金欣口服液对RSV诱导的TLR7信号转导通路的作用研究
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摘要
肺炎为小儿时期的常见病,多为支气管肺炎,好发于冬春季节或气候交替之际,是我国住院小儿死亡的第一原因,严重威胁儿童健康,被世界卫生组织列为全球三种重要儿科疾病之一,我国卫生部也将其列为重点防治的小儿四病之一。据统计,肺炎占目前城市医院儿科病区住院病例的25%-50%；而病毒性肺炎是小儿肺炎的常见类型,约占小儿肺炎总数的50%。在引起小儿病毒性肺炎的病原体中又以呼吸道合胞病毒(respiratory syncytial virus, RSV)为主,西医治疗小儿RSV肺炎缺乏有效的治疗手段,而中医药在治疗病毒感染性疾病方面有其独到的优势。
导师汪受传教授领衔的课题组完成的临床研究表明,具有开肺化痰解毒活血功效的金欣口服液是治疗小儿病毒性肺炎痰热闭肺证的有效方剂。前期实验研究结果表明,金欣口服液有明显抑制RSV的作用,主要通过抑制病毒的膜融合,其作用位点可能是病毒的F蛋白或其受体；可明显减低RSV感染后Hep-2细胞病变程度,对细胞有保护作用；具有诱导单个淋巴细胞产生IL-2、IFN-γ的作用,增强机体抗病毒免疫功能。
Toll样受体(Toll-like receptors, TLRs)是重要的模式识别受体(pattern recognition receptors, PRRs),可以识别不同微生物的病原相关分子模式(pathogen associated molecule pattem, PAMPs)。TLRs在RSV感染过程中发挥着重要的作用,是研究RSV感染的新的方向。导师汪受传教授带领的团队承担的国家自然科学基金项目“金欣口服液对RSV活化的TLRs信号转导通路作用机制研究(81072840)”前期研究成果已表明金欣口服液能调控RSV感染活化诱导的TLR3、TLR4及下游信号分子的表达水平。
RSV是单股负链RNA病毒,粘附融合宿主细胞后释放单链RNA,能被位于内体膜上的TLR7特异性识别,通过一系列信号传导途径,引起下游细胞因子的大量分泌,引起系统性炎症反应。鉴于中医药作用的多靶点性,本实验从TLR7及其介导的信号通路入手,探讨RSV感染时金欣口服液抗对TLR7信号转导通路的调控,及与其它TLRs的相关性。
目的
研究金欣口服液对RSV诱导的TLR7信号传导通路的调控作用,探讨其治疗RSV肺炎的可能免疫学机制。
方法
体外实验：RSV感染体外培养的RAW264.7细胞,采用金欣口服液含药血清进行干预,24h后收集细胞,real-time PCR法测定TLR7及其信号通路关键分子MyD88、NF-κB、IRF7mRNA的表达变化；Western Blot技术检测TLR7、 MyD88、NF-κB、IRF7蛋白表达,激光共聚焦技术检测TLR7在RSV感染RAW264.7细胞中的表达及分布；ELISA法检测细胞上清中TNF-α、IFN-α表达情况；并用TLR7siRNA干扰后检测上述指标,观察金欣口服液对RSV诱导的TLR7传导通路影响及与其它TLRs信号通路的相关性。
体内实验：RSV滴鼻感染BALB/c小鼠,金欣口服液进行干预,并于首次滴鼻后72、144小时,取小鼠肺组织,分别行病理组织切片评价肺部病变情况,real-time PCR法检测肺组织中TLR7、MyD88、NF-κB、IRF7mRNA表达情况,Western Blot法检测小鼠肺组织TLR7、MyD88、NF-κB、IRF7蛋白表达情况；取小鼠支气管肺泡灌洗液(BALF), ELISA法检测TNF-α、IFN-α表达情况。
结果
一、体外实验
1.TLR7siRNA转染前
(1)RSV感染RAW264.7细胞24h后,TLR7、NF-κB、IRF7mRNA表达明显升高(超过正常组2倍以上,与正常组比较,P<0.01),对MyD88mRNA的表达能提高,但没超过2倍：金欣口药血清组能明显降低TLR7、MyD88、NF-κB mRNA表达(P<0.01),对IRF7mRNA的表达下调作用不明显(P>0.05)。
(2)RSV感染RAW264.7细胞24h后,TLR7、MyD88、NF-κB蛋白表达量较正常组明显升高(P<0.01),而IRF7的蛋白表达无明显改变；金欣口服液含药血清组TLR7、MyD88、NF-κB蛋白表达量较RSV感染组明显降低(P<0.01)；对IRF7的蛋白表达无调控作用。
(3)RSV感染RAW264.7细胞24h后,细胞培养上清中IFN-α未能检测到；TNF-α表达量明显升高(与正常组比较,P<0.01)。金欣口服液含药血清显著降低TNF-α表达(P<0.01)。
2. TLR7siRNA转染后
(1) TLR7siRNA转染RAW264.7细胞后,TLR7mRNA的表达量下降46.6%,再用RSV感染,可见TLR7mRNA的表达又有明显增高(超过TLR7siRNA转染组的2倍以上),表明用筛选出的TLR7siRNA可干扰TLR7mRNA的生成。TLR7信号通路下游信号分子MyD88、NF-κB mRNA的表达也受到抑制,但其抑制率低于TLR7mRNA(分别为26.9%、27.3%),未成平行抑制关系,表明MyD88、NF-κB mRNA的表达可能还受其它因素的调控,特别是TLRs家族的其它成员。TLR7siRNA转染后IRF7mRNA的表达未受明显影响。
(2)金欣口药血清能明显降低转染及RSV感染后的TLR7、MyD88、NF-κB mRNA表达(P<0.01),对IRF7mRNA的表达下调作用不明显(P>0.05)。
(3)RSV感染RAW264.7细胞24h后,TLR7、MyD88、NF-κB蛋白表达量较正常组明显升高(P<0.01),而IRF7的蛋白表达无明显改变；金欣口服液含药血清组能明显下调TLR7、NF-κB蛋白表达量(P<0.01),但对MyD88的调控不明显(P>0.05)；对IRF7的蛋白表达无调控作用。
