| ObjectiveInfluenza A virus(IAV)is highly contagious,which can easily cause widespread influenza outbreaks and pose a serious threat to human life and health.Its high variability makes vaccines less effective or even ineffective,and at the same time it produces resistance to antiviral drugs,which brings serious trouble to clinical treatment.Endoplasmic reticulum stress and immune inflammatory response are important mechanisms for IAV to invade host cells.They are involved in the process of IAV infection-induced host cell apoptosis and inflammatory damage,and even induce cytokine storm,which greatly increases the difficulty of treatment.The previous research of the research group found that Liushen Pill and one of its main components,realgar,have significant anti-respiration virus replication effects in vitro.Clinical and experimental studies have found that bufonis venenum and its whole dried toad,which are also the main components of Liushen pills,are powerful detoxification drug and have good curative effect on viral infections.However,the ancient literature research on toad drugs is still blank,and the research on the antiviral effects of toad drugs is in the preliminary stage.To provide a theoretical basis of toad drugs for the prevention and treatment of influenza virus infection and other epidemic diseases,we further studied the dismantling of Liushen Pill on the basis of the previous work.The representative drugs of toads were traced from ancient treatises,the origin of the clinical application of toad and bufonis venenum.By using the new patented non-toxic dried toad and bufonis venenum,and the ERS pathway as the entry point,the effects of IAV-induced apoptosis and immune inflammatory response were observed in vitro and in vivo experiments.By exploring its protective effect and mechanism in the treatment of IAV infection,these researchs will provide data support for the development of Chinese herbs on the prevention and treatment of influenza virus infection.Methods1 Based on the database of Chinese Medical Dictionary,by setting keywords,we can search for the texts of herbal medicine and compound prescriptions containing keywords.With the help of EXCEL and SPSS Modeler 18.0,frequency statistics and association rule data mining were carried out in ancient documents on the collection,processing,medicinal properties,efficacy,and formula compatibility characteristics of toad and bufonis venenum.2 Preparation of the dilution of non-toxic dried toad water extract,non-toxic dried toad alcohol extract,bufonis venenum water extract,bufonis venenum alcohol extract and oseltamivir phosphate.Madin-Darby canine kidney(MDCK)were used as infected cells,and the maximum nontoxic concentration(TC0)and median toxic concentration(TC50)of the drugs were determined by CCK8 method.We used three different routes of administration,including direct inactivation,prophylaxis,and therapeutic administration.The CCK8 method was used to determine the effective rate(ER),half effective concentration(EC50)and therapeutic index(TI)of the drugs against influenza virus A/PR/8/34(H1N1)in vitro.We further selected the treatment methods with higher ER and TI.At 12 h,24 h and 48 h after administration,the apoptosis rates of cells in each group were detected by TUNEL method,and the mRNA expression levels of GRP78,CHOP,Caspase-12,Caspase-3 and Bcl-2 in the ERS pathway of cells in each group were detected by qPCR at different time points.3 Establishing a mouse model of influenza A virus pneumonia.After 1 st,3 rd and 5 th,the death protection rate,lung index,lung index inhibition rate,thymus index,spleen index,and colon length were measured.The levels of IL-6,MCP-1 and TNF-α in serum and lung tissue of mice were detected by CBA technique,and the pathological changes of lung tissue of mice were observed under light microscope.4 The mRNA and protein expression levels of GRP78,CHOP and Bcl-2 in the ERS pathway in mouse lung tissue were detected by qPCR and western blot,and the pathological changes of mouse lung tissue,the morphological changes of apoptosis of endoplasmic reticulum and other organelles were observed under electron microscope.Results1 Herbal properties of toads in ancient literature:There were 20 names of toad and 9 names of bufonis venenum.The Dragon Boat Festival was the best time to collect toads and bufonis venenum.The ancient methods of processing toads included drying in the shade,raw mashed juice,fried charcoal,fried coke,simmered in wine,and soaked in Huangjing juice and coated with crispy,clay burning or simmering,etc.The ancient processing methods of bufonis venenum mainly included wine,dairy,and vinegar.The properties and flavors of toads and bufonis venenum in ancient books were relatively consistent.Toad