| Respiratory syncytial virus (RSV) is the leading cause of low respiratory tract infection in infants which was in hospitalization. According to the report of the WHO, RSV shares 29 pecent of the pathogens of infantile pneumonia. The pathogenesis of RSV infection is not yet entirely clear. The modern medicine has no specific anti-RSV viral drug. Ribavirin was only approved by FDA to be available for RSV infection, but due to the higher incidence of side effects, the clinical use was limited. The development of RSV vaccine has not achieved a qualitative breakthrough, so the current vaccine is not yet available.At present, the treatment of viral diseases in Chinese medicine has a significant advantage. Jinxin Oral Liquid (JOL),that is Qingfei oral Liquid, is developed on the basis of MXSGT and clinical experience of Pro. Wang Shouchuan, a famous pediatrician of TCM in China and the doctoral tutor of Nanjing University of Chinese Medicine. JOL included Cortex Mori Radicis, Pepperweed Seed, Peucedani, Baical Skullcap Root, Rhizoma polygoni cuspidate, Main ephedra, Bitter apricot seed and gypsum. The main function of these herbs is heat-clearing and detoxifying, and relieving a cough and reducing phlegm based on the TCM theory. On the basis of the long-term clinical application and a large number of clinical studies, we found that JOL has significant effect on treating the respiratory diseases in children.Toll-like receptor 3 (TLR3) and retinoic acid inducible gene I (RIG-I) all belong to the innate immune pattern recognition receptors. They could identified the dsRNA which was produced in the multiplication process of RSV to activate TLR3 signaling pathway and RIG-I signaling pathway. Finally, the production of cytokine was promoted to play an anti-RSV effect. In this study, TLR3/RIG-I signaling pathway is the pointcut to study the molecular mechanisms of the JOL to treat the pneumonia in children with RSV.Objective:The treatment mechanism of JOL on the RSV-infected pneumonia was investigated by detecting the expression of the key signaling molecules of TLR3/RIG-I signaling pathway after RSV-infected and intervened by JOL.Methords:1. In vitro experiment1.1 Raw264.7 cells with RSV-infected were sampled once every two hours in the 24 hours. The expression trends of TLR3, IRF3 and IRF3 mRNA in cells were detected by Real time RT-PCR assay.1.2 Raw264.7 cells infected by RSV were intervened by JOL.12 hours after RSV infection, cells were lysed by RNA iso plus and the virus load in cell was detected by Real time RT-PCR assay.1.3 Raw264.7 cells infected by RSV were intervened by JOL.12 hours after RSV infection, cell culture supernatant and cell were collected respectively. The mRNA expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, IRF3, IFN-p and SOCS1 were detected by Real time RT-PCR assay. The protein expression of TLR3, RIG-I, IPS-I,TRAF3, TBK1, IRF3(P-IRF3), IFN-β and SOCS1 were detected by Western blot. Meanwhile, The protein expression of TLR3 was detected by immunocytochemistry and confocal laser scanning microscope. The expression of IFN-β in supernatant was detected by ELISA.1.4 siRNA labeled by fluorescent reagent was transfected into Raw264.7, and the transfection efficiency was detected by flow cytometry. Under the condition of MyD88 gene interference, Raw264.7 cells were infected by RSV and intervened by JOL.12 hours after RSV infection, cells were collected and lysed. The expression of MyD88, IRF3(P-IRF3) and NF-κB mRNA and protein were detected by Real time RT-PCR assay and Western blot respectively.2.1n vivo experiment2.1 The Balb/c mice were intranasally challenged with RSV and intervened by JOL and ribavirin respectively.2d,4d,7d after the first inoculation of RSV, the mice were sacrificed and the lungs of mice were collected, grinded and lysed respectively. The virus load of the lung was detected by Real time RT-PCR assay.2.2 The Balb/c mice were intranasally challenged with RSV and intervened by JOL and ribavirin respectively.2d,4d,7d after the first inoculation of RSV, the mice were sacrificed. The lungs of mice and bronchoalveolar lavage fluid (BALF) were collected respectively. The lungs were removed and fixed in 10% neutral buffered formalin. The tissue sample was sliced into 3μm section and stained by H & E staining. Tissue lesions and inflammatory cell infiltration were examined using microscope. The mRNA expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, IRF3, IFN-β and SOCS1 were detected by Real time RT-PCR assay. The protein expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, IRF3(P-IRF3), IFN-p and SOCS1 were detected by Western blot. The expression of IFN-p in BALF was detected by ELISA.Results:1. In vitro experiment1.1 During the 24 hours after RSV infection, the mRNA expression trends of TLR3, IRF3 and IFN-β were increased firstly and then decreased. The peak time of the mRNA expression of TLR3 was.12-14 hours after RSV infection. The peak time of