蒿芩清胆汤对病毒性呼吸道感染湿热证的免疫炎症机制研究
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摘要
病毒性呼吸道感染是临床常见病、多发病,几乎所有人群均易感。岭南地区,雨水充沛,气候炎热,病毒性呼吸道感染从中医辨证具有易化热夹湿的特点,湿热证侯表现突出,清热祛湿类方药应用广泛,且多获捷效。蒿芩清胆汤是清热祛湿法的代表方剂之一,具有清热祛湿、解表透邪之功,是治疗温病气分湿热证的主方。相关的临床研究已证明蒿芩清胆汤在治疗病毒性疾病方面有一定优势,因此,以该方为代表治疗岭南地区病毒性呼吸道感染湿热证的研究更具现实意义。本课题通过临床研究与实验研究相结合的思路,应用宏观与微观的观察方法,探讨蒿芩清胆汤对呼吸道病毒感染湿热证的临床疗效及其部分作用机制。
第一部分临床研究
目的:通过临床观察,分析蒿芩清胆汤对上呼吸道病毒感染湿热证患者中医症状改善情况及血清TNF-α、IL-6水平的影响,探讨蒿芩清胆汤治疗湿热证的临床疗效及其在免疫炎症方面的作用机制。
方法:符合纳入标准的上呼吸道病毒感染湿热证患者32例,随机分为蒿芩清胆汤治疗组和利巴韦林对照组。采用中医湿热证症状分级量表与利巴韦林进行中医症状疗效对比,ELISA法检测患者用药前后血清TNF-α、IL-6水平,进行治疗前后的自身对照和治疗后组间比较,观察蒿芩清胆汤对中医症状及血清TNF-α、IL-6水平的影响。
结果:两组在性别、年龄、病情程度方面比较无统计学差异,具有可比性。中医湿热证症状分级量表调查,蒿芩清胆汤组与利巴韦林组在改善中医湿热证主症(发热、恶寒、身重肢倦、胸闷脘痞、纳呆恶心欲吐、便溏、口干不欲饮)、次症(咳嗽、咽痛咽痒、鼻塞、咽部充血、头晕头重、小便短赤)各自治疗前后比较有统计学差异(P＜0.05),蒿芩清胆汤和利巴韦林均能改善湿热证症状。治疗后,蒿芩清胆汤在改善湿热证主症及次症方面与利巴韦林组比较均有统计学差异(P＜0.05),蒿芩清胆汤效果更显著。治疗前后两组患者血清TNF-α、IL-6水平比较均有统计学差异(P＜0.05)。治疗后,两组血清TNF-α、IL-6水平比较有统计学差异(P＜0.05),蒿芩清胆汤干预作用更明显。用药前后,两组实验各指标检测均正常。
结论:蒿芩清胆汤不仅能够改善上呼吸道病毒感染湿热证患者的临床症状,而且可以降低患者血清TNF-α、IL-6的水平。蒿芩清胆汤治疗上呼吸道病毒感染湿热证疗效确切,降低免疫炎症因子TNF-α、IL-6的释放可能是其作用机制之一。
第二部分实验研究
1、湿热证模型流感病毒感染量的探索
目的:探索复制小鼠病毒性肺炎湿热证模型所需的合适病毒感染量。
方法:实验小鼠分为8组,均给予内外湿热刺激(高脂饲料饲养、外湿热环境暴露),除内外湿热组外,其他7组分别感染不同浓度的流感病毒。观察各组小鼠症状、体征、存活率及肺脏病理改变。
结果:感染病毒前,小鼠具有体重增长迅速、饮水增加、排泄物增多等特征;病毒感染后出现呼吸急促,以腹式呼吸为主,活动量、进食及摄水量减少,体毛稀疏,爪甲紫绀,死亡等。病毒感染导致小鼠肺组织出现不同程度的间质性肺炎表现,病变程度与病毒感染浓度密切相关。120h内1LD_(50)浓度组小鼠存活率最高。
结论:复制小鼠病毒性肺炎湿热证动物模型的合适病毒感染量为2LD_(50)。
2、“肥甘饮食+外湿热环境+流感病毒感染”所致湿热证动物模型的研究
目的:探索“肥甘饮食+外湿热环境+流感病毒感染”综合因素干预下,小鼠病毒性肺炎湿热证动物模型的复制情况。
方法:实验小鼠分为4组,分别为正常对照组(A组)、内外湿热组(B组)、湿热证模型组(C组)、单纯病毒组(D组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达水平、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)病毒感染是动物死亡的直接原因。(2)在肺指数分析方面,A组、B组与C组或D组比较均有统计学差异(P＜0.05);A组与B组、C组与D组之间比较无统计学差异(P＞0.05)。(3)在病理形态学观察方面,A组、B组肺组织正常表现,C组、D组呈间质性肺炎表现;病变程度比较C组平均秩次大于D组;超微结构观察,A组肺组织正常表现,B组肺泡上皮胞浆内出现大量脂滴,C组除间质性肺炎表现外尚可见肺泡上皮胞浆内大量脂滴,D组仅见间质性肺炎表现。(4)在肺组织流感病毒基因表达水平方面,C组较D组有升高趋势,但无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,C组、D组较A组或B组表达水平增强,有统计学差异(P＜0.05);A组与B组、C组与D组之间比较均无统计学差异(P＞0.05)。(6)从各组平均秩次变化趋势推断,C组、D组肺组织的TNF-α、IL-6蛋白表达水平高于A组、B组,B组高于A组,C组高于D组。
结论:小鼠病毒性肺炎湿热证模型具备了湿热病证的部分临床特征,如纳差、便溏、消瘦等,且肺炎症状表现典型。湿热证动物模型具有脂肪代谢紊乱、免疫功能失调、炎症因子释放增加等特性。内外湿热干预对小鼠病毒性肺炎湿热证的免疫炎症反应可能具有促进作用,增加TNF-α、IL-6的释放可能是其机制之一。
3、蒿芩清胆汤对小鼠病毒性肺炎湿热证的免疫炎症作用机制探讨
目的:选用不同剂量的蒿芩清胆汤干预湿热证动物模型,探讨该方在抑制免疫炎症方面的作用机理。
