Research On Compatibility Attenuation Of Wu Tou Decoction And RA-HFLS PI3K/Akt Induction Of TNF-α Signaling Pathways Of Regulation

Background and ObjectiveRheumatoid arthritis is an inflammation of the synovial membrane as the main manifestations of autoimmune diseases. Literature, Aconite Decoction for treatment of arthralgia syndrome curative effect exact. Wu Tou decoction recorded in Jin Kui Yao Lue. Which was created by Zhang zhongjing the Eastern Han dynasty famous physician, This prescription is composed of radix aconite preparata, ephedra, peiny, astragalus menbranaceus, and honey-fired licorice root, with the efficacy of warming meridian and dispelling cold, dispelling wind and dampness and relieving pain, attending cold and dampness. Famous doctor of Mr Li Ke added black beans and radix saposhnikoviae on the basis of the original prescription, the purpose of restricting aconiti toxicity, while keeping the original formula’s effectiveness.The previous we has carried out the experiment of compatibility for attenuation synergia, spectral studies and the study on Aconitum alkaloids of Wu Tou decoction on proliferation effect of human rheumatoid arthritis synovial cells, on the basis of exploration, we do further study of compatibility attenuation of Wu Tou decoction, Wu Tou decoction alkaloids inhibit rheumatoid arthritis synovial fibroblasts (HFLS) proliferation of mechanisms for research, Providing a scientific basis for Wu Tou decoction of clinical medication and further development.Methods1 Study on compatibility attenuation of Wu Tou decoctionOn the basis of Radix Aconite Preparata in the laboratory, using HPLS-ESI-MS technical analysis of crude Radix Aconiti single decoction, Radix Aconiti Preparata single decoction, Wu Tou decoction, Jin Kui Wu Tou decoction, and Li Ke improved Wu Tou decoction of Aconitum alkaloids in composition to study the influence on Aconitine alkaloids after the processing and compatibility, meanwhile, the use of UPLC-MS/MS determination in crude Radix Aconiti single decoction, Radix Aconiti Preparata single decoction, Wu Tou decoction, Jin Kui Wu Tou decoction, and Li Ke improved Wu Tou decoction of six kinds of Aconitum aconitine in soup, summarize the law of Aconitum attenuated.2. Ephedra alkaloids of Wutou decoction on RA-HFLS PI3K/Akt induction of TNF-a signaling pathways of regulationThis study is operated by culturing rheumatoid arthritis synovial fibroblasts to screen the effect of anti-inflammatory with ingredients including Aconiti Preparat aconitine, ephedrine aconitine, and total alkaloid of WTD. In the part of anti-inflammatory effects in vitro screening, it was conducted by thiazole blue colorimetry cytotoxicity experiment (MTT method), that analyzing proliferation inhibitory rate of human rheumatoid arthritis synovial fibroblasts.Analysis showed that ephedrine alkaloids on proliferation inhibition effect of RA-HFLS the best, therefore, studies of ephedra alkaloids, further explore the mechanism of ephedra alkaloids inhibit the proliferation of RA-HFLS. By ELISA method for the determination of TNF-a-induced RA-HFLS cell culture supernatant found that ephedra alkaloids of RA-HFLS observation in the effects of TNF-a, IL-1-Beta expression. While, using Western Blot detection of alkaloids in ephedra in different concentration on TNF-a induced phosphorylation of Akt and RA-HFLS non-phosphorylated and expression of NF-κB proteins.ResultsWu Tou decoction attenuated study, comparing crude aconite decoction and Radix Aconiti Preparata in single decoction of aconitine alkaloids content of six aconitine found that Processing of radix aconiti process primarily through dual-mono-alkyl Ester-hydrolysis of single estered aconitine work.Compared crude Radix Aconiti single decoction, Radix Aconiti Preparata single decoction, Wu Tou decoction, Jin Kui Wu Tou decoction, and Li Ke improved Wu Tou decoction found that, after compatibility, hydrolysis reduction toxicity of role has very weak, Mostly single estered aconitine loss play a role in lowering alkali content of aconitine-type alkaloids, and Li Ke improved Wu Tou decoction of role effect to is superior to Jin Kui Wu Tou decoction.Wu Tou decoction of ephedra alkaloids RA-HFLS PI3K/Akt induction of TNF-a signaling pathways regulate research, ephedra alkaloids in vitro pharmacodynamic screening studies have shown that inhibitory effects on proliferation of RA-HFLS is stronger than soup with aconite Total alkaloids and Aconiti alkaloids. ELISA tests showed ephedra alkaloids can reduce RA-HFLS in TNF-a, IL-1β expression levels in a dose-dependent manner. Analysis of the Western Blot method showed varying doses of ephedra alkaloids were able to downgrade RA-HFLS phosphorylated Akt expression and NF-K.b protein, in a dose-dependent manner, but RA-HFLS non-phosphorylated Akt expression and NF-κb protein had no inhibitory effect.ConclusionsWu Tou decoction mainly through the combined attenuated single estered loss effect of alkaloids from Aconitum, and dual mono-alkyl Ester-hydrolysis of aconitine aconitine-type occurs mainly in the course of processing and compatibility play a role in the process is very weak.0.25mg/ml-l. OOmg/ml ephedra alkaloids can lower RA-HFLS expression of TNF-α, IL-1β levels, and reducing the level of phosphorylation of Akt, reduced phosphorylation of NF-κb proteins and inhibit the proliferation of RAFLS, play an apoptotic and anti-inflammatory effects.