HNRNPC promotes collagen fiber alignment and immune evasion in breast cancer via activation of the VIRMA-mediated TFAP2A/DDR1 axis.

BACKGROUND: Cancers aggressively reorganize collagen in their microenvironment, leading to the evasion of tumor cells from immune surveillance. However, the biological significance and molecular mechanism of collagen alignment in breast cancer (BC) have not been well established. METHODS: In this study, BC-related RNA-Seq data were obtained from the TCGA database to analyze the correlation between DDR1 and immune cells. Mouse BC cells EO771 were selected for in vitro validation, and dual-luciferase experiments were conducted to examine the effect of TFAP2A on DDR1 promoter transcription activity. ChIP experiments were performed to assess TFAP2A enrichment on the DDR1 promoter, while Me-RIP experiments were conducted to detect TFAP2A mRNA m6A modification levels, and PAR-CLIP experiments were conducted to determine VIRMA's binding to TFAP2A mRNA and RIP experiments to investigate HNRNPC's recognition of m6A modification on TFAP2A mRNA. Additionally, an in vivo mouse BC transplant model and the micro-physiological system was constructed for validation, and Masson staining was used to assess collagen fiber arrangement. Immunohistochemistry was conducted to identify the number of CD8-positive cells in mouse BC tumors and Collagen IV content in ECM, while CD8 + T cell migration experiments were performed to measure CD8 + T cell migration. RESULTS: Bioinformatics analysis showed that DDR1 was highly expressed in BC and negatively correlated with the proportion of anti-tumor immune cell infiltration. In vitro cell experiments indicated that VIRMA, HNRNPC, TFAP2A, and DDR1 were highly expressed in BC cells. In addition, HNRNPC promoted TFAP2A expression and, therefore, DDR1 transcription by recognizing the m6A modification of TFAP2A mRNA by VIRMA. In vivo animal experiments further confirmed that VIRMA and HNRNPC enhanced the TFAP2A/DDR1 axis, promoting collagen fiber alignment, reducing anti-tumor immune cell infiltration, and promoting immune escape in BC. CONCLUSION: This study demonstrated that HNRNPC promoted DDR1 transcription by recognizing VIRMA-unveiled m6A modification of TFAP2A mRNA, which enhanced collagen fiber alignment and ultimately resulted in the reduction of anti-tumor immune cell infiltration and promotion of immune escape in BC.

Gut microbiota and thyroid-associated ophthalmopathy. / è‚ é“èŒç¾¤ä¸Žç”²çŠ¶è ºç›¸å ³çœ¼ç— .

Thyroid-associated ophthalmopathy (TAO) is a multifactorial-mediated autoimmune orbital disease with the highest incidence of orbital disease in adults. Due to the complex clinical manifestations and prolonged course,TAO seriously affect the physical and mental health of patients.The pathogenesis of TAO has not been fully elucidated and the treatment lacks specificity. Therefore, in-depth research on the pathogenesis of TAO is to find effective treatments. In recent years, studies have suggested that there is gut microbiota disorder in TAO, and the risk factors of TAO can promote gut microbiota disorder. Disordered gut microbiota can participate in the occurrence and development of TAO via influencing T cell differentiation, mimicking autoantigens, and influencing host non-coding RNA expression. Modulating the gut microbiota also has therapeutic effects on TAO and is a promising therapeutic approach.

Research progress in Th17 cells and the relevant cytokines in Graves' ophthalmopathy. / Th17ç»†èƒžåŠå ¶ç›¸å ³å› å­åœ¨Gravesçœ¼ç— ä¸­çš„ç ”ç©¶è¿›å±•.

Graves' ophthalmopathy is the most common clinical orbital disease, and T helper (Th) cells play an important role in the development of Graves' ophthalmopathy. Th17 cells are a major subpopulation of Th cells and abnormally highly expressed in patients with Graves' ophthalmopathy. Th17 cells and the related cytokines interleukin (IL)-17A, IL-21 and IL-23 are involved in regulating the inflammatory response, fibrosis and adipogenesis. Th17 cells are unstable and exhibit a degree of plasticity, and they can differentiate into IL-17A and interferon (IFN)-γ dual-producing Th17.1 cells, which exacerbate the pathogenicity of Th17 cells. In addition, Th17 cells and the relevant factors are strongly associated with disease activity and severity in Graves' ophthalmopathy.

