扶正解毒化瘀方及有效部位干预RSV肺炎小鼠的作用机制研究
发布时间：2018-10-19 18:38
【摘要】：病毒性肺炎(VP)是临床常见的呼吸道感染性疾病,多见于儿童、老年人、免疫低下宿主及合并心肺基础病患者。近年来,随着社会老龄化加速、免疫损伤宿主增加、病毒变异和新病毒的产生,包括呼吸道合胞病毒(RSV)肺炎在内的VP的发病率明显增高,严重威胁着人类健康。目前,其治疗尚缺乏专属性强的特效治疗药物。中医学以整体观、辨证论治为特色,具有多靶点、多途径、多环节作用的特点,在防治病毒感染性疾病方面具有明显优势。导师临证数十载,长期致力于中医药防治呼吸道感染性疾病的研究,认为"虚、毒、痰、瘀"是病毒性肺炎的核心病机关键,治当以扶正、解毒、化瘀为大法。遂拟定了扶正解毒化瘀方,长期临床实践证实,安全可靠。该方由西洋参、黄芩、连翘、漏芦、赤芍、瓜萎、败酱草和生薏苡仁组成,具有扶正祛邪,清热解毒,活血化瘀之功,主要用于治疗多种病原体所致的肺部感染。前期研究表明,扶正解毒化瘀方可有效减轻肺炎症状、促进炎症吸收,控制疾病进展。对RSV所致的肺部感染疗效显著,但其治疗的物质基础和作用机制尚不清楚。本研究以RSV肺炎为切入点,探讨扶正解毒化瘀方及有效部位干预病毒性肺炎的作用机制。本论文包括综述和实验研究两部分。综述部分,系统阐述了中医学及现代医学对RSV肺炎的认识及研究进展,为实验研究提供了理论基础。实验部分主要从组织形态学,细胞分子生物学等角度探讨扶正解毒化瘀方及有效部位抗病毒作用机制。目的:从凋亡基因、PI3K/Akt信号通路相关调控蛋白、细胞因子及肺组织病理变化等多角度探讨扶正解毒化瘀方及有效部位干预RSV肺炎小鼠的作用机制。方法:将180只雌性BALB/c小鼠按感染后第1、3、5天三个时间点,随机分为3个大组,每组60只。每个大组分为正常组、模型组、全方组、总苷组、挥发油组及利巴韦林组6个小组,每组10只。RSV液滴鼻感染BALB/c小鼠,用扶正解毒化瘀方及有效部位进行干预,并于感染后第1、3、5天分别取小鼠肺组织,计算各组肺指数。并做病理组织切片,采用光学显微镜和电子显微镜观察小鼠肺组织病变情况。Real-time PCR法检测肺组织中Fas、P53及caspase-12 mRNA表达。免疫组化法检测肺组织中Akt、GSK-3 β及NF-κB p65蛋白的表达。ELISA法检测肺组织中IL-1β、IL-8、IFN-α、TLR3和TLR7的表达。结果:1.RSV感染BALB/c小鼠肺指数感染后不同时间点,模型组肺指数均有不同程度增高,且呈递增趋势;与正常组比较,均有显著差异(P0.01)。感染后第1、3、5天各给药组均可有效降低肺指数,与模型组比较差异显著(P0.05)。感染后第3、5天全方组与利巴韦林组疗效相当,且优于总苷组和挥发油组(P0.05)。全方组对肺指数抑制作用较总苷组、挥发油组有一定优势,感染后第3天最明显。2.RSV感染BALB/c小鼠肺病理改变RSV感染后主要表现为肺间质炎症,肺泡壁充血、水肿,明显增厚;肺间质有大量炎症细胞浸润;部分肺泡壁断裂,肺泡腔融合增大。电镜下可见Ⅰ型肺泡上皮细胞肿胀,含大量吞饮小泡,内皮结构欠完整;Ⅱ型肺泡细胞核膨大,内部充满大量的圆形、椭圆形病毒颗粒,核内结构模糊;胞浆内细胞器崩解,内质网,线粒体,核糖体等模糊不清。肺部病变在感染后第3、5天较为严重。感染后不同时间点,扶正解毒化瘀方及有效部位组肺病变均有不同程度减轻。3.RSV感染BALB/c小鼠肺组织凋亡基因表达RSV感染后第1、3天,与模型组比较,各治疗组均能下调小鼠肺组织中Fas的表达,其中总苷组和挥发油组下调作用显著(P0.01)。感染后第1天,模型组与各给药组P53表达水平均显著增高(P0.01);全方组、总苷组及挥发油组均能显著下调肺组织中P53、caspase-12的表达(P0.01),其中总苷组和挥发油组下调作用明显。感染后第3天,总苷组及挥发油组对caspase-12表达仍有显著的下调作用(P0.05)。4.RSV感染BALB/c小鼠肺组织PI3K/Akt通路相关蛋白表达RSV感染后第3天,与模型组比较,全方组肺组织Akt表达显著增加(P0.01),而在第5天明显下降。感染后第3、5天,全方组和总苷组GSK-3β表达明显增高(P0.01),与第1天相比则明显下调。全方组在感染后第1、5天可明显下调NF-kB P65的表达,而挥发油组仅在第1天有明显下调作用(P0.05)。5.RSV感染BALB/c小鼠肺组织细胞因子表达感染后不同时间点,模型组肺组织IL-8表达均高于正常组,其中感染后第1天明显(P0.01);总苷组与挥发油组可明显下调IL-8表达水平(P0.05)。IL-1β在各组中的表达于感染后第1天最高,随着时间推移,有下降趋势;感染后第1天,全方、总苷及挥发油组可明显下调IL-1 β表达(P0.01);总苷组在第3天仍有显著下调作用。感染后第1天,与模型组比较,三中药组对IFN-α表达均有上调作用,但无统计学差异;感染后第3天,全方组可显著上调IFN-α表达(P0.05)。对于TLR7的影响,仅在感染后第1天,模型组、总苷组和挥发油组较正常组有所上调(P0.05)。在RSV感染的整个过程中,扶正解毒化瘀方及有效部位对TLR3的表达未见明显调控作用。结论:1.扶正解毒化瘀方及有效部位对RSV感染BALB/小鼠肺指数有明显抑制作用;2.扶正解毒化瘀方及有效部位能够改善RSV感染BALB/c小鼠肺部病变;3.扶正解毒化瘀方及有效部位可下调Fas、P53与caspase-12凋亡基因表达水平;4.扶正解毒化瘀方及有效部位对PI3K/Akt通路相关蛋白Akt、GSK-3β及NF-κB的调控作用,在感染后不同时间呈现出不同特点。5.扶正解毒化瘀方及有效部位能够显著下调IL-1 β、IL-8的表达,并能上调IFN-α的表达;对TLR3与TLR7的表达无明显调控作用。6.扶正解毒化瘀方及有效部位是治疗RSV感染的有效药物,其作用机制可能是通过调节细胞凋亡基因、PI3K/Akt通路相关蛋白及诸多细胞因子的表达而实现的。
[Abstract]:Viral pneumonia (VP) is a common respiratory infectious disease, mostly in children, the elderly, the immunocompromised host, and the combined cardiopulmonary basic disease. In recent years, the incidence of VP, including respiratory syncytial virus (RSV) pneumonia, is significantly increased with the acceleration of aging society, the increase of immune damage host, virus variation and the generation of new virus, which seriously threatens human health. At present, there is a lack of specific therapeutic drugs with strong specificity in the treatment. Traditional Chinese medicine is characterized by holistic view, syndrome differentiation and differentiation, and has the characteristics of multi-target, multi-pathway and multi-link effect, and has obvious advantages in preventing and treating viral infection diseases. A study on the prevention and treatment of respiratory tract infectious diseases by traditional Chinese medicine has been carried out in a long period of time. "Virtual, toxic, phlegm, blood stasis" It is the key to the disease of viral pneumonia. It is mainly used for strengthening the body, removing toxic materials and removing blood stasis. Then developed the Fuzheng Jiedu Huayu Recipe, confirmed the long-term clinical practice, and is safe and reliable. The Chinese medicinal composition has the functions of strengthening body resistance, dispelling pathogenic wind, clearing away heat and toxic materials, promoting blood circulation and removing blood stasis, and is mainly used for treating pulmonary infection