| 王乙连,何晨晨,张珊苑,等.宽胸气雾剂通过调控TLR4/MyD88/NLRP3/Caspase-1通路减轻心梗诱导的大鼠心肌细胞损伤[J].浙江中医药大学学报,2024,48(1):122-130. |
| 宽胸气雾剂通过调控TLR4/MyD88/NLRP3/Caspase-1通路减轻心梗诱导的大鼠心肌细胞损伤 |
| Kuanxiong Aerosol Regulates TLR4/MyD88/NLRP3/Caspase-1 Pathway to Reduce Cardiomyocyte Injury in Myocardial Infarction Rats |
| DOI：10.16466/j.issn1005-5509.2024.01.021 |
| 中文关键词: 宽胸气雾剂 心肌梗死 焦亡 炎症 TLR4 NLRP3 |
| 英文关键词: Kuanxiong aerosol myocardial infarction pyroptosis inflammation TLR4 NLRP3 |
| 基金项目:福建省科技计划项目(2021L3014) |
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| 中文摘要: |
| [目的] 探索宽胸气雾剂(Kuanxiong aerosol，KXA)对异丙肾上腺素(isoproterenol，ISO)诱导的心肌梗死(myocardial infarction，MI)大鼠心肌炎症和细胞焦亡的影响，并分析其对焦亡关键通路Toll样受体4(Toll-like receptor 4，TLR4)/髓样分化因子(myeloid differentiation primary response gene 88，MyD88)/NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain containing 3，NLRP3)/胱天蛋白酶-1(cysteinyl aspartate specific proteinase-1，caspase-1)的作用。[方法] 按体质量将30只雄性Wistar大鼠随机分为5组:对照组(0.9%氯化钠溶液)、模型组(ISO 120 mg·kg-1)、单硝酸异山梨酯组(ISO 120 mg·kg-1＋单硝酸异山梨酯5 mg/kg·d)、KXA低剂量组(ISO 120 mg·kg-1＋KXA 0.1 mL/kg·d)和KXA高剂量组(ISO 120 mg·kg-1＋KXA 0.3 mL/kg·d)，每组6只，连续灌胃14 d，并在第13、14天腹腔注射ISO。末次干预结束后，麻醉采血、分离心脏，以酶联免疫吸附试验(enzyme-linked immunosorbent assay，ELISA)检测各组血清肌酸激酶同工酶(creatine kinase-MB，CK-MB)和心肌肌钙蛋白T(cardiac troponin T，cTnT)水平，以及血清白细胞介素-6(interleukin-6，IL-6)、白细胞介素-18(interleukin-18，IL-18)、白细胞介素-1β(interleukin-1β，IL-1β)和肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)水平；苏木精-伊红(hematoxylin-eosin，HE)染色检测心肌病理变化；转录组测序分析各组心肌基因表达差异；免疫印迹检测焦亡通路相关蛋白的表达情况。[结果] 与对照组比较，模型组cTnT和CK-MB检测显著升高，差异具有统计学意义(P<0.01)，表明MI大鼠模型构建成功。HE染色显示，KXA和单硝酸异山梨酯干预后心肌纤维排列断裂、紊乱情况明显减轻。转录组测序显示，对照组和模型组之间存在2 646个差异基因；模型组和KXA组之间存在714个差异基因，进一步分析发现130个基因表达上调，7个基因表达下调，其中炎症相关性通路TLR4通路被显著富集。与模型组比较，KXA高剂量组、KXA低剂量组和单硝酸异山梨酯组血清IL-6、IL-18、IL-1β和TNF-α表达显著减少，差异具有统计学意义(P<0.01，P<0.05)。免疫印迹显示，模型组TLR4、MyD88、磷酸化核因子-κB p65(phospho-nuclear factor-κB，p-NF-κB )p65、NLRP3、切割的胱天蛋白酶-1(cleaved caspase-1)和消皮素D蛋白N端结构域(Gasdermin D-N，GSDMD-N)表达显著高于对照组；而KXA高剂量组、KXA低剂量组和单硝酸异山梨酯组的表达显著低于模型组(P<0.05，P<0.01)。[结论] KXA可以改善心肌缺血，减轻心脏损伤，抑制心肌细胞焦亡和炎症反应，其机制可能与调节TLR4/MyD88/NLRP3/caspase-1信号通路有关。 |
| 英文摘要: |
| [Objective] To investigate the effects of Kuanxiong aerosol(KXA) on pyroptosis and inflammatory response in isoproterenol(ISO)-induced myocardial infarction(MI) rats， and its effect on the key pathway of pyroptosis Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/NOD-like receptor pyrin domain containing 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1) . [Methods] Thirty male Wistar rats were randomly divided into five groups，6 rats in each group， as control group(0.9% sodium chloride solution)， model group(ISO 120 mg·kg-1)， isosorbide mononitrate(IMSN) group(ISO 120 mg·kg-1＋IMSN 5 mg/kg·d)， KXA low dose group(ISO 120 mg·kg-1＋KXA 0.1 mL/kg·d)， and KXA high dose group(ISO 120 mg·kg-1＋KXA 0.3 mL/kg·d)， gave continuous intragastric administration for 14 days， and intraperitoneal injection of ISO on the 13th and 14th day. After the last intervention， collected heart tissues and blood under anesthesia. Enzyme-linked immunosorbent assay(ELISA) was performed to investigate the expression of creatine kinase-MB(CK-MB) and cardiac troponin T(cTnT)， as well as serum inflammatory indicators such as interleukin-1β(IL-1β)， interleukin-18(IL-18)， interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α). The histopathological changes in heart tissue were evaluated using hematoxylin-eosin(HE) staining， and RNA-sequencing was used to detect the differential expression genes among groups. And the expression of the pyroptosis relevant protein was detected by Western blot. [Results] The results of the ELISA showed that CK-MB and cTnT expression in model group were significantly higher than those in control group(P<0.01)， which meant successful model construction. Pathological staining results showed disordered and fractured muscle fibers were significantly improved after KXA and IMSN intervention. RNA-seq results showed there were 2 646 different genes between model group and control group， while 714 other genes were in KXA and model group. After analyzing these two compared groups， it found that there were 130 up-regulated genes and 7 down-regulated genes; among them， inflammation related TLR4 pathway was significantly enriched. Furthermore， compared with model group， the expression of inflammatory factors IL-1β， IL-18， IL-6 and TNF-α decreased significantly in KXA groups and IMSN group(P<0.01， P<0.05)， and Western blot showed that the protein expression of TLR4， MyD88， phospho-nuclear factor-κB(p-NF-κB) p65， NLRP3， cleaved cysteinyl aspartate specific proteinase-1(cleaved caspase-1) and Gasdermin D-N(GSDMD-N) increased significantly in model group while significantly down-regulated in KXA groups and IMSN group(P<0.05， P<0.01). [Conclusion] KXA can improve myocardial ischemia, reduce cardiac damage, and inhibit cardiomyocyte pyroptosis and inflammatory response, the mechanism may be related to regulating the TLR4/MyD88/NLRP3/caspase-1 signaling pathway. |
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