荆防药对正丁醇提取部位抗炎作用的NF-κB信号通路机制研究

Anti-inflammatory effect of n-butanol extracts from couplet medicinals of Jingjie and Fangfeng: a study on NF-κB signal pathway mechanism

目的观察荆防药对正丁醇提取部位对气管滴注脂多糖(LPS)所致小鼠急性肺损伤(ALI)模型及LPS刺激诱导的小鼠单核巨噬白血病细胞(RAW264. 7细胞)炎症模型的保护作用,探讨其抗炎效应的核转录因子-κB(NF-κB)信号通路机制。方法 (1)小鼠ALI模型:将雄性昆明种小鼠分为空白对照组,假手术组,模型组,地塞米松(5 mg/kg)+LPS组,荆防药对正丁醇部位低、中、高剂量(3. 33、6. 67、10. 01 g/kg)+LPS组,除地塞米松组于实验第2、4、5天腹腔注射给药1次外,其余各组动物连续灌胃给药5 d,1次/d,空白对照组、假手术组和模型组给予等体积0. 5%吐温溶液。末次给药30 min后,除空白对照组和假手术组外其余各组小鼠气管滴注LPS 100μL(5 mg/kg)制备小鼠ALI模型,假手术组小鼠行麻醉、气管滴注操作,但给予无菌生理盐水。造模后5 h各组再次给药1次,给予LPS后6 h处死小鼠,取小鼠肺组织进行病理形态学观察,Real-Time PCR法检测小鼠肺组织NF-κB p65、NF-κB p50、IκBαmRNA表达水平,Western-Blot法检测小鼠肺组织NF-κB p50蛋白表达水平。(2)RAW264. 7细胞炎症模型:取对数期RAW264. 7细胞悬液接种、培养24 h贴壁后,设空白对照组,LPS组(1 mg/L),LPS+DMSO对照组,LPS+地塞米松(5 mg/L)组,LPS+荆防药对正丁醇部位高、低剂量组(50、25 mg/L),LPS+5-O-甲基维斯阿米醇苷高、低剂量组(50、25 mg/L),LPS+橙皮苷组(50μg/L),LPS+迷迭香酸组(5 mg/L)。受试药物预处理3 h,除空白对照组外其余各组均加入1 mg/L LPS刺激12 h造模,吸取细胞上清,Griess法测定一氧化氮(NO)含量,ELISA法测定白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平;裂解细胞,Real-Time PCR法检测细胞NF-κB p65、NF-κB p50和IκBαmRNA表达水平。结果 (1)与LPS所致小鼠ALI模型组比较,荆防药对正丁醇部位各剂量组可减少ALI小鼠肺组织嗜中性粒细胞计数,其中低剂量组降低作用显著(P <0. 01);荆防药对正丁醇部位中、低剂量组显著下调ALI小鼠肺组织NF-κB p65mRNA及NF-κB p50蛋白表达水平(P <0. 01),中剂量组亦明显下调NF-κB p50 mRNA表达水平(P <0. 05)。(2)与细胞炎症模型组比较,所有受试药物均能明显降低细胞上清IL-6水平(P <0. 01或P <0. 05),荆防药对正丁醇部位(50、25 mg/L)、5-O-甲基维斯阿米醇苷低剂量组可显著降低细胞上清NO含量(P <0. 01或P <0. 05),荆防药对正丁醇部位高剂量组有减少细胞上清TNF-α含量的趋势;荆防药对正丁醇部位低剂量组显著下调细胞NF-κB p65、NF-κB p50、IκBαmRNA表达水平(P <0. 05或P <0. 01),高剂量组明显抑制细胞NF-κB p50、IκBαmRNA表达(P <0. 05或P<0. 01),5-O-甲基维斯阿米醇苷和橙皮苷亦可显著下调细胞NF-κB p50 mRNA表达水平(P <0. 05),迷迭香酸明显抑制模型细胞NF-κB p50和IκBαmRNA表达的上调(P <0. 05)。结论荆防药对正丁醇提取部位具有抗急性炎症作用,抗炎作用发挥与抑制NF-κB信号通路中关键因子NF-κBp65、NF-κB p50、IκBα基因及NF-κB p50蛋白表达有关,5-O-甲基维斯阿米醇苷、橙皮苷与迷迭香酸可能为其主要有效物质基础。