(4) TLR7siRNA转染后继以RSV感染RAW264.7细胞24h后,细胞培养上清中IFN-α仍未能检出；TNF-α表达量明显升高(与正常组比较,P<0.01),金欣口服液含药血清显著降低TNF-α表达(P<0.01)。
二、体内试验
(1)RSV感染BALB/c小鼠后肺部病理改变主要表现为肺间质性病变,肺泡壁血管扩张充血,水肿增厚,肺泡壁及间质炎性细胞浸润；肺泡腔无明显渗出物；支气管腔内无显著炎性渗出物,上皮细胞无显著变性、坏死。随着感染时间的延长,肺部病变程度逐渐加重。金欣口服液组小鼠肺内炎症均有不同程度减轻,病理评分结果显示治疗组各时间点与RSV感染组相比均有统计学显著性差异。
(2)RSV感染BALB/c小鼠72h后,肺组织中TLR7、MyD88、NF-κB mRNA表达显著增高(与正常组比较,P<0.01),而144h组随着感染时间的延长,TLR7、 MyD88、NF-κB mRNA的表达则下降。金欣口服液组TLR7、MyD88、NF-κB mRNA表达较RSV感染组不同程度降低(P<0.01或P<0.05)。
RSV感染BALB/c小鼠72、144h后,肺组织中IRF7mRNA表达量与正常组比较无统计学差异(P>0.05),金欣口服液高剂量组在感染72h能下调IRF7mRNA表达(P<0.05),金欣口服液高剂量组、等效剂量组在144h能上调IRF7mRNA表达,但无统计学差异(P>0.05)。
(3)RSV感染BALB/c小鼠72h后肺组织内TLR7、MyD88、NF-κB蛋白表达量较正常组高(P<0.01或P<0.05),144h表达下降与正常组无明显差异。金欣口服液组TLR7、MyD88、NF-κB蛋白表达量于72h较RSV感染组不同程度降低(P<0.01或P<0.05),感染144h金欣口服液组TLR7、MyD88、NF-κB表达量下降较RSV组差异无统计学意义(P>0.05)。
RSV感染BALB/c小鼠72h后肺组织内IRF7蛋白表达量较正常组无显著差异(P>0.05),144h表达较正常有所上升(P>0.05)。金欣口服液组IRF7蛋白表达量于72h较RSV感染组无统计学差异,感染144h金欣口服液组能上调IRF7蛋白表达(P<0.01)。
(4)RSV感染BALB/c小鼠72h后,BALF中TNF-α、IFN-α表达量均明显升高(与正常组比较,P<0.01);144h, BALF中TNF-α、IFN-α下降。72h,金欣口服液组小鼠BALF中TNF-α、IFN-α表达较感染组显著上升,随着感染时间的延长,144h金欣组TNF-α、IFN-α表达明显下降。
结论
1.金欣口服液能明显减轻RSV感染小鼠的肺部炎症。
2.金欣口服液对TLR7信号通路TLR7、MyD88、NF-κB mRNA及蛋白表达有明显的调控作用,对IRF7的表达在RSV感染早期无明显调控,晚期可上调IRF7的表达。
3.金欣口服液能够显著上调RSV感染初期TLR7介导的信号通路下游TNF-α、IFN-α等炎症细胞因子的表达；随着感染时间的延长,金欣口服液能适当下调TNF-α、IFN-α的表达,具有双向动态调节作用。
4.金欣口服液是治疗RSV感染的有效药物,其作用是通过调节RSV诱导的TLR7信号通路实现的,主要依赖的是TLR7/MyD88/NF-κB/TNF-α途径,在我们的实验中发现TLR7/MyD88/IRF7/IFN-α途径在RSV感染早期作用不明显。
5. TLR7siRNA转染后,TLR7mRNA及蛋白表达明显受抑,但MyD88、 NF-κB mRNA及蛋白的抑制率低于TLR7,表明MyD88、NF-κB的表达还受其它TLRs调控,从而说明TLR7信号通路只是RSV感染后的一个通路,而金欣口服液亦可通过其它通路调控下游信号分子生成,体现了中药复方发挥作用的多靶点效应；TLR7siRNA转染与否对IRF7的表达无明显影响。
Pneumonia, especially bronchial pneumonia, is a major childhood diseases, which mainly occurs in winter, spring or during climate changes. Seriously threatened children's health, pneumonia has become the top death cause of the hospitalized children. The World Health Organization list its name as one of the three major global pediatric diseases, and Chinese Ministry of Health as one of the four pediatric diseases. According to statistics, of all the urban hospital pediatric ward inpatients, pneumonia accounted for25%to50%, among which viral pneumonia is a common type, accounting for about50%of the total cases. Respiratory syncytial virus (RSV) is the main pathogen that causes viral pneumonia in children, and western medicine therapy shows little effect in treating RSV pneumonia, while Chinese medicine has its unique advantages in treating viral infectious diseases.