caused by various pathogens. Previous research shows that Fuzheng Jiedu Huayu can effectively reduce the symptoms of pneumonia, promote the absorption of inflammation and control the progress of disease. The pulmonary infection caused by RSV is significant, but the material basis and mechanism of treatment are not clear. This study takes RSV pneumonia as the entry point, and probes into the mechanism of the role of Fuzheng Jiedu Huayu Recipe and effective site in the intervention of viral pneumonia. This thesis includes two parts: review and experimental research. In this paper, the cognition and research progress of the traditional Chinese medicine and modern medicine to RSV pneumonia are expounded, and the theoretical foundation is provided for the experimental research. The experimental part mainly discusses the mechanism of anti-virus action of Fuzheng Jiedu Huayu Recipe and effective part from the aspects of tissue morphology, cell molecular biology and so on. Objective: To investigate the effect mechanism of Fuzheng Jiedu Huayu Recipe and effective site on RSV pneumonia mice from the aspects of apoptosis gene, p38/ Akt signal pathway related regulatory proteins, cytokines and pathological changes of lung tissue. Methods: 180 female BALB/ c mice were randomly divided into 3 groups at the first, third and fifth days after infection. Each of the major components was the normal group, the model group, the whole group, the total alkaloids group, the volatile oil group and the Linlin group, with 10 rats in each group. BALB/ c mice infected with RSV droplets were treated with Fuzheng Jiedu Huayu Recipe and effective site, and the lung tissues of mice were taken to calculate the lung index of each group on Days 1, 3 and 5 after infection. The pathological tissue sections were observed, and the pathological changes of lung tissue in mice were observed by optical microscope and electron microscope. Detection of Fas, P53 and caspase-12 mRNA in lung tissues by Real-time PCR. Immunohistochemical method was used to detect the expression of Akt, OPG-3 and NF-cyclin B p65 protein in lung tissue. The expression of IL-1, IL-8, IFN-jun, TLR3 and TLR7 in lung tissue was detected by ELISA. Results: 1. The lung index of the model group increased in different time points after the infection of the lung index of the BALB/ c mice infected with RSV, and there was an increasing tendency. Compared with the normal group, there was a significant difference (P0.01). Compared with the model group, the lung index could be effectively decreased after the first, 3rd and 5th day after infection (P <0.05). In the third and fifth day after infection, the efficacy of group 3 and 5 was comparable to that of the group of essential oil, and it was superior to that of the total alkaloids group and the essential oil group (P0.05). The results showed that the lung function of RSV infected BALB/ c mice showed pulmonary interstitial inflammation, alveolar wall congestion, edema and obvious thickening after RSV infection, and there was a large number of inflammatory cells infiltration in the interstitial lung. The alveolar wall was broken and the fusion of alveolar cavity increased. Under the electron microscope, I can see the swelling of alveolar epithelial cells of type 鈪，
本文编号：2282014
[Abstract]:Viral pneumonia (VP) is a common respiratory infectious disease, mostly in children, the elderly, the immunocompromised host, and the combined cardiopulmonary basic disease. In recent years, the incidence of VP, including respiratory syncytial virus (RSV) pneumonia, is significantly increased with the acceleration of aging society, the increase of immune damage host, virus variation and the generation of new virus, which seriously threatens human health. At present, there is a lack of specific therapeutic drugs with strong specificity in the treatment. Traditional Chinese medicine is characterized by holistic view, syndrome differentiation and differentiation, and has the characteristics of multi-target, multi-pathway and multi-link effect, and has obvious advantages in preventing and