was pungent and cool,and belong to the stomach meridian of foot Yangming,while bufonis venenum was pungent and warm or hot,and can pass through twelve meridians or belong to Taiyin Lung Meridian or foot Shaoyin Kidney Meridian,etc.The functions of toad included dispelling external pathogens,clearing heat and detoxifying,dispelling stagnation,killing insects and eliminating chancre,diverting water and reducing swelling,calming the liver and relieving wind,etc.While the effects of bufonis venenum included detoxification,warding off evil spirits and opening orifices,astringent essence,helping yang,eliminating accumulation and killing insects,eliminating stasis and removing blood stasis,etc.At the same time,the research showed that the toxicity of toad was low,and its poison was mainly in bufonis venenum.The bufonis venenum poisoning ways were mostly reactions of the eye,nose,skin and other pores.2 General characteristics of the toads prescription in ancient literature:A total of 887 prescriptions were included,among them,there were 337 toad prescriptions,including 228 for oral administration,108 for external use,and 1 for buccal administration.Among 550 bufonis venenum prescriptions,including 258 for oral administration,263 for external use,and 29 for buccal administration.Most of the oral toad prescriptions,the oral bufonis venenum prescriptions,and the buccal prescriptions of bufonis venenum were pills,while the external prescriptions and the buccal prescriptions of toad were mainly powders.There were 19 and 4 kinds of excipients for the oral and external prescriptions of toad,and no excipients were used in the buccal prescriptions of toad.There were 16,18 and 3 kinds of excipients for oral administration,external administration,and buccal administration of bufonis venenum respectively.Among them,plant accessories such as noodles,rice,honey pills and animal accessories such as animal bile,chicken white,human milk,snail juice and toad juice were mainly used.There were 25 and 5 ways of administration for the oral and external toad prescriptions and 24,9,and 2 ways of administration for the oral,external,and buccal bufonis venenum prescriptions respectively.Among them,rice drink or porridge drink,wine,green onion soup,and traditional Chinese medicine decoction such as Jingjie soup and Bohe soup appeared more frequently.3 Indications and compatibility characteristics of toads prescriptions in ancient literature:There were 14 types of indications for internal and external toad prescriptions.Among them,the internal prescriptions were mainly used to treat chancre syndrome,epidemic diseases and children’s convulsions,the external prescriptions were mainly for oral diseases and sores,and the containing prescription was for oral diseases.There were 16,20,and 4 types of indications in the oral,external,and buccal bufonis venenum prescriptions.Among them,the internal prescriptions were mainly used for sores,chancre syndrome,infantile convulsions and epidemic diseases,the external prescriptions and the oral prescriptions were mainly for sores,oral diseases,male reproductive diseases and epidemic diseases.Oral and external toad prescriptions were most easily compatible with heat-clearing drugs,and oral bufonis venenum prescriptions were most frequently compatible with orifice drugs,and external bufonis venenum prescriptions were most usually compatible with drugs for attacking poison,killing insects,and relieving itching.Compatible drugs were mostly bitter,acrid,warm or cold in nature and flavor,and the spleen and liver meridians were the main meridians.Through the analysis of association rules,it was found that the core compatible drugs of the oral toad recipe were Shexiang,Zhusha,Huhuanglian and Xionghuang,etc.The core compatible drugs of the external bufonis venenum prescription were Shexiang,Xionghuang,Ruxiang and Moyao,etc.The core compatible drugs of the internal bufonis venenum prescription were Shexiang,Zhusha,Xionghuang,Niuhuang,etc.And the core compatible medicines of the external bufonis venenum prescription were Moyao,Ruxiang,Shexiang and Dingxiang,etc.4 Cell experiments:In the three modes of action,each drug had a certain anti-influenza virus PR8 effect,and showed a concentration-dependent relationship,but the anti-viral effects of non-toxic dried toad water extract and non-toxic dried toad alcohol extract were not significant under the preventive action mode.The non-toxic dried toad water extract,non-toxic dried toad alcohol extract,bufonis venenum water extract and bufonis venenum alcohol extract had the highest ER and TI for influenza virus PR8 under the direct action mode.When the concentration was TC0,the ER of the four groups under the direct action mode was 60.94%,63.65%,86.20%,72.07%,and