Effect of bone marrow mesenchymal stem cells on paraquat-induced pulmonary fibrosis in rats

Abstract

Objective: To study the effect of the injected bone marrow mesenchymal stem cells (BMSC) on rats with pulmonary fibrosis induced by paraquat (PQ) during different poisoning periods and explore the potential mechanism. Methods: From October to December 2018, BMSCs of SPF SD rats were isolated and purified by whole-bone marrow adherent culture method and cultured to the Third Generation (P3) . The surface antigens CD29, CD90, CD45 and CD34 of P3 BMSC were detected by Flow cytometry, the formation of alkaline phosphatase (ALP) , calcium nodules and fat droplets were observed by ALP, Alizarin Red staining and oil red O staining. At the same time, 36 SPF male rats were randomly divided into 6 groups: NC Group (Blank Control Group, injected with the same amount of saline) and PQ group (PQ model group, injected with 20% PQ solution 18 mg/kg intraperitoneally) , bMSC-A group, BMSC-B group, BMSC-C group and BMSC-D group were injected with BMSC suspension 1×10(6) cells/mice at 3 h、3 d、7 d and 14 d after PQ poisoning. After 28 days, the rats were killed, the lung organ coefficients were calculated, the hydroxyproline (HYP) content in lung tissue was calculated by alkaline hydrolysis, and the lung injury and fibrosis were observed by HE and Masson staining, serum TGF-1、TNF-α、MMP-9 and TIMP-1 were detected by Elisa. Results: High Purity BMSCs were successfully isolated and obtained. The P3 BMSC generation was positive expression of CD29、CD90、and negative expression of CD34、CD45, and had the potential of osteogenic and adipogenic differentiation. The results of HE staining and Masson staining showed that the alveolar structure in NC group was intact and homogeneous, in PQ group, the alveolar structure was severely damaged and a lot of collagen fibers and fibroblasts were deposited, and the degrees of lung injury in each BMSC intervention group were obviously less than in PQ group, in BMSC-A group and BMSC-B group, the degrees of reduction were obvious. Compared with NC group, the Lung organ coefficient, HYP content in lung tissue and TGF-β1, TIMP-1 levels in serum were significantly higher in PQ group (P<0.05) , while TNF-α and MMP-9 had no significant difference (P>0.05) . Compared with PQ group, the lung organ Coefficients, HYP, TGF-1 and TIMP-β1 in BMSC-A and BMSC-B groups were lower than those in PQ group (P<0.05) . The Lung organ coefficients, TGF-β1 and TIMP-1 in BMSC-C and BMSC-D groups were lower than those in PQ group, there was no significant difference (P>0.05) . Conclusion: Early BMSC injecting can alleviate pulmonary fibrosis induced by PQ. The mechanism may be that BMSC can reduce pulmonary fibrosis through reducing the level of TGF-β1 and regulating the balance of TIMP-1/MMP-9, threrby reducing inflammatory damage and increasing the degradation of extracellular matrix (ECM) .