Objective To investigate the synergistic effect of methotrexate (MTX) and cyclophos phamide (CTX) on cell cycle and cyclin D1 of periphery blood lymphocytes (PBLs) and bone marrow byflow cytometry. Methods Wistar rats were randomly divided into four groups including normal control, MTX and cyclophosphamide combination group, MTX and CTX only treatment groups respectively. PBLs were isolated for flowcytometry analysis for the changes of cell cycle and the expression of cyclin D1 at week 0, week 3,week 9, week18 and week 27. Mice were dissected and the changes of lymphoeytes cell cycle and the expressions of cyclin D1 in the bone marrow were measured at week 0, week 3, week 9, week 18 and week 27 increased and the ratio of phase S cells was decreased (P>0.05). In the CTX treatment group, there was no statistical difference in ratios of each phase. In the MTX and CTX combination treatment group, the proportion of phase G0/G1 cells decreased significantly and the percentage of phase S cells increased in both PBLs and bone marrow ceils (P<0.05). And there was no statistical significant difference in different time points after marrow between different groups or different dissecting time points. Conclusion MTX combined with CTX has been shown to have antagonistic effect on cell cycle. However, this effect is not via the cyclin DI pathway.

Objective To investigate the prevalence of fatigue in rheumatoid arthritis (RA) and its relationship with other clinical and functional parameters used for the evaluation of disease activity and health related quality of life. Methods The fatigue was assessed in 230 patients with RA using visual analogue scale (VAS). The correlation between fatigue and the clinical disease activity including morning stiffness, pain, PGA, physician's global assessment, TJC, TJI, SJC, SJI, DAS28, HAQ and health-related quality of life were assessed. Results The prevalence of fatigue was 85.7% and the fatigue score of 51.7% patients was higher than 50 mm. After controlled for the possible confounding factors such as age, gender and disease duration, it was found that fatigue was highly correlated with pain, disease activity, functional disability, physical health and mental health. Conclusion Fatigue is an important symptom of RA and is correlated with pain, disease activity, functional disability and health-related quality of life.

Objective To evaluate the clinical efficacy and safety of methotrexate(MTX),cyclophosphamide(CTX)and MTX plus CTX in patients with active rheumatoid arthritis(RA).Methods In a randomized,single-blinded,controlled study,90 patients were randomly assigned to receive MTX(10～15 mg/w)or CTX(400 mg/2～3 w)or MTX plus CTX(MTX 10～15 mg/w+CTX 400 mg/2～3 w).The primary end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria(achieving an ACR20 response,)at week 24.The secondary end points were responses of the ACR50 and ACR70 improvement criteria,and the European League Against Rheumatism(EULAR)response criteria.The change from baseline in duration of pain,patient's global assessment,physician's global assessment,tender joint count/index,swollen joint count/index,health assessment questionnaire(HAQ),erythrocyte sedimentation rate(ESR)were also evaluated.The clinical efficacy and safety were analyzed at baseline,6,12 and 24 weeks respectively.Results The ACR response rate was significantly higher in the MTX plus CTX treatment group compared with MTX or CTX group at week 24.The MTX plus CTX group,MTX group and CTX group showed 81%,56% and 35% in ACR20,58%,41% and 12% in ACR50 and 19%,11% and 0 in ACR70,.respectively.At week 24,the proportion of patients achieving the EULAR moderate response in those who received combination treatment were significantly higher than those who received either MTX or CTX.The incidence of adverse events(AEs)was not significantly higher in MTX plus CTX group than MTX or CTX group.Conclusion MTX plus CTX effectively reduces the signs and symptoms of RA and is generally well tolerated by patients without significant increase in the rate of adverse events compared with monotherapy.

