基于“肺合大肠”理论的敷胸巴布贴干预Ⅳ诱导小鼠肺炎免疫机制研究
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摘要
目的:从“肺合大肠”的古今论述,提取肺与大肠的生理、病理的关系,探讨敷胸巴布贴组方应用的理论基础;通过克隆测序基因比对方法鉴定流感病毒(IV)诱导小鼠致肺炎的动物模型,为中医药防治IV实验研究提供可靠而稳定的动物模型;通过皮肤刺激实验对敷胸巴布贴进行安全性评价,为临床推广应用提供安全用药依据;将传统敷胸膏与巴布贴进行比较研究,通过不同剂量巴布贴对IV诱导小鼠肺炎的药效学及免疫机制研究,探讨其对IV肺炎小鼠的治疗作用,试阐明敷胸巴布贴治疗IV肺炎的作用机制,从而验证“肺合大肠”为其应用的理论基础,为中药新药研发提供实验基础和理论依据;采用高效液相色谱法(HPLC法)测定敷胸巴布贴中药物含量并进行透皮吸收实验,以期明确其治疗IV肺炎的物质基础。
材料与方法:
1.采用鼻腔接种流感病毒FM1株诱导昆明种小鼠致肺炎,观察小鼠在一般状态、肺组织的病理组织学等方面的差异,并应用RT-PCR技术检测病毒载量、克隆测序基因比对等多角度进行模型鉴定。
2.通过皮肤刺激实验观察敷胸巴布贴对完整皮肤和破损皮肤的刺激作用。
3.将传统敷胸膏与不同剂量的敷胸巴布贴以外敷在小鼠背部(相当于肺腧穴、膏肓穴等)为给药途径治疗小鼠IV肺炎,观察各组药物对小鼠一般状态、死亡保护作用、肺指数、肺悬液血凝滴度的影响。
4.采用免疫组织化学方法和RT-PCR技术检测IV诱导小鼠肺炎的肺肠组织中促炎因子TNF-α、抗炎因子IL-10的蛋白及mRNA的表达水平,比较传统敷胸膏与不同剂量敷胸巴布贴对IV肺炎小鼠肺肠组织中TNF-α、IL-10改变的影响并对同一指标进行相关性分析。
5.采用免疫组织化学方法和RT-PCR技术检测IV诱导小鼠肺炎的肺肠组织中转录因子NF-κB P65蛋白及mRNA的表达水平,比较传统敷胸膏与不同剂量敷胸巴布贴对IV肺炎小鼠肺肠组织中NF-κB P65改变的影响并进行相关性分析。
6.应用HPLC法测定敷胸巴布贴中大黄的五种活性成分含量。
7.以离体大鼠皮肤为透皮屏障,采用改良的Franz扩散装置,应用HPLC法测定大黄素、大黄酚的经皮渗透量,考察敷胸巴布贴透皮吸收情况。
结果:
1.采用鼻腔接种流感病毒FM1株诱导昆明种小鼠致肺炎,模型组病理改变有肺泡间隔增厚、水肿,内含有大量炎细胞浸润,肺泡壁毛细血管扩张,间见大量红细胞。小鼠感染IV第3～10天肺组织中病毒核酸阳性检出率达100%。将模型组小鼠肺组织的IV RT-PCR扩增产物,采用克隆测序方法测序,与美国国立生物信息中心已知的IV cDNA全序列比较,吻合率达到99.06%。
2.敷胸巴布贴在完整皮肤用药部位未出现红斑、水肿等皮肤刺激反应;而对破损皮肤,用药部位出现轻度刺激反应。
3.死亡保护作用结果:低、高剂量巴布贴组对IV感染小鼠的死亡保护率分别是25%、40%;延长生命率是33.33%、58.52%。与模型组比较,死亡率明显降低,平均存活天数明显延长(P<0.01)。可见高剂量巴布贴的死亡保护率最高、平均存活时间最长。
4.体重结果:正常组体重逐日增长;模型组体重逐日下降,第3天开始直至第7天较正常组明显下降(P<0.05);敷胸巴布贴组和敷胸膏组较模型组体重下降变慢,从第6天起体重有所增长,敷胸巴布贴组第6天和第7天体重较模型组明显升高(P<0.01)。
5.肺指数、肺组织血凝滴度结果:模型组较正常组肺指数明显升高,低、高剂量巴布贴组和敷胸膏组较模型组肺指数明显降低(P<0.05)。低、高剂量巴布贴组分别与敷胸膏组比较,均有显著性差异(P<0.05)。低、高剂量巴布贴组和敷胸膏组的血凝滴度较模型组明显降低(P<0.05),且高剂量巴布贴组疗效最佳。
6.IV诱导小鼠肺炎的肺肠组织中TNF-α蛋白表达和mRNA表达水平及相关性分析:
肺组织:正常组可见少许表达于支气管上皮细胞,可以检测到少量的TNF-α蛋白表达和mRNA表达水平。模型组TNF-α蛋白表达和mRNA含量最高,与正常组比较,有显著性差异(P<0.01);与赋形剂组相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组TNF-α蛋白表达和mRNA表达水平均高于正常组,但明显低于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组与正常组比较,无显著性差异(P>0.05)。高剂量巴布贴组TNF-α蛋白表达和mRNA表达水平最低。
肠组织:正常组可见少许表达于肠上皮细胞,可以检测到少量的TNF-α蛋白和mRNA表达水平。模型组TNF-α蛋白表达和mRNA含量最高,明显高于正常组,有显著性差异(P<0.05);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组TNF-α蛋白表达和mRNA表达水平均高于正常组,且明显低于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组与正常组比较,无显著性差异(P>0.05)。高剂量巴布贴组TNF-α蛋白表达和mRNA表达水平最低。
相关性分析:正常组肺肠组织TNF-α蛋白表达和mRNA表达水平未出现相关性(P>0.05);模型组、赋形剂组、敷胸膏组、高剂量巴布贴组出现正相关(P<0.05);低剂量巴布贴组未出现相关性(P>0.05)。高剂量巴布贴组肺肠组织相关性均高于敷胸膏组。7.IV诱导小鼠肺炎的肺肠组织中IL-10蛋白和mRNA表达水平及相关性分析:
肺组织:正常组可以检测到少量的IL-10蛋白表达和mRNA表达水平。模型组IL-10蛋白表达和mRNA含量均高于正常组,具有显著性差异(P<0.05);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组IL-10蛋白表达和mRNA表达水平均明显高于正常组,但高于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组IL-10蛋白和mRNA表达水平最高。
肠组织:正常组可以检测到少量的IL-10蛋白表达和mRNA表达水平。模型组IL-10蛋白表达和mRNA含量均高于正常组,具有显著性差异(P<0.05～0.01);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组IL-10蛋白表达和mRNA表达水平均明显高于正常组,且高于模型组(P<0.05～0.01);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组IL-10蛋白表达和mRNA表达水平最高。
相关性分析:正常组肺肠组织IL-10蛋白和mRNA表达水平未出现相关性(P>0.05);模型组、赋形剂组、敷胸膏组、高剂量巴布贴组出现正相关(P<0.05);低剂量巴布贴组未出现相关性(P>0.05)。相关性排序:高剂量巴布贴组>模型组>赋形剂组>敷胸膏组。
8.IV诱导小鼠肺炎的肺肠组织中NF-κB蛋白表达和mRNA表达水平及相关性分析:
肺组织:正常组可以检测到少量的NF-κB P65蛋白表达和mRNA表达水平。模型组NF-κB P65蛋白表达和mRNA表达水平最高,与正常组比较,具有显著性差异(P<0.05);赋形剂组表达相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组的NF-κB P65蛋白表达和mRNA表达水平均高于正常组,且明显低于模型组(P<0.05～0.01);两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组NF-κB P65蛋白表达和mRNA表达水平最低。
