羟基红花黄素A对脑卒中相关性肺炎大鼠肺组织的保护作用及机制
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摘要
目的探讨羟基红花黄素A(HSYA)对脑卒中相关性肺炎(SAP)大鼠肺组织的保护作用及可能的机制。方法随机选取SD大鼠10只作为假手术组,其余大鼠采用四动脉阻断法制备SAP模型。将造模成功的SAP模型大鼠随机分为4组,分别用生理盐水(假手术组、SAP组)、Janus激酶2特异性抑制剂N-苄基-3,4-二羟基亚苄基氰基乙酰胺(AG490,5 mg/kg,AG490组)、低剂量(8 mg/kg,L-HSYA组)及高剂量HSYA(16 mg/kg,H-HSYA组)对大鼠进行治疗,连续给药7 d。用HE染色法观察各组肺组织病理变化;测量肺组织湿重量(W)、干重量(D),计算W/D值;用比色法检测肺组织中髓过氧化物酶(MPO)活性;用酶联免疫吸附法检测支气管肺泡灌洗液(BALF)中炎性细胞因子水平及肺组织核提取物中核因子κB(NF-κB)结合活性; Western blotting法检测肺组织核提取物中磷酸化Janus激酶2(p-JAK2)、磷酸化信号传导子及转录激活子3(p-STAT3)、核因子κB p65(NF-κB p65)蛋白表达。结果与假手术组比较,SAP组肺损伤评分、肺组织W/D值、MPO活性高,BALF中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-18水平及NF-κB DNA结合活性高,NF-κB p65、p-JAK2、p-STAT3蛋白表达高(P均<0. 05);与SAP组比较,AG490组、L-HSYA组及H-HSYA组肺损伤评分、肺组织W/D值、MPO活性低,BALF中TNF-α、IL-1β、IL-18水平及NF-κB DNA结合活性低,NF-κB p65、p-JAK2、p-STAT3蛋白表达低(P均<0. 05);且AG490组、HHSYA组以上指标变化最明显(P均<0. 05)。结论 HSYA能够减轻SAP大鼠肺组织的损伤及炎症反应,该作用可能与抑制肺组织中JAK2/STAT3信号通路激活有关。
Objective To investigate the protective effect of hydroxysafflor yellow A( HSYA) on lung tissues of stroke-associated pneumonia( SAP) rats and to explore its possible mechanism. Methods Ten SD rats were randomly selected as the sham operation group,and the other rats were prepared for the SAP models by 4-artery occlusion method. After modeling,the rats were treated with normal saline( sham group,SAP group),Janus kinase 2( JAK2) specific inhibitor N-benzyl-3,4-two hydroxybenzyl cyanoacetamide AG490( 5 mg/kg,AG490 group),low-dose HSYA( 8 mg/kg L-HSYA group) and high-dose HSYA( 16 mg/kg,H-HSYA group) for 7 consecutive days. Histopathological analysis was used to observe the lung injury. We measured the wet weight( W) and dry weight( D) of the lung tissue and calculated the W/D value. The activity of myeloperoxidase( MPO) in the lung tissues was detected by colorimetry. The level of inflammatory cytokines in bronchoalveolar lavage fluid( BALF) was detected by enzyme-linked immunosorbent assay. Enzyme linked immunosorbent assay was used to detect nuclear factor κB( NF-κB) binding activity in the lung tissues. Western blotting was used to analyze the expression of phosphorylated Janus kinase 2( p-JAK2),phosphorylated signal transducer and activator of transcription 3( p-STAT3),nuclear factor κB p65( NF-κB p65) in the nuclear extracts of the lung tissues. Results Compared with the sham group,the lung injury score,the W/D,MPO of the lung tissues,the levels of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and IL-18 in BALF,the activity of NF-κB in p65 subunit,and the expression of p-JAK2 and p-STAT3 protein in the SAP group increased significantly( all P < 0. 05). Compared with the SAP group,the lung injury score,the W/D of the lung tissues,the levels of TNF-α,IL-1β,and IL-18 in BALF,the activity of NF-κB in p65 subunit,and the expression of p-JAK2 and p-STAT3 protein decreased significantly in the AG490 group,L-HSYA group and H-HSYA group( all P < 0. 05). Conclusion HSYA can attenuate the lung tissue damage and inflammatory response in SAP rats,which may be related to the inhibition of JAK2/STAT3 signaling pathway activation in the lung tissues.
