| BackgroudRheumatoid arthritis(RA)is a common autoimmune disease.Studies have shown that B cells play an important role in the pathogenesis of RA.Abnormal activation of B cells will produce a large number of autoantibodies,which can induce or aggravate the disease of RA and bone destruction.Immunoglobulin Fc receptor IIb(Fc?RIIB)is an important transmembrane receptor that negatively regulates B cell receptor(BCR)signaling.Its main function is to inhibit the activation of B cells and induce peripheral immune tolerance.However,the Fc?RIIB-ile232Thr gene variant attenuates its ability to inhibit B cell activation.More importantly,Fc?RIIB has been shown to be regulated,with dysfunction in patients with active RA and normal function in patients with inactive RA whose disease is under control,making it a promising new target for RA therapy.Our previous study found that Fc?RIIB-I232T gene variation was more frequent in patients with renal deficiency than in patients without renal deficiency RA,indicating that renal deficiency and Fc?RIIB gene variation had a certain correlation.Based on the above research,Professor Chen Guangxing,our tutor,combined with the understanding of RA in traditional Chinese medicine,and established Yishen-tongbi Decoction to treat kidney deficiency syndrome(kidney deficiency type RA)with the method of treating from kidney.The research group's preliminary clinical study found that Yishen Tongbi Decoction can significantly relieve the clinical symptoms of active RA patients,such as joint swelling and pain,morning stiffness,improve joint function and reduce the level of inflammatory factors in active RA patients.However,the molecular mechanism of its treatment of renal deficiency RA is still unclear.Therefore,this study focuses on the Fc?RIIB gene variation to study the intervention mechanism of Yishen Tongbi Decoction on rheumatoid arthritis with kidney deficiency,aiming to explain the molecular mechanism of Yishen Tongbi Decoction in the treatment of RA with kidney deficiency.ObjectiveBy observing the effect of Yishen-tongbi Decoction on CD19/Fc?RIIb-Lin-SHP-1signal pathway,and the inhibitory effect of Chinese medicine on inflammation and bone destruction in the Collagen-induced arthritis(CIA)rat model,the specific molecular mechanism of Chinese medicine in the treatment of kidney deficiency RA was explained,providing scientific data for the treatment of RA with Chinese medicine characteristics.Methods1.Animal experiment:Inhibitory effect of Yishen Tongbi Decoction on inflammation and bone destruction in CIA rat model(1)SPF female Wistar rats were selected and randomly divided into blank group,model group,Yishen Tongbi decoction low-dose group,Yishen Tongbi decoction medium-dose group,Yishen Tongbi decoction high-dose group and MTX group,with 10rats in each group.Each concentration group of Yishen Tongbi Decoction was intragastric with Yishen Tongbi Decoction solution of 4.5,9 and 18g·kg-1·d-1,MTX group was intragastric with methotrexate solution of 1.35mg·kg-1·w-1,and the other two groups were intragastric with 0.9%Na Cl solution of equal volume.Body mass and foot swelling were measured every 3 days during the experiment,and the arthritis index(AI)was evaluated according to the foot swelling.(2)After Yishen Tongbi Decoction was administered ingastric for 34 days,X-ray was performed on the limbs and joints of the rats,and the radiologists performed blind evaluation on the X-ray films of the limbs,including bone erosion score and joint space narrowing(JSN)score.(3)After 34 days of intragastrization of Yishen Tong Bi Decoction,HE staining was performed on the left forelimb and the third toe joint of the right hind limb.He pathological films of the toe joint were evaluated by blind method,including inflammatory cell infiltration score,synovial tissue hyperplasia score,and joint destruction score.2.Cell experiment:The effects of Yishen Tongbi Decoction on the proliferation,apoptosis and activation of ST486 cells,as well as the regulatory effect of Yishen Tongbi Decoction on the activation related signal pathway CD19/Fc?RIIB-LYN-SHP-1 of ST486cells.