摘要目的 观察饥饿素(Ghrelin)对血小板源生长因子BB(PDGF-BB)刺激后体外培养人肝星状细胞(HSC-LX2)前胶原Ⅰ(procollagen type Ⅰ)合成及α平滑肌肌动蛋白(α-SMA)生成的影响,同时探讨PI3K-AKT通路在其中发挥的作用.方法 依不同浓度饥饿素与PDGF联合干预,以及设立空白对照分为6组:空白对照组、0.10 μmol/L饥饿素组、10 μg/L PDGF组、0.05 μmol/L饥饿素+10 μg/L PDGF组、0.10 μmol/L饥饿素+10 μg/L PDGF组、0.15 μmol/L饥饿素+10 μg/L PDGF组.体外培养HSC-LX2,分别行饥饿素、PDGF干预和两者按饥饿素不同浓度共同干预细胞.采用定量PCR方法检测各组前胶原Ⅰ mRNA,Western blot方法检测相应组别α-SMA以及AKT的表达变化.PI3K特异性抑制剂LY294002预处理LX2细胞以阻断PI3K-AKT通路,分为3组:PDGF组、饥饿素+PDGF组以及LY294002+饥饿素+PDGF组.采用定量PCR法检测各组前胶原Ⅰ mRNA及Western blot法检测α-SMA表达变化.结果 定量PCR结果显示,PDGF组前胶原Ⅰ mRNA为(6.91±0.46),较空白对照组(1.00±0.08)明显升高(P＜0.05).饥饿素组前胶原Ⅰ mRNA为(0.60±0.13),与空白对照组比较差异无统计学意义(P＞0.05).不同浓度饥饿素+PDGF共同处理组前胶原Ⅰ mRNA表达(依次为:3.11±0.28、2.03±0.23、0.70±0.06)与PDGF组比较显著降低,差异均有统计学意义(P均＜0.05),且随饥饿素浓度的增加其抑制作用增强,呈浓度依赖性关系.Western blot结果显示,饥饿素+PDGF组α-SMA较PDGF组表达降低.饥饿素+PDGF组AKT表达较PDGF组上调,提示PI3K-AKT可能参与了饥饿素对LX2细胞抗纤维化作用的调节.PI3K特异性抑制剂拮抗PI3K-AKT预处理后,LY 294002+饥饿素+PDGF组前胶原Ⅰ mRNA为(4.13±0.21),较饥饿素+PDGF组(2.34±0.25)明显上升(P＜0.05);Western blot结果亦显示α-SMA表达水平前者较后者明显升高,提示PI3K阻断后,饥饿素的抗纤维化作用下调.结论 PDGF刺激肝星状细胞活化后,饥饿素通过激活PI3K-AKT通路抑制前胶原Ⅰ及α-SMA生成从而发挥抗肝纤维化作用,可能成为今后防治肝纤维化的新途径.

abstractsObjective To observe the effect of ghrelin on the expression of procollagen type Ⅰ and alpha smooth muscle actin (α-SMA) synthesis in vitro cultured human hepatic stellate cell (HSC-LX2) stimulated by Platelet-derived growth factor-BB (PDGF-BB).Besides,the effect of PI3K-AKT pathway was studied.Methods Cultured LX2 were intervented and jointing intervented according to the different ghrelin concentration by ghrelin and PDGF:control group,0.1 μmol/L Ghrelin group,10 μg/L PDGF group,0.05 μmol/L Ghrelin +10 μg/L PDGF group,0.1 μmol/L Ghrelin + 10 μg/L PDGF group,0.15 μmol/L Ghrelin + 10 μg/L PDGF group.Culture HSC-LX2 in vitro,joint intervention cells with different concentrations.Procollagen Ⅰ mRNA expression were detected by Polymerase chain reaction (PCR),besides,α-SMA and AKT expression were detected by Western blot in each groups.After treatment by PI3K specific inhibitor LY294002 in LX2,three groups were divided into PDGF,Ghrelin + PDGF and LY294002 + Ghrelin +PDGF.Procollagen Ⅰ mRNA expression were detected by PCR,and α-SMA was detected by Western blot.Results PCR results showed that procollagen Ⅰ expression in PDGF treated group was significantly higher than the control group ((6.91 ± 0.46) vs.(1.00 ± 0.08),P ＜ 0.05),so PDGF can promote the expression of procollagen type Ⅰ.Procollagen Ⅰ mRNA expression between ghrelin group(0.60±0.13) and blank control group had no significant change(P＞0.05).Procollagen Ⅰ mRNA expression between different concentrations of Ghrelin and PDGF (3.11 ± 0.28,2.03 ±0.23,0.70 ± 0.06) was significantly reduced than PDGF group.The difference was statistically significant (P ＜0.05),and with the increase of the ghrelin concentration of the inhibition,the effect was more obvious in a concentration dependent manner.Western blot showed that α-SMA expression was lower in Ghrelin +PDGF group than PDGF group.AKT expression was higher in Ghrelin +PDGF group than PDGF group,indicating that PI3K-AKT may participate in the anti-fibrosis effect of ghrelin in LX2.After treatment of PI3K specific inhibitor,procollagen Ⅰ expression in LY294002+Ghrelin +PDGF group was significantly higher than Ghrelin +PDGF group((4.13±0.21) vs.(2.34±0.25),P＜0.05).Western blot also showed that α-SMA expression was higher in LY294002 + Ghrelin + PDGF group than Ghrelin + PDGF group.It was suggested that after inhibitation of PI3K,the anti-fibrosis effect of ghrelin in LX2 was attenuated.Conclusion After stimulated by PDGF in hepatic stellate cell,ghrelin can inhibit procollagen type Ⅰ and alpha-SMA synthesis in the process of hepatic fibrosis via PI3K-AKT pathway,thus,ghrelin may become one of the new ways of prevention and treatment of liver fibrosis.