Functional Mechanism On Tujia Medicine Toddalia Asiatica Inhibit RA Synovial Membrane Proliferation Based On TLR/NF-?B Signal Pathway

Objective: The study of functional mechanism on Tujia medicine Toddalia asiatica inhibit RA synovial membrane proliferation based on TLR/NF-?B signal pathwayMethods: To investigate the effect of Toddalia asiatica alcohol extracts on inhibiting the growth of MH7 A cells: CCK-8 method detect a variety of Toddalia asiatica alcohol extracts(0,50,37.5,18.75?g/ml)working on the effect of inhibiting the growth of MH7 A cells.Make use of real-time unmarked cell functional analyzer to Toddalia asiatica alcohol extracts with a variety of concentration(0,50,37.5,18.75?g/ml)working on the effect of inhibiting the growth of MH7 A cells in different periods of time.Make use of Flow cytometric method to detect Toddalia asiatica alcohol extracts with a variety of concentration(0,50,37.5,18.75?g/ml)working on the effect of inducing MH7 A cells apoptosis at 3h,6h,12 h.To detect Toddalia asiatica alcohol extracts working on gene transcription levels of NF-?B,IL-6,IKK,IRAK-1m RNA in MH7 A cells at 3h,6h,12h: Make use of RT-PCR method to detect Toddalia asiatica alcohol extracts with a variety of concentration(0,50,37.5,18.75?g/ml)working on the transcriptional levels of NF-?B,IL-6,IKK,IRAK-1 change in MH7 A cells at 3h,6h,12 h.Results: 1 The effect of Toddalia asiatica alcohol extracts on inhibiting the growth of MH7 A cells: With the same working time,the inhibiting effects enhance with the increase of Toddalia asiatica concentration;with the same Toddalia asiatica alcohol extracts concentration,the inhibiting effects enhance with the increase of working time.2 The result of Real-time unmarked cell functional analyzer to Toddalia asiatica alcohol extracts with a variety of concentration working on the effect of inhibiting the growth of MH7 A cells in different periods of time: adding different concentration of Toddalia asiatica alcohol extracts,then detect MH7 A cells for 80 hours,finally,the drug concentration of 600?g/ml,300?g/ml,150?g/ml and 75?g/ml are with great poisonousness,the drugs with the concentration of 50?g/ml,37.5?g/ml,18.75?g/ml are obvious on the inhibiting MH7 A cells proliferation.3 Flow cytometry detect Toddalia asiatica alcohol extracts affecting MH7 A cells apoptosis: drug with concentration of 0-50?g/ml stimulates MH7 A cells for 3h,6h,12 h,apoptosis rate of 3h are 6.50%,11.7%,18.2%,23.2%;apoptosis rate of 6h are 4.73%,10.2%,17.4%,29.7%;apoptosis rate of 12 h are 5.45%,13.5%,15.0%,29.4%;the differences between experimental groups and control group are with statistical significance(P<0.05).4 RT-PCR detect Toddalia asiatica alcohol extracts working on gene transcription levels of NF-?B,IL-6,IKK,IRAK-1m RNA in MH7 A cells at 3h,6h,12 h : Toddalia asiatica alcohol extracts with concentration of 0-50?g/ml work on MH7 A cells stimulated by LPS for 3h,6h,12 h,the IRAK-1,IKK,NF-?B,IL-6m RNA expressions of blank control group in the three time points have no significant changes;the IRAK-1,IKK,NF-?B,IL-6m RNA expressions of MH7A+LPS group are higher obviously than that of blank group and MH7A+LPS+drug group,(P<0.05);and the expression of IRAK-1,IKK,NF-?B,IL-6m RNA in MH7A+LPS+drug group decreases mostly with concentration of 50?g/ml.the expression has decrease trend with concentration of 37.5?g/ml,which is not obvious as the concentration of 50?g/ml.the expression of IRAK-1,IKK,NF-?B,IL-6m RNA in MH7A+LPS+drug group does not change obviously,there is not statistical difference with blank control group.The differences between experimental groups and control group are with statistical significance(P<0.01).Conclusion: 1 Toddalia asiatica is able to inhibit the proliferation of NH7 A cells,with the concentration of 0-50?g/ml,inhibition ratio increases with the rise of Toddalia asiatica concentration and the prolong of working time.2 Toddalia asiatica is able to induce the apoptosis of MH7 A cells.From the results of FCM,Toddalia asiatica alcohol extracts is able to induce the apoptosis of MH7 A cells,apoptosis rate grows with the rise of medicine concentration.3 The possible mechanisms of Toddalia asiatica inhibiting proliferation and inducing apoptosis on MH7 A cells may be concerned with inhibit the expression of NF-?B,IL-6,IKK,IRAK-1m RNA genetic transcription,Toddalia asiatica may inhibit transit of NF-?B,restrain the release of inflammatory factors through restraining related cell factors of TLR/NF-?B signal pathway,achieve the final aim of inhibiting RA synovial membrane proliferation.