Effect of supernatant of human amnion homogenate on lipopolysaccharide induced pulmonary microvascular endotheli-al cells injury and their proliferation and expression of proinflammatory factors in rats
Chen Yunpeng
Wang Lei
Tong Yalin
Liu Liang
Lü Lu
Mo Yongliang
Zhan Qiu
Yang Qiqiong
Liang Jing
Zhu Fujun
Gong Zhenyu
Xin Haiming
Abstract：Objective:To investigate the protective effect of supernatant of human amnion homogenate (hAHS)on proliferation and expression of proinflammatory mediators by lipopolysaccharide (LPS)induced inj ured pulmonary microvascular endothelial cells of rats (RPMVECs).Methods:hAHS was prepared from fresh human amnion. The total protein content and the content of epithelial growth factor (EGF),basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF),interleukin-4 (IL-4),IL-10,angiogenin-1 (Ang-1),humanβ-defensin2 (HBD2)of hAHS were determined with Coomassie blue staining and ELISA. The effect of 0,10%,15%, 20%,25% hAHS on cell proliferation activity of RPMVECs was respectively determined with MTT assay,in or-der to determine the optimal concentration of hAHS on promoting RPMVECs proliferation. According to different co-culture conditions,RPMVECs were randomly divided into 4 groups:group N (cultured with 10%FBS+DMEM/F12),group A(10%FBS+DMEM/F12+15%hAHS),group B (10%FBS+DMEM/F12+LPS),and group C (10%FBS+DMEM/F12+15%hAHS+LPS). At 0,12,24,48,72 hours after culturing with the corre-sponding medium of each group,optical density values (A values)of each group were determined respectively with MTT assay to determine the proliferation activity,and the contents of IL-6,IL-8,TNF-αlevels in the culture su-pernates were also determined by ELISA at 6,8,10,12 and 24 hours. Results:The total protein concentration of hAHS was (725.125±12.625)mg/L,and levels of EGF,bFGF,VEGF,IL-4,IL-10,Ang-1,HDB2 were re-spectively(504.785±4.665)ng/L,(4.426±0.138)ng/L,(0.185±0.006)ng/L,(25.650±4.104)ng/L,(13.733 ±2.197)ng/L,(15.561±0.496)ng/L,(4.763±0.714)ng/L.10%-20% hAHS was shown to promote prolifer-ation of RPMVECs,and 15% hAHS,and the best result was observed on 7 and 9 days. The proliferation rate of RPMVECs in 25% hAHS group at 7,9 and 11 days was lower than those in the 0%hAHS group (P<0.05).The proliferation activity (A value )of group A was greater than that of group N at 48 and 72 hours,and it was lower in group B significantly as compared with that of group N at 24,48 and 72 hours (P<0.05),while it was signifi-cantly higher in group C compared with that of the group B at 24,48 and 72 hours (P<0.05). ELISA result showed that the contents of IL-6 and TNF-αin group B were higher than those of group N(P<0.05). IL-8 levels of group B at 8,10,12 and 24 hours were higher than those of group N (P<0.05). In group C,the level of IL-6 was significantly lower than that of group B at 10 and 12 hours,IL-8 level at 8,10,12 and 24 hours (P<0.05),and TNF-αcontent at each time point (P<0.05).Conclusion:hAHS are rich in factors which can promote tissues repair,regulate immune response,reduce vascular permibility,promote hAHS on LPS induced injury of the proliferation of RPMVECs after being inj ured by LPS,and reduce the secretion of inflammatory cytokines after inj ury by LPS challenge.
Keywords：Supernatant of human amnion homogenateRatPulmonary microvascular endothelial cellsLipopolisac-charideCell proliferationAcute lung inj ury
Publication Date：2015-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 10-16 )