摘要
目的:绿原酸具有抗菌、抗炎、抗病毒等多种生理活性,研究显示绿原酸可缓解急性肺损伤(acute lung injury,ALI)小鼠机体炎症反应,但具体作用机制尚不明确。本研究旨在探讨绿原酸是否通过调控微RNA-223(microRNA-223,miR-223)/核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)轴来减轻脂多糖(lipopolysaccharide,LPS)诱导的小鼠ALI。方法:将SPF级BALBc雄性小鼠随机分为对照组、模型组、绿原酸组、绿原酸+miR-223阴性对照(miR-223 NC)组、绿原酸+miR-223抑制物(miR-223 antagomir)组,每组10只,除对照组,其余各组均通过气道滴入4 mg/kg的LPS制备ALI小鼠模型,造模成功后绿原酸组小鼠使用绿原酸(100 mg/kg)连续灌胃7 d。绿原酸+miR-223 NC组、绿原酸+miR-223 antagomir组每天灌胃100 mg/kg的绿原酸后分别经尾静脉注射10μL的miR-223 NC(0.5 nmol/μL)、miR-223 antagomir(0.5 nmol/μL),连续7 d。对照组和模型组用生理盐水替代。取小鼠肺组织,测定肺湿/干重比(W/D);收集小鼠肺泡灌洗液,采用ELISA试剂盒测定炎症因子TNF-α、IL-6、IL-1β的水平;于光学显微镜下统计嗜酸性粒细胞(eosinophils,EOS)、淋巴细胞、中性粒细胞的数量;行HE染色后观察小鼠肺组织病理学变化并进行肺损伤评分;采用qRT-PCR法测定小鼠肺组织中miR-223的表达水平,蛋白质印迹法测定小鼠肺组织中NLRP3蛋白质的表达水平;采用荧光素酶报告实验分析miR-223与NLRP3的靶向关系。结果:与对照组相比,模型组小鼠肺W/D值、肺损伤评分、肺泡灌洗液中炎症因子水平显著升高(均P<0.05),肺组织炎症细胞浸润严重,肺泡空间明显增加,肺泡壁显著增厚,肺泡灌洗液中EOS、淋巴细胞、中性粒细胞数量显著增加(均P<0.05),肺组织中miR-223表达水平显著降低、NLRP3蛋白质表达水平显著升高(均P<0.05)。与模型组相比,绿原酸组、绿原酸+miR-223 NC组、绿原酸+miR-223 antagomir组小鼠肺W/D值、肺损伤评分、肺泡灌洗液中炎症因子水平显著降低(均P<0.05),肺组织损伤减轻,肺泡灌洗液中EOS、淋巴细胞、中性粒细胞数量显著减少(均P<0.05),肺组织中miR-223表达水平显著升高,NLRP3蛋白质表达水平显著降低(均P<0.05)。与绿原酸组相比,绿原酸+miR-223 antagomir组小鼠肺W/D值、肺损伤评分、肺泡灌洗液中炎症因子水平显著升高(均P<0.05),肺组织损伤加重,肺泡灌洗液中EOS、淋巴细胞、中性粒细胞数量显著增加(均P<0.05),肺组织中miR-223表达水平显著降低,NLRP3蛋白质表达水平显著升高(均P<0.05)。荧光素酶报告实验结果显示miR-223与NLRP3存在靶向关系。结论:绿原酸可能通过提高miR-223水平,靶向抑制NLRP3表达,降低LPS诱导的ALI小鼠炎症反应,缓解肺组织病理损伤。
Objective: Chlorogenic acid has various physiological activities such as antibacterial, antiinflammatory, and antiviral activities. Studies have shown that chlorogenic acid can alleviate the inflammatory response of mice with acute lung injury(ALI), but the specific mechanism is still unclear. This study aims to investigate whether chlorogenic acid attenuates lipopolysaccharide(LPS)-induced ALI in mice by regulating the micro RNA-223(mi R-223)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)axis.Methods: SPF grade BALBc male mice were randomly divided into a control group, a model group, a chlorogenic acid group, a chlorogenic acid+mi R-223 negative control(mi R-223 NC) group, and a chlorogenic acid+mi R-223 inhibitor(mi R-223 antagomir) group, 10mice in each group. Except the control group, the other groups were instilled with 4 mg/kg LPS through the airway to establish the ALI mouse model. After the modeling, the mice in the chlorogenic acid group were continuously given chlorogenic acid(100 mg/kg) by gavage for 7 d. The chlorogenic acid+mi R-223 NC group and the chlorogenic acid+mi R-223 antagomir group were given 100 mg/kg chlorogenic acid by gavage every day, and then were injected with 10 μL of mi R-223 NC(0.5 nmol/μL) and mi R-223 antagomir(0.5 nmol/μL) respectively for 7 consecutive days.The control group and the model group were replaced with normal saline. The lung tissues of mice were taken to measure the ratios of lung wet to dry weight(W/D). The bronchoalveolar lavage fluid of mice was collected to measure the levels of TNF-α, IL-6, and IL-1β by ELISA kit and to count the number of eosinophils(EOS), lymphocytes, neutrophils