Role Of JAK2/STAT3 Signaling Pathway-induced Inflammatory Response In Pulmonary Tissue Fibrosis In Coal Workers’ Pneumoconiosis

Objective:In this study,a rat model of coal worker’s pneumoconiosis was established by exposure to coal dust,coal silica dust and quartz dust,and the characteristics of inflammatory reaction and EMT changes in rat lung tissue caused by dust exposure at different time points were observed,and the role of JAK2/STAT3 signal pathway in mediating inflammatory reaction in the EMT of rat model of coal worker’s pneumoconiosis was discussed.Methods:96 healthy male SD rats were randomly divided into 1,3,and 6-month-old control groups and dust-exposed groups according to body weight,including 12 groups of coal dust group,coal-silica dust group and quartz group.And 8 rats in each group.After one week of adaptive feeding,the rats in each dust-exposed group were instilled into the trachea（1ml/rat）at one time using the non-exposed trachea perfusion method.The quartz dust suspension was 50g/L,and the control group was injected with 1m L of 0.9%sodium chloride solution.The experimental indicators were tested at 1,3,and 6 months after dust exposure.Small animal pulmonary function tester for detecting lung function.Whole-lung bronchoalveolar lavage was carried out,and lung tissue and alveolar lavage fluid were collected for follow-up experiments.Alkaline hydrolysis was used to detect the content of hydroxyproline（HYP）in lung tissue.Hematoxylin-eosin staining and Masson staining were used to observe the pathological changes of lung tissue.Detection of inflammasome（NLRP3）,interleukin 1β（IL-1β）,interleukin 18（IL-18）,transforming growth factor（TGF-β1）,interleukin 10（IL-10）,interleukin 4（IL-4）content.The expression levels of JAK2,STAT3,P-JAK2,P-STAT3,E-Cadherin,α-SMA and CollagennⅠproteins in rat lung tissue were detected by Western blot.Results:1.Body mass and organ coefficient:In dust exposure treatment and dust exposure time,there was an interaction between rat body weight and lung organ coefficient（P<0.05）.With the prolongation of dust exposure time,the body weight first increased and then slightly decreased,the lung organ coefficient decreased and then increased,and the lung wet weight increased.Compared with the control group at the same time point,in terms of lung wet weight and lung viscera coefficient,the dust-contaminated group was the highest in the quartz group,followed by the silica dust group and the coal dust group,and there was no statistically significant difference in body mass（P<0.05）.2.Lung function test results:There were interactions between forced vital capacity and forced expiratory volume in 0.2 seconds in each group of rats in terms of treatment and treatment time between groups（P<0.05）.With the increase of dust exposure time,FVC,FEV_0.2and MVV all decreased,and the difference was statistically significant（P<0.05）.Compared with the control group at the same time point,among the dust-exposed groups,the lung function index of the rats was the lowest in the quartz group,followed by the silica dust group and the coal dust group.Among them,the change trend of FEV_0.2and MVV indexes in the quartz group was the most obvious.3.Pathological morphology staining of lung tissue:The results of HE staining showed that the lung tissue structure of rats in the control group was clear,and there was no inflammatory cell infiltration;with the increase of dust exposure time,inflammatory cells and red blood cell infiltration appeared in the dust exposure group,and the alveolar interval became wider;the coal dust group small amount of coal dust particles appeared in the first month,a large number of coal dust clusters appeared in the third month,and the structure of the lung tissue around the coal spots was broken in the sixth month;a small amount of coal dust particles appeared in the coal silica dust group in the first month,and alveoli appeared in the third and sixth months.Structural collapse or even severe damage,coal dust particles and coal-silicon nodules appeared;the alveolar cavity of the quartz group expanded significantly at 1 month,with inflammatory cell lesions;the alveolar cavity disappeared or fused at 3 and 6 months,and there were small clusters lung nodules.The results of Masson staining showed that there were a small amount of blue-stained areas in the lung tissue of the control group,and no fibrous tissue hyperplasia.With the increase of dust exposure time,the blue Collagenn fibers in the coal dust group,coal silica dust group,and quartz group gradually increased,surrounded by coal spots.A large number of blue Collagenn fibers and blue fibrous nodules appeared,and small blue Collagenn fiber clusters appeared in the quartz group at 6 months.4.The content of hydroxyproline（HYP）in lung tissue:The HYP content in the lung tissue of the rats in each group had an interaction effect on the treatment and treatment time between the groups（P<0.05）.Compared with the control group after being exposed to dust for 6 months,the HYP content in the lung