Anti-rheumatoid Arthritis Mechanism Of Active Constituents From Vitex Negundo L. Seeds

Huang-Jing-Zi is the ripe seeds of Vitex negundo L.(Verbenaceae) and is taken to dispel the wind regulate qi, and relieve pains. It has been widely used in folk medicine for anti-rheumatoid arthritis. In the previous study, researchers paid more attention to its anti-inflammatory, analgesic, anti-tumor effects while its effective material basic and the mechanism of anti-rheumatoid arthritis are not clear. So, it is quite necessary to trengthen the research work about the possible mechanism of Fructus Viticis Negundo against rheumatoid arthritis (RA), which will lay the foundation for the abundant development of Huang-Jing-Zi.The therapeutic effects of the extract of Vitex negundo seeds (EVNS) on adjuvant arthritis (AA) rats was investigated. Our results revealed that EVNS significantly inhibited the paw edema and decreased the arthritis score and spleen index of AA rats. The weight loss of AA rats was also reversed by administration of EVNS. The remarkable decrement of serum inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukinl P (IL-1β) and interleukin6(IL-6), were observed in EVNS-treated rats, whereas, as an anti-inflammatory cytokine, interleukin10(IL-10) was found to be significantly increased. The expressions of cyclooxygenase-2(COX-2) and5-lipoxygenase (5-LOX) in peripheral blood mononuclear cells (PBMC) were also inhibited by administration of EVNS. Histopathological studies showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the ankle of EVNS-treated animals. Thus, our results demonstrated that EVNS improved the inflammatory damage in the pathogenesis of RA through immune regulation. EVNS maintained cytokine network relative balance and improved joint cartilage destruction. So we conjecture that EVNS played the role of anti-RA by inhibiting the inflammatory damage and improving the joint bone destruction.The main coumponds of EVNS including lignans (VEL) and terpenoids (VET) were evaluated for their effects of inhibiting nitric oxide (NO) level on LPS-stimulated RAW264.7cells. The lignans mainly includes6-Hydroxy-4β-(4-hydroxy-3-methoxyphenyl)-3a-hydroxymethyl-7-methoxy-3,4-dihydro-2-naphthaldehyde (VEL-Ⅰ), Vitedoin A (VEL-Ⅱ), Vitedoamine A (VEL-Ⅲ), Vitexdoin A (VEL-Ⅳ), Vitrofolal F (VEL-Ⅴ) and the terpenoids include Oleanolic acid (VET-Ⅰ), Negundol (VET-Ⅱ), Vitexilactone (VET-Ⅲ), Ursolic acid (VET-Ⅳ). The results showed that all the coumpounds except VEL-II significantly inhibited the levels of NO. Based on the vertical analysis, lignin compounds were evaluated for their effect of inhibiting TNF-a and IL-1β levels on LPS-stimulated RAW264.7cells. Our data demonstrated that pretreatment with phloretin had inhibitory effect on TNF-a and IL-1β and in LPS-stimulated RAW264.7cells, which suggested the lignans may the main anti-inflammatory active ingredient.The lignans and terpenoids of Fructus Viticis Negundo were evaluated for their effects of proliferation and alkaline phosphatase (ALP) of rat osteoblast-like cells (UMR106). The results showed that VEL-II, VET-Ⅲ and VET-IV can increased the UMR106proliferation and ALP activity.The multinucleated osteoclasts were derived from rat marrow cells induced by1,25-(OH)2-D3and dexamethasone. Among the nine active compounds, VEL-Ⅴ, VET-Ⅲ and VET-Ⅳ showed the strongest suppressing effect on the activity of TRAP. Therefore, these three active compounds from Fructus Viticis Negundo were choosed to study the effects and action mechanism on bone resorption.The Western-blot experiment results show that VEL-I, VET-Ⅱ and VET-Ⅱ inhibited the LPS-induced up-regulation of nitric oxide synthase (iNOS) and cyclooxygenase-2(COX-2) protein level in RAW264.7cells. VEL-Ⅲ and VEL-Ⅴ suppressed LPS-induced iNOS while VET-Ⅰ selectively reduced LPS-induced COX-2. The lignans VEL-I, VEL-Ⅲ, VEL-Ⅴ and the terpenoids VET-Ⅱ, VET-Ⅲ suppressed LPS-induced NF-κB p65.Osteoblast involved in the fromation, differentiation and mature of osteoclast through OPG/RANKL. Result showed that VEL-Ⅱ and VET-Ⅳ can improve the ratio of OPG/RANKL. In the study of the osteoclast RANK signal transduction system research, we found that LPS-induced activation of P-JNK、P-ERK、P-P38and P-IκB were suppressed significantly by VEL-Ⅴ and VET-Ⅳ.In study of pharmacological effect of VEL on AA rats, significantly inhibition of paw edema and decrease of the arthritis score were observed. The weight loss of AA rats was also reversed by administration of VEL. VEL significantly improved the enlarged thymus and spleen in a dose-dependent manner and suppressed the expression of COX-2and5-LOX. VEL downregulated the level of IL-1β while upregulared the level of IL-10. The levels of TNF-a and IL-6were downregulared by VEL at the concentration of340mg/kg. Histopatholo gical studies showed a marked decrease of synovial inflammatory infiltration and synovial lining hyperplasia in the ankle of VEL-treated animals.In conclution, our study demonstrated that the main active coumpounds of Fructus Viticis Negundo including lignans and terpenoids played their roles of anti-RA by inhibiting the inflammatory damage and improved the joint bone destruction in the patho genesis of RA.