摘要
呼吸道病毒感染为临床常见病和多发病,其中以流感病毒对人类的健康影响最为严重,且近几年发生呈活跃势态。病毒性疾病临床多表现湿热证特性,尤其是在岭南地区由于受气候地理环境,饮食习惯等影响,呼吸道病毒感染性疾病湿热特征更为明显。湿热证与水液代谢关系密切,而水通道蛋白(aquaporin,AQP)是人体内调节水液代谢的重要物质,因此湿热证与水通道蛋白之间的关系也引起了学者的广泛关注。同时,根据中医理论,湿热病邪蒸腾氤氲,弥漫三焦,对全身脏腑水液代谢都产生影响,因此在探讨水通道蛋白变化过程中应注意从整体观入手,观察和水液代谢相关脏腑的变化。前期实验和临床实践显示蒿芩清胆汤治疗岭南呼吸道病毒感染湿热证有显著疗效,本实验通过观察湿热因素对流感病毒感染湿热证小鼠水通道蛋白的影响,和蒿芩清胆汤及其拆方对此的干预作用,以期探讨湿热证在水液代谢异常方面的物质基础以及蒿芩清胆汤在调节水液代谢方面的作用。
目的:
本研究以湿热环境+肥甘饮食+致病因子建立流感病毒感染湿热证小鼠动物模型,观察内外湿热环境对小鼠肺组织病毒表达的影响以及对小鼠肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2的影响,探讨湿热证在水液代谢异常方面的物质基础。同时通过蒿芩清胆汤及其拆方对流感病毒感染湿热证小鼠动物模型小鼠肺组织病毒表达的影响以及对小鼠肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2的影响,探讨蒿芩清胆汤治疗病毒性疾病湿热证在“和胃化痰祛湿”方面作用的机制和作用靶点。
方法:
1.内外湿热对流感病毒感染湿热证小鼠多脏器水通道蛋白表达的影响作用:50只BALB/c小鼠随机分为4组:正常对照组、单纯湿热组(湿热环境+肥甘饮食)、湿热模型组(湿热环境+肥甘饮食+病毒感染)、单纯病毒组(病毒感染),观察小鼠宏观症状表现,处死后计算小鼠肺指数,免疫组化法检测各组小鼠肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2变化,FQ-PCR法检测各组小鼠肺组织流感病毒、肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2变化。
2.蒿芩清胆汤及拆方对小鼠流感病毒感染湿热证水通道蛋白的作用:115只BALB/c小鼠随机分为8组:正常对照组、湿热模型组、利巴韦林组、蒿芩清胆汤组、拆方1组、拆方2组、拆方3组、拆方4组,观察小鼠宏观症状表现,计算小鼠肺指数,免疫组化法检测各组小鼠肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2变化,FQ-PCR法检测各组小鼠肺组织流感病毒、肺AQP1、AQP5,胃AQP4,结肠AQP1及肾AQP2变化。
结果:
1湿热模型组死亡率(26.7%)略高于单纯病毒组(20.0%)。湿热模型组肺指数较单纯病毒组无统计学差异(P>0.05)。
2湿热模型组小鼠肺AQP1、肺AQP5、胃AQP4、肠AQP1、肾AQP2表达与正常对照组相比均有显著性差异,单纯湿热组与正常对照组比较各脏器水通道蛋白表达无显著性差异(P>0.05),与湿热模型组比较有显著性差异(P<0.01或P<0.05)。单纯病毒组与正常对照组比较肺AQPl、胃AQP4、肠AQP1、肾AQP2表达无显著性差异(P>0.05),肺AQP5表达有显著性差异(P<0.05),与湿热模型组比较肺AQP1、肺AQP5表达无显著性差异(P>0.05),胃AQP4、肠AQP1、肾AQP2表达有显著性差异(P<0.01)
3蒿芩清胆汤组、利巴韦林组和拆方3组与湿热证模型组比较流感病毒mRNA表达有显著性差异(P<0.01或P<0.05)。
4蒿芩清胆汤组、拆方3组、拆方4组与湿热模型组比较,小鼠肺AQP1、肺AQP5、胃AQP4、肠AQP1、肾AQP2表达均有显著性差异(P<0.01或P<0.05);拆方1组与湿热模型组比较胃AQP4、肠AQP1、肾AQP2表达均有显著性差异(P<0.01或P<0.05);拆方2组与湿热模型组比较胃AQP4、肾AQP2表达均有显著性差异(P<0.01或P<0.05);
5蒿芩清胆汤组与拆方3组、拆方4组比较,小鼠肺AQP1、肺AQP5、胃AQP4、肠AQP1、肾AQP2表达均无显著性差异(P>0.05);蒿芩清胆汤组与拆方1组比较,肺AQP1、肺AQP5、胃AQP4表达均有显著性差异(P<0.01或P<0.05);蒿芩清胆汤组与拆方2组比较,肺AQP1、胃AQP4、肠AQP1、肾AQP2表达均有显著性差异(P<0.01或P<0.05)。
结论:
1湿热模型组和单纯病毒组比较,肺组织病毒含量无明显差异,说明内外湿热因素并不能影响流感病毒在体内的复制。
2内外湿热与流感病毒感染共同作用,会影响机体的水液代谢,出现全身水液代谢紊乱,而单纯的内外湿热因素或单纯病毒感染无此作用。
3湿热证动物模型小鼠感染流感病毒后,病位主要在肺,却表现出的多部位的水通道蛋白表达异常,与中医学湿热弥漫三焦的理论契合。提示水通道蛋白的表达异常可能是湿热证的物质基础之一,同时,对水通道蛋白与湿热证关系的研究应从整体观入手,不应局限在一脏一腑。
4蒿芩清胆汤能够抑制流感病毒感染湿热证小鼠流感病毒的复制,其中竹茹、半夏、茯苓、滑石、甘草等化痰和胃祛湿类药物对抑制病海复制也起到一定作用,推测其原因可能是通过调节机体内水液代谢紊乱状态,从而有利于青蒿、黄芩、大青叶、板蓝根等药物对病毒的抑制。
5蒿芩清胆汤能够调节流感病毒感染湿热证小鼠肺AQP1、肺AQP5、胃AQP4、肠AQP1、肾AQP2表达的紊乱,这可能是蒿芩清胆汤“祛湿”作用的物质基础,且其作用是全身性的。