肠组织:正常组可以检测到少量的NF-κB P65蛋白表达和mRNA表达水平。模型组NF-κB P65蛋白表达和mRNA表达水平最高,与正常组比较,具有显著性差异(P<0.05);与赋形剂组表达相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组的NF-κB P65蛋白表达和mRNA表达水平均高于正常组(P<0.01),且敷胸膏组、高剂量巴布贴组明显低于模型组(P<0.01),而低剂量巴布贴组的NF-κB P65蛋白表达和mRNA表达水平与模型组比较,无显著性差异(P>0.05)。三组药物组两两比较,均有显著性差异(P<0.05),高剂量巴布贴组NF-κB P65蛋白表达mRNA表达水平最低。
相关性分析:正常组肺肠组织中NF-κB P65蛋白表达和mRNA表达水平未出现相关性(P>0.05),模型组、赋形剂组、敷胸膏组、高剂量巴布贴组均出现正相关(P<0.05),低剂量巴布贴组未出现相关性(P>0.05)。相关性排序:高剂量巴布贴组>模型组>赋形剂组>敷胸膏组。
9.每片敷胸巴布贴中游离总蒽醌的含量是11.9023mg,每克敷胸膏中游离总蒽醌的含量是5.8702mg,赋形剂中未检出游离蒽醌。
10.敷胸巴布贴和敷胸膏中主要的有效成分是有差异的,敷胸膏中除大黄酸含量低于巴布贴外,大黄素、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚含量均不同程度的高于敷胸巴布贴。
11.敷胸巴布贴的主要成分大黄素、大黄酚从给药4 h起,在各个时点的单位面积累积渗透量均高于敷胸膏,并随着时间的延长,单位面积累积渗透量的差值越来越大。
结论:
1.敷胸巴布贴对IV诱导小鼠肺炎具有一定的死亡保护作用;可明显缓解体重的降低,并减轻其肺部炎性病变,有一定的抑制鼠肺IV增殖作用。
2.IV肺炎的发病机制可能与促炎因子TNF-α、抗炎因子IL-10异常升高及转录因子NF-κB P65的过度活化有关;敷胸巴布贴治疗IV肺炎可能与降低TNF-α、提高IL-10及抑制NF-κB P65表达水平有关。
3.推测当敷胸巴布贴药量达到一定剂量,使肺肠组织中同一指标具有相关性时,即可能达到肺肠同治的治疗效果,验证了“肺合大肠”理论。
4.HPLC法可作为敷胸巴布贴质量控制及含量测定可靠的方法。
5.敷胸巴布贴是一种安全可靠的中药外用制剂,且有较好的经皮渗透性,剂型改良可行。
Purpose:From the " lung associating with large intestine " the ancient and modern discourse, extract the physiology and pathology relationship between lung and colon, to explore the application rationale of fuxiong- cataplasms. Through the method of cloning and sequencing to identify the influenza A virus induced animal model to provide a reliable and stable animal model for the influenza A virus prevention of traditional Chinese medicine.Through cutaneous stimulation experiment to evaluate the safety of fuxiong- cataplasms to provide the medication evidence for clinical implication.Comparing with the fuxiong-masticp and fuxiong- cataplasms, through the study of pharmacodynamics and immunologic mechanism of differnet doses of fuxiong- cataplasms on IV induced mice pneumonia,to explore the treatment of fuxiong- cataplasms and validate the rationale of " lung associating with large intestine ",to provie the experiment and theory foundation for the traditional chinese medicine research;Through high efficiency liquid chromatography (HPLC) to determine the active component contents of fuxiong- cataplasms and percutaneous permeability experiment to identify its material foundation of IV infected pneumonia treatment.
Material and method:
1.Using influenza A virus FM1 to infect mice to mice pneumonia, observed the general status、the differences of lung tissue, applied RT-PCR method to detect IV-RNA viral load、clone sequencing gene correlation to accredit the animal model.
2.Through cutaneous stimulation experiment to observe the stimulatory function of fuxiong- cataplasms on intact and breakage skin.
3.Put the fuxiong-masticp and different doses of fuxiong- cataplasms on the back of the mice(equal to Lung acupoints and Gaohuangshu points) to treat the mice pneumonia and observe the general state、mortality、lung index、hemagglutination titer of lung suspension.