Single­cell RNA­seq necroptosis­related genes predict the prognosis of breast cancer and affect the differentiation of CD4+ T cells in tumor immune microenvironment.

Breast cancer (BC) is one of the most prevalent types of malignancy and a major cause of cancer-related death. The purpose of the present study was to identify prognostic models of necroptosis-related genes (NRGs) in BC at the single-cell RNA-sequencing level and reveal the role of NRGs in tumour immune microenvironment (TIME). A risk model was constructed based on Cox regression and LASSO methods. Next, high-scoring cell populations were searched through AUCell scores, and cell subtypes were then analyzed by pseudotime analysis. Finally, the expression level of the model genes was verified by reverse transcription-quantitative (RT-qPCR). A new prognostic model was constructed and validated based on five NRGs (BCL2, BIRC3, AIFM1, IFNG and VDAC1), which could effectively predict the prognosis of patients with BC. NRGs were found to be highly active in CD4+ T cells and differentially expressed in their developmental trajectories. Finally, the RT-qPCR results showed that most of the model genes were significantly overexpressed in MDA-MB-231 and MCF-7 cells (P<0.05). In conclusion, an NRG signature with excellent predictive properties in prognosis and TIME was successfully established. Moreover, NRGs were involved in the differentiation and development of CD4+ T cells in TIME. These findings provide potential therapeutic strategies for BC.

Investigating the impact of elevated urinary trace elements on non-alcoholic fatty liver disease using vibration-controlled transient elastography.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is a global public health concern. However, limited data are available on urinary trace elements and NAFLD caused by various exposure factors. This study aimed to investigate the relationship between the presence of 16 trace elements in urine and NAFLD using data from the National Health and Nutrition Examination Survey (NHANES). Methods: By utilizing the NHANES data from 2017 to 2018, 1613 participants who fulfilled the research criteria were identified from the initial pool of 2979 participants with available urine trace element detection data. Among them, 706 individuals had been diagnosed with NAFLD based on a coefficient of attenuation parameter (CAP) value of at least 274 db/m, determined using vibration-controlled transient elastography (VCTE); whereas the remaining 907 participants were classified as non-NAFLD. The data obtained were used to construct univariate and multivariate logistic regression models and restricted cubic spline models (RCS) analyses. Results: The presence of arsenic, iodine, barium, cesium, molybdenum, lead, tin, and tungsten in the urine of individuals with NAFLD showed a positive correlation with the likelihood of developing NAFLD. The risk of NAFLD had a non-linear dose-dependent relationship with urinary iodine, molybdenum, barium, and cesium. NAFLD was also associated with elevated levels of barium and cesium in urine, which were identified as significant risk factors. Conclusion: These findings suggest a positive association between exposure to trace elements in the urine and the risk of NAFLD. Specifically, urinary barium and cesium appeared to have the greatest impact on the risk of NAFLD. These results provide novel insights into the diagnosis and treatment of NAFLD.

Shared genetic architecture between hypothyroidism and rheumatoid arthritis: A large-scale cross-trait analysis.

BACKGROUND: In recent years, mounting evidence has indicated a co-morbid relationship between hypothyroidism and rheumatoid arthritis (RA), however, the shared genetic factors underlying this association remain unclear. This study aims to investigate the common genetic architecture between hypothyroidism and RA. METHODS: Genome-wide association study (GWAS) summary statistics from recently published studies were utilized to examine the genetic correlation, shared genetic loci, and potential causal relationship between hypothyroidism and RA. Statistical methods included linkage disequilibrium score regression (LDSC), high-definition likelihood (HDL), cross-trait meta-analyses, colocalization analysis, multi-marker analysis of genomic annotation (MAGMA), tissue-specific enrichment analysis (TSEA), functional enrichment analysis, and latent causal variable method (LCV). RESULTS: Our study demonstrated a significant genetic correlation between hypothyroidism and RA(LDSC:rg=0.3803,p=7.23e-11;HDL:rg=0.3849,p=1.02e-21). Through cross-trait meta-analysis, we identified 1035 loci, including 43 novel genetic loci. By integrating colocalization analysis and the MAGMA algorithm, we found a substantial number of genes, such as PTPN22, TYK2, and CTLA-4, shared between the two diseases, which showed significant enrichment across 14 tissues. These genes were primarily associated with the regulation of alpha-beta T cell proliferation, positive regulation of T cell activation, positive regulation of leukocyte cell-cell adhesion, T cell receptor signaling pathway, and JAK-STAT signaling pathway. However, our study did not reveal a significant causal association between the two diseases using the LCV approach. CONCLUSION: Based on these findings, there is a significant genetic correlation between hypothyroidism and RA, suggesting a shared genetic basis for these conditions.