under light microscope. After HE staining, the pathological changes of lung tissues were observed and lung injury was scored. q RT-PCR method were used to determine the expression levels of mi R-223 in lung tissues. Western blotting was used to determine the expression levels of NLRP3 protein in mouse lung tissues. Luciferase reporter assay was used to analyze the targeting relationship of mi R-223 to NLRP3.Results: Compared with the control group, the lung W/D value, the lung injury score and the level of inflammatory factors in the bronchoalveolar lavage fluid were significantly increased in the model group(all P<0.05);the infiltration of inflammatory cells in the lung tissue was severe;the alveolar space was significantly increased;the alveolar wall was significantly thickened;the number of EOS, lymphocytes, and neutrophils in the bronchoalveolar lavage fluid was significantly increased(all P<0.05);the expression levels of mi R-223 in lung tissue were significantly decreased(P<0.05);and the protein expression levels of NLRP3 were significantly increased(P<0.05). Compared with the model group,the W/D value of lungs, lung injury score, and levels of inflammatory factors in bronchoalveolar lavage fluid were significantly decreased in the chlorogenic acid group,the chlorogenic acid+mi R-223 NC group, and the chlorogenic acid+mi R-223 antagomir group(all P<0.05);lung tissues damage was alleviated;the numbers of EOS, lymphocytes,and neutrophils in bronchoalveolar lavage fluid were significantly decreased(all P<0.05);the expression levels of mi R-223 in lung tissues were significantly increased(P<0.05);and the expression levels of NLRP3 protein were significantly decreased(P<0.05). Compared with the chlorogenic acid group, the lung W/D value, lung injury score, and inflammatory factor levels in the bronchoalveolar lavage fluid were significantly increased in the chlorogenic acid+mi R-223 antagomir group(all P<0.05);lung tissue damage was aggravated;the number of EOS, lymphocytes and neutrophils in bronchoalveolar lavage fluid significantly increased(all P<0.05);the expression levels of mi R-223 in lung tissues were significantly decreased(P<0.05);and the expression levels of NLRP3 protein were significantly increased(P<0.05). The results of luciferase reporter assay showed that mi R-223 had a targeting relationship with NLRP3.Conclusion: Chlorogenic acid may increase the level of mi R-223, target the inhibition of NLRP3 expression, reduce LPS-induced inflammatory response in ALI mice, and alleviate pathological damage of lung tissues.
作者
刘畅
程晓丹
孙家安
张少华
张强
LIU Chang;CHENG Xiaodan;SUN Jia’an;ZHANG Shaohua;ZHANG Qiang(Department of Emergency,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450007;Department of Emergency,Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen Guangdong 518107,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2022年第3期280-288,共9页
Journal of Central South University ：Medical Science
基金
河南省医学科技攻关计划联合共建项目(LHGJ20191051)
河南省高等学校重点科研项目(20B320024)。
