对比剂激活NLRP3炎症体通路诱导肾小管上皮细胞凋亡

Contrast induces kidney epithelial cell apoptosis through NRLP3 inflammasome pathway

目的探讨核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎症体在对比剂所致肾小管上皮细胞凋亡过程中的作用及其相关机制。方法将传代培养的人肾小管上皮细胞（HK-2）随机分为5组：对照组（Con组）；碘海醇组（O组）；NLRP3基因沉默＋碘海醇组（si—NLRP3＋O组）；凋亡相关斑点样蛋白（ASC）基因沉默＋碘海醇组（si-ASC＋O组）；甘露醇组（M组）。将不同浓度的低渗对比剂碘海醇加入HK．2细胞培养皿中分别孵育24、48、72h。流式AnnexinV-FITC／PI双染法检测细胞凋亡；RT-PCR法检测细胞内NLRP3、ASCmRNA相对表达量；Western印迹法检测HK一2细胞内NLRP3、ASC、凋亡相关调控蛋白半胱氨酸天冬氨酸蛋白酶8（caspase-8）／活化型caspase-8（cleavedcaspase-8）、B细胞淋巴瘤2蛋白（Bcl-2）／Bcl-2相关X蛋白（Bax）、caspase-1／cleavedcaspase，1、caspase-3／cleavedcaspase-3蛋白相对表达量；ELISA法检测细胞培养上清中白细胞介素（IL）1β、IL-18水平。结果与对照组相比，碘海醇组HK-2细胞凋亡率升高；细胞内NLRP3、ASCmRNA相对表达量增多；NLRP3、ASC、cleavedcaspase-1蛋白相对表达量增多；细胞上清中IL-1β、IL-18含量升高（均P〈0．05）。提示碘海醇组HK-2细胞NLRP3炎症体被激活。与对照组相比，碘海醇组促凋亡蛋白cleavedcaspase-8、Bax、cleavedcaspase-3表达量增多；抗凋亡蛋白Bcl-2表达量降低（均P〈0．05）。与碘海醇组相比，si-NLRP3＋碘海醇组、si．ASC＋碘海醇组HK-2细胞凋亡率显著降低；cleavedcaspase-1表达量减少；促凋亡蛋白Bax、cleavedcaspase-3表达减少；抗凋亡蛋白Bcl-2表达量增多；细胞上清中IL-1β、IL-18含量降低（均P〈0．05）。结论对比剂可激活HK-2细胞中NLRP3通路，诱导细胞凋亡，阻断NLRP3通路可减轻细胞凋亡。

Objective To investigate the effect of pyrin domain 3 （NLRP3） inflammasome in the process of contrast induced human kidney cell apoptosis. Methods Human kidney 2 （HK-2） cells were cultured in DMEM- F12 medium with 5% FBS. Cells were divided into control group, Contrast group （O group）, NLRP3 - siRNA ＋ Iohexol group （si - NLRP3 ＋ O group）, ASC - siRNA ＋ Iohexol group （si-ASC＋O group）, and mannitol group （M group）. Different concentrations of hypotonic contrast agent were added to HK-2 cell culture plates for 24, 48 and 72 h. Flow cytometry was used to detect apoptosis. NLRP3 and ASC mRNA expressions were detected by RT-PCR. The expressions of NLRP3, ASC, caspase- 8/cleaved caspase- 8, Bcl- 2/Bax, caspase- 1/cleaved caspase- 1, and caspase- 3/cleaved caspase - 3 protein were detected by Western blot. The levels of interleukin （IL） 1β and IL - 18 in supernatant were detected by ELISA. Results Compared with the control group, the rate of apoptotic ceils, as well as the expressions of NLRP3, ASC and cleaved caspase- 1 proteins were increased in HK-2 cells of contrast group. The expressions of NLRP3 and ASC mRNA in the contrast group also increased, so did IL- 1β and IL- 18 levels （all P〈0.05）, suggesting that NLRP3 inflammasome in HK- 2 ceils was activated by contrast. Compared with the control group, the expressions of cleaved caspase- 8, Bax and cleaved caspase- 3 protein were increased, and the expression of anti- apoptotic protein Bcl-2 was decreased （all P 〈 0.05）. Compared with the contrast group, the rate of apoptotic cells in the si- NLRP3 ＋contrast group and si- ASC ＋ contrast group was significantly decreased; the expression of cleaved caspase- 1 was decreased; the expressions of Bax and cleaved caspase-3 were decreased, and Bcl-2 level was increased. The expressions of IL-1β and IL-18 in the supematant of cells were decreased （all P 〈 0.05）. Conclusion Contrast agent can activate the NLRP3 pathway in HK- 2 cells and induce apoptosis, which could be reduced by blocking the NLRP3 pathway.