高良姜素通过调控NLRP3炎性小体抑制巨噬细胞焦亡

Galangin inhibits the pyroptosis of macrophages mediated by NOD-like receptor proteins 3

目的探讨高良姜素对骨髓来源的巨噬细胞(BMDMs)焦亡的影响。方法将体外培养的BMDMs分为空白对照组、模型组、不同浓度高良姜素组;采用脂多糖(LPS)+三磷酸腺苷(ATP)构建细胞焦亡模型用细胞增殖与毒性检测试剂盒8(CCK-8)测定不同浓度高良姜素对BMDMs增殖的影响;采用蛋白质免疫印迹试验(Western blotting)检测BMDMs培养上清液中天冬氨酸特异性半胱氨酸蛋白酶1p10亚基(caspase-1p10)、白细胞介素-1β(IL-1β)及细胞中NOD样受体蛋白3(NLRP3)的蛋白表达水平;采用酶联免疫吸附试验(ELISA)测定BMDMs上清液中IL-1β含量;采用碘化丙啶(PI)染色观察细胞死亡情况;采用高通量测序比较模型组和高良姜素20μmol/L组的差异基因表达。结果5、10、20、40、60、80μmol/L高良姜素组BMDMs的增殖水平比较差异均无统计学意义(均P>0.05)说明上述浓度高良姜素对BMDMs增殖无明显影响,故本研究选择5、10、20μmol/L高良姜素和处理1、2、4h观察不同浓度及时间对BMDMs焦亡的影响。与空白对照组比较,模型组caspase-1p10、成熟IL-1β蛋白表达和IL-1β含量均明显升高(均P<0.05);与模型组比较,高良姜素5、10、20μmol/L各组上清液中caspase-1p10、成熟IL-1β蛋白表达和IL-1β含量均明显降低[IL-1β蛋白表达(灰度值):0.155±0.006、0.113±0.006、0.111±0.007比1.000±0.000,caspase-1p10蛋白表达(灰度值):0.207±0.044、0.160±0.008、0.082±0.008比1.000±0.000,IL-1β(μg/L):99.80±10.36、85.21±8.78、26.53土4.56比494.10±35.47,均P<0.05],不同浓度高良姜素组间比较差异无统计学意义(均P>0.05),但随着高良姜素处理时间的延长,抑制作用增强,以高良姜素20μumol/L组作用4h的抑制作用最明显[IL-1β蛋白表达(灰度值):0.186±0.004比1.000±0.000,caspase-1p10蛋白表达(灰度值):0.247±0.009比1.000±0.000,IL-1β(μg/L):173.80±10.56比653.80±76.02,均P<0.05];20μmol/L高良姜素作用4h可减少焦亡细胞数量(个/视野:23.00±3.61比67.67±15.63,P<0.05),抑制NLRP3的蛋白表达水平(灰度值:0.178土0.025比0.406±0.066,P<0.05)。高通量测序显示,与模型组比较,高良姜素下调了Nlrp3、Nod2、IL-1β等基因,上调了Skp2(又称Fbxl1)、Fbxl20、Fbxl4、Fbxo32和Fbxw7等基因水平。结论高良姜素通过调控巨噬细胞中NLRP3炎性小体来抑制细胞焦亡。

Objective To investigate the effect of Galangin on pyroptosis of bone marrow derived macrophages(BMDMs).Methods BMDMs were cultured in vitro and divided into blank control group,model group and Galangin group with different concentrations.Lipopolysaccharide(LPS)and adenosine triphosphate(ATP)were used to construct the pyroptosis model.The effect of different concentrations of Galangin on the proliferation of BMDMs was detected by cell counting Kit-8(CCK-8).The level of cysteinyl aspartate specific proteinase-1 pl0 subunit(caspase-1 p10),interleukin-1β(IL-1β)in supernatant and intracellular nucleotide NOD-like receptor protein 3(NLRP3)were detected by Western blotting.IL-1βin supernatant was detected by enzyme-linked immunosorbent assay(ELISA).The cell death was observed by propidium iodide(PI)staining.High-throughput sequencing was used to compare the gene expression in the model group and Galangin groups at 20μmol/L.Results There was no statistically significant difference between the 5,10,20,40,60,80μmol/L Galangin groups on the proliferation level of BMDMs(all P>0.05),indicating that no significant effect of Galangin at 5,10,20,40,60,80μmol/L was observed on the proliferation of BMDMs.So we selected Galangin at 5,10,20μmol/L and treatment for 1,2 and 4 hours as the effects of different concentrations and time on the pyroptosis of BMDMs.Compared with blank control group,the expression of caspase-1 pl0 and mature IL-1βprotein and IL-1βin supernatant in model group were significantly increased(all P<0.05).Compared with model group,the expression of caspase-1 p10 and mature IL-1βprotein and IL-1βin supernatant of Galangin at 5,10 and 20μmol/L were significantly decreased[IL-1βprotein expression(gray value):0.155±0.006,0.113±0.006,0.111±0.007 vs.1.000±0.000,caspase-1 p10 protein expression(gray value):0.207±0.044,0.160±0.008,0.082±0.008 vs.1.000±0.000,IL-1β(μg/L):99.80±10.36,85.21±8.78,26.53±4.56 vs.494.10±35.47,all P<0.05].There was no significant difference between the different concentration groups(all P>0.05),but with the extension of treatment time of Galangin,the inhibitory effect was enhanced.The inhibitory effect of Galangin at 20μmol/L for 4 hours was the most obvious[1L-1βprotein expression(gray value):0.186±0.004 vs.1.000±0.000,caspase-1 p10 protein expression(gray value):0.247±0.009 vs.1.000±0.000,IL-1β(μg/L):173.80±10.56 vs.653.80±76.02,all P<0.05].Treatment with 20μmol/L Galangin for 4 hours could reduce the number of pyroptotic cell deaths(number of view:23.00±3.61 vs.67.67±15.63,P<0.05)and inhibited the expression of NLRP3 protein(gray value:0.178±0.025 vs.0.406±0.066,P<0.05).High-throughput sequencing showed that,compared with the model group,Galangin down-regulated the genes of Nlrp3,Nod2,IL-1βand up-regulated genes of Skp2(also known as Fbxll),Fbxl20,Fbxl4,Fbxo32 and Fbxw7.Conclusion Galangin inhibited pyroptosis mediated by NLRP3 inflammasome in macrophages.