| Background and Objective:Pulmonary fibrosis(PF)is the common outcome of interstitial lung disease(ILD)which has different causes.Many patients with interstitial lung disease were proved to be related to connective tissue disease which was known as connective tissue disease associated interstitial lung disease(CTD-ILD).The prevalence of ILD in systemic sclerosis is 60%to 70%.The prognosis of patients with connective tissue disease associated pulmonary fibrosis is poor,and they may be died of severe infection and/or respiratory failure in not long time.Nowadays,the etiology and pathogenesis of pulmonary fibrosis remains unknown,and effective therapeutic agents have not yet been discovered.Currently,comprising corticosteroids with immunosuppressive agents is the most frequently used therapeutic methods,but it has many side effects,adverse reactions and could not improve the poor prognosis.So the study about the pathogenesis and treatment is particularly important.In recent years,researches on the pathogenesis and treatment of pulmonary fibrosis are more,and oxidative stress is one of the most important mechanisms.Oxidative stress indicates an imbalance state between production of reactive oxygen species(ROS)and antioxidant defenses oxidative stress,hence results in all the molecular,cellular and tissue abnormalities.The lungs are particularly vulnerable to oxidative stress compared to other organs because they are exposed to the highest levels of oxygen.The oxygen pressure of inhaled air is 150mmHg,and in alveolar air it is 100 mmHg while in venous blood returning from various other organs it ranges from a high of?45 mm Hg to a low of?1 mmHg.N-acetylcysteine which is a precursor to the antioxidant is partial protection from pulmonary fibrosis.Quercetin(QUE)which present in plants widely,is a representative member of flavonols.It possesses the ability of scavenging the free radicals and chelating the transition metal ions in order to prevent the oxidation of lipoproteins.Therefore,it is believed that quercetin can be a promising agent for the treatment of free radical induced diseases like cancer,chronic inflammation and improve the prognosis of pulmonary fibrosis.The present study was performed to evaluate the therapeutic effects of quercetin in bleomycin(BLM)induced pulmonary fibrosis in Sprague-Dawley rats and effects on oxidative stress;meanwhile,MMP-1,TIMP-1,miR-21 and TGF-?1/Smad signaling passway which are related to oxidative stress were also studied.Methods:Divide 60 Sprague-Dawley rats into 3 groups at random,which were control group,bleomycin group(BLM group)and bleomycin+quercetin group(BLM+QUE group),with 20 rats in each group.Rats in BLM group were given bleomycin(5mg/kg)intratraeheally on the first day to establish pulmonary fibrosis models.Control group were given saline intratraeheally with the same amount.BLM+QUE group were given bleomycin(5mg/kg)intratracheally on the first day and quercetin(3mg/kg)intraperitoneally once a day until they were sacrificed.On day 12,24 and 36,6 rats were sacrificed respectively.Part One:Therapeutic effects of quercetin on bleomycin induced pulmonary fibrosis in rats1.We assessed the general conditions,weight of rats and lung,then calculated the lung coefficient.We also evaluated the degree of inflammation and fibrosis by haematoxylin&eosin(H&E)staining and Masson trichrome staining and calculated the total fibrosis score.2.The total cells in the bronchoalveolar lavage fluid(BALF)were counted,and the different cell types were also counted by Wright-Giemsa staining under microscope.3.We detected the total protein concentration in cell-free BALF and hydroxyproline(HYP)in lung tissue.Part Two:Effects of quercetin on oxidative stress of lung in rats with pulmonary fibrosisMalondialdehyde(MDA)which is the marker of oxidative stress,reduced glutathione,oxidized glutathione,and total antioxidant capacity(T-AOC)of lung tissue were tested by corresponding kits.Part Three:Effects of quercetin on TGF-?1,MMP-1 and TIMP-1 of lung in rats with pulmonary fibrosis1.Total RNA was isolated.The abundance of matrix