(1)Constructing Fc?RIIB-I232T gene defective cell lineHuman Burkitt B lymphoma cell line ST486(lacking endogenous Fc?RIIB)was transfected with Fc?RIIB-vector(no load),Fc?RIIB-WT(wild type),and Fc?RIIB-I232T(mutant type)plasmids,respectively,to construct a gene deficient cell model.RT-q PCR and Western Blot were used to verify the transfection efficiency from m RNA and protein levels,respectively.The gene defective cell model ST486 was successfully constructed and used for subsequent cell experiments.(2)Effects of different concentrations of Yishen Tongbi Decoction on the proliferation,apoptosis and activation of ST486 cellsST486 cells were treated with 0ng/ml,125ng/ml,250ng/ml,500ng/ml,1000ng/ml and2000ng/ml for 48h,respectively,and cell proliferation was detected by CCK-8 method.The chloroform fraction of Yishen Tongbi Decoction with concentrations of 250ng/ml,500ng/ml and 1000ng/ml was used to intervene ST486 cells for 48h,and the total apoptosis rate of each group was detected by flow cytometry.The chloroform parts of Yishen Tongbi Decoction at concentrations of 250ng/ml,500ng/ml and 1000ng/ml were used to intervene ST486 cells,and the dynamic changes of Ca2+flux in ST486 cells were observed dynamically by flow cytometry.(3)The regulation effect of chloroform fractions of Yishen Tongbi Decoction at different concentrations on CD19/Fc?RIIB-LYN-SHP-1 signaling pathway related to ST486 cell activation.The chloroform fractions of Yishen Tongbi Decoction were treated with concentrations of 250ng/ml,500ng/ml and 1000ng/ml respectively for 48h,and the protein expressions of CD19,Fc?RIIB,Lyn and SHP-1 in each group were detected by Western Blot.Results1.Animal experiments(1)Changes in general condition of ratsIn model group,the hair was dull,the dorsal arch was curled up,the activity was reduced,and there were thin stools.Mental state and behavioral activity of rats in Yishen Tongbi decoction group and methotrexate group were improved compared with model group.(2)Changes in body weight of ratsAfter the establishment of CIA model,compared with the blank group,the body weight of the other 5 groups was significantly decreased(P<0.01);Compared with model group,the body weight of rats in different concentration groups of Yishen Tongbi Decoction and MTX group were higher on 25-34d,and the difference was statistically significant(P<0.01,P<0.01,P<0.01,P<0.01).(3)Comparison of podocytosis values in ratsThe peak value of CIA rats'foot swelling was reached on the 7th day after modeling.Compared with blank group,podocysis value in model group was significantly increased(P<0.01);Compared with the model group,the high dose group of Yishen Tongbi Decoction significantly decreased the foot swelling value from the 4th day(P<0.01),and the MTX group significantly decreased the foot swelling value from the 10th day(P<0.01).(4)Comparison of arthritis index scores in ratsCompared with the model group,the AI score of Yishen Tongbi Decoction groups continued to decrease,especially the middle and high dose groups.Compared with the model group,the AI score of Yishen Tongbi Decoction medium and high dose group was significantly decreased on the 10th day(P<0.01,P<0.01),and the AI score of MTX group was significantly decreased on the 16th day(P<0.05).(5)Comparison of bone erosion scores in ratsCompared with model group,the bone erosion scores of Yishen Tongbi decoction medium-high dose group were significantly decreased,with statistical significance(P<0.01,P<0.01);Compared with MTX group,the bone erosion score of Yishen Tongbi Decoction medium and high dose group was decreased,but the difference was not statistically significant(P>0.05,P>05).(6)Comparison of JSN scores in ratsCompared with the model group,the JSN scores of Yishen Tongbi Decoction medium-dose and high-dose groups were significantly decreased,with statistical significance(P<0.01,P<0.01).Compared with MTX group,the JSN scores of Yishen Tongbi Decoction medium-dose and high-dose groups were higher,but the difference was not statistically significant(P>0.05,P>05).