Single-cell RNA transcriptomic and plasma Lipidomic reveal the potential mechanisms of a Methotrexate-based therapy against Rheumatoid Arthritis.

OBJECTIVE: To assess whether a Methotrexate-based therapy could achieve more clinical benefit, we arranged a Simon 2-Stage Phase 1 Trial. Single-cell RNA sequencing and lipidomic profiling were performed to reveal the potential mechanisms. METHODS: Patients were enrolled in an open-label, Simon 2-stage, single-center, single-arm trial at Guangdong Provincial Hospital of Chinese Medicine. Main inclusion criteria were defined as follows: Aged 18 to 70, low to medium disease activity, fulfilled the RA classification criteria of EULAR/ACR 2010. Patients received the oral medication of MTX 10-15 mg weekly and natural product granules twice a day. Primary outcome was the American College of Rheumatology (ACR) 20% preliminary definition of improvement. Single-cell RNA sequencing(scRNA-seq) on peripheral blood mononuclear cells (PBMCs) was used to show the aberrant metabolism before and after the trial. Plasma lipidomic profiling quantified the lipid changes caused by this MTX-based therapy. Finally, post-hoc analysis on responders and non-responders were used for further analysis. RESULTS: Between October 2020 and June 2022, 46 patients received treatment, while 42 finished follow-ups. 27 of 46 (58.70%) patients achieved ACR20, and significant changes were observed in several secondary outcomes. Comparative scRNA-seq analysis before and after the treatment revealed that lipidomic metabolism was broadly downregulated. Plasma lipidomic profiling reveals that 40 lipids were observed significantly changed. Post-hoc analysis showed the lipid changes were separately linked to clinical parameters in responders and non-responders. CONCLUSION: The study reveals that the combination therapy of HQT+MTX is effective and has a certain correlation with lipid metabolism, but more rigorous study design is still needed to confirm this speculation.

The monocyte to high-density lipoprotein cholesterol ratio and outcomes in type 2 diabetes mellitus patients with non-ST-segment elevation acute coronary syndrome.

BACKGROUND: The monocyte to high-density lipoprotein cholesterol ratio (MHR) has been demonstrated as a new marker of inflammation. However, at present, the prognostic value of MHR in type 2 diabetes mellitus (T2DM) accompanied with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) undergoing percutaneous coronary intervention (PCI) is unclear. METHODS: T2DM patients with NSTE-ACS undergoing PCI were consecutively enrolled from January 1, 2010 to December 31, 2014 and divided according to MHR value tertiles. Baseline, procedural, and follow-up data were collected. The primary outcomes were in-hospital major adverse clinical events (MACE). The prespecified secondary outcomes included any bleeding [as indicated by Bleeding Academic Research Consortium definition (BARC) grades 1-5] and death during follow-up. RESULTS: Of the 1,405 enrolled patients, the rates of in-hospital MACE (0.2%, 0.2%, and 1.3%, P=0.043) and bleeding (12.4%, 12.2%, and 17.1%, P=0.048) increased significantly in high MHR tertiles. After 1 year of follow-up, the rates of bleeding (15.0%, 14.5%, and 22.2%, P=0.002) and all-cause death (1.5%, 1.7%, and 4.3%, P=0.010) were higher in higher MHR tertiles. Our results also suggested that MHR was an independent predictor of in-hospital MACE [adjusted odds ratio =8.36; 95% confidence interval (CI): 1.57-44.47; P=0.013] and long-term bleeding (adjusted hazard ratio =1.21; 95% CI: 1.07-1.37; P=0.002). Receiver-operating characteristic curve analysis indicated that MHR >0.022 had a sensitivity of 75.0% and specificity of 72.7% for predicting in-hospital MACE [area under the curve (AUC) =0.722; 95% CI: 0.51-0.933; P=0.040]. Furthermore, Kaplan-Meier curves showed that a higher risk of all-cause death in long-term follow-up was prevalent in patients with high MHR (P=0.033). CONCLUSIONS: The increased level of MHR was related to in-hospital MACE and long-term bleeding events in T2DM patients with NSTE-ACS undergoing PCI.

