Objective To investigate the effect of leflunomide on the superficial costimulatory molecules expression of T lymphocytes in patients with lupus nephritis ( LN ). Methods The peripheral blood mononuclear cells (PBMCs) of female active LN patients and healthy female were separated by density gradient centrifugation, and cultured by phytohemagglutinin (PHA) or leflunomide active metabolites(A771726).The CD28, CD40L, CTLA-4 and LFA-1a expressions on the peripheral blood T lymphocytes were detected by double-colored flow cytometry. The differences of the means were tested by analysis of variance( ANOVA ) and SNK q test. Results Comparing with healthy controls, there were significantly higher expressions of CD28,CD40L, LFA-1a and CTLA-4 on the peripheral blood T cells in active LN patients (CD28:33.4±6.5 vs14.4±3.2; CD40L: 13.2±3.2 vs 5.4±2.3; LFA-1a: 8.5±2.3 vs2.2±1.1; CTLA-4:4.6±1.5 all P＜0.01) as well as CD28 and CD40L expression on the peripheral blood T cells from healthy controls induced by PHA (CD28:26.8±6.7 vs14.4±3.2; CD40L: 13.9±4.9 vs 5.4±2.3 all P＜0.01 ), but not CTLA-4 and LFA-1a expression.However, CD28, CD40L, LFA-1a and CTLA-4 expressions on T cells stimulated by PHA increased in active LN patients(all P＜0.05 ). A771726 could significantly inhibit over-expression of LFA-1a and CD40L on the T cells from active LN patients (CD40L:8.2±2.0 vs13.3±3.2;LFA-1a:5.1±1.3 vs8.5±2.3 all P＜0.01 ), but not CD28 and CTLA-4 expression. A771726 did not inhibit CD28, CD40L, LFA-1a and CTLA-4 expression on the T cells in healthy controls. Furthermore, A771726 could markedly inhibit the over-expression of all of the above molecules induced by PHA on the T cells of active LN patients (all P＜0.05). Conclusion One of the major mechanisms for LEF treatment of LN is that LEF can down-regulate CD40L and LFA-1a expression but not CD28 and CTLA-4 expression on the peripheral blood T cells in active LN patients.

Objective To analyze the outcomes and prognostic factors associated with the death of systemic lupus erythematosus (SLE) patients admitted to the intensive care unit (ICU). Methods During June 1996 to June 2007, all SLE patients admitted to the ICU were included. Patients were excluded if the diagnosis of SLE was established at or after ICU admission. A multivariate logistic regression model was applied using variables that were associated with death in the univariate analysis. Results A total of 101 patients meeting the criteria were included. The mortality rate was 48.6%. The most common causes of admission was lung disorder with acute respiratory distress syndrome (ARDS). Multivariate logistic regression analysis suggested that SLICC/ACR DI>7.7 (OR=6.87), APACHE Ⅲ≥21 (OR=29.8), lung disorders with ARDS (OR =55.81 ), septic shock (OR =32.22 ), intracranial haemorrhage (OR =57.35 ), hypocytopenia (OR = 5.89), mean equivalent prednisone dose>25 mg/d (OR=7.65) and prolonged tracheal intubation (OR=5.98) were signi-ficantly associated with death. Whereas sex, age, SLEDAI >27, gastrointestinal bleeding, the cumulative dosage of CTX higher than 1.0 g, pulse intravenous methylprednisolone therapy were not associated with death. Conclusion The mortality rate of critically ill SLE patients in ICU is very high. SLICC/ACR DI> 7.7, APACHE Ⅲ≥21, lung disorders with ARDS, septic shock, intracraniai haemorrhage, average prednisone equivalent dosage higher than 25mg/d and prolonged tracheal intubation (longer than 4 days) are negative prognostic factors in SLE patients admitted to the ICU.