4.By immunohistochemistry and RT-PCR detection of lung pro-inflammatory factors in intestinal tissue TNF-α, anti-inflammatory factor IL-10 protein and mRNA expression levels, compare with the variance of TNF-α, IL-10 in IV induced mice pneumonia lung and intestine issues of fuxiong-masticp and different doses of fuxiong- cataplasms,and the dependablity analysis was available for the same index.
5.By immunohistochemistry and RT-PCR method to detect lung and intestine transcription factor NF-κB P65 protein and mRNA expression levels, compare with the variance of NF IV -κB P65 in IV induced mice pneumonia lung and intestine issues of fuxiong-masticp and different doses of fuxiong- cataplasms and the dependablity analysis was available.
6.Application of HPLC method to determin the five active components in the fuxiong- cataplasms.
7. The skin excised rat was used as transdermal barrier, using the modified Franz diffusion devices,HPLC was used to determin the percutaneous permeation centent of emodin and rhein to investigate the percutaneous permeation conditon of fuxiong- cataplasms.
Results:
1.Intranasal inoculation with influenza virus FM1 strain caused pneumonia in mice were induced, pathological changes in the model group had alveolar septal thickening, edema, containing a large number of inflammatory cell infiltration, alveolar wall telangiectasia, a large number of red blood cells between the see. Mice were infected IV 3 to 10 days of virus DNA in lung tissue positive detection rate of 100%. The model mice lung tissue IV RT-PCR amplification, cloning and sequencing methods using DNA sequencing, and the United States National Center for Biotechnology Information known IV cDNA sequences comparison with the rate of 99.06%。
2.External using the fuxiong- cataplasms and the excipient, there is no skin stimulus response on the intact skin, on the contrary, external using on the damaged skin,has mild irritation response.
3.Protective effect of death results: low dose and high dose group barb post death in mice infected with influenza virus protection rate was 25%, 40%; prolong life rate is 33.33%, 58.52%. Compared with model group, mortality was reduced, the average survival time was significantly longer (P<0.01).the barb post can be seen affixed to the death of high doses of the highest protection rate, the average survival time is the longest。
4.Weight results: growth of normal body weight daily; model group decreased body weight daily, 3 day until day 7 was significantly lower than normal (P<0.05); dressing fuxiong- cream paste group and apply weight loss group than in model group Slow, from 6 days increased body weight, dressing fuxiong- cataplasms Group 6 and 7 days was significantly higher body weight compared with model group (P <0.01)。
5.Lung index, the virus titer Results: the model group was significantly higher than normal increase in lung index, deposited fuxiong-cream group and low and high dose group than in model group, fuxiong- cataplasms the lung index was significantly lower (P<0.05). Low and high dose groups were posted and deposited fuxiong-cream group, there were significant differences (P<0.05). Apply ointment group and the low thoracic and high dose groups hemagglutination barb posted significantly lower than the model (P<0.05), and the efficacy of high dose group posted the best.
