| Paraquat(PQ)stands out as a herbicide with high efficiency and low cost,which has been extensively applied in agricultural production throughout the world.At the present time,it still remains in use in agricultural production in over 100 countries and regions such as the United States,Japan,Australia and New Zealand.As a result of improper storage or use,PQ poisoning has proven to be a common cause of pesticide poisoning fatalities in major agricultural countries.Since 2020,in spite of the fact that the ban on the sale of PQ in China has brought significant results,patients with PQ poisoning have still been identified clinically as a result of mixing PQ with diquat,glyphosate and other herbicides or being used under the guise of PQ.Given the uncertainty about the mechanism of PQ poisoning and the deficiency of specific antidotes,the death rate of PQ poisoning is exceptionally high.In this context,PQ poisoning has turned out to be a major public health issue in major agricultural countries worldwide,as well as an issue in urgent need of solution in the medical field.With regard to the surviving patients with PQ poisoning who were successfully resuscitated in the Emergency Department and the Intensive Care Medicine Department,most of them fell into the category of moderate to heavy patients who ingested PQ at doses of 20-40 mg/kg.The persistent hypoxemia and respiratory failure as a result of PQ-induced progressive pulmonary fibrosis in the late stages of the lung remained the primary cause of death in this group of patients.At the present time,there has no effective therapeutic strategy to ameliorate the poor prognosis of PQ-induced pulmonary fibrosis,which has imposed a tremendous economic burden on society,while also inflicting a heavy emotional blow to the family members of patients.As a consequence,an effective therapeutic strategy for targeted palliation of PQ-induced pulmonary fibrosis would usher in new hope for enhancing the survival rate of patients with moderate to severe PQ poisoning.The essence of pulmonary fibrosis caused by PQ poisoning lies in the fact that PQ leads to the death of a substantial number of normal alveolar epithelial cells,destruction of normal alveolar tissue structure,lung tissue undergoing abnormal repair,the differentiation of alveolar mononuclear fibroblasts into myofibroblasts,and excessive deposition of extracellular matrix(ECM),which eventually results in abnormal lung tissue structure and irreversible impairment of lung function as characterized by pulmonary fibrosis remains unchanged.For this reason,the reduction of PQ-induced massive alveolar epithelial cell death represents an effective strategy to alleviate PQ-induced pulmonary fibrosis.The prior studies have identified PQ-induced mitochondrial dysfunction in lung epithelial cells as the primary cause of lung epithelial cell death,while targeted improvement of mitochondrial dysfunction would salvage lung epithelial cell death.In the preliminary experiments of this study,it was explored that lung tissue suffered from T3 deficiency after PQ-induced pulmonary fibrosis occurred in mice.In conjunction with the available studies,it has been demonstrated that thyroid hormone(TH)exhibits the capability to induce mitochondrial biogenesis,while exerting positive anti-fibrotic effects on critical organs.In this study,the focus lies on the improvement of mitochondrial dysfunction in lung epithelial cells,for which an innovative research was conducted to examine the effect of T3 on PQ-induced pulmonary fibrosis,as well as to probe the mechanism by which the thyroid hormone receptorα(THRβ)downstream of T3 serves as a transcription factor to regulate the transcription of downstream target genes.Ultimately,it was elucidated that T3 could activate THRβ to promote peroxisome proliferatoractivated receptor-γ coactivator-1α(PGC-1α)expression could mitigate PQ-induced pulmonary fibrosis.In the in vitro study,this project clarified that PQ induction provoked mitochondrial dysfunction and apoptosis in mice lung epithelial MLE-12 cells by assaying cellular ATP levels,mitochondrial membrane potential by TMRM staining,apoptosis pathway proteins BAX and BCL-XL expression by Western bolt,and apoptosis rate by TUNEL method,while T3 could mitigate the PQ-induced cell injury.In the in vivo study,it was well defined that T3 nebulization could mitigate PQ-induced pulmonary fibrosis in mice by HE staining of lung tissue,Masson staining,α-SMA expression,and hydroxyproline levels.With regard to the upstream mechanism,on the basis of the prior studies,it has confirmed