Study On The Effects And Mechanism Of The Active Ingredient Resibufogenin In Bufonis Venom To Inhibit LPS-Induced Macrophage Inflammation

Toad venom(Bufonis Venenum)is a precious traditional Chinese medicine which consists of the self-defensive secretions from the auricular and skin glands of either Bufo bufo gargarizans Cantor or Bufo melanostictus Schneide.With the effects of detoxification,pain relief and resuscitation,toad venom has a long history in treating infectious and inflammatory diseases,such as carbuncle,sore throat,pharyngitis,heatstroke,abdominal pain and vomiting.For example,Liushen Pill,a Chinese patent medicine famous for treating sore throat,is made of toad venom and other components.Modern researches have demonstrated that toad venom possesses a variety of pharmacological activities such as cardiotonic,hypotensive,antioxidant,anti-tumor,etc.Among them,its anti-tumor activity has attracted widespread attention and is favored by the clinic.Cinobufotalin injection,which is mainly prepared from toad venom,has been used in the adjuvant treatment of various malignant tumors.In fact,inflammation is associated with the development and deterioration of most cancers,many anti-tumor targets are also the anti-inflammatory targets,and not a few antitumor agents also possess anti-inflammatory capacity.This study started with the evaluation of the anti-inflammatory activity of toad venom.We prepared the alcohol extract and water extract of toad venom.In LPS-activated macrophages,both the alcohol extract and water extract could effectively inhibit NO and IL-6 production,indicating that the anti-inflammatory components of toad venom are not limited to a certain polar part,and there are anti-inflammatory components could be found both in its small polar part and large polar part.Through the preliminary comparative analysis of the monomer components of toad venom,resibufogenin(RBG),a low polar small molecule which showed a prominent NO inhibition,was finally selected as the final object to carry out the follow-up research.RBG is an ingredient isolated from toad venom,which is listed as one of the index ingredients of toad venom by the Chinese Pharmacopoeia.RBG belongs to Bufadienolides,and can be further subdivided into Bufogenins for not linking organic acids.In endotoxemia mice,a single intraperitoneal administration of RBG(10 mg/kg,20 mg/kg)significantly lowered serum TNF-α,IL-6 and MCP-1 levels.In LPS-stimulated RAW264.7 cell line and mouse peritoneal macrophages(MPMs),RBG decreased LPS-induced pro-inflammatory mediators’ productions(e.g.,iNOS,IL-6,TNF-α and MCP-1)in a concentration-dependent manner.Further results showed that RBG suppressed NO production not by changing the activity of iNOS,the upstream synthase of NO,but through the inhibition on iNOS transcription and translation.qPCR results showed that RBG effectively reduced the mRNA expression level of IL-6 and TNF-α and MCP-1.Further mechanism studies showed that RBG hindered IκBα phosphorylation,suppressed IκBα degradation and prevented nuclear translocation of p65,thus reducing the transcription activity of NF-κB.Concurrently,RBG also dampened the transcription activity of AP-1 signaling through inhibiting the phosphorylation levels of JNK and ERK,but not p38.Besides LPS(TLR4 ligand)model,we stimulated macrophages with Pam3CSK4(TLR2 ligand),RBG also concentration-dependently suppressed NF-κB transcription activity and reduced the production of NO,IL-6,TNF-α,and MCP-1.And in poly I:C(TLR3 ligand)-induced model,NF-κB transcription activity also inhibited by RBG.The inhibitory effect of RBG on inflammation induced by different stimulators shows that:①RBG is effective in suppressing inflammation induced by different TLRs,not limited to the inflammation caused by LPS;②RBG can suppress the inflammation caused by either extracellular(TLR2,TLR4)or intracellular(TLR3)pattern recognition receptors,its anti-inflammatory target(s)should be located in the cytoplasm,and RBG indirectly affects NF-κB and/or AP-1 signals through its target(s).