| 1 Objective To research changes of Immune globulin in Rheumatoid arthritis(RA)patients and rats. Based on Xin’an medical theory, evaluate the protective effect of XFC, investigate the mechanism for improving immune globulin in RA.2 Methods2.1 Clinical Research2.1.1 60 cases of RA patients which choosed from Anhui Provincial Hospital of Chinese Medicine were randomly divided into observer group(XFC, 30 cases),control group(LEF, 30 cases) and normal group(20 cases). To observe changes of age, gender, course, quality of life, mental health and other laboratory indicators of 60 cases RA patients.2.1.2 Using the biochemical analyzer instrument to detect RA patients’ basic biochemical indexes, ELISA detect Ig G subtype(Ig G1, 2, 3, 4) and B lymphocyte activating factor, B lymphocyte activating factor receptor detected by flow cytometry; detection of peripheral blood B lymphocyte microtubule associated protein 1 in the light chain of 3- II(LC3- II) expression in RA patients with Western Blotting.2.1.3 To analyze the improvement of immune globulin RA, compare DAS28,ACR20/50/70, SF36, SAS, SDS scores, and related laboratory parameters(ESR,etc.) before and after treatment. Using statistical software of SPSS19.0 to analyze the relationship between those indexes.2.2 Experimental research2.2.1 60 male SD rats,150±20g in weight, were randomly divided into those five groups: normal control group(NC), model control group(MC), Xinfeng Capsule dose group(XFC), leflunomide control group(LEF), Triptolide control group(TPS), with 12 in each group. Except the NC group, each rat was injected in theright rear paw intradermally with Freund’s complete adjuvant to induce inflammation and thus obtain AA model, and strengthen the immune in the 10 th.The rats were given the dose from the 19 th days after inflammation, and the dosage was equivalent to 10 times the clinical dosage. The dosage of each group was as follows: NC group and MC group: saline was given(9mg/1ml/100g);XFC-M group:(0.034g/1ml/100g); TPS group:(50ug/kg/100g); LEF group:(0.5mg/1ml/100g); all the rats were continuously dosed once per day for 30 days.2.2.2 To observe the activity state and behavior of AA rats, measure body mass,paw swelling and arthritic index, immunoglobulin subtypes, cytokines(IL-1 、TNF-α、IL-4、IL-10), expression of LC3-Ⅱ, Beclin1,PI3-kinase p110δ,p-Akt1/2/3and m TORC1 in synovium, thymus and spleen tissues; autophagy related gene(atg5\atg7\atg12) of rat synovium thymus; electron microscope observation of autophagy AA of rat spleen, thymus and synovium; observe rat synovium, spleen,thymus tissue pathological changes were observed under the microscope. To analyze the effects of XFC on AA rats.3 Results3.1 Clinical studies3.1.1 Immunoglobulin, subtypes of immunoglobulin and BAFF/BAFF-R changes in RA patients Detection of immunoglobulin in 20 cases of normal persons and 60 cases of patients with RA. Compared with the normal range, the amount of Ig A higher than the normal range(0.7-4.06g/L)is 15 people, accounting for 25% of all patients; the amount of Ig G higher than the normal range(6.8-14.5 g/L) is 26 people,accounting for 43.3%;the amount of Ig M(0.4-2.5 g/L) anomaly is 3 people,accounting for 5%. Compared with the normal group, RA group’s immunoglobulin Ig A, Ig G, Ig M were increased,the Ig G is most obviously(P<0.01). Compared with the normal group, Ig subtype Ig G1 of the RA group were increased significantly(P <0.01).Detected BAFF/BAFF-R in 20 cases of normal people and 43 cases of patients with RA. Compared with the normal group, the RA group BAFF/BAFF-R were increased significantly, where the BAFF-R is obviously increased(P <0.01).3.1.2 Study on the relationship between the immune globulin, Ig G subtype and BAFF/BAFF-R in the RA patients Correlation analysis showed that the negative correlation between immune globulin G subtype Ig G1 and BAFF-R(P <0.05). BAFF-R and WBC, PLT, CRP,RF and ASO were positively correlated, Ig A was negatively