that the biological effects exerted by TH are predominantly mediated by the thyroid hormone receptor(THR).In this project,the protective effect of T3 on PQ-induced MLE-12 cells in vitro experiments was clarified to be achieved by activating and regulating THRβ through the THRβ-specific antagonist NH-3 and the assays on the variations of THRβ protein expression.Meanwhile,the experimental results that nebulization with THRβ-specific agonist Sobetirome can mitigate PQ-induced pulmonary fibrosis in mice were verified in the in vitro experiments.With regard to the downstream mechanism,this study elucidated the binding of transcription factor THRβ to the promoter region of PGC1α gene by transcription factor database screening,dual luciferase reporter gene experiment,ChIPqPCR,and other experiments,while also elucidating the binding sites of-2045 bp to-2031 bp,and-1058 bp to-1044 bp(transcription start site was+1).Furthermore,it was clarified by RT-qPCR and Western bolt that T3 contributed to PGC1α expression by activating THRβ.A PGC1α overexpression lentivirus was constructed to establish a steadytransformation cell line of PGC1α overexpression MLE-12 cells,so as to clarify the protective effect of PGC1α overexpression on PQ-induced MLE-12 cells in in vitro experiments.In in vivo experiments,PGC1α overexpression adeno-associated virus was transfected by tracheal drip to accomplish PGC1α overexpression in mice lung tissues,which in turn clarified that PGC1α overexpression alleviated PQ-induced pulmonary fibrosis in mice.Lastly,a "rescue assay" was designed by means of PGC1α knockdown lentivirus and PGC1α knockout mice to elucidate that T3 protects PQ-induced MLE-12 cells through the THRβ/PGC1α axis in vitro experiments,as well as to verify that T3 alleviates PQ-induced pulmonary fibrosis in mice through the THRβ/PGC1α axis in vivo experiments.In this study,on the basis of exploring the T3 deficiency in the lung tissue of PQinduced pulmonary fibrosis in mice through various experimental techniques both in vitro and in vivo,it was identified that T3 alleviates PQ-induced pulmonary fibrosis in mice through the THRβ/PGC1 axis,as well as elucidating at the mechanistic level that T3 activates THRβ as a transcription factor to promote PGC1α transcription,in addition to elucidating the binding sites of the transcription factor THRβ to the promoter region of PGC1α gene.This project has pioneered to uncover the protective effect and mechanism of T3 on PQ-induced pulmonary fibrosis.The research results will furnish a pre-theoretical basis for the clinical translation of T3 nebulization for the treatment of patients with pulmonary fibrosis caused by PQ poisoning,while further animal experiments as well as pre-clinical studies are likely to introduce new treatment strategies for patients with PQ poisoning.Part Ⅰ.T3 Activates and Regulates THRβ to Mitigate Paraquat-Induced Pulmonary Fibrosis in MiceObjective:1.To probe whether there exists T3 deficiency in lung tissue during paraquat(PQ)induced pulmonary fibrosis in mice.2.To examine whether T3 can protect PQ-induced lung epithelial cells(MLE-12 cells)in mice in in vitro experiments,and to elucidate the potential mechanism.3.To exploit whether T3 nebulization can mitigate PQ-induced pulmonary fibrosis in mice in in vivo experiments,and to elucidate its potential mechanism.Methods:1.To establish a PQ-induced pulmonary fibrosis model in mice:The 7-week-old C57 male mice were separated into two groups of 6 mice each,with PQ(20 mg/kg)and saline injected intraperitoneally on Day 1 in each group,and paraffin-embedded sections of lung tissues were taken from two groups of mice on Day 29,while the establishment PQ-induced pulmonary fibrosis model in mice was clarified to be a success by detecting HE staining,Masson staining,α-SMA expression and hydroxyproline level in lung tissues.2.To investigate the presence of T3 deficiency in lung tissues when PQ-induced pulmonary fibrosis occurs in mice:a PQ-induced pulmonary fibrosis model in mice was established,another group of mice was injected intraperitoneally with saline for control,with the tissue taken on the Day 29.Western blot was performed to assay the protein expression of type 2 iodothyronine deiodinase(DIO2)of lung tissue in mice.Meanwhile,the serum T3 level and HE staining of thyroid tissue of mice were detected.3.To establish a PQ-induced lung epithelial cell(MLE-12 cell)injury model in mice:MLE-12 cells were induced with PQ at different concentrations(100,200,400 and 800 μM)for 24 hours,the cell viability was detected by means of CCK8 method,while the