Study On The Effect And Mechanism Of Polydatin On The Pulmonary Fibrosis In Rats Induced By Bleomycin

ObjectiveTo study the effect and mechanism of polydatin(PD)on bleomycin-induced pulmonary fibrosis in rats.MethodsSD rats were randomly divided into blank control group,model control group,low dose group of polydatin(20mg/kg),medium dose group of polydatin(40mg/kg),high dose group of polydatin(80mg/kg),low dose group of resveratrol(20mg/kg),medium dose group of resveratrol(40mg/kg),high dose group of resveratrol(80mg/kg),pirfenidone group(50mg /kg),A total of 9 groups.Bleomycin(BLM)was intratracheally instilled into lung tissue to establish rat pulmonary fibrosis model.After the instillation of BLM,body weight changes were recorded every day.Four rats were randomly sacrificed on the 7th and 14 th days.The lung tissues were preserved in 4% paraformaldehyde,followed by Hematoxylin-Eosin(HE)staining and Masson staining.On the 28 th day,the remaining rats were sacrificed,lung tissues were taken,some of them were stained with HE staining and Masson staining,to observe the changes of pulmonary fibrosis and inflammation when compared with the 7th and 14 th days;Other lung tissue was used to measure the content of hydroxyproline(HYP)with the alkaline water method to reflect the degree of pulmonary fibrosis.The rats in the latter group were grouped and model in the same way of previous group.the rats were sacrificed in the fourth week,and the lungs were removed.The levels of inflammatory cytokine Tumor necrosis factor-?(TNF-?)and interleukin-6(IL-6)were determined by enzyme-linked immunosorbent assay(ELISA))to reflect the effect of polydatin and resveratrol on inflammation;The content of Malondialdehyde(MDA)in lung tissue was measured by TBA method.The content of myeloperoxidase(MPO)in lung tissue was detected by kit to reflect the effect of polydatin and resveratrol in oxidative stress.By themethod of Real time PCR(RT-PCR),the expression of E-calcium adhesion proteins(E-cadherin,E-cad)and Alpha Smooth muscle actin(?-SMA),Vimentin,?type Collagen(Collagen ?)mRNA were detected to reflect the effect of polydatin on the process of epithelial mesenchymal transformation(EMT).Western Blot(WB)was used to detect the protein expression of transforming growth factor beta 1(TGF-? 1),phosphorylated Smad3(P-Smad3)and phosphorylated extracellular regulated protein kinase 1/2(ERK1/2),To investigate whether polydatin can regulate the EMT process by regulating the classical TGF-? 1/Smad signaling pathway and the TGF-?1 non Smad signaling pathway.ResultsAfter intratracheal instillation of bleomycin,lung sections were observed by HE staining and Masson staining.The model group showed more obvious inflammatory reaction on 7 days,mainly for neutrophils and macrophages infiltration.With the passage of time,on 14 days?28 days,the degree of inflammation showed a slowing trend;After instillation,the degree of fibrosis showed a gradual increase with time,manifested as significant damage to the alveolar structure,alveolar wall thickening,alveolar septum thickening,alveolar cavity diminished or even disappeared,by Masson staining,collagen content that were stained as light blue increased significantly.When compared with the model group,the treatment of polydatin(20mg/kg,40mg/kg,80mg/kg),resveratrol(20mg/kg,40mg/kg,80mg/kg)and pirfenidone(50mg/kg)show a effect of slowing the development of inflammation and fibrosis.On 28 days,HYP content in lung tissue of rat was detected,and the HYP content in the control group was significantly higher than that in the blank control group(P< 0.01).the low,medium and high dose group of polydatin and resveratrol show a effect that could significantly reduce the content of HYP caused by BLM(P< 0.05).In addition,compared with resveratrol,in the corresponding dose group,the polydatin showed better performance in reducing HYP content,but did not show significant statistical difference(P>0.05).And the effects of polydatin and resveratrol on the prevention and treatment of pulmonary fibrosis were demonstrated.In the aspect of mechanism research,on 28 days,the inflammatory factor TNF-? and IL-6 were measured.BLM instillation significantly increased the content of TNF-? and IL-6(P<0.01).The administration of low,middle and high doses of polydatin and resveratrol,pirfenidone could significantly reduce the level of TNF-?and IL-6(P <0.01).The contents of MDA and MPO in lung tissue of BLM rats were significantly increased after four weeks(P<0.01).Low,medium and high doses of polydatin and resveratrol,and pirfenidone intervention,showed a significant decrease in MDA and MPO levels,with statistical significance(P<0.01).In the effects on TNF-?,IL-6,MDA and MPO,the relative doses of polydatin and resveratrol showed similar effects(P>0.05).The mRNA expression of EMT related protein was detected by RT-PCR.It was found that in BLM induced rats,the expression of E-cadherin gene in lung tissue increased significantly(P<0.01),and the gene expression of alpha-SMA,Vimentin and Collagen I decreased significantly(P<0.01).The administration of low,medium and high doses of polydatin and pirfenidone can significantly reverse the the up-regulation of the expression of E-cadherin,the down-regulation of the expression of ?-SMA,Vimentin and Collagen I(P<0.01).Thus the EMT process is slowed down.The detection of the expression of key protein of TGF-?1,p-Smad3 and P-ERK1/2 in TGF-?1signaling pathway found that BLM could significantly increase the expression of TGF-?1,p-Smad3 and P-ERK1/2(P <0.01).And low,medium and high doses of polydatin and pirfenidone showed a significant inhibitory effect on the expression of TGF-?1,p-Smad3 and P-ERK1/2(P <0.01).Conclusion1.BLM tracheal instillation can cause obvious fibrotic lesions in rats,in the course of fibrosis caused by it,the inflammation in the early stage is more serious.With the passage of time,the inflammation gradually slows down,and the pathological changes of fibrosis become more serious.Pulmonary fibrosis was the main disease in the later stage.Polydatin,resveratrol and pifenidone can slow the process of fibrosis and play a preventive and therapeutic role.The content of HYP suggests that polydatin and resveratrol may play a similar role in the improvement of pulmonary fibrosis.2.In BLM-induced pulmonary fibers,accompanied by inflammation and oxidative stress,polydatin and resveratrol can play a similar role in slowing the inflammatory reaction and oxidative stress in the fibrosis and play a role in inhibiting the pulmonary fibrosis.3.Polydatin can reduce the production of fibroblasts / myofibroblasts by inhibiting the epithelial-interstitial transformation in pulmonary fibrosis,thereby reducing the production and accumulation of extracellular matrixproteins and improving the fibrosis symptoms.It can inhibit the process of epithelial-mesenchymal transformation by inhibiting the activation of Smad3 signaling pathway and ERK1/2 signaling pathway mediated by TGF-?1.