方法:实验小鼠分为6组,分别为正常对照组(A组)、湿热证模型组(C组)、利巴韦林组(E组)、蒿芩清胆汤高、中、低剂量组(F组、G组、H组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达水平、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)蒿芩清胆汤各剂量及利巴韦林均可在一定程度上减少动物的死亡率。(2)在肺指数分析方面,E、F和G各组与C组比较有统计学差异(P＜0.05),H组与C组比较无统计学差异(P＞0.05),F组肺指数降低趋势最明显。(3)药物治疗可在一定程度上减轻流感病毒感染所致间质性肺炎病理改变,从平均秩次的变化趋势推断F组干预作用最明显。(4)在肺组织流感病毒基因表达水平方面,E、F、G和H各治疗组与C组比较均有统计学差异(P＜0.05)。蒿芩清胆汤可下调流感病毒的基因表达水平,具有抑制流感病毒增殖作用。各治疗组间比较,E组作用最显著,F组次之,但均无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,E、F和G各组与C组比较均有统计学差异(P＜0.05),H组与C组比较无统计学差异(P＞0.05)。蒿芩清胆汤能下调肺组织TNF-α、IL-6基因表达的增强。各治疗组间比较,F组下调趋势最明显,但无统计学差异(P＞0.05)。(6)药物治疗均可不同程度下调肺组织TNF-α、IL-6蛋白表达的增强,从平均秩次的变化趋势推断F组作用最明显。
结论:蒿芩清胆汤对小鼠病毒性肺炎湿热证模型的干预发挥了整体保护作用,同时具有下调肺组织TNF-α、IL-6蛋白及基因表达增强的作用,且其干预作用与剂量相关,高剂量组效果最显著。
4、蒿芩清胆汤及其化裁方药对小鼠病毒性肺炎湿热证的免疫炎症作用机制探讨
目的:依据中药方剂君臣佐使的配伍关系,对蒿芩清胆汤在全方基础上进行化裁,观察不同的化裁方药对湿热证动物模型的免疫炎症作用机制。
方法:实验小鼠分为7组,分别为正常对照组(A组)、模型对照组(C组)、蒿芩清胆汤组(F组)、化裁1组(I组)、化裁2组(J组)、化裁3组(K组)、化裁4组(L组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)蒿芩清胆汤及各化裁药均可不同程度上减少动物的死亡率。(2)在降低肺指数方面,F组作用最明显。(3)在减轻肺组织病变程度方面,从平均秩次大小趋势推断,F组效果最佳,K组和L组次之,I组和J组较差。(4)在肺组织流感病毒基因表达水平方面,蒿芩清胆汤及各化裁组与C组比较均有统计学差异(P＜0.05)。蒿芩清胆汤及各化裁方均可下调流感病毒基因的表达水平,具有抑制流感病毒增殖作用。各治疗组间比较,F组下调趋势最显著,但无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,F、J、K、L各组与C组比较有统计学差异(P＜0.05),I组与C组比较无统计学差异(P＞0.05)。蒿芩清胆汤及各化裁药物均具有下调TNF-α、IL-6基因表达的增强作用,从下调趋势推断F组作用最显著。(6)蒿芩清胆汤及各化裁药物均具有下调TNF-α、IL-6蛋白表达的作用,从平均秩次的大小趋势推断F组作用最显著。
结论:蒿芩清胆汤及各化裁药物在减少动物死亡率、减轻肺组织炎症病变、抑制病毒增殖等方面均发挥积极作用,具有下调肺组织TNF-α、IL-6蛋白及基因表达增强的作用。干预作用以全方效果最显著,君药青蒿、黄芩在全方中作用较突出。根据病证结合、方证对应的原则,结合化裁各组药物的干预结果,推测小鼠病毒性肺炎湿热证模型热重于湿。
The viral infection of respiratory tract is a common and frequently-occurring clinical disease,and all of the people are susceptible to it.Because of abounding rain water and hot climate in LingNan region of China,the viral respiratory infection have the characteristics of transforming into heat and intermingling dampness from differentiating syndrome,some researchers believe that damp-heat syndrome was embodied in the disease.Some heat-clearing and dampness-dispelling medicinal were widely used.HaoQinQingDan(HQQD) decoction is a Chinese herbal formula with the effect of heat-clearing and dampness-dispelling.The warm disease of dampness-heat syndrome of qi aspect is its indication.Based on some related clinical research,HQQD decoction was effective in the treatment of viral disease.It is practical significant to carry out the clinical and experimental research about the formula in viral respiratory infection of damp-heat syndrome.On the basis of clinical and experimental research,we explore the immune inflammatory mechanism of HQQD decoction for treating damp-heat syndrome through the methods of macro-observation and microscopic analysis.
