刺络拔罐法对内毒素致热家兔的退热作用及机制研究
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摘要
目的:观察刺络拔罐法对内毒素致热家兔的退热作用,并探讨其作用机制。方法:将25只家兔随机分为正常组、模型组、刺络拔罐组、单纯拔罐组和西药(吲哚美辛)组,每组各5只,除正常组外,均采用大肠杆菌内毒素注射法复制发热家兔模型。模型建立后,西药组给予相应药物灌胃,剂量为10 mL/kg,其余4组分别给予相同容积生理盐水灌胃,灌胃后单纯拔罐组、刺络拔罐组给予相应治疗。造模后每1 h记录体温1次,连续测量温度8 h,观察各组动物体温变化后,采用酶联免疫吸附试验(ELISA法)检测家兔血清细胞因子(IL-4、IL-10、IFN-γ)水平,采用免疫印迹法(Western Blot法)检测家兔下丘脑组织诱导型一氧化氮合酶(iNOS)、环氧合酶(COX-1、COX-2)蛋白表达水平。结果:模型组家兔体温明显升高,与正常组相比(P<0.01),刺络拔罐组、单纯拔罐组、西药组兔体温升高明显低于模型组(P<0.05)。模型组家兔血清IL-4、IFN-γ水平均明显低于正常组(P<0.05),其血清IL-10水平与正常组比较无统计学差异(P>0.05),其家兔下丘脑组织iNOS,COX-2蛋白表达水平均有所升高(P<0.05);干预后,刺络拔罐组、单纯拔罐组家兔血清IL-4、IFN-γ水平较模型组明显被提升(P<0.05),下丘脑组织iNOS、COX-2蛋白表达下降(P<0.05),而COX-1蛋白表达水平没有明显的下降(P>0.05);西药组家兔血清IL-4、IL-10、IFN-γ水平相比均无明显提升(P>0.05),其下丘脑组织iNOS、COX-1、COX-2蛋白表达量均比模型组减少(P<0.05),提示经刺络拔罐和单纯拔罐法干预后,可提高内毒素致热家兔血清细胞因子中IL-4,IFN-γ水平,对于血清IL-10水平无明显提升作用,对下丘脑组织COX-1蛋白表达没有抑制作用,对COX-2蛋白表达有选择性抑制作用。结论:刺络拔罐法对内毒素致热家兔有良好的退热作用,其退热机制可能是通过提高抗炎因子水平,抑制中枢iNOS、COX-2蛋白表达水平来调节机体免疫功能实现的,其对中枢COX-2蛋白表达的选择性抑制可能是优于吲哚美辛类药物的根本体现。
Objective:To observe the antipyretic effect of acupuncturing collaterals and cupping therapy on endotoxin-induced fever in rabbits and explore its mechanism. Methods:Twenty-five rabbits were randomly divided into normal group, model group, acupuncturing collaterals and cupping therapy group, cupping therapy group and western medicine(indomethacin) group, 5 rabbits in each group. Except the normal group, escherichia coli endotoxin injection was used to replicate the fever rabbit model. After the establishment of the model, the western medicine group was given corresponding drugs by gavage with a dose of 10 mL/kg, and the other four groups were given normal saline of the same volume by gavage. After gavage, the cupping therapy group and the acupuncturing collaterals and cupping therapy group were given corresponding treatment. We recorded every 1 hour temperature after modeling and continuously measuring for 8 hours to observe each animal body temperature changes. ELISA was used to detect rabbit serum cytokines(IL-4,IL-10,IFN-γ) levels. Western blot method was used to detect rabbit hypothalamus tissues induced nitric oxide synthase(iNOS), the enzyme called cyclooxygenase(COX-1, COX-2) protein expression levels. Results:The temperature of rabbits in the model group increased significantly compared with that of the normal group(P<0.01). The temperature increase of the acupuncturing collaterals and cupping group, the cupping group and the western medicine group were obviously lower than that of the model group(P<0.05). The serum IL-4 and IFN-γ levels of the model group were significantly lower than those of the normal group(P<0.05), and the serum IL-10 levels were not statistically different from those of the normal group(P>0.05). The expression levels of iNOS and COX-2 protein in the hypothalamus of the model group were increased(P<0.05). After the intervention, serum IL-4 and IFN-γ levels of rabbits in the acupuncturing collaterals and cupping group and the cupping therapy group were significantly increased compared with those of the model group(P<0.05). The expressions of iNOS and COX-2 protein in the hypothalamus decreased(P<0.05), while the expression of COX-1 protein was not significantly decreased(P>0.05). Western medicine group's rabbit serum IL-4, IL-10 and IFN-γ levels had no significant increases(P>0.05). The organization of the hypothalamus iNOS, COX-1 and COX-2 protein expression quantity was less than that of the model group(P<0.05), suggesting the acupuncturing collaterals and cupping and pure cupping method after the intervention can improve the endotoxin induced by increasing rabbit serum cytokine IL-4 and IFN-γ levels. There was no obvious promotion effect on IL-10, no inhibition on COX-1. It had selective inhibition effect on COX-2 protein. Conclusion:Acupuncturing collaterals and cupping method for endotoxin has good antifebrile effect. Its mechanism may be through improving the level of anti-inflammatory factors, inhibiting central iNOS and COX-2 protein expression levels to regulate the body's immune function. Its selective inhibition of the central COX-2 protein expression may be superior to the basic revelation of indomethacin drugs.