Collagen-induced DDR1 upregulates CXCL5 to promote neutrophil extracellular traps formation and Treg infiltration in breast cancer.

Neutrophil extracellular traps (NETs) have been implicated in many cancers, but the regulatory mechanisms in the context of breast cancer have not been thoroughly discussed. This study proposed a mechanism based on collagen-activated DDR1/CXCL5 for NET formation in breast cancer. Through TCGA and GEO-based bioinformatics analysis, we examined the DDR1 expression and the correlation of CXCL5 with immune cell infiltration in breast cancer. It was found that high DDR1 expression was correlated with poor prognosis of patients with breast cancer, and CXCL5 was positively correlated with neutrophil and Treg infiltration. Expression of DDR1 and CXCL5 was determined in collagen-treated breast cancer cells, the malignant phenotypes of which were evaluated by ectopic expression and knockdown methods. Collagen-activated DDR1 upregulated CXCL5 expression, resulting in augmented malignant phenotypes of breast cancer cells in vitro. The formation of NETs caused promotion in the differentiation and immune infiltration of Tregs in breast cancer. A in situ breast cancer mouse model was constructed, where NET formation and lung metastasis of breast cancer cells were observed. The differentiation of CD4+ T cells isolated from the mouse model was induced into Tregs, followed by Treg infiltration assessment. It was further confirmed in vivo that DDR1/CXCL5 induced the formation of NETs to promote immune infiltration of Tregs, driving tumor growth and metastasis. Accordingly, our results provided new mechanistic insights for an understanding of the role of collagen-mediated DDR1/CXCL5 in formation of NETs and Treg infiltration, revealing potential targets for therapeutic intervention of breast cancer.

Looking Beyond Th17 Cells: A Role for Th17.1 Cells in Thyroid-associated Ophthalmopathy?

Thyroid-associated ophthalmopathy (TAO), an ordinary extrathyroid syndrome of Graves' disease (GD), is closely associated with immunity. T helper (Th) 17, Th1, and Th2 cells in Th lineages are thought to be related to the disease pathogenesis. Recently, there has been growing evidence that Th17.1 cells are involved in the development and progression of TAO. The characteristics of this pathology are similar to those of Th1 and Th17 lymphocytes, which secrete interferon (IFN)-γ and interleukin (IL)-17A. This paper reviews the potential role of the Th17.1 subgroup pathogenesis of TAO. The therapeutic effects of drugs that can modulate Th17.1 cell populations are also highlighted. Rich Th17.1 cells exist in peripheral blood and ocular tissues of patients suffering from thyroid eye disease (TED), especially those with severe or steroid-resistant TAO. The bias of Th17.1 cells to secrete cytokines partly determines the pathological outcome of TAO patients. Th17.1 cells are important in regulating fibrosis, adipocyte differentiation, and hyaluronic acid production. In summary, the Th17.1 subpopulation is essential in the onset and progression of TED, and targeting Th17.1 cell therapy may be a promising therapeutic approach.

Association of overweight, obesity and risk of urinary incontinence in middle-aged and older women: a meta epidemiology study.

Aims: The aim of this meta-analysis is to evaluate the potential correlation between obesity and overweight, and the vulnerability to urinary incontinence (UI) in women aged middle-aged and above. Methods: We searched PubMed, Cochrane Library, and Embase for observational studies published between the inception of the databases and April 25, 2023. A fixed-effects model was used when the P>0.1 and the I2 ≤ 50%. In cases where I2 ≥ 50% (indicating significant heterogeneity), a random-effects model was applied. For the purpose of evaluating publication bias, a funnel plot and Egger's test were used. Stata 14.0 was used for all statistical analyses. Findings: This meta-analysis includes 16 observational studies, covering29,618 individuals. The pooled analysis shows that being overweight(25 kg/m2≤BMI<30kg/m2) in middle-aged and elderly women is more likely to develop UI (OR=1.27; 95% CI: 1.17-1.37; I2 = 51.8%, P=0.013). Middle-aged and elderly women with obesity(30 kg/m2≤BMI<35 kg/m2) are significantly more likely to develop UI (OR=1.60; 95% CI: 1.42-1.81; I2 = 71.8%, P=0.000). In addition, the results indicated a higher probability of UI in middle-aged and older women with obesity class II (BMI≥35 kg/m2) (OR=1.85; 95% CI: 1.59-2.16; I2 = 48.1%, P=0.103). In subgroup analysis, there is no direct relationship between the obesity in middle-aged and elderly women and an increased risk of stress urinary incontinence (SUI) (OR=1.31; 95% CI: 0.99-1.74; I2 = 63.7%, P=0.011). In middle-aged and elderly women with obesity are more likely to develop urgent urinary incontinence (UUI) (OR=2.11; 95% CI: 1.54-2.89; I2 = 80.2%, P=0.000). Conclusion: In this meta-analysis, overweight and obesity are associated with an increased risk of UI in middle-aged and elderly women. Obesity and overweight are independent risk factors for UI, as demonstrated by this study. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42023421986.

