| Objective To observe the influence of angiopoietin-1(Ang-1) on the expression of inflammatory cytokines and apoptosis in cultured mice pulmonary microvascular endothelial cells(PMVEC) induced by lipopolysaccharide(LPS).We investigated wheth er Ang-1has direct anti-inflammatory and anti-apoptosis effects on PMVEC stimulat ed by LPS.Mean while the preliminary mechanism were investigated.Methods1.The neonatal BALB/c mice was used to isolate and delete culture t he pulmonary microvascular endothelial cells,then the PMVCE was used to passage d、purified and identified.2.CCK-8assay was used to observe the proliferation of L PS-induced PMVEC at different concentrations (Ong/ml、1ng/ml、10ng/ml、100ng/ml、1000ng/ml) or Ang-1-induced PMVEC at different concentrations (Ong/ml、1ng/ml、10ng/ml、100ng/ml、1000ng/ml).3.The PMVEC was cultured and equally divided i nto seven groups randomly:control group(A);LPS group(B);LPS+Ang-1group(C);LPS+Ang-1+LY294002group(D);Ang-1group(E);LY294002group(F);DMSO group(G).4. The cells in each group were harvested after24h.The levels of IL-8and TNF-a of PMVEC were detected by ELSIA technique.The early stage of apoptosis of PMVE C were detected by Flow cytometry;Annexin V/PI technique;Caspase-3levels of PM VEC were assessed by the caspase colorimetric assay;The expression of PI3K and P-AKT of PMVEC were examined by Western blotting.Results1.The cultured cells exhibited as a typical cobblestone-like and pavings tone shape under inverted microscope,Immunofluorescent assay staining revealed that the expression of CD31membrane antigen was positive.2.100ng/ml LPS significant ly inhibited the proliferation of PMVEC(OD:0.56±0.14)compared with control group (OD:1.08±0.22)(P<0.05);Otherwise,100ng/ml Ang-1significantly promoted the prolif eration of PMVEC(OD:1.44±0.28)compared with control group(OD:1.07±0.24)(P<0.05).3.The expression of IL-8、TNF-α、the early stage of apoptosis、the levels of Caspase-3were significantly increased in LPS group(780±22pg/ml、360±20pg/ml、13.00±0.58%、1.21±0.12)compared with the control group(220±16pg/ml、50±14p g/ml、4.50±0.44%、0.30±0.09)(all P<0.01);But the expression of IL-8, TNF-α, t he early stage of apoptosis、the levels of Caspase-3were significantly decreased in Ang-1+LPS group(450±17pg/ml、220±13pg/ml、6.10±0.58%、0.59±0.13)compared with LPS group (all P<0.05);The early stage of apoptosis(10.3±0.50%)and the lev els of Caspase-3(0.92±0.11) in LPS+Ang-1+LY294002group were significantly inc reased than in LPS+Ang-1group (P<0.05).The expression of PI3K and p-Akt of P MVEC were significantly decreased in LPS group compared with control group(P<0.05);and in Ang-1+LPS group,The expression of PI3K and p-Akt were significantly i ncreased compared with LPS group(P<0.05),also significantly increased compared wi th Ang-1+LPS+LY294002group(P<0.05);LY294002group significantly decreased the expression of PI3K and p-Akt compared with control group(P<0.05);But Ang-1gro up significantly increased the expression of PI3K and p-Akt compared with control group(P<0.05);There was no difference between the control group and DMSO group (P>0.05).Conclusion Lipopolysaccharide could induce inflammation and apoptosis of P MVEC.Angiopoietin-1protected PMVEC from LPS-induced injury via its anti-inflam matory and anti-apoptosis effect,but the protective effect could be blocked by the P I3K inhibitor,LY294002.Angiopoietin-1also could maintain the expression of PI3K and p-Akt in LPS-stimulated PMVEC.It is suggested that the roles angiopoietin-1p1ayed might be involving the PI3K/AKT pathway. |
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