The Effects Of HHGF Gene Modified Bone Marrow Mesenchymal Stem Cells On Pulmonary Fibrosis Following LPS-induced Acute Lung Injury In Rats

Objective To observe the effects of h HGF gene modified MSCs on pulmonary fibrosis following LPS-induced Acute Lung Injury in Rats.Methods（1）To evaluate pulmonary fibrosis（PF）following LPS-induced acute lung injury in rats.Twenty male sprague-dawley rats aged 7weeks,weighing 180-220 g,were randomly divided into 2 groups:Control group,and LPS group（LPS treated group）.Rats in the LPS group were established a PF model though the three-hit regimen of LPS.The control group was given equal volume of saline.The rats in LPS group were sacrificed in batch on the 7^th,14^th,21^st days after the third administration.Lung tissue was collected.HE staining and Masson staining were performed to observe the pathological changes.Ashcroft score was used to quantify the degree of fibrosis.Hydroxyproline（HYP）content in the lung tissues were detected using alkaline hydrolysis.（2）In order to observe the effect of h HGF-MSCs on lung fibrosis following LPS-induced acute lung injury in rats.Twenty-eight rats were randomly divided into 4 groups:Control group,LPS group,LPS+EGFP-MSCs group（cells transduced with empty vector）,LPS+h HGF-MSCs group（MSCs genetically modified hepatocyte growth factor）.PF model were established after the three-hit regimen of LPS.The control group were given equal volume of saline.On the first day after the third LPS administration,rats in the control group and LPS group were injected with 1ml DMEM via tail vein.Rats in LPS+EGFP-MSCs group and LPS+h HGF-MSCs group were injected 2×10⁶EGFP-MSCs or h HGF-MSCs in equal volume DMEM.Samples were collected at the 21^th day.HE staining and Masson staining were performed to observe the pathological changes.Ashcroft score was used to quantify the degree of fibrosis.Hydroxyproline（HYP）levels in the lung tissues were detected using alkaline hydrolysis.TGF-β,TNF-α,IL-6 and IL-10 in BALF were measured by ELISA.The expression of E-cadherin,Vimentin andα-SMA in the lung tissues was analyzed by Western blot.The oxidative stress was detected by malondialdehyde（MDA）concentration and superoxide dismutase（SOD）activity in the lung tissues.Result（1）After the LPS treatment,the pathological changes of pulmonary tissue in the 7^th day were mainly inflammatory response,while in the 21^st days were mainly collagen fiber deposition.Compared with the control group,LPS group had increased Ashcroft score and increased HYP content in lung tissue at the 14^th day and 21^st day（P<0.05）.（2）Compared with the control group,LPS group had severe pathological injury,increased Ashcroft score,increased HYP content,increased TGF–β,TNF–α,IL-6 and decreased IL-10 content in BALF,increased MDA levels and decreased SOD activities in lung tissue,increased expression of Vimentin andα-SMA protein,and decreased E-cadherin protein expression in lung tissue（P<0.05）.Compared with the LPS group,the contents of HYP and MDA,the contents of TGF-β,TNF-αand IL-6 in BALF,tee protein expressions of Vimentin andα-SMA in LPS+EGFP-MSCs group were significantly decreased,while the contents of IL-10,SOD activity and E-cadherin protein expression in LPS+EGFP-MSCs group were significantly increased（P<0.05）.Compared with LPS+EGFP-MSCs group,the content of HYP,Vimentin andα-SMA protein expression levels and the contents of TGF-β,TNF-α,IL-6 in BALF in LPS+HGF-MSCs group were significantly decreased,and the activity of SOD in lung tissue was significantly increased（P<0.05）.Conclusion（1）The pulmonary fibrosis model following acute lung injury in rats was established successfully through three-hit regimen of LPS.（2）HGF-MSCs have a better anti-fibrosis effect than EGFP-MSCs after acute lung injury,and its mechanism may be related to the reduction of inflammatory reaction and oxidative stress in lung and the inhibition of epithelial endothelial mesenchymal transition.