Jinxin oral liquid, developed to promote lung, resolve phlegm, detoxicate and remove blood stasis, proves an effective prescription in clinic which treats the viral pneumonia with phlegm-heat closed lung syndrome. Preclinic cases and experimental studies have shown that Jinxin oral liquid significantly inhibits RSV through depressing its membrane fusion, the target of which may lies in the virus'F protein or its receptor; Jinxin also sharply reduces the cytopathic effect (CPE) of Hep-2cell line infected with RSV; moreover, it induces single lymphocyte to produce IL-2, IFN-y to enhance antiviral immune function.
Toll like receptors (TLRs), an important pattern recognition receptors (PRRs), can identify different microbial pathogen associated molecular patterns (PAMPs) TLRs plays an important role in the process of RSV infection and becomes the new trend in this research field. Preliminary research results of "the effect and mechanism of Jinxin Oral Liquid on TLRs signal transduction pathway induced with RSV infection(81072840)" a National Natural Science Foundation project led by professor Wang Shou-chuan, have shown that Jinxin oral liquid can regulate the expression levels of TLR3, TLR4and downstream signaling molecules induced with RSV infection.
As a single-stranded negative-strand RNA (ss-RNA) virus, RSV adheres and fuses to the host cell and then ss-RNA is released, which can be recognized by TLR7's specific module in the inner body membrane, and through a series of signal transduction pathway, downstream cytokines secrete in high level and thus results in systemic inflammatory response. In view of the multiple target effect of Chinese medicine, this experiment starts with the TLR7and its signal transduction pathway, investigates the regulatory role of Jinxin oral liquid on the pathway induced by RSV infection, and researches the correlation to other TLRs pathways.
Objective
To study the role of Jinxin oral liquid on TLR7signal transduction pathway induced by RSV, and explore its possible immunological mechanism in treating RSV pneumonia.
Methods
Experiments in vitro:
1. The serum containing Jinxin oral liquid was applied to intervene RAW264.7cells infected with RSV, and after24hours, the cells were collected to detect the levels of TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay, and TLR7expression and distribution in cells by confocal laser scanning technology. And cell supernatants were collected to detect TNF-α, IFN-a levels by ELISA assay.
2. TLR7siRNA was used to interfere TLR7expression, and then detect the expression of above substances to observe the effect of Jinxin oral liquid on TLR7signal transduction pathway and the correlation with other TLRs pathways.
Experiments in vivo:
Jinxin oral liquid in different dosage was applied to treat BALB/c mice inoculated with RSV, and after72,144hours of the first inoculation, mice lungs were taken to evaluate lung lesions by histologic slide analysis, to detect the levels of TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and to examine the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay. The broncho alveolar lavage liquids (BALF) were collected to measure TNF-α, IFN-α levels by ELISA assay.
Results
Experiments in vitro:
A. Before TLR7siRNA transfection
1.24h after RAW264.7cells infected by RSV, expression of TLR7, NF-κB, IRF7mRNA significantly increased (more than2times more than the normal group, P<0.01), the expression of MyD88mRNA can improve, but was not more than two times than the normal group. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01), but the effect of down-regulating IRF7mRNA not obviously (P>0.05).
2.24h after RAW264.7cells infected by RSV, TLR7, MyD88, NF-κB protein expression were significantly higher than that in normal group (P<0.01), and the protein expression of IRF7had no obvious change. The serum containing Jinxin oral liquid down-regulated significantly the expression of TLR7, MyD88, NF-κB protein (P<0.01), and had no effect on IRF7expression.
3.24h after RAW264.7cells infected by RSV, in cell supernatants the expression of TNF-a was significantly increased (P<0.01), The serum containing Jinxin oral liquid significantly reduced TNF-a expressionThe serum containing Jinxin oral liquid.But IFN-a was not found.
