Therapeutical Effect And Its Mechanism Of Total Flavonoids Of Chrysanthemum On Adjuvant Arthritis Rats

Objective To observe the anti-inflammatory and immunomodulatory effect of totalflavonoids Chrysanthemum indicum (TFC) to adjuvant arthritis(AA) models. Westudied the effect on TFC to AA model rats several cytokines, ERK signal pathway onperitoneal macrophages (PMΦ), explored the mechanism related signal pathwaymechanism of TFC anti-inflammatory immunological activity on AA model rats. Tostudy TFC apoptosis effect and Caspase-3 molecule mechanism on synovium tissue andsynoviocytes with electron microscope, immunohistochemistry method, TUNELstaining, Hoechst33258 fluorescent staining, DNA fragment agarose gel electrophoresisand Western blot analyzed.Methods AA rat's model was induced by Freund's complete adjuvant to observe thepaw swelling, the arthritic index and the body weight of rats to evaluate the effect ofTFC. To observe expression of vascular endothelial growth factor(VEGF) in synovialmembrane vessel wall. Effect of TFC on splenocyte lymphocytes proliferation in AArats were stimulated by LPS and ConA.Change of cytokines were detched withradio-immunity, immunohistochemistry, RT-PCR methods. To explore the regulation ofTFC on p-ERK1/2 of AA rats PMΦ, which were stimulated by TFC in vitro or themacrophages were pretreated with PKC inhibitor Chelerythrine 2 hour beforestimulated by TFC. The expression of p-ERK1/2 of PMΦwas detected by usingWestern blot. We observed TFC's therapeutical effect through ultrastructure ofsynoviocytes in joint of AA rats. Synovial tissue of knee joint immunohistochemistrymethod and image analysis technique observed impacting on NF-κB, Bcl-2, Bax of TFC,and studied its internal link: Synoviocytcs were derived from explant culture of tissue fragment or collagenase-trypsin digest methods. Effect of synoviocytes apoptosisobserved by TUNEL staining, Hoechst33258 fluorescent, DNA fragment agarose gelelectrophoresis; Western blot analyzed the expression change of Caspase-3 activationfragment protein, Annexin V fluorecent staining detected Caspase-3 specificity blockingagent influenced on synoviocytes apoptosis, Through described above methods toapproach the mechanism of TFC on AA rats.Results TFC (84,168,336 mg.kg^-1, ig) could significantly inhibit secondary feetswelling, multiple arthritis, body weight loss. TFC could downregulate expression ofVEGF in synovial membrane vessel wall; lessened proliferation of blood vesselendothelium and neogenesis of pannus. So TFC could improve the symptom of AA rats.The results in vivo showed that the low response of splenocytes proliferation induced byConA and LPS and the decreased IL-2 synthesis were restored in AA rats treated withTFC(84,168,336 mg·kg^-1,ig), while the elevated IL-1, PGE₂, TNF-αproduction werealso reduced. TFC(0.005, 0.05, 0.5, 5, 50 mg·L^-1) not only increased ConA and LPSinduced splenocytes proliferation, but also reduced the elevated IL-1β, TNF-αproduction release from peritoned macrophate (PMΦ) in AA rats in vitro. Meanwhile,RT-PCR showed TFC could decrease mRNA expression of IL-1βand TNF-αin PMΦ.The p-ERK1/2 activity was low in AA model rats PMΦ. After the macrophages werestimulated by TFC, its protein expression level of p-ERK1/2 was obviously higher thanAA model group. After the macrophages were pretreated with Chelerythrine 2h, itsprotein expression level of phosphorylation of ERK1/2 was markedly lower thancompared with 50 mg·L^-1 TFC stimulating group. TFC could recovery secretoryfunction of synoviocytes and morphologic abnormality. TFC could inhibit theproliferation of synoviocytes measured with MTT assay and showed dose dependentand the IC₅₀ in 48h was 115mg·L^-1. TFC depressed activity of NF-κB, cut down theratio of Bcl-2/Bax, raised apoptosis index. DNA gel electrophoresis showed step strap,apoptotic body were observed in cell nucleus by Hoechst33258, TFC could promotc apoptosis of synoviocytes through TUNEL staining. The protein level of Caspase-3active fragments increased obviously and related with the concentration of TFC byWestern blot. Furthermore, the apoptotic cells were markedly decreased by theCaspase-3 specific inhibitor.Conclusion TFC could improve AA rat's model induced by Freund's complete adjuvantand adjust the abnormal activation of PMΦ. The results suggested the therapeutic actionof TFC on AA rats related to its immunoregulatory actions, balance cytokine, decreasedinflamed medium. It may be one of pathways TFC could activate p-ERK1/2 of PMΦthrough PKC. TFC could recovery the morphology of secretory function organell of injoint of AA rats, inhibit synoviocytes proliferation and promote apoptosis throughCaspase-3 pathway. The activation of Caspase-3 might be an important causation for theapoptosis of synoviocytes induced by TFC.