Objective To analyze the quality of life of patients with active rheumatoid arthritis (RA) and its relationship with other clinical and functional parameters used for the evaluation of disease activity. Methods The quality of life was assessed in 127 patients with active RA using SF-36 and was compared with non-active RA and the general population. The correlation between the quality of life and the clinical measures of disease, including morning stiffness, pain, fatigue, patient's global assessment (PGA) physician's global assessment , SJC/SJ1, TJC/TJI, DAS28, HAQ were measured. Results The patients with active RA reported significantly decreased scores in all dimensions of SF-36. Fatigue, PGA, physician's global assess-ment, DAS28 and HAQ significantly correlated with the scores in all dimensions of SF-36. Pain was correlated with the scores in all dimensions of SF-36 except RE. TJI was correlated with six dimensions. TJC was correlated with five dimensions. ESR was correlated with three dimensions. Conclusion The quality of life in patients with active RA is significantly declined compared with non-active RA and the general population. Disease activity and functional status of patients with active RA is closely correlated with the quality of life.

Objective To compare the correlation between anti-cyclic citrulinated peptide antibody (anti-CCP), rheumatoid factor (RF) and disease activity, functional capacity, bone erosion of rheumatoid arthritis (RA). Methods The correlation between anti-CCP, RF and disease activity score 28 (DAS28),health assessment questionnaire(HAQ) and bone erosion was assessed. Results Among the 218 RA patients,76% were anti-CCP-positive and 71% were RF-positive. The mean DAS28 score of RA patients with anti-CCP-positive and RF-positive was significantly higher than patients without anti-CCP and RF. The serum an-ti-CCP levels and RF levels showed a significant correlation with DAS28 score. However, the HAQ scores, ESR and CRP levels showed no significant difference between anti-CCP-positive and anti-CCP-negafive patients, RF-positive and RF-negative patients. Among anti-CCP-positive RA patients, the number of patients with erosive disease was significantly higher than patients without anti-CCP. However, the number of patients with erosive disease was not significantly different between RF-positive and RF-negative patients. Conclusion Anti-CCP and RF arc associated with disease activity. Anti-CCP is associated with bone erosion. No association is found between RF and bone erosion.

The pearly gallstone was discharged after Treatment with Magnetic Field (TMF). The pearly gallstones are measured with FT-IR spectrometer. The results demonstrate that mostly composition of this gall-stones is cholesterol and intermixture with some protean and inorganic calcium salt. Nature pearls is mostly composed with calcium carbonate. Their compositions are different. The pearly gall-stones show sandwich of cholesterol crystal in structure.

Objective To assess the correlation between sleep quality and disease activity in rheumatoid arthritis (RA) patients by comparing the sleep quality of RA patients with healthy control group.Methods One hundred and sixty cases of RA patients who were hospitalized in the Second Hospital of Shanxi Medical University from January 2015 to June 2017 were included into this study,and 90 age and gender matched healthy people were selectedas control group.Age,sex,therapeutic drugs,Pittsburgh Sleep Quality Index (PSQI),serum erythrocyte sedimentation rate (ESR),C-reactive protein (CRP),the 28-joint Disease Activity Score (DAS28),Visual Analogue Score (VAS) and Beck Depression Inventory second edition (BDI-Ⅱ) were detected and compared.According to the DAS28 scores,the relationship between the sleep quality of RA patients and the degree of disease activity was analyzed.Results One hundred and nine cases (68.1％) with sleep disorders in the RA group,which was higher than that in the healthy control group,in which 17 cases (18.9％) had sleep disorder.The total scores of the PSQI in the RA group were higher than those in the healthy control group,which were (7.2±3.2) scores vs (3.1±2.8) scores (t=10.636,P＜0.05) respectively.In the RA group,the average age of patients with sleep disorders (PSQI＞5) (56±12) years was higher than that of patients without sleep disorders (48±9) years (PSQI≤5).The VAS and BDI-Ⅱ score in RA patients with sleep distur-bance were higher than those with non-sleep disturbance,which were (5.8±2.2) scores vs (25.1 ±9.1) scores vs (2.1 ±2.1) scores vs (14.2±6.6) scores (P＜0.05) respectively.According to the results of Pearson correlation an-alysis,DAS28 was significantly positively correlated with BDI-Ⅱ (r=0.382,P=0.001),PSQI wassignificantly positively-correlated with BDI-Ⅱ (r=0.312,P=0.001).Subjective sleep quality,sleep time,sleep efficiency,sleep disorder,daytime dysfunction and the PSQI total score increased with the increase of RA activity.Conclusion The incidence of sleep disturbance in RA patients is higher than that in the normal population.The incidence of sleep disturbance,increases significantly accompanied by high disease activities in RA patients.