B. After TLR7siRNA transfection
1. After RAW264.7cells transfected by TLR7siRNA, TLR7mRNA expression decreased by46.6%, then infected by RSV, TLR7mRNA expression significantly increased (more than two times than siRNA transfection group, P<0.01). And showed that TLR7siRNA transfection interfered TLR7mRNA generation. The expression of MyD88, NF-κB mRNA were also inhibited, but the inhibition rate lower than that of TLR7mRNA (respectively26.9%,27.3%<46.6%), and that may show that MyD88, NF-κB mRNA expression may also be regulated by other factors, especially by other members of TLRs family. IRF7mRNA expression was unaffected after TLR7siRNA transfection.
2. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01) in RAW264.7cells treated by TLR7siRNA transfection and RSV infection, the down-regulation on IRF7mRNA expression was not obvious (P>0.05)
3. The expression of TLR7, MyD88, NF-κB protein significantly increased24h after RSV infection (P<0.01), while IRF7protein expression had no significant change. The serum containing Jinxin oral liquid significantly down-regulated TLR7, NF-κB protein expression (P<0.01), but had no obvious regulation on MyD88protein expression (P>0.05), and had no regulating effect on IRF7protein expression.
4.24h after TR7siRNA transfection followed with RSV infection, IFN-α failed to check out in the cell supernatant, and TNF-α expression significantly increased (P <0.01). The serum containing Jinxin oral liquid significantly reduced serum TNF-a expression (P<0.01)
Experiments in vivo:
1. Pulmonary pathological changes in BALB/c mice infected by RSV were mainly including interstitial inflammation, vascular dilatation and congestion, vascular wall edema and thickening, inflammatory cell infiltration in alveolar wall and interstitial lung. Alveolar cavity had no obvious exudation, and the bronchial cavity no significant inflammatory exudation, and bronchial epithelial cells were no significant degeneration and necrosis. With the RSV infection prolonged, the degree of lung lesions gradually worsened. Lung inflammation in mice treated by Jinxin oral liquid alleviate to some extent. The pathological scores of treatment group showed statistically significant differences at each time point compared with RSV infection group.
2.72h after RSV infecting BALB/c mice, TLR7, MyD88, NF-κB mRNA expression in lung tissue were significantly increased compared with the normal group (P<0.01), while at144h those expressions decreased. In Jinxin group TLR7, MyD88, NF-κB mRNA expression decreased compared with RSV group (P<0.01)
72h and144h after RSV infection, IRF7mRNA expression had no obvious difference compared with normal group (P>0.05). At72h, high-dosage Jinxin oral liquid significantly down-regulated IRF7mRNA expression (P<0.01). At144h, high-dosage and equivalent-dosage Jinxin oral liquid both up-regulated IRF7mRNA expression, but had no statistics difference compared with RSV group (P>0.05)
3.72h after RSV infection in BALB/c mice, TLR7、MyD88、NF-κB protein expression were higher than nomal group (P<0.01or P<0.05), and at144h the expression decreased and had no difference compared with normal group (P>0.05). TLR7、MyD88、NF-κB protein expression in Jinxin group at72h decreased significantly compared with RSV group (P<0.01or P<0.05), and at144h those expression decreased but had no obvious difference compared with RSV group (P>0.05)
72h after RSV infecting BALB/c mice, IRF7protein expression had no difference compared with normal group, and at144h the expression increased (P>0.05). At72h IRF7protein expression was no difference between in RSV group and Jinxin group. At144h, Jinxin oral liquid significantly increased IRF7protein expression (P<0.01)
4.72h after RSV infecting BALB/c mice, TNF-α and IFN-α expression in BALF were significantly higher than that in normal group (P<0.01) while at144h TNF-a and IFN-a expression decreased. At72h, TNF-a and IFN-a expression in Jinxin group significantly increased (P<0.01), and at144h the expression obviously reduced, but had no difference comared with RSV group.
Conclusions
1. Jinxin oral liquid significantly alleviate the lung inflammation of the RSV infected mice.
2. Jinxin oral liquid has an evident down-regulatory effects on TLR7, MyD88, NF-κB mRNA and protein expression in TLR7signal transduction pathway induced by RSV; in the late stage after SRV infection, Jinxin oral liquid can up-regulate IRF7mRNA and protein expression, while in the early stage, no obvious regulation effect can be observed.
3. Jinxin oral liquid can increase the levels of TNF-a, IFN-a in the early stage of RSV infection. During the infection prolonged period, it can down-regulate their expressions. The results show that Jinxin oral liquid has a bidirectional dynamic regulation on TNF-a and IFN-a expression.
4. Jinxin oral liquid is an effective drug in treating RSV infection, and its effect is realized by adjusting the TLR7signaling pathways induced by RSV, mainly relies on the TLR7/MyD88/NF-κB/TNF-α pathway. In the early stage of RSV infection, the effect on TLR7/MyD88/IRF7/IFN-α pathway is not obvious.
