Protective Effect Of MT2A On Lipopolysaccharide-induced Damage Of Human Pulmonary Microvascular Endothelial Cells

Objective To observe the cytoskeletal damage and inflammatory mediators of tumor necrosis factor in cultured human lung micro-vascular endothelial cells(HPMVECs)after stimulation with a certain concentration of lipopolysaccharide(LPS)solution.Tumor Necrosis Factor ?(TNF-?),Interleukin6(IL-6)release,Establishment of a lipopolysaccharide-mediated model of human pulmonary vascular endothelial cell injury.Grouping experiments were performed after the establishment of the injury model.The concentrations of metallothionein 2A(MT2A)were co-cultured with the control group.After a period of time,the release of inflammatory mediators and the changes in the morphology of HPMVECs in the fluorescence microscopy group were observed.After comparative analysis,the protective effect of MT2 A on LPS-mediated injury of human lung microvascular endothelial cells was verified.Methods1.LPS-induced damage to human pulmonary capillary endothelial cells Two groups of experimental group(U group)and control group(N group)were set up.A certain amount of LPS diluent(500 ng/ml)was added to the N medium,and the same amount of physiological saline was added to the U group.Under the same condition,5attached holes were set up in each group.The enzyme-linked immunosorbent assay(ELISA)was used to detect the content of IL-6 and TNF-? in the culture medium of each group at the fixed time.At the same time,the skeleton changes of endothelial cells in each group were observed and analyzed under fluorescence microscope.2.The protective effect of MT2 A on human pulmonary capillary endothelial cells Control group A(LPS + saline group),experimental group B1(LPS + MT2A50ng/ml group),B2 group(LPS + MT2 A 250ng/ml group),B3 group(LPS + MT2A500ng/ml group)contrast experiment,experiment After the group was added LPS,different concentrations of MT2 A were added for co-cultivation.After culture,the content of IL-6 and TNF-? in the culture supernatant of the experimental group and the control group was measured at a fixed time,and cytoskeletal injury was observed under a fluorescence microscope,then conducted comparative analysis.Results After a period of cultivation,the concentrations of TNF-? in the two groups were the lowest at 0 h,and then gradually increased,reaching a peak at 6 h.From each time point,except for 0h,there was no significant difference in treatment factors(t=-1.731,p=0.103).In the rest of the time points,U group was significantly higher than that of N group(All p<0.001);In N group,The concentration of IL-6 in the U group was the lowest at 0 h,then gradually increased,and reached the peak at 6 h.From each time point,except for 0h,there was no significant difference in treatment factors(t=-0.297,p=0.770).In the other time points,U group was significantly higher than that in group N(All p<0.001).The results of fluorescence microscopy showed that the distribution of F-actin in the cytoskeleton of N group was more dense,and a large number of stress fibers showed a regular radial arrangement;After 6 hours,the F-actin in group U was significantly deaggregated and distributed obviously,and the stress fibers arranged in disorder or disappeared..2.After cultured in Method 2,the concentrations of TNF-? in each group were the lowest at 0 h,gradually increased,and reached the peak at 6 h.From each time point,there was no significant difference in TNF-? concentrations except 0 h(F=0.717,p=0.549).At the other time points,B1,B2,and B3 were significantly higher than those of A group(All p<0.05).Compared with B1,the concentrations of TNF-? in B2 and B3 were higher than B1 at 2h,4h,and 6h(All p<0.05).Compared with B2,the concentrations of TNF-? in B3 was higher than B2 at 2h,4h,and 6h(All p<0.05).The concentration of IL-6 in all groups was the lowest at 0 h,and group A ?group B1 ?group B2 and B3 groups gradually increased from 0 h and reached the peak at 6 h.From the time point of view,except for 0h,there was no significant difference in treatment factors(F=2.341,p=0.092).At the other time points,B1,B2,and B3 were all lower than in group A(All p<0.05).Compared with B1,concentration of IL-6 in B2 and B3 were lower than B1 at 2h,4h,and 6h(All p<0.05).Compared with B2,concentration of IL-6 in B3 was lower than B2 at 4h and 6h(all p<0.05).By the fluorescence microscope,the fibrous actin(F-actin)was obviously depolymerization after 6 hours in A group,the distribution of the stress fiber was obviously reduced and the stress fibers were disarranged or disappearing.After 6 hours,the distribution of F-actin in group B1,group B2 and group B3 was significantly more than that in group A,and the stress fibers arranged more orderly.Conclusion LPS has obvious damage effect on human pulmonary capillary endothelial cells.After adding MT2 A,the release of inflammatory factors and cytoskeleton damage of cells are obviously reduced,indicating that MT2 A may have protective effect on pulmonary capillary injury mediated by LPS.