A case of acute myelogenous leukemia characterized by arterial and venous thrombosis.

Acute myelogenous leukemia (AML) is a malignant disease of the hematopoietic system, characterized by features of bone marrow insufficiency and organ infiltration by leukemic cells. Venous thrombosis in AML patients is uncommon, compared to bleeding; therefore in patients with AML, simultaneous occurrence of venous and arterial thrombosis is a rather rare presentation. We reported an unusual case of anti-phospholipid antibody syndrome secondary to AML characterized by venous and arterial thrombosis. A 70-year-old man with deep venous thrombosis (DVT) of the left leg confirmed by Doppler was seen in our clinic. During treatment with a Vitamin K antagonist (3 mg daily of Warfarin) and a low molecular weight heparin (LMWH), he developed an acute pulmonary embolism and an acute inferior wall ST elevation myocardial infarction (STEMI), a result of right coronary artery embolism. His full blood count showed leukocytosis and thrombocytopenia. Lupus anticoagulant and anti-cardiolipin antibodies were positive. A bone marrow aspirate test showed results consistent with AML (FAB class M1). A diagnosis of antiphospholipid antibody syndrome secondary to AML characterized by coronary artery embolism, pulmonary embolism and left leg DVT was eventually established. He received anticoagulation with a low dose of warfarin after refusing chemotherapy. He however died of cerebral hemorrhage despite the fact that the INR was in the normal therapeutic range. It is challenging to anticoagulated AML patients complicated by multiple vascular thromboses and thrombocytopenia.

The Role of Th17/Treg Axis in the Traditional Chinese Medicine Intervention on Immune-Mediated Inflammatory Diseases: A Systematic Review.

The Th17/Treg axis plays a crucial role in immune-mediated inflammatory diseases (IMID) and might represent an interesting drug target of treatment strategy for these diseases. Accumulating evidence suggests a role for traditional Chinese medicine (TCM) in the modulation of Th17/Treg axis, but a comprehensive overview which summarizes this field hitherto is lacked. This paper performs a systematic literature review of the regulatory effects of TCM on the imbalance of Th17/Treg axis and its potential mechanisms. In addition, the frequency analysis and network pharmacology for the collected TCM herbs from clinical trial data were performed. The studies reported the changes in the ratio of Th17 and/or Treg cells as well as their transcription factor and related cytokines were included. Frequency analysis of composition of the 39 assessed TCM prescriptions showed that Astragalus membranaceus var.mongholicus (5.20%), Glycyrrhiza uralensis (3.67%), Paeonia obovate (3.06%), Salvia digitaloides (3.06%), and Angelica sinensis (2.75%) were the top five herbal components, which were closely associated to the treatment of IMID. Network pharmacology showed that six target proteins (transforming growth factor (TGF)-beta receptor type-1, TGF-beta receptor type-2, retineic-acid-receptor-related orphan nuclear receptor gamma (ROR-gamma), TGFB2, IL-17 and IL-2, respectively) might be involved in the regulatory effects of TCM on Th17/Treg axis. Moreover, there were nine active ingredients (including Oxymatrine, Baicalin, Triptolide, Paeoniflorin, Sinomenine, Celastrol, Emodin, Diosgenin and Chlorogenic acid) originating from TCM reported to have an immunological regulation effect on the Th17/Treg axis. The highlight of this systematic review is to reveal the pharmacological basis of TCM treating IMID and is helpful for supporting future pharmacologic-driven studies. Further research elucidates the immune-modulating mechanisms on Th17/Treg axis by TCM might provide a broader insight for the treatment of IMID.

Advanced glycation end products induce proliferation, invasion and epithelial-mesenchymal transition of human SW480 colon cancer cells through the PI3K/AKT signaling pathway.

