Research Of The Regulatory Effect Of Cytosolic Phospholipase A₂ On Leptin Release In ECV-304 Cells

Leptin, the circulating hormone secreted primarily by adipose tissue , is an important regulator for weight balance and energy homeostasis. Recent studies have found that leptin may participate in metabolic disorders in injury and infection as a novel inflammatory cytokine. But whether leptin protects body from injury or promotes injury has not been confirmed. Cytosolic phospholipase A₂(cPLA₂) mediates inflammatory cytokines production and promotes inflammation as the key enzyme in inflammatory signal transduction. Because of the key role of cPLA₂ in inflammation, we hypothesize that it can mediate leptin release. Verification of the hypothesis will help to confirm the relationship between leptin and cPLA₂, and the role of leptin in inflammation. Therefore, we explore in this thesis the regulatory role of cPLA₂ in leptin release and the effect of leptin on inflammatory cytokines release in ECV-304 cells.Our study is devided into three parts. First, to observe the effect of cPLA₂ activation on leptin release in ECV-304 cells. Second, to observe the effect of cPLA₂ inhibition on leptin release in ECV-304 cells. Third, to observe the effect of leptin on IL-6 and IL-8 release. Detailed approaches are as follows:1. Lipopolysaccharide(LPS), platelet activating factor(PAF) and A₂₃₁₈₇ were used to activate cPLA₂ in ECV-304 cells, and leptin levels in the supernatant were detected. Results showed that LPS and PAF lowered leptin level in the supernatant significantly(P <0.05), but A₂₃₁₈₇ had no significant influence on leptin level in the supernatant. It suggests that the furthest cPLA₂ activation can reduce leptin release.2. MEK1/2 specific inhibitor U0126. cPLA2 specific inhibitor AAC0CF3 and CPLA2 antisense oligonucleotide were used to block CPLA2 activation and expression in LPS-induced ECV-304 cells, and leptin levels in the supernatant were detected. Results showed that cPLAi activity was inhibited respectively by U0126. AACOCF3 and antisense oligonucleotide dose-dependently, and leptin decrease in the supernatant was restored gradually when CPLA2 activity was suppressed in LPS-induced cells. It further suggests that CPLA2 mediates leptin release in ECV-304 cells.3. Different doses of leptin were administrated on ECV-304 cells, and IL-6 and IL-8 levels in the supernatant were detected. Results showed a trend that IL-6 and IL-8 concentrations decreased as leptin doses increased. It suggests that leptin has a potential role in inhibiting IL-6 and IL-8 release in ECV-304 cells.We draw a conclusion that CPLA2 has an important role in regulating leptin release, and leptin can regulate IL-6 and IL-8 release in ECV-304 cells.