Effects And Preliminary Mechanism Of Thyroid Hormone T3 On Lung Inflammation And Fibrosis In Mice Exposed To Silica

Silicosis is a systemic disease characterized by progressive pulmonary fibrosis caused by long-term inhalation of free silica during the production process.It is one of the most important occupational diseases in the world.However,the specific pathogenesis of silicosis is still unclear,and there is no effective treatment for silicosis.Thyroid hormone(TH)is involved in regulating immune system homeostasis and energy metabolism,and plays an important role in a variety of pathophysiological processes.Studies have shown that triiodothyronine T3(the main active form of TH)can inhibit inflammation and promote tissue repair.At present,research on the role of T3 in the occurrence and development of silicosis is very limited,and its underlying mechanism is still unclear.Therefore,the main purpose of this study is to explore the role of T3 in inflammation and fibrosis of lung tissue caused by silica and its possible mechanism.By constructing a model of silicosis in mice,the thyorid morphology and function of mice exposed to silica were first observed.Then,two methods of early supplementation(simultaneous silica exposure)and late supplementation(28 days after silica exposure)were used to observe the effect of T3 on lung inflammation and fibrosis caused by silica;and the mechanism of T3 is initially discussed from the perspective of mitochondrial biosynthesis and glycolysis,providing experimental evidence and theoretical basis for the prevention and treatment of silicosis.PartⅠ.The effect of silica exposure on thyroid morphology and function in mice Objective:To observe and analyze the changes of thyroid morphology and function of mice after exposure to silica at different times.Methods:The silicosis model of mice was constructed by single exposing tracheal instillation of silica suspension,and 48 C57BL/6 male mice were randomly divided into silica group and control group according to body weight,and were given 50 μ1 of dust suspension(containing 2.5mg of silica)and the same volume of normal saline,respectively.In each group,8 mice were randomly killed after 7 days,28 days and 84 days of silica exposure,and mouse serum,lung tissue and thyroid tissue were collected.Hematoxylin-eosin(HE)staining,Masson staining for pathological detection,and immunohistochemical detection of lung tissue α-smooth muscle actin(α-SMA)protein content to evaluate the effectiveness of silicosis model.HE staining was used to observe the morphological changes of thyroid tissue in mice exposed to silica;enzyme-linked immunosorbent assay(ELISA)was used to detect the content of T3,free T3(FT3),thyroxine(T4),free T4(FT4)and thyroid-stimulating hormone(TSH)in the serum of mice;Real-time fluorescence quantitative PCR(qRT-PCR)and immunohistochemistry were used to detect the mRNA and protein expression levels of thyroid hormone receptorα(TRα)and thyroid hormone receptor β(TRβ)in the lung tissue of mice.Results:(1)Establishment of mouse silicosis model:Compared with the control group,after 7 days of single tracheal instillation of silica,the inflammatory response of the lung tissue of mice increased significantly,and the inflammatory response remained at a high level after 28 and 84 days of silica exposure;With the prolongation of the modeling time,the fibrosis reaction in the lung tissue of mice gradually increased,and the protein content ofα-SMA increased progressively.The above results indicate that the mouse silicosis model was successfully established.(2)Effects of silica exposure on thyroid morphology in mice:At various time points after silica exposure,the thyroid tissue of the control group showed that the size and shape of the follicles were basically the same,the epithelial cells were cubic,and the glial globulin in the follicular cavity was rich and uniform;In Silica group,follicular epithelial cells in thyroid tissue were diffusely proliferated,and the colloid in the follicular cavity was uneven.The morphology of epithelial cells tended to be flattened.Some follicles were fused or even vacuolated in the later stage of silica exposure.