The Chemical And Anti-Inflammatory Activity Mechanism Research Of Indolealkylamines In Toad Skin

Background:Toad skin is the dried skin of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider.As a traditional Chinese medicine,it has the effect of clearing heat,detoxifying and diminishing swelling,usually used for the treatment of gangrene,swollen venom,boils,tumors,bloated bloating and chronic bronchitis.The main components in toad skin include hydrophilic nucleosides,amino acids,IAAs and lipophilic bufadienolide.Among them,bufadienolide are the main material basis for antitumor effects.However,water extraction of toad skin usually used in traditional application or preparation of clinical preparations,such as Huachansu injection,Huachansu oral liquid,Huachansu tablets,etc.,they contain a large amount of hydrophilic ingredients.Specifically,IAAs is one of the important hydrophilic active ingredients,but its pharmacological activity has not been reported.A large number of studies have shown that alkaloids containing indole rings exert good anti-inflammatory activity.Therefore,it is speculated that IAAs in toad skin may have anti-inflammatory activity.In view of the fact that the traditional Chinese medicinal preparation of toad skin contains a large amount of hydrophilic components such as IAAs,the systematic research and analysis of these components can provide a scientific basis for guiding the clinical use of toad skin.Objective:In this study,a qualitative and quantitative analysis of hydrophilic chemical components in toad skin was performed to clarify the basis of its hydrophilic substance.LPS stimulated transgenic fluorescent neutrophil zebrafish and primary neutrophils from mouse were used as inflammation models.The anti-inflammatory activity of the main hydrophilic component IAAs was evaluated in vivo and in vitro,providing a theoretical basis for guiding the clinical application of toad skin.Methods:(1)This study uses modern classical phytochemical separation technology to separate and purify IAAs.The structures of IAAs were identified by nuclear magnetic resonance(1H-NMR 13C-NMR)and high-resolution mass spectrometry(UHPLC-HR-MS).(2)In this study,UHPLC-Orbitrap-MS technology was used to analyze and summarize the mass spectrometry of the 3 different structure of IAAs for the purpose of deriving unknown alkaloids.At the same time,the other hydrophilic components were identified based on standards and the data reported from previous literature.On the other hand,UPLC-QqQ-MS/MS technology was used to systematically investigate and determine the main peaks of the hydrophilic part of toad and toad.(3)The LPS-stimulated transgenic zebrafish inflammation model was used to evaluate the anti-inflammatory activity of IAAs.Secondly,real-time fluorescence quantitative PCR(RT-PCR)was used to detect the RNA level of inflammatory factors and key proteins in related inflammation pathways.(4)Targeted and non-targeted lipidomics techniques was carried out to profile the samples thus to screen out potential lipid markers related to inflammation.(5)Using network pharmacology technology to screen proteins closely related to differentially metabolized lipids,so as to speculate possible targets for IAAs.(6)Enzyme-linked immunosorbent assay(ELISA)was used to detect changes in neutrophil proinflammatory factors(IL-6,TNF-?,IL-1?)in mice induced by LPS after IAAs intervention;using Annexin V/PI double Staining detects cell apoptosis.At the same time,Western blot was used to detect apoptosis-related proteins(Bcl-2,Bax,Cleaved caspase-3)and autophagy-related proteins(Beclin 1,LC3-?,LC3-?).Results:(1)Extraction and separation of hydrophilic components from toad skin:six IAAs were isolated and identified from toad skin,which were identified by nuclear magnetic field and high resolution mass spectrometry as serotonin,N-methyl serotonin,bufotenidine,bufotenine,dehydrotryptamine and bufothionine,respectively.(2)Quantitative analysis of hydrophilic components from toad skin and toad venom:A total of 38 hydrophilic components were identified from the toad skin and toad venom,with 36 in toad skin(17 IAAs)and 22 in toad venom(10 IAAs),including amino acids,nucleosides,polypeptide and IAAs.There are three new IAAs were found in the toad skin for the first time.According to the qualitative results,11 hydrophilic compounds with high content in 11 batches of toad skin and 4 batches of toad venom samples were quantitatively analyzed,including 6 IAAs.The results showed that the IAAs were the main hydrophilic components in toad skin and toad venom.Quantitative results by multivariate statistical analysis,5 compounds with large differences were screened out,among which 4 were IAAs,which could be used as the difference compounds in the hydrophilic components of toad skin and toad venom.(3)The anti-inflammatory activity evaluation of IAAs in LPS-mediated genetically modified fluorescent zebrafish:the anti-inflammatory activity of five IAAs were obatained LPS induced zebrafish inflammation model.According to the results of RT-PCR,the RNA level of inflammation factor(IL-6,TNF-?,IL-1?,IFN-? and IL-10)and inflammatory pathways related protein(I?B-?,p65,ERK1,JNK1 and p38a)were regulatied toward a normal level after five IAAs treatment.(4)Targeted lipid omics eventually screened out 33 potential pathological lipid markers,including 14 SM,6 Cer,2 GlcCer and 11 PC.These lipid markers were mainly involved in sphingolipid metabolism and glycerophospholipid metabolism.Eighteen potential lipid markers including 15 TG,2 SM and 1 Cer were identified by untargeted lipid omics.(5)Using network pharmacology,a total of 26 predicted targets were screened out based on topological characteristics(which play a key role in the network)to participate in lipid marker regulation.Among these enzymes,PLA2 and LPCAT are related to PC metabolism.LBP and SMPD are shown to be associated with SM and Cer metabolism.SGMS is related to the metabolism of PC,SM and Cer.(6)Effect of IAAs on the function of primary neutrophils in mice:after 2 hours of treatment with 6 IAAs,the inflammatory factors(IL-6,TNF-?)in primary neutrophils in mice induced by LPS have a significant decrease.Among them,the expression of apoptosis-related proteins bcl-2/Bax was significantly increased after the treatment of by bufotenine for 16 h,while the expression of autophagy-related proteins beclin-1 and LC3-?/LC3-? showed no significant changes.Conclusion:(1)IAAs were one of the main hydrophilic components in toad skin and toad venom.Among them,most were free IAAs.The content of IAAs in toad venom was much higher than that of in toad skin.(2)IAAs have obvious anti-inflammatory effects on LPS-stimulated transgenic zebrafish inflammation models.IAAs could reduce the RNA level of pro-inflammatory factor like IL-6 TNF-? and IL-1? through inhibit TLR4-MyD88-NF-?B and TLR4-MyD88-MAPK inflammation pathway.Lipid homeostasis regulation,such as SM,Cer,PC metabolic regulation may be another mechanism by which IAAs exert anti-inflammatory effect.enzymes like LBP,PLA2 CERK,SMPD,and SGMS may be key targets regulated by IAAs.(3)IAAs can down-regulate increased secretion of inflammatory factors caused by LPS in neutrophil.The mechanism may be related to promoting mitochondrial-mediated endogenous apoptosis pathways.