穴位注射联合中药贴敷对支气管哮喘大鼠的作用机制研究

发布时间：2018-05-06 08:33

本文选题：支气管哮喘 + 穴位注射　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:观察穴位注射草分枝杆菌与中药穴位贴敷对支气管哮喘大鼠血清免疫球蛋白E(Ig E)水平、肺组织白介素10(IL-10)、白介素17(IL-17)、细胞粘附因子1(ICAM-1)表达的影响,探讨穴位注射、穴位贴敷中医外治法对支气管哮喘的作用机制,为中医外治法治疗支气管哮喘的临床应用提供有力依据及参考。方法:SPF级Wistar雄性大鼠60只适应性喂养一周,随机分为对照组、模型组、草分枝杆菌穴位注射组(穴注组)、中药贴敷组(贴敷组)、和穴位注射联合中药贴敷组(穴注+贴敷组),每组12只。复制卵蛋白(OVA)大鼠哮喘模型,分为致敏阶段与激发阶段进行。除对照组,其余大鼠分别于第1天、第8天腹腔注射OVA、氢氧化铝混悬溶液(10%的OVA及10%的氢氧化铝)1ml致敏,对照组注射同量的生理盐水。第15天,各组均给予密闭雾化,大鼠置于密闭雾化箱中,以2%OVA溶液超声雾化激发,对照组以生理盐水代替,每天一次,每次30min;相比对照组观察雾化时各组大鼠行为变化,出现烦躁、搔鼻、咳嗽、呼吸频率加快且幅度加大、明显的腹式呼吸、点头呼吸,甚至四肢瘫软俯伏不动、大小便失禁等现象视为激发成功。连续激发7天,后改为每周激发2次。连续激发哮喘发作7天后,对照组正常喂养,其余各组进行治疗。取穴大椎、双定喘、双肺俞、双脾俞、双肾俞,穴注组行草分枝杆菌穴位注射,五组穴位每日轮流取穴注射;贴敷组行贴敷2号穴位贴敷,每贴6h;穴注+贴敷组穴位注射后随即进行中药贴敷6h;对照组取穴同治疗组,以同量的生理盐水及空贴代替。隔天操作一次,3次为一疗程,共进行四个疗程。肺组织HE染色观察气道炎症情况,ELISA法检测血清Ig E、肺组织匀浆IL-10及IL-17的含量;免疫组织化学(SABC)法检测肺组织ICAM-1的表达。结果:1各组大鼠行为学表现:对照组大鼠呼吸平稳,雾化时可见蜷缩抱团休息现象,无喘促等特殊表现,雾化后精神、饮食正常;模型组大鼠雾化激发时不同程度出现呼吸频率快,张口呼吸、点头呼吸,喷嚏,挠鼻,烦躁不安,腹肌抽动,口鼻及爪甲处紫绀,甚至行动缓慢,瘫软,大小便失禁等,随激发次数增加而症状逐步加重,其中由于哮喘发作死亡1只,数次激发后大鼠活动、饮食明显减少,且有毛色无华,竖毛等表现,大鼠模型具备哮喘特征。穴注组、贴敷组、穴注+贴敷组上述症状表现不明显,可见烦躁不安、点头样呼吸、搔鼻,但口唇爪甲紫绀较轻,个别可见伏地不动,大小便失禁,但随激发次数减少及治疗次数增多,激发表现逐渐好转,穴注+贴敷组好转较明显,治疗组雾化后饮食活动未见明显变化。2 HE染色:对照组:细支气管上皮完整,形态规则,支气管管壁未见增生,肺泡形态规则,肺泡壁规整,未见增生及炎性浸润。模型组:气管周围及肺组织大量炎性细胞浸润,支气管狭窄变形,腔内可见脱落上皮细胞,黏膜皱襞增多,黏膜水肿,肺泡壁及肺间隔明显增厚、充血,肺泡腔明显变小甚至消失。穴注组:与模型组相比,炎性细胞浸润减轻,支气管形态稍有狭窄等改变,肺泡壁及肺间隔增厚减轻,肺泡形态欠规则。贴敷组:与模型组相比,细支气管形态较规则,腔内未见明显上皮细胞脱落,管壁有炎性细胞浸润,平滑肌有所增厚,肺泡壁及肺间隔可见增厚减轻,肺泡形态欠规则。穴注+贴敷组:细支气管形态较规则,管周可见炎性细胞,管壁轻度增生,黏膜较正常,肺泡腔形态尚可,个别肺泡壁有所增厚及炎性细胞增多。3 Ig E含量测定结果:模型组血清Ig E明显高于对照组,有显著性差异(P0.01);穴注组、贴敷组、穴注+贴敷组三组大鼠的Ig E含量均低于模型组,有显著差异(P0.01);穴注+贴敷组Ig E含量较穴注组、贴敷组低(P0.05);穴注组与贴敷组Ig E含量无明显差异(P0.05)。4 IL-10含量测定结果:模型组肺组织IL-10含量明显低于对照组,有显著性差异(P0.01),穴注组、贴敷组、穴注+贴敷组三组大鼠的IL-10含量均高于模型组,差异显著(P0.01),穴注+贴敷组IL-10含量较穴注组、贴敷组高,差异有统计学意义(P0.05),穴注组与贴敷组IL-10含量无明显差异(P0.05)。5 IL-17含量测定结果:模型组肺组织IL-17含量明显高于对照组,有显著性差异(P0.01);与模型组相比,穴注组、贴敷组、穴注+贴敷组三组大鼠的IL-17含量减低,有显著差异(P0.01);穴注+贴敷组IL-17含量较穴注组、贴敷组低,差异有统计学意义(P0.05),穴注组与贴敷组IL-17含量无明显差异(P0.05)。6 ICAM-1表达:免疫组化可见,对照组支气管周围可见少量阳性细胞;模型组支气管周围以及支气管分泌物及肺间质、肺泡壁棕黄色颗粒表达较多;穴注组和贴敷组支气管周围、肺间质肺泡壁棕黄色颗粒较模型组少;穴注+贴敷组支气管周围、肺间质、肺泡壁棕黄色阳性细胞表达不明显。各组平均光密度值比较:模型组肺组织ICAM-1表达明显高于对照组,有显著性差异(P0.01),穴注组、贴敷组、穴注+贴敷组三组大鼠的ICAM-1表达均低于模型组,差异显著(P0.01),穴注+贴敷组ICAM-1表达较穴注组、贴敷组低,差异有统计学意义(P0.05),穴注组与贴敷组ICAM-1表达无明显差异(P0.05)。结论:1根据模型组大鼠症状表现以及各项检测指标,HE染色提示,支气管哮喘大鼠造模成功。2支气管哮喘大鼠血清Ig E的含量增多,肺组织IL-10含量减少、IL-17含量增多,ICAM-1在肺组织的表达增多,并与大鼠激发哮喘症状以及肺脏病理表现变化一致,提示IL-10、IL-17、Ig E、ICAM-1均参与了哮喘的发病。3穴位注射草分枝杆菌、中药贴敷可能是通过降低哮喘大鼠Ig E水平,提高哮喘大鼠肺组织中IL-10表达,降低IL-17表达,降低ICAM-1的表达,来减轻气道炎症的发生,调节免疫平衡,两种方法综合应用较单独应用效果更佳。
[Abstract]:Objective: To observe the effect of acupoint injection of Mycobacterium Mycobacterium and traditional Chinese medicine acupoint application on serum immunoglobulin E (Ig E) level, interleukin 10 (IL-10), interleukin 17 (IL-17), and cell adhesion factor 1 (ICAM-1) expression in the lung tissue, and explore the mechanism of acupuncture point injection and acupoint application of traditional Chinese medicine on bronchial asthma. The clinical application of external treatment for bronchial asthma provides a strong basis and reference. Methods: 60 Wistar male rats of grade SPF were fed for one week. They were randomly divided into control group, model group, acupoint injection group (point injection group), Chinese medicine sticking group (Acupoint Application Group), and Acupoint Injection group (acupoint + adherent group), 12 rats in each group. The asthma model of replicating ovalbumin (OVA) rats was divided into the sensitization stage and the excitation stage. In addition to the control group, the other rats were injected OVA in first days, eighth days in the abdominal cavity, and the aluminum hydroxide suspension solution (10% OVA and 10% aluminum hydroxide) were sensitized by 1ml, and the control group was injected with the same amount of raw saline. All groups were treated with closed atomization for fifteenth days. Rats were placed in close density. In the closed nebulization box, the control group was stimulated by ultrasonic atomization of 2%OVA solution, and the control group was replaced by physiological saline, once a day, each time, 30min each time. Compared with the control group, the behavior changes of rats in each group were observed, such as irritability, scratching nose and coughing, the respiratory rate was accelerated and the amplitude was increased, the obvious abdominal breathing, nodding breathing, even the limbs were paralyzed and inactive, and the size and stool were lost. The forbidden phenomenon was regarded as the excitation success. 