Mechanism Research On Anti-inflammation Of Caulophyllum Robustum And Its Characteristic Constituents Based On NF-?B And MAPK Signal Pathway

C.robustum Maxim is a traditional medicine of treating rheumatoid arthritis,and it has both the anti-inflammation and analgesic action,and immune regulation.Previous studies had found that the saponins and alkaloids play an important role in anti-inflammation and immunization.However,the role of specific link and the target of them are not clear.This article established the inflammation model by inducing RAW264.7 cells with lipopolysaccharide from the perspective of anti-inflammatory mechanism.The preliminary anti-inflammatory mechanism of C.robustum Maxim and its characteristic are carried at the levels of cell,gene,protein,and the molecular.Objective:C.robustum Maxim and its characteristic constituents were used as model drugs in this paper.We screened compositions with much stronger anti-inflammatory activity for the further molecular mechanism study in RAW264.7 cells induced by lipopolysaccharides.Methods:a.MTT method was used to detect the cytotoxicity of the total extract and 10 monomers of C.robustum Maxim,in order to determine the drug concentrations for the subsequent experiment,b.A stable cell inflammation model was constructed.RAW264.7 cells induced by lipopolysaccharide produced inflammation.And the anti-inflammatory activity of the total extract and 10 monomers of C.robustum Maxim were detected by the Griess reagent method,comparing results of experiments to determine the main active ingredients of C,robustum Maxim,c.We used the real-time PCR to detect the expressions of inflammatory factors,including TNF-?,MCP-1 and IL-10.d.Using flow cytometry assay,we detected the expression of cell surface auxiliary molecules CD14 and TLR4 after treatment.e.Western blot assay was used to detect the protein expression of IKK?,I?B?and NF-?B in NF-?B signaling pathway and pJNK,pERK and p-p38 in MAPK signal pathway.Results:Results of MTT showed that when the concentrations of CLD,CLG,CLH,Leonticin D,hederagenin and N-methylcytisine were 100?M,the cell survival rates were higher than 50%.The IC50 of Magnoflorine,oleanolic acid,echinocystic acid,CLC and the total extract of C.robustum Maxim are 78.772?M,46.234?M,13.539?M,11.500?M and 0.499 mg/ml at 24h.By Griess reagent method,we detected the anti-inflammatory activity of the total extract and 10 monomers of C.robustum Maxim,screening out CLC and the total extract of C.robustum Maxim for the following research.Real-time PCR results showed that the expression of TNF-a and MCP-1 was decreased obviously,the expression of IL-10 was obviously higher after treatment with CLC and the total extract of C.robustum Maxim.By flow cytometry,the expression of cell surface helpers CD14 and TLR4 were reduced after treated.The levels of the relative proteins of the MAPK signal pathway and the NF-?B pathway were rised or reduced in varying degrees.We found that CLC inhibited the protein expression of pERK,pJNK,p-p38,IKKp and NF-?B p65,and the inhibition of pERK,p-p38 was presented dosage dependence.CLC increased the expression of I?B?.Total extract of C.robustum Maxim inhibited the protein expression of pERK,pJNK,p-p38 and IKK?,reduced the protein expression of NF-?B p65 at high doses,reduced the protein expression of IkB?.Conclusion:A stable cell inflammation model was established successfully in vitro,and found that the total extract and 10 monomers of C.robustum Maxim had different anti-inflammatory effects.Among them,CLC and the total extract of C.robustum Maxim had the best anti-inflammatory effect.Through the mechanism study,we preliminarily determined the targets of MAPK and NF-?B signal pathway on which CLC and the total extract of C.robustum Maxim had effect.So this paper provided the basis of the feasibility for illustrating the anti-inflammatory mechanism of C.robustum Maxim,and also provided technologies and methods for the mechanism research of inflammation.