concentration of PQ-induced MLE-12 cell injury model was screened.4.To probe whether T3 exerts a protective effect on PQ-induced MLE-12 cells:By means of detecting cellular ATP levels,mitochondrial membrane potential by TMRM staining,expression of apoptotic pathway proteins BAX and BCL-XL by Western bolt and apoptosis rate by TUNEL method,it was clarified that PQ induced mitochondrial dysfunction and apoptosis in MLE-12 cells,so as to explore whether T3 could mitigate the PQ-induced cell injury.5.To probe whether T3 nebulization can mitigate PQ-induced pulmonary fibrosis in mice:On the basis of establishing a PQ-induced pulmonary fibrosis model in mice,mice in the treatment group were administered T3 nebulization inhalation,while the HE staining,Masson staining,α-SMA expression and hydroxyproline level of lung tissue were detected to ascertain whether T3 nebulization can mitigate PQ-induced pulmonary fibrosis in mice.6.To probe the protective mechanism of T3 on PQ-induced MLE-12 cells in in vitro experiments:In PQ-induced MLE-12 cells,NH-3,an antagonists of THRβ,was applied on top of T3 treatment to observe whether the protective effect of T3 on MLE12 cells disappeared when NH-3 and T3 processed the cells concurrently,while THRβexpression variations were observed by Western bolt to examine whether T3 exerted cytoprotective effects by activating and regulating THRβ.7.To exploit the protective mechanism of T3 on PQ-induced pulmonary fibrosis in mice in an in vivo experiment:A PQ-induced pulmonary fibrosis model in mice was established to observe whether nebulization with THRβ agonist Sobetirome could mitigate PQ-induced pulmonary fibrosis in mice.Results:1.PQ-induced pulmonary fibrosis model in mice could be established by intraperitoneal injection of PQ(20 mg/kg)for 28 days.2.Following PQ-induced pulmonary fibrosis in mice,thyroid follicular epithelial cells were gravely damaged,DIO2 expression in lung tissue was elevated,and serum T3 levels were declined.3.The MLE-12 cells treated with PQ 800 μM for 24 hours could establish a PQ cell injury model.4.T3 protected PQ-induced MLE-12 cells by ameliorating mitochondrial dysfunction and anti-apoptotic cell death.5.T3 nebulization mitigated PQ-induced pulmonary fibrosis in mice by ameliorating lung tissue structure in mice,diminishing collagen deposition in lung tissue,and decreasing the differentiation of fibroblasts to myofibroblasts.6.T3 promoted THRβ expression,which partially reversed the PQ-induced decrease in THRβ expression.The mitochondrial protective and anti-apoptotic effects of T3 on PQ-induced MLE-12 cells were lost,when NH-3 and T3 processed cells concurrently.7.Sobetirome nebulization alleviated PQ-induced pulmonary fibrosis in mice by ameliorating lung tissue structure in mice,diminishing collagen deposition in lung tissue,as well as decreasing the differentiation of fibroblasts to myofibroblasts.Conclusion:1.There probably exists T3 deficiency in the lung tissue of mice following PQ-induced pulmonary fibrosis.2.T3 protects PQ-induced MLE-12 cells by activating and promoting THRβ expression.3.T3 mitigates PQ-induced pulmonary fibrosis in mice by activating THRβ.Part Ⅱ.The Mechanism of Activation of THRβ and Promotion of PGC1α Transcription by T3Objective:1.To probe whether T3 in PQ-induced MLE-12 cells can promote THRβ into the nucleus.2.To examine whether THRβ as a transcription factor binds to the promoter region of PGC1α gene,and to elucidate the binding sites of transcription factor THRβ to the promoter region of PGC1α gene.3.To specify that T3 increases PGC1α expression by activating THRβ.Methods:1.To probe whether T3 in PQ-induced MLE-12 cells can promote THRβ into the nucleus,MLE-12 cells were classified into 4 groups:Control group,T3 group:(T3 treated with 15 μg/ml for 24 hours);PQ group:(PQ treated with 800 μM for 24 hours);PQ+T3 group:(PQ treated with 800 μM for 24 hours,replaced T3 treated with 15μg/ml treatment for 24 hours).Cytoplasmic proteins and cytosolic proteins were extracted separately from each group of cells,while THRβ expression was assayed by Western blot in each group of cells.2.RT-qPCR was performed to assay the effect of T3 on the expression of genes related to mitochondrial quality control in MLE-12 cells,as well as to predict the possible target genes of transcription factor THRβ by JASPER database.3.To examine whether THRβ binds to the promoter region of PGC1α gene and the binding sites by dual luciferase reporter gene assay,and to verify whether THRβ can enrich the DNA fragment of the promoter region of PGC1α gene at the corresponding sites