PartⅠClinical Research
Objective:To observe the effect of HQQD in the improving symptoms of TCM and the serum levels of tumor necrosis factor-α(TNF-α) and Interleukin-6(IL-6) of patients in treating viral upper respiratory infection of damp-heat syndrome.And to explore the clinical efficacy and the mechanism in the immune inflammatory.
Methods:32 cases are selected and divided into two groups at random:HQQD group and Ribavirin group.Comparing the effect before and after the treatment interclass to know the effect of symptoms of damp-heat syndrome and the serum levels of TNF-αand IL-6 by the estimate tables of TCM symptom schedule and Enzyme-linked immunosorbent assay(ELISA).
Results:There are no significant differences in sex,age and serious extent between HQQD group and Ribavirin group,so the two groups are comparable.It has statistical significance(P＜0.5) that both HQQD and Ribavirin can improve main and minor symptoms of damp-heat syndrome before and after treating.Seven main symptoms(fever,aversion to cold,heavy body and lassitude of the four limbs,oppression in the chest and gastric stuffiness,torpid intake and nausea and vomiting,sloppy stool,and dry mouth but not desire for drinking water.) and six minor symptoms(cough,pharyngeal itching pharyngodynia,nasal congestion,congestive throat,vertigo and head heaviness,and reddish scanty urine) of damp-heat syndrome can be relieved visibly after using HQQD(P＜0.05) than Ribavirin.Both HQQD and Ribavirin can decrease the serum levels of TNF-αand IL-6 of patients before and after treating(P＜0.05).The serum levels of TNF-αand IL-6 of patients are significant between HQQD group and Ribavirin group(P＜0.05).HQQD can decrease the serum levels better than Ribavirin.The indexes of laboratory for patients both HQQD group and Ribavirin group are normal after treating.HQQD is safety for patients.
Conclusions:HQQD can not only improve the symptoms of viral upper respiratory infection of damp-heat syndrome but also decrease the serum levels of TNP-αand IL-6 for patients.HQQD is very effective in treating viral upper respiratory infection of damp-heat syndrome,and it may be its' mechanism suppressing the immune inflammatory.
PartⅡExperimental research
1.To explore the concentration of influenza A(H_1N_1) virus on mouse infected for experimental animal models
Objective:Explore an appropriate virus infection dose of the production of animal models of damp-heat.
Methods:The mice are randomly divided into eight groups and were given richly fatty and sweet diet and humid heat environment:dampness and heat group,seven groups with different concentrations(1 LD_(50)、5 LD_(50)、10 LD_(50)、20 LD_(50)、30 LD_(50)、 40 LD_(50)、50 LD_(50)).The symptoms,sign,survival rate of mice and pathogenic change of lung tissue were observed in the experimental process.
Results:Dampness and heat group showed some features such as rapid gain weight, drinking more water,more plentiful excretion,etc.The mice infected influenza virus of clinical manifestations showed pneumonia.The symptoms and pathogenic change were closely related to virus infection.The survival rate of 1LD_(50) group was the highest one.
Conclusions:The appropriate virus infection dose of the production of animal models of damp-heat is 2LD_(50).
2.The research on animal models of damp-heat syndrome by adopting a richly fatty and sweet diet,humid heat environment and influenza virus infected
Objective:To produce animal models of damp-heat in the multiple factors.
Methods:The mice were randomly divided into four groups,control group(A group),dampness and heat group(B group),model group of damp-heat syndrome (C group),and influenza virus group(D group).Analyzing and comparing death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α,IL-6 protein and mRNA in the lung tissue.
