藿朴夏苓汤对葡聚糖硫酸钠诱导的小鼠炎症性肠病的作用及机制

The Study of Huo Pu Xia Ling Tang on Ameliorating Dextran Sulfate Sodium-induced Colitis in Mice

目的探讨藿朴夏苓汤(HPXL)对葡聚糖硫酸钠(DSS)诱导的小鼠炎症性肠病(IBD)的药效作用及机制。方法体外研究:采用荧光素酶报告基因测定HPXL对LS174T细胞中核转录因子-κB(NF-κB)活性的影响;采用LPS刺激细胞,荧光定量PCR和ELISA法测定细胞因子IL-1β、IL-6、TNF-ɑ表达与释放,研究HPXL对NF-κB炎症通路的调控作用。体内研究:采用饮用4%DSS的方法建立IBD小鼠模型。将C57BL/6小鼠随机分成6组(n=10):正常组,模型组,HPXL高、中、低剂量组和柳氮磺胺吡啶(SASP)组。除正常组外,其他组小鼠饮用水中加入4%DSS造模,各给药组于造模前2 d开始灌胃给药,共9 d,每天1次。于给药第9天处死小鼠,取结肠做HE染色并进行组织病理学研究;ELISA方法检测小鼠结肠黏膜髓过氧化物酶(MPO)活性以及IL-1β、IL-6、TNF-ɑ炎症因子的含量。采用Western Blot法检测结肠组织中NF-кB、磷酸化NF-кB(p-NF-кB)、核转录因子抑制蛋白(IкBα)和磷酸化IкBα(p-IкBα)的蛋白表达水平。结果 HPXL显著抑制TNF-ɑ诱导的NF-кB活性;抑制LPS诱导的炎症因子IL-1β、IL-6、i NOS和COX-2的m RNA表达水平(P<0.01),并降低细胞中TNF-α和IL-1β含量的释放(P<0.05)。与模型组比较,HPXL能显著对抗DSS诱导的模型小鼠体质量降低和便血。HPXL通过降低结肠组织中NF-кB和IкBα的磷酸化蛋白表达水平,降低结肠组织中TNF-α、IL-1β和IL-6的含量以及MPO的活性,改善DSS诱导的IBD症状。结论 HPXL通过抑制NF-κB调节的炎症通路,改善DSS诱导的IBD症状。

Objective To explore the effect of Huo Pu Xia Ling Tang（HPXL） on inflammatory bowel disease（IBD）through blocking nuclear factor-kappa B（NF-κB）pathway in colitis mice induced by dextran sulfate sodium（DSS）. Methods In vitro study,LS174 T cells were transiently transfected with p CMV6-entry,p GL4.32 [luc2 P/NF-κB-RE/Hygro] NF-κB reporter plasmid and p RL-TK lenilla plasmid using Fu GENE 6 for 12 h;and cells were treated with HPXL（0,0.25,0.5 and 1 mg·m L-1）and dexamethasone（DEX,10 μM）for 12 h and then followed by additional TNF-α（20 ng·m L-1）for another 12 h. A standard Dual-Glo luciferase reporter assay was used to determine NF-κB-luc activity. The m RNA expression for IL-1β,IL-6,i NOS and COX-2 and the contents of TNF-ɑ and IL-1β in LS174 T cells were determined by real-time PCR and ELLISA method,after treatment with HPXL for 24 h and following induction with LPS for 24 h. In vivo study,C57 BL/6 mice were treated with 4% DSS to induce IBD model. Mice were randomized into normal group,model group,high,medium and low dose of HPXL（2,1 and 0.5 g·kg-1·d-1,respectively）and salazosulfapyridine（SASP）group. After treatment with HPXL for 9 d,the body weight changes,bloody diarrhea,myeloperoxidase（MPO）activity and contents of IL-1β,IL-6 and TNF-ɑ in inflamed colon and histopatholigical changes were examined. Moreover,protein expression levels of NF-кB,pNF-кB,IкBα and p-IкBα from inflamed colon were determined using Western Blot method. Results Treatment with HPXL significantly decreased TNF-ɑ induced NF-кB-luc activity（P 〈 0.01）. The m RNA expression of IL-1β,IL-6, i NOS and COX-2 and the contents of TNF-ɑ and IL-1β were also inhibited after treatment with HPXL in LS174 T cells（P 〈 0.05）. HPXL treatment obviously decreased the loss of body weight and the ratio of bloody diarrhea induced by DSS（P 〈 0.01）. Additionally, HPXL treatment suppressed DSS-induced inflammation in inflamed colon by inhibiting the production of TNF-ɑ,IL-6,IL-1β and MPO activity（P 〈 0.01）. Furthermore,HPXL treatment also blocked the increased protein expression levels of p-NF-кB and p-IкBα without changes of total protein expression levels of NF-кB and IкBα in inflamed colon（P 〈 0.01）,resulting the inflammation relieving of colon tissue histo-pathological changes. Conclusion HPXL has protective effect on DSS-induced colitis via suppressing NF-κB inflammatory pathway in mice.