Biejiajian Pill Ameliorates Diabetes-Associated Atherosclerosis through Inhibition of the NLRP3 Inflammasome.

Objective: To research the efficacy of Biejiajian pill (BJJ) on diabetes-associated atherosclerosis and explore its subsequent mechanisms. Methods: Diabetes-associated atherosclerosis (AS) was established in apolipoprotein E knockout (ApoE -/- ) mice using high-fat diet and streptozotocin. Atorvastatin (ATV, 10 mg/kg/day) or BJJ-L (BJJ low-dose, 0.9 g/kg/day), BJJ-M (BJJ medium-dose, 1.8 g/kg/day), and BJJ-H (BJJ high-dose, 3.6 g/kg/day) were administered to diabetic ApoE -/- mice for 12 continuous weeks. The normal control group consisted of 10 male C57BL/6J mice. Atherosclerosis plaques, vascular endothelial function, fasting blood glucose, lipid metabolism, inflammatory factors, NLRP3 inflammasome expression, and mitochondria and autophagy changes were evaluated. Results: The atherosclerotic lesions areas in the aortas were analyzed through Oil Red O and H&E staining, and they were reduced in the BJJ-H and BJJ-M groups. In the BJJ group, endothelin-1 (ET-1) levels were decreased, whereas endothelial nitric oxide synthase (eNOS) was increased. Fasting blood glucose levels in the BJJ and ATV groups were gradually decreased. Lipid metabolism parameters such as TG, TC, and LDL-C were reduced, while HDL-C was elevated in BJJ groups. The serum IL-1ß and IL-18 were decreased under BJJ therapy. The aortic mRNA and protein expressions of NF-κB, TXNIP, NLRP3, ASC, caspase-1, and IL-1ß were inhibited in BJJ-H and BJJ-M groups, especially in the BJJ-H group. Electron microscopy revealed an increase in autophagy in each treatment group. Conclusions: The findings reveal that BJJ could alleviate diabetic atherosclerosis in diabetic ApoE -/- mice by inhibiting NLRP3 inflammasome.

[Simple obesity of stomach heat and damp obstruction treated with acupoint thread embedding therapy: a randomized controlled trial].