metalloproteinase-1(MMP-1)and tissue inhibitor of metalloproteinase-1(TIMP-1)mRNA was detected by real time quantative polymerase chain reaction(RT-PCR).2.The expression of MMP-1 and TIMP-1 protein in lung tissue was detected by immunohistochemistry.Part Four:Effects of quercetin on miR-21 and TGF-?1/Smad signaling passway of lung in rats with pulmonary fibrosis1.The abundance of miR-21 was detected by RT-PCR2.The abundance of transforming growth factor-?1(TGF-?1)and Smad7 mRNA was detected by RT-PCR.3.The expression of TGF-?1 and Smad7 protein in lung tissue was detected by western blot.Statistical analysis:All data analysis was performed using SPSS 20.0 software.The results are presented as the mean ? SD.Student's t test was used to compare the data between two groups and one way analysis of variance(ANOVA)was used compare the data between the groups.The differences were considered statistically significant at P<0.05.Results:Part One:1.The general condition of the rats:the fur,activity,appetite and breathing was normal in control group;the body weight increased steadily.The fur,activity,appetite and breathing in BLM group was worse than control group;the body weight decreased significantly on day 12,and increased after day 24,but still significantly lower than control group(day 12,24 and 36:P<0.01).One rat died on day 7 in BLM group.The fur,activity,appetite and breathing in BLM+QUE group was better than BLM group,and body weight was heavier than BLM group(day 12,24 and 36;P<0.01).2.Lung tissue:the appearance,texture and elasticity of lung was good in control group.In BLM group,we found swelling signif-icantly and focal hemorrhage in early stage,and consolidation in late stage.BLM+QUE group was in between.Lung coefficient in BLM group was increased significantly compared with control group(day 12,24 and 36:P<0.05).In BLM+QUE group it was lower than BLM group after day 36(P<0.05).3.Histological examination:we didn't found obvious inflammatory cell infiltration and deposition of collagen at different time in control group.In BLM group,there are a large amount of inflammatory cells infiltration,alveolar septum and a small amount of collagen deposition in early stage;and inflammatory cells infiltration significantly reduced,alveolar structure damaged obviously and interstitial collagen deposition increased in late stage;BLM+QUE group was in between.The total fibrosis score in BLM group was higher than BLM group(day 12,24 and 36:P<0.01);and in BLM+QUE group it was lower than BLM group(day 12:P<0.05,day 24 and 36:P<0.01).4.BALF total cell counts and different cell type counts:change of total cell counts in control group at different time were not obviously.Total cell counts in BLM group were significantly higher than that in control group(day 12,24 and 36:P<0.01).In BLM+QUE group they were significantly lower than that in BLM group(day 12,24 and 36:P<0.05).By different cell type counts we found that macrophages,neutrophils and lymphocytes in control group at different time showed no obvious change.The neutrophil ratio in BLM group was higher than control group,the difference was statistically significant after day 36(P<0.05);the lymphocyte ratio was higher too,the difference was statistically significant after day 12(day 12,24 and 36:P<0.05);the macrophage ratio was significantly lower after day 12(day 12,24 and 36:P<0.05).The neutrophil ratio in BLM+QUE group was significantly lower than BLM group after day 36(P<0.05);the lymphocyte ratio was significantly lower after day 24(day 24 and 36 P<0.05);the macrophage ratio was significantly higher after day 36(P<0.05).5.Total protein in BALF and HYP in lung tissue:no obvious change of total protein was found in control group at different time.Total protein in BLM group significantly increased on day 12,and then decreased gradually,but it was still significantly higher than the control group(day 12 and 24:P<0.05,day 36:P<0.01).After day 24,total protein in BLM+QUE group was significantly lower than that in BLM group(day 24 and 36:P<0.01).No obvious change of HYP was found in control group at different