6.TNF-αprotein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:The normal group showed little expression in bronchial epithelial cells, and could detect small amounts of TNF-αprotein expression and mRNA expression levels.The expression of TNF-αprotein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01) ; with the excipient group rather,there was no significant difference (P>0.05).Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of TNF-αprotein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group and normal group, no significant difference (P>0.05). High dose cataplasms group the lowest TNF-αprotein expression and mRNA expression levels.
Intestinal tissue: The normal group showed little expression in intestinal epithelial cells, and could detect small amounts of TNF-αprotein expression and mRNA expression levels. The expression of TNF-αprotein and mRNA were the highest concentrations in model group,with the excipient group,no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of TNF-αprotein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group and normal group,no significant difference (P>0.05). High dose cataplasms group the lowest TNF-αprotein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, TNF-αprotein expression and mRNA expression levels did not appear correlated (P>0.05); the model group, the excipient groups, fuxiong-cream group, the high dose cataplasms group, TNF-αexpression in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlated in the low dose cataplasms group(P>0.05).The correlation of the lung and intestinal tissue in the high dose cataplasms group were higher than fuxiong-cream group. 7.IL-10 protein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:The normal group could detect small amounts of IL-10 protein expression and mRNA expression levels.IL-10 protein expression and mRNA levels in the model group were higher than the normal group,with significant differences(P<0.05);with the excipient group,no significant difference(P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of IL-10 protein and mRNA were significantly higher than the normal group,and higher than the model group(P<0.05); and the three pairwise comparisons were significantly different(P<0.05).High dose cataplasms group the highest IL-10 protein expression and mRNA expression levels.
Intestinal tissue: The normal group could detect small amounts of IL-10 protein expression and mRNA expression levels. IL-10 protein expression and mRNA levels in the model group were higher than the normal group ,with significant differences(P<0.05～0.01); with the excipient group, no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of IL-10 protein and mRNA were significantly higher than the normal group,and higher than the model group(P<0.05); and the three pairwise comparisons were significantly different(P<0.05).High dose cataplasms group the highest IL-10 protein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, IL-10 protein expression and mRNA expression levels did not appear correlated (P>0.05); In the model group, the excipient group, fuxiong-cream group,the high dose cataplasms group, IL-10 protein expression and mRNA expression levels in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlatedin the low dose cataplasms group (P>0.05). Sort by Relevance:the high dose cataplasms group > the model group > the excipient group >fuxiong-cream group.
8.NF-κB P65 protein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:In the normal group could detect small amounts of NF-κB P65 protein expression and mRNA expression levels.The expression of NF-κB P65 protein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01);with the excipient group rather,there was no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of NF-κB P65 protein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group the lowest NF-κB P65 protein expression and mRNA expression levels.
Intestinal tissue:In the normal group could detect small amounts of NF-κB P65 protein expression and mRNA expression levels. The expression of NF-κB P65 protein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01) ;with the excipient group,no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of NF-κB P65 protein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group the lowest NF-κB P65 protein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, NF-κB P65 protein expression and mRNA expression levels did not appear correlated (P>0.05); the model group, the excipient groups, fuxiong-cream group, the high dose cataplasms group, NF-κB P65 expression in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlated in the low dose cataplasms group(P>0.05).Sort by Relevance:the high dose cataplasms group>the model group>the excipient group>fuxiong-cream group.
9.The content of The total free anthraquinones in the fuxiong- cataplasms was 11.9023mg esch,that in fuxiong- cream is 5.8702mg each, there was no the total free anthraquinones in excipient.
10.It is different Active ingredient from the fuxiong- cataplasms and the fuxiong- cream, the content of rhein in cream is lower than that in cataplasms, but the contents of emodin、rhein、aoe-emodin、chrysophanol and physcion in cream are higher.
11.Starting from the forth hour, the Major components- emodin、chrysophanol in fuxiong-cataplasms,that permeation in unit area are higher than fuxiong- cream, as time been longer, as the difference value of permeation in unit area is bigger.
Conclusion:
1.The fuxiong- cataplasms reduced mortality of influenza virus to infect mice pneumonia;and reduced losses in transit visibly, its eased Lung inflammation, and been Inhibition of influenza viral to infect Rat lung replication。
2.Pathogenesis of influenza viral Pneumonia maybe relate to excessive increasing inflammatory and activation of transcription factor NF-κB P65;The treatment of fuxiong- cataplasms maybe related to degrade TNF-α、raise IL-10 and interruption of NF-κB P65 expreesion.
3.When the dose of fuxiong- cataplasms was up to a certain dose, the same indicator in the pulmonary and intestinal organizations were relevant to the treatment effect,which was correspond to the theory of“lung associating with large intestine”.
4.HPLC become a mature technology to determin contents and quality control.
