| Section One:The dynamic activation pattern of Hegdghog signaling pathway in paraquat induced pulmonary fibrosis in ratsBackgroud and aimsParaquat is a highly toxic herbicide that selectively devastates the lung through the oxidative and inflammatory processes,which irreversibly progresses to extensive pulmonary fibrosis.The mechanism of lung injury and pulmonary fibrosis is not entirely clear.Studies have shown that the pathogenesis of pulmonary fibrosis include oxidative stress,alveolar inflammation,cytokines,signal network,connective tissue hyperplasia,protease imbalances,abnormal gene expression,and calcium overload.Paraquat-induced pulmonary fibrosis is mainly manifested as abnormal proliferation of pulmonary interstitial fibroblasts and collagen deposition in the extracellular matrix,and affects alveolar epithelial cells and pulmonary vessels in a few cases.Studies have shown that transforming growth factor-betal(TGF-β1)is the key cytokine of paraquat-induced pulmonary fibrosis,and its involvement in ERK and Smad-related pathways plays an important role in the development of paraquat pulmonary fibrosis.The role of Hegdgehog(Hh)signaling pathway in hepatic fibrosis has been studied.This study was to investigate the expression of Hh signaling pathway in rats with paraquat-induced lung fibrosis and to explore the role of Hh signaling pathway in the pathogenesis of paraquat pulmonary fibrosis.Methods6-8 weeks Wister rats were randomized into normal control group(n=30)and paraquat groups(n=30*3,20%paraquat dose,10/50/100 mg/kg).The normal control group(Cont)was fed with distilled water,while the paraquat groups were treated with different doses of paraquat for gavage to imulat the pulmonary fibrosis caused by oral paraquat.We found the suitable dose(50mg/kg,PQ group)for paraquat-induced pulmonary fibrosis by observating the general condition(mental state,feeding,coat color,activity,weight,etc.).The rats in PQ group and Cont group were sacrificed respectively on the 1st,3rd,7th,14th,21th,28th day to collect serum specimens for monitoring SOD,MDA,GSH and lung tissues for hydroxyproline(Hyp)level.The degree of pulmonary fibrosis of rats in different periods was evaluated by HE staining and Masson staining.The methods of immunofluorescence,immunohistochemistry,PCR and Westeon-blot were applied to analyze the lung tissue factor Gli1,Shh,ERK,Smad3 and TGF-β1 in every group.Results1.The average body weight of control group rats increased steadily with feeding time,while the weight of PQ group rats increased very slowly from the 3rd to the 7th day,and had statistical difference(P<0.05)to the control group.The weight of the PQ group rats began to increase after the 14th day,When there was no statistical difference(P>0.05)from the 21st day to the 28th day.2.Gavaged the volume fraction of 20%paraquat dose 50 mg/kg to rats can simulate paraquat poisoning process,which underwent the period of alveolar congestion edema,acute alveolitis,and pulmonary fibrosis.This experiment showed that the blood SOD,GSH content of PQ group decreased obviously from the 1st day to the 7th day compared with the Cont group,which had statistical difference(P<0.05),but there was no statistical difference(P>0.05)from the 14th day to the 28th day.The blood MDA content of PQ group obviously increased,compared with the Cont group,from the 1st day to the 7th day,and the difference was statistically significant(P<0.05).The MDA content had a drop on the 14th day,then there was no statistical difference from the 14th day to the 28th day(P>0.05).3.Compared with the control group,the lung Hyp content of PQ group increased from the 3rd day(P>0.05),continued to rise on the 7th day(P<0.05),and increased significantly from the 14th day to the 28th day,which was consistent with the morphological observation.4.HE staining showed that the lung tissue structure of control group was normal,but the lung tissue of PQ group showed the dynamic changes from the alveolar inflammation to fibrosis in chronological order.Early poisoning symptoms were characterized by severe alveolar inflammatory exudation,and a large number of inflammatory cells infiltrating alveolar interval from the 1st day to the 7th day.Late poisoning symptomswere characterized by the alveolar atelectatic and disappearing,the gradual thickening of alveolar interval,and the mass proliferation of fibroblasts from the 14th day to the 28th day.Masson staining showed that the lung tissue structure of control group was normal,and obvious hyperplasia of collagen fibers,fibrous thickening,and collagen fiber irregular