Objective To investigate the protective effect of hydroxysafflor yellow A( HSYA) on lung tissues of stroke-associated pneumonia( SAP) rats and to explore its possible mechanism. Methods Ten SD rats were randomly selected as the sham operation group,and the other rats were prepared for the SAP models by 4-artery occlusion method. After modeling,the rats were treated with normal saline( sham group,SAP group),Janus kinase 2( JAK2) specific inhibitor N-benzyl-3,4-two hydroxybenzyl cyanoacetamide AG490( 5 mg/kg,AG490 group),low-dose HSYA( 8 mg/kg L-HSYA group) and high-dose HSYA( 16 mg/kg,H-HSYA group) for 7 consecutive days. Histopathological analysis was used to observe the lung injury. We measured the wet weight( W) and dry weight( D) of the lung tissue and calculated the W/D value. The activity of myeloperoxidase( MPO) in the lung tissues was detected by colorimetry. The level of inflammatory cytokines in bronchoalveolar lavage fluid( BALF) was detected by enzyme-linked immunosorbent assay. Enzyme linked immunosorbent assay was used to detect nuclear factor κB( NF-κB) binding activity in the lung tissues. Western blotting was used to analyze the expression of phosphorylated Janus kinase 2( p-JAK2),phosphorylated signal transducer and activator of transcription 3( p-STAT3),nuclear factor κB p65( NF-κB p65) in the nuclear extracts of the lung tissues. Results Compared with the sham group,the lung injury score,the W/D,MPO of the lung tissues,the levels of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and IL-18 in BALF,the activity of NF-κB in p65 subunit,and the expression of p-JAK2 and p-STAT3 protein in the SAP group increased significantly( all P < 0. 05). Compared with the SAP group,the lung injury score,the W/D of the lung tissues,the levels of TNF-α,IL-1β,and IL-18 in BALF,the activity of NF-κB in p65 subunit,and the expression of p-JAK2 and p-STAT3 protein decreased significantly in the AG490 group,L-HSYA group and H-HSYA group( all P < 0. 05). Conclusion HSYA can attenuate the lung tissue damage and inflammatory response in SAP rats,which may be related to the inhibition of JAK2/STAT3 signaling pathway activation in the lung tissues.
引文
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[10]雷传江,焦艳,王关嵩,等.受体相互作用蛋白140在脓毒症急性肺损伤大鼠肺泡巨噬细胞中的表达及其作用[J].第三军医大学学报,2015,37(9):851-857.
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[13]Liu S,Su X,Pan P,et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury[J]. Sci Rep,2016,6(10):37252-37263.
[14]方靖,杨雅婷,刘景云,等.黄芪多糖通过抑制中性粒细胞活化减轻脂多糖诱导的大鼠肺损伤[J].细胞与分子免疫学杂志,2017,33(8):1020-1023,1029.
[15]Jiang S,Shi Z,Li C,et al. Hydroxysafflor yellow A attenuates ischemia/reperfusion-induced liver injury by suppressing macrophage activation[J]. Int J Clin Exp Pathol,2014,7(5):2595-2608.
[16]程石,杨彬,闫文貌,等.抗TNF-α治疗对重症急性胰腺炎肺损伤NF-κB信号通路的影响[J].中国普通外科杂志,2008,17(3):219-223.
[17]Lv Y,Qian Y,Ou-Yang A,et al. Hydroxysafflor yellow A attenuates neuron damage by suppressing the lipopolysaccharide-induced TLR4 pathway in activated microglial cells[J]. Cell Mol Neurobiol,2016,36(8):1241-1256.
[18]朱子诚,吴章友,温跃春,等. AG490抑制STAT3信号通路活性对豚鼠形觉剥夺性近视巩膜重塑的调控[J].中华实验眼科杂志,2015,33(6):493-499.
[19]谢秀芳,李多,熊彬,等. Ghrelin对脓毒症小鼠肺脏炎症及JAK/STAT通路的影响[J].国际呼吸杂志,2016,36(10):730-734.
[20]王立,张裕祥,杜萍,等.不同肺表面活性物质对兔胸部撞击肺损伤中促炎性细胞因子的影响[J].广西医学,2015,37(3):302-304.
[21]徐志红,白永愉,黄新策,等.垂盆草提取物经JAK2/STAT3信号通路途径改善大鼠重症急性胰腺炎肺损伤的研究[J].肝胆胰外科杂志,2014,26(5):398-402.
