Effect Of Bone Marrow-derived Mesenchymal Stem Cells On Apoptosis,Autophagy,and Pyroptosis In Silicosis Rats

BackgroundSilicosis is systemic disease induced by long-term silica inhalation in occupational environment.When the lungs are exposed to silica,it is first recognized and engulfed by lung macrophages.Since immune cells cannot dissolve and digest silica dust,it can destroy lysosomes,which further causes cell damage and finally leads to the death of lung tissue cells.Cell death includes necrosis,apoptosis,autophagy,pyroptosis,ferroptosis,necroptosis,and oncosis.According to the available literature,apoptosis,autophagy,and pyroptosis play significant roles in silicosis development.Bone marrow-derived mesenchymal stem cell（BMSC）is one of the ideal target cells for cell therapy,which provides a new idea and possibility for the treatment of silicosis.Previous studies have proved that MSCs might inhibited apoptosis and autophagy in pulmonary fibrosis,which indicates the regulating effect of BMSCs on cell death.But,whether BMSCs can protect the lung against pyroptosis has not been investigated in the rat silicosis models.Therefore,transplatation of BMSCs was applied intravenously in pulmonary fibrosis induced by silica in rats.Thus,the silicosis model was established and the changes of apoptosis,autophagy,and pyroptosis were investigated after exposing to silica and BMSCs administration,which provide a novel idea for silicosis treament and silicosis.Materials and Methods1.Culture and identification of BMSCsBMSCs of SD rats were purchased from Cyagen Biosciences,Guangzhou.BMSCs were cultured in T25 flasks with complete medium in an incubator with 37℃)and 5%CO₂.Specific cell morphology,surface markers,and differentiation abilities of the BMSCs were detected by inverted phase contrast microscope,flow cytometry,and adipogenic and osteogenic differentiation kits in fifth generation,respectively.2.Influence of BMSCs on pulmonary fibrosis of silicosis in rats30 SD rats weighting 150-180 g were randomly divided into control group,silica group,and silica+BMSC group（n=10 rats per group）.Firstly,by intratracheal instillation,500 ul silica suspension（100 mg/ml）was administered to the rats,and rats of the control group were 500 ul saline.1 ml saline with 2×10⁶BMSCs was injected into the tail vein of the rats on 14th,28th,and 42nd days after silica exposure.And rats in the control and silica group were injected with 1 ml saline.All rats were sacrificed on the 56th day after silica exposure.Lung coefficients and HYP content were detected on the 56th day after silica exposure.Lung inflammatory infiltrates and collagen deposition were analyzed by H&E and Masson’s trichrome staining,respectively.The m RNA and protein levels of fibrosis-related markers（Fibronectin and Collagen I）were detected with RT-PCR and western bloting.3.Influence of BMSCs on apoptosis,autophagy,and pyroptosis of silicosis in ratsImmunohistochemistry（IHC）staining was used to detect the level of Cleaved caspase 3 and P62.Immunofluorescence staining was used to detect the expression of Beclin 1 and Cleaved caspase 1.The content of IL-1βand IL-18 was measured using ELISA kits.Western blot was used to detect the levels of fibrosis-related proteins （Collagen 1 and Fibronectin）,apoptosis-related proteins（Cleaved caspase 3,Bax,and Bcl2）,autophagy-related proteins（Beclin 1,P62,and LC3）,and pyroptosis-related proteins（Cleaved caspase 1 and Nlrp3）.4.Statistical analysisAll data were analyzed and processed using SPSS 21.0 software and are presented as the mean±standard deviation（SD）.One-way ANOVA was performed for multiple comparisons,and the differences between any two groups were compared by least-significant difference（LSD）.Differences were considered significant at p<0.05.Results1.Identification of BMSCsBMSCs were spindle morphology and swirl-shaped colonies.The following surface markers of BMSCs:CD90（97.5%）,CD29（98.9%）,CD45（0.23%）and CD11b（0.18%）.Adipogenic and osteogenic differentiation showed that adipogenic cells stained Oil Red O and calcium deposition stained Alizarin red.2.Effect of BMSCs on pulmonary fibrosis of silicosis in ratsCompared to the rats from the control group,the lung coefficients,hydroxyproline content,the modified Ashcroft semi-quantitative score and the levels of fibrosis-related proteins（Fibronectin and Collagen I）of rats from the silica group was significantly elevated（p<0.05）,and inflammatory infiltrates,excessive collagen deposition,and silicon nodules were evident in rats after exposure to silica.However,the Lung coefficients,hydroxyproline content,the modified Ashcroft semi-quantitative score and the levels of fibrosis-related proteins（Fibronectin and Collagen I）of rats from the BMSCs group was significantly lower than rats from the silica group（p<0.05）.After injection of BMSCs,the pathological changes of lung tissues were significantly relieved compared to the silica group.3.Effect of BMSCs on apoptosis of silicosis in ratsCompared to the rats from the control group,in rats after exposure to silica,the Cleaved caspase 3,Bax levels were significantly increased,and Bcl-2 levels were significantly decreased.Compared to the silica group,the protein levels of Cleaved caspase 3 and Bax were significantly reduced（p<0.05）,Bcl-2 expression was insignificantly upregulated（p>0.05）,and the Bcl-2/Bax ratio was significantly upregulated in the BMSCs group（p<0.05）.4.Effect of BMSCs on autophagy of silicosis in ratsCompared to the rats from the control group,in rats after exposure to silica,the P62 level was significantly decreased,LC3Ⅱ/LC3Ⅰratio was significantly increased（p<0.05）,and the Beclin 1 expression had no significant change（p>0.05）.Compared to the silica group,the P62,LC3 and Beclin 1 levels had no significant difference between the silica group and the BMSCs group（p>0.05）.5.Effect of BMSCs on pyroptosis of silicosis in ratsCompared to the rats from the control group,in rats after exposure to silica,the expression of Cleaved caspase 1,Nlrp3,IL-1βand IL-18 were significantly increased（p<0.05）;BMSCs could decrease these proteins level（p<0.05）.ConclusionRepeated administration of BMSCs can protect lung tissue from fibrosis induced by silica in a rat model.According to our results,the treatment of BMSCs can inhibit apoptosis,and pyroptosis but not autophagy.