脂氧素A4对脓毒症肥胖大鼠肝脏TLR4、TRAF6表达的影响

Effect of lipoxin A4 on the expression of Toll-like receptor 4 and TNF receptorassociated factor 6 in the liver of obese rats with sepsis

目的探讨脂氧素A4(LXA4)对脂多糖诱导肥胖大鼠感染脓毒症的保护作用及其对肝脏Toll样受体4(TLR4)、肿瘤坏死因子受体相关因子6(TRAF6)表达的影响。方法选取3周龄雄性SpragueDawley大鼠60只随机分为普通组和肥胖组(n=30),建立高脂饮食诱导的肥胖大鼠模型,将两组大鼠再随机分成对照组、脓毒症组、脂氧素组,每组各取8只大鼠。对照组、脓毒症组、脂氧素组分别予生理盐水、脂多糖、脂氧素A4+脂多糖腹腔注射,观察12 h后心脏采血并采集肝脏组织。ELISA法检测血清白细胞介素6(IL-6)及肿瘤坏死因子-α(TNF-α)水平;蛋白免疫印迹法检测肝脏组织TLR4、TRAF6蛋白水平;实时荧光定量PCR法检测TLR4 m RNA、TRAF6 m RNA的表达水平。结果高脂饮食喂养6周后,肥胖组大鼠的平均体重、Lee's指数明显高于普通组(P<0.05)。与普通组比较,肥胖组血清IL-6、TNF-α水平明显增高(P<0.05);同一饮食组内,脓毒症组较对照组血清IL-6、TNF-α水平明显增高(P<0.05),脂氧素组较脓毒症组血清IL-6、TNF-α水平显著降低(P<0.05)。与普通组比较,肥胖组大鼠肝脏组织中TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6m RNA表达水平上调(P<0.05);同一饮食组内,脓毒症组较对照组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量明显增高(P<0.05),而与脓毒症组比较,脂氧素组TLR4、TRAF6蛋白及TLR4 m RNA、TRAF6 m RNA表达量显著降低(P<0.05)。结论 LXA4能降低血清IL-6、TNF-α水平,减轻炎症反应。LXA4能抑制TLR4、TRAF6在脓毒症大鼠肝脏中的表达,可能是通过抑制TLR4的信号传导途径来实现的。

Objective To study the protective effect of lipoxin A4（LXA4） against sepsis induced by lipopolysaccharide（LPS） in rats with obesity and its effect on the expression of Toll-like receptor 4（TLR4） and TNF receptor-associated factor 6（TRAF6） in the liver. Methods A total of 60 male Sprague-Dawley rats aged three weeks were randomly divided into a normal group and an obesity group, with 30 rats in each group. A rat model of obesity was established by high-fat diet. Each of the two groups was further randomly divided into control group, sepsis group, and LXA4 group, and 8 rats were selected from each group. The rats in the control, sepsis, and LXA4 groups were treated with intraperitoneal injection of normal saline, LPS, and LXA4＋LPS respectively. Twelve hours later, blood samples were collected from the heart and liver tissue samples were also collected. ELISA was used to measure the serum levels of interleukin-6（IL-6） and tumor necrosis factor-α（TNF-α）. Western blot was used to measure the protein expression of TLR4 and TRAF6 in liver tissue. Quantitative real-time PCR was used to measure the m RNA expression of TLR4 and TRAF6. Results After being fed with high-fat diet for 6 weeks, the obesity group had significantly higher average weight and Lee＇s index than the normal group（P〈0.05）. Compared with the normal group, the obesity group had significant increases in the serum levels of IL-6 and TNF-α（P〈0.05）. In the normal group or the obesity group, the sepsis subgroup had significant increases in the serum levels of IL-6 and TNF-α compared with the control subgroup（P〈0.05）, while the LXA4 subgroup had significant reductions in the two indices compared with the sepsis subgroup（P〈0.05）. Compared with the normal group, the obesity group had significant increases in the protein and m RNA expression of TLR4 and TRAF6（P〈0.05）. In the normal group or the obesity group, the sepsis subgroup had significant increases in the protein and m RNA expression of TLR4 and TRAF6 compared with the control subgroup（P〈0.05）. Compared with the sepsis subgroup, the LXA4 subgroup had significant reductions in the protein and m RNA expression of TLR4 and TRAF6（P〈0.05）. Conclusions LXA4 can reduce the serum levels of IL-6 and TNF-α and alleviate inflammatory response. LXA4 can inhibit the expression of TLR4 and TRAF6 in the liver of septic rats, possibly by inhibiting the TLR4 signaling pathway.