arrangement presented were observed in paraquat group.5.The expression of lung tissue ERK,TGF-betal,Smad3 and Gli1 in paraquat group was significantly increased by immunofluorescence and immunohistochemistry.The results of RT-PCR and Western-Blot showed that the lung tissue Glil,Shh,ERK,TGF-beta 1 and Smad3 protein in paraquat group were higher than that in control group,and the highest expression of Gli1 and Shh was on the 21st day,but the highest expression of TGF-betal,Smad3,ERK was on the 14th day,which was statistically different to the control group(P<0.05).Conclusion1.We successfully reproduced pulmonary fibrosis model by gavaging 50mg/kg 20%concentration paraquat into the male wister rats(6-8weeks old).The rats poisoned by paraquat experenced the early alveolar inflammation and the late hyperplasia and fibrosis.2.Hh signaling pathway was quiescent in the nomarl rats,but it was activated after the rats poisoned by paraquat.The activation of Hh pathway was associated with the progress of pulmonary fibrosis,so we inferred that Hedgehog patheway has an important and potential role in the development of pulmonary fibrosis.Section two:The preventative and therapeutic effects of cyclopamine and Gant61 in paraquat induced pulmonary fibrosis in ratsBackground and aimsCyclopamine is a specific inhibitor of Hh signaling pathway,which has the effect of anti-fibrosis and anti-tumor.Gant61 can suppress Gli1,Gli2 transcription and the binding ability of Glil,thus can inhibite the Hedgehog signal pathway.The purpose of the experiment was to explore whether cyclopamine and Gant61 could ameliorate pulmonary fibrosis induced by paraquat in rats,and study whether their protective effects was achieved by inhibiting the pathologically activated Hh signaling patheway,and investigate the relationship between Hh and ERK/Smad pathways.Methods6-8 weeks Wister male rats were randomly assigned to 5 groups:the normal control group(Cont,n=30),paraquat group(PQ,n=30),Cyclopamine treatment group(Cyc,n=30),Gant61 treatment group(Gant,n=30)and SB431542 treatment group(SB,n=30).Each intervention group was given a concentration of 20%,the dose of 50mg/kg of paraquat for one-time gavage to establish the pulmonary fibrosis rat model.Then the preventive groups respectively received cyclopamine(5mg/kg/d),Gant61(200ug/kg/d),SB431542(100ug/kg/d)intraperitoneal injection.All the rats were sacrified respectively on the 1st,3rd,7th,14th,21th,28th day to collect serum specimens for monitoring SOD,MDA,GSH and lung tissues for hydroxyproline(Hyp)level.The degree of pulmonary fibrosis of rats in different periods was evaluated by HE staining and Masson staining.The methods of immunofluorescence,immunohistochemistry,PCR and Western-blot were applied to analyze the lung tissue factor Gli1,Shh,ERK,Smad3 and TGF-β1 in every group.Results1.The initial average body weight of every group had no difference,but weight increasing very slowly occurred from the 3rd day to the 7th day after the rats poisoned by paraquat.Compared with PQ group,the body weight of every preventive group inceased slowly on the 7th day(P<0.05),and reached to the control level on the 21st day(P>0.05).2.The concentration of blood MDA of every preventive group increased unconspicously.The level of MDA in SB group and Cyc group on the 1st day,and in Cyc group and Gant group on the 3rd day had statistical difference,compared with PQ group(P<0.05).The content of SOD and GSH became lower in every preventive group than the control group,but still higher than the PQ group,and the decreasing duration was shorter than the PQ group.Compared with the paraquat group,there was statistical difference on the 3rdday and the 7th day(P<0.05),but there was no statistical difference on the 21st day and the 28th day(P>0.05).3.The lung tissue hydroxyproline of every preventive group in rats has a similar trend with the paraquat group,but after treatment,the increased Hyp level of every preventive group was slower than the PQ group.Compared with the control group,there was no statistical difference on the 3rd day in every preventive group(P>0.05),while there was statistical difiference on the 7th day,the 14th day and the 28th day(P<0.05).Compared with the PQ group,the Hyp level of every preventive group had no statistical difference(P>0.05)on the 3rd day and the 7th day;but has statistical difference on thel4th day and on the 28th day(P<0.05).4.HE and Masson staining showed that the trend pathology changes were similar between every preventive group and the PQ group,and it also showed a dynamic change from alveolar