OBJECTIVE: To explore the therapeutic effect and safety of acupoint thread embedding therapy in treatment of simple obesity of stomach heat and damp obstruction. METHODS: A total of 144 patients with simple obesity of stomach heat and damp obstruction were randomized into an acupoint thread embedding group (72 cases, 3 cases dropped off and 1 case removed) and a sham-embedding group (72 cases, 6 cases dropped off and 3 cases removed). On the base of the lifestyle adjustment, the acupoint thread embedding therapy with PGLA thread was applied to Tianshu (ST 25), Zhongwan (CV 12), Ganshu (BL 18), Shuidao (ST 28), etc. in the acupoint thread embedding group, while in the sham-embedding group, the acupoint selection and operation were all same as the acupoint thread embedding group, but without PGLA thread embedded. In either group, the treatment was given once every 2 weeks, consecutively for 12 weeks and the follow-up was conducted for 3 months after treatment. Separately, before and after treatment as well as in follow-up, the obesity indices (body mass index [BMI], waist circumference [WC], waist-to-hip ratio [WHR] and fat percentage [F%]) were observed in the two groups. Before and after treatment, the indices of blood glucose and insulin (fasting blood glucose [FBG], fasting insulin [FINS] and insulin resistance index [HOMA-IR]), adipocyte factor indices (adiponectin, leptin [LP] and serine protease inhibitor [Vaspin]) and inflammatory factor indices (tumor nercosis factor [TNF-α], interleukin-1ß [IL-1ß] and interleukin-6 [IL-6]) were observed separately in the two groups. The therapeutic effect and safety were compared between the two groups. RESULTS: After treatment and in follow-up, except WC and WHR in the sham-embedding group, BMI, WC, WHR and F% were all reduced as compared with those before treatment in the two groups (P<0.01, P<0.05), and the values in the acupoint thread embedding group were lower than the sham-embedding group (P<0.01). After treatment, except FBG, LP and Vaspin in the sham-embedding group, FBG, FINS, HOMA-IR, LP and Vaspin were all reduced as compared with those before treatment in the two groups (P<0.01, P<0.05), and adiponectin was increased as compared with that before treatment (P<0.01, P<0.05); the improvements in the acupoint thread embedding group were more significant than the sham-embedding group (P<0.01). After treatment, the levels of serum TNF-α, IL-1ß and IL-6 in the acupoint thread embedding group were reduced as compared with the values before treatment and those in the sham-embedding group separately (P<0.01). The total effective rate was 89.7% (61/68) in the acupoint thread embedding group, higher than 19.0% (12/63) in the sham-embedding group (P<0.01). There was no severe adverse reaction reported in the two groups. CONCLUSION: Acupoint thread embedding therapy with PGLA thread can alleviate obesity, regulate glucose metabolism and adipocyte factors activity, improve insulin resistance and inhibit the expression of pro-inflammatory factors in the patients with simple obesity with stomach heat and damp obstruction, and this therapy presents a satisfactory safety in treatment.

Potential Mechanisms of Biejiajian Pill in the Treatment of Diabetic Atherosclerosis Based on Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation.

Background: Biejiajian pill (BJJP), a classical traditional Chinese formula, has been reported that it has an effective treatment for diabetic atherosclerosis in recent years, but its underlying mechanisms remain elusive. The study aimed to explore the potential mechanisms of BJJP on diabetic atherosclerosis by integrating network pharmacology, molecular docking, and molecular dynamics simulation. Methods: The active components of BJJP were collected by TCMSP and TCMID, and then the potential targets were obtained from the SwissTargetPrediction database. The targets related to diabetic atherosclerosis were identified from the GeneCards and OMIM databases. The intersection of the potential targets regulated by active components of BJJP and the targets of diabetic atherosclerosis were common targets, which were visualized by the Venn diagram. The common targets were imported into the STRING database to construct a protein-protein interaction (PPI) network. The network of "Medicine-Compound-Target" was constructed with Cytoscape 3.7.1 software. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed using the DAVID database and visualized through bioinformatics. The intersecting targets were input into Cytoscape 3.7.1 software, and the Network Analyzer tool was employed to screen out the key targets. Then molecular docking was used to verify the binding affinity between the active compounds and the key targets, and molecular dynamics simulation was used to investigate the stability of the binding models. Results: A total of 81 active components, 186 targets of BJJP, and 4041 targets of diabetic atherosclerosis were obtained. Furthermore, 121 overlapping targets were identified. GO functional enrichment analysis revealed that these targets were correlated with the oxidation-reduction process, negative regulation of apoptotic process, inflammatory response, and other biological processes. The results of the KEGG pathway enrichment analysis showed that the common targets mainly participated in proteoglycans in cancer, PPAR signaling pathway, adherens junction, insulin resistance, HIF-1 signaling pathway, PI3K-Akt signaling pathway, etc. The results of molecular docking confirmed that the core active components in BJJP could bind well to the key targets. Results from molecular dynamics simulation showed that the binding energies of AKT1-Luteolin, MMP9-quercetin, and MMP9-luteolin complexes were -28.93 kJ·mol-1, -37.12 kJ·mol-1, and -62.91 kJ·mol-1, respectively. Conclusion: The study revealed that BJJP is characterized as multicomponent, multitarget, and multipathway to treat diabetic atherosclerosis, which is helpful to provide ideas and a basis for pharmacological research and clinical application in the future.

Ultrasensitive electrochemical detection of CYFRA 21-1 via in-situ initiated ROP signal amplification strategy.