6蒿芩清胆汤中青蒿、黄芩与竹茹、半夏、茯苓、滑石、甘草的组合在调节水液代谢方面起到了重要作用,而陈皮、枳壳、大青叶、板蓝根在水液代谢调节方面作用不明显。
Viral infections of respirovirus are common and frequently occurring diseases, in which the influenza was most severe and broke out actively recent years. Viral diseases clinical manifestations of damp-heat syndrome characteristics, especially in the south of the Five Ridges area because of the climate and geographical environment, eating habits and other effects, viral infection of the respiratory tract are more obvious. Damp-heat syndrom and fluid metabolism are closely related, and aquaporin (AQP) is the important material of body's regulation of water metabolism, so the damp-heat syndrome and aquaporin relationship also caused the extensive concern of scholars. At the same time, according to Chinese medicine theory, damp-heat pathogen diffuse the whole organs, cause body fluid metabolism disfunction, therefore to observe the aquaporin changes, we should observe all water metabolism related organs changes. Preliminary experimental and clinical practice showed Haoqin Qingdan Decoction in treating virus infection of respirovirus infection in south of the Five Ridges of damp-heat syndrome have a significant effect, through observe the effects of damp-heat factor on aquaporin in damp-heat syndrome of mouse by influenza virus infection, and Haoqin Qingdan Decoction and its disassembled prescription to this intervention effect, to discuss the material foundation of damp-heat in body fluid metabolism disfunction and Haoqin Qingdan Decoction in regulating the metabolism of body fluid.
Object ive:
We use heat and humid environment+richly fatty and sweet diet+pathogenic factor to establish the animal model of influenza virus infect mouse with damp-heat syndrome. Observe endogenous and exogenous damp-heal factors'effects on the expressions of influenza virus in lung, AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in mice, to discuss the material foundation of damp-heat in body fluid metabolism disfunction. At the same time, Observe Haoqin Qingdan Decoction and its disassembled prescription effects on the expressions of influenza virus in lung, AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in mice, to discuss the treatment mechanisms of Haoqin Qingdan Decoction in viral diseases in damp-heat syndrome by eliminating dampness.
