| BackgroundRheumatoid arthritis(RA)is a chronic,systemic autoimmune disease characterized by aggressive,symmetric polyarthritis.Fibroblast-like synoviocytes(FLSs)plays an important role in articular cartilage and bone destruction by over-activated and enhanced migration and invasion ability.Therefore,the inhibition of its excessive activation and migration,invasion ability is vital to the treatment of RA.RA is equal to Bi syndrome in TCM.After long-term theoretical research and clinical practice,my supervisor,Professor Lin Changsong concluded a basic pathogenesis of RA is "exo pathogens of wind,cold and wet cause qi and blood stagnation and further damage bones and tendons",and a principle of treatment "nourishing kidney,eliminating dampness and promoting blood circulation" as RA.The use of the DTYMD to treat RA,has received good clinical results,but its mechanism has not been thoroughly elucidated.In the early stage,the research team has studied the effects and mechanisms of related monomers,such as Leonurine and kaempferol on synovial cell’s function.And there needs further research on the DTYMD.This topic is a part of the National Natural Science Foundation of China(no 81573930),based on network pharmacology to study the integration mechanism of the Duanteng Yimu Decoction in the treatment of rheumatoid arthritis.This thesis intends to systematically study the quality standard of the DTYMD production,the effect and mechanism of the DTYMD on FLSs’ activation,migration and invasion,and the therapeutic effect of the DTYMD on the CIA mouse.Eventually,to provide a scientific basis for the DTYMD’s clinical use.Part Ⅰ A preliminary study on quality standard of DTYMDObjective1.To establish a Thin-Layer Chromatography(TLC)method for the identification the lyophilized powder of duanteng yimu decoction2.To construct the fingerprint of lyophilized powder of duanteng yimu decoctionMethodsWater decoction was used to extract the effective components of the decoction,and the freeze-dried powder was prepared by the vacuum freeze-drying mechanism.The drug composition of lyophilized powder of begonia sinensis was identified by TLC.The method was investigated by precision test,stability test and repeatability test.The six reproducible samples were analyzed by chromatography to find out the common peaks,determine the reference peaks,and classify the partial peaks by distraction.Resu!tsConstruction of Kunming Shan-hai-tang thin layer identification method,and the chromatographic spots were clear.All of these samples can get good separation effect in different temperature(8℃,20℃),different humidity(18%,72%),and different manufacturers of thin layer plate.In the sample chromatography,the same color spots appeared in the corresponding positions with the control medicine chromatography.The method of identification with radix dipsaci thin layer was built.The chromatographic spots were clear,and no interference from the negative control.All of these samples can get good separation effect in different temperature(8℃,27℃),different humidity(18%,57%),and different manufacturers of thin layer plate.In the sample chromatography,the same color spots appeared in the corresponding positions with the control medicine chromatography.To establish the TLC identification method for motherwort,the identification method under the 290-page motherwort[identification]in the 2015 edition of Chinese pharmacopoeia was referred to,but the spots were not clear.On the basis of consulting relevant literature,the experiment was conducted to improve the identification method.As a result the chromatographic spots were clear and the negative control without interference,(8℃,27℃)at different temperatures and different humidity(18%,65%),thin layer plate of different manufacturers,such as conditions,all can get good separation effect.In the sample chromatography,the same color spots appeared in the corresponding positions with the control medicine chromatography.The precision test showed that the similarity of chromatogram fingerprints for 6 consecutive times was greater than 0.99,indicating that the instrument had good precision.The stability test showed that the samples were injected at 0,2,4,6,8,10,24 and 48h,and the obtained chromatogram fingerprint similarity was greater than 0.99.The results of the reproducibility test showed that the 6 solutions were tested,and the similarity of chromatogram fingerprints was greater than 0.98.The results showed that the method had good reproducibility.The precision test,stability test and repeatability test show that the experimental method is stable and reliable.Chromatogram analysis results:34 stability of common peak is determined,including 34 chuan radix dipsaci saponins Ⅵ separation degree is high,good stability,so choose GaiFeng as reference peak;No.3,4,5,8,9,11,14,18,19,20,6,7,12,28,29,30 belong to dipsacus,no.1,2,13,21,22,23,24,25,26,27,31 belong to herba leonuri,no.10 is the common peak of