(3)Effect of silica exposure on thyroid function in mice:At each time point after dust exposure,compared with the control group,the levels of serum T3,FT3,T4 and FT4 in the Silica group were significantly reduced,and the differences were statistically significant(P<0.05);The mRNA and protein expression levels of TRα and TRβ in the lung tissue of mice in the Silica group decreased significantly,and the differences were statistically significant(P<0.05).After 7 and 28 days of silica exposure,there was no significant difference in the serum TSH content of the Silica group compared with the control group at the same time point(P>0.05).After 84 days of silica exposure,the TSH content of the Silica group mice was significantly higher than that in the control group,the difference was statistically significant(P<0.05).Conclusion:Silica exposure is accompanied by changes in thyroid tissue morphology and function during the process of causing lung tissue inflammation and fibrosis,and the levels of serum TH(T3,FT4,T4 and FT4)level decreased at different stages after silica exposure,and the expression level of TRs in lung tissue decreased.It suggests that silica exposure can cause impaired thyroid function and the steady-state imbalance of TH.PartⅡ.The effect of exogenous T3 supplementation on the lung inflammation and fibrosis in mice exposed to silicaObjective:To explore the role of early supplementation of T3(supplemented with modeling at the same time)and late supplementation of T3(supplemented after 28 days of modeling)in the inflammatory response and fibrosis of lung tissue in mice exposed to silica.Methods:The preliminary experiment initially determines the appropriate dosage range of supplementary T3(12.5～100μg/kg),and the formal experiment is divided into two parts:Ⅰ.Early supplementation of T3:168 C57BL/6 male mice were randomly divided into 7 groups according to body weight,namely saline control group,Silica group,Silica+T3(12.5μg/kg)group,Silica+T3(25μg/kg)group,Silica+T3(50μg/kg)group,Silica+T3(100μg/kg)group and simple T3(100μg/kg)group.The control group and the simple T3(100μg/kg)group were instilled with 50 μl saline in the trachea,and the remaining groups were infused with an equal volume of dust suspension(containing 2.5mg silica),And the mice in each group were injected intraperitoneally with the corresponding dose of T3(T3 supplement group and simple T3 group)or solvent(control group and Silica group)according to body weight from the day of silica contamination.In each group,8 mice were randomly killed at 7d,28d and 84d,and the mouse serum,alveolar lavage fluid(BALF)and lung tissue were collected.(1)Monitor mice’s general performance,body weight,respiratory rate,heart rate,water intake and food intake,and detect serum T3,glucose(GLU),triglyceride(TG),total cholesterol(TC),high-density lipoprotein(HDL-C)and low-density lipoprotein(LDL-C)content.(2)Detection of lung injury and inflammatory response indicators:The level of total protein(TP)in BALF and the activities of lactate dehydrogenase(LDH),acid phosphatase(ACP)and alkaline phosphatase(AKP)were detected by enzyme-labeled plate method;HE staining was used to observe the degree of inflammatory response in lung tissue;ELISA was used to detect the expression levels of inflammatory factors IL-1β,TNF-α and IL-6 in BALF.(2)Detection of lung fibrosis-related indicators:Masson staining method was used to observe the degree of lung fibrosis;ELISA was used to detect the expression level of TGF-β1;qRT-PCR was used to detect the mRNA relative expression levels of fibrosis-related genes type I collagen-1(COL1A1),type III collagen-1(COL3A1)and fibronectin-1(Fn1)in mouse lung tissue;Western blotting was used to detect the protein expression levels of COL1A1 and Fn-1 in mouse lung tissue;immunohistochemistry was used to detect the protein content of α-SMA in mouse lung tissue.Ⅱ.Late T3 supplementation:32 C57BL/6 male mice were randomly divided into 4 groups according to body weight,namely saline control group,Silica group,Silica+T3(100μg/kg)group and simple T3(100μg/kg)group.Silica group and Silica+T3(100μg/kg)group were exposed to tracheal instillation to give 50 μ1 of suspension(containing 2.5mg of silica),and the remaining groups were given 50 μ1 of normal saline in the same way.Silica+T3(100μg/kg)group and simple T3(100μg/kg)group received daily intraperitoneal injection of T3 100μg/kg body weight after 28 days of silica exposure(fibrosis has formed),and the control group and Silica group received daily intraperitoneal injection of the same amount of solvent as a control.All mice were killed after 84 days of silica exposure.Collect mouse serum,BALF and lung tissue,the detection index is the same as in Method Ⅰ.Results:Ⅰ.Early supplementation of T3:(1)The effect of T3 on systemic