5. After TLR7siRNA transfection, the expression of TLR7mRNA and protein were inhibited, and the inhibition rate is higher than those of MyD88, NF-κB mRNA and protein, which shows that the expression of MyD88, NF-κB are also controlled by other TLRs and TLR7signal transduction pathway is one of all pathways. Whether TLR7siRNA transfection or not has no significant effects on IRF7expression.
导师汪受传教授领衔的课题组完成的临床研究表明,具有开肺化痰解毒活血功效的金欣口服液是治疗小儿病毒性肺炎痰热闭肺证的有效方剂。前期实验研究结果表明,金欣口服液有明显抑制RSV的作用,主要通过抑制病毒的膜融合,其作用位点可能是病毒的F蛋白或其受体；可明显减低RSV感染后Hep-2细胞病变程度,对细胞有保护作用；具有诱导单个淋巴细胞产生IL-2、IFN-γ的作用,增强机体抗病毒免疫功能。
Toll样受体(Toll-like receptors, TLRs)是重要的模式识别受体(pattern recognition receptors, PRRs),可以识别不同微生物的病原相关分子模式(pathogen associated molecule pattem, PAMPs)。TLRs在RSV感染过程中发挥着重要的作用,是研究RSV感染的新的方向。导师汪受传教授带领的团队承担的国家自然科学基金项目“金欣口服液对RSV活化的TLRs信号转导通路作用机制研究(81072840)”前期研究成果已表明金欣口服液能调控RSV感染活化诱导的TLR3、TLR4及下游信号分子的表达水平。
RSV是单股负链RNA病毒,粘附融合宿主细胞后释放单链RNA,能被位于内体膜上的TLR7特异性识别,通过一系列信号传导途径,引起下游细胞因子的大量分泌,引起系统性炎症反应。鉴于中医药作用的多靶点性,本实验从TLR7及其介导的信号通路入手,探讨RSV感染时金欣口服液抗对TLR7信号转导通路的调控,及与其它TLRs的相关性。
目的
研究金欣口服液对RSV诱导的TLR7信号传导通路的调控作用,探讨其治疗RSV肺炎的可能免疫学机制。
方法
体外实验：RSV感染体外培养的RAW264.7细胞,采用金欣口服液含药血清进行干预,24h后收集细胞,real-time PCR法测定TLR7及其信号通路关键分子MyD88、NF-κB、IRF7mRNA的表达变化；Western Blot技术检测TLR7、 MyD88、NF-κB、IRF7蛋白表达,激光共聚焦技术检测TLR7在RSV感染RAW264.7细胞中的表达及分布；ELISA法检测细胞上清中TNF-α、IFN-α表达情况；并用TLR7siRNA干扰后检测上述指标,观察金欣口服液对RSV诱导的TLR7传导通路影响及与其它TLRs信号通路的相关性。
体内实验：RSV滴鼻感染BALB/c小鼠,金欣口服液进行干预,并于首次滴鼻后72、144小时,取小鼠肺组织,分别行病理组织切片评价肺部病变情况,real-time PCR法检测肺组织中TLR7、MyD88、NF-κB、IRF7mRNA表达情况,Western Blot法检测小鼠肺组织TLR7、MyD88、NF-κB、IRF7蛋白表达情况；取小鼠支气管肺泡灌洗液(BALF), ELISA法检测TNF-α、IFN-α表达情况。
结果
一、体外实验
1.TLR7siRNA转染前
(1)RSV感染RAW264.7细胞24h后,TLR7、NF-κB、IRF7mRNA表达明显升高(超过正常组2倍以上,与正常组比较,P<0.01),对MyD88mRNA的表达能提高,但没超过2倍：金欣口药血清组能明显降低TLR7、MyD88、NF-κB mRNA表达(P<0.01),对IRF7mRNA的表达下调作用不明显(P>0.05)。
(2)RSV感染RAW264.7细胞24h后,TLR7、MyD88、NF-κB蛋白表达量较正常组明显升高(P<0.01),而IRF7的蛋白表达无明显改变；金欣口服液含药血清组TLR7、MyD88、NF-κB蛋白表达量较RSV感染组明显降低(P<0.01)；对IRF7的蛋白表达无调控作用。
(3)RSV感染RAW264.7细胞24h后,细胞培养上清中IFN-α未能检测到；TNF-α表达量明显升高(与正常组比较,P<0.01)。金欣口服液含药血清显著降低TNF-α表达(P<0.01)。
2. TLR7siRNA转染后
(1) TLR7siRNA转染RAW264.7细胞后,TLR7mRNA的表达量下降46.6%,再用RSV感染,可见TLR7mRNA的表达又有明显增高(超过TLR7siRNA转染组的2倍以上),表明用筛选出的TLR7siRNA可干扰TLR7mRNA的生成。TLR7信号通路下游信号分子MyD88、NF-κB mRNA的表达也受到抑制,但其抑制率低于TLR7mRNA(分别为26.9%、27.3%),未成平行抑制关系,表明MyD88、NF-κB mRNA的表达可能还受其它因素的调控,特别是TLRs家族的其它成员。TLR7siRNA转染后IRF7mRNA的表达未受明显影响。
(2)金欣口药血清能明显降低转染及RSV感染后的TLR7、MyD88、NF-κB mRNA表达(P<0.01),对IRF7mRNA的表达下调作用不明显(P>0.05)。
(3)RSV感染RAW264.7细胞24h后,TLR7、MyD88、NF-κB蛋白表达量较正常组明显升高(P<0.01),而IRF7的蛋白表达无明显改变；金欣口服液含药血清组能明显下调TLR7、NF-κB蛋白表达量(P<0.01),但对MyD88的调控不明显(P>0.05)；对IRF7的蛋白表达无调控作用。