malus malus and dipsacus.Conclusion:The quality standard of the lyophilized powder of DTMD was preliminarily established by means of thin-layer scanning and fingerprint technology.Part Ⅱ Study on the effects of DTYMD on the activation,migration and invasion of RA FLSsObjectiveTo study the effects of duanteng yimu decoction on the activation,migration and invasion of RA FLSs.MethodsFresh synovial tissue of patients with knee arthroscopy or joint replacement was taken and primary culture of human fibroblast-like synovial cells were conducted by tissue block culture method.The cells were cultured for 3-6 generations and identified by cell immunofluorescence for subsequent experiments.Cck-8 experiment was conducted to detect the effect of the DTYMD on the survival rate of RA FLSs.Elisa method was used to investigate the regulatory effect of the DTYMD on the expression levels of RA FLSs inflammatory factors and MMPs.Real Time PCR was used to study the regulation effect of the DTYMD on the expression levels of RA FLSs inflammatory factors and MMPs mRNA.The effect of the DTYMD on the migration function of RA FLSs was studied by scratch test and migration test.Invasion was adopted to detect the effect of invasion board on the migration function of RA FLSs.ResultsPrimary cultured cells were observed under light microscope and the morphology was consistent with FLSs.Cck-8 results showed that in DTYMD concentration groups of 200ug/ml,400ug/ml,600ug/ml and 800ug/ml,MTX 10uM intervention for 24h and 48h had no significant effect on the activity of RA FLSs cells.Both DTYMD 1200ug/ml group and MTX group for 24h and DTYMD 1000ug/ml for 48h could significantly inhibit the activity of RA FLSs cells(P<0.01).The results of Elisa experiment showed that in the TNF-α group,the levels of inflammatory factors,IL-1,IL-6,IL-8 and MMP1,MMP3 and MMP9 in RA FLSs cells were significantly higher than those in the blank control group(P<0.01).The low,medium and high dose DTYMD group could significantly down-regulate the expression levels of inflammatory cytokines IL-1,IL-6,IL-8 and MMP1,MMP3 and MMP9 in a dose-dependent manner.MTX can down-regulate the expression of inflammatory factors and MMPs.The results of Real Time PCR showed that the mRNA levels of RA FLSs inflammatory factors IL-1,IL-6,IL-8 and MMP1,MMP3 and MMP9 were significantly higher in the TNF-α group than in the blank control group(P<0.01).The mRNA expression levels of inflammatory cytokines IL-1,IL-6,IL-8 and MMP1,MMP3 and MMP9 in the low,medium and high dose groups of DTYMD were significantly down-regulated in a dose-dependent manner.MTX has a downregulation effect on the mRNA expression levels of inflaumatory cytokines and MMPs.The results of scratch test and migration test showed that,both the low,medium and high dose DTYMD group and the MTX group could reduce the migration ability of RA FLSs in vitro(P<0.01),compared with the blank control group.The inhibitory effect of DTYMD on the migration function of RA FLSs was dose-dependent.The invasion experiment results showed that,the invasion ability of RA FLSs was decreased in both the low,medium and high dose DTYMD group and the MTX group(P<0.01),compared with the blank control group.ConclusionDTYMD can down-regulate the protein and mRNA expression levels of TNF-α-stimulated elevated inflammatory cytokines IL-1,IL-6,IL-8,and MMP1,MMP3,MMP9.It can also inhibit the migration and invasion of RA FLSs in vitro.Part Ⅲ The molecular mechanism of the regu I ation of RA FLSs act i vat ion,migration and invasion by DTYMDObjectiveTo investigate the molecular mechanism of duanteng yimu decoction on the activation,migration and invasion of RA FLSs.MethodsWestern blot method was used to detect the effect of DTYMD on the phosphorylation of p65,IKK a/p and IKB a in the NFkB signaling pathway of RA FLSs,and the effect of NFkB inhibitor on the activation of NFkB signaling pathway.Real Time PCR was used to investigate the regulation of NFkB inhibitor PDTC on the mRNA expression levels of RA FLSs inflammatory cytokines IL-1β,IL-6,IL-8 and metalloproteinases MMP1,MMP3 and MMP9.The effects of NFkB inhibitor PDTC on the migration and invasion of RA FLS were detected by transwell migration plate and Invasion plate.ResultsWestern blotting showed that the levels of phosphorylation of p65,IKK/and IKB in RA FLSs were significantly increased after TNF-α stimulation(P<0.01).The phosphorylation of p65 and IKB induced by TNF-α was significantly down-regulated in the low,medium and high dose groups group of DTYMD(P<0.01),but the phosphorylation of IKK/induced by TNF-α was not affected(P>0.05).The results of Real Time PCR showed that NFkB inhibitor could significantly down-regulate the expression levels of the inflammatory cytokines IL-1β,IL-6,IL-8 and matrix metalloproteinases MMP1,MMP3 and MMP9 stimulated by TNF-α(P<0.05).Transwell migration assay and Invasion invasion assay showed that NFkB inhibitor significantly down-regulated the migration