Objective To observe the protective effect of n-butanol extracts( NBE) from couplet medicinals of Jingjie( Schizonepeta,Herba Schizonepetae) and Fangfeng( Ledebouriella Root,Radix Ledebouriellae,JF-couplet) on mouse model of acute lung injury( ALI) induced by lipopolysaccharide( LPS) and on RAW264. 7 cell inflammation induced by LPS,and investigate the anti-inflammatory mechanism NF-κB signal pathway. Methods(1) ALI model: male Kunming mice were divided into blank group,sham-operation group,model group,dexamethasone + LPS group( 5 mg/kg,DXM group)and low-dose,mid-dose and high-dose NBE-JF-couplet + LPS groups( 3. 33,6. 67,10. 01 g/kg,low-dose,mid-dose and high-dose NBE-JF-couplet groups). DXM group was given intraperitoneal injection of DXM once on the 2 nd,4 thand 5 thd,NBE-JF-couplet groups were orally given corresponding medicinals for 5 d( once a day),and blank group,sham-operation group and model group were given equivalent Tween solution( 0. 5%). After the last medication for 30 min,except of blank group and sham-operation group,other groups were given intratracheal drip of LPS( 100 μL,5 mg/kg) for establishing ALI model,and sham-operation group was given anesthesia and intratracheal drip of saline solution. All groups were medicated once more after modeling for 5 h,and mice were executed after LPS administration for 6 h. The samples of lung tissue were collected from mice for pathological histomorphological observation. The mRNA expressions of lung NF-κB p65,NF-κB p50 and IκBα were detected by using RT-PCR. The expression of NF-κB p50 protein was detected by using Western blotting assay.(2)Inflammatory model of RAW264. 7 cells: RAW264. 7 cell suspension in logarithmic term was inoculated and cultured for 24 h,the cells were divided into blank group,LPS group( 1 μg/m L),DMSO group,DXM group( 5 mg/L),high-dose and low-dose NBE-JF-couplet groups( 50 mg/L,25 mg/L),high-dose and low-dose 5-O-methylvisamminol groups( 50,25 mg/L),hesperidin group( 50μg/L) and rosmarinic acid group( 5 mg/L). After pretreatment with medicinals for 3 h,all groups,except of blank group,were given LPS( 1 mg/L) for 12 h to establish model and absorb supernatant.The content of NO was detected by using Griess method,and levels of IL-6 and TNF-α were detected by using ELISA. The mRNA expressions of NF-κB p65,NF-κB p50 and IκBα were detected by using RT-PCR. Results(1)The count of neutrophils decreased in all NBE-JF-couplet groups compared with model group, which was more significant in low-dose NBE-JF-couplet group( P < 0. 01). The expressions of lung NF-κB p65 mRNA and NF-κB p50 protein were down-regulated in mid-dose and low-dose NBE-JF-couplet group( P < 0. 01),and expression of NF-κB p50 mRNA was significantly down-regulated in mid-dose NBE-JF-couplet group( P < 0. 05). The level of IL-6 decreased significantly in all medicated groups compared with model group( P < 0. 01 or P < 0. 05). The content of NO decreased significantly in high-dose and low-dose NBE-JF-couplet groups and low-dose 5-Omethylvisamminol groups( P < 0. 01 or P < 0. 05). The level of TNF-α had a descending trend in high-dose NBE-JF-couplet group. The mRNA expressions of NF-κB p65,NF-κB p50 and IκBα were down-regulated significantly in low-dose NBE-JF-couplet group( P < 0. 05 or P < 0. 01). The mRNA expressions of NF-κB p50 and IκBα were down-regulated significantly in high-dose NBE-JF-couplet group( P < 0. 05 or P < 0. 01). The expressions of NF-κB p50 mRNA was down-regulated significantly in high-dose and low-dose 5-O-methylvisamminol groups and hesperidin group( P < 0. 05). The mRNA expressions of NF-κB p50 and IκBα were down-regulated significantly in rosmarinic acid group( P < 0. 05). Conclusion The n-butanol extracts from couplet medicinals of Jingjie and Fangfeng has effects against acute inflammation,which is related to inhibition of mRNA and protein expressions of NF-κB p65,NF-κB p50,IκBα and NF-κB p50 in NF-κB signaling pathway,and 5-O-m-ethylvisamminol,hesperidin and rosmarinic acid may be the main material basis.