5.The fuxiong- cataplasms is a safe and reliable Chinese herb preparations, and there is good percutaneous penetration, improved formulations possible.
材料与方法:
1.采用鼻腔接种流感病毒FM1株诱导昆明种小鼠致肺炎,观察小鼠在一般状态、肺组织的病理组织学等方面的差异,并应用RT-PCR技术检测病毒载量、克隆测序基因比对等多角度进行模型鉴定。
2.通过皮肤刺激实验观察敷胸巴布贴对完整皮肤和破损皮肤的刺激作用。
3.将传统敷胸膏与不同剂量的敷胸巴布贴以外敷在小鼠背部(相当于肺腧穴、膏肓穴等)为给药途径治疗小鼠IV肺炎,观察各组药物对小鼠一般状态、死亡保护作用、肺指数、肺悬液血凝滴度的影响。
4.采用免疫组织化学方法和RT-PCR技术检测IV诱导小鼠肺炎的肺肠组织中促炎因子TNF-α、抗炎因子IL-10的蛋白及mRNA的表达水平,比较传统敷胸膏与不同剂量敷胸巴布贴对IV肺炎小鼠肺肠组织中TNF-α、IL-10改变的影响并对同一指标进行相关性分析。
5.采用免疫组织化学方法和RT-PCR技术检测IV诱导小鼠肺炎的肺肠组织中转录因子NF-κB P65蛋白及mRNA的表达水平,比较传统敷胸膏与不同剂量敷胸巴布贴对IV肺炎小鼠肺肠组织中NF-κB P65改变的影响并进行相关性分析。
6.应用HPLC法测定敷胸巴布贴中大黄的五种活性成分含量。
7.以离体大鼠皮肤为透皮屏障,采用改良的Franz扩散装置,应用HPLC法测定大黄素、大黄酚的经皮渗透量,考察敷胸巴布贴透皮吸收情况。
结果:
1.采用鼻腔接种流感病毒FM1株诱导昆明种小鼠致肺炎,模型组病理改变有肺泡间隔增厚、水肿,内含有大量炎细胞浸润,肺泡壁毛细血管扩张,间见大量红细胞。小鼠感染IV第3～10天肺组织中病毒核酸阳性检出率达100%。将模型组小鼠肺组织的IV RT-PCR扩增产物,采用克隆测序方法测序,与美国国立生物信息中心已知的IV cDNA全序列比较,吻合率达到99.06%。
2.敷胸巴布贴在完整皮肤用药部位未出现红斑、水肿等皮肤刺激反应;而对破损皮肤,用药部位出现轻度刺激反应。
3.死亡保护作用结果:低、高剂量巴布贴组对IV感染小鼠的死亡保护率分别是25%、40%;延长生命率是33.33%、58.52%。与模型组比较,死亡率明显降低,平均存活天数明显延长(P<0.01)。可见高剂量巴布贴的死亡保护率最高、平均存活时间最长。
4.体重结果:正常组体重逐日增长;模型组体重逐日下降,第3天开始直至第7天较正常组明显下降(P<0.05);敷胸巴布贴组和敷胸膏组较模型组体重下降变慢,从第6天起体重有所增长,敷胸巴布贴组第6天和第7天体重较模型组明显升高(P<0.01)。
5.肺指数、肺组织血凝滴度结果:模型组较正常组肺指数明显升高,低、高剂量巴布贴组和敷胸膏组较模型组肺指数明显降低(P<0.05)。低、高剂量巴布贴组分别与敷胸膏组比较,均有显著性差异(P<0.05)。低、高剂量巴布贴组和敷胸膏组的血凝滴度较模型组明显降低(P<0.05),且高剂量巴布贴组疗效最佳。
6.IV诱导小鼠肺炎的肺肠组织中TNF-α蛋白表达和mRNA表达水平及相关性分析:
肺组织:正常组可见少许表达于支气管上皮细胞,可以检测到少量的TNF-α蛋白表达和mRNA表达水平。模型组TNF-α蛋白表达和mRNA含量最高,与正常组比较,有显著性差异(P<0.01);与赋形剂组相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组TNF-α蛋白表达和mRNA表达水平均高于正常组,但明显低于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组与正常组比较,无显著性差异(P>0.05)。高剂量巴布贴组TNF-α蛋白表达和mRNA表达水平最低。
肠组织:正常组可见少许表达于肠上皮细胞,可以检测到少量的TNF-α蛋白和mRNA表达水平。模型组TNF-α蛋白表达和mRNA含量最高,明显高于正常组,有显著性差异(P<0.05);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组TNF-α蛋白表达和mRNA表达水平均高于正常组,且明显低于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组与正常组比较,无显著性差异(P>0.05)。高剂量巴布贴组TNF-α蛋白表达和mRNA表达水平最低。
相关性分析:正常组肺肠组织TNF-α蛋白表达和mRNA表达水平未出现相关性(P>0.05);模型组、赋形剂组、敷胸膏组、高剂量巴布贴组出现正相关(P<0.05);低剂量巴布贴组未出现相关性(P>0.05)。高剂量巴布贴组肺肠组织相关性均高于敷胸膏组。7.IV诱导小鼠肺炎的肺肠组织中IL-10蛋白和mRNA表达水平及相关性分析:
肺组织:正常组可以检测到少量的IL-10蛋白表达和mRNA表达水平。模型组IL-10蛋白表达和mRNA含量均高于正常组,具有显著性差异(P<0.05);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组IL-10蛋白表达和mRNA表达水平均明显高于正常组,但高于模型组(P<0.05);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组IL-10蛋白和mRNA表达水平最高。
肠组织:正常组可以检测到少量的IL-10蛋白表达和mRNA表达水平。模型组IL-10蛋白表达和mRNA含量均高于正常组,具有显著性差异(P<0.05～0.01);与赋形剂组比较,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组IL-10蛋白表达和mRNA表达水平均明显高于正常组,且高于模型组(P<0.05～0.01);且两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组IL-10蛋白表达和mRNA表达水平最高。
