KangBingDu Oral Liquid Reduces The Susceptibility To Influenza Virus In Restraint-stressed Mice Through Mitochondrial Mfn2/MAVS Antiviral Signaling Pathways

Aim: Kang Bing Du?KBD? oral liquid, a Chinese patent medicine?CPM?, which is modified based on the TCM formulations of "Bai Hutang" and "Qing Wen Bai Du Yin", is widely used for clinical treatment of virus infection, especially influenza virus. This study was designed to investigate the effect of Kang Bing Du?KBD? oral liquid on decreasing susceptibility of influenza and its related mechanisms by employing restraint stress to set up a disease susceptible animal model.Methods: Mice were randomly divided into 6 groups: Control, Virus, Model?restraint stress + virus?, Oseltamivir?restraint stress + virus + 30.8 mg/kg/d oseltamivir?, KBD-L?restraint stress + virus + 12.3 m L/kg/d KBD? and KBD-H?restraint stress + virus + 24.6 m L/kg/d KBD?. In Model and drug treatment groups, mice were physically restrained in a 50 m L polypropylene restraint tube with holes for 22 h. After recovering for 1 day, mice were inoculated intranasally with a double amount of LD50 dose of virus?35 ?L? under anesthetization by diethyl ether vapor. The oral administration of oseltamivir and KBD started from the first day of stress and lasted for 7 days?twice a day?. The body weight, survival, and several typical symptoms of illness were recorded for 21 days or until death. Morbidity rate, mean time to sickness, survival rate and MDD were consequently calculated.Further experiment was performed to evaluate the anti-influenza virus mechanisms of KBD in mice loaded with restraint stress. The animal group and drug administrations were the same as the first experiment. Five days after virus infection, mice were weighed and anesthetized by ethyl ether. The lungs were removed, cleaned and weighed. Lung index was calculated as a parameter of inflammatory edema according to the following formula: Lung index?mg/g? = lung weight/body weight. Lung tissues were harvested for histopathologic and inflammatory markers?NF-kB, IL-1b and TNF-a? examination. Moreover, susceptivity markers?IFN-?, IFITM3?, virus load?NP? and antiviral signaling proteins?Mfn2, MAVS, and p-IRF3? in lung were also determined by reverse transcription polymerase chain reaction?RT-PCR? and Western Blotting analysis.The 3^th experiment was performed to examine the effect of KBD on plasma CORT level and MAVS in mice loaded with restraint stress. The mice were randomly divided into 4 groups with 25 mice in each group: Control, Restraint stress, KBD-L?restraint stress + 12.3 m L/kg/d KBD? and KBD-H?restraint stress + 24.6 m L/kg/d KBD? groups. Mice except for Control group were physically restrained in a 50 m L polypropylene restraint tube with holes for 22 h. Mice were orally administered with KBD for 4 days?twice a day?. On the 3^th day after stress, mice were anesthetized by ethyl ether. Blood samples were collected from heart, and plasma CORT level was determined by high performance liquid chromatography?HPLC?. Lung tissues were harvested for the protein expression of Mfn2 and MAVS by Western Blotting method.The chemical fingerprint of KBD was determined using HPLC and gas chromatography?GC? analysis. In order to identify the active components in KBD and mimic the stress-induced susceptibility to influenza virus infection in vitro, we use CORT, an indicator of stress response, to establish "CORT + Virus" A549 cell model. The main compounds identified in KBD??R, S?-goitrin, mangiferin, forsythoside A, forsythin? were pretreated for 2 h, then cells were challenged with CORT and virus. Cells were harvested to determine the protein expressions of NP, IFN-? and IFITM3 by Western Blotting.Results: Results demonstrated that treatment with KBD alleviated influenza symptoms, significantly reduced morbidity, improved the survival rate and prolonged the mean time to sickness and mean day to death of virus-infected mice loaded with restraint stress. The administration of KBD was effective in limiting IL-1? and TNF-? production by down-regulating NF-?B, as well as inflammatory cells infiltration. KBD increased the IFN-? responses to boost resistance to influenza through mitochondrial anti-viral signaling pathway independent of non-specific anti-stress effect.Conclusion: In conclusion, the present study demonstrated that restraint-stress mice model is effective in the evaluation of the antiviral activity of KBD. It can reduce the susceptibility to influenza via regulating mitochondrial anti-viral signaling. In addition, KBD could also down-regulate NF-?B protein expression to reduce the level of pro-inflammatory cytokines. The results of the current study provided scientific data for the use and development of KBD and other anti-influenza TCM.