by ChIP-qPCR.4.NH-3 and T3 were treated with MLE-12 cells,to clarify whether T3 promotes PGC1αexpression through activation of THRβ by Western blot assay.Results:1.T3 promoted THRβ transfer from cytoplasm to nucleus,while T3 in PQ-induced MLE-12 cell could still promote THRβ transfer from cytoplasm to nucleus.2.The mRNAs of AMPK,PINK1,PGC1α,TFAM,NRF1 and UCP2 were elevated after T3 treated with MLE-12 cells for 12 hours,among which the mRNA of PGC1α was elevated about 1.56-fold.3.The transcription factor THRβ could bind to the promoter region of PGC1α gene,with two binding sites of THRβ to the promoter region of PGC1α gene,namely-2045 bp to-2031 bp,and-1058 bp to-1044 bp(the start site of transcription was+1).4.T3 increased PGC1α protein expression in MLE-12 cells,while NH-3 could block the effects of T3-promoted PGC1α expression in MLE-12 cells.Conclusion:1.T3 promotes THRβ cytoplasmic transfer into the nucleus,while T3 also promotes THRβ transfer from the cytoplasm into the nucleus during PQ induction.2.The transcription factor THRβ binds to the promoter region of PGC1α gene with binding sites being-2045 bp to-2031 bp,and-1058 bp to-1044 bp(the start site of transcription is+1).3.T3 increases PGC1α protein expression by activating THRβ.Part Ⅲ.T3 Activates THRβ to Promote PGC1α Expression to Mitigate Paraquat-Induced Pulmonary Fibrosis in MiceObjective:1.To explore whether PGC1α overexpression exerts a protective effect on PQ-induced MLE-12 cells in in vitro experiments.2.To explore whether PGC1α overexpression can mitigate PQ-induced pulmonary fibrosis in mice in in vivo experiments.3.To elucidate whether T3 protects PQ-induced MLE-12 cells through THRβ/PGC1αaxis by in vitro rescue assay.4.To elucidate whether T3 can mitigate PQ-induced pulmonary fibrosis in mice through THRβ/PGC1α axis by in vivo rescue assay.Methods:1.To explore whether PGC1α overexpression can protect PQ-induced MLE-12 cells in in vitro experiments:MLE-12 cells were transfected by PGC1α overexpression lentivirus,while MLE-12 cells were also screened for stable strains of PGC1αoverexpression.A model of PQ-induced cell injury was established,in which cellular ATP levels,mitochondrial membrane potential by TMRM staining,apoptosis pathway proteins BAX and BCL-XL expression by Western bolt,and apoptosis rate by TUNEL assay were detected.2.To explore whether PGC1α overexpression can mitigate PQ-induced pulmonary fibrosis in mice in an in vivo experiment:PGC1α overexpression in lung tissues of mice was achieved by transfection with PGC1α overexpressing adeno-associated virus.A PQ-induced pulmonary fibrosis model in mice was established,in which HE staining,Masson staining,α-SMA expression and hydroxyproline levels of lung tissues were examined.3.With a view to elucidating T3 protects PQ-induced MLE-12 cells through THRβ/PGC1α axis,cells were transfected by PGC1α knockdown lentivirus,and MLE-12 cells were screened for stable strains of PGC1α knockdown.Furthermore,through in vitro "rescue assay":to probe whether PGC1α overexpression could protect PQ-induced MLE-12 cells on the basis of THRβ inhibitor NH-3 treatment;as well as to explore whether the protective effect of T3 on PQ-induced MLE-12 cells disappeared following PGC1α knockdown on the basis of T3 treatment.4.With a view to elucidating whether T3 alleviates PQ-induced pulmonary fibrosis through the THRα/PGC1α axis,it was explored in an in vivo experiment whether T3 can mitigate PQ-induced pulmonary fibrosis in PGC1α knockout mice.Results:1.PGC1α overexpression could ameliorate mitochondrial dysfunction and anti-apoptotic cell death to protect PQ-induced MLE-12 cells.2.PGC1α overexpression could ameliorate lung tissue structure,minimize lung tissue collagen deposition,decrease the differentiation of fibroblasts to myofibroblasts,as well as mitigate PQ-induced pulmonary fibrosis in mice.3.On the basis of NH-3 treatment,PGC1α overexpression could still ameliorate mitochondrial dysfunction and anti-apoptotic cell death to protect PQ-induced MLE12 cells;following PGC1α knockdown,the protective effect of T3 on PQ-induced MLE-12 cells did not recur.4.The protective effect of T3 nebulization in mitigating PQ-induced pulmonary fibrosis in mice failed to be reproduced in PGC1α knockout mice.Conclusion:1.PGC1α overexpression protects PQ-induced MLE-12 cells.2.PGC1α overexpression mitigates PQ-induced pulmonary fibrosis in mice.3.T3 protects PQ-induced MLE-12 cells through THRβ/PGC1α axis.4.T3 mitigates PQ-induced pulmonary fibrosis in mice through THRβ/PGC1α axis. |
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