The cytokeratin19 fragment (CYFRA 21-1) is an essential biomarker for non-small cell lung cancer (NSCLC). This work proposed a novel electrochemical immunosensor with a high selective and sensitive detection of CYFRA 21-1via the ring-opening polymerization (ROP) signal amplification strategy. Specifically, 3-mercaptopropionic (MPA) was employed as a cross-linking agent to immobilize cAb on the electrode surface for subsequent specific capture of CYFRA 21-1. After CYFRA 21-1 bound to cAb, the amino groups of them were blocked with acrolein. Then, the sandwich-type compositions were formed via the specific recognition between detection antibody (dAb) and CYFRA 21-1. Finally, the ROP was triggered by the amino group on dAb and the polymers containing a large number of ferrocene electroactive molecules were in situ grown on the electrode surface, thereby outputting a high sensing signal. Under optimal conditions, the fabricated immunosensor showed an ultrasensitive and highly selective with a linear range of 1 pg/mL ∼1 µg/mL, and the detection limit down to 9.08 fg/mL. Furthermore, a bright correlation was obtained for CYFRA 21-1 detection in the clinical serum samples. By merits of its ease of operation, environmental friendliness and low cost, this method had considerable potential application in bioanalytical for the ultrasensitive quantitation of biological molecules.

Effect of testosterone on the proliferation and collagen synthesis of cardiac fibroblasts induced by angiotensin II in neonatal rat.

The objective is to explore the effect of testosterone on the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts (CF) induced by Angiotensin II (Ang II) and the underlying mechanisms. Derived from neonatal rats, the CFs were divided into 4 groups: the control group, Ang II group, testosterone group, and testosterone + Ang II group in vitro. Cell cycle distribution, collagen counts, and phosphorylated extracellular signal-regulated kinase (ERK1/2) (p - ERK1/2) expression were assessed by flow cytometry, VG staining, and immunocytochemistry, respectively. The Ang II group had a much higher proportion of cells in the S-phase, higher collagen contents, and a higher p - ERK1/2 expression level than either the control or testosterone group. However, these factors were significantly reduced in the testosterone + Ang II group as compared to the Ang II group. In terms of cells in the S-phase and the collagen contents, there was not a significant difference between the testosterone group and the control. However, the protein expression of p-ERK1/2 was significantly increased in the testosterone group as compared to the control. Testosterone inhibits the proliferation and collagen synthesis of CF induced by Ang II. The underlying mechanism may involve the ERK1/2 signaling pathway.

Effects of technetium-99 methylenediphosphonate on cytokine-induced activation of retro-ocular fibroblasts from patients with Graves' ophthalmopathy.

OBJECTIVE: To explore the effects of technetium-99 methylenediphosphonate (99Tc-MDP) on cell proliferation, hyaluronic acid (HA) synthesis and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) on cultured retro-ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy. METHODS: After two to seven passages, cultured RFs were incubated for 72 h with interferon-γ (100 U/ml), interleukin-1 (100 U/ml) or tumour necrosis factor-α (100 U/ml) in the presence of 99Tc-MDP. Flow cytometry was used to investigate the expression of HLA-DR and ICAM-1. RF proliferation was assessed by 3H-thymidine incorporation assay. HA synthesis was measured by radioimmunoassay. RESULTS: At base conditions, the percentage of positive cells of HLA-DR and ICAM-1 on RFs was 6.70±3.06% and 5.29±3.02%, respectively, and the synthesis of HA was 337.8±42.7 ng/ml. Compared with basal values, 72-h incubation with cytokine significantly enhanced the expression of HLA-DR and ICAM-1, and HA synthesis. 99Tc-MDP (1 ng/ml) had little effect on cytokine-induced HLA-DR and ICAM-1 expression, and HA synthesis. When the concentration ranged from 10 to 100 ng/ml, 99Tc-MDP inhibited cytokine-induced RF activation in a dose-dependent manner. 99Tc-MDP also inhibited the proliferation of RFs in a dose-dependent manner. It was also found that 99Tc-MDP had the same effect on cytokine-induced RFs and skin fibroblasts from patients with normal individual conditions. CONCLUSIONS: 99Tc-MDP could inhibit cytokine-induced activation of RFs derived from patients with Graves' ophthalmopathy.

Inhibitory effects of Triptolide on interferon-gamma-induced human leucocyte antigen-DR, intercellular adhesion molecule-1, CD40 expression on retro-ocular fibroblasts derived from patients with Graves' ophthalmopathy.