effects in mice:At various time points after silica exposure,the serum T3 content of mice in each T3 supplement group was significantly higher than that in the Silica group,the difference was statistically significant(P<0.05).The serum T3 level of Silica+T3(100μg/kg)group was not higher than that of the normal control group(P>0.05);the serum T3 level of mice in the simple T3(100μg/kg)group was slightly higher than that of the normal control group,but no obvious symptoms of hyperthyroidism were observed.There were no significant differences in body weight,basal metabolism(drinking water and food intake),respiratory rate and heart rate,and glucose and other biochemical indicators.(2)T3 reduces lung damage and inflammation in mice exposed to silica:At various time points after silica exposure,compared with the control group and the simple T3 group,the content of TP in the BALF and activities of LDH,ACP and AKP in Silica group mice were significantly increased,the differences were statistically significant(P<0.05);HE staining results showed that the lung tissue of Silica group showed septal defect,alveolar wall thickening,a large number of inflammatory cell infiltration and nodular changes,and as the time of silica exposure increased,the inflammatory score was more obvious than the control group,and the expression levels of IL-1β,TNF-α and IL6 in BALF of the Silica group were higher than those of the control group and the simple T3 group,and the differences were statistically significant(P<0.05).Compared with the Silica group,each T3 supplementation group can reduce the expression of injury indicators(TP,LDH,ACP,and AKP)and inflammatory factors(IL-1 β,TNF-α and IL6)in BALF of mice to varying degrees,and lower the inflammatory score of lung tissue,and the effect of Silica+T3(100μg/kg)group is more obvious.(3)T3 inhibits lung tissue fibrosis in mice exposed to silica:At various time points after silica exposure,Masson staining results show that,compared with the control group and the simple T3 group,the Silica group showed blue collagen deposition in the lung interstitial,and the collagen deposition increased continuously with the extension of the silica exposure time,and the fibrosis score increased significantly.Each T3 supplementation group can reduce the blue collagen deposition and fibrosis score in the interstitium to varying degrees;after 7 days of silica exposure,the differences in lung fibrosis-related indicators(TGF-β1,COL1A1,COL3A1,and Fn-1)in each group were not statistically significant(P>0.05);after 28 days and 84 days of silica exposure,compared with the control group and the simple T3 group,the expression level of TGFβ1 in BALF and the relative mRNA expression levels of COL1A1,COL3A1 and Fn-1 in lung tissue of the Silica group mice were increased,and the differences were statistically significant(P<0.05).Compared with the Silica group,each T3 supplementation group can reduce the expression level of COL1A1,COL3A1 and Fn-1 to different degrees.After 84 days of silica exposure,the protein expression levels of COL1A1 and Fn-1 in the lung tissue and the protein content of α-SMA in Silica group were increased compared with the control group and the simple T3 group,and the differences were statistically significant(P<0.05);Compared with the Silica group,each T3 supplement group can reduce the protein content of COL1A1,Fn-1 and α-SMA in lung tissue,and among all the fibrosis indicators,the decrease in Silica+T3(100μg/kg)group was more obvious.Ⅱ.Late supplementation of T3 can also lower the levels of lung injury,inflammation and fibrosis in mice exposed to silica:The pathological results of HE and Masson showed that after 84 days of dust exposure,the inflammatory cell infiltration and collagen deposition in the lung tissue of the Silica+T3(100μg/kg)group were significantly lower than that of the Silica group,and the inflammatory and fibrosis scores decreased,the difference was statistically significant(P<0.05).Compared with the Silica group,the indexes of lung injury(TP and ACP)and inflammation(IL-1 β and IL-6)in BALF of Silica+T3(100μg/kg)group were decreased;the mRNA levels of COL1A1,COL3A1 and Fn-1 and the protein content of COL1A1,Fn-1 and α-SMA in the lung tissues of the mice were reduced,the differences were statistically significant(P<0.05).Conclusion:In the dose range of 12.5～100μg/kg,no obvious toxic and side effects of T3 were observed.Supplementation of different doses of T3 on silica contaminating can