相关性分析:正常组肺肠组织IL-10蛋白和mRNA表达水平未出现相关性(P>0.05);模型组、赋形剂组、敷胸膏组、高剂量巴布贴组出现正相关(P<0.05);低剂量巴布贴组未出现相关性(P>0.05)。相关性排序:高剂量巴布贴组>模型组>赋形剂组>敷胸膏组。
8.IV诱导小鼠肺炎的肺肠组织中NF-κB蛋白表达和mRNA表达水平及相关性分析:
肺组织:正常组可以检测到少量的NF-κB P65蛋白表达和mRNA表达水平。模型组NF-κB P65蛋白表达和mRNA表达水平最高,与正常组比较,具有显著性差异(P<0.05);赋形剂组表达相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组的NF-κB P65蛋白表达和mRNA表达水平均高于正常组,且明显低于模型组(P<0.05～0.01);两两比较,均有显著性差异(P<0.05)。高剂量巴布贴组NF-κB P65蛋白表达和mRNA表达水平最低。
肠组织:正常组可以检测到少量的NF-κB P65蛋白表达和mRNA表达水平。模型组NF-κB P65蛋白表达和mRNA表达水平最高,与正常组比较,具有显著性差异(P<0.05);与赋形剂组表达相当,无显著性差异(P>0.05)。低、高剂量巴布贴组、敷胸膏组的NF-κB P65蛋白表达和mRNA表达水平均高于正常组(P<0.01),且敷胸膏组、高剂量巴布贴组明显低于模型组(P<0.01),而低剂量巴布贴组的NF-κB P65蛋白表达和mRNA表达水平与模型组比较,无显著性差异(P>0.05)。三组药物组两两比较,均有显著性差异(P<0.05),高剂量巴布贴组NF-κB P65蛋白表达mRNA表达水平最低。
相关性分析:正常组肺肠组织中NF-κB P65蛋白表达和mRNA表达水平未出现相关性(P>0.05),模型组、赋形剂组、敷胸膏组、高剂量巴布贴组均出现正相关(P<0.05),低剂量巴布贴组未出现相关性(P>0.05)。相关性排序:高剂量巴布贴组>模型组>赋形剂组>敷胸膏组。
9.每片敷胸巴布贴中游离总蒽醌的含量是11.9023mg,每克敷胸膏中游离总蒽醌的含量是5.8702mg,赋形剂中未检出游离蒽醌。
10.敷胸巴布贴和敷胸膏中主要的有效成分是有差异的,敷胸膏中除大黄酸含量低于巴布贴外,大黄素、大黄酸、芦荟大黄素、大黄酚、大黄素甲醚含量均不同程度的高于敷胸巴布贴。
11.敷胸巴布贴的主要成分大黄素、大黄酚从给药4 h起,在各个时点的单位面积累积渗透量均高于敷胸膏,并随着时间的延长,单位面积累积渗透量的差值越来越大。
结论:
1.敷胸巴布贴对IV诱导小鼠肺炎具有一定的死亡保护作用;可明显缓解体重的降低,并减轻其肺部炎性病变,有一定的抑制鼠肺IV增殖作用。
2.IV肺炎的发病机制可能与促炎因子TNF-α、抗炎因子IL-10异常升高及转录因子NF-κB P65的过度活化有关;敷胸巴布贴治疗IV肺炎可能与降低TNF-α、提高IL-10及抑制NF-κB P65表达水平有关。
3.推测当敷胸巴布贴药量达到一定剂量,使肺肠组织中同一指标具有相关性时,即可能达到肺肠同治的治疗效果,验证了“肺合大肠”理论。
4.HPLC法可作为敷胸巴布贴质量控制及含量测定可靠的方法。
5.敷胸巴布贴是一种安全可靠的中药外用制剂,且有较好的经皮渗透性,剂型改良可行。
Purpose:From the " lung associating with large intestine " the ancient and modern discourse, extract the physiology and pathology relationship between lung and colon, to explore the application rationale of fuxiong- cataplasms. Through the method of cloning and sequencing to identify the influenza A virus induced animal model to provide a reliable and stable animal model for the influenza A virus prevention of traditional Chinese medicine.Through cutaneous stimulation experiment to evaluate the safety of fuxiong- cataplasms to provide the medication evidence for clinical implication.Comparing with the fuxiong-masticp and fuxiong- cataplasms, through the study of pharmacodynamics and immunologic mechanism of differnet doses of fuxiong- cataplasms on IV induced mice pneumonia,to explore the treatment of fuxiong- cataplasms and validate the rationale of " lung associating with large intestine ",to provie the experiment and theory foundation for the traditional chinese medicine research;Through high efficiency liquid chromatography (HPLC) to determine the active component contents of fuxiong- cataplasms and percutaneous permeability experiment to identify its material foundation of IV infected pneumonia treatment.
