【摘要】：病毒性肺炎(VP)是臨床常見的呼吸道感染性疾病,多見于兒童、老年人、免疫低下宿主及合并心肺基礎(chǔ)病患者。近年來,隨著社會(huì)老齡化加速、免疫損傷宿主增加、病毒變異和新病毒的產(chǎn)生,包括呼吸道合胞病毒(RSV)肺炎在內(nèi)的VP的發(fā)病率明顯增高,嚴(yán)重威脅著人類健康。目前,其治療尚缺乏專屬性強(qiáng)的特效治療藥物。中醫(yī)學(xué)以整體觀、辨證論治為特色,具有多靶點(diǎn)、多途徑、多環(huán)節(jié)作用的特點(diǎn),在防治病毒感染性疾病方面具有明顯優(yōu)勢(shì)。導(dǎo)師臨證數(shù)十載,長(zhǎng)期致力于中醫(yī)藥防治呼吸道感染性疾病的研究,認(rèn)為"虛、毒、痰、瘀"是病毒性肺炎的核心病機(jī)關(guān)鍵,治當(dāng)以扶正、解毒、化瘀為大法。遂擬定了扶正解毒化瘀方,長(zhǎng)期臨床實(shí)踐證實(shí),安全可靠。該方由西洋參、黃芩、連翹、漏蘆、赤芍、瓜萎、敗醬草和生薏苡仁組成,具有扶正祛邪,清熱解毒,活血化瘀之功,主要用于治療多種病原體所致的肺部感染。前期研究表明,扶正解毒化瘀方可有效減輕肺炎癥狀、促進(jìn)炎癥吸收,控制疾病進(jìn)展。對(duì)RSV所致的肺部感染療效顯著,但其治療的物質(zhì)基礎(chǔ)和作用機(jī)制尚不清楚。本研究以RSV肺炎為切入點(diǎn),探討扶正解毒化瘀方及有效部位干預(yù)病毒性肺炎的作用機(jī)制。本論文包括綜述和實(shí)驗(yàn)研究?jī)刹糠帧＞C述部分,系統(tǒng)闡述了中醫(yī)學(xué)及現(xiàn)代醫(yī)學(xué)對(duì)RSV肺炎的認(rèn)識(shí)及研究進(jìn)展,為實(shí)驗(yàn)研究提供了理論基礎(chǔ)。實(shí)驗(yàn)部分主要從組織形態(tài)學(xué),細(xì)胞分子生物學(xué)等角度探討扶正解毒化瘀方及有效部位抗病毒作用機(jī)制。目的:從凋亡基因、PI3K/Akt信號(hào)通路相關(guān)調(diào)控蛋白、細(xì)胞因子及肺組織病理變化等多角度探討扶正解毒化瘀方及有效部位干預(yù)RSV肺炎小鼠的作用機(jī)制。方法:將180只雌性BALB/c小鼠按感染后第1、3、5天三個(gè)時(shí)間點(diǎn),隨機(jī)分為3個(gè)大組,每組60只。每個(gè)大組分為正常組、模型組、全方組、總苷組、揮發(fā)油組及利巴韋林組6個(gè)小組,每組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進(jìn)行干預(yù),并于感染后第1、3、5天分別取小鼠肺組織,計(jì)算各組肺指數(shù)。并做病理組織切片,采用光學(xué)顯微鏡和電子顯微鏡觀察小鼠肺組織病變情況。Real-time PCR法檢測(cè)肺組織中Fas、P53及caspase-12 mRNA表達(dá)。免疫組化法檢測(cè)肺組織中Akt、GSK-3 β及NF-κB p65蛋白的表達(dá)。ELISA法檢測(cè)肺組織中IL-1β、IL-8、IFN-α、TLR3和TLR7的表達(dá)。結(jié)果:1.RSV感染BALB/c小鼠肺指數(shù)感染后不同時(shí)間點(diǎn),模型組肺指數(shù)均有不同程度增高,且呈遞增趨勢(shì);與正常組比較,均有顯著差異(P0.01)。感染后第1、3、5天各給藥組均可有效降低肺指數(shù),與模型組比較差異顯著(P0.05)。感染后第3、5天全方組與利巴韋林組療效相當(dāng),且優(yōu)于總苷組和揮發(fā)油組(P0.05)。全方組對(duì)肺指數(shù)抑制作用較總苷組、揮發(fā)油組有一定優(yōu)勢(shì),感染后第3天最明顯。2.RSV感染BALB/c小鼠肺病理改變RSV感染后主要表現(xiàn)為肺間質(zhì)炎癥,肺泡壁充血、水腫,明顯增厚;肺間質(zhì)有大量炎癥細(xì)胞浸潤(rùn);部分肺泡壁斷裂,肺泡腔融合增大。