Objective:To observe the antipyretic effect of acupuncturing collaterals and cupping therapy on endotoxin-induced fever in rabbits and explore its mechanism. Methods:Twenty-five rabbits were randomly divided into normal group, model group, acupuncturing collaterals and cupping therapy group, cupping therapy group and western medicine(indomethacin) group, 5 rabbits in each group. Except the normal group, escherichia coli endotoxin injection was used to replicate the fever rabbit model. After the establishment of the model, the western medicine group was given corresponding drugs by gavage with a dose of 10 mL/kg, and the other four groups were given normal saline of the same volume by gavage. After gavage, the cupping therapy group and the acupuncturing collaterals and cupping therapy group were given corresponding treatment. We recorded every 1 hour temperature after modeling and continuously measuring for 8 hours to observe each animal body temperature changes. ELISA was used to detect rabbit serum cytokines(IL-4,IL-10,IFN-γ) levels. Western blot method was used to detect rabbit hypothalamus tissues induced nitric oxide synthase(iNOS), the enzyme called cyclooxygenase(COX-1, COX-2) protein expression levels. Results:The temperature of rabbits in the model group increased significantly compared with that of the normal group(P<0.01). The temperature increase of the acupuncturing collaterals and cupping group, the cupping group and the western medicine group were obviously lower than that of the model group(P<0.05). The serum IL-4 and IFN-γ levels of the model group were significantly lower than those of the normal group(P<0.05), and the serum IL-10 levels were not statistically different from those of the normal group(P>0.05). The expression levels of iNOS and COX-2 protein in the hypothalamus of the model group were increased(P<0.05). After the intervention, serum IL-4 and IFN-γ levels of rabbits in the acupuncturing collaterals and cupping group and the cupping therapy group were significantly increased compared with those of the model group(P<0.05). The expressions of iNOS and COX-2 protein in the hypothalamus decreased(P<0.05), while the expression of COX-1 protein was not significantly decreased(P>0.05). Western medicine group's rabbit serum IL-4, IL-10 and IFN-γ levels had no significant increases(P>0.05). The organization of the hypothalamus iNOS, COX-1 and COX-2 protein expression quantity was less than that of the model group(P<0.05), suggesting the acupuncturing collaterals and cupping and pure cupping method after the intervention can improve the endotoxin induced by increasing rabbit serum cytokine IL-4 and IFN-γ levels. There was no obvious promotion effect on IL-10, no inhibition on COX-1. It had selective inhibition effect on COX-2 protein. Conclusion:Acupuncturing collaterals and cupping method for endotoxin has good antifebrile effect. Its mechanism may be through improving the level of anti-inflammatory factors, inhibiting central iNOS and COX-2 protein expression levels to regulate the body's immune function. Its selective inhibition of the central COX-2 protein expression may be superior to the basic revelation of indomethacin drugs.