(7)Comparison of scores of inflammatory cell infiltration in ratsCompared with model group,lymphocyte infiltration score of Yishen Tongbi decoction medium-dose and high-dose group was significantly decreased,with statistical significance(P<0.01,P<0.01);Compared with the MTX group,the scores of inflammatory cell infiltration in the high-dose group of Yishen Tongbi Decoction were higher,but the difference was not statistically significant(P>0.05,P>05).(8)Comparison of synovial tissue hyperplasia scores in ratsCompared with model group,lymphocyte infiltration score of Yishen Tongbi decoction high-dose group was significantly decreased,with statistical significance(P<0.01);Compared with MTX group,the synovial tissue hyperplasia score of Yishen Tongbi decoction medium and high dose group was higher,but the difference was not statistically significant(P>0.05,P>05).(9)Comparison of joint destruction scores in ratsCompared with model group,joint destruction scores of Yishen Tongbi decoction medium-dose and high-dose groups were significantly decreased,with statistical significance(P<0.05,P<0.01).Compared with MTX group,the bone destruction scores of Yishen Tongbi Decoction medium and high dose group were higher,but the difference was not statistically significant(P>0.05,P>05).2.Cell experiments(1)Compared with blank group,the expression levels of Fc?RIIB m RNA and protein in WT group were significantly increased(P<0.01,P<0.01),and those in Mut group were significantly increased(P<0.01,P<0.01).Compared with WT group,the expression levels of Fc?RIIB m RNA and protein in Mut group were significantly increased(P<0.01,P<0.01),suggesting that the Fc?RIIB gene defective cell model was successfully constructed.(2)The chloroform fraction of Yishen Tongbi Decoction with different concentrations had significant inhibitory effect on the proliferation of ST486 cells in a concentration-dependent manner(P<0.01,P<0.01,P<0.01).The chloroform fraction of Yishen Tongbi Decoction with a concentration of 500ng/ml could reach 50%inhibition rate of ST486 cells for 48h.(3)Compared with Mut group,low concentration of chloroform fraction of Yishen Tongbi Decoction had no significant effect on apoptosis of ST486 cells(P>0.05);The chloroform fraction of Yishen Tongbi Decoction at medium and high concentration significantly promoted the apoptosis of ST486 cells(P<0.01,P<0.01).(4)Flow cytometry dynamic observation results showed that,compared with Mut group,different concentrations of chloroform fractions of Yishu Tongbi Decoction had significant inhibitory effect on Ca2+flux in ST486 cells(P<0.05,P<0.01,P<0.01),suggesting that the chloroform fractions of Yishu Tongbi Decoction could inhibit the activation of ST486 cells.(5)Changes in CD19-related activation signaling pathways:Compared with the Mut group,the high concentration of Yishu Tongbi Decoction chloroform fraction could reduce the phosphorylation level of CD19 in ST486 cells(P<0.05),suggesting that the high dose of TCM compound group could inhibit the activation of ST486 cells by reducing the phosphorylation level of CD19.(6)Changes in inhibitory signaling pathway of Fc?RIIB:Compared with Mut group,medium and high concentration of Yishen Tongbi Decoction chloroform fraction could up-regulate the protein level of Fc?RIIB in ST486 cells(P<0.05,P<0.05);High concentration of Yishen Tongbi Decoction could up-regulate the protein level of Lyn in ST486 cells(P<0.05).High concentration of Yichen Tongbi Decoction chloroform fraction can up-regulate the protein level of SHP-1 in ST486 cells(P<0.05),suggesting that high dose of TCM compound group can inhibit the activation of ST486 cells by activating Fc?RIIB/Lyn/SHP-1 pathway.Conclusion1.Yishen Tongbi Decoction can reduce the inflammatory response and inhibit bone destruction in CIA rats.2.The chloroform fraction of Yishen Tongbi Decoction can inhibit the proliferation,activation and apoptosis of ST486 cells(Fc?RIIB gene deficient cell model),and the mechanism may be related to the CD19/Fc?RIIB-LYN-SHP-1 signaling pathway. |
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