黄芩汤提取物加减滴鼻液对实验性变应性鼻炎豚鼠模型的影响

【作者】 谢铮；

【导师】 傅延龄；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2010， 博士

【摘要】 变应性鼻炎(Allergic Rhinitis, AR),又称过敏性鼻炎,是特应性个体接触致敏原后由IgE介导的介质释放为开端的、有多种免疫活性细胞和细胞因子等参与的鼻黏膜慢性炎症反应性疾病,临床主要表现为打喷嚏、流涕、鼻塞和鼻痒。变应性鼻炎已成为全球性普遍存在的疾患,尤其是近20年其患病率明显呈增加趋势,给患者的工作、学习和生活带来了严重影响,并且加重了个人和整个社会的经济负担,还可诱发支气管哮喘、鼻窦炎、鼻息肉、中耳炎等疾病,或与变应性结膜炎同时发生。目前西医在临床上治疗AR主要选用5种类型药物,即抗组胺药、减充血药、抗胆碱药、肥大细胞稳定剂和皮质类固醇药,存在停药后复发率高,长期维持治疗的副作用大,给患者带来沉重经济负担等问题。根据AR的临床表现,当属于中医的鼻鼽、鼽嚏、鼽水、鼽喷、鼽、嚏等范畴。近年来,中医对本病研究的报道逐年增加,各医家运用传统中医理论和中医方法治疗本病,积累了丰富的经验,取得了较为满意的效果,且副作用较少,复发率低,展现了中医药治疗变应性鼻炎的较大优势和广阔前景。目的1将历代有关AR资料作系统整理,揭示古代中医在AR诊治方面的规律和成就,为在临床上更好的应用中医理论和方法治疗AR提供理论和文献依据；2阐释AR与肺和胃肠之热的关系,探讨“以清肠以泻肺”的治法为指导思路治疗AR的科学性与可行性,为变应性鼻炎的治疗提供了新的思路；3建立豚鼠AR模型,应用现代科学技术的研究方法,多层次、多角度探讨黄芩汤提取物加减滴鼻液对实验性AR豚鼠模型的疗效及作用机理,为临床防治AR提供实验依据。方法1理论研究查阅古代中医文献,系统总结历代医家对鼻鼽、鼽嚏、鼽水、鼽喷、鼽、嚏诸病的认识,探讨其诊治规律。探讨“以清肠以泻肺”的治法为指导思路治疗AR的科学性与可行性,并进一步阐释黄芩汤治疗AR的理论依据。2实验研究2.1 AR豚鼠模型的建立与评价健康清洁级豚鼠60只,采取卵白蛋白(OVA)全身致敏加局部致敏的方法复制豚鼠AR模型,对实验组的豚鼠进行腹腔注射：注射液为卵白蛋白(OVA) 30mg,氢氧化铝Al(OH)330mg,将其共同溶解于1ml生理盐水中形成混合悬液,二日一次,共7次；雾化吸入0.5%的OVA生理盐水溶液,连续5天,每天1次；间隔3天后,用2%OVA生理盐水液进行滴鼻,每日1次,连续4天。正常组以生理盐水代替OVA溶液。以行为症状学观察及光镜下病理形态学观察评价模型。2.2黄芩汤提取物加减滴鼻液对实验性AR的疗效观察和作用机制研究健康清洁级豚鼠60只,适应性饲养一周后,按体重随机分成A组空白对照组,B组AR模型组,C组中药滴鼻剂低频率组,D组中药滴鼻剂中频率组,E组中药滴鼻剂高频率组,F组富马酸酮替芬滴鼻液阳性对照组,每组10只。A、B二组分别给予生理盐水10μl滴鼻,2h一次,给药3周；C组给予中药滴鼻剂10μl滴鼻,6h一次；D组给予中药滴鼻剂10μl滴鼻,4h一次；E组给予中药滴鼻剂10μl滴鼻,2h一次,均给药3周；F组给与富马酸酮替芬滴鼻液10μl滴鼻,2h一次,连续给药3周。观察豚鼠的活动表现、摄食量、进水量及精神状态。末次给药后记录5min内动物搔鼻、喷嚏次数,1小时后麻醉,处死所有动物,断头后从上腭进入鼻腔取双侧鼻粘膜组织,用冰生理盐水冲洗,固定的选取左侧,固定于4%中性多聚甲醛溶液固定48h,常规组织脱水、透明、浸蜡、包埋、切片,石蜡切片用于HE染色；另一侧置液氮冻存,以备提取SP-R mRNA。并抽取腹主动脉动脉血3ml,用生化法测定各组豚鼠血清中Histamine, IL-4, IL-5, TNF-α的含量；RT-PCR方法测定鼻粘膜组织中SP-RmRNA的表达。结果1理论研究在历代相关文献研究的基础上,发现AR的病位在鼻,与肺和胃肠关系密切,基本病机为“肺和胃肠有热,上蒸于鼻”。随着人们生活水平的提高,物质生活的极大丰富,生活饮食结构的调整,今人多食肥甘厚味,辛辣烟酒之品,呆脾滞胃,蕴生湿热,湿热内生,循经上蒸鼻窍而发病；而今人多骨弱而皮肤盛,易受外邪侵袭,外邪化火,上犯鼻窍亦致此病。治疗当以“清肺和胃肠之热”为基本原则,随证加减。黄芩汤方中以黄芩之苦寒清利湿热,芍药敛阴合营,甘草与芍药相伍取芍药苷草汤之意,大枣之甘以调中,并与甘草相调益气和中,四味药并用,正是清胃肠湿热的有效方剂,肺与大肠相表里,黄芩汤可以通过泻肠清肺的方法治疗AR,而且根据现代研究黄芩汤各单位药的有效成分具有很好的抗过敏作用,这些都是其治疗AR的理论依据。2实验研究2.1模型评价通过对OVA全身致敏和局部致敏相结合方法复制的豚鼠AR模型的观察,发现各模型组喷嚏和搔鼻次数与正常组相比显著增加(P<0.05),光镜下出现变应性鼻炎的特征性病理表现。卵白蛋白逐步致敏造成的豚鼠的变应性鼻炎的模型,方法简单易行,模型生存率高,可达到90%,病变率100%,符合变应性鼻炎的典型临床特征,因此是变应性鼻炎的较好的动物模型的制备方法。2.2黄芩汤提取物加减对AR豚鼠模型的治疗作用与模型组相比,各治疗组喷嚏和搔鼻次数有明显降低,黄芩汤提取物加减滴鼻液高、中、低频率治疗组效果明显(均为P<0.05),三组均能明显改善豚鼠模型鼻粘膜的病理组织学损伤,减少炎细胞计数,光镜下炎细胞评分与模型组相比明显降低(均为P<0.05)。2.3黄芩汤提取物加减对AR豚鼠动物模型血清中Histamine的影响AR模型组豚鼠血清中Histamine含量与正常组相比明显升高(P<0.001)。黄芩汤各频率组均能显著降低豚鼠血清中Histamine含量(P<0.001),其中高、中频率组优于低频率组,以中频率组疗效最佳。2.4黄芩汤提取物加减滴鼻液对AR豚鼠动物模型血清中IL-4和IL-5的影响AR模型组豚鼠血清中IL-4、IL-5含量与正常组相比明显升高(P<0.001,P<0.05)。黄芩汤各频率组均能显著降低豚鼠血清中IL-4、IL-5含量(P<0.001,P<0.01),在降低IL-5含量方面,黄芩汤各频率组优于西药组。2.5黄芩汤提取物加减滴鼻液对AR豚鼠动物模型血清中TNF-α的影响AR模型组豚鼠血清中TNF-a含量与正常组相比明显升高(P<0.001)。黄芩汤各频率组均能显著降低豚鼠血清中TNF-α含量(P<0.001),其中高、中频率组优于低频率组,以中频率组疗效最佳。2.6黄芩汤提取物加减滴鼻液对AR豚鼠动物模型鼻粘膜SP-R mRNA表达的影响AR模型组豚鼠鼻粘膜SP-R mRNA的表达量与正常组相比明显升高(P<0.001)。黄芩汤各频率组均能显著降低鼻粘膜SP-R mRNA的表达量(P<0.001),其中高、中频率组优于低频率组,以中频率组疗效最佳。结论1变应性鼻炎的病机特点为热,清肺和胃肠之热治疗本病科学可行；2卵白蛋白致敏的造模方法是研究AR较为理想的动物造模方法；3 AR发病时,鼻粘膜组织可观察到明显病理形态学改变,主要是鼻粘膜上皮细胞的破坏,血管的充血,腺体水肿,嗜酸性粒细胞浸润等；4黄芩汤提取物加减治疗变应性鼻炎的机制可能为：4.1显著降低血清中Histamine的水平；4.2显著降低血清中IL-4、IL-5的水平；4.3显著降低血清中TNF-α的水平；4.4显著抑制鼻粘膜中SP-R的基因表达。综上所述,黄芩汤提取物加减滴鼻液可以有效治疗实验性AR,可能是通过调节免疫调节机制和神经调节机制来达到其治疗效果的；从中医角度来看,黄芩汤是通过泻肠清肺治疗AR的,其中高、中、低频率组均有明显效果,但综合比较,以中频率组效果最佳。更多还原