(4) TLR7siRNA转染后继以RSV感染RAW264.7细胞24h后,细胞培养上清中IFN-α仍未能检出；TNF-α表达量明显升高(与正常组比较,P<0.01),金欣口服液含药血清显著降低TNF-α表达(P<0.01)。
二、体内试验
(1)RSV感染BALB/c小鼠后肺部病理改变主要表现为肺间质性病变,肺泡壁血管扩张充血,水肿增厚,肺泡壁及间质炎性细胞浸润；肺泡腔无明显渗出物；支气管腔内无显著炎性渗出物,上皮细胞无显著变性、坏死。随着感染时间的延长,肺部病变程度逐渐加重。金欣口服液组小鼠肺内炎症均有不同程度减轻,病理评分结果显示治疗组各时间点与RSV感染组相比均有统计学显著性差异。
(2)RSV感染BALB/c小鼠72h后,肺组织中TLR7、MyD88、NF-κB mRNA表达显著增高(与正常组比较,P<0.01),而144h组随着感染时间的延长,TLR7、 MyD88、NF-κB mRNA的表达则下降。金欣口服液组TLR7、MyD88、NF-κB mRNA表达较RSV感染组不同程度降低(P<0.01或P<0.05)。
RSV感染BALB/c小鼠72、144h后,肺组织中IRF7mRNA表达量与正常组比较无统计学差异(P>0.05),金欣口服液高剂量组在感染72h能下调IRF7mRNA表达(P<0.05),金欣口服液高剂量组、等效剂量组在144h能上调IRF7mRNA表达,但无统计学差异(P>0.05)。
(3)RSV感染BALB/c小鼠72h后肺组织内TLR7、MyD88、NF-κB蛋白表达量较正常组高(P<0.01或P<0.05),144h表达下降与正常组无明显差异。金欣口服液组TLR7、MyD88、NF-κB蛋白表达量于72h较RSV感染组不同程度降低(P<0.01或P<0.05),感染144h金欣口服液组TLR7、MyD88、NF-κB表达量下降较RSV组差异无统计学意义(P>0.05)。
RSV感染BALB/c小鼠72h后肺组织内IRF7蛋白表达量较正常组无显著差异(P>0.05),144h表达较正常有所上升(P>0.05)。金欣口服液组IRF7蛋白表达量于72h较RSV感染组无统计学差异,感染144h金欣口服液组能上调IRF7蛋白表达(P<0.01)。
(4)RSV感染BALB/c小鼠72h后,BALF中TNF-α、IFN-α表达量均明显升高(与正常组比较,P<0.01);144h, BALF中TNF-α、IFN-α下降。72h,金欣口服液组小鼠BALF中TNF-α、IFN-α表达较感染组显著上升,随着感染时间的延长,144h金欣组TNF-α、IFN-α表达明显下降。
结论
1.金欣口服液能明显减轻RSV感染小鼠的肺部炎症。
2.金欣口服液对TLR7信号通路TLR7、MyD88、NF-κB mRNA及蛋白表达有明显的调控作用,对IRF7的表达在RSV感染早期无明显调控,晚期可上调IRF7的表达。
3.金欣口服液能够显著上调RSV感染初期TLR7介导的信号通路下游TNF-α、IFN-α等炎症细胞因子的表达；随着感染时间的延长,金欣口服液能适当下调TNF-α、IFN-α的表达,具有双向动态调节作用。
4.金欣口服液是治疗RSV感染的有效药物,其作用是通过调节RSV诱导的TLR7信号通路实现的,主要依赖的是TLR7/MyD88/NF-κB/TNF-α途径,在我们的实验中发现TLR7/MyD88/IRF7/IFN-α途径在RSV感染早期作用不明显。
5. TLR7siRNA转染后,TLR7mRNA及蛋白表达明显受抑,但MyD88、 NF-κB mRNA及蛋白的抑制率低于TLR7,表明MyD88、NF-κB的表达还受其它TLRs调控,从而说明TLR7信号通路只是RSV感染后的一个通路,而金欣口服液亦可通过其它通路调控下游信号分子生成,体现了中药复方发挥作用的多靶点效应；TLR7siRNA转染与否对IRF7的表达无明显影响。
Pneumonia, especially bronchial pneumonia, is a major childhood diseases, which mainly occurs in winter, spring or during climate changes. Seriously threatened children's health, pneumonia has become the top death cause of the hospitalized children. The World Health Organization list its name as one of the three major global pediatric diseases, and Chinese Ministry of Health as one of the four pediatric diseases. According to statistics, of all the urban hospital pediatric ward inpatients, pneumonia accounted for25%to50%, among which viral pneumonia is a common type, accounting for about50%of the total cases. Respiratory syncytial virus (RSV) is the main pathogen that causes viral pneumonia in children, and western medicine therapy shows little effect in treating RSV pneumonia, while Chinese medicine has its unique advantages in treating viral infectious diseases.
