人诱导多能间充质干细胞来源外泌体对肺泡巨噬细胞焦亡的抑制作用

Exosomes derived from human-induced pluripotent mesenchymal stem cells inhibit the pyrolysis of alveolar macrophages

目的探讨人诱导多能间充质干细胞来源外泌体(iMSC-Exos)对肺泡巨噬细胞(AM)焦亡的作用及机制。方法通过旋转超滤法提取人诱导多能间充质干细胞(iMSC)培养上清液中的外泌体,并利用透射电镜、蛋白质免疫印迹试验(Western blotting)、高分辨率可调电阻脉冲对提取的外泌体进行鉴定。体外培养大鼠AM细胞株NR8383,取对数生长期细胞分为3组:对照组在AM上清液中加入等量磷酸盐缓冲液(PBS);脂多糖/三磷酸腺苷(LPS/ATP)组AM经500μg/L的LPS刺激23 h后再加入5 mmol/L的ATP刺激1 h诱导细胞焦亡;iMSC-Exos组将100 mg/L iMSC-Exos与NR8383细胞共孵育3 h后再给予LPS和ATP刺激。采用细胞增殖与毒性检测试剂盒(CCK-8)和乳酸脱氢酶(LDH)分析检测细胞毒活性;采用免疫荧光法观察细胞凋亡和天冬氨酸特异性半胱氨酸蛋白酶-1(caspase-1)表达;采用酶联免疫吸附试验(ELISA)检测AM释放炎性因子白细胞介素(IL-1β、IL-18)水平;采用Western blotting法检测NOD样受体蛋白3(NLRP3)炎症小体途径及焦亡相关蛋白消皮素D(GSDMD)表达。结果提取的外泌体经透射电镜观察为圆形囊泡,Western blotting显示外泌体标志物CD63、CD9呈阳性表达,高分辨率可调电阻脉冲检测显示粒子平均直径130 nm,提示外泌体提取成功,能被AM吞噬。与对照组相比,LPS和ATP刺激后,细胞活性下降〔(0.56±0.05)%比(1.06±0.07)%,P<0.01〕,坏死物质LDH释放增加(U/L:1218.86±22.73比188.30±1.61,P<0.01),炎性因子分泌增加〔IL-1β(ng/L):958.91±32.78比194.63±5.14,IL-18(ng/L):870.89±21.86比288.85±24.48,均P<0.01〕,细胞凋亡率〔(55.35±6.19)%比(12.01±1.32)%,P<0.01〕及caspase-1表达均升高(荧光强度:41.06±3.65比2.80±0.54,P<0.01),提示LPS联合ATP可成功诱导细胞焦亡。与LPS/ATP组相比,给予iMSC-Exos预处理后,AM活性增加〔(0.81±0.05)%比(0.56±0.05)%,P<0.01〕,LDH释放减少(U/L:535.05±42.55比1218.86±22.73,P<0.01),炎性因子分泌减少〔IL-1β(ng/L):381.82±19.50比958.91±32.78,IL-18(ng/L):533.77±31.54比870.89±21.86,均P<0.01〕,细胞凋亡率〔(19.74±2.96)%比(55.35±6.19)%,P<0.01〕和caspase-1表达均下降(荧光强度:12.16±1.31比41.06±3.65,P<0.01),同时NLRP3炎症小体途径及焦亡相关蛋白GSDMD表达水平受到显著抑制〔NLRP3蛋白(NLRP3/β-actin):0.62±0.06比1.89±0.11,活化的caspase-1蛋白(cleaved caspase-1/β-actin):0.42±0.07比1.22±0.17,GSDMD蛋白(GSDMD/β-actin):0.57±0.05比1.22±0.05,均P<0.01〕。结论iMSC-Exos抑制了LPS/ATP诱导的AM焦亡和炎性因子表达,可能与靶向抑制NLRP3炎症小体途径有关,提示iMSC-Exos能抑制AM焦亡,发挥抗炎效应。

Objective To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells(iMSC-Exos)on alveolar macrophages(AM)pyroptosis.Methods The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells(iMSC)were extracted by rotating ultrafiltration,and the extracted exosomes were identified by transmission electron microscopy,Western blotting and high-resolution adjustable resistance pulse.The rat alveolar macrophage cells(NR8383 cells)were cultured in vitro and the logarithmic growth phase cells were divided into three groups:the control group was added with an equal volume of phosphate buffered saline(PBS)in the AM supernatant;in LPS/ATP group AM cells were stimulated with 500μg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis;iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP.The cytotoxic activity was detected by cell counting kit-8(CCK-8)and lactate dehydrogenase(LDH)analysis,the apoptosis and the expression of caspase-1 were observed by immunofluorescence,the levels of inflammatory factors interleukins(IL-1βand IL-18)released by AM were detected by enzyme linked immunosorbent assay(ELISA),the NOD-like receptor protein 3(NLRP3)inflammasome pathway and the expression level of pyroptosis related protein gasdermin D(GSDMD)were detected by Western blotting.Results The extracted exosomes were observed by transmission electron microscopy as round vesicles,expressing exosomal markers CD63 and CD9 showed by Western blotting,high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm,and could be uptaken by AM.Compared with the control group,the cell activity decreased[(0.56±0.05)%vs.(1.06±0.07)%,P<0.01],the release of necrotic substance LDH increased(U/L:1218.86±22.73 vs.188.30±1.61,P<0.01),the expression levels of inflammatory factors increased[IL-1β(ng/L):958.91±32.78 vs.194.63±5.14,IL-18(ng/L):870.89±21.86 vs.288.85±24.48,both P<0.01],and the apoptosis rate[(55.35±6.19)%vs.(12.01±1.32)%,P<0.01]and caspase-1 expression(fluorescence intensity:41.06±3.65 vs.2.80±0.54,P<0.01)elevated in the AM after LPS/ATP stimulation,suggesting that LPS combined with ATP successfully induced alveolar pyroptosis.Compared with the LPS/ATP group,AM pretreated with iMSC-Exos showed increased cell viability[(0.81±0.05)%vs.(0.56±0.05)%,P<0.01],decreased LDH secretion(U/L:535.05±42.55 vs.1218.86±22.73,P<0.01),decreased expression of inflammatory factors[IL-1β(ng/L):381.82±19.50 vs.958.91±32.78,IL-18(ng/L):533.77±31.54 vs.870.89±21.86,both P<0.01],and decreased apoptosis rate[(19.74±2.96)%vs.(55.35±6.19)%,P<0.01]and caspase-1 expression(fluorescence intensity:12.16±1.31 vs.41.06±3.65,P<0.01).At the same time,the expression of NLRP3 inflammasome pathway[NLRP3 protein(NLRP3/β-actin):0.62±0.06 vs.1.89±0.11;cleaved caspase-1 protein(cleaved caspase-1/β-actin):0.42±0.07 vs.1.22±0.17,both P<0.01]and pyrolysis-related protein was significantly inhibited[GSDMD protein(GSDMD/β-actin):0.57±0.05 vs.1.22±0.05,P<0.01].Conclusion iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP,which may be due to the targeted inhibition of NLRP3 inflammasome pathway,suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.