注射用红花黄色素经TLR-NF-κB炎症通路抗大鼠MIRI的保护作用研究
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摘要
该研究探讨注射用红花黄色素调节炎症反应抗大鼠心肌缺血再灌注损伤的作用机制。将雄性Wistar大鼠随机分为假手术组,模型组,合贝爽组,注射用红花黄色素高、中、低剂量组,结扎冠状动脉左前降支建立大鼠心肌缺血/再灌注损伤模型。用HE染色观察心肌组织病理变化; TTC染色法检测心肌梗死面积;生化法或酶联免疫分析(ELISA)法检测血清肌酸激酶(CK)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)含量及肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)的变化;蛋白质免疫印迹(Western blot)法测定心肌组织中Toll样受体4(TLR4)、核转录因子-κB(NF-κB p65)蛋白的表达。结果显示,与假手术组相比,模型组心肌排列紊乱,间质严重水肿,心肌梗死面积明显增大,血清中AST,CK,LDH活性升高,TNF-α和IL-6含量显著升高,心肌组织中TLR4和NF-κB(p65)蛋白的表达量也升高;与模型组相比,合贝爽组、注射用红花黄色素高、中、低剂量组心肌组织排列较整齐,水肿明显减轻;心肌梗死面积显著缩小,血清中AST,CK,LDH活性及TNF-α,IL-6含量显著降低,心肌组织中TLR4,NF-κB(p65)蛋白的表达量降低;与合贝爽组相比,注射用红花黄色素高、中、低剂量组心肌梗死面积更大,血清中AST,CK,LDH活性较高,血清中TNF-α,IL-6含量更高,心肌组织中TLR4,NF-κB(p65)蛋白的表达水平未见明显统计学差异。提示,注射用红花黄色素具有显著抗心肌缺血再灌注损伤作用,其机制可能与调节TLR-NF-κB通路抑制炎症反应相关。
This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.
This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.
引文
[1]汪刚,刘莹,侯雪峰,等.川芎提取物通过激活Nrf2通路对抗心肌缺血大鼠氧化应激损伤[J].中国中药杂志,2017,42(24):4834.
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[3] Vilahur G,Badimon L. Ischemia/reperfusion activates myocardial innate immune response:the key role of the toll-like receptor[J].Front Physiol,2014,5(5):496.
[4]瞿城,唐于平,史旭芹,等.基于化学计量学和多指标综合指数法比较研究丹参-红花药对不同制法活血化瘀作用[J].中国中药杂志,2017,42(15):3017.
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[7]陈珊滢.探讨红花黄色素对急性冠脉综合征患者PCI术后心肌保护作用及可能作用机制[D].石家庄:河北医科大学,2016.
[8] Zirpoli H,Abdillahi M,Quadri N,et al. Acute administration of n-3 rich triglyceride emulsions provides cardioprotection in murine models after ischemia-reperfusion[J]. PLoS ONE,2015,10(1):e0116274.
[9] Hu G,Huang X,Zhang K,et al. Anti-inflammatory effect of B-type natriuretic peptide postconditioning during myocardial ischemia-reperfusion:involvement of PI3K/Akt signaling pathway[J]. Inflammation,2014,37(5):1669.
[10]郑晴,包怡敏.中药对心肌缺血再灌注损伤过程中自噬的调控[J].中国中药杂志,2017,42(15):2925.
[11]李彦,焦旭,胥华泰,等.注射用盐酸地尔硫卓的药理分析及临床应用总结[J].现代养生,2017,6(3):88.
[12] Liu L,Gu H L,Liu H M,et al. Protective effect of resveratrol against IL-1β-induced inflammatory response on human osteoarthritic chondrocytes partly via the TLR4/My D88/NF-κB signaling pathway:an"in vitro study"[J]. Int J Mol Sci,2014,15(4):6925.
[13]杨茹茜,徐倩,皇甫志敏,等.黄芪甲苷对肾缺血再灌注后纤维化小鼠Toll样受体通路的作用研究[J].中国中药杂志,2018,43(18):3729.
[14] Zhou S,Wang G,Zhang W. Effect of TLR4/My D88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats,via regulation of macrophage activation and inflammatory response[J]. Exp Ther Med,2018,15(4):3376.
[15]钦丹萍,周毅骏,张绍珠,等.雷公藤多苷抗巨噬细胞炎症及对TLR4/NF-κB调控炎症作用的研究[J].中国中药杂志,2015,40(16):3256.
[16]庾国桢,彭皓均,黄秀芳,等.黄连素对Aβ25-35诱导的HT22细胞TLR4/NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(24):164.
[17]吕敬雷,王鹏,隋雪琴,等.缺血后处理对脑缺血再灌注大鼠TNF-α和IL-1β表达影响[J].齐鲁医学杂志,2012,27(4):319.
