基于NLRP3炎性体信号通路研究桂枝芍药知母汤对尿酸钠诱导的大鼠中性粒细胞炎性信号表达的影响  

Effect of Guizhishaoyaozhimu decoction on expression of neutrophil inflammatory signal in monosodium urate-induced rats based on NLRP3 inflammasome signaling pathway

作　　者：房树标[1] 王永辉[2] 李艳彦[2] 周然[2] 

机构地区：[1]湖北中医药大学基础医学院,武汉430065 [2]山西中医学院中医系

出　　处：《中国药物与临床》2016年第2期170-175,共6页Chinese Remedies & Clinics

基　　金：山西省科技创新团队建设项目(2012081018)

摘　　要：目的基于核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性体信号通路观察桂枝芍药知母汤(GD)对尿酸钠诱导的大鼠中性粒细胞炎性信号表达的影响,以期探明其抗炎的药效作用机制。方法健康雄性SD大鼠30只,按体质量分为5组,每组6只,GD高、中、低剂量组(4,8,16 g/kg)、秋水仙碱阳性对照组(3×10-4g/kg)均灌胃给药,正常组给予等容积的蒸馏水,每天1次,连续给药7 d。最后1次灌胃1 h后,所有大鼠乙醚麻醉,取血清备用。SD雄性大鼠20只,参照文献方法提取分离大鼠中性粒细胞,接种培养。细胞试验分为2个实验组(不加受体抑制剂,加NALP3受体抑制剂),每个实验组均含6个组,正常对照组加入正常大鼠血清,模型对照组,高、中、低剂量组,秋水仙碱组全部滴加200 mg/L的尿酸钠混悬液造模,同时滴加含药血清,置于CO2细胞培养箱中,孵育12 h取出。酶联免疫吸附实验(ELISA)法测定炎性因子白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)的表达,DNA-蛋白质互作ELISA(DPI-ELISA)方法检测核因子-κB(NF-κB)活性;蛋白印迹法检测凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-12(caspase-12)信号衔接蛋白表达水平;反转录聚合酶链反应(RTPCR)观测NLRP3炎性体m RNA的表达。结果细胞造模12 h后,与正常组比较,模型组大鼠中性粒细胞IL-1β、IL-6、TNF-α、NF-KB、ASC(除了加NALP3受体抑制剂组)、NALP3 m RNA表达水平明显升高(P<0.05),Caspase-12表达水平明显降低(P<0.05);与模型组比较,给药各组IL-1β、IL-6、TNF-α、NALP3 m RNA及GD中、高剂量组NF-κB、ASC表达均明显降低(P<0.05),GD高、中剂量组caspase-12表达明显升高(P<0.05),而秋水仙碱组caspase-12表达无明显变化(P>0.05)。结论 GD抗炎作用机制可能与降低中性粒细胞NLRP3、ASC表达,抑制IL-1β分化成熟及NF-KB活化,降低NLRP3炎性体信号通路炎性因子表达有关。与秋水仙碱不同的是,GD能够增加caspase-12表达Objective To investigate the effect of Guizhishaoyaozhimu decoction（GD） on expression of neutrophil inflammatory signal in monosodium urate-induced rats based on NACHT-LRR-PYD-containing proteins 3（NLRP3） inflammasome signaling pathway, and to determine its anti-inflammatory mechanism. Methods Thirty rats were randomly divided into 5 groups according to the weight（n=6 each）. The high-, medium-, and low-dose GD groups（4,8, 16 g/kg） and Colchicine positive control group（3 ×10^-4g/kg） received gastric administration, and the normal group was given equal volume of distilled water once daily for continuous 7 days. At 1h after the last gastric administration,all rats were anesthetized with ether, and the serum was collected. According to literature, neutrophils were extracted and isolated from 20 male SD rats, and cultured. The cell test included 2 experimental groups（one without receptor inhibitor, and one with NALP3 receptor inhibitor）. Each experimental group included 6 subgroups. The normal control group was added with normal rat serum. The high-, medium- and low-dose model control groups and Colchicine group were dropwise added with 200 mg/L monosodium urate suspension for modeling with serum containing medicine. All groups were cultured in the CO2 cell incubator for 12 hour. Enzyme-linked immunosorbent assay（ELISA） was used determine the expression of interleukin-1 beta（IL-1β）, interleukin-6（IL-6）, and tumornecrosis factor-α（TNF-α）. DNAprotein interaction-ELISA（DPI-ELISA） was used to determine nuclear factor-kappa B（NF-κB） activity. Western Blot was used to determine the expression levels of apoptosis-associated speck-like protein containing a CARD（ASC） and Csapase-12 signaling adapter protein. Reverse transcription-PCR（RT-PCR） was used to determine the expression of NLRP3 inflammasome m RNA. Results At 12 h after cell modeling, the expression levels of IL-1β, IL-6, TNF-α, NF-κB, ASC（except the group with NALP3 receptor inhibitor）, and NALRP3 m R

关 键 词：中性粒细胞 桂枝芍药知母汤 NLRP3炎性体 作用机制 

分 类 号：R285.5[医药卫生—中药学]