Jinxin oral liquid, developed to promote lung, resolve phlegm, detoxicate and remove blood stasis, proves an effective prescription in clinic which treats the viral pneumonia with phlegm-heat closed lung syndrome. Preclinic cases and experimental studies have shown that Jinxin oral liquid significantly inhibits RSV through depressing its membrane fusion, the target of which may lies in the virus'F protein or its receptor; Jinxin also sharply reduces the cytopathic effect (CPE) of Hep-2cell line infected with RSV; moreover, it induces single lymphocyte to produce IL-2, IFN-y to enhance antiviral immune function.
Toll like receptors (TLRs), an important pattern recognition receptors (PRRs), can identify different microbial pathogen associated molecular patterns (PAMPs) TLRs plays an important role in the process of RSV infection and becomes the new trend in this research field. Preliminary research results of "the effect and mechanism of Jinxin Oral Liquid on TLRs signal transduction pathway induced with RSV infection(81072840)" a National Natural Science Foundation project led by professor Wang Shou-chuan, have shown that Jinxin oral liquid can regulate the expression levels of TLR3, TLR4and downstream signaling molecules induced with RSV infection.
As a single-stranded negative-strand RNA (ss-RNA) virus, RSV adheres and fuses to the host cell and then ss-RNA is released, which can be recognized by TLR7's specific module in the inner body membrane, and through a series of signal transduction pathway, downstream cytokines secrete in high level and thus results in systemic inflammatory response. In view of the multiple target effect of Chinese medicine, this experiment starts with the TLR7and its signal transduction pathway, investigates the regulatory role of Jinxin oral liquid on the pathway induced by RSV infection, and researches the correlation to other TLRs pathways.
Objective
To study the role of Jinxin oral liquid on TLR7signal transduction pathway induced by RSV, and explore its possible immunological mechanism in treating RSV pneumonia.
Methods
Experiments in vitro:
1. The serum containing Jinxin oral liquid was applied to intervene RAW264.7cells infected with RSV, and after24hours, the cells were collected to detect the levels of TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay, and TLR7expression and distribution in cells by confocal laser scanning technology. And cell supernatants were collected to detect TNF-α, IFN-a levels by ELISA assay.
2. TLR7siRNA was used to interfere TLR7expression, and then detect the expression of above substances to observe the effect of Jinxin oral liquid on TLR7signal transduction pathway and the correlation with other TLRs pathways.
Experiments in vivo:
Jinxin oral liquid in different dosage was applied to treat BALB/c mice inoculated with RSV, and after72,144hours of the first inoculation, mice lungs were taken to evaluate lung lesions by histologic slide analysis, to detect the levels of TLR7, MyD88, NF-κB, IRF7mRNA by real-time PCR assay, and to examine the protein expression of TLR7, MyD88, NF-κB, IRF7by western blot assay. The broncho alveolar lavage liquids (BALF) were collected to measure TNF-α, IFN-α levels by ELISA assay.
Results
Experiments in vitro:
A. Before TLR7siRNA transfection
1.24h after RAW264.7cells infected by RSV, expression of TLR7, NF-κB, IRF7mRNA significantly increased (more than2times more than the normal group, P<0.01), the expression of MyD88mRNA can improve, but was not more than two times than the normal group. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01), but the effect of down-regulating IRF7mRNA not obviously (P>0.05).
2.24h after RAW264.7cells infected by RSV, TLR7, MyD88, NF-κB protein expression were significantly higher than that in normal group (P<0.01), and the protein expression of IRF7had no obvious change. The serum containing Jinxin oral liquid down-regulated significantly the expression of TLR7, MyD88, NF-κB protein (P<0.01), and had no effect on IRF7expression.
3.24h after RAW264.7cells infected by RSV, in cell supernatants the expression of TNF-a was significantly increased (P<0.01), The serum containing Jinxin oral liquid significantly reduced TNF-a expressionThe serum containing Jinxin oral liquid.But IFN-a was not found.
B. After TLR7siRNA transfection
1. After RAW264.7cells transfected by TLR7siRNA, TLR7mRNA expression decreased by46.6%, then infected by RSV, TLR7mRNA expression significantly increased (more than two times than siRNA transfection group, P<0.01). And showed that TLR7siRNA transfection interfered TLR7mRNA generation. The expression of MyD88, NF-κB mRNA were also inhibited, but the inhibition rate lower than that of TLR7mRNA (respectively26.9%,27.3%<46.6%), and that may show that MyD88, NF-κB mRNA expression may also be regulated by other factors, especially by other members of TLRs family. IRF7mRNA expression was unaffected after TLR7siRNA transfection.