the mRNA expression of IRF3 was 10-12 hours after RSV infection. The peak time of the mRNA expression of IFN-β was 12-16 hours after RSV infection.1.212 hours after Raw264.7 cell infected by RSV, the level of viral loads were significantly increased by JOL(P<0.01).1.312 hours after Raw264.7 cell infected by RSV, compared with the NC group, the mRNA expression of TLR3, RIG-I, IPS-I, TBK1, IRF3 and IFN-β in the MC group was significantly increased(all P<0.01). JOL could all prevent expression of TLR3, RIG-I, IPS-I, TBK1, IRF3 and IFN-β in Raw264.7 cells with RSV infection(all P<0.01). However, there were no significant difference in the mRNA expression of TRAF3 between NC and MC group (P>0.05). Compared with the NC group, the mRNA expression of SOCS1 in the MC group was significantly decreased(P<0.01), but JOL could upregulate the level of the mRNA expression of SOCS1 significantly (P<0.01).1.412 hours after Raw264.7 cell infected by RSV, compared with the NC group, the protein expression of TLR3, RIG-I, IPS-I, TBK1, P-IRF3 and IFN-β in the MC group was significantly increased(all P<0.01). JOL could all downregulate the expression of TLR3, RIG-I, IPS-I, TBK1, P-IRF3 and IFN-β in Raw264.7 cells with RSV infection(all P<0.01). However, the difference of IRF3 protein expression was not significant among these groups. Meanwhile, there were no significant difference in the expression of TRAF3 between NC and MC group (P >0.05), but JOL could downregulate the level of the expression of TRAF3 significantly. The protein expression of SOCS1 in the MC group was significantly decreased(P<0.01), but JOL could upregulate the level of the expression of SOCS1 significantly (P<0.01).1.5 After MyD88 gene interference, the difference of IRF3 protein expression was not significant among these groups (P>0.05). However, after MyD88 gene interference, compared with the NC group, the expression of NF-κB in the NC-siRNA group was significantly decreased(P<0.01). Meanwhile, compared with the MC group, the expression of NF-κB in the MC-siRNA group was also significantly decreased(P<0.01). However, JOL can’t regulate this change(P>0.05).2. In vivo experiment2.1 the level of viral loads were significantly increased on 2nd and 4th day after infection, but relatively decreased on 7th day when compared with those on 2nd and 4th day. At the three different time-point, the RSV-M mRNA could not be determined by Real-time RT-PCR in the NC group, but viral loads in the MC group were significantly higher than those in NC group (all P<0.01). On 2nd and 7th day after infection, viral load in the JOL and Ribavirin treated group were all lower than that of the MC group (all P<0.01), but there were no significant difference in viral load among JOL-HD, JOL-LD and Ribavirin (P>0.05). On 4th day after infection, viral loads in the JOL and Ribavirin treated groups were also significantly lower than that of the MC group (all P<0.01), but viral loads in the JOL-HD and JOL-LD were lower than that of Ribavirin treated group (all P<0.01).2.2 On day 2,4,7 after infected RSV, the lung sections form uninfected mice exhibited no or little histopathology. In contrast, lung histopathology of the RSV-infected mice was the congestion and edema of pulmonary in interstitial around vessels, macrophages and mononuclear cell infiltrates in foci scattered throughout the lumen of alveoli, both within and adjacent to many bronchi and bronchioles, and compensatory emphysema. Especially, there were numerous inflammatory cells infiltration (e.g., lymphocytes, macrophages and polymorphonuclear), a large amount of exudates in the airway lumen, and alveolar wall thickening and edema. However, compared with the RSV-infected group, pulmonary section in the JOL-treated groups exhibited fewer foci of inflammatory cells, less edema and fewer bronchioles or bronchi.2.3 On 2rd days after RSV infection, compared with the NC group, the mRNA expression of TLR3, RIG-I, IPS-I, IRF3,SOCS1 and IFN-β in the MC group was significantly increased(all P<0.01). Compared with the MC group, the different doses of JOL could all down-regulate the expression of TLR3, RIG-I, IPS-I, SOCS1 and IFN-β (all P<0.01), but JOL-LD can’t regulate the mRNA expression of IRF3(.P>0.05). There were no difference significantly in the mRNA expression of TRAF3 and TBK1 between the NC and MC group (P>0.05). On 2rd days after RSV infection, compared with the NC group, the protein expression of TLR3, RIG-I, IPS-I, TRAF3, P-IRF3, SOCS1 and IFN-β in the MC group was significantly increased(all P<0.01). Compared with the MC group, the different doses of JOL could all down-regulate the expression of TLR3, RIG-I, IPS-I, TRAF3, P-IRF3, SOCS1 and IFN-β (all P<0.01). However, the difference of IRF3 protein expression was not significant among these groups (P>0.05). Compared with the NC group, the protein expression of TBK1 in the MC group was significantly decreased(P<0.01), and JOL-HD could down-regulate the expression of