tissue of the dust exposure group increased,and the difference was statistically significant（P<0.05）.The quartz group had the highest HYP content,followed by the coal silica dust group,coal dust group Group.5.Expression of EMT protein in rat lung tissue:Compared with the control group after being exposed to dust for one month,the expression levels of E-cadherin protein decreased by 20.51%,34.80%,and 64.76%in the coal dust group,coal silica dust group,and quartz group,and the expression levels ofα-SMA protein increased 19%,36%,89%（P<0.05）.After being exposed to dust for 3 months,compared with the control group,the expression of E-cadherin protein in the coal silica dust group decreased by 29.49%,decreased by 31.67%in the quartz group,and the expression ofα-SMA protein in the quartz group increased by 30%（P<0.05）.Compared with the control group after dust exposure for 6 months,the expression of E-cadherin protein in the quartz group decreased by 61.87%（P<0.05）,and the expression ofα-SMA protein in the quartz group increased by 167%（P<0.05）.Coal lung had obvious EMT changes at 1 month,coal silicosis had EMT changes at 1 and 3 months,and silicosis had obvious EMT changes at 1,3,and 6 months.It shows that the coal workers pneumoconiosis model has EMT changes in the early stage,and the silicosis model has the most serious and persistent EMT changes,followed by coal silicosis and coal lung.6.Expression of ECM protein in rat lung tissue:Compared with the control group after dust exposure for 6 months,the expression of CollagenΙprotein in the coal silica dust group and the quartz group increased by 38%and 61%（P<0.05）.Significant changes in ECM Collagen deposition in rat lung tissue at 6 months,suggesting that the coal worker’s pneumoconiosis rat model needs a long time for ECM deposition to occur.7.Content of inflammatory factors in rat alveolar lavage fluid:The pro-inflammatory factors and anti-inflammatory factors of the rats in each group had interaction effects in treatment and treatment time between groups（P<0.05）,indicating that dust exposure treatment and dust exposure time could aggravate the changes of pro-inflammatory factors and anti-inflammatory factors.Compared with the control group at the same time point,the contents of NLRP3,IL-1β,and IL-18 increased,and the difference was statistically significant（P<0.05）.The content of pro-inflammatory factors in each dust-contaminated group was the highest in the quartz group,followed by the coal-silica dust group and the coal dust group.Compared with the control group at the same time point,the contents of TGF-β1 and IL-10 increased,while IL-4 decreased,and the difference was statistically significant（P<0.05）.The content of anti-inflammatory factors in each dust-contaminated group was the highest in the quartz group,followed by the coal-silica dust group and the coal dust group.IL-18 is suitable for monitoring coal silicosis,pro-inflammatory response of silicosis and late pro-inflammatory response of coal pneumonia;NLRP3 continues to increase with dust exposure time in the progress of coal pneumonia,and can be used to observe the changes of pro-inflammatory factors in the early progression of coal pneumonia;NLRP3,IL-1β,IL-18 were significantly changed at each time point of silicosis,suitable for monitoring the pro-inflammatory response of silicosis.TGF-β1 increases continuously and steadily with the dust exposure time in the progress of coal workers pneumoconiosis,which is suitable for monitoring the changes of anti-inflammatory responses of various types of coal workers pneumoconiosis;IL-4 is significantly increased in coal workers pneumoconiosis at 1 month,which can be used to observe early anti-inflammatory response of coal workers pneumoconiosis.8.Expression of JAK2/STAT3 signal pathway protein in rat lung tissue:Compared with the control group,the ratio of P-JAK2/JAK2 protein expression was increased by39%,77%,and 91%in the coal dust group,coal silica dust group,and quartz group,respectively（P<0.05）.It shows that the phosphorylation of JAK2 protein in coal lung,coal silicosis and silicosis occurs after 6 months of dust exposure.Compared with the control group after being exposed to dust for 1 month,the ratio of P-STAT3/STAT3protein expression in the quartz group increased by 1.38 times（P<0.05）.Compared with the control group after being exposed to dust for 6 months,the ratio of P-STAT3/STAT3protein expression in the coal silica dust group and the quartz group increased by 1.10times and 1.69 times（P<0.05）.It shows that STAT3 protein is activated by phosphorylation in the early stage of silicosis,and obvious phosphorylation occurs in coal silicosis and later stage of silicosis.Conclusions:The early inflammatory response of coal worker’s pneumoconiosis is mediated by phosphorylated STAT3,and the late stage is involved in the JAK2/STAT3 signaling pathway,thereby forming EMT and causing pulmonary dysfunction.Among them,silicosis is mainly caused by ventilation disorders,which eventually leads to pulmonary fibrosis.