【Abstract】 Allergic Rhinitis (abbreviated as AR) refers to chronic inflammation in nasal mucosa induced by allergen. And this kind of chronic inflammation is mediated by IgE and participated by immunocyte and cytokines. The disease often causes many symptoms such as rhinostegnosis, rhinocnesmus, sneezing, and mucus in the nose and so on. AR now is a common disease in the world and recently the prevalence rate of AR is increasing year by year. And allergic asthma, sinusitis, rhinopolypus and tympanitis could be related with severe AR, which has caused serious effect in daily life, study and work to us. In Western Medicine, antihistaminics, decongestant drug, anticholinergics, mast cell stabilizer and corticosteroids are applied to the treatments of AR. However, there are many problems in these drugs such as the high relapse rate after treatments, side effects and expensive costs because of long-term treatments and so on. According to their clinical symptoms, AR could belong to "bi qiu", "qiu ti", "qiu shui", "qiu pen", "qiu", "pen" in Traditional Chinese Medicine (abbreviated as TCM). Recently, TCM researches of AR are increasing significantly. TCM treatments have accumulated rich experiences and achieved good effects, less side effects and lower recurrence under the supervision of TCM theories. TCM therapy has potential advantages and bright future in the treatment of AR, which has laid a good groundwork for the further study and the mechanism of TCM.Objective1 Putting the ancient literatures about AR in order, revealing the law and achievements in diagnosis and treatment of TCM, providing evidence for better treatment of TCM in AR.2 Discussing the relationship between AR and "Heat of lung and stomach, intestine" and feasibility of applying "clearing the heat of intestine and lung" to treat AR.3 Copying AR model of guinea pig, and applying the modern scientific and technological research methods to study the effect and mechanism of HQT modified extract drops on experimental guinea pig AR model, in order to provide experimental basis for prevention and treatment of AR.Method1 Theoretical StudyAccess to the ancient Chinese medical literatures, the discuss of "bi qiu", "qiu ti", "qiu shui", "qiu pen", "qiu", "pen" was put in order and the law of their diagnosis and treatment were revealed. And the feasibility of applying "clearing the heat of intestine and lung" to treat AR and explain the theoretical basis for the AR was well considered and discussed. 2 Experimental Study2.1 The copy and evaluate the guinea pig models of AR60 healthy guinea pigs were used to copy the guinea pigs models of AR though ovalbumin (OVA) sensitization. The experimental groups of guinea pigs were injected by a mixed suspension:ovalbumin (OVA) 30mg, Al(OH)330mg, and lml saline, one time two days for 7 times; inhalation of 0.5% OVA saline solution for 5 consecutive days, one time a day; 3 days later, nasaldrops with 2% OVA saline solution, one time a day for 4 days. Normal group used saline instead of OVA. The models were observed and evaluated through the changes of behavioral symptoms and of nasal mucosa (both macroscopic and microscopic).2.2 Pharmacodynamics Study and Mechanism Study of HQT Modified extract DropsAfter adaptive feeding one week,60 healthy guinea pigs were randomly divided into control group