Objective To investigate the inlfuence of overexpression of miR-26b on the secretion of adipokines dur-ing human adipocyte differentiation. Methods Human preadipocytes were infected with the hsa-miR-26b over-expressing lentivirus and were induced to differentiate, and then the levels of adipokines (IL-6, leptin, resistin, TNF-α) at different time points during differentiation were measured by ELISA. Results Compared with control group, decreased secretions of both IL-6 and leptin, and increased secretion of resistin were found during the differentiation of human adipocytes in miR-26b overexpressed group. However, the secretion of TNF-αwas not measured in both groups. Conclusion The miR-26b can improve the inlfammation and insulin resistance of human adipocytes, which will provide potential targets for obesity treat-ment.

Objective To investigate the expression of lipid rafts (LRs) and actin cytoskeleton (F-actin) in the peripheral blood B lymphocytes of patients with systemic lupus erythematosus (SLE).Methods Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque.B lymphocytes were isolated by positive selection from PBMCs.Membrane staining for LRs was achieved with FITC-conjugated cholera toxin B (CTB).The level and distribution of LRs were studied by flow cytometry and confocal microscopy.Staining for F-actin was carried out with Rhodamine phalloidin.The expression of F-actin was analyzed by confocal microscopy.In an in vitro examination,the effect of Leflunomide on lipid rafts in B lymphocytes from SLE was analyzed.Disease carried out was measured using the SLE disease activity index (SLEDAI).Analysis of the enumerical data was performed using ANOVA or paired-samples t test.Correlation was examined by Pearson's rank correlation test.Results The number of CTB-binding lipid rafts in B cell from active SLE patients or from SLE patients in disease remission.who were treated with immunosuppressive drugs was higher than B cells from healthy controls [(59+4)％,(51±5)％,(33±4)％,F=9.21,P=0.001].Confocal microscopy revealed that in B cell from healthy controls,lipid raft was found to be small and uniformly distributed on the plasma membrane.F-actin was found mainly in the cortical region of the cells.This pattern was different from the pattern seen in B cells from patients with SLE,which presented with stronger staining and irregular large clustering of LRs,with a decrease in F-actin levels.In addition,the number of CTB-binding LRs in B cells from the active SLE patients was correlated significantly with the SLEDAI score (r=0.632,P=0.028).Furthermore,thein vitro results showed that leflunomide treatment reduced the number of CTB-binding LRs in B cell from SLE patients [(48±5)％ vs (39±5)％,t=2.29,P=0.048].Conclusion The altered expression of Lipid raft and F-actin can been seen in B lymphocytes in SLE,and modulation of LRs and F-actin expression may be a potential approach in the treatment of SLE.

Objective To investigate the effect of glucocorticoid and bisphosphonate on Hedgehog signaling pathway in human bone mesenchymal stem cells (hBMSCs) and bone tissue.Methods ① Bone biopsy test:forty cases of systemic lupus erythematosus (SLE) patients were divided into 2 groups:20 cases in newly diagnosis group,20 cases in the GCs group (the dosage of glucocorticoids was higher than 1 mg·kg-1·d-1).Patients in the GCs group was randomly divided into 2 groups:10 cases in the control group were without anti-osteoporosis treatment,10 cases in treatment group received alendronate 70 mg once a week.All of the patients had bone marrow puncture after24 weeks,and the value of average optical density of Gli1 was tested with immunohistochemistry.② Cell culture:hBMSCs cultured in normal medium were intervened with rh-SHHN and methylprednisolone (10-3 mol/L,10-5 mol/L) after successfully identified.Quantitative polymerase chain reaction (PCR) was used to detectthe mRNA expression of Gli1.The final calcium nodules was detected by Alizarin red staining.hBMSCs cultured in osteogenesis medium were intervened with bisphosphonate and methylprednisolone (10-3 mol/L) after successfully identified.Quantitative PCR was used to detect the expression of Gli1 mRNA.Alizarin red staining was used to detect the final calcium nodules.Comparisons between the two groups were carried out using t-test while the difference analysis of multi-groups were tested by factorial analysis.Results The average optical density of Gli1 in the GCs group (47±7) was less than the newly diagnosed group (61±12) (t=4.442,P＜0.01),and it was less in the control group (51±6) than in the treatment group (42±6) (t=3.701,P=0.002).When normally cultured,high and moderate concentration of methylprednisolone suppressed the mRNA (0.38±0.04; 0.68±0.24) (F=8.748,P＜0.01) expression of Gli1 which was initially stimulated by rh-SHHN (2.01 ±0.38).And the final calcium nodules in groups which contained methylprednisolone were much less than rh-SHHN only group.When hBMSCs were cultured in osteogenesis medium,compared with the methylprednisolone group (0.024±0.011),the expression of Gli1mRNA(0.034-0.006) (t=7.62,P＜0.01) and the final calcium nodules were significantly improved by bisphosphonate.Conclusion High and moderate doses of glucocorticoids inhibit hBMSC osteogenesis by inhibiting Gli1.Low concentration of alendronate can not only stimulate hBMSC osteogenesis differentiation but also can remit the inhibition effects of GCs through the way of Hedgehog.