correlated with CRP(P <0.05 or P <0.01). Joint pain, morning stiffness, subcutaneous nodules were positively correlated with Ig G(P <0.05); less gas lazy words, joint heavy was positively correlated with Ig G1; joint tenderness was positively correlated with BAFF. Quality of life integral scores and Ig G positively correlated(P <0.05),social function score was positively correlated with Ig G3(P <0.05). DAS28, Ig G and Ig A were positively correlated(P <0.01); Ig M and SAS/SDS were positively correlated(P <0.05).3.1.3 Clinical therapeutic effect of Xinfeng Capsule in the treatment of RA ACR20 of LEF group and XFC group after treatment for fourth weeks,eighth weeks, Twelfth weeks were 15% and 25%, 45% and 60%, 85% and 85%,ACR50 were 5% and 5%, 10% and 20%, 40% and 70%, ACR70 were 0% and 0%,5% and 5%, 15% and 25%, chi square test the P value of >0.05, explains the difference between the two groups has no statistical significance between the ACR20/70; ACR50 of 12 weeks was P<0.05, XFC group was higher than LEF group.3.1.4 Comparison of Xinfeng capsule to the immunoglobulin in the RA patients Ig A, Ig G, Ig M, and Ig G subtype Ig G1, Ig G2, Ig G3, Ig G4 in two groups before and after drug treatment were not significantly reduced, but after 12 weeks of treatment, the Ig G1 in LEF group and XFC group was statistical difference(P<0.05). Xinfeng Capsule can significantly decreased the expression of Ig G1, while LEF increased slightly(P >0.05).3.1.5 Comparison of BAFF/BAFF-R by Xinfeng Capsule treat RA patients The two groups before and after the drug treatment of BAFF were not significantly reduced, but after 12 weeks of treatment, LEF group and XFC group BAFF numerical with statistical difference(P <0.05), can significantly from that on the expression of BAFF-R, XFC group decreased significantly after 12 weeks(P <0.01).3.1.6 Changes of serum laboratory indexes of RA patients treated by Xinfeng Capsule The HGB before and after the Xinfeng capsule and leflunomide treatment increased significantly(P <0.01), PLT decreased significantly(P <0.05 or 0.01);ESR decreased significantly(P <0.01), CCP increased significantly(P <0.05)in LEF group. There was no statistically significant difference after 12 weeks by treatment of each index in LEF group and XFC group.3.1.7 The changes of quality life in RA petients treated by Xinfeng capsule The symptom integral of traditional Chinese medical syndrome and total score in two groups after treatment were significantly lower(P <0.01 or P <0.05).Compared with LEF group, XFC group after treatment, less gas lazy words, loose stool, loss of appetite decreased significantly(P <0.01 or P <0.05); joint pain,swelling, tenderness, night pain, morning stiffness decreased slightly, but no significant differences(P >0.05). The two groups after treatment were significantly lower in the quality of life of integral(P <0.01 or P <0.05).Compared with LEF group, XFC group after treatment, psychological function,social function of healthy self cognition improved more obviously(P <0.01). The two groups after treatment HAQ, DAS28, SAS, SDS were significantly lower(P<0.01 or P <0.05). Compared with LEF group after treatment, after treatment the XFC group, SDS decreased significantly(P <0.01 or P <0.05).3.1.8 The changes of markers of autophagy LC3- II in RA patients By Western blot testing found that: compared with the normal control group,RA group B lymphocyte LC3-Ⅱ which is in blood decreased expression(P <0.01 or P <0.05).3.2 Experimental results3.2.1 Effect of XFC for AA rats’ body weight, toes swelling degree and AI Prior to administration, compared with the NC group, body weight was reduced in MC rats, and paw swelling degree, AI were significantly higher than NC rats(P <0.01).After treatment, compared with MC group, XFC can significantly increase the body mass, reduce paw swelling and AI(P <0.05 or P <0.01).3.2.2 Effect of XFC on AA rats articular pathological Compared with NC group, the