骆驼乳清蛋白对热应激大鼠肝脏炎症及高迁移率族蛋白B1/Toll样受体4/核转录因子-κB信号通路的调节

Effects of Camel Whey Protein on Hepatic Inflammation and High Mobility Group Protein B1/Toll Like Receptor 4/Nuclear Factor-κB Signaling Pathway in Heat Stressed Rats

本试验旨在研究骆驼乳清蛋白(CWP)对热应激(HS)所致大鼠肝脏炎症及相关信号通路的调节作用。选取6周龄SD大鼠,适应性饲养2周后随机分为6组,每组设置5个重复,每个重复6只,试验期22 d。正常对照组(Control组)饲喂基础饲粮;CWP对照组(CWP组)每天灌服400 mg/kg BW CWP;HS致肝脏损伤组(HS组)饲喂基础饲粮并从第15天开始每天进行2 h HS处理,连续8 d;CWP低、中、高剂量干预组(L、M和H组)每天灌服100、200和400 mg/kg BW CWP,且从第15天开始每天进行2 h HS处理,连续8 d。酶联免疫吸附试验(ELISA)检测肝脏肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和白细胞介素-10(IL-10)含量,免疫组织化学及免疫印迹(Western blotting)方法检测肝脏高迁移率族蛋白B1(HMGB1)、Toll样受体4(TLR4)和磷酸化核转录因子κB p65(p-NF-κB p65)蛋白表达。结果表明:1)与HS组相比,400 mg/kg的CWP干预显著降低了HS大鼠肝脏TNF-α、IL-1β、IL-6及IL-8含量(P<0.05),显著提高了IL-10含量(P<0.05)。2)与HS组相比,CWP以剂量依赖方式极显著降低了HS大鼠肝脏HMGB1蛋白核转位(P<0.01),但对其总蛋白表达水平没有显著影响(P>0.05),同时极其显著降低了TLR4蛋白表达水平(P<0.001),极显著降低了p-NF-κB p65蛋白水平(P<0.01)及核转位。与Control组相比,400 mg/kg的CWP干预效果最好。由此可见,HS前灌服400 mg/kg的CWP可调节HS大鼠肝脏HMGB1/TLR4/NF-κB信号通路并缓解炎症反应。

This experiment was aimed to investigate the regulatory effects of camel whey protein(CWP)on hepatic inflammatory and relative signaling pathways in rats induced by heat stress(HS).SD rats at 6 weeks of age were selected and randomly divided into six groups after 2 weeks of adaptive feeding,with five replicates in each group,six in each replicate,and test period was 22 d.The normal control group(Control group)was fed a basal diet.The CWP control group(CWP group)fed 400 mg/kg BW CWP daily.HS induced liver injury group(HS group)was fed the basal diet and subjected to 2 h HS treatment daily from day 15 for 8 d.CWP low-,medium-,and high-dose intervention groups(L,M and H groups)were received 100,200,and 400 mg/kg BW CWP daily and subjected to 2 h HS treatment daily for 8 consecutive days starting at day 15.Hepatic tumor necrosis factor-α(TNF-α),interleukins-1β(IL-1β),interleukins-6(IL-6),interleukins-8(IL-8),and interleukins-10(IL-10)contents were measured by enzyme linked immunosorbent assay(ELISA),and hepatic high mobility group protein B1(HMGB1),Toll like receptor 4(TLR4),and phosphorylated nuclear factor-κB(p-NF-κB p65)protein expression was detected by immunohistochemistry and Western blotting.The results showed as follows:1)compared with the HS group,CWP intervention at 400 mg/kg effectively reduced hepatic TNF-α,IL-1β,IL-6 and IL-8 contents(P<0.05)and increased IL-10 content in HS rats(P<0.05).2)Compared with the HS group,CWP reduced the nuclear translocation of HMGB1 protein in the liver of HS rats in a dose-dependent manner(P<0.01),but had no effect on the total protein expression(P>0.05),while it extremely significantly reduced TLR4 protein expression(P<0.001),significantly reduced p-NF-κB p65 protein expression(P<0.01)and nuclear translocation.Compared with the Control group,CWP intervention at 400 mg/kg had the best effect.Thus,it is concluded that CWP at 400 mg/kg administered before HS modulates the hepatic HMGB1/TLR4/NF-κB signaling pathway and alleviates inflammatory responses in HS rats.