Study On Treatment Of Rheumatoid Arthritis With Melittin Combined With Flavonoids Of Eucommia Ulmoides

IntroductionRheumatoid arthritis(RA)is a chronic inflammation of synovial joints mainly involved in facet joints,which belongs to the category of autoimmune diseases.RA often lead to joint swelling and destruction of articular cartilage,joint space narrowing,joint deformities and other symptoms.If the patients did not received diagnose and therapy timely,RA will cause varying degrees of disability[1].The disease belongs to Bi syndrome of traditional Chinese medicine,there are mainly 4 kinds of common disease type,the wind cold and dampness type,the wind dampness and heat type,phlegm and blood stasis type,yin deficiency of liver and kidney type etc.The early pathological changes of bone and joint in RA patients are osteoporosis(OP).Some studies indicate that bone loss is common phenomenon in patients with RA.Wnt signal pathway is the Wnt family proteins through autocrine or paracrine and cell membrane receptor binding and activation of intracellular signaling molecules,signal transduction pathways promote downstream gene transcription.Studies have shown that the Wnt signaling pathway is closely related to the occurrence and development of RA.Melittin,the main active substance in bee venom,which has anti-inflammatory,antibacterial and analgesic effect.Studies have shown that melittin can inhibit the development of a variety of pathogenic microorganisms.Using Bee venom to treat diseases which have a history of thousands of years,especially for the treatment of arthritis and other inflammatory pain."Shen Nong's herbal classic" wrote Eucommia can treat waist and knee pain,invigorating spleen-stomach and replenishing qi,strengthening bone and musculature,improve the condition of vulva itching.Eucommia is one of Chinese unique and widely used in clinical medicinal materials.Modern pharmacological studies have found that Eucommia has different degrees of regulation of the central nervous system,circulatory,immune,endocrine,urinary system.A large number of studies show that Eucommia extract has more remarkable antioxidant hemolysis in vitro and protect the role of red blood cells.This research focuses on the liver and kidney yin deficiency type of rheumatoid arthritis clinical incidence and associated with osteoporosis;and from the cell protein,animal model levels of melittin,Eucommia flavonoids and the best combined drug concentration and response to treatment and how to avoid the adverse reaction of melittin is easy to cause hemolysis.This study divided into three parts,as follows:The first part:Study on the mechanism of melittin combined with flavonoids from Eucommia ulmoides on inhibiting the growth of synovial fibroblasts in rheumatoid arthritis by WNT signaling pathwayObjective:To explore the melittin combined with eucommia flavonoids through the pathway of Wnt on Wnt-1,Dkk-1,C-myc,?-catenin protein inhibits rheumatoid arthritis synovial fibroblast growth mechanism.Methods:Rheumatoid arthritis synovial fibroblasts(RA-FLS)were cultured for 3-7 generations.The cell surface labeled antibodies were examined by flow cytometry and verified as RA-FLS cells.After the appropriate number of cell were inoculated in 6-well plates,then adding 10ng/mL cell proliferation by TNF-a so as to induce RA-FLS cells into rheumatoid arthritis cell model.The cellular proliferation was detected using MTT after cell model treated with different concentrations of melittin,flavonoids of Eucommia ulmoides Oliver and melittin combined with flavonoids of Eucommia ulmoides.The expressions of Wnt-1,?-catenin,C-myc and DKK-1 protein in different treatment groups were detected by Western Blot.All the experimental results were collated and analyzed by SPSS 19.0.Results:The concentration of 1,10,100,1000ng/?L melittin and Eucommia flavonoids whether used alone or mixed use can effectively reduce the survival rate of RA-FLS.The melittin combined with eucommia flavonoids 1000ng/p.L made the rate of cell survival lowest,and the inhibition rate was 89.73%.1 and the 1000ng/?L concentration of melittin and mixed administration on the growth inhibition of cells were significantly better than Eucommia flavonoids group.However,there was no significant difference between the melittin group and the mixed administration group(p>0.05).The expression of Wnt-1,?-catenin,C-myc and DKK-1 protein was significantly lower than that of TNF-a induced RA-FLS cell model,and the protein content of melittin combined with Eucommia flavonoids was the lowest.