A, AR model group B, HQT Modified extract Drops low frequency group C, HQT Modified extract Drops middle frequency group D, HQT Modified extract Drops high frequency group E, ketotifen fumarate Drops positive control group F,10 guinea pigs in each group. A, B two groups were given intranasal 10μl saline, one time 2h; C group were given intranasal 10μl HQT Modified extract Drops, one time 6h; D group were given intranasal 10μl HQT Modified extract Drops, one time 4h; E group were given intranasal 10μl HQT Modified extract Drops, one time 2h, F group were given intranasal 10μl ketotifen fumarate, one time 2h. All the groups were administered for 3 weeks The activity, food and water intake, and spirit of guinea pigs were obseverd. The count of scratch nose, sneezing after administration in 5 min was recorded. After anaesthetized, all animals were sacrificed 24 hours after last administration. Peeling nasal mucosa off from nasal cavities and select the left nasal mucosa fixed in 4% neutral paraformaldehyde solution for 48h, and dehydration, clearing, paraffin, embedded, sliced, paraffin sections for HE staining. The other side of the nasal mucosa was put into liquid nitrogen to freeze and prepare for extraction of SP-R mRNA. The blood sample was drawn from abdominal aorta to be detected Histamine,IL-4, IL-5, TNF-a levels with a biochemical method. RT-PCR method was detected SP-R mRNA expression of nasal tissue.Results1 Theoretical ResearchIn the study of ancient literatures about AR, the location of the disease was founded in the nose, and "lung" and "stomach, intestine" are closely related to AR. The basic mechanism is that the heat of lung and stomach intestine steam up to the nose. With the improvement of living standard, the great wealth of material life, and life diet adjustment, more and more people prefer to have fatty and spicy food, tobacco and alcohol products, and this could make spleen and stomach stagnation, so heat and dampness produce. Heat and wet through the meridians could steam up to the nose lead to AR. So "clearing the lungs and gastrointestinal heat" as the basic principles. HQT treatment prescription could clear dampness and heat. And according to modern research, active ingredients of herbs in HQT prescription have a good anti-allergic effect. These could be the theoretical basis of its treatment of AR2 Experimental Research2.1 Model EvaluationThough the observation of OVA sensitization method to copy guinea pig AR model, the number of sneezing and nasal scratching compared with the control group was significantly increased (P<0.05); and allergic characteristic pathology syptom of rhinitis was appeared under the light microscope. OVA progressively sensitized guinea pigs lead to allergic rhinitis model was easy to copy, and model survival rate can reach 90%, and caused disease rate was 100%. Therefore this model should be the good rhinitis animal model.2.2 The effect of modified HQT extract drops on guinea pigs AR model.Compared with model group, the number of sneezing and nasal scratching in the treatment groups were significantly decreased (P<0.05). Modified HQT extract drops high, medium and low frequency groups can improve model of pathological tissue damage in nasal mucosa, and reduce inflammatory cell. Inflammatory