Effect Of Let-7c On Macrophage Polarization Induced EMT In The Pathogenesis Of Coal Workers’ Pneumoconiosis In Rats

Objective:In this study,we used coal dust and Mirna let-7c inhibitor containing different free silicon dioxide to intervene in vivo and in vitro cell experiments,to investigate the role of let-7c in the pathogenesis of coal worker’s pneumoconiosis in rats.Methods:1.Animal experiment:8W rats were subjected to intratracheal instillation of dust.The experimental groups were: blank control group,coal dust group(free silicon dioxide content 5%),coal silica group(free silicon dioxide content 30%),and silica group(free silicon dioxide content 99.9%).Macrophages and lung tissue were extracted at the time point of intervention 1 and 3 months,and the degree of pulmonary fibrosis was observed using Masson and HE staining;Using immunofluorescence to detect macrophage polarization;Western blotting was used to detect the expression levels of TGF-β1,vimentin and E-cadherin.PCR was used to detect the expression levels of Let-7c and c/EBP-δ.2.Cell experiment:NR8383 rat alveolar macrophages were cultured in vitro.NR8383 was induced to polarize to M1 and M2 types in M1 type polarization environment(100ng/m L LPS)and M2 type polarization environment(20ng/m L IL-4),respectively.The experimental groups were: control group,coal silica dust group,IL-4+coal silica dust group.PCR was used to detect the expression levels of Let-7c and VIc/EBP-δ.Then,Transfection of mi RNA let-7c inhibitor into NR8383 cells inhibited the expression of let-7c.The experimental groups were: control group,IL-4 group,IL-4+coal silica dust group,Let-7c inhibitor group,IL-4+coal silica dust+Let-7c inhibitor group.Immunofluorescence was used to detect the polarization of macrophages,and NR8383 cells and RLE-6TN cells were co cultured in Transwell.After treatment according to the above groups,PCR was used to detect the expression levels of Let-7c and c/EBP-δ,Western blotting was used to detect the expression levels of TGF-β1 in NR8383 cells,vimentin and E-cadherin in RLE-6TN cells.Results:1.Animal experiment(1)The effect of coal dust on the morphology of lung tissue,the lung tissue morphology of rats in the experimental group was significantly different from that in the control group.The surface of the lungs in the control group was smooth,pink,and soft;In the coal dust group,obvious black spots can be seen on the surface of the lungs,some of which spread throughout the entire lung lobe and are soft and black in color;In the coal silicosis group,there were obvious "coal siliceous nodules" on the lung surface,which were large,round,irregular,and hard in texture;Large white nodules can be seen in the silica dust group.(2)The effect of coal dust on pulmonary fibrosis,in HE staining,after one month of exposure to dust(1M),the lung tissue structure of rats in the control group was clear,and the alveolar walls were intact.There were coal plaques in the coal dust group and the coal silica dust group,while the alveolar walls in the silica dust group became narrower;After three months of dust exposure(3M),coal spots were formed in the coal dust group and the coal siliceous dust group,and silicon nodules were formed in the high-dose group.In Masson staining,at 1 M time point,the lung tissue structure of the control group rats was clear,and there was no blue fibrosis in the alveoli.In the coal dust group and the coal silica dust group,it is obvious that there are blue fibers surrounding the coal spots,while in the silica dust group,there are more blue fibers;At 3M time point,the blue fibers around the coal spots in the coal dust group and the coal silica dust group increased more significantly than in January,and there were silicon nodules formed in the silica dust group.(3)The effect of coal dust on alveolar macrophage polarization in rats,the immunofluorescence results showed that there was significant interaction between treatment and time on CD86(P<0.05).Compared with the control group,the expression of CD86 in the coal dust group,the coal silica dust group and the silica dust group increased gradually at 1m time(P<0.05)and decreased gradually at 3m time(P<0.05).Compared with the control group,the expression of CD206 in the coal dust group,the coal silica dust group and the silica dust group increased gradually at 1m time point and3 m time point(P<0.05),especially in the coal silica dust group and the silica dust group(P<0.05).Further analysis showed that the ratio of CD206 to CD86(CD206/CD86)increased at 1m time point and 3m time point(P<0.05).(5)The effect of coal dust on EMT of lung tissue in rats,western blot results showed that Treatment and time had a significant interaction on the expression of TGF TGF-β1and vimentin(P<0.05).At 1 M time point,compared with the control group,there was no significant change in TGF-β1 expression levels in the coal dust group and the coal silica dust group(P>0.05),while the silica dust group significantly increased(P<0.05);Compared with the control group,the expression levels of E-cadherin in coal dust group,coal silica dust group,and silica dust group were significantly decreased(P<0.05);Compared with the control group,the expression levels of vimentin in the coal dust group,the coal silica dust group,and the silica dust group were significantly increased(P<0.05).At 3M time point,compared with the control group,the TGF-β1 of the coal dust group,the coal silica dust group,and the silica dust group were significantly increased(P<0.05);Compared with the control group,the expression levels of E-cadherin in the coal dust group,coal silica dust group,and silica dust group were significantly decreased(P<0.05);Compared with the control group,the expression levels of vimentin in the coal dust group,the coal silica dust group,and the silica dust group were significantly increased(P<0.05).