(p<0.05)Conclusion:Melittin combined with eucommia flavonoids can effectively inhibit TNF-?induced RA-FLS growth,1000ng/?L drug concentration was best.The mechanism may be through inhibiting the expression of Wnt signaling pathway of Wnt-1,?-catenin,C-myc,DKK-1 protein.The second part:The treatment of melittin combined with Eucommia flavonoids on CIA model miceObjective:To observe the therapeutic response of melittin and flavonoids from Eucommia combined with coating on collagen induced arthritis in mice.Methods:According to the collagen induced(CIA)model mice preparation method of arthritis in mice,then dividing them into control group,melittin group,Eucommia flavonoids group and mixed drug group.Different groups were given different drugs.Each paw of mouse was given 5?L drug lasting for 15 days,PBS control group was given the same amount of PBS.The thickness of the foot pad and the width of the ankle were measured every day.X-ray examination was performed before and after administration.ELISA kit was used to evaluate the relative indexes and calculate the degree of joint swelling and spleen index.Results:On the naked eye visible,regardless of melittin,Eucommia flavonoids or mixed paw swelling of mice to the medicine group were lighter than the control group without administration of PBS.Comparing the foot pad thickness,width of ankle and foot swelling measurements found that melittin group,Eucommia flavone group,mixed group of mice results the swelling were relieved,the mixed drug group in improving joint is better than the other groups.Detected by Elisa found that mixed drug group immunological indexes of mice TNF-?,IL-1?,IL-2,GM-CSF were better than other groups.Conclusion:melittin,Eucommia ulmoides,melittin combined with eucommia flavonoids mixture can treat the CIA model mice joint swelling and inflammatory factor levels,the mixed drug group can decrease the level of inflammation best.The third part:The clinical research of relationship between the liver and kidney yin deficiency in rheumatoid arthritis and osteoporosis.Objective:To investigate the incidence of secondary osteoporosis in RA patients with liver-kidney-yin deficiency,and to analyze the correlation among rheumatoid arthritis related indicators,acute inflammation index,bone metabolic index and bone mineral density.Method:All the rheumatoid arthritis inpatients were in Shanghai Guanghua Hospital from October 2015 to May 2016.Excluding the relevant taboos,patients who participated in the study never had used formal anti-osteoporosis treatment.All the women were postmenopausal patients and men were older than 50 years of age.They should complete the relevant questionnaire,including the blood routine test,acute inflammation indexes erythrocyte sedimentation rate(ESR),C-reactive protein(CRP)and rheumatoid factor(IgMRF,IgARF,IgGRF)and anti cyclic citrullinated peptide antibody(anti-CCP antibody),osteocalcin(OT);type ? collagen crosslinked C end peptide(CTX);I collagen N terminal peptide(PINP);parathyroid hormone(PTH);1,25(OH)2D3),lumbar spine and left hip test value of routine bone mineral density(BMD)values.All the experimental results were analyzed and collated by SPSS 19.0.Results:A total of 64 RA patients were enrolled in the study,of whom 30 were diagnosed with osteoporosis(46.88%).There was statistical difference between the normal bone mass and bone mass reduction,normal bone mass and osteoporosis group between the hemoglobin values.The bone mass normal group had higher levels of hemoglobin.There was a significant negative correlation between anti-CCP and IgMRF and hip BMD levels.Age,anti-CCP antibody titer,and ESR were all risk factors for secondary osteoporosis.Patients with older than 65 years of age are twice as likely to have osteoporosis compared with other patients.The incidence of OP in the lumbar vertebrae in patients with high titer anti-CCP antibody is 13 times higher than that in other patients;the occurrence of hip OP in patients with high titer anti-CCP Patients more than 4 times;erythrocyte sedimentation in patients with lumbar spine and hip OP occurred more than 9 times the other patients.Conclusions:Age,ESR and anti-CCP antibody can predict secondary osteoporosis risk factors and occurrence.Liver and kidney yin deficiency type in RA patients who are older than 65 years old,have high ESR and anti-CCP antibody level as well as low HB level are more prone to secondary osteoporosis.