time.HYP in BLM group was obviously higher than that in control group(day 12:P<0.05,day 24 and 36:P<0.01).After day 24,HYP in BLM+QUE group decreased significantly compared with BLM group(day 24 and 36:P<0.01).Part Two:1.No obvious change of MDA was found in control group at different time.On day 12,MDA in the three groups had no statistical significance;after day 24,MDA in BLM group were significantly higher than control group(day 24 and 36:P<0.01).After day 36,MDA in BLM+QUE group was significantly lower than BLM group(P<0.01).2.No obvious change of GSH/GSSH ratio was found in control group at different time.GSH/GSSH ratio in BLM group decreased significantly compared with the control group after day 24(day 24 and 36:P<0.01).In BLM+QUE group,GSH/GSSH ratio was significantly higher than that in BLM group after day 24(day 24 and 36:P<0.01).3.No obvious change of T-AOC was found in control group at different time.T-AOC in BLM group was significantly lower than that in control group after day 24(day 24:P<0.05,day 36:P<0.01).T-AOC in BLM+QUE group increased significantly compared with BLM group after day 24(day 24 and 36;P<0.05).Part Three:1.By RT-PCR we found no significant changes of MMP-1 and TIMP-1 mRNA in control group at different time.MMP-1 and TIMP-1 mRNA in BLM group was significantly higher than that in control group(day12,24 and 36 MMP-1:P<0.01;day 12 TIMP-1:P<0.05,day 24 and 36 TIMP-1:P<0.01).MMP-1 and TIMP-1 mRNA in BLM+QUE group decreased significantly compared with BLM group after day 24(day 24 and 36 MMP-1:P<0.05;day 24 TIMP-1:P<0.05,day 36 TIMP-1:P<0.01).2.By immunohistochemistry we found that MMP-1 and TIMP-1 protein expression in control group at different time did not change significantly.The expression of MMP-1 and TIMP-1 in BLM group increased significantly(day 12,24 and 36 MMP-1:P<0.01;day 12 and 24 TIMP-1:P<0.05,day 36 TIMP-1:P<0.01).MMP-1 in BLM+QUE group decreased significantly compared with BLM group after day 24(day 24:P<0.01,day 36:P<0.05),and TIMP-1 in BLM+QUE group decreased significantly after day 36(P<0.05).Part Four:1.By RT-PCR we found no significant changes of miR-21 level in control group at different time.MiR-21 in BLM group was significantly higher than that in control group(day 12:P<0.05,day 24 and 36:P<0.01).MiR-21 in BLM+QUE group decreased significantly compared with BLM group after day 24(day 24:P<0.05,day 36:P<0.01).2.By RT-PCR we found no significant changes of TGF-?1 and Smad7 mRNA in control group at different time.Compared with control group,TGF-?1 increased significantly(day 12,24 and 36:P<0.01),and Smad7 decreased significantly(day P<0.05,day 24 and 36:P<0.01).Compared with BLM group,TGF-?1 decreased significantly(day 12,24 and 36:P<0.05),and Smad7 increased significantly after day 24(day 24 and 36:P<0.05).3.By western blot we found no significant changes of TGF-?1 and Smad7 protein in control group at different time.Compared with control group,TGF-?1 increased significantly(day 12:P<0.05,day 24 and 36:P<0.01),and Smad7 decreased significantly after day 24(day 24 and 36:P<0.05).Compared with BLM group,TGF-?1 decreased significantly after day 24(day 24 and 36:P<0.05),and Smad7 increased significantly after day 24(day 24 and 36:P<0.05).Conclusions:1.The model of pulmonary fibrosis can be successfully established by intratracheal instillation of BLM in rats.2.Quercetin can obviously improve the general situation of rats with pulmonary fibrosis,alleviate inflammatory damage and reduce collagen deposition of lung in rats with pulmonary fibrosis.3.Quercetin can inhibit the generation of reactive oxygen species and enhance the antioxidant capacity,which indicates that it may exert its antipulmonary fibrosis ability through inhibiting oxidative stress.4.Quercetin can inhibit the expression of TIMP-1 and MMP-1,promote degradation of extracellular matrix,reduce collagen deposition,and prevent pulmonary fibrosis.5.Quercetin can inhibit the expression of TGF-?1 and miR-21,increase the expression of Smad7,interfere with TGF-?1/Smad signaling passway,and prevent pulmonary fibrosis. |
PDF Full Text Request