Material and method:
1.Using influenza A virus FM1 to infect mice to mice pneumonia, observed the general status、the differences of lung tissue, applied RT-PCR method to detect IV-RNA viral load、clone sequencing gene correlation to accredit the animal model.
2.Through cutaneous stimulation experiment to observe the stimulatory function of fuxiong- cataplasms on intact and breakage skin.
3.Put the fuxiong-masticp and different doses of fuxiong- cataplasms on the back of the mice(equal to Lung acupoints and Gaohuangshu points) to treat the mice pneumonia and observe the general state、mortality、lung index、hemagglutination titer of lung suspension.
4.By immunohistochemistry and RT-PCR detection of lung pro-inflammatory factors in intestinal tissue TNF-α, anti-inflammatory factor IL-10 protein and mRNA expression levels, compare with the variance of TNF-α, IL-10 in IV induced mice pneumonia lung and intestine issues of fuxiong-masticp and different doses of fuxiong- cataplasms,and the dependablity analysis was available for the same index.
5.By immunohistochemistry and RT-PCR method to detect lung and intestine transcription factor NF-κB P65 protein and mRNA expression levels, compare with the variance of NF IV -κB P65 in IV induced mice pneumonia lung and intestine issues of fuxiong-masticp and different doses of fuxiong- cataplasms and the dependablity analysis was available.
6.Application of HPLC method to determin the five active components in the fuxiong- cataplasms.
7. The skin excised rat was used as transdermal barrier, using the modified Franz diffusion devices,HPLC was used to determin the percutaneous permeation centent of emodin and rhein to investigate the percutaneous permeation conditon of fuxiong- cataplasms.
Results:
1.Intranasal inoculation with influenza virus FM1 strain caused pneumonia in mice were induced, pathological changes in the model group had alveolar septal thickening, edema, containing a large number of inflammatory cell infiltration, alveolar wall telangiectasia, a large number of red blood cells between the see. Mice were infected IV 3 to 10 days of virus DNA in lung tissue positive detection rate of 100%. The model mice lung tissue IV RT-PCR amplification, cloning and sequencing methods using DNA sequencing, and the United States National Center for Biotechnology Information known IV cDNA sequences comparison with the rate of 99.06%。
2.External using the fuxiong- cataplasms and the excipient, there is no skin stimulus response on the intact skin, on the contrary, external using on the damaged skin,has mild irritation response.
3.Protective effect of death results: low dose and high dose group barb post death in mice infected with influenza virus protection rate was 25%, 40%; prolong life rate is 33.33%, 58.52%. Compared with model group, mortality was reduced, the average survival time was significantly longer (P<0.01).the barb post can be seen affixed to the death of high doses of the highest protection rate, the average survival time is the longest。
4.Weight results: growth of normal body weight daily; model group decreased body weight daily, 3 day until day 7 was significantly lower than normal (P<0.05); dressing fuxiong- cream paste group and apply weight loss group than in model group Slow, from 6 days increased body weight, dressing fuxiong- cataplasms Group 6 and 7 days was significantly higher body weight compared with model group (P <0.01)。
5.Lung index, the virus titer Results: the model group was significantly higher than normal increase in lung index, deposited fuxiong-cream group and low and high dose group than in model group, fuxiong- cataplasms the lung index was significantly lower (P<0.05). Low and high dose groups were posted and deposited fuxiong-cream group, there were significant differences (P<0.05). Apply ointment group and the low thoracic and high dose groups hemagglutination barb posted significantly lower than the model (P<0.05), and the efficacy of high dose group posted the best.
6.TNF-αprotein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:The normal group showed little expression in bronchial epithelial cells, and could detect small amounts of TNF-αprotein expression and mRNA expression levels.The expression of TNF-αprotein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01) ; with the excipient group rather,there was no significant difference (P>0.05).Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of TNF-αprotein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group and normal group, no significant difference (P>0.05). High dose cataplasms group the lowest TNF-αprotein expression and mRNA expression levels.