Results:(1) Virus infection directly caused death.(2)The lung indexes number of C group or D group are higher than A group and B group(P＜0.05).There are not significant difference between A group and B group(P＞0.05),the same to between C group and D group(P＞0.05).(3)A group and B group show normal lung tissue,but pathogenic changes of C group and D group show interstitial pneumonia caused by virus infection.C group of mean rank is higher than D group for the degree of pathological changes.Ultrastructural observation: A group of lung tissue is normal;a large number of lipid droplets in the cytoplasm of alveolar epithelial are found in B group;C group show pathogenic changes of interstitial pneumonia and a large number of lipid droplets in the cytoplasm of alveolar epithelial are found;D group only show pathogenic changes of interstitial pneumonia.(4) Compared to D group,the expressions of influenza virus mRNA of C group increase in the lung tissue,but the difference is not significant(P＞0.05).(5) Compared to A group or B group, the expressions of cytokine TNF-αand IL-6 mRNA of C group and D group raise up in the lung tissue,and there are significant(P＜0.05).There are not significant difference between A group and B group(P＞0.05),the same to between C group and D group(P＞0.05).(6) Mean ranks of C group and D group are obviously higher than A group and B group for the expressions of cytokine TNF-αand IL-6 protein in the lung tissue.Mean rank of B group is higher than A group,and mean rank of C group is higher than D group
Conclusions:Animal model of damp-heat syndrome have partial clinical characteristics of damp-heat syndrome,such as torpid intake,sloppy stool, etc.;but clinical manifestation show prominent pneumonia symptoms.Animal model have characteristics such as fat metabolism disorders,immune dysfunction,inflammatory factors increasing,etc.Internal and external humid heat environment may play an active role in the inflammatory cytokine secreted of TNF-αand IL-6.
3.The research of the immune inflammatory mechanism of HQQD decoction on animal models of damp-heat syndrome
Objective:The mice were treated by the different doses of HQQD in order to explore the immune inflammatory mechanism and find the most effective dose. Methods:The mice were randomly divided into six groups,control group(A group),model group of damp-heat syndrome(C group),Ribavirin group(E group), high,middle and low dose HQQD groups(F group,G group and H group).Analyzed and compared death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α, IL-6 protein and mRNA in the lung tissue.
Results:(1)Each medicine can decrease the mortality rate of animals and the lung inflammatory reaction to some extent.(2)Compared with C group,the lung index number of E,F and G groups are significant difference except for H group (P＜0.05).The lung index number of F group is most obviously decreased. (3)Each medicine can decrease degree of pathological changes to some extent. We can surmise that the effect of high dose group is mostly obvious from the trend from the order of smallest mean rank.(4)Compared with C group,E,F, G and H groups are significant for the expressions of influenza virus mRNA in lung tissue(P＜0.05).The decrease of influenza virus may have a role in the mechanism of HQQD.The effect of E group is better than other groups,but the difference is not significant(P＞0.05).(5) Compared with C group,E, F and G groupsare significant for the expressions of cytokine TNF-αand IL-6 mRNA in lung tissue(P＜0.05).There are not significant difference between H group and C group(P＞0.05).HQQD can decrease the expressions of cytokine TNF-αand IL-6 mRNA.F group is obvious,but the difference is not significant (P＞0.05).(6)HQQD can decrease the expressions of cytokine TNF-αand IL-6 protein in the lung tissue to some extent.We can infer that the effect of F group is mostly obvious from the trend of mean rank order.
Conclusions:HQQD may play an overall protection role in viral pneumonia of damp-heat syndrome of mouse,and decrease the expressions of cytokine TNF-αand IL-6 mRNA and protein in the lung tissue.The effect is closely related to dose,and high dose group is mostly obvious.
4.The research of the immune inflammatory mechanism of HQQD prescription on animal models of damp-heat syndrome
Objective:To explore the immune inflammatory mechanism of HQQD prescription according to the relation of formula of sovereign,minister,minister and courier medicinal.
Methods:The mice were randomly divided into seven groups,control group(A group),model group of damp-heat syndrome(C group),high dose HQQD groups(F group),first group of prescription(I group),second group of prescription (J group),third group of prescription(K group),forth group of prescription (L group).Analyzed and compared death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α,IL-6 protein and mRNA in the lung tissue.
Results:(1) Each TCM prescription can decrease the mortality rate of animals and the lung inflammatory reaction to some extent.(2)Compared with C group, the lung index number of F group is most obviously decreased,and it is better than I and J groups.(3)We infer that the effect of F group is mostly obvious in decreasing the degree of lung pathology from the mean rank trend.Group F is best,K and L groups are followed,but I and J groups are not obvious. (4)Compared with C group,HQQD prescription groups are significant for the expressions of influenza virus mRNA in lung tissue(P＜0.05).F group is better than other groups,but the difference is not significant(P＞0.05).The decrease of influenza virus may have a role in the mechanism of HQQD prescription.(5) Compared with C group,E,J,K and L groups are significant for the expressions of cytokine TNF-αand IL-6 mRNA in lung tissue(P＜0.05). There are not significant difference between I group and C group(P＞0.05). HQQD prescriptions can decrease the expressions of cytokine TNF-αand IL-6 mRNA.(6) HQQD prescriptions can decrease the expressions of cytokine TNF-αand IL-6 protein in the lung tissue to some extent.We can infer that the effect of F group is mostly obvious from the trend of mean rank order.