The aim of the present study was to determine the mechanism by which advanced glycation end products (AGEs) induce proliferation, invasion and epithelial-mesenchymal transition (EMT) of human colon cancer SW480 cells. SW480 cells were divided into groups as follows: i) Control; ii) cells treated with AGEs alone; and iii) cells treated with AGEs combined with LY294002. Proliferation, cell cycle progression, apoptosis, invasion and migration of SW480 cells were assessed using an MTT assay, flow cytometry, Transwell assays and a wound healing assay, respectively. The protein expression levels of PI3K, AKT and epithelial cadherin (E-cadherin) were examined by western blot analysis in SW480 cells treated with various concentrations of AGEs. Proliferation, invasion and migration were enhanced, cell cycle progression was increased and apoptosis was decreased in SW480 cells treated with AGEs compared with the control. The PI3K inhibitor, LY294002, reversed the effects of AGEs. Western blot analysis data demonstrated that AGEs increased the protein expression levels of PI3K and AKT, and decreased the expression of E-cadherin. The results suggested that AGEs exert a positive effect on the proliferation, invasion and EMT in SW480 cells through the PI3K/AKT signaling pathway.

Hepatitis B Virus X Protein-Induced RORγ Expression to Promote the Migration and Proliferation of Hepatocellular Carcinoma.

Aberrant expression of RORγ is implicated in cancer development. A previous study identified that RORγ functions as a tumor promoter to drive hepatocellular carcinoma (HCC) growth. However, its expression and significance in HCC remain unclear. The central finding of this work is that RORγ was overexpressed in HCC due to its dysfunction of promoter methylation, and hepatitis B virus X protein (HBx) can remarkably induce the expression of RORγ in hepatocellular carcinoma through enhancing the transcriptional function. Also, the HBx-induced RORγ could promote the migration and proliferation of hepatoma cells. Hence, these results suggest that RORγ was an important regulator in HCC, and our finding provides new insights into the significance of RORγ in HCC.

Long noncoding RNA NEAT1 sponges miR­125a­5p to suppress cardiomyocyte apoptosis via BCL2L12.

Increasing evidence has suggested that long non­coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has critical roles in multiple biological processes; however, few studies have reported on its function in heart disease. The present study indicated that NEAT1 expression is markedly downregulated in cardiomyocytes following ischemia/reperfusion injury in vivo and hydrogen peroxide treatment in vitro. Further experiments suggested that ectopic overexpression of NEAT1 suppresses cardiomyocyte apoptosis induced by hydrogen peroxide, as assessed by TUNEL assay and flow cytometry. In addition, using a dual­luciferase reporter assay, NEAT1 was demonstrated to directly interact with microRNA (miR)­125a­5p and overexpression of miR­125a­5p efficiently reversed the stimulatory effect of NEAT1 on B­cell lymphoma­2­like 12 (BCL2L12) expression. Furthermore, the results indicated that NEAT1 inhibits cardiomyocyte apoptosis via regulating the expression of BCL2L12, which appeared to be mediated via miR­125a­5p. In conclusion, the present study suggested that NEAT1 functions as a miR sponge to inhibit cardiomyocyte apoptosis and may be a novel therapeutic target for cardiomyocyte apoptosis­associated heart diseases.

Hemoglobin A1c and risk of left atrial thrombus and spontaneous echo contrast in non-valvular atrial fibrillation patients.

OBJECTIVES: To evaluate the relationship between hemoglobin A1c (HbA1c) and risk of left atrial thrombus/spontaneous echo contrast (LAT/SEC) in non-valvular atrial fibrillation (AF) patients. METHODS: In this retrospective study, 1158 consecutive non-valvular AF patients undergoing transesophageal echocardiography prior to radiofrequency catheter ablation or electric cardioversion were enrolled. Baseline characteristics were collected and analyzed. RESULTS: There were 87 (7.5%) patients with LAT/SEC. The HbA1c levels in the patients with LAT/SEC were significantly higher than that in patients without LAT/SEC (6.13 ± 0.41 vs. 5.89 ± 0.45 µmol/L, P < 0.001). The optimal cut-off point for HbA1c predicting LAT/SEC was 6.1% determined by receiver-operating characteristic curve. The area under the curve is 0.788 (95% confidence interval: 0.764-0.812). HbA1c ≥6.1% was an independent risk factor for LAT/SEC (odds ratio, 1.74; 95% confidence interval, 1.01-2.98; P = 0.045). CONCLUSIONS: Elevated HbA1c indicated a significantly increased risk for LAT/SEC in non-valvular AF patients. HbA1c might have significance in predicting the risk for prothrombotic state in non-valvular AF patients.