PURPOSE: To explore the effects of Triptolide, the principal active diterpenoid from the Chinese Medicinal Herb Tripterygium Wilfordii Hook F that has immunosuppressive and anti-inflammatory properties, on cell proliferation, hyaluronic acid (HA) synthesis, and the expressions of human leucocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) and CD40 on cultured retro-ocular fibroblasts (RFs) from patients with Graves' ophthalmopathy. METHODS: After two to five passages, cultured RFs were incubated for 48 h within a medium alone or in the presence of recombinant human interferon-gamma (IFN-gamma) and various concentrations of Triptolide. Cell viability was assessed by MTT (3-[4.5-dimethylahiazol-2-yl]-2,5-diphenyltetrazolium Bromide). RFs proliferation was assessed by [(3)H]-thymidine incorporation assay. Flow cytometry was used to investigate the amount of HLA-DR, ICAM-1 and CD40. HA synthesis was measured by radioimmunoassay. RESULTS: Cell viability was not detrimentally affected when incubated with Triptolide from 0.01 microg/L to 10 microg/L for 48 h, and decreased with 20 microg/L Triptolide. The incorporation of [(3)H]-thymidine of RFs was 55 476 +/- 15 842 cpm incubated with medium alone or 18 352 +/- 3568 cpm with 10 microg/L Triptolide (t = 5.600, P < 0.01). Initially, the percentage of positive cells of HLA-DR, ICAM-1 and CD40 on RFs were 4.75 +/- 2.13%, 17.53 +/- 10.12% and 6.38 +/- 2.23%, respectively, and the synthesis of HA was 100 +/- 12%. Compared with basal values, 48-h incubation with IFN-gamma (100 U/mL) significantly enhanced the amount of HLA-DR, ICAM-1 and CD40, and HA synthesis. The values were 60.58 +/- 10.12% (t = 13.224, P < 0.01), 62.66 +/- 18.17% (t = 5.315, P < 0.01), 57.67 +/- 13.61% (t = 9.110, P < 0.01) and 164 +/- 22% (t = 9.238, P < 0.01), respectively. Triptolide 0.01 microg/L had little effect on IFN-gamma-induced HLA-DR, ICAM-1 and CD40 amounts, as well as HA synthesis. When the concentration ranged from 0.1 microg/L to 10 microg/L, Triptolide inhibited IFN-gamma-induced RFs activation in a dose-dependent manner. It was also found that Triptolide had the same inhibiting effects on IFN-gamma-induced RFs and skin fibroblasts from patients with normal individual conditions. CONCLUSIONS: Triptolide could inhibit IFN-gamma-induced activation of RFs derived from patients with Graves' ophthalmopathy.

Effect of angiotensin II receptor 1 antisense oligodoexynucleotides on physiological and pathophysiological growth of cardiomyocytes.

AIM: To evaluate the role of angiotensin II receptor 1 antisense oligodexynucleotides (AT1R-AS-ODNs) on physiological and pathophysiological growth of cardiomyocytes from normotensive rats. METHODS: Cardiomyocytes were transfected with AT1R-AS-ODNs (200 nmol/L) followed by treatment with or without angiotensin II (1 micromol/L). In situ hybridization and Western blot were used for AT1R mRNA and protein detection, respectively. c-Jun N-terminal protein kinase (JNK) activity was characterized by immune complex kinase assay. c-Jun protein expression was examined by immunocytochemistry. DNA content was detected by flow cytometric assay. Atrial natriuretic factor (ANF) expression was identified by radioimmunoassay. RESULTS: Treatment with AT1R-AS-ODNs for 24 h resulted in 51.2 % decrease in AT1R mRNA and 60.7 % in protein (P<0.05 vs control). However, the basal level of JNK activity, c-Jun protein expression, and DNA content were not altered by AT1R-AS treatment in absence of overactive hormonal system. After treatment with angiotensin II for 30 min, both p46JNK and p54JNK were robustly activated. By 2 h, c-Jun protein expression was increased. By 24 h, angiotensin II caused a marked increase both in G0/G1 and G2/M DNA content, and increased ANF expression by 1.8-fold. All these were inhibited by AT1R-AS-ODNs pretreatment. In contrast, sense sequence was ineffective. CONCLUSION: Decrease of AT1R expression by AS-ODNs did not interfere with normal growth, but protected cardiomyocytes from angiotensin II-dependent pathophysiological growth.