Anti-Influenza And Anti-Inflammatory Mechanisms Of Phamacodynamic Fraction Of Laggera Pterodonta

Background:Laggera pterodonta produced in Yunnan is commonly used as Chinese medicine for the clinical treatment of influenza.The composition of Laggera pterodonta is complex and its antiviral substance basis and pharmacological mechanism are unclear.A previous study has found that ethanol extracts had anti-influenza virus effects in vivo.Our recent work showed the ethyl acetate fraction?the pharmacodynamic fraction?had the antiviral activity in vitro,and the anti-influenza virus efficacy was better than ethanol extract.In order to further clarify the antiviral substance basis of Laggera pterodonta,this study conducted an in vitro and in vivo study to determine the efficacy of Laggera pterodonta based on the previous work.In addition,the previous study found that Pterodontic acid isolated by petroleum ether has been proved to have obvious antiviral and anti-inflammatory effects and was found in the phamacodynamic fraction.Therefore,the Pterodontic acid was used to study the antiviral target and anti-inflammatory mechanism.Objective:Our study aimed to obtain and analyze the pharmacodynamic fraction of Laggera pterodonta to investigate the effect of anti-virus and anti-inflammation and use the effective compound of sesquiterpenoids:Pterodontic acid to explain the anti-influenza virus and anti-anti-inflammatory mechanism,providing a scientific theoretical basis for its clinical application.Methods:?1?The pharmacodynamic fraction of Laggera pterodonta was obtained by ethanol and ethyl acetate.The Pterodontic acid was isolated from C8 by petroleum ether.The pharmacodynamic fraction and Pterodontic acid were analyzed systematically by UHPLC-Q-TOF-MS.?2?The cytotoxicity of the fraction was detected by MTT method and the antiviral activity of different subtypes of influenza virus?A/PR/8/34?H1N1?,A/GZ/GIRD07/09?H1N1?,A/HK/4801?H3N2?,A/Aichi/2/1968?H3N2?,A/HK/Y280/97?H9N2??B/Lee/1940?FLU B??were determined by the virus plaque reduction method.Furthermore,the progeny virus reduction method was used to further determine the antiviral effect of the fraction on H1N1 and H3N2 subtypes.The messenger RNA?mRNA?expressions of influenza virus-induced inflammatory factors treated with the compounds were detected by real-time fluorescent quantitative PCR.The antiviral efficacy in vivo of the compounds was studied by the mice and ferret pneumonia model induced by influenza infection.?3?The anti-influenza target of Pterodontic acid was detected by the viral growth curve experiment and luciferase reporter assay.The mRNA expression of influenza virus-induced inflammatory factors treated with Pterodontic acid was detected by real-time fluorescent quantitative PCR,such as RIG-I,TRIAL,Fasl,IFN-?,IFN-?,PD-L1 and PD-L2.The host signal pathways of influenza infection were examined by western blot?WB?,including RIG-I,NF-KB/ERK,STAT1 signal pathway.Results:?1?Extraction,and Identification of the fraction and Isolation of Pterodontic acidA total of 119 chemical components are sesquiterpenes?such as Pterodontic acid?and flavones,phenolic acids,organic acids,lactones and other ingredients,laying a foundation for the experiments in vivo and in vitro of anti-influenza virus and the mechanism of anti-virus and anti-inflammation.?2?Analysis of anti-influenza virus efficacy in vivo and in vitro of the fractionThe fraction had an antiviral effect on influenza A virus A/PR/8/34?H1N1?,A/GZ/GIRD07/09?H1N1?and A/Aichi/2/1968?H3N2?in vitro.IC₅₀ were22.1±2.08?g/ml,11.22±1.31?g/ml,44.3±2.1?g/ml,SI were 3.26,6.42 and 1.38;The progeny virus yield reduction method further confirmed that A/PR/8/34?H1N1?and A/Aichi/2/1968?H3N2?were strongly inhibited at 17.5?g/ml?P<0.05?,35?g/ml?P<0.05?and 