TGF-β1 and Smad3 were significantly decreased after tacrolimus treatment(P<0.05).After treatment with LY2109761,the expression level of TGF-β1 gradually decreased with time;at 8h and 16h,compared with paraquat group,the expression level of TGF-β1 in LY2109761 group was significantly decreased(P<0.05).Compared with paraquat group,Smad3 mRNA expression level was also decreased after LY2109761 treatment for 4h,8h and 16h(P<0.05).Compared with the control group,the expression of Smad7 mRNA in RLE-6TN cells was significantly increased after paraquat exposure(P<0.05).After tacrolimus pretreatment,Smad7 expression level was significantly up-regulated compared with paraquat group.Compared with paraquat and control group,Smad7 mRNA expression level was also significantly increased after treatment with LY2109761 for 1 h,4 h,8 h and 16 h(P<0.05).Compared with the control group,the expression level of CTGF in paraquat treated cells was significantly increased(P<0.05);compared with the control group and paraquat group,tacrolimus pretreatment significantly decreased the expression of CTGF mRNA.Compared with paraquat group and control group,the CTGF expression level of LY2109761 treatment group was significantly lower than that of paraquat group and control group(P<0.05).Conclusion Tacrolimus can significantly reduce the level of inflammatory factors in the TGF-β1/Smads signaling pathway of paraquat exposed rat alveolar type Ⅱ epithelial cells,and increase the level of inflammatory inhibitory factors.Tacrolimus has a protective effect on paraquat exposed alveolar type Ⅱ epithelial cells.Section Two Effect of tacrolimus on TGF-β1/Smads signaling pathway in paraquat induced pulmonary fibrosis in ratsBackground and aims Paraquat poisoning often causes severe damage to multiple organs such as lung,digestive tract,liver,kidney,heart and brain.Lung injury is the most obvious,the early performance of alveolar epithelial cells damage,inflammatory infiltration,late stage of pulmonary interstitial fibrosis,and eventually died of respiratory failure.Many of the surviving patients have pulmonary fibrosis.It is characterized by epithelial cell injury,myofibroblast aggregation and excessive deposition of extracellular matrix elements such as collagen,which leads to the loss of lung function.The differentiation of lung fibroblasts into muscle fibroblasts is a key step in the development of tissue fibroblasts.The pathogenesis of pulmonary fibrosis involves many signal systems,such as transforming growth factor-β1(TGF-β1)/Smads pathway,MAPK pathway,PI3K/Akt pathway and so on.Among them,TGF-β1/Smads signaling pathway plays an important role.In this study,the effect of tacrolimus on TGF-β1/Smads signaling pathway in paraquat induced pulmonary fibrosis rats was observed to explore the mechanism of tacrolimus in the treatment of pulmonary fibrosis.Methods 100 healthy male SD rats were randomly divided into control group,PQ group,TAC low group,TAC medium group and TAC high group with 20 rats in each group.PQ poisoning model was established by intragastric administration of 20%PQ solution at a dose of 50 mg/kg,while the control group was given 1 ml normal saline.The low,medium and high dose tacrolimus groups were given 20%PQ solution by gavage at a dose of 50 mg/kg to establish the PQ poisoning model.Then,tacrolimus was given 0.2 mg/kg,0.5 mg/kg and 0.8 mg/kg by gavage once a day.Rats in each group were killed 7,14,21 and 28 days after paraquat poisoning.The levels of serum TGF-β1,Smad3 and Smad7 were detected by ELISA;the lung tissue structure and collagen deposition were observed by HE staining and Masson staining;the ultrastructural changes of lung were observed by transmission electron microscope;the protein expressions of TGF-β1,Smad3 and Smad7 were detected by Western blot and immunofluorescence.Results After paraquat poisoning,the serum levels of TGF-β1 and Smad3 increased.After tacrolimus treatment,the levels of TGF-β1 and Smad3 decreased,while the level of Smad7 increased.With the increase of tacrolimus concentration,the difference was statistically significant(P<0.05).HE staining,Masson staining and electron microscopy showed that the early pathological manifestations were congestion and edema of alveolar wall,exudation of red blood cells in alveolar cavity,infiltration of neutrophils and other inflammatory cells,vacuolization of lamellar bodies,swelling of mitochondria,proliferation of fibroblasts and deposition of matrix collagen,destruction of normal structure of lung tissue,widening of alveolar septum and collapse of alveoli.After treatment with different doses of tacrolimus,it was reduced compared with paraquat group at the same time.Western blot results showed that compared with the control group,the expression levels of TGF-β1 and Smad3 in paraquat group and tacrolimus group were increased at 7,14,21,28 days(P<0.05).The expression of TGF-β1 and Smad3 protein increased with the increase of poisoning days.The expression of TGF-β1 and Smad3 decreased after tacrolimus treatment.The expression of Smad7 increased with the increase of tacrolimus dosage and days.Immunofluorescence results showed that TGF-β1,Smad3 and Smad7 were expressed in cytoplasm and nucleus,mainly in cytoplasm.The expression of TGF-β1 and Smad3 in paraquat group increased significantly with time.Tacrolimus reduced the protein expression of TGF-β1 and Smad3.The expression of Smad7 increased with time.Tacrolimus up regulated the expression of Smad7.Conclusion Tacrolimus can reduce paraquat induced pulmonary fibrosis,and the effect is more obvious with the increase of dosage.The mechanism may be related to the inhibition of TGF-β1 and Smad3 and the increase of Smad7 level.