A meta-analysis of association of vitamin D receptor BsmI gene polymorphism with the risk of type 1 diabetes mellitus.

Association between vitamin D receptor (VDR) BsmI (rs1544410) gene polymorphism and the risk of type 1 diabetes mellitus (T1DM) from the published reports are still conflicting. This study was conducted to evaluate the relationship between VDR BsmI gene polymorphism and the risk of T1DM using meta-analysis method. The association studies were identified from PubMed, and Cochrane Library on 1 December 2013, and eligible investigations were included and synthesized using meta-analysis method. Twenty-three reports were recruited into this meta-analysis for the association of VDR BsmI gene polymorphism with T1DM susceptibility. In overall populations, bb genotype was associated with T1DM, but the B allele and BB genotype were not. In Asians and Latino population, B allele and bb genotype were associated with TIDM risk, but BB genotype was not. In Caucasians, VDR BsmI gene polymorphism was not associated with the T1DM risk. In Africans, B allele and BB genotype were associated with T1DM risk, but the bb genotype was not. However, the sample size for Latino population and Africans was small. In conclusion, VDR BsmI B allele, bb genotype was associated with T1DM risk in Asians, and bb genotype was associated with T1DM risk in overall populations. However, more studies should be conducted to confirm it.

ß-Elemene enhances susceptibility to cisplatin in resistant ovarian carcinoma cells via downregulation of ERCC-1 and XIAP and inactivation of JNK.

ß-Elemene is a promising new plant-derived drug with broad-spectrum anticancer activity. It also increases cisplatin cytotoxicity and enhances cisplatin sensitivity in resistant human carcinoma cells. However, little is known about the mechanism of its action. To explore the potential therapeutic application of ß-elemene as a drug-resistance modulator, this study investigated the underlying mechanism of ß-elemene activity in cisplatin-resistant ovarian cancer cells. ß-Elemene enhanced cisplatin sensitivity to a much greater extent in chemoresistant A2780/CP70 and MCAS human ovarian carcinoma cells compared to the chemosensitive parental cell line A2780. The dose-modifying factors for cisplatin were between 35 and 60 for A2780/CP70 cells and between 1.6 and 2.5 for A2780 cells. In the cisplatin-resistant ovarian carcinoma cells, ß-elemene abrogated cisplatin­induced expression of excision repair cross-complementation group­1 (ERCC-1), a marker gene in the nucleotide excision repair pathway that repairs cisplatin-caused DNA damage. In addition, ß-elemene not only reduced the level of X-linked inhibitor of apoptosis protein (XIAP), but also downregulated cisplatin-mediated XIAP expression in chemoresistant cells. Furthermore, ß-elemene blocked the cisplatin-stimulated increase in the level of phosphorylated c-Jun NH2-terminal kinase (JNK) in these cells. These novel findings suggest that the ß-elemene enhancement of cisplatin sensitivity in human chemoresistant ovarian cancer cells is mediated at least in part through the impairment of DNA repair activity and the activation of apoptotic signaling pathways, thereby making resistant ovarian cancer cells susceptible to cisplatin-induced cell death.

ß-Elemene promotes cisplatin-induced cell death in human bladder cancer and other carcinomas.