Objective:To study the expression of receptor activator of nuclear factor-κB ligand (RANKL) induced by interleukin-6 (IL-6) in fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLS) and its signal pathway, to clarify the synergistic mechanism of methotrexate (MTX) combined with cyclophosphamide (CTX) in inhibiting RA bone erosion.Methods:The synovial tissues of 6 patients with active RA were collected, and RA-FLS were cultured in vitro. IL-6/sIL-6R and drug intervention were given successively. All RA-FLS were divided into five groups, including blank control group, IL-6/sIL-6R group, CTX group, MTX group and MTX+CTX group. The expression levels of RANKL and signal pathway protein in RA-FLS were detected by real-time quantitative polymerase chain reaction (qPCR), Western blotting and cell-based enzyme-linked im-munosorbent assay (ELISA). Single factor analysis of variance was used to compare in the comparison of multiple groups, LSD- t test or Dunnett's T3 test was used in the comparison of two groups, and 2×2 factorial analysis of var-iance was used in the interaction of two drugs. Results:① There were statistically significant differences in the mRNA and protein levels of RANKL in each group ( F=26.246, P<0.01; F=4.565, P=0.023). The highest level of RANKL mRNA (2.14±0.40) and protein (2.33±0.39) was found in IL-6/sIL-6R group, and the lowest level of RANKL mRNA (0.10±0.08) and protein (0.75±0.21) in MTX+CTX group. ② The difference of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) level in each group was statistically significant ( F=18.151, P<0.01). The level of p-STAT3 (0.328±0.073) was the highest in IL-6/sIL-6R group and the lowest (0.178±0.022) in MTX+CTX group. There was no significant difference in STAT3 in each group ( F=3.173, P=0.051). ③ MTX combined with CTX had interaction on the expression of RANKL mRNA and protein ( F=33.932, P<0.01; F=16.265, P<0.01), and had interaction on the expression of p-STAT3 ( F=16.477, P=0.01), but had no interaction on the expression of STAT3 ( F=0.471, P=0.500). Conclusion:IL-6 can up-regulate the expression of RANKL in RA-FLS through JAK2/STAT3 signal pathway. MTX combined with CTX can down-regulate the expression of RANKL by inhibiting the phosphorylation of STAT3, and this combination is synergistic.

Objective We investigated the correlation between inflammatory cytokines and drugresi-stant proteins. Methods Fourteen DBA1 mice were successfully induced by collagen and Freund's adjuvant. According to the scores of synovial pathology, the collagen-induced arthritis (CIA) group was divided into mild, moderate-severe groups, another four mice were selected as controls. The mRNA expressions of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MRP1) in splenic lymphocyte cells were measured by reverse transcription-polymerase chain reaction (RT-PCR). The concentrations of inter-leukin (IL)-1β, IL-2, IL-6, IL-10, umor necrosis factor (TNF)-α, IL-17 in serum were detected by Cytometric Bead Array (CBA). The correlation between different inflammatory cytokines and these proteins were analyzed, then one of the proteins which were most related with cytokines by immunohisto-chemical (IHC) in the synovium was studied. Data were analyzed by the Mann-Whitney U test, Kruskal-Wallis H test and spearman analysis. Results ①Compared with normal controls, the levels of IL-6 and TNF-α in the serum of mild CIA group, moderate-severe CIA group were significantly increased {IL-6 controls: [7.75 (5.14, 9.17) pg/ml];mild CIA: [25.31 (15.15, 29.27) pg/ml]; modeate-severe CIA: [45.03 (38.87, 64.02) pg/ml]. TNF-α: controls: [22.81 (20.84, 28.17) pg/ml]; mild CIA: [45.00(32.76, 58.51) pg/ml]; modeate-severe CIA: [45.00(39.78, 8.95) pg/ml]}(Z=14.383, P<0.05; Z=8.375, P<0.05). Compared with the mild CIA group, the level of IL-6 in serum was significantly increased in the moderate-to-severe CIA group (P<0.05), but there was no signigicant difference in the TNF-α level (P>0.05). ② In the spleen lymphocytes, there was no significant difference in the mRNA expression level of P-gp and BCRP among the groups, but the mRNA expression level of MRP1 was significantly increased (Z=12.634, P<0.05). Compared with the mild CIA group, the MRP1 mRNA in the moderate-severe CIA group was higher, the difference was significant (Z=12.634, P<0.05). There was a correlation between mRNA expression of MRP1 and P-gp (r=0.635, P=0.015). ③ The mRNA expression of MRP1 was positively correlated with IL-6 level (r=0.711, P=0.004). ④ The expression of MRP1 in normal group, low-level IL-6 group and high- IL-6 level group were as follows: [1.08 (0.65, 1.30)], [1.32 (1.08, 1.49)], [2.07 (1.77, 2.22)] respectively.⑤Compared with the controls, the cytoplasm/membrane of the knee and ankle joint synovial tissue in the CIA group was yellowish-brown, which indicated that MRP1 expression was positive. Conclusion In the CIA arthritis model, synovial tissue lesion is not only related to inflammatory cytokines, but also related to MRP1 expression in the ATP-binding cassette (ABC) transport protein family, and it is proved that IL-6 is highly correlated to MRP1.