synovium in MC rats can found a large number of inflammatory cells infiltration, and multiple vasculars hyperplasis in synovial layered, fibroplasia, macrophage-like type-A cell hyperplasia etc.After treatment, the inflammatory cells infiltration, and multiple vasculars hyperplasis in synovial layered were decreased. Compared with the control group,the XFC group’s synovial cells mitochondrial swelling reduced obviously.3.2.3 Effect of serum cytokines IL-1β, IL-4, IL-10, TNF-α in AA rats after treated by XFC capsule Compared with NC group, MC group IL-1 beta, TNF- alpha was significantly increased, IL-4, IL-10 decreased significantly.Compared with NC group, the control groups IL-1 beta and TNF- alpha was no significant difference, IL-4, IL-10 decreased significantly; compared with the model group(P <0.05 or P <0.01).3.2.4 Effect of XFC on serum immunoglobulin and Ig G1 subtypes, Ig G2 a,BAFF, kappa, lambda and ratio in AA rats Compared with NC group, MC group BAFF, Ig G, Ig A, Ig M, Ig G1, Ig G2aincreased significantly(P <0.01). Compared with MC group, control groups BAFF,Ig G, Ig G2 a, Ig G2a/, Ig G1 decreased significantly, Ig M increased significantly in group LEF, Ig G1 increased significantly, while XFC group decreased significantly;there was no significant difference in Ig A(P <0.01 or P <0.05).Compared with NC group, Ig G light chain of lambda of MC group significantly increased in synovium, kappa, kappa / lambda decreased significantly(P <0.01 or P <0.05). Compared with MC group, XFC group kappa,kappa / lambda significantly increased(P <0.05), no significant differences between LEF group.3.2.5 Effect of XFC capsules to AA rat about synovial, spleen, thymus autophagic bodies Seen under electron microscope: in NC group, mitochondrial without deformation, swelling in synovial cells, abundant rough endoplasmic reticulum,nuclear membrane clear boundary, evenly distributed chromatin, crista ordered,autophagic double membrane structure visible containing organelles and a plurality of autophagy lysosome; MC group synovial cells were deformed,swelling, mitochondrial swelling and hypertrophy and destroy, damage reduction,rough endoplasmic reticulum, nuclear membrane was not intact, the boundary is not clear, chromatin distribution is uneven, irregular cristae arrangement, part of autophagic vacuoles disappeared, fewer and smaller, irregular shape. Control groups were not obvious clear nuclear membrane, swelling of mitochondria,rough endoplasmic reticulum is complete, autophagy and autophagic lysosome increased slightly.Seen under electron microscope: in NC group, spleen, thymus tissue mitochondria swelling, no deformation, abundant rough endoplasmic reticulum,nuclear membrane clear boundary, evenly distributed chromatin, crista ordered,autophagic double membrane structure visible containing organelles and a plurality of autophagy lysosome; MC group spleen weaving boundaries are notclear, the cell organelles the serious damage of autophagic vacuoles, fewer and smaller, irregular shape. Control groups organizational boundaries can be resolved,autophagy and autophagic lysosome increased slightly.3.2.6 The effect of XFC on autophagy related gene ATG5/7/12 m RNA expression in AA rats synovial, thymus, spleen tissue Compared with NC group, MC group Atg12 m RNA and Atg5 m RNA of synovial decreased significantly, no significant difference of Atg7 m RNA; spleen Atg5 m RNA decreased significantly, Atg7 m RNA and Atg12 m RNA increased significantly; thymus Atg12 m RNA increased significantly, Atg5 m RNA and Atg7 m RNA had no obvious difference(P <0.05 or P <0.01).Compared with MC group, LEF group Atg12 m RNA and Atg5 m RNA of synovial decreased significantly, Atg7 m RNA increased significantly; Atg5 m RNA, Atg7 m RNA in spleen, thymus Atg12 m RNA decreased significantly,Atg5 m RNA decreased significantly, Atg7 m RNA, Atg12 m RNA had no obvious difference; XFC group synovial Atg12 m RNA decreased significantly; the