In order to study the role of the bone marrow-derived mesenchymal stem cells(MSCs)transplanted with or without bone marrow(BM)in the treatment of lupus mice and the effect of MSCs in the onset of systemic lupus erythematosus(SLE).Method Twenty 12-week-old female MRL/lpr mice were randomly divided into four groups,including simple bone marrow transplantation group(SG,BM 1×107),united group-1(UG1,BM 1×107+MSCs 1×104),united group-2(UG2,BM 1×107+MSCs 1×106),the positive control group(PG,no transplantation).BALB/c mice were used as the negative control group(NG,no transplantation).MSCs which were amplified from the bone marrow of male BALB/c mice in vitro were transplanted into the female MRL/lpr mice with or without BM.One month later Y chromosome was detected to confirm if the transplantation was successful or not.The change of weight, white blood cells,urine protein,anti-dsDNA antibody,the pathology and immunofluorescence of renal were observed to evaluate the therapeutic efficacy.Results Y chromosome was detected in all transplanted female mice.Compared with PG,urine protein concentration in SG,UG1 and UG2 significantly decreased 30 days after transplantation(P<0.05).40 days after transplantation,the rite of anti-dsDNA antibodies in sG(0.91±0.27)was still higher than NG,which OD value wag 0.47 s0.10(P<0.05),but there was no statistical difference among UG1(0.76±0.28),uG2(0.73±0.10)and NG(P>0.05).However,50 days after transplantation,there was no marked difference of the tite of anti-dsDNA antibodies in SG(0.55±0.15),UG1(0.57±0.14)and UC2(0.58±0.05)compared with NG(P>0.05).After transplantation there was no vasculitis.no inflammatory cell infiltration in matrix and no obvious intercapillary cells proliferation in the kidney.The immunofluoroscence became negative or weakly positive.Conclusion MSCs transplantation with or without BM Can both improve the pathogenetic condition of MRL/lpr mice.MSCs can accelerate the clearance of anti-dsDNA antibody and promote the restoration of injured organs.We presume that MSCs are important immunological regulation cells in SLE.

Objective To explore the application value of magnetic resonance molecular functional imaging diffusion weighted imaging(DWI) in the identification of pancreatic carcinoma and mass-type pancreatitis of animal model.Methods Each 8 cases of laboratory pancreatic head transplantation tumor model,chronic mass-type pancreatitis model and normal rabbits were selected and performed the MR DWI molecular functional imaging,the b values were 333,667,1 000 s/mm2 respectively.The apparent diffusion coefficients(ADC) of pancreatic carcinoma model,mass-type pancreatitis model and normal pancreas under different b values were observed.Then the change situation of ADC values of pancreatic carcinoma model,mass-type pancreatitis model and normal pancreas under different b values and difference of ADC(DADC) was analyzed.Moreover the differences in molecular diffusion,tissue perfusion among various groups were observed.Results Throughout the study period,the mortality rate of pancreatic head transplantation tumor model was 50%;the mass-type pancreatitis model and 8 normal rabbits were normally survival.The ADC value of pancreatic carcinoma under the same b value was significantly lower than that of chronic inflammation and normal pancreas area.The ADC value in each group was decreased with the increase of b value,and there was significant difference in ADC value when the b value was 333 s/mm2(F=6.662,P=0.014),in the pairwise comparison among groups,the difference between pancreatic cancer and pancreatitis (t=6.773,P=0.003) and between pancreatic cancer and normal pancreas(t=5.883,P=0.016) had statistical significance (P<0.05).The b value was increased,DADC was smaller,the difference change of DADC between pancreatic cancer area and chronic pancreatitis mass area,between pancreatic cancer area and normal pancreatic head area had statistical significance (P<0.05).Conclusion Rationally selecting the molecular functional imaging DWI technology of b value can better distinguish pancreatic cancer from mass-type pancreatitis,which may be promoted and applied in the evaluation of animal pancreatic head cancer model.