[18]范茜茜.电针对缺血再灌注脑损伤大鼠炎症细胞因子IL-4、TNF-α的影响[D].南京:南京中医药大学,2017.
[19] Nizamutdinova Irina T,Guleria Rakeshwar S,Singh Amar B,et al. Retinoic acid protects cardiomyocytes from high glucose-induced apoptosis via inhibition of sustained activation of NF-κB signaling[J]. J Cell Physiol,2013,228(2):380.
[20]朱开权.瑞舒伐他汀钙片对大鼠心肌梗死后炎症反应及NF-kB的影响[J].中西医结合心血管病电子杂志,2016,4(27):2.
[2] Anzell A R,Maizy R,Przyklenk K,et al. Mitochondrial quality control and disease:insights into ischemia-reperfusion injury[J]. Mol Neurobiol,2018,55(3):2547.
[3] Vilahur G,Badimon L. Ischemia/reperfusion activates myocardial innate immune response:the key role of the toll-like receptor[J].Front Physiol,2014,5(5):496.
[4]瞿城,唐于平,史旭芹,等.基于化学计量学和多指标综合指数法比较研究丹参-红花药对不同制法活血化瘀作用[J].中国中药杂志,2017,42(15):3017.
[5] Shi X M,Zhang H,Zhou Z J,et al. Effects of safflower yellow on beta-amyloid deposition and activation of astrocytes in the brain of APP/PS1 transgenic mice[J]. Biomed Pharmacother,2017,98:553.
[6]陈培栋,房利勤.红花注射液和注射用红花黄色素药理作用研究[J].世界中医药,2016,11(2):308.
[7]陈珊滢.探讨红花黄色素对急性冠脉综合征患者PCI术后心肌保护作用及可能作用机制[D].石家庄:河北医科大学,2016.
[8] Zirpoli H,Abdillahi M,Quadri N,et al. Acute administration of n-3 rich triglyceride emulsions provides cardioprotection in murine models after ischemia-reperfusion[J]. PLoS ONE,2015,10(1):e0116274.
[9] Hu G,Huang X,Zhang K,et al. Anti-inflammatory effect of B-type natriuretic peptide postconditioning during myocardial ischemia-reperfusion:involvement of PI3K/Akt signaling pathway[J]. Inflammation,2014,37(5):1669.
[10]郑晴,包怡敏.中药对心肌缺血再灌注损伤过程中自噬的调控[J].中国中药杂志,2017,42(15):2925.
[11]李彦,焦旭,胥华泰,等.注射用盐酸地尔硫卓的药理分析及临床应用总结[J].现代养生,2017,6(3):88.
[12] Liu L,Gu H L,Liu H M,et al. Protective effect of resveratrol against IL-1β-induced inflammatory response on human osteoarthritic chondrocytes partly via the TLR4/My D88/NF-κB signaling pathway:an"in vitro study"[J]. Int J Mol Sci,2014,15(4):6925.
[13]杨茹茜,徐倩,皇甫志敏,等.黄芪甲苷对肾缺血再灌注后纤维化小鼠Toll样受体通路的作用研究[J].中国中药杂志,2018,43(18):3729.
[14] Zhou S,Wang G,Zhang W. Effect of TLR4/My D88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats,via regulation of macrophage activation and inflammatory response[J]. Exp Ther Med,2018,15(4):3376.
[15]钦丹萍,周毅骏,张绍珠,等.雷公藤多苷抗巨噬细胞炎症及对TLR4/NF-κB调控炎症作用的研究[J].中国中药杂志,2015,40(16):3256.
[16]庾国桢,彭皓均,黄秀芳,等.黄连素对Aβ25-35诱导的HT22细胞TLR4/NF-κB信号通路的影响[J].中国实验方剂学杂志,2018,24(24):164.
[17]吕敬雷,王鹏,隋雪琴,等.缺血后处理对脑缺血再灌注大鼠TNF-α和IL-1β表达影响[J].齐鲁医学杂志,2012,27(4):319.
[18]范茜茜.电针对缺血再灌注脑损伤大鼠炎症细胞因子IL-4、TNF-α的影响[D].南京:南京中医药大学,2017.
[19] Nizamutdinova Irina T,Guleria Rakeshwar S,Singh Amar B,et al. Retinoic acid protects cardiomyocytes from high glucose-induced apoptosis via inhibition of sustained activation of NF-κB signaling[J]. J Cell Physiol,2013,228(2):380.
[20]朱开权.瑞舒伐他汀钙片对大鼠心肌梗死后炎症反应及NF-kB的影响[J].中西医结合心血管病电子杂志,2016,4(27):2.