cell score in light microscopy was significantly decreased compared with the model group (P<0.05)2.3 The influence of modified HQT extract drops on content in serum Histamine of the guinea pig animal AR modelThe content of serum Histamine in AR guinea pig model group was significantly increased compared with the normal group (P<0.001). Modified HQT extract drops (high, medium and low frequency) can significantly reduce serum Histamine content in guinea pig (P<0.001), in which high and middle frequency group was better than low frequency group. Middle frequency group had best effect in Histamine reduction.2.4 The influence of modified HQT extract drops on content in serum IL-4, IL-5 of the guinea pig animal AR modelThe content of serum IL-4, IL-5 in AR guinea pig model group was significantly increased compared with the normal group (P<0.001, P<0.01). Modified HQT extract drops (high, medium and low frequency) can significantly reduce serum Histamine content in guinea pig (P<0.001, P<0.01), which were better than ketotifen fumarate Drops positive control group.2.5 The influence of modified HQT extract drops on content in serum TNF-a of the guinea pig animal AR modelThe content of serum TNF-a in AR guinea pig model group was significantly increased compared with the normal group (P<0.001). Modified HQT extract drops (high, medium and low frequency) can significantly reduce serum TNF-a content in guinea pig (P<0.001), in which high and middle frequency group was better than low frequency group. Middle frequency group had best effect in TNF-a reduction.2.6 The influence of modified HQT extract drops on SP-R mRNA expression in nasal mucosa of the guinea pig animal AR modelThe expression of s SP-R mRNA in nasal mucosa of AR guinea pig model group was significantly increased compared with the normal group (P<0.001). Modified HQT extract drops (high, medium and low frequency) can significantly reduce SP-R mRNA expression in nasal mucosa (P<0.001), in which high and middle frequency group was better than low frequency group. Middle frequency group had best effect in SP-R mRNA expression reduction in nasal mucosa.Conclusion1 Mechanism of allergic rhinitis is characterized as heat, and clearing the lungs and gastrointestinal heat is the way to treat AR.2 OVA sensitization is an ideal method to establish the RE model.3 Significant morphological changes, such as the destruction of nasal epithelial cells, vascular congestion, glandular swelling, and infiltration of eosinophils could be observed in the nasal mucosa of AR animals.4 The followings could be the mechanism of modified HQT extract drops treating AR.4.1 Significantly decreasing the content of Histamine in serum.4.2 Significantly decreasing the content of IL-4、IL-5 in serum.4.3 Significantly decreasing the content of TNF-a in serum.4.4 Significantly decreasing the content of SP-R mRNA expression in nasal mucosa.In summary, modified HQT extract drops treat AR probably by regulating the immune and neural regulatory mechanisms to achieve the therapeutic effect; from TCM point of view, HQT treat AR though clearing the lungs and gastrointestinal heat. Modified HQT extract drops (high, medium and low frequency) have a significant effect on AR. Middle frequency group has best affect.更多还原