Conclusions:HQQD prescription can decrease mortality rate of animals,lung inflammatory reaction,reproduction of influenza virus and expressions of cytokine TNF-αand IL-6 protein and mRNA in the lung tissue.The integrated HQQD prescription is the most effective.According to the results of HQQD prescription separated,sovereign medicinal(QingHao and HuangQin) is very important in the formula.Furthermore we infer that the mouse model of damp-heat syndrome have the characteristics of damp prevailing heat.
第一部分临床研究
目的:通过临床观察,分析蒿芩清胆汤对上呼吸道病毒感染湿热证患者中医症状改善情况及血清TNF-α、IL-6水平的影响,探讨蒿芩清胆汤治疗湿热证的临床疗效及其在免疫炎症方面的作用机制。
方法:符合纳入标准的上呼吸道病毒感染湿热证患者32例,随机分为蒿芩清胆汤治疗组和利巴韦林对照组。采用中医湿热证症状分级量表与利巴韦林进行中医症状疗效对比,ELISA法检测患者用药前后血清TNF-α、IL-6水平,进行治疗前后的自身对照和治疗后组间比较,观察蒿芩清胆汤对中医症状及血清TNF-α、IL-6水平的影响。
结果:两组在性别、年龄、病情程度方面比较无统计学差异,具有可比性。中医湿热证症状分级量表调查,蒿芩清胆汤组与利巴韦林组在改善中医湿热证主症(发热、恶寒、身重肢倦、胸闷脘痞、纳呆恶心欲吐、便溏、口干不欲饮)、次症(咳嗽、咽痛咽痒、鼻塞、咽部充血、头晕头重、小便短赤)各自治疗前后比较有统计学差异(P＜0.05),蒿芩清胆汤和利巴韦林均能改善湿热证症状。治疗后,蒿芩清胆汤在改善湿热证主症及次症方面与利巴韦林组比较均有统计学差异(P＜0.05),蒿芩清胆汤效果更显著。治疗前后两组患者血清TNF-α、IL-6水平比较均有统计学差异(P＜0.05)。治疗后,两组血清TNF-α、IL-6水平比较有统计学差异(P＜0.05),蒿芩清胆汤干预作用更明显。用药前后,两组实验各指标检测均正常。
结论:蒿芩清胆汤不仅能够改善上呼吸道病毒感染湿热证患者的临床症状,而且可以降低患者血清TNF-α、IL-6的水平。蒿芩清胆汤治疗上呼吸道病毒感染湿热证疗效确切,降低免疫炎症因子TNF-α、IL-6的释放可能是其作用机制之一。
第二部分实验研究
1、湿热证模型流感病毒感染量的探索
目的:探索复制小鼠病毒性肺炎湿热证模型所需的合适病毒感染量。
方法:实验小鼠分为8组,均给予内外湿热刺激(高脂饲料饲养、外湿热环境暴露),除内外湿热组外,其他7组分别感染不同浓度的流感病毒。观察各组小鼠症状、体征、存活率及肺脏病理改变。
结果:感染病毒前,小鼠具有体重增长迅速、饮水增加、排泄物增多等特征;病毒感染后出现呼吸急促,以腹式呼吸为主,活动量、进食及摄水量减少,体毛稀疏,爪甲紫绀,死亡等。病毒感染导致小鼠肺组织出现不同程度的间质性肺炎表现,病变程度与病毒感染浓度密切相关。120h内1LD_(50)浓度组小鼠存活率最高。
结论:复制小鼠病毒性肺炎湿热证动物模型的合适病毒感染量为2LD_(50)。
2、“肥甘饮食+外湿热环境+流感病毒感染”所致湿热证动物模型的研究
目的:探索“肥甘饮食+外湿热环境+流感病毒感染”综合因素干预下,小鼠病毒性肺炎湿热证动物模型的复制情况。
方法:实验小鼠分为4组,分别为正常对照组(A组)、内外湿热组(B组)、湿热证模型组(C组)、单纯病毒组(D组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达水平、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)病毒感染是动物死亡的直接原因。(2)在肺指数分析方面,A组、B组与C组或D组比较均有统计学差异(P＜0.05);A组与B组、C组与D组之间比较无统计学差异(P＞0.05)。(3)在病理形态学观察方面,A组、B组肺组织正常表现,C组、D组呈间质性肺炎表现;病变程度比较C组平均秩次大于D组;超微结构观察,A组肺组织正常表现,B组肺泡上皮胞浆内出现大量脂滴,C组除间质性肺炎表现外尚可见肺泡上皮胞浆内大量脂滴,D组仅见间质性肺炎表现。(4)在肺组织流感病毒基因表达水平方面,C组较D组有升高趋势,但无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,C组、D组较A组或B组表达水平增强,有统计学差异(P＜0.05);A组与B组、C组与D组之间比较均无统计学差异(P＞0.05)。(6)从各组平均秩次变化趋势推断,C组、D组肺组织的TNF-α、IL-6蛋白表达水平高于A组、B组,B组高于A组,C组高于D组。
结论:小鼠病毒性肺炎湿热证模型具备了湿热病证的部分临床特征,如纳差、便溏、消瘦等,且肺炎症状表现典型。湿热证动物模型具有脂肪代谢紊乱、免疫功能失调、炎症因子释放增加等特性。内外湿热干预对小鼠病毒性肺炎湿热证的免疫炎症反应可能具有促进作用,增加TNF-α、IL-6的释放可能是其机制之一。
3、蒿芩清胆汤对小鼠病毒性肺炎湿热证的免疫炎症作用机制探讨
目的:选用不同剂量的蒿芩清胆汤干预湿热证动物模型,探讨该方在抑制免疫炎症方面的作用机理。