Objective:To explore the risk factors for 30-day death in emergency department patients, and then construct a prediction model and validate it using nomogram.Methods:A retrospective cohort study was conducted. The clinical data of 1 091 patients admitted to the emergency department of the First People's Hospital of Changde from January 1 to June 30, 2021 was collected, including 741 patients from January 1 to March 31 in the development group and 350 patients from April 1 to June 30 in the validation group. General information, first vital signs admitted to the emergency department, and laboratory results were collected, the modified early warning score (MEWS) was calculated, and 30-day outcomes were recorded. Univariate and multivariate Logistic regression analysis was used to screen out the risk factors of 30-day death. According to the results of multivariate analysis, the nomogram was used to construct a 30-day death prediction model. The receiver operator characteristic curve (ROC curve) was used to evaluate the consistency of the prediction model, the calibration of the prediction model was evaluated by the Hosmer-Lemeshow goodness of fit test.Results:A total of 1 091 patients were enrolled. There were 741 patients in the development group, including 356 males and 385 females, aged (51.42±17.33) years old, and the 30-day mortality was 28.88%. There were 350 patients in the validation group, including 188 males and 162 females, aged (52.88±16.11) years old, and the 30-day mortality was 24.00%. The results of the univariate analysis showed that age, primary diagnosis on admission, consciousness, respiratory rate (RR), systolic blood pressure (SBP), heart rate (HR), pulse oxygen saturation (SpO 2), MEWS score, erythrocyte sedimentation rate (ESR), procalcitonin (PCT) and body mass index (BMI) might be the risk factors for 30-day death in patients in the emergency department. The results of the multivariate analysis showed that the MEWS score [odds ratio ( OR) = 14.22, 95% confidence interval (95% CI) was 1.46-138.12], ESR ( OR = 46.71, 95% CI was 20.48-106.53), PCT ( OR = 4.97, 95% CI was 2.46-10.02), BMI (24.0-27.9 kg/m 2: OR = 37.82, 95% CI was 14.69-97.36; ≥28.0 kg/m 2: OR = 62.11, 95% CI was 25.77-149.72) were independent risk factors for 30-day death in the emergency department (all P < 0.05). Using the four variables with the results of multivariate analysis to construct a nomogram prediction model, the area under the ROC curve (AUC) was 0.974 (95% CI was 0.753-0.983) for the development group, and the AUC was 0.963 (95% CI was 0.740-0.975) for the validation group. The Hosmer-Lemeshow test showed no statistically significant difference between the predicted outcome of the nomogram prediction model and the actual occurrence ( χ2 = 1.216, P = 1.270). Conclusion:The prediction model developed by the MEWS score combined with BMI, ESR and PCT can scientifically and effectively predict the 30-day outcome of emergency department patients.

Objective:To explore the changes of disease structure in a tertiary general hospital during the COVID-19 pandemic.Methods:A database of 783 diagnosis-related groups(DRG) patients in a tertiary general hospital from 2017 to 2020 was used. The rank sum test was used to compare the number of patients among different years, and the Chi-square test was used to compare the composition of patients among different years. With the patient composition ratio as the main index, the thermal cluster analysis was used to analyze the changes of disease structure during the COVID-19 pandemic, from the perspectives of major diagnostic categories(MDC) and the key DRG(the number of patients in any year more than 2 000)respectively. All analyses were performed in R software, with P<0.05 indicating significance. Results:There were significant differences in the number and composition of patients in MDC groups and key DRG groups among different years( P<0.05). The results of thermal clustering analysis showed that the MDC composition of patients in 2020 was significantly different from those in 2017 to 2019; the 26 MDC groups were classified into four main categories. The results of thermal clustering analysis also showed that the DRG composition of patients in 2020 were significantly different from those in 2017 to 2019; The RU14 group and the other 19 key DRG groups were classified into different groups; and the other 19 key DRG groups except RU14 were classified into five main categories. Conclusions:The disease structure of tertiary general hospitals has changed significantly during the COVID-19 pandemic.