[2] He Y,Liu Q,Li Y,et al. Protective effects of hydroxysafflor yellow A(HSYA)on alcohol-induced liver injury in rats[J]. J Physiol Bioche,2015,71(1):69-78.
[3]刘金连,王海芳,马淑惠,等.羟基红花黄色素A通过抑制动脉内皮细胞中TNFR1/NF-κB信号通路而发挥抗炎作用[J].细胞与分子免疫学杂志,2015,31(7):945-948.
[4]刘慧,陈根成,孙瑛.电针对脑卒中相关性肺炎大鼠干预效应实验研究[J].新中医,2014,46(4):191-193.
[5]Zhang S,Liu X,Goldstein S,et al. Role of the JAK/STAT signaling pathway in the pathogenesis of acute myocardial infarction in rats and its effect on NF-κB expression[J]. Mol Med Rep,2013,7(1):93-98.
[6]刘永刚,李芳君,汤芳.羟基红花黄色素A对大鼠心肌细胞缺血再灌注损伤的保护作用[J].中药药理与临床,2015,31(1):71-74.
[7]Fukumoto J,Fukumoto I,Parthasarathy PT,et al. NLRP3 deletion protects from hyperoxia-induced acute lung injury[J]. Am J Physiol Cell Physiol,2013,305(2):182-189.
[8]秦莎莎,余梦黎,廖金明,等.羟基红花黄色素A与芍药苷联合用药对大鼠脑缺血再灌注损伤的保护作用[J].广东药学院学报,2016,32(6):747-751.
[9]许航,丁一,刘文星,等.黄芪甲苷和羟基红花黄色素A配伍抗脑缺血再灌注损伤的协同作用及机制研究[J].中华神经外科疾病研究杂志,2017,16(5):427-431.
[10]雷传江,焦艳,王关嵩,等.受体相互作用蛋白140在脓毒症急性肺损伤大鼠肺泡巨噬细胞中的表达及其作用[J].第三军医大学学报,2015,37(9):851-857.
[11]Takahara M,Aoyama-Ishikawa M,Shuno K,et al. Role of endogenous IL-18 in the lung during endotoxin-induced systemic inflammation[J]. Acu Med Sur,2014,1(1):23-30.
[12] Yang CM,Luo SF,Hsieh HL,et al. Interleukin-1beta induces ICAM-1 expression enhancing leukocyte adhesion in human rheumatoid arthritis synovial fibroblasts:involvement of ERK,JNK,AP-1,and NF-kappaB[J]. J Cel Physiol,2010,224(2):516-526.
[13]Liu S,Su X,Pan P,et al. Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury[J]. Sci Rep,2016,6(10):37252-37263.
[14]方靖,杨雅婷,刘景云,等.黄芪多糖通过抑制中性粒细胞活化减轻脂多糖诱导的大鼠肺损伤[J].细胞与分子免疫学杂志,2017,33(8):1020-1023,1029.
[15]Jiang S,Shi Z,Li C,et al. Hydroxysafflor yellow A attenuates ischemia/reperfusion-induced liver injury by suppressing macrophage activation[J]. Int J Clin Exp Pathol,2014,7(5):2595-2608.
[16]程石,杨彬,闫文貌,等.抗TNF-α治疗对重症急性胰腺炎肺损伤NF-κB信号通路的影响[J].中国普通外科杂志,2008,17(3):219-223.
[17]Lv Y,Qian Y,Ou-Yang A,et al. Hydroxysafflor yellow A attenuates neuron damage by suppressing the lipopolysaccharide-induced TLR4 pathway in activated microglial cells[J]. Cell Mol Neurobiol,2016,36(8):1241-1256.
[18]朱子诚,吴章友,温跃春,等. AG490抑制STAT3信号通路活性对豚鼠形觉剥夺性近视巩膜重塑的调控[J].中华实验眼科杂志,2015,33(6):493-499.
[19]谢秀芳,李多,熊彬,等. Ghrelin对脓毒症小鼠肺脏炎症及JAK/STAT通路的影响[J].国际呼吸杂志,2016,36(10):730-734.
[20]王立,张裕祥,杜萍,等.不同肺表面活性物质对兔胸部撞击肺损伤中促炎性细胞因子的影响[J].广西医学,2015,37(3):302-304.
[21]徐志红,白永愉,黄新策,等.垂盆草提取物经JAK2/STAT3信号通路途径改善大鼠重症急性胰腺炎肺损伤的研究[J].肝胆胰外科杂志,2014,26(5):398-402.