Cisplatin-based combination treatment is the most effective systemic chemotherapy for bladder cancer; however, resistance to cisplatin remains a significant problem in the treatment of this disease. ß-Elemene is a new natural compound that blocks cell-cycle progression and has a broad spectrum of antitumor activity. This study was conducted to explore the potential of ß-elemene as a chemosensitizer for enhancing the therapeutic efficacy and potency of cisplatin in bladder cancer and other solid carcinomas. ß-Elemene not only markedly inhibited cell growth and proliferation but also substantially increased cisplatin cytotoxicity towards human bladder cancer 5637 and T-24 cells. Similarly, ß-elemene also enhanced cisplatin sensitivity and augmented cisplatin cytotoxicity in small-cell lung cancer and carcinomas of the brain, breast, cervix, ovary, and colorectal tract in vitro, with dose-modifying factors ranging from 5 to 124. ß-Elemene-enhanced cisplatin cytotoxicity was associated with increased apoptotic cell death, as determined by DNA fragmentation, and increased activities of caspase-3, -7, -8, -9, and -10 in bladder cancer cell lines. Collectively, these results suggest that ß-elemene augments the antitumor activity of cisplatin in human bladder cancer by enhancing the induction of cellular apoptosis via a caspase-dependent mechanism. Cisplatin combined with ß-elemene as a chemosensitizer warrants further pre-clinical therapeutic studies and may be useful for the treatment of cisplatin-resistant bladder cancer and other types of carcinomas.

Enhancement of cisplatin-induced apoptosis by ß-elemene in resistant human ovarian cancer cells.

ß-Elemene is a new anticancer compound extracted from the Chinese medicinal herb Rhizoma zedoariae. We have shown previously that ß-elemene increases cisplatin cytotoxicity and enhances cisplatin sensitivity via blocking cell cycle progression at G2/M phase in resistant ovarian tumor cells. In the current study, we asked whether ß-elemene-augmented cisplatin activity in ovarian carcinoma cells is mediated through the induction of apoptosis. Here, we show that ß-elemene triggered apoptotic cell death in chemoresistant human ovarian cancer A2780/CP and MCAS cells in a dose- and time-dependent fashion, as assessed by six different apoptosis assays. Intriguingly, ß-elemene was a stronger inducer of apoptosis than cisplatin in this model system, and a synergistic effect on induction of cell death was observed when the tumor cells were treated with both agents. Furthermore, ß-elemene plus cisplatin exposure significantly disrupted the mitochondrial transmembrane potential (ΔΨ (m)) and increased the release of cytochrome c from mitochondria into the cytoplasm. The combination treatment with both compounds also induced increases in caspase-3/8/9 activities and caspase-9 cleavage, enhanced protein expression of Bax and phosphorylation of Bcl-2 at Ser-70, and reduced the protein levels of Bcl-2 and Bcl-X(L) in the platinum-resistant ovarian cancer cells. Taken together, these data indicate that ß-elemene sensitizes chemoresistant ovarian carcinoma cells to cisplatin-induced apoptosis and that the augmented effect of ß-elemene on cisplatin cytotoxicity and sensitivity in resistant ovarian tumor cells is mediated through a mitochondria- and caspase-dependent cell death pathway.

Anticancer activity of ß-Elemene and its synthetic analogs in human malignant brain tumor cells.

Malignant brain tumors are aggressive in both children and adults. Despite recent improvements in diagnostic techniques, therapeutic approaches remain disappointing and unsuccessful. There is an urgent need for promising anticancer agents to improve overall survival of patients with brain cancer. ß-Elemene has been shown to have antiproliferative effects on many types of carcinomas. In this study, we compared the cytotoxic efficacy of ß-elemene and its synthetic analogs in the brain tumor cell lines A172, CCF-STTG1, and U-87MG. ß-Elemene exhibited cytotoxicity towards the tumor lines, effectively suppressing tumor cell survival. The inhibitory effect of ß-elemene was mediated by the induction of apoptosis, as demonstrated by three assays. The annexin V assay showed that ß-elemene increased the percentage of early- and late-apoptotic cells. Apoptotic nuclei were detected in cancer cells in situ by the terminal deoxynucleotidyltransferase-mediated deoxy-UTP-fluorescein nick end labeling (TUNEL) staining, and the number of TUNEL-positive cells was significantly increased at 24-72 h following drug treatment of the cell lines. Cell death enzyme-linked immunosorbent assay (ELISA) gave similar results. Furthermore, ß-elemene increased caspase-3/7/10 activity, up-regulated protein expression of BAX, and down-regulated the one of BCL-2, BCL-XL, and of X-linked inhibitor of apoptosis (XIAP) in the cells, suggesting that apoptotic signaling pathways are involved in the responses triggered by ß-elemene. Compared with ß-elemene, only three of the 10 synthetic ß-elemene analogs studied here, exerted comparable cytotoxic efficacy towards the three brain tumor lines: the analogs Lr-1 and Lr-2 had the same antitumor efficacy, while Lr-3 was less potent than ß-elemene. Thus, some synthetic analogs of ß-elemene may inhibit brain cancer cell growth and proliferation, and the synthetic analogs Lr-1 and Lr-2 may have great potential as alternatives to ß-elemene for anticancer therapy. Overall, this study provides, to our knowledge, the first evidence showing that synthetic analogs of ß-elemene hold promise for patients with brain tumors.