and invasion ability of RA FLSs induced by TNF-α.Conclusion:DTYMD may regulate the activation,migration and invasion of RA FLSs by NFkB signaling pathway.Part IV The therapeutic effect of DTYMD on collagen-induced miceObjectiveTo observe the therapeutic effect of DTYMD on collagen-induced arthritis(CIA)mice.MethodsDBA/lJ mouse were selected to construct the classic RA model,collagen-induced arthritis(CIA)mice,by twice immunization method.A total of 48 mouse were randomly divided into 6 groups:normal group,model group,MTX group(2mg/kg)and DTYMD different dose groups(7.50g/kg,15mg/kg,30mg/kg).Except the normal group,the other five groups constructed the CIA model.On the same day after enhanced immunization,each group was given appropriate intervention.The therapeutic effects of DTYMD on RA model mouse were measured by observing the general condition of mouse before and after treatment,the degree of swelling of ankle joints in CIA mouse,arthritis score,HE staining and pathological score.The effects of DTYMD on immune function and reproductive system of CIA mouse were observed by calculating the spleen index and testis index of CIA mouse.ResultsThe mouse in each group had a good general condition before constructed.After the initial immunization,the CIA mouse were swollen in the tail,some had ulcers,and even the tail was broken.The body weight of the mouse generally began to decrease,the activity of the mouse,and mice’ s activity also decreased.After booster immunization,most of the mouse in the model group and the DTYMD high-dose group were under-glossy and less active,and the testicular atrophy was observed in the high dose of DTYMD.The mouse began to have joint swelling one week after the booster immunization,and the front and rear limbs could be swollen.Two weeks after the booster immunization,most of the mouse developed joint swelling,and then there were joint swelling in the mouse.Usually,the mice begin to have joint swelling one week after the booster immunization,and the front and rear limbs can be swollen.Two weeks after the booster immunization,most of the mice developed joint swelling,and and later some mice showed joint swelling.The results showed that:compared with the normal group,the model group joint swelling,severe tetanus.Comparedwith the model group,the mice in the DTYMTD dose group and MTX group had less joint redness and swelling,and their joint activities were basically normal.In this experiment,the effect of DTYMD on the scores of arthritis in the front and back of mice was evaluated by dynamic observation.About 3 weeks after the administration(day 41),the arthritis scores of each group showed a downward trend.Compared with the model group,the arthritis scores of the DTYMD medium,high dose group and MTX group were significantly decreased(P<0.05 or P<0.01)at 2 weeks(day33);the time points after 2 weeks of administration(day37,Day41,day45),arthritis scores of DTYMD low,medium and high dose groups and MTX group were significantly lower than the model group(P<0.01).The pathological results of synovial membrane of the ankle joint of CIA mice showed that the normal group had complete ankle joint structure,no proliferation of synovial membrane,no infiltration of inflammatory cells,smooth articular cartilage surface and no destruction of articular cartilage and bone.In the model group,ankle joint structure was destroyed by inflammation,inflammatory cells infiltration was obvious,pannus formation invaded into cartilage and caused cartilage and bone destruction.In each treatment group,the ankle joint was relatively complete,synovial thickening was not obvious,and a small amount of inflammatory cells infiltrated.Compared with the normal group,the inflammatory score,cartilage destruction score and total pathological score of the model group were significantly increased(P<0.01).Compared with the model group,the scores of DTYMD groups and MTX groups were significantly reduced after intervention(P<0.01).The results of CIA mice spleen index and testis index showed that the spleen index of the model group was higher than that of the normal group(P<0.01).Compared with the model group,the spleen index of DTYMD dose group and MTX group showed a decreasing trend(P<0.01).Compared with the normal group,the spleen index of the model group was significantly higher(P<0.01).Compared with the model group,the spleen index of the DTYMD low,medium and high dose groups and the MTX group was significantly decreased(P<0.05 or P<0.01).Compared with the normal group,there was no statistical difference in the testicular index of the model group;compared with the model group,the testicular index of the DTYMD high-dose group was significantly decreased(P<0.01)..ConclusionDTYMD can effectively relieve joint swelling in CIA mice,reducing inflammatory indexes,preventing joint and bone destruction,and has an exact therapeutic effect on RA mouse model. |
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