TBK1, but JOL-LD could up-regulate the expression of TBK1.2.4 On 4th days after RSV infection, compared with the NC group, the mRNA expression of TLR3, RIG-Ⅰ, IPS-Ⅰ, TBK1, IRF3,SOCS1 and IFN-β in the MC group was significantly increased(all P<0.01). Compared with the MC group, the different doses of JOL could all down-regulate the expression of TLR3, RIG-Ⅰ, IPS-Ⅰ, TBK1, IRF3,SOCS1 and IFN-β (all P< 0.01). Compared with the NC group, the mRNA expression of TRAF3 in the MC group was significantly decreased(P<0.01), but the different doses of JOL could all up-regulate the expression of TRAF3 (all P<0.01). On 4th days after RSV infection, compared with the NC group, the protein expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, P-IRF3, SOCS1 and IFN-βin the MC group was significantly increased(all P<0.01). Compared with the MC group, the different doses of JOL could all down-regulate the expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, P-IRF3, SOCS1 and IFN-β (all P<0.01). However, the difference of IRF3 protein expression was not significant among these groups (P>0.05).2.5 On 7th days after RSV infection, compared with the NC group, the mRNA expression of TLR3, RIG-I, IPS-I, TRAF3, TBK1, IRF3 and IFN-β in the MC group was significantly decreased(all P<0.01). Compared with the MC group, the different doses of JOL could all up-regulate the expression of TLR3, IPS-Ⅰ, TBK1, IRF3 and IFN-β (all P<0.01). And JOL-LD could up-regulate the expression of RIG-Ⅰ (P<0.01), but JOL-HD could not regulate the expression of RIG-I (P>0.05). Compared with the NC group, the mRNA expression of SOCS1 has no significant change (P>0.05), but the different doses of JOL could all down-regulate the expression of SOCS1 (all P<0.01). On 7th days after RSV infection, compared with the NC group, the protein expression of TLR3, RIG-I, IPS-I, TRAF3, P-IRF3, SOCS1 and IFN-β in the MC group was significantly decreased(all P<0.01). Compared with the MC group, the different doses of JOL could all up-regulate the expression of TLR3, RIG-Ⅰ, IPS-Ⅰ, TRAF3, TBK1, P-IRF3 and IFN-β (all P<0.01), but the different doses of JOL could all down-regulate the expression of SOCSl(all P<0.01). However, the difference of IRF3 protein expression was not significant among these groups (P>0.05).Conclusion:(1) JOL could all reduce the virus loads of the lungs of mice and R264.7 cells significantly. Therefore, JOL could inhibit directly the proliferation of RSV.(2) JOL could alleviate lung histopathology of the RSV-infected mice. Pulmonary section in the JOL-treated groups exhibited fewer foci of inflammatory cells, less edema and fewer bronchioles or bronchi.(3) During the 24 hours after Raw264.7 with RSV infection, the mRNA expression trends of TLR3, IRF3 and IFN-β were increased firstly and then decreased. The peak time of the mRNA expression of TLR3, IRF3 and IFN-β was 10-14 hours after RSV infection.(4) The main chemical components of JOL was analyzed by ultra performance liquid technical, including sinapine thiocyanate, polydatin, scutellarin, resveratrol, baicalin, emodin-8-O-β-glucoside, baicalein and wogonin.(5) TLR3/RIG-I signaling pathway was activated by RSV infection. In the early stages of RSV infection, the mRNA and protein expression of TLR3, RIG-Ⅰ, IPS-Ⅰ, TRAF3, TBK1 and IRF3 (P-IRF3) were increased significantly. Therefore, JOL could inhibit the activation of TLR3/RIG-I signaling pathway in the early stages. However, the mRNA and protein expression of TLR3, RIG-Ⅰ, IPS-Ⅰ, TRAF3, TBK1 and IRF3 (P-IRF3) were decreased significantly in the late stages. Therefore, JOL could promote the activation of TLR3/RIG-I signaling pathway in the late stages. The expression trends of the key signal molecules of TLR3/RIG-I signaling pathway were increased firstly and then decreased.(6) The expression of SOCS1, that is negative regulatory factors of TLR3 signaling pathway, was induced by RSV infection. However, with prolonged of infection, the expression of SOCS1 gradually decreased. JOL can continue to inhibit the expression of SOCS1 to relieve inhibition effect to TLR3 signaling pathway.(7) The expression of IFN-β was induced by RSV infection. The expression trends of IFN-β was increased firstly and then decreased. In the early stage of RSV infection, JOL can inhibit the over-expression of IFN-βto reduce inflammation damage. In the late stage, JOL can promote the expression of IFN-β to continue to maintain the antiviral effect.(8) After MyD88 gene interference, the expression of IRF3 induced by RSV infection was not significantly affected. However, the expression of NF-κB was decreased significantly, and JOL could not regulate the change. Therefore, the interference of MyD88 gene has no effect on the TLR3 signaling pathway, but inhibit the activation of MyD88-dependent signaling pathway. Meanwhile, it is confirmed that the action site of JOL should be located in the upstream of MyD88. |
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