The Protective Effects Of HAHS On LPS-induced Rat AT-â…¡ Injury

Objective(1) To observe the effects of different concentrations of human Amniotic Homogenates Supernatant (hAHS) on cultured rat Alveolar Type Ⅱ cell (AT-Ⅱ) proliferation.(2) To investigate the protective effect of hAHS on Lipopolysaccharide (LPS)-induced rat AT-Ⅱ 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 Special culture medium into EP tube 1:1 by volume, then homogenate.Transfer the homogenates into a new EP tube and add Special culture medium 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 Special culture medium with PBS. The total protein content of hAHS was detected by Coomassie blue staining. Use ELISA to detect the contents of cytokines:EGF、 bFGF、HGF、IL-4、 IL-10、 HBD2.(2) To observe the effects of different concentrations of hAHS on cultured rat AT-Ⅱ proliferation:Digest the first AT-Ⅱ generation after cultured 6 days,2×104cell/mL,200μL/well AT-Ⅱ 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. Special culture medium meant a percentage of them in the total volume of the culture medium in each group):0%hAHS: 10%FBS+90% Special culture medium,10%hAHS:10%FBS + 80% Special culture medium+ 10%hAHS,20%hAHS:10%FBS+70% Special culture medium+20% hAHS,40%hAHS: 10%FBS+50% Special culture medium+40% hAHS,80%hAHS:10%FBS+10% Special culture medium+80% 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 days. Draw growth curve and calculate the rate of cell proliferation.(3) The protective effect of hAHS on LPS-induced rat AT-Ⅱ injury: Digest the first AT-Ⅱ generation which fused 90%. 1×105 cell/mL,200μL/well AT-Ⅱ were seeded in 96-well plates. After incubation 24h, the medium was changed with Special culture medium containing 10% FBS. Depend on the different components of culture medium to divide them into five groups (The concentrations of FBS, hAHS, Special culture medium 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):FBS group(control group):10%FBS+90% Special culture medium, hAHS group:10%FBS + 50% Special culture medium+40%hAHS, LPS group:10%FBS + 90% Special culture medium + 40μg/mLLPS, LPS+hAHS group: 10%FBS + 50% Special culture medium + 40%hAHS + 40μg/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),24th,48th,72nd h, collecte cell culture supernatant at2nd,4th,6th, 12th,24th h, use ELISA to detect the contents of Rat IL-1β、Rat TNF-α in the cell culture supernatant.Results(1) The total protein concentration of hAHS is 725.125±12.625 mg/L and some of the cytokines in hAHS are EGF (504.785±4.665) ng/L、bFGF (4.426±0.138) ng/L、HGF (20.763±3.396) ng/L, IL-4 (25.650±4.104) ng/L、IL-10 (13.733±2.197) ng/L、HBD2 (4.763±0.714) ng/L.(2) The promotion of hAHS effect on AT-Ⅱ:The growth curve trend of each group-0%hAHS, the growth curve trend of AT-Ⅱ is in lag phase at 0～1st days, the 3rd～5th days in logarithmic growth phase. The all of experimental groups has a short lag phase, then into the logarithmic growth phase earlier than the 0% group, their logarithmic growth phase at 1st～5th days.Compare the OD values of AT-Ⅱ with each group:The OD values of Control group are less than the all of experimental groups at 1st,3rd,5th days with statistically significant difference (P<0.05). The OD values of 10%hAHS are less than 40% hAHS at 1st,3rd,5th days with statistically significant difference (P<0.05), while at3rd,5th days was less than20%, and 80% hAHS with statistically significant difference (P<0.05). The OD values of 20%hAHS are statistically significant difference with 40% hAHS at 3rd,5th days, and statistically significant for less than 80% at 3rd day was statistically difference (P<0.05), slightly more than 80% in 5 days hAHS concentration group (P>0.05).The OD values of 40%hAHS are statistically significant difference 10%hAHS at (P<0.05), and statistically significant in3rd,5th days was greater than hAHS groups and statistically significant (P<0.05)Compare the proliferation rate of AT-Ⅱ with each group:The proliferation rate of 10%hAHS is less than 20% hAHS at 5th day (P<0.05). The proliferation rate of 20%hAHS is less than 40% hAHS at 3rd,5th days,and statistically significant less than 80% at 3rd day group (P <0.05).The proliferation rate of 40% hAHS is more than 10% hAHS at lst,3rd,5th days (p< 0.05),and statistically significant more than 20%,80% at 3rd,5th days (P<0.05)(3) The protective effect of hAHS on LPS-induced Rat AT-Ⅱ Injury:The OD values of Group hAHS are greater than Group FBS at 24th,48th,72ndH with statistically significant difference (P<0.05),The content of, TNF-α in hAHS are not statistically significant difference with Group FBS at each time point(P>0.05). The OD values of Group LPS are less than Group FBS at 48th,72nd h with statistically significant difference (p<0.05).The content of IL-1β in hAHS are greater than Group FBS with statistically significant difference at 2nd,4thH (P< 0.05).The content of TNF-a in hAHS are greater than Group FBS with statistically significant difference at 24th,48th,72nd H (P<0.05). The OD values of Group LPS+hAHS are greater than Group LPS at 2nd,4th,6th,12th H with statistically significant difference (P<0.05). IL-1β in hAHS are less than Group LPS with statistically significant difference at2nd,4thH(P<0.05).The content of TNF-α in hAHS are less than Group LPS with statistically significant difference at 2nd,4th,6th,12thH (P<0.05)Conclusions(1) hAHS contains a certain amount of EGF, bFGF, HGF, IL-4, IL-10, HBD2.(2) hAHS plays an promoterole in the proliferation of rat AT-Ⅱ.especially 40% hAHS is the most obvious in vitro.(3) hAHS plays a protective effect on LPS-induced rat AT-Ⅱ injury and reduces the expression of pro-inflammatory cytokines IL-1β, TNF-a in LPS-induced rat AT-Ⅱ.