方法:实验小鼠分为6组,分别为正常对照组(A组)、湿热证模型组(C组)、利巴韦林组(E组)、蒿芩清胆汤高、中、低剂量组(F组、G组、H组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达水平、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)蒿芩清胆汤各剂量及利巴韦林均可在一定程度上减少动物的死亡率。(2)在肺指数分析方面,E、F和G各组与C组比较有统计学差异(P＜0.05),H组与C组比较无统计学差异(P＞0.05),F组肺指数降低趋势最明显。(3)药物治疗可在一定程度上减轻流感病毒感染所致间质性肺炎病理改变,从平均秩次的变化趋势推断F组干预作用最明显。(4)在肺组织流感病毒基因表达水平方面,E、F、G和H各治疗组与C组比较均有统计学差异(P＜0.05)。蒿芩清胆汤可下调流感病毒的基因表达水平,具有抑制流感病毒增殖作用。各治疗组间比较,E组作用最显著,F组次之,但均无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,E、F和G各组与C组比较均有统计学差异(P＜0.05),H组与C组比较无统计学差异(P＞0.05)。蒿芩清胆汤能下调肺组织TNF-α、IL-6基因表达的增强。各治疗组间比较,F组下调趋势最明显,但无统计学差异(P＞0.05)。(6)药物治疗均可不同程度下调肺组织TNF-α、IL-6蛋白表达的增强,从平均秩次的变化趋势推断F组作用最明显。
结论:蒿芩清胆汤对小鼠病毒性肺炎湿热证模型的干预发挥了整体保护作用,同时具有下调肺组织TNF-α、IL-6蛋白及基因表达增强的作用,且其干预作用与剂量相关,高剂量组效果最显著。
4、蒿芩清胆汤及其化裁方药对小鼠病毒性肺炎湿热证的免疫炎症作用机制探讨
目的:依据中药方剂君臣佐使的配伍关系,对蒿芩清胆汤在全方基础上进行化裁,观察不同的化裁方药对湿热证动物模型的免疫炎症作用机制。
方法:实验小鼠分为7组,分别为正常对照组(A组)、模型对照组(C组)、蒿芩清胆汤组(F组)、化裁1组(I组)、化裁2组(J组)、化裁3组(K组)、化裁4组(L组)。实验结束后,分析比较各组死亡率、肺指数、病理形态学、肺组织流感病毒基因表达、肺组织TNF-α、IL-6蛋白及基因的表达水平。
结果:(1)蒿芩清胆汤及各化裁药均可不同程度上减少动物的死亡率。(2)在降低肺指数方面,F组作用最明显。(3)在减轻肺组织病变程度方面,从平均秩次大小趋势推断,F组效果最佳,K组和L组次之,I组和J组较差。(4)在肺组织流感病毒基因表达水平方面,蒿芩清胆汤及各化裁组与C组比较均有统计学差异(P＜0.05)。蒿芩清胆汤及各化裁方均可下调流感病毒基因的表达水平,具有抑制流感病毒增殖作用。各治疗组间比较,F组下调趋势最显著,但无统计学差异(P＞0.05)。(5)在肺组织TNF-α、IL-6的基因表达水平方面,F、J、K、L各组与C组比较有统计学差异(P＜0.05),I组与C组比较无统计学差异(P＞0.05)。蒿芩清胆汤及各化裁药物均具有下调TNF-α、IL-6基因表达的增强作用,从下调趋势推断F组作用最显著。(6)蒿芩清胆汤及各化裁药物均具有下调TNF-α、IL-6蛋白表达的作用,从平均秩次的大小趋势推断F组作用最显著。
结论:蒿芩清胆汤及各化裁药物在减少动物死亡率、减轻肺组织炎症病变、抑制病毒增殖等方面均发挥积极作用,具有下调肺组织TNF-α、IL-6蛋白及基因表达增强的作用。干预作用以全方效果最显著,君药青蒿、黄芩在全方中作用较突出。根据病证结合、方证对应的原则,结合化裁各组药物的干预结果,推测小鼠病毒性肺炎湿热证模型热重于湿。
The viral infection of respiratory tract is a common and frequently-occurring clinical disease,and all of the people are susceptible to it.Because of abounding rain water and hot climate in LingNan region of China,the viral respiratory infection have the characteristics of transforming into heat and intermingling dampness from differentiating syndrome,some researchers believe that damp-heat syndrome was embodied in the disease.Some heat-clearing and dampness-dispelling medicinal were widely used.HaoQinQingDan(HQQD) decoction is a Chinese herbal formula with the effect of heat-clearing and dampness-dispelling.The warm disease of dampness-heat syndrome of qi aspect is its indication.Based on some related clinical research,HQQD decoction was effective in the treatment of viral disease.It is practical significant to carry out the clinical and experimental research about the formula in viral respiratory infection of damp-heat syndrome.On the basis of clinical and experimental research,we explore the immune inflammatory mechanism of HQQD decoction for treating damp-heat syndrome through the methods of macro-observation and microscopic analysis.
