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Objective:To investigate the effect of rapamycin on the proliferation and apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) and its mechanism.Methods:Synovial tissues were collected from patients with RA during joint replacement surgery, and primary synovial fibroblasts were extracted by trypsin digestion. The effect of rapamycin on the proliferation of RA-FLS was detected by cell counting kit (CCK-8) method. RA-FLS were divided into the control group and the rapamycin group (10 nmol/L). The effect of rapamycin on apoptosis of RA-FLS cells was detected by flow cytometry. The mRNA expres-sion levels of mammalian target of rapamycin (mTOR), serine/threonine-protein kinase AKT, B lymphocy-toma-2 (Bcl-2) associated X gene (Bax) and Bcl-2 were detected by RT-PCR. The protein expression levels of Bax, Bcl-2, mTOR, p-mTOR (2448), AKT, p-AKT and mTORC1 downstream related molecules protein S6 kinase 1(S6K1), p-S6K1, eukaryotic translation initiation factor-binding protein 1 (4EBP1) and p-4EBP1 were detected by Western blot. Differences between the two groups were compared using two independent samples t-test. Results:The results showed that the proliferation efficiency of RA-FLS treated with rapamycin was significantly weaker than that of the control group, and the drug inhibition rate of rapamycin increased with the increase of rapamycin concentration. The apoptosis rate of rapamycin group was significantly higher than that of the control group (5.31±0.59)% vs (3.49±0.40)%, t=7.83, P=0.001). The expression of Bax mRNA in rapamycin group was significantly increased (1.35±0.04 vs 1.00±0.00, t=15.60, P=0.004), while the expression of Bcl-2 mRNA (0.790±0.003 vs 1.000±0.000, t=85.50, P=0.007), mTOR mRNA (0.41±0.08 vs 1.00±0.00, t=14.37, P=0.044) and AKT mRNA (0.59±0.08 vs 1.00±0.00, t=7.54, P=0.017) were decreased, and the differences were statistically significant when compared with the control group. Compared with the control group, the protein expression of Bax in rapamycin group was significantly increased (0.75±0.10 vs 0.48±0.09, t=4.04, P=0.007), and the expression levels of Bcl-2 (0.632±0.055 vs 0.758±0.020, t=7.35, P=0.002), p-AKT/AKT(0.61±0.07 vs 0.88±0.04, t=5.61, P=0.005), p-mTOR/mTOR(0.92±0.12 vs 1.28±0.09, t=5.05, P=0.002), p-S6K1/S6K1(0.884±0.020 vs 1.023±0.058, t=4.52, P=0.004) and p-4EBP1/4EBP1 were decreased(0.86±0.05 vs 1.11±0.05, t=6.00, P=0.004). Conclusion:Rapamycin may inhibit the proliferation and induce apoptosis of RA-FLS cells by inhibiting AKT/mTORC1 pathway.
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Objective:To explore the link between the differentially expressed long non-coding RNAs (lncRNAs) and the number of regulatory T cells (Tregs) by detecting the lncRNAs expression profiles in patients with systemic lupus erythematosus (SLE), then analyze the correlation between Tregs and lncRNAs and the clinical features of SLE patients. We also predict the mechanism by which lncRNAs regulate the differentiation and development of Tregs, and provid new approach for the treatment of SLE.Methods:Peripheral blood of 9 active SLE patients was collected and mononuclear cells (PBMCs) were extracted. The lncRNAs expression profiles of PBMCs was analyzed by whole transcriptome sequencing. Nine healthy people served as controls to screen the differentially expressed lncRNAs, and to analyze the correlation between lncRNAs and Tregs number. Pearson test was used to analyze the correlation between lncRNA and the number of Tregs, and the correlation between Treg-associated lncRNAs and systemic lupus erythematosus disease activity index(SLEDAI) score, erythrocyte sedimentation rate (ESR), C3, C4 in SLE patients. The targeted genes of Treg asso-ciated lncRNAs were predicted with miRcode and Targetscan databases and co-expression network.Results:There were 240 differentially expressed lncRNAs in SLE patients compared with healthy controls, including 134 highly expressed lncRNAs ( P<0.05) and 106 low expressed lncRNAs ( P<0.05). The expression of ANKRD44-AS1 ( r=0.74, P=0.022), LINC00200 ( r=0.70, P=0.037), AP001363.2 ( r=0.78, P=0.014) and LINC02824 (r=0.79, P=0.011) were positively correlated with the number of Tregs, and the expression of AP000640.1 ( r=-0.72, P=0.028), AC124248.1 ( r=-0.77, P=0.016), LINC00482 ( r=-0.83, P=0.005) and MIR503HG ( r=-0.96, P<0.001) were negatively correlated with the number of Tregs. Among these eight Tregs associated lncRNAs, the expression of LINC00482 ( r=-0.73, P<0.001) and MIR503HG ( r=-0.76, P<0.001) were negatively correlated with C3. LINC00200, ANKRD44-AS1 and AP000640.1 related to Tregs regulated the expression of STAT5, PLD1, HOPX and RUNX3 through competitively binding of miRNA or transregulatory mechanism, thereby regulating the differentiation and development of Tregs. Conclusion:The lncRNAs expression profiles are changed in SLE patients, the differentially expressed lncRNAs are associated with abnormal number and function of Tregs in SLE patients, and Treg associated lncRNAs are associated with SLE disease activity, which may affect the expression of STAT5, PLD1, HOPX, RUNX3 and regulate Tregs function and participate in the pathogenesis and progression of SLE by competitively binding to miRNAs or trans-regulatory mechanism.