(6)The effect of coal dust on let-7c and c-EBP-δ in rat alveolar macrophage,the PCR results showed that Treatment and time had a significant interaction on the expression of let-7c and c/EBP-δ(P<0.05).At 1 M time point,compared with the control group,there was no significant change in the expression level of let-7c protein in the coal dust group and the coal silica dust group(P>0.05),while the expression level of let protein in the silica dust group was significantly increased(P<0.05);Compared with the control group,the expression level of c/EBP-δ in coal dust group,coal silica dust group,and silica dust group was significantly lower(P<0.05);At 3M time point,compared with the control group,the let-7c table of the coal dust group,the coal silica dust group,and the silica dust group were significantly increased(P<0.05);Compared with the control group,the expression level of c/EBP-δ in coal dust group,coal silica dust group,and silica dust group was significantly lower(P<0.05).2.Cell experiment:(1)The effect of coal silica dust on the expression of let-7c and C/EBP-δ in NR8383 cells,the PCR results showed that compared with the control group,there was no significant change in the expression of let-7c in the coal silica dust group and the LPS+coal silica group(P>0.05),while the expression of let-7c in the IL-4+coal silica group increased(P<0.05).Compared with the control group,there was no significant change in the expression of c/EBP-δ in the coal silica dust group and the LPS+coal silica group(P>0.05),while the expression of c/EBP-δ in the IL-4+coal silica group decreased(P<0.05).(2)After transfection of let-7c inhibitor into NR8383 cells,the effect of let-7c on the expression of let-7c and C/EBP-δ in NR8383 cells after coal silica dust treatment,the PCR results showed that compared with the control group,the expression of let-7c in the IL-4 group and the IL-4+coal silica dust group gradually increased(P<0.05),while the expression of let-7c in the let-7c inhibitor group,the IL-4+coal silica dust group and the let-7c inhibitor group decreased(P<0.05).However,compared with the let-7c inhibitor group,the expression of let-7c in the IL-4+coal silica dust group and the let-7c inhibitor group increased(P<0.05).Compared to the control group,the IL-4 group and the IL-4+coal silica dust group had c/EBP-δ The expression of let-7c gradually decreased(P<0.05),while the expression of let-7c in the let-7c inhibitor group and the IL-4+coal silica dust group+let-7c inhibitor group increased(P<0.05),but compared to the let-7c inhibitor group,the expression of let-7c in the IL-4+coal silica dust group+let-7c inhibitor group increased(P<0.05).(3)The effect of let-7c on the polarization of NR8383 cells after coal silica dust treatment,immunofluorescence and PCR results showed that compared with the control group,the expression of CD206 in the IL-4 group and the IL-4+coal silica dust group gradually increased(P<0.05),while the expression of CD206 in the let-7c inhibitor group and the IL-4+coal silica dust+let-7c inhibitor group decreased(P<0.05).However,compared to the let-7c inhibitor group,the expression of CD206 in the IL-4+coal silica dust+let-7c inhibitor group increased(P<0.05).Compared with the control group,there was no significant change in the expression of CD86 in the IL-4 group,IL-4+coal silica dust group,let-7c inhibitor group,and IL-4+coal silica dust group+let-7c inhibitor group(P>0.05).(4)The effect of let-7c on the expression of E-cadherin and vimentin in NR8383 cells and RLE-6NT cells exposed to coal silica,western blot results showed that compared with the control group,the expression levels of TGF-β1 and Vimentin in the IL-4 group and IL-4+coal silica dust group gradually increased(P<0.05),while the expression levels of TGF-β1 and Vimentin in the let-7c inhibitor group and the IL-4+coal silica dust group+let-7c inhibitor group decreased(P<0.05),but compared to the let-7c inhibitor group,the expression levels of TGF-β1 and Vimentin in the IL-4+coal silica dust+let-7c inhibitor group increased(P<0.05);Compared with the control group,the expression of E-cadherin in the IL-4 group and the IL-4+coal silica dust group gradually decreased,while the expression of E-cadherin in the let-7c inhibitor group and the IL-4+coal silica dust group+let-7c inhibitor group increased,but compared to the let-7c inhibitor group,the expression of E-cadherin in the IL-4+coal silica dust+let-7c inhibitor group decreased(P<0.05)Conclusions:1.Coal dust can promote pulmonary fibrosis in rats and promote the progression of CWP.2.Coal dust can affect the expression of TGF-β1,E-cadherin,and vimentin by affecting the polarization of macrophages.3.Let-7c can promote epithelial mesenchymal transformation by regulating macrophage polarization,thereby participating in the formation of pulmonary fibrosis.