Study On The Anti-rheumatoid Arthritis Material Basis And Mechanism Of Dendropanax Dentiger And Mechanism Of Dendropanax Dentiger

Rheumatoid arthritis(RA)is a global and autoimmune chronic disease with great harm,but safe and effective anti-RA drugs are extremely scarce.Cyclooxygenase 2(COX-2)plays an important role in the occurrence and development of RA.Furthermoe,COX-2 and human RA fibroblast-like synovial cell(MH7A cell)are the key target enzyme and cell for the development of anti-RA drugs,respectively.RA belongs to the category of"Bizheng"and"external wind"in traditional She medicine,and is a"cold"disease.As a result,based on the basic treatment principle of"the cold is hot,and the hot is cold"and the concept of"strengthening the body and eliminating the evil",She medicine used the"Yang”medicine of dispelling wind and removing dampness for treatment of RA in clinic.Dendropanax dentiger(Harms)Merr.belongs to the Araliaceae family.The root of D.dentiger,belongs to“Yang”medicine,is one of the 30 She medicines frequently used for thousand of years to treat RA in Zhejiang,Jiangxi and other provinces.Neverthless,its material basis and action mechanism are still unclear so far.In addition,affinity ultrafiltration coupled chromatography-mass spectrometry(AUF-LC-MS)technology can be used to quickly screen the anti-RA active constituents from traditional Chinese medicines(TCMs).Therefore,based on COX-2 combined with AUF-LC-MS technology,along with MH7A cells to quickly screen the anti-RA active constituents and their mechanisms from the roots of D.dentiger has important scientific value and social benefits.This research paper uses the complete Freund's adjuvant-induced arthritis(AIA)model rat,COX-2,AUF-LC-MS,MH7A cells,Western blot and other technologies to systematically elucidate the anti-RA material basis and mechanism of the roots of D.dentiger.The results are as follows:(1)The roots of D.dentiger has significant anti-RA effect in AIA model rats.Using AIA model rats,the model rats'arthritis index,body weight,thymus and spleen index,foot and ankle joint pathology observations and changes in key inflammatory factors were used to investigate the ethanolic extract of D.dentiger roots(DD,1 g/kg of low dose,2 g/kg of medium dose,4 g/kg of high dose,crude drug dose)anti-RA effect.The results showed that the high and medium doses of ethanolic extract from the roots of D.dentiger can significantly reduce the arthritis index,thymus and spleen index of model rats,increase the body weight of model rats,and improve the pathological damage of model rats'ankle joints.In addition,the high and medium doses of ethanolic extract from the roots of D.dentiger can remarkably decreased rat rheumatoid factor(RF),C-reactive protein(CRP),tumor necrosis factor?(TNF-?),and interleukin 1?(IL-1?),IL-6 and IL-17 levels in the serum of model rats,increase the levels of IL-4 and IL-10 in serum,and down-regulated the levels of COX-2,5-lipoxygenase(5-LOX)and matrix metalloproteinase 2(MMP-2)in peripheral blood mononuclear cells.These findings demonstrated that the roots of D.dentiger could effectively block the inflammatory response and suppress the abnormal immune response of the immune organs by inhibiting the vicious cycle of inflammatory storm factors,thereby achieving the anti-RA effect.(2)The roots of D.dentiger have chemical components with various structural types.Using ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS)technology,a total of 81 phytochemicals in the ethanolic extract of D.dentiger roots were identified using positive and negative ion modes,including 18 phenylpropanoids,15 alkaloids,14 flavonoids,14 fatty acids,6phenols,4 steroids,4 cyclic peptides,4 terpenes and 2 others.Among them,78chemical constituents were reported from D.dentiger for the first time,which greatly enriched the chemical constituents of D.dentiger.The results laid the foundation for the subsequent in-depth material basis research of D.dentiger root.(3)The COX-2 inhibitors in the roots of D.dentiger have important development and application value.The COX-2 kit was used to investigate the COX-2 inhibitory activity of the ethanolic extract of D.dentiger root and its different polar extraction parts.The results show that the ethanolic extract of D.dentiger root showed prominent COX-2inhibitory effect,and its active ingredients mainly enriched in ethyl acetate and n-butanol fractions with half inhibitory concentrations(IC₅₀)were 77.2±4.2,40.7±3.5 and 28.6±1.9?g/m L,respectively.The AUF-LC-MS technology was used to rapid identify the COX-2 inhibitors in the ethanolic extracts of D.dentiger roots and their ethyl acetate and n-butanol fractions.A a result,a total of 13 chlorogenic acids(belonging to phenylpropanoids)were identified,indicating that phenylpropanoids are the main active ingredients against COX-2 effect of D.dentiger roots.(4)The material basis of the anti-RA of the root of D.dentiger has important development and application value.Using a variety of column chromatography techniques,a total of 34(1-34)target monomer compounds,including 20 phenylpropanoids(1-20),11 simple phenols(21-31)and 3 other types(32-34).Among them,there is 1 new compound(1),and there are 18,5 and 1 compounds isolated for the first time from Araliaceae,Dendropanax dentiger genus,and Dendropanax dentiger plants,respectively.(5)D.dentiger root inhibits inflammatory response by inhibiting NF-?B,Akt and JNK signaling pathways to achieve anti-RA effects.Using COX-2 kit,1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)method and TNF-?-induced MH7A cell model,the anti-RA effects of 34 target monomer compounds were evaluated.The results show that compounds 6-8,14-21 and 27 have strong COX-2 inhibitory activity,and their IC₅₀ values are all less than 20?M;compounds 15-21 have strong antioxidant effects,stronger than the positive drug vitamin C(IC₅₀=33.9±1.1?M);Compounds 1,13,28 and 30 have an effect on nitric oxide(NO),IL-2,IL-6,IL-8 and TNF-?which released by TNF-?induced MH7A cells has a strong inhibitory effect.In addition,compounds 1,13,28 and 30 at a concentration of 10?M have significant inhibitory effects on the nuclear transcription factor-kappa B(NF-?B),protein kinase B(Akt)and c-Jun amino-terminal kinase(JNK)signaling pathways in MH7A cells induced by TNF-?.The research results of this paper can not only clarify the material basis and mechanism of action of the anti-RA of Dendropanax dentiger roots,but also provide high-quality lead compounds for the development of natural-source anti-RA drugs,and provide rapid targeted screening of the material basis of other anti-RA Chinese herbal medicine example.