扶正解毒化瘀方及最佳有效部位群干预呼吸道合胞病毒融合性病变形成期的作用机制研究

Respiratory syncytial virus (RSV) is a common pathogen that leads to lower respiratory tract infection. Preliminary study supported by National TCM Project Application in the 11th Five-Year Period confirms that Fuzheng Jiedu Huayu decoction can relieve the symptoms and control the progression of RSV infection in aged pneumonia patients. But the mechanism is not clear. In the early stage of RSV infection, the PI3K/Akt pathway plays a central role in signaling pathways activated by RSV. It causes the virus replication by inhibiting cell apoptosis and promoting the integration of lesion formation.The action time of Chinese herbal compound is at the early stage of virus infection. It is believed by traditional Chinese medicine that the early stage of RSV infection belongs to the deficiency of heat toxin stasis lung syndrome of wind-warm disease with lung heat, which puts forward the hypothesis “the phase of deficiency of heat toxin stasis lung syndrome in wind-warm disease with lung heat is the replicative phase of RSV fusion lesions”. This study intends to establish the compound prescription of Jiedu Huayu and the fingerprint of effective fractions of the overall composition in vitro and in vivo on the basis of pharmacodynamics, effective fractions and ingredients. At the same time , material basis and mechanism of action of Fuzheng Jiedu Huayu Decoction of anti RSV are illustrated through observing PI3K/Aktpathway on cell apoptosis and inflammatory reaction. A wider application prospects will be brought to the prevention and treatment of traditional Chinese Medicine on RSV.

呼吸道合胞病毒（RSV）是导致下呼吸道感染的常见病原体，前期十一五课题研究证实扶正解毒化瘀方可减轻RSV感染老年肺炎的症状，控制疾病发展，但机理尚不明确。在RSV感染早期，PI3K/Akt通路在RSV激活的多条信号通路中发挥中心作用，可通过抑制细胞凋亡，促进融合性病变形成，有利于病毒复制。中药复方发挥药效时间恰是感染早期，中医认为RSV感染早期属于风温肺热病的正虚热毒瘀肺证，由此提出“风温肺热病正虚热毒瘀肺的时期正是RSV融合病变形成的复制期”假说。本课题以药效作用为基础、有效部位群为整体、体内外成分为引导，运用化学计量学、药效学和谱学相结合的方法，建立扶正解毒化瘀方总方、各有效部位、有效部位配伍组方体内外指纹图谱，同时观察其通过PI3K/Akt通路对细胞凋亡和炎性反应的影响，阐明扶正解毒化瘀方抑制RSV融合性病变形成的物质基础和作用机制，为中医药防治RSV带来更广阔的应用前景。

体外实验表明扶正解毒化瘀方全方、总苷提取物、挥发油提取物和扶正解毒化瘀方全方含药血清、总苷提取物含药血清、挥发油提取物含药血清在体外对RSV均有抑制作用，呈一定的量-效关系。但是，含药血清对RSV所致CPE的抑制作用与空白血清相比无明显差异，而且空白血清作用RSV感染HEp-2细胞的干扰因素比较多，因此，在后续的研究中主要选择扶正解毒化瘀方全方、总苷提取物、挥发油提取物原药进行研究。在采用荧光定量RT-PCR实验中，扶正解毒化瘀方全方组和总苷提取物的抑制RSV N基因的程度与阳性对照药物利巴韦林组相当，因此，扶正解毒化瘀方全方组和总苷提取物可通过直接抑制RSV复制而起到抗病毒的作用。在RSV感染早期，AKT、GSH-3β和NF-B被激活，蛋白表达量明显增加，扶正解毒化瘀方全方和总苷提取物组可通过减少AKT、GSH-3β和NF-B的表达，发挥抑制炎性反应的作用。同时，在RSV感染早期全方组、总苷组可通过增加IFN-水平，提高免疫功能，通过降低IL-1β、IL-8、RANTES、TLR3和TLR7抑制RSV感染所致炎性反应。再通过增加促凋亡基因fas mRNA和trial mRNA的表达，抑制抑凋亡基因bax mRNA的表达，从而在RSV感染早期诱导感染细胞凋亡，从而发挥更好的抑制RSV复制的作用。通过液相质谱分析表明，扶正解毒化瘀方全方体外原药的主要成分是以苷类为主，扶正化瘀方总提取物组入血成分共鉴别出3个化学成分，包括芍药苷，连翘酯苷H，人参皂苷Rb1。因此，扶正解毒化瘀方可能通过其主要的苷类成分发挥的抗RSV的作用。体内实验结果提示RSV感染后不同时间点，扶正解毒化瘀方及有效部位具有调节细胞凋亡、抑制病毒复制，发挥抗病毒作用，同时也能下调炎症因子过度表达，抑制炎症反应，减轻病理损伤。

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