Anti-rheumatoid Arthritis Effects Of Iridoid Glycosides From The Root Of Morinda Officinalis How.

Rheumatoid arthritis(RA),which is a chronic progressive autoimmune disease,are characterized as multiple joints lesions,sustained synovial inflammation with recurrence and difficulty to cure,leading to destruction of cartilage and bone in the joints.In traditional Chinese medicine,the etiology and pathogenesis of RA are related with loss of liver and kidney and also cold-dampness blocking collaterals.Therefore,the treatment methods of RA often include tonifying liver and kidney,removing wind and dampness,and promoting blood circulation.The Bajitian,a commonly used traditional Chinese medicine,comes from the root of Morinda officinalis How.(MO),is naturally warm,pungent,taste sweet,contribute to liver and kidney,and has the action of tonifying kidney-yang,strengthening bones and muscles and removing wind and dampness.The Iridoid glycosides from MO have been shown to possess the activities of anti-inflammation.Our previous study showed that ethanol extract of MO can effectively inhibit acute inflammatory reaction of ankle joint in adjuvant arthritis rats,significantly reduce the levels of serum IL-1? and TFN-?,while the Iridoid glycosides from MO are responsible for the activities of anti-inflammation and antirheumatoid arthritis.Objective: To elucidate the anti-rheumatoid arthritis effects and mechanism of iridoid glycosides from MO to provide scientific basis for the utilization and development of new drugs for anti-rheumatoid arthritis.Methods 1.HPLC method was developed to determine the content of iridoid glycosides,including monotropein,deacetyl asperulosidic acid,asperulosidic acid and asperuloside,in mo and total iridoid glycosides(MOIG).The macroporous adsorption resins were used to enrich iridoid glycosides from MO to obtain total iridoid glycosides(MOIG).2.The acute toxitic effects of MOIG were evaluated on mice with single intragastric administration maximum volume and the maximum concentration of drug dissolved through observing the toxicity and mortality of mice within 14 days after administration.The acetic acid writhing test in mice,bladder granuloma test in mice,cotton ball granuloma test in rats were conducted to investigate the anti-inflammatory and analgesic effects of MOIG.The RAW264.7 cell stimulated with LPS were used to preliminary study the mechanism of anti-inflammation of MOIG.3.Adjuvant rheumatoid arthritis in rats model,and ovariectomized and collageninduced rheumatoid arthritis model in mice were used to evaluate the anti-rheumatoid arthritis activities of MOIG through examining the swelling of foot,index of arthritis,biochemical parameters in serum and alteration of tissue structure of synovium in joint.The TNF-? stimulated synovial cells were used to explore mechanism of MOIG on the antirheumatoid arthritis.Results 1.The developed HPLC method can be used to determine the contents of iridoid glycosides from MO.Monotropein,deacetyl asperulosidic acid,asperulosidic acid and asperuloside showed good linearity(r>0.9995)in the ranges of 0.375-12 ?g,0.13-4.16 ?g,0.016-0.516 ?g,0.012-0.384 ?g with their corresponding peak areas,respectively.Repeatability,precision,recovery and stability were validated according to the requirements of China Pharmacopoeia.The content of iridoid glicosides in MOIG obtained through enrichment of macroporous adsorption resin reached to 62%.2.The results of acute toxicity indicate that MOIG did not show any toxitic effects on mice.MOIG at dose of 50mg/kg,100mg/kg and 200mg/kg significantly decreased the number of writhings in the acetic acid writhing test,reduced the wet and dry weight of granuloma in mouse bladder granuloma test and rat cotton ball granuloma test,indicating that MOIG had anti-inflammatory and analgesic effect.Furthermore,MOIG and monotropein significantlyinhibited the production of the IL-1?,IL-6 and NO,the expression of iNOS and COX-2 protein,the protein expression of NF-?B inflammatory pathway in LPS-stimulated RAW264.7 cells,suggesting that MOIG maybe exerted anti-inflammatory effects through NF-?B pathway.3.MOIG can abate the paw swelling of arthritis rats induced with Freund's complete adjuvant;significantly reduce the levels of serum inflammatory cytokines IL-6,IL-1? and IL-17?,exhibiting that MOIG had potential anti-rheumatoid arthritis effect.In rheumatoid arthritis mice induced with ovariectomy and collagen ?,MOIG can relieve the paw swelling of mice,significantly reduce the serum levels of inflammatory cytokines IL-6 and IL-10,and decrease the synovial hyperplasia in joint tissue,indicating that MOIG can prevent rheumatoid arthritis.In addition,MOIG can increase the levels of serum bone formation markers osteocalcin,reduce serum alkaline phosphatase activity(ALP),significantly decrease the levels of serum bone resorption parameters,such as DPD(Deoxypyridinoline)and RANKL(receptor activator of NF-?B ligand),significantly increase the femur bone mineral density and bone mineral content,enhance the number of trabecular bone,reduce the degree of trabecular bone separation,and improve bone microstructure,indicating that MOIG can decrease bone loss and alleviate the destruction of bone tissue in joint of rheumatoid arthritis mice induced with ovariectomy and collagen?.MOIG and monoteoprin significantly reduced the production of IL-6 and IL-8,and significantly inhibited the proliferation of Fibrous synovial cells stimulated by TNF-?.Conclusion MOIG had definitely anti-inflammatory analgesic and anti-rheumatoid properties.This will provide scientific basis for the utilization of MO and development of new drugs for the prevention and treatment of rheumatoid arthritis.