Objective:To investigate the level of peripheral blood regulatory T cells in rheumatoid arthritis (RA) patients with cardiovascular disease (CVD) and its clinical significance.Methods:A total of 191 patients with RA in the Department of Rheumatology and Immunology, the Second Affiliated Hospital of Shanxi Medical University and 86 healthy controls (HCs) were enrolled from January 2019 to January 2021. All peripheral blood CD4 + T lymphocyte subsets of participants were assessed by flow cytometry. Patients were divided into RA-CVD group ( n=71) and RA only group ( n=120) and their clinical data were recorded. The differences between the groups were analyzed by Independent-Samples t test, Mann-Whitney U test or χ2 test, and risk factors that affected CVD were analyzed using Logistic regression. Results:① The age of patients and the proportion of male patients in the RA-CVD group were significantly higher than those in the RA only group [age: (64±10) years old vs (56±12) years old, t=-4.16, P<0.001; male patients: 35 cases vs 31 cases, χ2=10.86, P=0.001]. ② The level of Treg cells in the peripheral blood of patients with RA only and RA-CVD groups was significantly lower than that of HCs ( Z=-4.14, P<0.001; Z=-6.27, P<0.001), while the numbers of peripheral Th17 cells in the two groups of patients were not significantly different from those of HCs ( P>0.05). The ratios of Th17/Treg cells in the two group patients were higher than those of HCs, but only the difference between RA-CVD patients and HCs was significant ( Z=-5.49, P<0.001). ③ Compared with the RA only group, the absolute number of Treg cells in peripheral blood of RA-CVD group was significantly lower [19.00(13.62, 26.73) vs 24.94 (19.32, 34.12), Z=-3.19, P=0.001], the level of Th17 cells was significantly higher [absolute number: 7.77 (3.86, 13.64) cell/μl vs 5.59 (3.49, 8.91) cells/μl, Z=-2.14, P=0.033; percentage: 1.37%(0.78, 2.00)% vs 0.80%(0.56, 1.24)%, Z=-4.20, P<0.001], and the ratio of Th17/Treg cells was significantly higher [0.40(0.24, 0.62) vs 0.23(0.14, 0.35), Z=-4.46, P<0.001]. ④ Logistic regression analysis showed that Treg cell [ OR(95% CI)=0.934 (0.903, 0.967)] was a protective factor, while elder age [ OR(95% CI)=1.038(1.003, 1.074), male [ OR(95% CI)=2.450(1.005, 5.973)], hypertension [ OR(95% CI)=2.654 (1.219, 5.779)] and Th17 cell [ OR (95% CI)=1.066 (1.019, 1.116)] were risk factors of RA complicated with CVD. Conclusion:The level of Treg cells in peripheral blood of RA patients with CVD decreases significantly, and the immune imbalance of Th17/Treg is more singificant than that of RA patients without CVD. It is suggested that the immune imbalance and dysfunction caused by the number and/or functional deficiency of Treg cells may be involved in the occurrence and development of RA complicated with CVD.