Type 1 receptor parathyroid hormone (PTH1R) influences breast cancer cell proliferation and apoptosis induced by high levels of glucose.

Increased breast cancer incidence parallels the increase in cases of type 2 diabetes. We investigated the effect of type 1 receptor parathyroid hormone (PTH1R) expression on viability and apoptosis of breast cancer cells exposed to high levels of glucose. Upregulation of PTH1R was detected in patients with invasive ductal carcinoma of the breast and diabetes. In vitro, PTH1R silencing suppressed cell proliferation and apoptosis induced by high levels of glucose by regulating Bax/Bcl-2 expression. These results suggest PTH1R silencing may represent a novel treatment approach for patients diagnosed with invasive ductal carcinoma of the breast who are also managing diabetes.

Diagnostic value of 16 cellular tumor markers for metastatic thyroid cancer: an immunohistochemical study.

BACKGROUND: The prognosis for thyroid cancer differs between metastatic and non-metastatic cases. To identify biomarkers useful for thyroid cancer diagnosis and to establish a marker panel for the early detection of metastatic thyroid carcinoma, this study compared histomorphological features and biomarker expression profiles in thyroid carcinomas according to pathological diagnoses. PATIENTS AND METHODS: Thyroid carcinoma samples were obtained from 113 consecutive patients who underwent resection at multiple centers between 2001 and 2008. These cases included 63 metastatic thyroid tumors (34 papillary carcinomas, 20 follicular carcinomas, 9 undifferentiated carcinomas) and 50 non-metastatic thyroid tumors (36 papillary carcinomas, 14 follicular carcinomas). Tissue microarrays constructed using the 113 samples were analyzed by immunohistochemistry for the expression of 16 protein markers: MMP9, VEGF-C, E-cadherin, MMP2, PPARγ, PCNA, CXCR4, PTEN, C-myc, PTTG, HBME-1, p16, p53, FHIT, bFGF and hTERT. The clinicopathological variables with diagnostic significance were determined by multivariate analysis, and the predictive values of the identified biomarkers for metastasis in thyroid carcinoma were determined by receiver operating characteristic (ROC) curve analysis. RESULTS: The expression of six proteins, VEGF-C, MMP2, CXCR4, PTTG, HBME-1 and bFGF, was up-regulated in metastatic compared to non-metastatic thyroid carcinoma. Multiple factor binary ordinal logistic regression analysis showed that MMP2, PTTG, VEGF-C, CXCR4 and bFGF were independent factors associated with the metastatic status of thyroid carcinoma. ROC curve analysis of these five proteins revealed that VEGF-C and bFGF were the most useful protein markers for the diagnosis of metastatic thyroid cancer. CONCLUSION: MMP2, PTTG, VEGF-C, CXCR4 and bFGF are potential cellular tumor markers for identifying thyroid cancer with greater risk for metastasis and the novel combination of VEGF-C and bFGF as biomarkers may improve the accuracy of early detection and the differential diagnosis between metastatic and non-metastatic thyroid carcinoma.

Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo.