電鏡下可見Ⅰ型肺泡上皮細(xì)胞腫脹,含大量吞飲小泡,內(nèi)皮結(jié)構(gòu)欠完整;Ⅱ型肺泡細(xì)胞核膨大,內(nèi)部充滿大量的圓形、橢圓形病毒顆粒,核內(nèi)結(jié)構(gòu)模糊;胞漿內(nèi)細(xì)胞器崩解,內(nèi)質(zhì)網(wǎng),線粒體,核糖體等模糊不清。肺部病變?cè)诟腥竞蟮?、5天較為嚴(yán)重。感染后不同時(shí)間點(diǎn),扶正解毒化瘀方及有效部位組肺病變均有不同程度減輕。3.RSV感染BALB/c小鼠肺組織凋亡基因表達(dá)RSV感染后第1、3天,與模型組比較,各治療組均能下調(diào)小鼠肺組織中Fas的表達(dá),其中總苷組和揮發(fā)油組下調(diào)作用顯著(P0.01)。感染后第1天,模型組與各給藥組P53表達(dá)水平均顯著增高(P0.01);全方組、總苷組及揮發(fā)油組均能顯著下調(diào)肺組織中P53、caspase-12的表達(dá)(P0.01),其中總苷組和揮發(fā)油組下調(diào)作用明顯。感染后第3天,總苷組及揮發(fā)油組對(duì)caspase-12表達(dá)仍有顯著的下調(diào)作用(P0.05)。4.RSV感染BALB/c小鼠肺組織PI3K/Akt通路相關(guān)蛋白表達(dá)RSV感染后第3天,與模型組比較,全方組肺組織Akt表達(dá)顯著增加(P0.01),而在第5天明顯下降。感染后第3、5天,全方組和總苷組GSK-3β表達(dá)明顯增高(P0.01),與第1天相比則明顯下調(diào)。全方組在感染后第1、5天可明顯下調(diào)NF-kB P65的表達(dá),而揮發(fā)油組僅在第1天有明顯下調(diào)作用(P0.05)。5.RSV感染BALB/c小鼠肺組織細(xì)胞因子表達(dá)感染后不同時(shí)間點(diǎn),模型組肺組織IL-8表達(dá)均高于正常組,其中感染后第1天明顯(P0.01);總苷組與揮發(fā)油組可明顯下調(diào)IL-8表達(dá)水平(P0.05)。IL-1β在各組中的表達(dá)于感染后第1天最高,隨著時(shí)間推移,有下降趨勢(shì);感染后第1天,全方、總苷及揮發(fā)油組可明顯下調(diào)IL-1 β表達(dá)(P0.01);總苷組在第3天仍有顯著下調(diào)作用。感染后第1天,與模型組比較,三中藥組對(duì)IFN-α表達(dá)均有上調(diào)作用,但無統(tǒng)計(jì)學(xué)差異;感染后第3天,全方組可顯著上調(diào)IFN-α表達(dá)(P0.05)。對(duì)于TLR7的影響,僅在感染后第1天,模型組、總苷組和揮發(fā)油組較正常組有所上調(diào)(P0.05)。在RSV感染的整個(gè)過程中,扶正解毒化瘀方及有效部位對(duì)TLR3的表達(dá)未見明顯調(diào)控作用。結(jié)論:1.扶正解毒化瘀方及有效部位對(duì)RSV感染BALB/小鼠肺指數(shù)有明顯抑制作用;2.扶正解毒化瘀方及有效部位能夠改善RSV感染BALB/c小鼠肺部病變;3.扶正解毒化瘀方及有效部位可下調(diào)Fas、P53與caspase-12凋亡基因表達(dá)水平;4.扶正解毒化瘀方及有效部位對(duì)PI3K/Akt通路相關(guān)蛋白Akt、GSK-3β及NF-κB的調(diào)控作用,在感染后不同時(shí)間呈現(xiàn)出不同特點(diǎn)。5.扶正解毒化瘀方及有效部位能夠顯著下調(diào)IL-1 β、IL-8的表達(dá),并能上調(diào)IFN-α的表達(dá);對(duì)TLR3與TLR7的表達(dá)無明顯調(diào)控作用。6.扶正解毒化瘀方及有效部位是治療RSV感染的有效藥物,其作用機(jī)制可能是通過調(diào)節(jié)細(xì)胞凋亡基因、PI3K/Akt通路相關(guān)蛋白及諸多細(xì)胞因子的表達(dá)而實(shí)現(xiàn)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，

本文編號(hào)：2282014