70?g/ml?P<0.05?of the fraction.The mRNA levels of the inflammatory factor were found to inhibit the overexpression of MCP-1?P<0.05?,IP-10?P<0.05?,IL-8?P<0.05?and IL-1??P<0.05?at 60?g/ml of the fraction.Viral pneumonia of mice infected by A/Aichi/2/1968?H3N2?and A/PR/8/34?H1N1?were improved by the fraction.For mice infected with A/Aichi/2/1968?H3N2?,the body weight change,lung index?P<0.05?,and virus titer?P<0.05?of the mice in the treatment group of 400 mg/kg and 600 mg/kg were decreased.In the treatment group of 600mg/kg,BALF and serum were found to inhibit the overexpression of IL-6?P<0.05?,MCP-1?P<0.05?,RANTES?P<0.05?in protein levels.The lung pathology of A/Aichi/2/1968?H3N2?infected mice showed bronchial epithelium shedding,necrosis,infiltration of peripheral lymphocytes,widening of adjacent lung tissue and pulmonary interstitial diseases,infiltration of lymphocytes,histiocytes,vascular edema,and infiltration of perivascular inflammation cells.The pathology of the mice treated with the fraction was improved compared with the virus group.For mice infected with A/PR/8/34?H1N1?,the body weight changes,lung index?P<0.05?,and virus titer?P<0.05?of mice in the fraction of 400mg/kg,600mg/kg group decreased too.The overexpression of IL-6?P<0.05?and RANTES?P<0.05?protein in serum was effectively inhibited by the fraction of600mg/kg.In addition,the fraction has a positive effect on viral infection pneumonia of ferrets caused by A/Guangzhou/GIRD/07/09?H1N1?.The symptoms of influenza virus infection in ferrets were alleviated by the fraction of 250mg/kg,including relief of weight loss,increased body temperature,decreased activity,and nasal symptoms.?3?The antiviral mechanism of the effective component:Pterodontic acidAccording to the virus growth curve,it was found that 100?g/ml of Pterodontic acid effectively inhibited A/PR/8/34?H1N1?in 24 hours?P<0.05?,48 hours?P<0.05?,and 72 hours?P<0.05?after influenza virus infection.But for H3N2,in addition to the inhibition of 100?g/ml Pterodontic acid in 24 hours?P<0.05?after infection,it cannot be inhibited for 48 hours and 72 hours.Our study suggested that the target of Pterodontic acid for H1N1 is PB1,PB2,PA,NP,M,NS proteins by generating the recombinant virus through reverse genetic system.Moreover,it was found that50?g/ml?P<0.05?,100?g/ml?P<0.05?,200?g/ml?P<0.05?of Pterodontic acid can inhibit A/PR/8/34?H1N1?polymerase activity,but not A/China/LZP/2017?H3N2?by luciferase report assay,which further demonstrated that PB1,PB2,PA,NP were the target of Pterodontic acid on H1N1.As for the host inflammatory signal pathway,100?g/ml of Pterodontic acid can usefully inhibit the overexpression of the RIG-I at mRNA levels?P<0.05?and protein levels.Then,real-time quantitative PCR was used to verify that 100?g/ml Pterodontic acid can decrease the overexpression of the TRAIL/Fasl induced by the NF-?B pathway in the inflammatory pathway of A549 cells?P<0.05?.100?g/ml Pterodontic acid can inhibit the phosphorylation of inflammation-related proteins NF-?B,ERK,and overexpression of caspase 3/7.In addition,100?g/ml Pterodontic acid can reduce the mRNA overexpression of STAT?P<0.05?,IFN-??P<0.05?,and IFN-??P<0.05?by real-time quantitative PCR and can inhibit STAT protein phosphorylation in the interferon pathway by Western blot.Finally,100?g/ml Pterodontic acid can inhibit the mRNA overexpression of PD-L1?P<0.05?,PD-L2?P<0.05?by real-time quantitative PCRConclusion:The fraction has a total of 119 chemical components and have anti-influenza virus activity in vitro and in vivo.The mechanism of Pterodontic acid is the antiviral effect which of target is PB1,PB2,PA or NP of H1N1.Another one is the anti-inflammatory and antioxidant effects through the inhibition of RIG-I/NF-?B/STAT1/I interferon and PD-L1/2.