引文
[1] 邢歆莉,王远照.负压罐配合中药足浴辅助治疗小儿外感发热疗效观察[J].上海针灸杂志,2018,37(6):653-656.
[2] 李莉,杨保林,王兰.刺血拔罐退热法治疗急诊内科发热患者的临床研究[J].现代中医临床,2017,24(1):12-14.
[3] 方成华,段亚平,曾贤,等.大椎穴位刺血拔罐治疗风热感冒发热疗效观察[J].亚太传统医药,2016,12(7):124-125.
[4] 魏伟,吴希美,李元建.药理实验方法学[M].北京:人民卫生出版社,2010:68-73.
[5] 杨果杰,陈秀莲,邵萍,等.内毒素法兔发热动物模型及其标准化的研究[J].中国实验动物学报,2001(4):57-60.
[6] 郭义.实验针灸学[M].北京:中国中医药出版社,2008:
[7] 中国畜牧兽医学会.中国兽医针灸学[M].北京:农业出版社,1984.
[8] 陈强威,唐春萍,王秋新,等.蟾酥注射液对脂多糖发热模型大鼠解热及体外抑菌作用的研究[J].中药药理与临床,2018,34(2):58-62.
[9] 朱洋波,韩永生,范西真.热速清颗粒联合阿莫西林治疗外感发热的疗效及对炎性反应的影响[J].世界中医药,2018,13(10):2421-2424.
[10] 张翠平,邵长周.炎症细胞因子与肺部感染关系的研究进展[J].中国临床医学,2018,25(1):132-136.
[11] 李志祥.开颅术后发热患者血清及脑脊液中降钙素原、C-反应蛋白和一氧化氮的水平及临床意义[J].河北医药,2018,40(5):752-754,758.
[12] 王靳琎,杨晓梅,苏丽万,等.海带多糖对脂多糖致慢性炎症大鼠血管内皮的保护作用[J].中国药理学通报,2018,34(5):651-656.
[13] 袁小媚,雷寒,夏勇.HSP70抑制剂PFT对干扰素诱导的iNOS 表达的研究[J].中国药理学通报,2016,32(3):390-394.
[14] 韦卓,袁冰,马琴,等.玻璃酸钠对兔骨关节炎早期iNOS炎症途径及炎性细胞因子表达影响的实验研究[J].生物骨科材料与临床研究,2017,14(4):15-19,23,82.
[15] 王晶,丁少帧,胡翠.同型半胱氨酸对实验性结肠炎模型中COX-2及5-LOX表达的影响[J].中国药理学通报,2017,33(11):1627-1628.
[16] 舒文娜.针刺、艾灸对慢性萎缩性胃炎大鼠胃黏膜COX-2及炎症细胞因子表达影响的对比研究[D].长沙:湖南中医药大学,2018.
[17] 毛近隆,李晓宇,孙蓉.环氧合酶(COX-2)抑制剂提高心血管安全性的抗炎机制探讨[J].中国中药杂志,2014,39(20):4054-4059.
[18] Fitzgerald GA,Patrono C.The coxibs,selective inhibitors of cyclooxygenase -2[J].New Eng J Med,2001,345(6):433-442.
[19] Cannon CP,Cannon PJ.Physiology.COX-2 inhibitors and cardiovascular risk[J].Science,2012,336(6087):1386.
[20] Kerr DJ,Dunn JA,Langman MJ,et al.Rofecoxib and cardiovascular adverse events in adjuvant treatment of colorectal cancer[J].N Engl J Med,2007,357(4):360.
[21] 王富春.刺法灸法学[M].上海:上海科学技术出版社,2009:87.
[22] 张立文.大椎穴刺血拔罐疗法治疗小儿风热型外感发热的临床研究[D].广州:广州中医药大学,2013.
[23] 郑亚超,孟向文,马佳佳,等.近10年拔罐疗法在治未病中应用现状[J].辽宁中医药大学学报,2018,20(5):98-100.