Intestinal tissue: The normal group showed little expression in intestinal epithelial cells, and could detect small amounts of TNF-αprotein expression and mRNA expression levels. The expression of TNF-αprotein and mRNA were the highest concentrations in model group,with the excipient group,no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of TNF-αprotein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group and normal group,no significant difference (P>0.05). High dose cataplasms group the lowest TNF-αprotein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, TNF-αprotein expression and mRNA expression levels did not appear correlated (P>0.05); the model group, the excipient groups, fuxiong-cream group, the high dose cataplasms group, TNF-αexpression in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlated in the low dose cataplasms group(P>0.05).The correlation of the lung and intestinal tissue in the high dose cataplasms group were higher than fuxiong-cream group. 7.IL-10 protein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:The normal group could detect small amounts of IL-10 protein expression and mRNA expression levels.IL-10 protein expression and mRNA levels in the model group were higher than the normal group,with significant differences(P<0.05);with the excipient group,no significant difference(P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of IL-10 protein and mRNA were significantly higher than the normal group,and higher than the model group(P<0.05); and the three pairwise comparisons were significantly different(P<0.05).High dose cataplasms group the highest IL-10 protein expression and mRNA expression levels.
Intestinal tissue: The normal group could detect small amounts of IL-10 protein expression and mRNA expression levels. IL-10 protein expression and mRNA levels in the model group were higher than the normal group ,with significant differences(P<0.05～0.01); with the excipient group, no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of IL-10 protein and mRNA were significantly higher than the normal group,and higher than the model group(P<0.05); and the three pairwise comparisons were significantly different(P<0.05).High dose cataplasms group the highest IL-10 protein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, IL-10 protein expression and mRNA expression levels did not appear correlated (P>0.05); In the model group, the excipient group, fuxiong-cream group,the high dose cataplasms group, IL-10 protein expression and mRNA expression levels in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlatedin the low dose cataplasms group (P>0.05). Sort by Relevance:the high dose cataplasms group > the model group > the excipient group >fuxiong-cream group.
8.NF-κB P65 protein expression and mRNA expression levels in the lung and intestinal tissue and correlation analysis:
Lung tissue:In the normal group could detect small amounts of NF-κB P65 protein expression and mRNA expression levels.The expression of NF-κB P65 protein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01);with the excipient group rather,there was no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of NF-κB P65 protein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group the lowest NF-κB P65 protein expression and mRNA expression levels.
Intestinal tissue:In the normal group could detect small amounts of NF-κB P65 protein expression and mRNA expression levels. The expression of NF-κB P65 protein and mRNA were the highest concentrations in model group, with the normal group, there was significant difference (P<0.01) ;with the excipient group,no significant difference (P>0.05). Apply the low and high doses fuxiong- cataplasms group and fuxiong-cream group, the expression of NF-κB P65 protein and mRNA were higher than the normal group,but significantly lower than the model group (P<0.05); and the three pairwise comparisons were significantly different (P<0.05). High dose cataplasms group the lowest NF-κB P65 protein expression and mRNA expression levels.
Correlation analysis: The lung and intestinal tissue in the normal groups, NF-κB P65 protein expression and mRNA expression levels did not appear correlated (P>0.05); the model group, the excipient groups, fuxiong-cream group, the high dose cataplasms group, NF-κB P65 expression in the lung and intestinal tissue showed positive correlation (P<0.05) and did not appear correlated in the low dose cataplasms group(P>0.05).Sort by Relevance:the high dose cataplasms group>the model group>the excipient group>fuxiong-cream group.
9.The content of The total free anthraquinones in the fuxiong- cataplasms was 11.9023mg esch,that in fuxiong- cream is 5.8702mg each, there was no the total free anthraquinones in excipient.
10.It is different Active ingredient from the fuxiong- cataplasms and the fuxiong- cream, the content of rhein in cream is lower than that in cataplasms, but the contents of emodin、rhein、aoe-emodin、chrysophanol and physcion in cream are higher.
11.Starting from the forth hour, the Major components- emodin、chrysophanol in fuxiong-cataplasms,that permeation in unit area are higher than fuxiong- cream, as time been longer, as the difference value of permeation in unit area is bigger.
Conclusion:
1.The fuxiong- cataplasms reduced mortality of influenza virus to infect mice pneumonia;and reduced losses in transit visibly, its eased Lung inflammation, and been Inhibition of influenza viral to infect Rat lung replication。
2.Pathogenesis of influenza viral Pneumonia maybe relate to excessive increasing inflammatory and activation of transcription factor NF-κB P65;The treatment of fuxiong- cataplasms maybe related to degrade TNF-α、raise IL-10 and interruption of NF-κB P65 expreesion.
3.When the dose of fuxiong- cataplasms was up to a certain dose, the same indicator in the pulmonary and intestinal organizations were relevant to the treatment effect,which was correspond to the theory of“lung associating with large intestine”.
4.HPLC become a mature technology to determin contents and quality control.
5.The fuxiong- cataplasms is a safe and reliable Chinese herb preparations, and there is good percutaneous penetration, improved formulations possible.
引文
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