2. The serum containing Jinxin oral liquid significantly reduced TLR7, MyD88, NF-κB mRNA expression (P<0.01) in RAW264.7cells treated by TLR7siRNA transfection and RSV infection, the down-regulation on IRF7mRNA expression was not obvious (P>0.05)
3. The expression of TLR7, MyD88, NF-κB protein significantly increased24h after RSV infection (P<0.01), while IRF7protein expression had no significant change. The serum containing Jinxin oral liquid significantly down-regulated TLR7, NF-κB protein expression (P<0.01), but had no obvious regulation on MyD88protein expression (P>0.05), and had no regulating effect on IRF7protein expression.
4.24h after TR7siRNA transfection followed with RSV infection, IFN-α failed to check out in the cell supernatant, and TNF-α expression significantly increased (P <0.01). The serum containing Jinxin oral liquid significantly reduced serum TNF-a expression (P<0.01)
Experiments in vivo:
1. Pulmonary pathological changes in BALB/c mice infected by RSV were mainly including interstitial inflammation, vascular dilatation and congestion, vascular wall edema and thickening, inflammatory cell infiltration in alveolar wall and interstitial lung. Alveolar cavity had no obvious exudation, and the bronchial cavity no significant inflammatory exudation, and bronchial epithelial cells were no significant degeneration and necrosis. With the RSV infection prolonged, the degree of lung lesions gradually worsened. Lung inflammation in mice treated by Jinxin oral liquid alleviate to some extent. The pathological scores of treatment group showed statistically significant differences at each time point compared with RSV infection group.
2.72h after RSV infecting BALB/c mice, TLR7, MyD88, NF-κB mRNA expression in lung tissue were significantly increased compared with the normal group (P<0.01), while at144h those expressions decreased. In Jinxin group TLR7, MyD88, NF-κB mRNA expression decreased compared with RSV group (P<0.01)
72h and144h after RSV infection, IRF7mRNA expression had no obvious difference compared with normal group (P>0.05). At72h, high-dosage Jinxin oral liquid significantly down-regulated IRF7mRNA expression (P<0.01). At144h, high-dosage and equivalent-dosage Jinxin oral liquid both up-regulated IRF7mRNA expression, but had no statistics difference compared with RSV group (P>0.05)
3.72h after RSV infection in BALB/c mice, TLR7、MyD88、NF-κB protein expression were higher than nomal group (P<0.01or P<0.05), and at144h the expression decreased and had no difference compared with normal group (P>0.05). TLR7、MyD88、NF-κB protein expression in Jinxin group at72h decreased significantly compared with RSV group (P<0.01or P<0.05), and at144h those expression decreased but had no obvious difference compared with RSV group (P>0.05)
72h after RSV infecting BALB/c mice, IRF7protein expression had no difference compared with normal group, and at144h the expression increased (P>0.05). At72h IRF7protein expression was no difference between in RSV group and Jinxin group. At144h, Jinxin oral liquid significantly increased IRF7protein expression (P<0.01)
4.72h after RSV infecting BALB/c mice, TNF-α and IFN-α expression in BALF were significantly higher than that in normal group (P<0.01) while at144h TNF-a and IFN-a expression decreased. At72h, TNF-a and IFN-a expression in Jinxin group significantly increased (P<0.01), and at144h the expression obviously reduced, but had no difference comared with RSV group.
Conclusions
1. Jinxin oral liquid significantly alleviate the lung inflammation of the RSV infected mice.
2. Jinxin oral liquid has an evident down-regulatory effects on TLR7, MyD88, NF-κB mRNA and protein expression in TLR7signal transduction pathway induced by RSV; in the late stage after SRV infection, Jinxin oral liquid can up-regulate IRF7mRNA and protein expression, while in the early stage, no obvious regulation effect can be observed.
3. Jinxin oral liquid can increase the levels of TNF-a, IFN-a in the early stage of RSV infection. During the infection prolonged period, it can down-regulate their expressions. The results show that Jinxin oral liquid has a bidirectional dynamic regulation on TNF-a and IFN-a expression.
4. Jinxin oral liquid is an effective drug in treating RSV infection, and its effect is realized by adjusting the TLR7signaling pathways induced by RSV, mainly relies on the TLR7/MyD88/NF-κB/TNF-α pathway. In the early stage of RSV infection, the effect on TLR7/MyD88/IRF7/IFN-α pathway is not obvious.
5. After TLR7siRNA transfection, the expression of TLR7mRNA and protein were inhibited, and the inhibition rate is higher than those of MyD88, NF-κB mRNA and protein, which shows that the expression of MyD88, NF-κB are also controlled by other TLRs and TLR7signal transduction pathway is one of all pathways. Whether TLR7siRNA transfection or not has no significant effects on IRF7expression.
引文
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