CAJ | 학술논문

目的观察高浓度胆汁成分触发前炎症反应的途径.方法分离培养大鼠外周血单核细胞,分别与5％胆汁、内毒素(LPS)、三磷酸腺苷(ATP)、高K+培养液接触,采用乳酸脱氢酶(LDH)检测细胞毒性,酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-1β含量,Western blot法检测半胱氨酸天冬氨酸特异性蛋白酶-1(Caspase-1)、炎症小体NALP3表达并定量分析.结果单纯5％胆汁未能诱导IL-1β、Caspase-1、NALP3表达.LPS+胆汁组及ATP+胆汁组IL-1β浓度分别为[(769.8 ±29.4)、(788.6±32.4) ng/L],均远高于单用LPS组[(88.1±6.7) ng/L] (P＜0.01).培养6h后LPS预处理组IL-1β浓度均明显升高(871.1 ±69.7) ng/L.LDH值与IL-1β的变化间无相关.LPS+胆汁组在1.0 ×104和1.2 ×105附近见Caspase-1和NALP3蛋白表达条带.通过图片的定量研究显示LPS+胆汁组Caspase-1和NALP3明显升高(0.69±0.10、1.86±0.33).高K+培养基培养6h后,LPS+胆汁组IL-1β浓度降至(638.2±26.2)ng/L.结论进入血循环的高浓度胆汁合并细菌感染通过炎症小体途径触发早期炎症反应,细胞外高K+能抑制此过程.
목적 관찰고농도담즙성분촉발전염증반응적도경.방법 분리배양대서외주혈단핵세포,분별여5％담즙、내독소(LPS)、삼린산선감(ATP)、고K+배양액접촉,채용유산탈경매(LDH)검측세포독성,매련면역흡부시험(ELISA)검측백세포개소(IL)-1β함량,Western blot법검측반광안산천동안산특이성단백매-1(Caspase-1)、염증소체NALP3표체병정량분석.결과 단순5％담즙미능유도IL-1β、Caspase-1、NALP3표체.LPS+담즙조급ATP+담즙조IL-1β농도분별위[(769.8 ±29.4)、(788.6±32.4) ng/L],균원고우단용LPS조[(88.1±6.7) ng/L] (P＜0.01).배양6h후LPS예처리조IL-1β농도균명현승고(871.1 ±69.7) ng/L.LDH치여IL-1β적변화간무상관.LPS+담즙조재1.0 ×104화1.2 ×105부근견Caspase-1화NALP3단백표체조대.통과도편적정량연구현시LPS+담즙조Caspase-1화NALP3명현승고(0.69±0.10、1.86±0.33).고K+배양기배양6h후,LPS+담즙조IL-1β농도강지(638.2±26.2)ng/L.결론 진입혈순배적고농도담즙합병세균감염통과염증소체도경촉발조기염증반응,세포외고K+능억제차과정.
Objective To investigate the signal pathway of high concentration bile triggering monocyte proinflammatory response.Methods The peripheral blood monouclear cells (PBMCs) of rats were isolated and cultured in vitro.The concentration of 5％ bile in rats was prepared.The PBMCs were cocultured with 5％ bile,endotoxin (LPS),adenosine-triphosphate (ATP),or high K+ culture fluid.The cytotoxicity was detected by lactate dehydrogenase (LDH) after 1,6,12,and 24 h.The contents of interleukin (IL)-1β were determined by using enzyme linked immunosorbent assay (ELIAS),and the expression of Caspase-1 and NALP3 was detected by using Western blotting and analyzed quantitatively.Results 5％ bile only was failed to induce expression of interleukin (IL)-1β,Caspase-1 or NALP3.But the levels of IL-1β were increased to (769.8 ± 29.4) and (788.6 ± 32.4) ng/L after pretreatment with LPS or ATP,which were significantly higher than single LPS group [(88.1 ±6.7) ng/L,P ＜0.01].The levels of IL-1β reached the peak [(871.1 ± 69.7) ng/L] at 6th h after pretreatment with LPS.There was no correlation between LDH and IL-1β.Western blotting analysis demonstrated that the Caspase-1 protein generated a band about 1.0 × 104 and a broad band about 1.2 × 105 of NALP3 in LPS + bile group.The quantitative analysis showed that the expression of Caspase-1 and NALP3 was higher obviously in LPS + bile group than that in single LPS group.Concentration of IL-1β was decreased in LPS + bile group after culture with high K+ substratum for 6 h.Conclusion The high concentration of bile in the blood circulation combined with bacterial infection induces proinflammatory responses via activation of the NLRP3 inflammasome.The potassium efflux is required for the procedure.