绿原酸通过ROS/TXNIP/NLRP3信号通路介导的细胞焦亡途径减轻脓毒症小鼠急性肺损伤

Chlorogenic acid attenuates acute lung injury in septic mice via ROS/TXNIP/NLRP3 signaling pathway mediated pyrocytosis pathway

目的:探究绿原酸对脓毒症小鼠急性肺损伤的影响及活性氧/硫氧还蛋白互作蛋白/NOD样受体蛋白3(ROS/TXNIP/NLRP3)信号通路在其中的作用。方法:建立脓毒症小鼠模型,随机分为模型组、阳性对照(地塞米松)组及低剂量(5 mg/kg)、中剂量(10 mg/kg)、高剂量(20 mg/kg)绿原酸组,每组各12只,另取12只正常小鼠作为对照组。计数肺泡灌洗液中炎症细胞数量;测定肺组织湿重/干重(W/D)比值;HE染色观察肺组织病理改变;TUNEL法检测肺组织细胞凋亡;ELISA法测定肺泡灌洗液中白细胞介素(IL)-1β、IL-6和肿瘤坏死因子α(TNF-α)含量,相应试剂盒检测肺组织中丙二醛(MDA)、超氧化物歧化酶(SOD)和活性氧(ROS)水平;Westernblot法检测TXNIP、NLRP3蛋白、caspase-1及细胞焦亡关键蛋白GasderminD的N末端片段(GSDMD-N)的表达水平。结果:相较于对照组,模型组肺泡间隔增厚,肺泡内及肺泡间质水肿,炎症细胞数量显著增加,IL-1β、IL-6和TNF-α含量、W/D比值、细胞凋亡率、MDA和ROS水平以及TXNIP、NLRP3、caspase-1和GSDMD-N蛋白水平均显著升高(P<0.05);而模型组SOD水平较对照组显著降低(P<0.05)。相较于模型组,绿原酸低、中、高剂量组肺泡间隔增厚及水肿情况有所减轻,炎症细胞数量显著减少,IL-1β、IL-6和TNF-α含量、W/D比值、细胞凋亡率、MDA和ROS水平以及TXNIP、NLRP3、caspase-1和GSDMD-N蛋白水平显著降低(P<0.05);而模型组SOD水平较对照组显著升高(P<0.05);绿原酸高剂量组与阳性对照组上述指标差异无统计学意义(P>0.05)。结论:绿原酸可能通过下调ROS/TXNIP/NLRP3信号通路表达水平,抑制炎症分子产生及细胞焦亡发生,有效减轻脓毒症小鼠肺损伤。

AIM:To explore the effect of chlorogenic acid on acute lung injury and the role of reactive oxygen species/thioredoxin interacting protein/NOD-like receptor protein 3(ROS/TXNIP/NLRP3)signaling pathway in septic mice.METHODS:Sepsis mouse model was established and the mice were randomly divided into model group,positive control(dexamethasone)group,low-dose(5 mg/kg),medium-dose(10 mg/kg)and high-dose(20 mg/kg)chlorogenic acid groups,12 rats in each group,and another 12 normal mice were selected as controls.The number of inflammatory cells in bronchoalveolar lavage fluid(BALF)was counted.The ratio of wet weight(W)/dry weight(D)of lung tissue was measured.The pathological changes of lung tissue were observed by HE staining.Apoptosis rate was detected by TUNEL staining.The levels of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)in BALF and the levels of malondialdehyde(MDA),superoxide dismutase(SOD)and reactive oxygen species(ROS)in lung tissue were measured.The protein levels of TXNIP,NLRP3,caspase-1 and N-terminal fragment of gasdermin D(GSDMD-N)were determined by Western blot.RESULTS:Compared with control group,the alveolar septum of model group was thickened,there was edema in the alveoli and the interstitium of alveoli,and the number of inflammatory cells was increased significantly in model group,the contents of IL-1β,IL-6 and TNF-α,W/D ratio,the apoptosis rate,the levels of MDA and ROS,and the protein levels of TXNIP,NLRP3,caspase-1 and GSDMD-N were significantly increased(P<0.05),while the level of SOD was significantly decreased(P<0.05).Compared with model group,the thickening and edema of alveolar septum in low-,medium-and high-dose chlorogenic acid groups were reduced,and the number of inflammatory cells was significantly reduced,the contents of IL-1β,IL-6 and TNF-α,W/D ratio,the apoptosis rate,the levels of MDA and ROS,and the protein levels of TXNIP,NLRP3,caspase-1 and GSDMD-N were significantly decreased(P<0.05),while the level of SOD was significantly increased(P<0.05).No significant difference between high-dose chlorogenic acid group and positive control group was observed(P>0.05).CONCLUSION:Chlorogenic acid inhibits the production of inflammatory molecules and the occurrence of pyrocytosis by down-regulating the ROS/TXNIP/NLRP3 signaling pathway,effectively alleviates acute lung injury of septic mice.