Material Basis And Evaluation Of Ruyi Jinhuang Powder In Treating Necrotizing Fasciitis

Background:Necrotizing fasciitis(NF)is a diffuse soft tissue infection.At that develops rapidly and is life threatening,NF is rare but mortality is very high.It is tissue necrosis due to one or more bacterial infections.According to traditional Chinese medicine,is the pathogenesis of this disease.NF falls into the category of "rotten furuncle" which is an acute suppurative disease that occurs between the skin and the flesh accompanied by caries.It is characterised by local redness,swelling,thermal pain,caries and systemic symptoms leading to death.The basic pathogenesis of the disease is that the heat toxin flourishes,burrows into the skin,attacks the viscera internally,leading to blood and gas failure,necrosis of bones and muscles.Ruyi Jinhuang Powder(RYJHP)comes from the "Authentic Surgery" of Chen Shigong,a celebrated surgeon of Chinese traditional medicine in Ming Dynasty,and is included as part of the first volume of Chinese Pharmacopoeia(2020 edition).It is made up of ten herbs:Trichosanthes root,Phellodendron amurense,Rhubarb,Turmeric,Angelica dahurica,Magnolia officinalis,Tangerine peel,Licorice,Atractylodes lancea,and Rhizoma Arisaematis.The prescription has the effects of alleviating swelling and pain,and can be used for treating traumatic injury,carbuncle and abscess on the back,various furuncles and swellings,damp-phlegm and toxic fluid,red swelling of the skin,swelling of the head,carbuncle of breast in women,erysipelas in children,etc.Modern studies have found that RYJHP has antibacterial and anti-inflammatory effects,and is used for the treatment of perianal abscesses,phlebitis,erysipelas,and other diseases.The efficacy of this formula includes features of NF,but it has not been reported to have a therapeutic application.Objectives:In order to explore the material base of the RYJHP,to assess the therapeutic effect of NF,and to investigate its therapeutic mechanism.To provide a theoretical basis and experimental basis for the clinical application of RYJHP in the treatment of NF,and for excavating the effective components of traditional Chinese medicine for the treatment of NF targeting specific routes from the RYJHP,in order to provide research ideas for the innovative application and development of traditional Chinese medicine.Methodologies and Results:1.Analysis of RYJHP’s Effective Material Basis by UPLC-Q/TOF-MSUPLC-Q/TOF-MS used to analyse and detect the active ingredients of the RYJHP.Using Superstar discovery platform,Pubmed platform,the Chinese traditional medicine systematic pharmacology database and the analytical platform,Pubchem database and the Reaxys database,taking the names of ten medicinal plants constituting RYJHP as the subject words,obtaining structures of relevant active ingredients to build a theoretical database of RYJHP active ingredients,and import it into the UNIFI automatic analysis platform to perform a high throughput full-molecular matching screen on the active ingredients according to specified errors.191 compounds are obtained by standard reference analysis,including 42 flavonoids,14 triterpenes,12 lignans,18 coumarins,16 anthraquinones,10 steroids,and 14 alkaloids.Based on the types of compounds obtained through analysis and associated research reports,RYJHP has been hypothesized to have antibacterial,anti-inflammatory,and other therapeutic effects and may be used in the treatment of NF.2.To assess the therapeutic effect of RYJHP on NFMRS A established the model of NF and GAS.The dose of mice was calculated based on the dose in adults.The treatment group consisted of RYJHP mixed with olive oil,which was applied to the affected area,and an alginate silver ion dressing was administered for external use following disinfection and drainage of pus with iodophor as a positive control group.Olive oil solvent was used for external purposes as a solvent control group.It is found that RYJHP can reduce the rate of death of mice with NF,increase the quality of life of the mouse,reduce the area of necrotic skin damage,relieving pathological lesions of the skin,living and kidney and improving liver and kidney functions by measuring the state of weight change,survival curve and skin lesion area of the mice after administration,observing the pathological changes by using HE staining skin tissues and liver and kidney slices,to detect indices of liver and kidney function using a fully automatic biochemical analyzer and the like,has an integral effect of treatment on NF mice.RYJHP can reduce the bacterial burden in the blood and skin tissue of mice with NF,and has been shown to have antibacterial activity in vivo.In the anti-inflammatory aspect,RYJHP can reduce the level of TNF-α,IL-1β,COX-2 