| ObjectiveSilicosis is an occupational disease caused by long-term inhalation of dust with a high content of free silica,which progresses quickly and causes serious harm.At present,the pathogenesis of silicosis has not been fully elucidated,and there is no effective treatment.New treatment methods are urgently needed to prevent and improve.It is generally believed that chronic inflammation and epithelialmesenchymal transition(EMT)are important links in the development of silicosis.Stem cell therapy is an emerging therapy.Mesenchymal stem cells(MSCs)are adult stem cells with self-renewal ability and multi-lineage differentiation potential.Due to their low immunogenicity,multi-differentiation potential and paracrine function,abundant sources and easy in vitro isolation and culture,and it is widely used in many diseases.Studies have shown that bone marrow-derived mesenchymal stem cells(BMSCs)can improve organ fibrosis by regulating the EMT process,but there are few studies on the application of silicosis.This study established an experimental silicosis model in rats and injected BMSCs by tail vein,exploded the effects of BMSCs on silicosis,and further explored the molecular mechanism.In order to provide new ideas for the treatment of silicosis.Methods1.Culture and identification of BMSCsBMSCs were purchased from Saiye Biology and cultured routinely.Observe the cell morphology under a microscope.Take the third generation of BMSCs for adipogenesis and osteoinduction experiments.Flow cytometry identified cell surface molecular markers CD90,CD29,CD45 and CDllb.2.Animal groupingSixty male SD rats(150-180 g)were randomly divided into Control group,Silicosis group and BMSCs group(20 rats per group).Rats in the Silicosis group and BMSCs group were received non-exposed intratracheal instillation with 0.5 mL silica suspension(100 mg/mL).Rats in the Control group were admitted with 500 μL sterile saline.Interfered with BMSCs on the 14,28 and 42 days after silica infusion.The BMSCs group was injected with 1 mL BMSCs suspension(2×10~6 cells/mL)through tail vein,and the Control group and the Silicosis group were injected with 1 mL saline.10 rats in each group were sacrificed on the 28th and 56th day after silica perfusion and lung tissue and bronchoalveolar lavage fluid(BALF)were collected.3.The effect of BMSCs on experimental silicosisDetermine the content of hydroxyproline;calculate the lung coefficient;H&E and Masson staining to observe the inflammatory infiltration and collagen deposition of lung tissue.4.The effect of BMSCs on inflammatory factorsqRT-PCR,Western blot and ELISA were used to detect the expression levels of TNF-α,IL-1β and IL-6.5.The effect of BMSCs on EMT and ECM related markersThe mRNA expression levels of E-cadherin,Vimentin and ECM-related markers Fibronectin and Collagen I were detected by qRT-PCR;the protein expression levels of E-cadherin,Vimentin,Fibronectin and Collagen I were detected by Western blot and immunohistochemistry respectively.6.The effect of BMSCs on TGF-β/Smad pathwayqRT-PCR,Western blot and ELISA were used to detect the the expression level of TGF-β1;the protein expression level of p-Smad2,Smad2,p-Smad3,Smad3 and Smad7 was detected by Western blot.7.Statistical analysisData analysis used SPSS 27.0.The data are all expressed as mean ± standard deviation(x±s).The comparison of the difference between the two groups used Student’s t test.A one-way analysis of variance(ANOVA)was used when comparing between multiple groups.Ispection level of alpha=0.05.Results1.Identification of BMSCsBMSCs were spindle-shaped,and the cells grow and converge in a whirlpool shape;lipid droplets were visualized by Oil Red O staining after adipocyte induction,red calcium nodules were visualized by Alizarin Red staining after osteogenic induction.the results of flow cytometry showed that the expressions of CD90 and CD29 were 98.6%and 99.0%,respectively,while the expressions of CD45 and CD11b were all below lower than 1%.2.BMSCs improved experimental silicosisCompared with the control group,the lung injury in the silicosis group was obvious.Compared with the silicosis group,the lung injury of the BMSCs group was reduced,the degree of inflammatory infiltration and collagen deposition was reduced,the lung coefficient,HYP content and the expression of Fibronectin and Collagen I was reduced.The difference was statistically significant(P<0.05).On the 28th day,the difference was not statistically significant.3.BMSCs reduced inflammationCompared with the Control group,the expressions of inflammatory factors TNFα,IL-1β and IL-6 in the Silicosis group and the BMSCs group were increased,while the BMSCs group was lower than that of the silicosis group,and the difference was statistically significant(P<0.05).4.BMSCs inhibited EMTCompared with the Control group,the expression of E-cadherin increased and the expression of Vimentin decreased in the Silicosis group and the BMSCs group,while the expression of E-cadherin increased and the expression of Vimentin decreased in the BMSCs group compared with the Silicosis group.The difference was statistically significant(P<0.05).5.BMSCs blocked the TGF-β/Smad pathwayCompared with the Silicosis group,the expression of TGF-β1,p-Smad2/Smad2,p-Smad3/Smad3 in the BMSCs group decreased,and the expression of Smad7 increased,and the difference was statistically significant(P<0.05).ConclusionBMSCs may alleviate experimental silicosis in rats by reducing inflammation and inhibiting EMT,and this process may be related to inhibiting the activation of TGFβ/Smad pathway. |
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