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AIM: To guide the multiple imputation of missing data in clinical longitudinal studies and its sensitivity analyses, and highlight the importance of sensitivity analyses by taking the clinical trial of Qizhitongluo Capsule in treating ischemic stroke as an example. METHODS: To implement PROC MI process in SAS to perform multiple imputation and its sensitivity analysis. RESULTS: In the example, after multiple imputation, improvements in lower limb motor scores of the Qizhitongluo group were greater than those of the placebo group (all P<0.01), and the results of two sensitivity analyses under "missing not at random" were consistent with those under "missing at random". CONCLUSION: Multiple imputations combined with sensitivity analyses can ensure a robust result. It is recommended that clinical researchers perform sensitivity analyses after filling missing data.
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To study the effect of Tibetan medicine Zuotai on the activity, protein and mRNA expression of CYP1A2 and NAT2, three different doses (1.2, 3.8 and 12 mg x kg(-1)) of Zuotai were administrated orally to rats once a day or once daily for twelve days, separately. Rats were administrated orally caffeine (CF) on the second day after Zuotai administration, and the urine concentration of CF metabolite 5-acetylamino-6-formylamino-3-methyl-uracil (AFMU), 1-methyluric acid (1U), 1-methylxanthine (1X), 1, 7-dimethylxanthine (17U) at 5 h after study drug administration was determined by RP-HPLC. The activity of CYP1A2 and NAT2 was evaluated by the ratio of metabolites (AFMU+1X+1U)/17U and the ratio of AFMU/(AFMU+1X+1U), respectively. The protein and mRNA expression of CYP1A2 and NAT2 were determined by ELISA and RT-PCR method, respectively. After single administration of Zuotai 3.8 mg x kg(-1) and repeated administration of Zuotai 3.8 and 12 mg x kg(-1), the activity of CYP1A2 and NAT2 decreased significantly compared with control group and there was no significant difference between other dose group and control group. The protein expression of CYP1A2 was significant lower than that in control group after repeated administration of Zuotai 12 mg x kg(-1), and the mRNA expression of CYP1A2 decreased significantly compared with that of control group after single administration of Zuotai 3.8 mg x kg(-1) and repeated admistration of Zuotai 12 mg x kg(-1), separately. The protein expression of NAT2 decreased significantly compared with that of control group after single and repeated administration of Zuotai 3.8 mg x kg(-1), respectively, and the mRNA expression of CYP1A2 decreased significantly compared with control group after single administration of Zuotai 3.8 mg x kg(-1). This study found that Tibetan medicine Zuotai had significant effect on the activity, protein and mRNA expression of CYP1A2 and NAT2.
Subject(s)
Animals , Female , Male , Rats , Administration, Oral , Arylamine N-Acetyltransferase , Genetics , Metabolism , Caffeine , Metabolism , Urine , Cytochrome P-450 CYP1A2 , Genetics , Metabolism , Dose-Response Relationship, Drug , Drugs, Chinese Herbal , Pharmacology , Medicine, Tibetan Traditional , RNA, Messenger , Metabolism , Rats, Sprague-Dawley , Theophylline , Urine , Uracil , Urine , Uric Acid , Urine , Xanthines , UrineABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression.</p><p><b>METHODS</b>Affymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data.</p><p><b>RESULTS</b>Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression.</p><p><b>CONCLUSIONS</b>HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.</p>
Subject(s)
Humans , Carcinoma, Transitional Cell , Genetics , Pathology , Cell Cycle Proteins , Metabolism , Cell Line, Tumor , Cyclin D1 , Metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Physiology , Nuclear Proteins , Metabolism , Protein Serine-Threonine Kinases , Metabolism , Proteins , Genetics , Physiology , Urinary Bladder Neoplasms , Genetics , PathologyABSTRACT
<p><b>BACKGROUND</b>Periodontal disease is closely related to type 2 diabetes and is an important complication of diabetes. This study aimed to investigate the effect of periodontal treatment on levels of blood glucose (Glu) and glycosylated hemoglobin (HbA1c) among elderly patients with type 2 diabetes and periodontal disease.</p><p><b>METHODS</b>A total of 107 elderly patients with type 2 diabetes and periodontal disease were selected and divided into two groups according to their HbA1c levels. Group A was a well-controlled diabetic group and group B was uncontrolled. Their probing depth (PD), attachment loss (AL), the value of Glu and HbA1c were analyzed before periodontal treatment and 4 months later.</p><p><b>RESULTS</b>There was a significant difference in periodontal condition between groups A and B (P < 0.01). The periodontal condition for both groups was significantly (P < 0.01) improved after periodontal therapy. The effect of treatment in group A was more pronounced than group B, and the difference was significant (P < 0.01). After the periodontal treatment, Glu and HbA1c were reduced significantly in both groups (P < 0.05).</p><p><b>CONCLUSIONS</b>Periodontal condition is related to the control of Glu level among elderly patients with type 2 diabetes and periodontal disease. Periodontal treatment can effectively reduce the level of Glu and HbA1c as well as improve the periodontal condition in elderly type-2 diabetes patients with periodontal disease.</p>