and inhibit the overexpression of inflammation.3.Mechanism of RYJHP in Treating NF and Screening of Core Targets and Key Effective ComponentsBased on the results of Part 1,52 transdermal active ingredients were obtained through virtual active ingredient screening.The predicted active ingredient targets were collected from TCMSP,BATMAN-TCM and ETCM.According to the property theory of traditional Chinese medicine,herbs are broken down into three functional modules:"cold","mild",and "warm",The effective components and targets are classified.A database of NF related targets was obtained by intersecting the NF related targets included in public databases with the targets with significant expression differences found by clinical transcriptomics data mining.Using mapping analysis,"herb-component-target" action network was constructed,and it was found that 44 active ingredients in RYJHP acted on 53 targets for the treatment of NF.According to the function modules,protein interaction network analysis was performed,and 6 central targets were eliminated in the three function modules of "cold","mild",and "warm ".GO and KEGG enrichment analysis is used for the target of action of RYJHP for the treatment of NF and the key target obtained by analysis of protein interaction networks,the result shows that RYJHP deals with NF through activation of lymphocytes to recognize and respond to invasion of bacteria and regulation of inflammation,most of the sites of enrichment occur in cell bodies,transcription factor complexes,and the like,and key molecular functions include transcription factor binding,serine/threonine kinase activity of proteins,kinase regulatory activity,and the like.NF-κB pathway is the central pathway of RYJHP in the treatment of NF.Mapping analysis has identified four core targets enriched in the NF-κB pathway and four pharmacodynamic components that are predicted to act on targets,but have not been clearly demonstrated.4.In Vitro Antibacterial and Anti-inflammatory Activities of RYJHP as well as Its Effective Components and Their MechanismsBased on the results of part 1 of the study and the literature search,the possible antibacterial components of RYJHP,as well as the discovery of the antibacterial activity of RYJHP against Staphylococcus aureus,MRSA and GAS was evaluated by inhibition zone method,but the antibacterial activity against Escherichia coli in vitro was poor.A two-fold broth dilution method is used for evaluating that emodin in the active ingredients has antibacterial activity on staphylococcus aureus,GAS and MRSA,and rhein and magnolol have antibacterial activity on staphylococcus aureus,escherichia coli,GAS and MRSA.A macrophage inflammation model induced by co-culture of MRSA and macrophages was constructed.RT-PCR revealed that α-spinasterol glucoside,wogonin,kaempferol,and naringenin were able to reduce the release of the pro-inflammatory factors COX-2,IL-1βand TNF-α in the cellular model.Using WB method as well as molecular docking,it was found that α-spinosterol glucoside may competitively bind to the S32 phosphorylation site of IκB protein and inhibit the activation of NF-κB pathway caused by the phosphorylation degradation of IκB,while wogonin,kaempferol and naringenin may inhibit the phosphorylation of IκB due to occupying the S32 and S36 phosphorylation binding sites of IκB protein and inhibiting the activation of NF-κB pathway caused by the phosphorylation degradation of IκB.Wogonin,kaempferol and naringenin can competitively bind to the SER536 phosphorylation site of the P65 protein,inhibit P65 phosphorylation,and reduce the inflammatory response.Conclusion:RYJHP contains 191 kinds of chemical components such as flavonoids,steroids,anthraquinones,coumarins,lignans,triterpenes,alkaloids,etc.That can treat NF through antibacterial and anti-inflammatory methods.RYJHP,emodin,rhein,magnolol,and other effective components have been shown to have antibacterial activity against the pathogenic bacteria of NF.RYJHP has the potential to improve the survival status of mice,decrease the mortality of mice,attenuate skin tissue damage,mitigate liver and kidney damage from excessive immune response,and reduce the release of inflammatory factors in vivo.One of the core mechanisms of therapeutic effect is to alleviate inflammatory reaction in vivo by inhibiting the activation of NF-κB pathway.The alpha-spinosterol glucoside,wogonin,kaempferol and naringenin serving as medicinal components can reduce inflammatory reaction by inhibiting the activation of an NF-kappa B pathway.These results can be used as the experimental basis for the clinical treatment of NF with RYJHP,and provide insights for the development and innovative application of compound prescription in traditional Chinese medicine.