PartⅠClinical Research
Objective:To observe the effect of HQQD in the improving symptoms of TCM and the serum levels of tumor necrosis factor-α(TNF-α) and Interleukin-6(IL-6) of patients in treating viral upper respiratory infection of damp-heat syndrome.And to explore the clinical efficacy and the mechanism in the immune inflammatory.
Methods:32 cases are selected and divided into two groups at random:HQQD group and Ribavirin group.Comparing the effect before and after the treatment interclass to know the effect of symptoms of damp-heat syndrome and the serum levels of TNF-αand IL-6 by the estimate tables of TCM symptom schedule and Enzyme-linked immunosorbent assay(ELISA).
Results:There are no significant differences in sex,age and serious extent between HQQD group and Ribavirin group,so the two groups are comparable.It has statistical significance(P＜0.5) that both HQQD and Ribavirin can improve main and minor symptoms of damp-heat syndrome before and after treating.Seven main symptoms(fever,aversion to cold,heavy body and lassitude of the four limbs,oppression in the chest and gastric stuffiness,torpid intake and nausea and vomiting,sloppy stool,and dry mouth but not desire for drinking water.) and six minor symptoms(cough,pharyngeal itching pharyngodynia,nasal congestion,congestive throat,vertigo and head heaviness,and reddish scanty urine) of damp-heat syndrome can be relieved visibly after using HQQD(P＜0.05) than Ribavirin.Both HQQD and Ribavirin can decrease the serum levels of TNF-αand IL-6 of patients before and after treating(P＜0.05).The serum levels of TNF-αand IL-6 of patients are significant between HQQD group and Ribavirin group(P＜0.05).HQQD can decrease the serum levels better than Ribavirin.The indexes of laboratory for patients both HQQD group and Ribavirin group are normal after treating.HQQD is safety for patients.
Conclusions:HQQD can not only improve the symptoms of viral upper respiratory infection of damp-heat syndrome but also decrease the serum levels of TNP-αand IL-6 for patients.HQQD is very effective in treating viral upper respiratory infection of damp-heat syndrome,and it may be its' mechanism suppressing the immune inflammatory.
PartⅡExperimental research
1.To explore the concentration of influenza A(H_1N_1) virus on mouse infected for experimental animal models
Objective:Explore an appropriate virus infection dose of the production of animal models of damp-heat.
Methods:The mice are randomly divided into eight groups and were given richly fatty and sweet diet and humid heat environment:dampness and heat group,seven groups with different concentrations(1 LD_(50)、5 LD_(50)、10 LD_(50)、20 LD_(50)、30 LD_(50)、 40 LD_(50)、50 LD_(50)).The symptoms,sign,survival rate of mice and pathogenic change of lung tissue were observed in the experimental process.
Results:Dampness and heat group showed some features such as rapid gain weight, drinking more water,more plentiful excretion,etc.The mice infected influenza virus of clinical manifestations showed pneumonia.The symptoms and pathogenic change were closely related to virus infection.The survival rate of 1LD_(50) group was the highest one.
Conclusions:The appropriate virus infection dose of the production of animal models of damp-heat is 2LD_(50).
2.The research on animal models of damp-heat syndrome by adopting a richly fatty and sweet diet,humid heat environment and influenza virus infected
Objective:To produce animal models of damp-heat in the multiple factors.
Methods:The mice were randomly divided into four groups,control group(A group),dampness and heat group(B group),model group of damp-heat syndrome (C group),and influenza virus group(D group).Analyzing and comparing death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α,IL-6 protein and mRNA in the lung tissue.
Results:(1) Virus infection directly caused death.(2)The lung indexes number of C group or D group are higher than A group and B group(P＜0.05).There are not significant difference between A group and B group(P＞0.05),the same to between C group and D group(P＞0.05).(3)A group and B group show normal lung tissue,but pathogenic changes of C group and D group show interstitial pneumonia caused by virus infection.C group of mean rank is higher than D group for the degree of pathological changes.Ultrastructural observation: A group of lung tissue is normal;a large number of lipid droplets in the cytoplasm of alveolar epithelial are found in B group;C group show pathogenic changes of interstitial pneumonia and a large number of lipid droplets in the cytoplasm of alveolar epithelial are found;D group only show pathogenic changes of interstitial pneumonia.(4) Compared to D group,the expressions of influenza virus mRNA of C group increase in the lung tissue,but the difference is not significant(P＞0.05).(5) Compared to A group or B group, the expressions of cytokine TNF-αand IL-6 mRNA of C group and D group raise up in the lung tissue,and there are significant(P＜0.05).There are not significant difference between A group and B group(P＞0.05),the same to between C group and D group(P＞0.05).(6) Mean ranks of C group and D group are obviously higher than A group and B group for the expressions of cytokine TNF-αand IL-6 protein in the lung tissue.Mean rank of B group is higher than A group,and mean rank of C group is higher than D group
Conclusions:Animal model of damp-heat syndrome have partial clinical characteristics of damp-heat syndrome,such as torpid intake,sloppy stool, etc.;but clinical manifestation show prominent pneumonia symptoms.Animal model have characteristics such as fat metabolism disorders,immune dysfunction,inflammatory factors increasing,etc.Internal and external humid heat environment may play an active role in the inflammatory cytokine secreted of TNF-αand IL-6.