[24] 李丹丹,孟向文,刘华朋,等.拔罐疗法作用机理研究概述[J].辽宁中医杂志,2014,41 (11):2506-2508.
[2] 李莉,杨保林,王兰.刺血拔罐退热法治疗急诊内科发热患者的临床研究[J].现代中医临床,2017,24(1):12-14.
[3] 方成华,段亚平,曾贤,等.大椎穴位刺血拔罐治疗风热感冒发热疗效观察[J].亚太传统医药,2016,12(7):124-125.
[4] 魏伟,吴希美,李元建.药理实验方法学[M].北京:人民卫生出版社,2010:68-73.
[5] 杨果杰,陈秀莲,邵萍,等.内毒素法兔发热动物模型及其标准化的研究[J].中国实验动物学报,2001(4):57-60.
[6] 郭义.实验针灸学[M].北京:中国中医药出版社,2008:
[7] 中国畜牧兽医学会.中国兽医针灸学[M].北京:农业出版社,1984.
[8] 陈强威,唐春萍,王秋新,等.蟾酥注射液对脂多糖发热模型大鼠解热及体外抑菌作用的研究[J].中药药理与临床,2018,34(2):58-62.
[9] 朱洋波,韩永生,范西真.热速清颗粒联合阿莫西林治疗外感发热的疗效及对炎性反应的影响[J].世界中医药,2018,13(10):2421-2424.
[10] 张翠平,邵长周.炎症细胞因子与肺部感染关系的研究进展[J].中国临床医学,2018,25(1):132-136.
[11] 李志祥.开颅术后发热患者血清及脑脊液中降钙素原、C-反应蛋白和一氧化氮的水平及临床意义[J].河北医药,2018,40(5):752-754,758.
[12] 王靳琎,杨晓梅,苏丽万,等.海带多糖对脂多糖致慢性炎症大鼠血管内皮的保护作用[J].中国药理学通报,2018,34(5):651-656.
[13] 袁小媚,雷寒,夏勇.HSP70抑制剂PFT对干扰素诱导的iNOS 表达的研究[J].中国药理学通报,2016,32(3):390-394.
[14] 韦卓,袁冰,马琴,等.玻璃酸钠对兔骨关节炎早期iNOS炎症途径及炎性细胞因子表达影响的实验研究[J].生物骨科材料与临床研究,2017,14(4):15-19,23,82.
[15] 王晶,丁少帧,胡翠.同型半胱氨酸对实验性结肠炎模型中COX-2及5-LOX表达的影响[J].中国药理学通报,2017,33(11):1627-1628.
[16] 舒文娜.针刺、艾灸对慢性萎缩性胃炎大鼠胃黏膜COX-2及炎症细胞因子表达影响的对比研究[D].长沙:湖南中医药大学,2018.
[17] 毛近隆,李晓宇,孙蓉.环氧合酶(COX-2)抑制剂提高心血管安全性的抗炎机制探讨[J].中国中药杂志,2014,39(20):4054-4059.
[18] Fitzgerald GA,Patrono C.The coxibs,selective inhibitors of cyclooxygenase -2[J].New Eng J Med,2001,345(6):433-442.
[19] Cannon CP,Cannon PJ.Physiology.COX-2 inhibitors and cardiovascular risk[J].Science,2012,336(6087):1386.
[20] Kerr DJ,Dunn JA,Langman MJ,et al.Rofecoxib and cardiovascular adverse events in adjuvant treatment of colorectal cancer[J].N Engl J Med,2007,357(4):360.
[21] 王富春.刺法灸法学[M].上海:上海科学技术出版社,2009:87.
[22] 张立文.大椎穴刺血拔罐疗法治疗小儿风热型外感发热的临床研究[D].广州:广州中医药大学,2013.
[23] 郑亚超,孟向文,马佳佳,等.近10年拔罐疗法在治未病中应用现状[J].辽宁中医药大学学报,2018,20(5):98-100.
[24] 李丹丹,孟向文,刘华朋,等.拔罐疗法作用机理研究概述[J].辽宁中医杂志,2014,41 (11):2506-2508.