Objective To investigate the effects and its mechanisms of bradykinin B2 receptor (B2R) on the growth and function of human extravillous trophoblast cells (HTR-8/SVneo cells).Methods B2R expression plasmid (pcDNA3.1-B2R) was constructed and B2R-specific small interfering RNA (siRNA) was synthesized.HTR-8/SVneo cells were divided into four groups and transfected with pcDNA-3.1 (blank plasmid group),pcDNA3.1-B2R (B2R expression plasmid group),siRNA negative control and B2R-specific siRNA,respectively.Quantitative real-time reverse transcription-polymerase chain reaction and Western blot were used to detect the changes in the expression of B2R,matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A at both mRNA and protein levels in HTR-8/SVneo cells.Cell counting kit-8 and flow cytometry were used to detect cell activity and cell cycle,respectively.Cell migration assay and cell invasion assay were used to detect cell migration and invasion,respectively.Tube formation assay was used to evaluate the tube formation abilities of HTR-8/SVneo cells.All data were analyzed with t test.Results (1) Compared with the blank plasmid group,expression of B2R in HTR-8/SVneo cells in the B2R expression plasmid group were significantly increased at both mRNA (5.06±0.49 vs 1.00±0.28,t=7.226,P=0.002) and protein levels (1.34 ± 0.07 vs 1.00± 0.05,t=3.727,P=0.006).And the expression of B2R in HTR 8/SVneo cells transfected with B2R-specific siRNA were significantly reduced at both mRNA (0.34±0.05 vs 1.00±0.17,t=3.667,P=0.021) and protein levels (0.74±0.03 vs 1.00±0.05,t=4.097,P=0.006) comparing with the siRNA negative control group.(2) Compared with the blank plasmid group,HTR-8/SVneo cells being transfected with B2R expression plasmid showed a higher proliferation activity (1.50 ±0.03 vs 1.34± 0.04) promoting G0/G1 to S phase transition;compared with the siRNA negative control group,B2R-specific siRNA inhibited the proliferation of HTR-8/SVneo cells (1.06 ± 0.04 vs 1.20± 0.02) and arrested the cell cycle at G0/G 1 phase (all P＜0.05).(3) Compared with the blank plasmid group,B2R expression plasmid significantly increased the HTR-8/SVneo cell migration distance [(80.67±0.33) vs (41.33±5.24) μm],the number of cells penetrating matrigel gel (360.70 ±12.33 vs 268.70 ±14.45) and the number of cells having tube-like structures (28.20 ± 2.47 vs 14.00± 1.67),while significantly decrease was shown in these three parameters in B2R-specific siRNA group comparing with the siRNA negative control group [HTR-8/SVneo cell migration distance:(56.00±3.51) vs (87.00±1.53) μ m,number of cells penetrating matrigel gel:143.30± 12.91 vs 252.30± 17.07;number of tube-like structures:6.25±1.49 vs 15.75 ±2.02;all P＜0.05].(4) Expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 at mRNA level,and expression of cyclin D1 and vascular endothelial growth factor-A increased in the B2R expression plasmid group than in the blank plasmid group,and decreased in the B2R-specific siRNA group than in the siRNA negative control group at both mRNA and protein levels (all P＜0.05).Conclusions B2R might enhance the activity,migration,invasion and tube formation ability of human extravillous trophoblast cells through promoting the expression of matrix metalloproteinase-2,matrix metalloproteinase-9,cyclin D1 and vascular endothelial growth factor-A.