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大鼠巨噬细胞炎性信号表达的影响

Research on The Effect of Guizhishaoyaozhimu Decoction on The Expression of Inflammatory Signal in Macrophage Induced with Monosodium Urate Crystals Based on NLRP3 Inflammasomes Signaling Pathway

目的:基于NLRP3炎性体(NACHT-LRR-PYD-containing proteins 3 inflammasome)信号通路观察桂枝芍药知母汤(GD)对尿酸钠诱导的大鼠巨噬细胞炎性信号表达的影响,以期探明其抗炎作用机制。方法:大鼠30只按体质量随机分为5组各6只,GD高、中、低剂量组(4、8、16 g·kg-1)、秋水仙碱阳性对照组(3×10-4g·kg-1)均灌胃给药,正常组给予等容积蒸馏水,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巨噬细胞接种培养12 h。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200μg·L-1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2 h取出。酶联免疫吸附实验(ELISA)法测定炎性因子白介素-1β(interleukin-1 beta,IL-1β)、白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumornecrosisfactor-a,TNF-α)的表达,DNA-蛋白质互作ELISA(DPI-ELISA)方法检测核因子-KB(nuclear factor-kappa B,NF-KB)活性;Western免疫印迹(Western Blot)检测凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)、半胱天冬酶-12(Csapase-12)信号衔接蛋白表达水平;逆转录PCR(reverse transcription-PCR,RTPCR)观测NLRP3炎性体mRNA的表达。结果:细胞造模12 h后与正常组比较,实验模型组大鼠巨噬细胞IL-1β、IL-6、TNF-α、NFKB、ASC、NALP3 mRNA表达水平明显升高,Csapase-12表达水平明显降低;与模型组比较,给药各组IL-1β、IL-6、TNF-α、NALP3mRNA及GD中、高剂量组NF-KB、ASC表达均明显降低,GD高剂量组Csapase-12表达明显升高,而秋水仙碱组Csapase-12表达无明显增加。结论:GD抗炎作用机制可能与降低巨噬细胞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Capase-12表达,负反馈抑制NLRP3炎性体信号通路炎性因子表达,提示GD治疗GA新的抗炎机制。在加入NALP3受体抑制剂下,GD仍能降低模型大鼠巨噬细胞中TNF-α、NF-KB表达,提示GD可以通过另外信号通路发挥抗炎作用。

Objective：To study the effect of Guizhishaoyaozhimu Decoction （GD） on the expression of inflammatory signal in macrophage induced with monosodium urate crystals based on NLRP3 inflammasomes signaling pathway and explore the anti inflammation mechanism. Methods ：30 male SD rats were randomly divided into 5 groups with 6 rats each group according to weight. The high, medium,low dose group of GD （4, 8, 16 g mg · kg-1）and colchieine group （3 × 10-4 g· kg-1 ） were treated with medicine by gastric administration, the normal group were given equal volume of distilled water. Medicine or distilled water was given once daily for inhibitor seven consecutive days throughout the experiment. One hour after the last gastric administration, all rats were anesthetized with ether, and the serum was collected and incubated for 12 hour. Macrophage were isolated from 30 male SD rats and cultured according to the methods from literatures and randomly divided into 2 experiments（ no receptor inhibitors, plus blALP3 receptor inhibitors experiment）with 6 groups each experiment. The normal was added to normal rat serum,the model group, the high, medium and low dose group of GD and eolehicine group were added uric acid sodium suspension of 200 μg·L - 1 to induce into cell model with serum containing medicine. All cells were put into the CO2 cell incubator for 2 hour. The expression of Inflammatory cytokine such as interleukin-1 beta （IL-1） and interleukin-6 （IL-6）, tumor necrosis factor alpha （TNF）were detected with Enzyme linked immunosorbent assay （ELISA）, and activity of nuclear factor - KB （ nuclear factor kappa B （ NF KB） was detected with DNA-protein interaction-ELISA （DPI-ELISA）. The expression levels of apoptosis associated protein （ASC）, Capase-12 signaling proteins were detected by Western blot. The mRNA expression levels of NACHT-LRR-PYD-containing proteins 3 （NLRP3） inflammasome was detected with reverse transcription-PCR （RT-PCR）. Result： Compared with normal group after 12 hours,the expression of IL-1β, IL-6 ,TNF-α, NF-KB, ASC and NLRP3 inflammasomes in macrophage of the model group significantly increased, whereas Capase-12 significantly decreased. The expression of IL-1β,1L-6, TNF-α, NALP3 mRNA in all drug delivery groups,and NF-KB,ASC in medium and high dose group of GD significantly decreased than the model group,whereas Capase-12 in high dose group of GD increased and there was no significant increase in group of Colchicine. Conclusion ： The anti infammation mechanism of GD is related to decreasing the expression levels of NLRP3 and ASC and increasing the expression of Capase-12, and accordingly inhibiting the maturation of IL-1 β and the activation of NF- KB, reducing the expression of inflammatory factors in NLRP3 inflammasomes signaling pathway. Different from the colchicine,GD significantly increased the expression of Capase-12 to inhibit the expression of inflammatory factors in NLRP3 inflammasomes signaling pathway in Negative feedback mode that suggested a new anti-inflammatory mechanism of GD. GD can also reduced the expression of NF-KB and TNF- in macrophage In the case of applying NALP3 receptor inhibitor, suggesting that GD can exert anti-inflammatory effect through the other signaling pathways.