alleviate lung inflammation and fibrosis caused by silica to varying degrees,and the effect of T3(100μg/kg)group is the best;Supplementation with T3(100μg/kg)after the formation of fibrosis can also reduce the lung inflammatory reaction caused by silica and can inhibit the progress of pulmonary fibrosis.Part Ⅲ.Preliminary study on the mechanism of T3 intervention in silicosisinduced lung inflammation and fibrosis in miceObjective:To explore the protective mechanism of T3 in the lung inflammation and fibrosis of mice induced by silica from the perspective of mitochondrial biosynthesis and glycolysis.Methods:The normal saline control group,the Silica group,the Silica+T3(100μg/kg)group and the simple T3(100μg/kg)group at different time points in the early supplement T3 experiment were selected as the research objects.(1)Enzyme plate method was used to detect the content of ATP in lung tissue;qRT-PCR and immunohistochemistry were used to detect the mRNA and protein expression levels of TRα and TRβ in lung tissue;qRT-PCR and Western blotting were used to detect mitochondrial biosynthesis-related factors:peroxisome proliferator-activated receptorsy coactivator-1α(PGC-1α),nuclear respiration factor 1(NRF1),nuclear respiration factor 2(NRF2),mitochondrial transcription factor A(TFAM)and peroxisome proliferatoractivated receptor α(PPARα)mRNA or protein expression levels.(2)The enzyme plate method was used to detect the content of lactic acid in the lung tissue;qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of the glycolysis related indicators hypoxia-inducible factor-lα(HIF-1α),hexokinase 2(HK2)and M2 pyruvate kinase(PKM2).Results:(1)T3 promotes mitochondrial biosynthesis in lung tissue of mice exposed to silica:At various time points after silica exposure,compared with the control group and the simple T3 group,the Silica group lung tissue ATP content and mRNA and protein expression levels of TRα,TRβ and PGC-1α were significantly reduced,mitochondrial biosynthesis-related transcription factors NRF1,NRF2,TFAM and PPARα mRNA expression levels decreased significantly,the differences were statistically significant(P<0.05).Compared with the Silica group,the ATP content and expression levels of TRα,TRβ and PGC-1α and mitochondrial biosynthesis-related transcription factors were increased,and the differences were statistically significant(P<0.05).(2)T3 inhibits glycolysis in lung tissue of mice exposed to silica:At each time point after silica exposure,compared with the control group and the simple T3 group,the lactic acid content and the mRNA and protein expression levels of HIF-1α and HK2 in the lung tissue of the Silica group were increased,and the differences were statistically significant(P<0.05);Compared with Silica group,the lactic acid content and the mRNA and protein expression levels of HIF-1α and HK2 in the lung tissue of the Silica+T3(100μg/kg)group were increased,and the differences were statistically significant(P<0.05);after 7 and 28 days of silica exposure,compared with the control group and the T3 group alone,the mRNA and protein expression levels of PKM2 in the lung tissue of the Silica group were increased,and the difference was statistically significant(P<0.05);Compared with the Silica group,the mRNA and protein expression levels of PKM2 in the lung tissue of the Silica+T3(100μg/kg)group were increased,and the difference was statistically significant(P<0.05);The mRNA and protein expression levels of PKM2 were not statistically different after 84 days of silica exposure(P>0.05).Conclusion:At different stages after exposure to silica,it can reduce the ATP level and mitochondrial biosynthesis function of the lung tissue of mice and increase the lactic acid content and glycolysis level of the lung tissue.T3 may promote mitochondrial biosynthesis regulated by PGC-1α and inhibit the HIF-1α/glycolysis pathway to reduce silica-induced lung inflammation and fibrosis.In summary,this study found that the formation of silicosis in mice was accompanied by changes in thyroid tissue morphology and function,and serum TH level decreased significantly;supplementation of exogenous T3(early and late)can reduce lung inflammation and fibrosis in mice caused by silica;T3 may play an antiinflammatory and anti-fibrotic role by improving the imbalance of energy metabolism in the lung tissue of mice exposed to silica.Our results provide a theoretical basis for the application of thyroid hormone in the prevention and treatment of silicosis.