7 days of continuous stimulation, then 2 times a week. After 7 days of continuous stimulation of the asthma attack, the control group was fed with normal feeding, the rest of the other groups were treated. Taking the acupoint DZX, double asthma, double lung Yu, double Shenshu, double Shenshu, acupoint injection group acupoint injection of Mycobacterium tumefaciens, the five acupoints were taken every day to take the acupoint injection; the sticking group was put on the patch. 2 acupoint application was applied, each 6h was attached, and 6h was applied after acupoint injection in the acupoint + application group. The control group was taken with the same treatment group, with the same amount of physiological saline and empty paste. 3 times for a course of treatment and four courses of treatment. HE staining of lung tissue was used to observe the airway inflammation, the ELISA method was used to detect the serum Ig E and IL-1 in the lung tissue IL-1. 0 and IL-17 content; immunohistochemistry (SABC) method to detect the expression of ICAM-1 in the lung tissue. Results: 1 the behavior of rats in each group: the control group of rats breathing is stable, the nebulization of the crouching band rest phenomenon, no asthma and other special manifestations, after atomization, the diet is normal; model group rats at different levels of atomization and stimulation of respiratory frequency faster, Breathing, nod, sneezing, nose, irritability, irritability, abdominal muscles twitch, cyanosis in the mouth and the claws, even slow movement, limp, incontinence and so on, the symptoms gradually aggravated with the increase of the number of excuses, 1 of them died of asthma attack, after several times of stimulation, the diet decreased obviously, and the hair color was not Hua, erect hair and so on, and the hair color was not Hua, erect hair and so on. The rat model had the characteristics of asthma. The symptoms of the acupoint injection group, the sticking group and the acupoint injection + cladding group were not obvious, and the symptoms of irritability, nod like breathing, scratching nose were seen, but the cyanosis of the lip and claw was lighter, and the incontinence and incontinence could be seen individually, but the frequency of stimulation and the number of treatment increased, the performance of the stimulation gradually improved, and the acupoint injection + cladding group was better than that. Obviously, there was no obvious change of.2 HE staining in the diet after atomization in the treatment group: the control group: the bronchial epithelium was intact, the shape rule, the bronchial tube wall no hyperplasia, the rules of alveolus shape, the alveolar wall regularity, no hyperplasia and inflammatory infiltration. Exfoliated epithelial cells, mucous plica increased, mucous membrane edema, alveolar wall and pulmonary septum thickening obviously, congestion, alveolar cavity obviously smaller or even disappeared. Group: compared with the model group, inflammatory cells infiltration, bronchi shape slightly narrowing and other changes, alveolar wall and pulmonary septum reduction, alveolar morphology under rules. Patch group: with model group phase The morphology of bronchioles was more regular. There was no obvious epithelial cells falling off in the cavity. The wall of the tube was inflammatory cell infiltration, the smooth muscle was thickened, the alveolar wall and the pulmonary septum were thickening and lessened. The alveolar morphology was under rules. The shape of the bronchioles was more regular. The bronchioles were more regular, the tube wall was slightly proliferated, the mucosa was more normal, and the alveolar