摘要
本研究通过建立H9C2细胞缺氧复氧(hypoxia/re-oxygenation,H/R)损伤模型,探讨香青兰总黄酮(total flavonoids of Dracocephalum moldavica L.,TFDM)抗心肌缺血再灌注损伤炎症反应的机制。通过CCK-8(cell counting kit-8)法检测细胞活性;酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测乳酸脱氢酶(lactate dehydrogenase,LDH)和肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)活性;2′,7′-二氯二氢荧光素二乙酸酯(2′,7′-Dichlorodihydrofluorescein Diacetate,DCFH-DA)荧光探针法检测细胞内活性氧(reactive oxygen species,ROS)水平;蛋白免疫印迹(Western blot,WB)法检测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)、成孔蛋白D(Gasdermin D,GSDMD)及半胱氨酸天冬氨酸蛋白酶-1(cysteinyl aspartate specific proteinase 1,Caspase-1)、Toll样受体4(Toll-like receptor 4,TLR4)、磷酸化核因子κB P65(p-nuclear factor-κB P65,p-p65)、凋亡相关含有CARD结构域的斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)的表达水平;使用NLRP3激动剂进一步验证TFDM对炎症反应的抑制作用。实验结果显示,TFDM显著提高了H9C2细胞的活性,降低了LDH、CK-MB和ROS的表达水平,降低了NLRP3、GSDMD、Caspase-1、TLR4、p-p65、ASC等蛋白的表达水平。上述研究结果表明,香青兰总黄酮可能通过抑制NLRP3炎症小体的激活,改善心肌缺血再灌注损伤炎症反应,其机制可能与下调TLR4/NF-κB信号通路有关。
This study aims to investigate the mechanism of total flavonoids of Dracocephalum moldavica L.(TFDM)against inflammatory response to myocardial ischemia-reperfusion injury through the establishment of hypoxia/re-oxygenation(H/R)model in H9C2 cells.The cell viability was measured by cell counting kit-8(CCK-8).The lactate dehydrogenase(LDH)and creatine kinase isoenzyme(CK-MB)were detected by enzyme-linked immunosorbent assay(ELISA)kits.The level of intracellular reactive oxygen species(ROS)was detected by 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe.The protein levels of nucleotide-binding oligomerization structural domain-like receptor protein 3(NBFR3),Gasdermin D(GSDMD),Caspase-1,Toll-like receptor 4(TLR4),phosphorylated nuclear factorκB P65(P-NF-κB P65,p-p65),apoptosis-associated speck-like protein containing a CARD(ASC)were detected by Western blot.The NLRP3 agonist BMS-986299 was used to further validate the inhibitory effect of TFDM on inflammatory responses.The results showed that TFDM significantly increased the activity of H9C2 cells and decreased the expression levels of LDH,CK-MB and ROS.TFDM decreased the expression levels of NLRP3,GSDMD,Caspase-1,TLR4,p-p65,ASC.The above findings demonstrate that TFDM may improve the inflammatory response to myocardial ischemia-reperfusion injury by inhibiting the activation of NLRP3 inflammatory vesicles,and the mechanism may be related to the down-regulation of the TLR4/NF-κB signaling pathway.
作者
马祖文
张歆
张裴
张丽
杨婷
孟繁龙
MA Zu-wen;ZHANG Xin;ZHANG Pei;ZHANG Li;YANG Ting;MENG Fan-long(The Fifth Affiliated Hospital,Xinjiang Medical University,Urumqi 830011,China;Urumqi Friendship Hospital,Urumqi 830049,China)
出处
《天然产物研究与开发》
北大核心
2025年第8期1513-1521,共9页
Natural Product Research and Development
基金
新疆维吾尔自治区卫生健康青年医学科技人才专项科研项目(WJWY-202334)
新疆维吾尔自治区自然科学基金(2020D01C228)。
关键词
香青兰总黄酮
心肌缺血再灌注损伤
炎症反应
NLRP3炎症小体
total flavonoids of Dracocephalum moldavica L.
myocardial ischemia-reperfusion injury
inflammatory response
NLRP3 inflammasome