inflammation to pulmonary fibrosis in chronological order,but the degree of every preventive group is lighter than PQ group.The alveolar inflammation scores of SB group on the 3rd,7th day,Cyc group on the 1st,3rd,7th day,Gant group on the 3rd,7th,14th day were lower than the PQ group and the differences were statistically significant(P<0.05).While the statistical differences of pulmonary fibrosis score occurred on the 14th,21st,28th day between the preventive groups and the PQ group(P<0.05).5.The results of immunofluorescence,immunohistochemistry,RT-PCR and Western-blot showed the expression patterns of Glil,TGF-betal,ERK,Smad3,Shh of each preventive group were similar to the paraqut group,but the expression levels were downgraded.The Gli1 expression levels of each preventive group began to increase on the 7th day,and reached the peak on the 21St day,and the expression levels of every preventive group was lower than the paraquat group.The expression differences were statistically significant(P<0.05)between the control group and the paraquat group or the control group on the 7th,14th,21st,28th day.The TGF-betal,Smad3 and ERK expression levels began to increase on the 7th day,and reached the peak on the 14th day,but began to decrease on the 21st day.Conclusion1.Cyclopamine and Gant61 may delay the progress,and relieve the symptoms of pulmonary fibrosis induced by paraquat,and its mechanism is related to the inhibition of the abnormal activaty of Hh signaling pathway.Just like Cyclopamine and Gant61,SB431542 can also play a similar role by suppressing ERK/Smad signaling pathway mediated by TGF-betal.2.The results showed that the increase of Hyp content happened in the late stage of PQ poisoning.Cyclopamine,Gant61,SB431542 intervention can delay the hyp rise time,and reduce the degree of increase,suggesting that Cyclopamine,Gant61,SB431542 can effectively inhibit the proliferation of collagen fibers in lung tissue and the deposition of extracellular matrix,play an important role in anti-pulmonary fibrosis.3.The Hedgehog signaling regulated by Cyclopamine and Gant61 may have a certain interaction with ERK/Smad signaling pathway mediated by TGF-β1 in the prosess of paraquat lung fibrosis.The Hedghog signaling pathway and TGF-β1 signaling pathway are activated by paraquat,whereas Cyclopamine,Gant61 and SB431542 inhibit the effects of paraquat.Section there:The research between autophagy and pulmonary injury induced by paraquat in ratsBackground and aimsAutophagy is the natural,regulated,destructive mechanism of the cell that disassembles unnecessary or dysfunctional components.Autophagy rarely occurs in normal cells,which has the characteristics of conservatism and inducibility.It is inconclusive whether the role of autophagy in paraquat-induced lung injury is positive or negative,so it is worthy of further investigation.The LC3 proteins are the marker protein on the autophagic membrane,and Beclin-1 is an essential molecule in the process of autophagosome formation,so we selected the two proteins to study the autophagy of paraquat-induced lung injury.The effects of autophagy on the occurrence of pulmonary fibrosis induced by paraquat were observed by electron microscopy.MethodsRats were divided into normal control group(Cont,n = 30),paraquat group(PQ,n =30),6 subgroups per group,5 rats in each subgroup.The autophagy of lung tissue after paraquat exposure was observed on the 1st,3rd,7th,14th,21th,28th day by the transmission electron microscopy(TEM).The expression of Beclin-1 and LC3 was also assayed by Western-blot.Results1.There was no obvious abnormal change in lung ultrastructure of Cont group.Compared with the Cont group,the observation of PQ group showed that autophagosomal precursors and primary lysosomes were observed on the 7th day.Autophagosomal precursors and autophagosomes were visible on the 14tth day.A large number of autophagosomes and lysosomes appeared in the cells on the 21th day.The lysosomes became turbid,swollen and vacuolized on the 28th day.2.Compared with the Cont group,the expression of LC3 and Beclin-1 in PQ group gradually changed with time,which began to increase after exposure,reached the peak on the 14th day,and decreased on the 28th day,but still higher than the expression of Cont group.ConclusionCell autophagy is involved in the process of pulmonary fibrosis induced by paraquat in rats,which results in the pulmonary fibrosis aggravation in rats,and plays an important role in the occurrence and development of pulmonary fibrosis induced by paraquat. |
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