Emodin Reduces LPS-induced Inflammation In Adipocytes Through PTEN/PI3K/PKB Pathway

Objectives:The purpose of this research was to investigate whether emodin reduces inflammation in LPS-treated 3T3-L1 adipocytes. and to investigate the underlying molecular mechanisms of action of emodin in these cells.Methods:(1) 3T3-L1 adipocytes were treated with vehicle, emodin or cortisone in the presence or absence of LPS. After 24 hours,11β-HSD1 activity was measured using a Cortisol EIA kit:IL-6 secretion was assayed by ELISA; the levels of IL-6, IL-1β and TNF-α mRNA were measured by qRT-PCR.(2) LPS-treated 3T3-L1 adipocytes were treated with vehicle, emodin, cortisone in the presence or absence of wortmannin. IL-6 production was measured by ELISA; p-PKB (Ser473) and PKB were analysed by Western blot.(3) LPS-treated 3T3-L1 adipocytes were transfected with vehicle, emodin. 11β-HSD1 siRNA or PTEN siRNA, respectively. IL-6 production was measured by ELISA; the levels of 11β-HSD1, PTEN, IL-6, IL-1β and TNF-α mRNA were measured by qRT-PCR; 11β-HSD1 and PTEN were analysed by Western blot.(4) LPS-treated 3T3-L1 adipocytes were transfected with vehicle, emodin and Rictor siRNA, respectively. IL-6 production was measured by ELISA; the levels of p-PKB (Ser473), p-PKB (Thr308), PKB and Rictor were analysed by Western blot.(5) LPS-treated 3T3-L1 adipocytes were treated with vehicle, emodin, cortisone in the presence or absence of wortmannin, p-IκBα (Ser32/36), IκBαand NFκB p50 were analysed by Western blot.(6) LPS-treated 3T3-L1 adipocytes were treated with emodin or vehicle in the presence or absence of 250 μM palmitate. After 24 hours. IL-6 secretion was assayed by ELISA;PTEN was analysed by Western blot.Results:(1) Emodin significantly reduced 10ng/ml LPS-induced IL-6 productions in 3T3-L1 adipocytes; Emodin dose significantly inhibited the activity of 11β-HSD1 and the mRNA levels of IL-6, IL-1β and TNF-α in these cells. Conversely, 1pM cortisol promoted IL-6 production.(2) Emodin increased the ratio of p-PKB(Ser473)/PKB, whereas 1pM cortisone decreased the ratio of p-PKB(Ser473)/PKB. and emodin reversed the effects of cortisone on p-PKB(Ser473)/PKB levels. Similar to cortisone, wortmannin decreased the ratio of p-PKB(Ser473)/PKB and enhanced IL-6 production. emodin reversed the effects of wortmannin on p-PKB(Ser473)/PKB and IL-6 levels.(3) Emodin and 11β-HSD1 siRNA reduced the level of PTEN; In contrast, 1pM cortisone increased the level of PTEN; PTEN siRNA decreased the levels of IL-6, IL-1β and TNF-α mRNA and IL-6 production, but did not affect the level of 1 11β-HSD1.(4) Emodin didn’t affect the p-PKB (Thr308)/PKB ratio:depeltion of Rictor. a key component of mTORC2. by specific siRNA, reduced the level of Rictor and p-PKB (Ser473)/PKB ratio; emodin didn’t affect p-PKB (Ser473)/PKB ratio in Rictor-depleted adipocytes; Moreover, Rictor siRNA increase the level of IL-6, emodin didn’t affect IL-6 in Rictor-depleted adipocytes.(5) Emodin decreased p-IκBa (Ser32/36)/IκBα ratio, whereas wortmannin and 1pM cortisone increased p-IκBa (Ser32/36)/IκBα ratio. Moreover, emodin reduced the nuclear NF-κB p50 subunit, but1pM cortisone increased the nuclear NF-κB p50 subunit.(6) Emodin also significantly reduced palmitate-induced IL-6 and PTEN productions:but not the basal levels.Conclusion:Emodin inhibited LPS-induced inflammation by inhibiting the activity of 11β-HSD1 and reducing the level of PTEN, leading to activation of PI3K/PKB pathway, resulting in inhibition of NF-κB signalling.