目的 探討腸源性外泌體通過miR-146a調(diào)控NF-κB通路介導(dǎo)人腎小管上皮細(xì)胞HK-2的炎癥反應(yīng)。方法 Caco-2細(xì)胞分為對(duì)照組、模型［10 ng·mL^-1的脂多糖（LPS）刺激構(gòu)建腸上皮細(xì)胞炎癥模型］組、NC mimic組和miR-146a mimic組，提取各組外泌體（30 μg·mL^-1）孵育HK-2細(xì)胞，記為對(duì)照-Exos、LPS-Exos、NC-Exos、miR-146a-Exos組。轉(zhuǎn)錄組學(xué)測序?qū)?duì)照組與模型組差異基因進(jìn)行檢測；實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測miR-146a 表達(dá)；酶聯(lián)免疫吸附 （ELISA） 法檢測細(xì)胞培養(yǎng)基白細(xì)胞介素（IL）-1β、白細(xì)胞介素-6（IL-6）、腫瘤壞死因子α（TNF-α）水平；Western blotting法檢測細(xì)胞中IL-1β、IL-6、TNF-α和核因子-κB（NF-κB）、磷酸化-NF-κB（p-NF-κB）、Toll樣受體4（TLR4）炎性信號(hào)通路蛋白表達(dá)。結(jié)果 與對(duì)照組比較，模型組 Caco-2 細(xì)胞炎癥因子 IL-1β、IL-6、TNF-α 和 TLR4、NF- κ B、p-NF- κ B 蛋 白 表 達(dá) 明 顯增加（P＜0.05、0.01），培養(yǎng)基中 IL-1β、IL-6、TNF-α 水平顯著增加（P＜0.05），細(xì)胞及外泌體中 miR-146a表達(dá)顯著增加（P＜0.05、0.01）；miR-146a mimic組細(xì)胞及外泌體中miR-146a表達(dá)顯著增加（P＜0.01）。外泌體與HK-2細(xì)胞共孵育后，與對(duì)照-Exos組比較，LPSExos、miR-146a mimic組培養(yǎng)基中炎癥因子IL-1β、IL-6、TNF-α水平明顯增加（P＜0.05），細(xì)胞中炎癥因子IL-1β、IL-6、TNF-α及NF-κB炎癥通路相關(guān) TLR4、NF-κB、p-NF- κB 蛋白表達(dá)顯著增加（P＜0.05、0.01）。結(jié)論 腸源性外泌體通過 miR-146a調(diào)控NF-κB通路介導(dǎo)HK-2細(xì)胞炎癥反應(yīng)。

Objective To investigate the role of intestine-derived exosomes in regulating the inflammatory response of HK-2 cells through the NF- κB pathway regulated by miR-146a. Methods Caco-2 cells were divided into control group, model group (constructed intestinal epithelial cell inflammation model by stimulating with 10 ng·mL^-1 LPS), NC mimic group, and miR-146a mimic group. The exosomes (30 μg·mL^-1) from each group were extracted and incubated with HK-2 cells, respectively, and named as control-Exos, LPS-Exos, NC-Exos, and miR-146a-Exos group. Transcriptome sequencing was used to detect differential genes between the control group and the model group; real-time fluorescence quantitative PCR (qRT-PCR) was used to detect miR-146a expression; ELISA was used to detect the levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) in the cell culture supernatant; Western blotting was used to detect the expressions of IL-1β, IL-6, TNF-α, NF-κB, p-NF-κB, and Toll-like receptor 4 (TLR4) inflammatory signaling pathway proteins in the cells. Results Compared with the control group, the expressions of inflammatory factors IL-1β, IL-6, and TNF- α and TLR4, NF- κB, and p-NF- κB proteins in the model group were significantly increased (P <0.05, 0.01), the levels of IL-1β, IL-6, and TNF-α in the cell culture supernatant were significantly increased (P <0.05), and the expressions of miR-146a in the cells and exosomes were significantly increased (P <0.05, 0.01); the expressions of miR-146a in the cells and exosomes of the miR-146a mimic group were significantly increased (P <0.01). Compared with the control-Exos group, the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the culture supernatant of the LPS-Exos and miR- 146a mimic groups were significantly increased (P <0.05). Conclusion Intestine-derived exosomes regulate the inflammatory response of HK-2 cells through miR-146a-regulated NF-κb pathway.

R363.1

中山市社會(huì)公益與基礎(chǔ)研究項(xiàng)目（2020B3005）；中山市第三批社會(huì)公益與基礎(chǔ)研究項(xiàng)目（2021B3006）；天津市中醫(yī)處課題（2021163）；天津市醫(yī)藥科學(xué)研究所課題（S202304）