痛风宁含药血清对尿酸盐诱导THP-1中NALP3炎性体相关蛋白及炎症因子表达的影响

Effects of Serum Containing Tongfengning on the Expression of NALP3 Inflammasome Related Proteins and Inflammatory Cytokines in THP-1 Induced by Uric Acid

目的通过检测痛风宁含药血清对尿酸盐(monosodium urate,MSU)诱导人单核细胞株THP-1(human monocytic cell line,THP-1)中NALP3炎性体相关蛋白与mRNA及上清液中IL-1β、PGE_2表达的影响,探讨痛风宁对痛风性关节炎的抗炎机制。方法制备低、中、高剂量痛风宁含药血清。将THP-1随机分为空白组和MSU干预组,MSU干预组予200 mg/L MSU混悬液诱导12 h建立炎症细胞模型,模型鉴定成功后采用随机数字表法分为模型组及低、中、高剂量痛风宁含药血清组(以下简称低、中、高剂量痛风宁组)、阳性对照组,分别予10%空白血清,10%低、中、高剂量痛风宁含药血清,10%空白血清+Caspase-1抑制剂干预,无MSU干预的空白组予10%空白血清干预。24 h后,离心收集各组THP-1及细胞上清液。采用ELISA检测各组细胞上清液中IL-1β、PGE_2含量,Western Blot、RT-qPCR分别检测各组THP-1中NALP3、Caspase-1、ASC蛋白及mRNA表达。结果与空白组比较,模型组NALP3、Caspase-1、ASC蛋白与mRNA表达及IL-1β、PGE_2含量均升高(P<0.01);各剂量痛风宁组所有指标均低于模型组及阳性对照组(P<0.01),同时低、高剂量痛风宁组所有指标水平均高于中剂量痛风宁组(P<0.05,P<0.01);阳性对照组Caspase-1蛋白表达及IL-1β、PGE_2含量低于模型组(P<0.01),Caspase-1 mRNA、NALP3、ASC蛋白及mRNA表达与模型组差异无统计学意义(P>0.05)。结论痛风宁对AGA的抗炎机制可能与抑制NALP3炎性体相关蛋白及mRNA表达,减少炎症因子IL-1β、PGE_2的产生有关。

Objective To explore the anti-inflammatory mechanism of Tongfengning on gouty arthritis by detecting the effects of serum containing Tongfengning on the expression of NALP3 inflammasome related protein and mRNA and the levels of IL-1β, PGE2 in human monocytic cell line THP-1 induced by monosodium urate(MSU). Methods The low, medium, high dose serum containing Tongfengning were prepared. THP-1 was randomly divided into blank group and MSU intervention group. 200 mg/L MSU suspension induced the MSU intervention group for 12 h to establish the cell model of inflammation. After the successful identification of the model, THP-1 in the MSU intervention group was randomly divided into model group and low, medium, high dose serum containing Tongfengning group and positive control group. These groups respectively received 10% blank serum, 10% low, 10% medium, 10% high dose serum containing Tongfengning, 10% blank serum plus Caspase-1 inhibitor intervention, the blank group without MSU intervention received 10% blank serum intervention. After intervention for 24 h, THP-1 and cell supernatant were collected by centrifugation. ELISA was used to detect the levels of IL-1β and PGE2 in cell supernatant of all groups. Western Blot and RT-qPCR were used respectively to detect the protein and mRNA expression of NALP3, Caspase-1, ASC in THP-1 of all the groups. Results Compared with the blank group, the protein and mRNA expression of NALP3, Caspase-1, ASC and the levels of IL-1β, PGE2 in the model group were increased significantly(P<0.01). Compared with the model group and the positive control group, the levels of all indexes in each dose serum containing Tongfengning groups were decreased(P<0.01). The levels of all indexes in the low and high dose serum containing Tongfengning groups were higher than those in the medium dose serum containing Tongfengning group(P<0.05, P<0.01). The expression of Caspase-1 protein and levels of IL-1β, PGE2 in the positive control group were lower than those in the model group(P<0.01), and there was no significant difference in the expression of Caspase-1 mRNA, NALP3, ASC protein and mRNA between the two groups(P>0.05). Conclusion The anti-inflammatory mechanism of Tongfengning on AGA may be related to inhibiting the expression of NALP3 inflammasome related protein and mRNA, decreasing the levels of inflammatory cytokines IL-1β and PGE2.