Methods
1. To study the effect of exo-endo damp-heat factors on AQPs in different organ of mouse with damp-heat syndrome and influenza virus infection:50mice of BALB/c were devided into4group randomly (control group, damp-heat group, model group, viral group), then observed the symptom and sign of mice. After executions, observe lung index and the expression of Influenza virus in lung, AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in mice by immunohistochemical method and FQ-PCR.
2. To study the effect of Haoqin Qingdan Decoction and its disassembled prescription on AQPs in different organ of mouse with damp-heat syndrome and influenza virus infection:115mice of BALB/c were devided into8group randomly (control group, model group, Ribavirin group, HQQD group, decomposed1group, decomposed2group, decomposed3group, decomposed4group), then observed the symptom and sign of mice. After executions, observe lung index and the expression of Influenza virus in lung, AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in mice by immunohistochemical method and FQ-PCR.
Results:
1. The mortality of model group was a little higher than viral group. The lung index of model group is a little lower than viral group, but the difference wasn't significant (P>0.05).
2. The expression of AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in model group shows significant difference compaired with control group. The expression of AQPs shows no signi ficant difference between damp-heat group and control group (P>0.05). The expression of AQPs shows significant difference between damp-heat group and control group (P<0.01 或P<0.05). Viral group cmopaired with control group, the expression of AQP1in lung, AQP4in stomach, AQP1in colon and AQP2in kidney shows no significant difference (P>0.05), and the expression of AQP5in lung shows significant difference (P<0.05). Model group cmopaired with Viral group, the expression of AQPland AQP5in lung shows no significant difference (P>0.05), the expression of AQP4in stomach, AQP1in colon and AQP2in kidney shows significant difference (P<0.01).
3. The expression of influenza virus mRNA in HQQD group, Ribavirin group and decomposed3group shows significant difference compaired with Model group (P<0.01or P<0.05)
4. HQQD group, decomposed3group and decomposed4group compaired with Model group, the expression of AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney in model group shows significant difference (P<0.01or P<0.05). Decomposed1group compaired with Model group, the expression of AQP4in stomach, AQP1in colon and AQP2in kidney shows significant difference (P<0.01or P<0.05). Decomposed2group compaired with Model group, the expression of AQP4in stomach and AQP2in kidney shows significant difference (P<0.01or P<0.05)
5. HQQD group compaired with decomposed3group and decomposed4group, the expression of AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney shows no significant difference (P>0.05). HQQD group compaired with decomposed1group, the expression of AQP1、AQP5in lung, AQP4in stomach shows significant difference (P<0.01or P<0.05). HQQD group compaired with decomposed2group, the expression of AQP1in lung, AQP4in stomach, AQP1in colon and AQP2in kidney shows significant difference (P<0.01or P<0.05)
Conelusions:
1. Model group compaired with viral group, the expression of influenza virus shows no significant difference, it means that exo-endo damp-heat factors can not influence the copy of influenza virus.
2. The joint action of exo-endo damp-heat factors and influenza virus infection can cause body fluid metabolism disfunction, but the single factor exo-endo damp-heat or influenza virus infection shows no such effect.
3. The patho]ogical organ of model group is lung, but the expression of AQPs in many organ are abnormal, this result fits the theory of damp-heat diffuse triple energizer in TCM. This result indicates that abnormal expression of AQPs may be one of the important, material foundations of damp-heat syndrome. It also indicates that we should study the relationship between AQPs and damp-heat syndrome through the integral view.
4. Haoqin Qingdan Decoction can inhibite the copy of influenza virus in influenza virus infect mouse with damp-heat syndrome. Zhuru, Banxia, Puling, Huashi, Gancao in Haoqin Qingdan Decoction can inhibite the copy of influenza virus, we assume the reason is that this kind of herb can regulate the disfunction of body fluid metabolism, and coordinate the function of Qinghao, Huangqin, Daqingye, Banlangen.
5. Haoqin Qingdan Decoction can regulate the expression of AQP1、AQP5in lung, AQP4in stomach, AQP1in colon and AQP2in kidney of influenza virus infect mouse with damp-heat syndrome. This may be the material foundation of eliminating dampness caused by Haoqin Qingdan Decoction, and the effect influence the whole bady.
6. Qinghao, Huangqin, Zhuru, Banxia, Fuling, Huashi, Gancao in Haoqin Qingdan Decoction show important effect to regulate the disfunction of body fluid metabolism, Chenpi, Zhike, Daqingye, Banlangen in Haoqin Qingdan Decoction show no such effect.
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