the TI was 19.33,18.85,106.22,32.51 respectively.Among them,the ER of bufonis venenum water extract was comparable to that of oseltamivir(86.98%).TUNEL experiments showed that the apoptosis rate of each group increases gradually at 12 h,24 h and 48 h after administration.Except the non-toxic dried toad water extract group at 24 h,compared with the virus control group,the other drug groups could significantly reduce the apoptosis rate of MDCK cells.Among the traditional Chinese medicine extracts,the apoptotic rate of bufonis venenum water extract was the lowest,followed by the bufonis venenum alcohol extract or the non-toxic dried toad alcohol extract.The non-toxic dried toad alcohol extract and bufonis venenum water extract can significantly down-regulate GRP78 mRNA,CHOP mRNA,Caspase-12 mRNA,Caspase-3 mRNA or up-regulate Bcl-2 mRNA at 12 h,24 h or 48 h after PR8 infection.The degree of up-regulation or down-regulation in the bufonis venenum water extract was the most significant.The bufonis venenum alcohol extract can significantly down-regulate CHOP mRNA,Caspase-12 mRNA,Caspase-3 mRNA or up-regulate Bcl-2 mRNA at 12 h,24 h or 48 h after PR8 infection.The non-toxic dried toad water extract can only significantly down-regulate CHOP mRNA and Caspase-12 mRNA in cells at 12 h after PR8 infection or up-regulate Bcl-2 mRNA expression in cells at 24 h post-infection.5 Protective effect of non-toxic dried toad and bufonis venenum on lung injury in model mice:Compared with the model group,non-toxic dried toad,bufonis venenum and oseltamivir can significantly increase the body weight,food intake,curled up and chills of the mouse infected with IAV.They can significantly reduce the lung index of mice on the 3 rd or 5 th day after infection,improve the survival rate of mice,and ameliorate the inflammatory pathological damage of lung tissue on the 3 rd and 5 th day after infection,and reduce the inflammatory changes in the lung.Non-toxic dried toad can reduce the levels of IL-6 and TNF-α in lung tissue or serum on 5 th day after infection,and bufonis venenum can reduce IL-6,TNF-α,MCP in lung tissue or serum on 5 th day after infection,and it can significantly increase the thymus index on 5 th day after infection and the length of colon at 3 rd day after infection,but the effects on spleen index was not obvious.6 Regulation of the ERS pathway by non-toxic dried toad and bufonis venenum:Compared with the model group,for GRP78,non-toxic dried toad could significantly down-regulate the level of GRP78 mRNA in the lung tissue of mouse with influenza A virus pneumonia on the 5 th day after infection,and bufonis venenum could significantly down-regulate the level of GRP 78 mRNA and corresponding protein in lung tissue on the 5 th day after infection.For CHOP,non-toxic dried toad and bufonis venenum can significantly down-regulate CHOP mRNA in lung tissue on the 3 rd and 5 th day after infection,but there was no significant difference in the level of CHOP protein.For Bcl-2,non-toxic dried toad and bufonis venenum could reduce the levels of Bcl-2 mRNA and corresponding protein in lung tissue on the 3 rd and 5 th days after infection.The results of electron microscopy showed that after the intervention of non-toxic dried toad and bufonis venenum,the endoplasmic reticulum was less swollen,the ribosomes were clear and abundant,the organelle structure was relatively complete,some nucleoli showed chromatin edge aggregation,and lung interstitial exudation was less.The morphology of apoptosis was improved compared with the model group.Conclusion1 The efficacy and main symptoms of toad or bufonis venenum in ancient literature are more extensive than today.Through certain processing,appropriate dosage forms,excipients,administration methods and reasonable compatibility,they can reduce toxicity and increase efficiency.Therefore,it provides a reliable theoretical basis for the treatment of IAV infection and the promotion of the development of related traditional Chinese medicine preparations.2 The non-toxic dried toad water extract,non-toxic dried toad alcohol extract,bufonis venenum water extract and bufonis venenum alcohol extract have the best anti-viral effects in vitro under the direct inactivation method.The non-toxic dried toad alcohol extract and bufonis venenum water extract may play an anti-influenza virus-induced apoptosis effect by inhibiting the endoplasmic reticulum stress pathway,and the anti-apoptosis effect of the bufonis venenum water extract is the best.3 By inhibiting cell apoptosis caused by ERS and reducing immune inflammatory response,non-toxic dried toad and bufonis venenum can protect lung injury caused by influenza A virus infection. |
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