cavity was more normal. .3 Ig E content of the serum Ig E in the model group was significantly higher than that of the control group (P0.01). The content of Ig E in three groups of rats in the acupoint injection group, the sticking group and the acupoint injection + cladding group were lower than those in the model group (P0.01), and the Ig E content in the acupoint injection + cladding group was compared with the acupoint injection group. Group low (P0.05); there was no significant difference in the content of Ig E in the acupoint group and the application group (P0.05).4 IL-10 content determination results: the IL-10 content in the lung tissue of the model group was significantly lower than that of the control group (P0.01). The IL-10 content of the three groups of rats in the acupoint injection group, the sticking group and the acupoint injection + cladding group were all higher than the model group, the difference was significant (P0.01), and the IL-10 content of the acupoint injection + application group was significantly different. Compared with the acupoint injection group, the difference was statistically significant (P0.05). There was no significant difference in the content of IL-10 in the acupoint group and the application group (P0.05).5 IL-17 content determination results: the IL-17 content in the lung tissue of the model group was significantly higher than that of the control group (P0.01); the IL-17 content in three groups of rats was compared with the model group, the acupoint injection group, the acupoint injection group and the acupoint injection group. There was a significant difference (P0.01); the content of IL-17 in the acupoint + cladding group was lower than that in the acupoint group, and the difference was statistically significant (P0.05). There was no significant difference (P0.05).6 ICAM-1 expression in the acupoint group and the application group (P0.05).6 ICAM-1 expression: the immunization group was visible, the control group showed a small number of positive cells around the bronchus, and the peripheral bronchus and the bronchial secretion in the model group. The expression of brown and yellow granules in the alveolar wall of the material and pulmonary interstitium was more than that of the model group. The expression of brown yellow granules in the pulmonary interstitial lung wall was less than that in the model group, and the expression of the brown yellow positive cells of the pulmonary interstitial and alveolar wall was not obvious. The average density of the lung tissue in the model group was significantly higher than that in the model group. The ICAM-1 expression in the model group was significantly higher. In the control group, there were significant differences (P0.01). The expression of ICAM-1 in the three groups of rats in the acupoint injection group, the acupoint injection + cladding group were lower than the model group, the difference was significant (P0.01), the ICAM-1 expression in the acupoint injection + cladding group was lower than the acupoint group, and the difference was statistically significant (P0.05). There was no significant difference in the expression of ICAM-1 between the acupoint injection group and the application group (P0.05). Conclusion: 1 basis (P0.05). HE staining showed that the content of Ig E in serum of.2 bronchial asthma rats increased, IL-10 content in lung tissue decreased, IL-17 content increased, and the expression of ICAM-1 increased in lung tissue, and was consistent with the symptoms of irritable asthma and lung pathological changes in rats. IL-10, IL-17, Ig E, ICAM-1 all participated in the.3 point injection of Mycobacterium phlebius in the pathogenesis of asthma. The application of traditional Chinese medicine may be to reduce the expression of IL-10 in the lung tissue of asthmatic rats, reduce the expression of IL-17, reduce ICAM-1 expression, reduce the incidence of airway inflammation and regulate the immune balance. Two methods are used synthetically. The effect of individual application is better.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

【参考文献】