The Protective Effects Of HAHs On LPS-induced PMVEC Injury

Objective(1) To observe the effects of different concentrations of human Amniotic Homogenates Supernatant (hAHS) on cultured SD rat Pulmonary Microvascular Endothelial Cells (PMVEC) proliferation.(2)To investigate the protective effect of hAHS on Lipopolysaccharide(LPS)-induced SD rat PMVEC injury by cell proliferation and inhibition of the expression of cell proinflammatory.Methods(1) Prepare hAHS, measure the total protein and some cytokines of hAHS. Take health cesarean section membranes, wash it with PBS and peel human amniotic under sterile conditions. Identify its vitality. Cut human amniotic into pieces and take it into EP tube. Add DMEM/F12 into EP tube 1:1 by volume, then homogenate.Transfer the homogenates into a new EP tube and add DMEM/F12 5:1 by volume, then centrifugate. After centrifugation, the supernatants were filtered with a bacteria filter. Collect the filtrate, then we got hAHS (used in cell culture),-80℃ store. Referring to the above method, we got hAHS (used in detecting cytokines) by replacing DMEM/F12 with PBS. The total protein content of hAHS was detected by Coomassie blue staining. Use ELISA to detect the contents of cytokines:EGF, bFGF, VEGF, IL-4, IL-10, Ang-1, HBD2.(2) To observe the effects of different concentrations of hAHS on cultured SD rat PMVEC proliferation:Digest the first PMVEC generation after cultured 6 days, 1×104cell/mL,200 u L/well PMVEC were seeded in 96-well plates. According to the grouping, medium was replaced after 24h incubation and changed every other day. Depend on the different components of culture medium to divide them into five groups (The concentrations of FBS, hAHS, DMEM/F12 meant a percentage of them in the total volume of the culture medium in each group):0%hAHS:10%FBS+90% DMEM/F12,10%hAHS:10%FBS+ 80%DMEM/F12+10%hAHS,15%hAHS:10%FBS+75% DMEM/F12+15%hAHS, 20%hAHS:10%FBS+70%DMEM/F12+20%hAHS,25%hAHS:10%FBS+ 65%DMEM/F12+25%hAHS. Respectively at the first time the medium was changed in each group, use MTT assay to detect the OD values of each hole at 0 (that day),1st,3rd, 5th,7th,9th,11th days. Draw growth curve and calculate the rate of cell proliferation.(3) The protective effect of hAHS on LPS-induced SD rat PMVEC injury:Digest the first PMVEC generation which fused 80%.4×104cell/mL,200μL/well PMVEC were seeded in 96-well plates. After incubation 24h, the medium was changed with DMEM/F12 containing 10%FBS, and cultured cells to 2 days. Depend on the different components of culture medium to divide them into five groups (The concentrations of FBS, hAHS, DMEM/F12 meant a percentage of them in the total volume of the culture medium in each group, the concentrations of LPS meant the final concentrations of LPS in the culture medium):Group N:10%FBS+90%DMEM/F12, Group A:10%FBS+75%DMEM/F12+15%hAHS, Group B:10%FBS+90%DMEM/F12+20μg/mLLPS, Group C:10%FBS+ 75%DMEM/F12+15%hAHS+20μg/mL LPS. After replacing the medium in accordance with the experimental group, use MTT assay to detect the OD values of each hole at 0 (that hour),12th,24th,48th,72nd h, collecte cell culture supernatant at 6th,8th,10th,12th, 24th h, use ELISA to detect the contents of Rat-IL-6, Rat-IL-8, Rat-TNF-a in the cell culture supernatant.Results(1) The total protein concentration of hAHS was 725.125±12.625 mg/L and some of the cytokines in hAHS were EGF (504.785±4.665)ng/L、bFGF (4.426±0.138)ng/L、VEGF (0.185±0.006)ng/L, IL-4 (25.650±4.104)ng/L、IL-10 (13.733±2.197) ng/L、Ang-1 (15.561±0.496)ng/L、HBD2 (4.763±0.714) ng/L.(2) The promotion of hAHS effect on PMVEC:The growth curve trend of each group-0%hAHS, the growth curve trend of PMVEC was in lag phase at 0-4th days, in logarithmic growth phase at 4th-7th days, in plateau phase at 7th～10th days, then entered the decline phase.10%hAHS, the growth curve trend of PMVEC was similar with 0%hAHS at the first 4 days, in logarithmic growth phase at 4th～8th days, in plateau phase at 8th-10th days, then entered the decline phase.15%hAHS, the growth curve trend of PMVEC was similar with 0%hAHS at the first 4 days, in logarithmic growth phase at 4th-7th days, in plateau phase at 7th～9th days, then entered the decline phase.20%hAHS, the growth curve trend of PMVEC was similar with 0%hAHS.25%hAHS, the growth curve trend of PMVEC was similar with 15%hAHS. Compare the OD values and the proliferation rate of PMVEC with each group-The OD values of 10%hAHS were greater than 0%hAHS at 1st,3rd,5th days with no statistically significant difference (P>0.05), while at 7th,9th,11th days with statistically significant difference (P<0.05). The proliferation rate of 10%hAHS was greater than 0% hAHS at 7th day (P<0.05), while at the other time point with no statistically significant difference (P>0.05). The OD values of 15%hAHS were greater than 0%hAHS at 1st,3rd days with no statistically significant difference (P>0.05), while at 5th,7th,9th,11th days with statistically significant difference (P<0.05). The proliferation rate of 15%hAHS was greater than 0% hAHS at 5th,7th days (P<0.05), while at the other time point with no statistically significant difference (P>0.05).The OD values of 20%hAHS were not statistically significant difference with 0%hAHS at each time point(P>0.05). The proliferation rate of 20%hAHS was less than 0%hAHS at 9th day (P<0.05), while at the other time point with no statistically significant difference (P>0.05). The OD values of 25%hAHS were not statistically significant difference with 0%hAHS at 1st,3rd,5th days (P> 0.05), which were less than 0%hAHS at 7th,9th,11th days (P<0.05). The proliferation rate of 25%hAHS was less than 0%hAHS at 7th,9th,11th days (P<0.05), while at the other time point with no statistically significant difference (P>0.05)(3) The protective effect of hAHS on LPS-induced SD Rat PMVEC Injury:The OD values of Group A were greater than Group N at 48th,72nd h with statistically significant difference (P<0.05).The content of IL-6,IL-8,TNF-a in hAHS were not statistically significant difference withGroup N at each time point (P>0.05). The OD values of Group B were less than Group N at 24th,48th,72nd h with statistically significant difference (P< 0.05).The content of IL-6,IL-8 (except 6h),TNF-a in hAHS were greater than Group N with statistically significant difference at each time point (P<0.05). The OD values of Group C were greater than Group B at 24th,48th,72nd h with statistically significant difference (P<0.05).The content of IL-6 (except 6th,8th,24th h),IL-8 (except 6th h),TNF-a in hAHS were less than Group B with statistically significant difference at each time point (P<0.05)Conclusions(1) hAHS were rich in cytokines, such as EGF, bFGF, VEGF, IL-4, IL-10, HBD2, Ang-1.(2) 10、15% hAHS promoted PMVEC growth, especially 15% hAHS was the most obvious in vitro. However,25%hAHS played an inhibited role in the proliferation of SD rat PMVEC.(3) hAHS played a protective effect on LPS-induced SD rat PMVEC injury and reduced the expression of pro-inflammatory cytokines IL-6, IL-8, TNF-a in LPS-induced SD rat PMVEC.