3.The research of the immune inflammatory mechanism of HQQD decoction on animal models of damp-heat syndrome
Objective:The mice were treated by the different doses of HQQD in order to explore the immune inflammatory mechanism and find the most effective dose. Methods:The mice were randomly divided into six groups,control group(A group),model group of damp-heat syndrome(C group),Ribavirin group(E group), high,middle and low dose HQQD groups(F group,G group and H group).Analyzed and compared death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α, IL-6 protein and mRNA in the lung tissue.
Results:(1)Each medicine can decrease the mortality rate of animals and the lung inflammatory reaction to some extent.(2)Compared with C group,the lung index number of E,F and G groups are significant difference except for H group (P＜0.05).The lung index number of F group is most obviously decreased. (3)Each medicine can decrease degree of pathological changes to some extent. We can surmise that the effect of high dose group is mostly obvious from the trend from the order of smallest mean rank.(4)Compared with C group,E,F, G and H groups are significant for the expressions of influenza virus mRNA in lung tissue(P＜0.05).The decrease of influenza virus may have a role in the mechanism of HQQD.The effect of E group is better than other groups,but the difference is not significant(P＞0.05).(5) Compared with C group,E, F and G groupsare significant for the expressions of cytokine TNF-αand IL-6 mRNA in lung tissue(P＜0.05).There are not significant difference between H group and C group(P＞0.05).HQQD can decrease the expressions of cytokine TNF-αand IL-6 mRNA.F group is obvious,but the difference is not significant (P＞0.05).(6)HQQD can decrease the expressions of cytokine TNF-αand IL-6 protein in the lung tissue to some extent.We can infer that the effect of F group is mostly obvious from the trend of mean rank order.
Conclusions:HQQD may play an overall protection role in viral pneumonia of damp-heat syndrome of mouse,and decrease the expressions of cytokine TNF-αand IL-6 mRNA and protein in the lung tissue.The effect is closely related to dose,and high dose group is mostly obvious.
4.The research of the immune inflammatory mechanism of HQQD prescription on animal models of damp-heat syndrome
Objective:To explore the immune inflammatory mechanism of HQQD prescription according to the relation of formula of sovereign,minister,minister and courier medicinal.
Methods:The mice were randomly divided into seven groups,control group(A group),model group of damp-heat syndrome(C group),high dose HQQD groups(F group),first group of prescription(I group),second group of prescription (J group),third group of prescription(K group),forth group of prescription (L group).Analyzed and compared death rate,lung index number,pathogenic change,expressions of influenza virus mRNA in the lung tissue,and expressions of cytokine TNF-α,IL-6 protein and mRNA in the lung tissue.
Results:(1) Each TCM prescription can decrease the mortality rate of animals and the lung inflammatory reaction to some extent.(2)Compared with C group, the lung index number of F group is most obviously decreased,and it is better than I and J groups.(3)We infer that the effect of F group is mostly obvious in decreasing the degree of lung pathology from the mean rank trend.Group F is best,K and L groups are followed,but I and J groups are not obvious. (4)Compared with C group,HQQD prescription groups are significant for the expressions of influenza virus mRNA in lung tissue(P＜0.05).F group is better than other groups,but the difference is not significant(P＞0.05).The decrease of influenza virus may have a role in the mechanism of HQQD prescription.(5) Compared with C group,E,J,K and L groups are significant for the expressions of cytokine TNF-αand IL-6 mRNA in lung tissue(P＜0.05). There are not significant difference between I group and C group(P＞0.05). HQQD prescriptions can decrease the expressions of cytokine TNF-αand IL-6 mRNA.(6) HQQD prescriptions can decrease the expressions of cytokine TNF-αand IL-6 protein in the lung tissue to some extent.We can infer that the effect of F group is mostly obvious from the trend of mean rank order.
Conclusions:HQQD prescription can decrease mortality rate of animals,lung inflammatory reaction,reproduction of influenza virus and expressions of cytokine TNF-αand IL-6 protein and mRNA in the lung tissue.The integrated HQQD prescription is the most effective.According to the results of HQQD prescription separated,sovereign medicinal(QingHao and HuangQin) is very important in the formula.Furthermore we infer that the mouse model of damp-heat syndrome have the characteristics of damp prevailing heat.
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