treating viral infection diseases. A study on the prevention and treatment of respiratory tract infectious diseases by traditional Chinese medicine has been carried out in a long period of time. "Virtual, toxic, phlegm, blood stasis" It is the key to the disease of viral pneumonia. It is mainly used for strengthening the body, removing toxic materials and removing blood stasis. Then developed the Fuzheng Jiedu Huayu Recipe, confirmed the long-term clinical practice, and is safe and reliable. The Chinese medicinal composition has the functions of strengthening body resistance, dispelling pathogenic wind, clearing away heat and toxic materials, promoting blood circulation and removing blood stasis, and is mainly used for treating pulmonary infection caused by various pathogens. Previous research shows that Fuzheng Jiedu Huayu can effectively reduce the symptoms of pneumonia, promote the absorption of inflammation and control the progress of disease. The pulmonary infection caused by RSV is significant, but the material basis and mechanism of treatment are not clear. This study takes RSV pneumonia as the entry point, and probes into the mechanism of the role of Fuzheng Jiedu Huayu Recipe and effective site in the intervention of viral pneumonia. This thesis includes two parts: review and experimental research. In this paper, the cognition and research progress of the traditional Chinese medicine and modern medicine to RSV pneumonia are expounded, and the theoretical foundation is provided for the experimental research. The experimental part mainly discusses the mechanism of anti-virus action of Fuzheng Jiedu Huayu Recipe and effective part from the aspects of tissue morphology, cell molecular biology and so on. Objective: To investigate the effect mechanism of Fuzheng Jiedu Huayu Recipe and effective site on RSV pneumonia mice from the aspects of apoptosis gene, p38/ Akt signal pathway related regulatory proteins, cytokines and pathological changes of lung tissue. Methods: 180 female BALB/ c mice were randomly divided into 3 groups at the first, third and fifth days after infection. Each of the major components was the normal group, the model group, the whole group, the total alkaloids group, the volatile oil group and the Linlin group, with 10 rats in each group. BALB/ c mice infected with RSV droplets were treated with Fuzheng Jiedu Huayu Recipe and effective site, and the lung tissues of mice were taken to calculate the lung index of each group on Days 1, 3 and 5 after infection. The pathological tissue sections were observed, and the pathological changes of lung tissue in mice were observed by optical microscope and electron microscope. Detection of Fas, P53 and caspase-12 mRNA in lung tissues by Real-time PCR. Immunohistochemical method was used to detect the expression of Akt, OPG-3 and NF-cyclin B p65 protein in lung tissue. The expression of IL-1, IL-8, IFN-jun, TLR3 and TLR7 in lung tissue was detected by ELISA. Results: 1. The lung index of the model group increased in different time points after the infection of the lung index of the BALB/ c mice infected with RSV, and there was an increasing tendency. Compared with the normal group, there was a significant difference (P0.01). Compared with the model group, the lung index could be effectively decreased after the first, 3rd and 5th day after infection (P <0.05). In the third and fifth day after infection, the efficacy of group 3 and 5 was comparable to that of the group of essential oil, and it was superior to that of the total alkaloids group and the essential oil group (P0.05). The results showed that the lung function of RSV infected BALB/ c mice showed pulmonary interstitial inflammation, alveolar wall congestion, edema and obvious thickening after RSV infection, and there was a large number of inflammatory cells infiltration in the interstitial lung. The alveolar wall was broken and the fusion of alveolar cavity increased. Under the electron microscope, I can see the swelling of alveolar epithelial cells of type 鈪，
本文编号：2282014
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