摘要
目的研究千金子制霜前后提取物对Toll样受体4(TLR4)/核转录因子-κB(NF-κB)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路的影响差异。方法42只BALB/c小鼠按体质量随机分为空白对照组,千金子生品低、中、高剂量组(10.49、20.98、41.96 g/kg)和千金子霜品低、中、高剂量组(10.49、20.98、41.96 g/kg),每组6只,连续灌胃7 d。酶联免疫吸附测定法检测血清白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的含量,蛋白质印迹法和实时荧光PCR法检测结肠组织TLR4、NF-κB p65、NLRP3蛋白和mRNA表达。30只BALB/c小鼠按体质量随机分为空白对照组、千金子生品组、千金子生品+TAK-242组、千金子霜品组、千金子霜品+TAK-242组,每组6只。各给药组灌胃相应药液(20.98 g/kg),连续7 d,千金子生品+TAK-242组和千金子霜品+TAK-242组于给药第1、5、6、7天给药前30 min腹腔注射TAK-242溶液(3 mg/kg)。采用蛋白质印迹法和实时荧光PCR法检测结肠组织NLRP3、NF-κB p65蛋白表达,以及NLRP3、IL-1βmRNA表达。结果千金子生品可导致IL-1β和TNF-α大量释放,上调TLR4、NF-κB p65、NLRP3蛋白和mRNA表达(P<0.05,P<0.01),千金子制霜后对IL-1β、TNF-α分泌,以及TLR4、NF-κB p65、NLRP3蛋白和mRNA表达的促进作用减弱(P<0.05,P<0.01)。加入TLR4的阻断剂TAK-242后,可抑制NLRP3、NF-κB p65蛋白表达,以及NLRP3、IL-1βmRNA表达(P<0.01)。结论千金子生品可能通过激活TLR4介导的NF-κB/NLRP3通路,促进TNF-α和IL-1β释放,诱导结肠组织产生炎性反应,而制霜后对TLR4/NF-κB/NLRP3通路传导及促炎因子分泌的上调作用明显减弱,提示其制霜减毒的作用机制可能与致炎能力下降及干预TLR4/NF-κB/NLRP3信号通路有关。
Objective To investigate the effects of the extracts from Euphorbiae Semen before and after frosting on the toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3)signaling pathway.Methods According to the body weight,42 BALB/c mice were randomly divided into a blank control group,Euphorbiae Semen low-,medium-,and high-dose groups(10.49,20.98,and 41.96 g/kg,respectively)and Euphorbiae Semen Pulveratum low-,medium-,and high-dose groups(10.49,20.98,and 41.96 g/kg,respectively),with six mice in each group.Mice received continuous gavage for 7 d.The contents of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum of mice were determined by enzyme linked immune sorbent assay.The protein and the mRNA expression of TLR4,NF-κB p65,and NLRP3 in colon tissues of mice were detected by Western blotting and real-time PCR.According to the body weight,30 BALB/c mice were randomly divided into a blank control group,Euphorbiae Semen group,Euphorbiae Semen+TAK-242 group,Euphorbiae Semen Pulveratum group,and Euphorbiae Semen Pulveratum+TAK-242 group,with six mice in each group.The administration groups were respectively given 20.96 g/kg of Euphorbiae Semen or Euphorbiae Semen Pulveratum,and the continuous gavage time was 7 d.The Euphorbiae Semen+TAK-242 group and Euphorbiae Semen Pulveratum+TAK-242 group were intraperitoneally injected with TAK242(3 mg/kg)30 min before and on Day 1,5,6,and 7 of administration.The protein expression of NLRP3 and NF-κB p65,and the mRNA expression of NLRP3 and IL-1βin colon tissues of mice were assessed by Western blotting and real-time PCR.Results Euphorbiae Semen not only significantly promoted the release of IL-1βand TNF-α,but it also significantly increased the protein and the mRNA expression of TLR4,NF-κB p65 and NLRP3(P<0.05,P<0.01).However,upregulation of IL-1βand TNF-αsecretion,and the protein and mRNA expressions of TLR4,NF-κB p65,NLRP3 were decreased by Euphorbiae Semen Pulveratum(P<0.05,P<0.01).NLRP3 and NF-κB p65 protein expression and NLRP3 and IL-1βmRNA expression were inhibited after the intervention with the TLR4 inhibitor TAK-242(P<0.01).Conclusion Euphorbiae Semen could induce an inflammatory response of the colon tissue by activating the TLR4-mediated NF-κB/NLRP3 pathway,thus promoting the release of TNF-αand IL-1β.However,upregulation of the TLR4/NF-κB/NLRP3 signaling pathway and pro-inflammatory factor secretion were significantly alleviated by Euphorbiae Semen Pulveratum,indicating that the detoxification mechanism of Euphorbiae Semen frosting may be related to the decreased ability to cause inflammation and the intervention of TLR4/NF-κB/NLRP3 signaling pathway.
作者
王慧楠
姜明瑞
王志成
岳珠珠
魏晓彤
王梦琳
石双慧
张婧秋
陈梦雨
刘芊芊
王英姿
WANG Huinan;JIANG Mingrui;WANG Zhicheng;YUE Zhuzhu;WEI Xiaotong;WANG Menglin;SHI Shuanghui;ZHANG Jingqiu;CHEN Mengyu;LIU Qianqian;WANG Yingzi(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China)
出处
《北京中医药大学学报》
CAS
CSCD
北大核心
2023年第6期780-789,共10页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金项目(No.82074021)。