spleen Atg5 m RNA, Atg7 m RNA and Atg12 m RNA increased significantly; the thymus Atg5 m RNA significantly increased, Atg7 m RNA significantly decreased, no significant differences in Atg12 m RNA. TP group Atg7 m RNA and Atg12 m RNA in synovium decreased significantly, Atg5 m RNA had no obvious change; spleen Atg5 m RNA, Atg7 m RNA and Atg12 m RNA decreased significantly; Atg5 m RNA and Atg7 m RNA in thymus decreased significantly, Atg12 m RNA increased significantly(P <0.05 or P <0.01).3.2.7 Effect of XFC capsule on LC3- II, Beclin1 expression in AA rats synovial, thymus, spleen tissue In synovial tissue, compared with NC group, MC group LC3- II, Beclin1 decreased significantly(P <0.01); compared with MC group, control groups LC3-II and Beclin1 increased significantly(P <0.01 or P <0.05).In the spleen tissues, compared with NC group, MC group LC3- II andBeclin1 decreased significantly(P <0.01); compared with MC group, control groups LC3- II and Beclin1 increased significantly(P <0.01 or P <0.05).In thymus tissue, compared with NC group, MC group LC3- II and Beclin1 decreased significantly(P <0.01); compared with MC group, control groups LC3-II and Beclin1 increased significantly(P <0.01).3.2.8 Effect of XFC capsule on on PI3K/AKT/m TOR expression in AA rats synovial, thymus, spleen tissue In synovial tissue, compared with NC group, MC group PI3K/AKT/m TOR increased significantly(P <0.01). Compared with MC group, control groups PI3K/AKT/m TOR decreased significantly(P <0.01).In the spleen tissues, compared with NC group, MC group PI3K/AKT/m TOR increased significantly(P <0.01). Compared with MC group, control groups PI3K/AKT/m TOR decreased significantly(P <0.01).In thymus tissue, compared with NC group, MC group PI3K/AKT/m TOR increased significantly(P <0.01). Compared with MC group, PI3K/AKT/m TOR decreased significantly in LEF group and XFC group, AKT/m TOR decreased significantly of TP group(P <0.01 or P <0.05).4 Conclusion4.1 In RA patients, Ig A, Ig G and Ig M increased, especially Ig G, which is related with BAFF/BAFF-R.4.2 XFC can significantly reduce the BAFF-R expression in RA patients.4.3 XFC could significantly increase the immunoglobulins levels and Ig G1 in RA patients, improve immune function, better than the control group.4.4 XFC can significantly modulate RA TCM symptoms and signs and the life quality of RA patients and depression emotion.4.5 XFC has no adverse effect on AA rat body weight growth, the body weight growth and survival rate than the control group; in the improvement of rat paw swelling degree, reduce arthritis index is as good as that of control group.4.6 XFC can reduce AA rat BAFF, Ig G, Ig G2 a, Ig G2a/Ig G1 levels, increased Ig M,Ig G1 level; AA rats synovial kappa, kappa / lambda level and the serum cytokines IL-4, IL-10 level increase;reduce the autophagy related gene IL-1 beta, TNFalpha level and regulating PI3K/AKT/m TOR signaling pathway in spleen, thymus and synovial, decrease the level of autophagy in AA rats.4.7 The mechanism of XFC improvement immunoglobulin in RA patients4.7.1 Through the various drug, cell immunity and humoral immune function is regulating, reduce the humoral immune activation,enhance immunoglobulin secretion and anti-inflammatory effects;4.7.2 Inhibit immunoglobulin secreting plasma cell, reduce the level of immunoglobulin by down regulating the expression of BAFF.4.7.3 The maintenance of peripheral tolerance, balance the secretion regulation of cytokines, inhibit excessive secretion of lymphocytes, immune complexes and infiltration of synovial tissue, reduce the level of immunoglobulin, relieve joint damage through the up regulation of Atg5 m RNA, Atg7 m RNA, Atg12 m RNA,LC3- II, Beclin1 expression.4.7.4 Enhance autophagy in vivo and improve the level of Foxp3, reduced BAFF stimulated B lymphocytes activation of secretory immunoglobulin via regulated the PI3K/Akt/m TOR pathway. |
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