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor in cells, and the interaction of RAGE with ligands results in pro-inflammatory gene activation. Aberrant RAGE activation was reported to promote the pathogenesis of colorectal cancer. This study aimed to investigate the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis, as well as the underlying molecular mechanisms regulating these interactions in colorectal cancer cells. The RAGE mRNA and protein were evaluated in five colorectal cancer cell lines and in 45 cases of colorectal cancer tissue specimens (using immuohistochemistry). RAGE expression was then knockdown using RAGE shRNA for assessing cell viability and invasion assays as well as for tube formation and CAM assays in human umbilical vein endothelial cells and chick embryos, respectively. RAGE was highly expressed in colorectal cancer tissues, and was associated with increased microvessel density. Two of the four RAGE shRNA constructs were able to significantly knockdown RAGE expression in SW480 cells. RAGE knockdown inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells and angiogenesis of chicken embryos. Knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells. In summary, these data suggest that silence of RAGE expression could effectively inhibit colorectal cancer angiogenesis in vitro and in vivo.

Palmitic acid-induced apoptosis in pancreatic ß-cells is increased by liver X receptor agonist and attenuated by eicosapentaenoate.

Saturated fatty acids are implicated in the development of diabetes via the impairment of pancreatic islet ß-cell viability and function. Liver X receptors (LXRs) and eicosapentaenoate (EPA) are known regulators of fatty acid metabolism. However, their roles in the pathogenesis of diabetes remain incompletely understood. The aim of this study was to determine the effects of EPA and the LXR agonist T0901317 on saturated fatty acid (palmitic acid)-induced apoptosis in the insulinoma ß-cell line INS-1, a model for insulin-secreting ß-cells. T0901317 significantly promoted palmitic acid-induced apoptotic cell death in the INS-1 cells. Consistent with these results, caspase-3 activity and BAX and sterol regulatory element binding protein-1c (SREBP-1c) mRNA levels were markedly increased in INS-1 cells co-administered palmitic acid and T0901317. The production of reactive oxygen species was considerably higher in the cells cultured concurrently with T0901317 and palmitic acid than in the cells incubated with either agent alone. EPA treatment attenuated the cellular death promoted by palmitic acid and T0901317 in the INS-1 cells, disclosing a possible mediating mechanism involving the inhibition of SREBP-1c. Finally, T0901317 up-regulated the palmitic acid-induced expression of p27(KIP1), transforming growth factor beta 1, and SMAD3 proteins in INS-1 cells. These results demonstrate that palmitic acid-induced apoptosis in ß-cells is enhanced by T0901317 via the activation of LXRs and is blocked by EPA via the inhibition of SREBP-1c, suggesting that the regulation of lipogenesis and lipotoxicity affecting pancreatic ß-cell viability and insulin production may be a unique strategy for diabetes therapy.

[Construction and identification of siRNA recombinant expression vector targeting parathyroid hormone 1 receptor gene and its effect on the cell cycle of INS-1 cells].

OBJECTIVE: To construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells. METHODS: The sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry. RESULTS: RT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase. CONCLUSION: The successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.

Assessment of biomarkers for clinical diagnosis of papillary thyroid carcinoma with distant metastasis.

Early diagnosis and treatment of thyroid cancers are critical for better prognosis and better survival rates. The purpose of this study was to identify potential diagnostic markers for papillary thyroid carcinomas with distant metastasis. Fifty-eight papillary thyroid tumor specimens (27 papillary thyroid carcinomas with distant metastasis and 31 without metastasis) were examined, and protein expression of pituitary tumor-transforming gene (PTTG), E-cadherin, p27kip1, vascular endothelial growth factor (VEGF)-C, metalloproteinase (MMP) 2, MMP9, chemokine receptor CXCR4, and basic fibroblast growth factor (bFGF) in these tumors was assessed by immunohistochemistry. The clinicopathological variables with diagnostic significance were determined by multivariate analysis, and their diagnostic values were evaluated by ROC curve analysis. PTTG, VEGF-C, MMP2, MMP9, CXCR4, and bFGF were overexpressed in metastatic papillary thyroid carcinomas, whereas p27kip1 expression was elevated only in carcinomas lacking metastasis. Multiple-factor binary ordinal logistic regression analysis revealed that PTTG, VEGF-C, MMP2, and bFGF were independently related to biological metastatic behavior in thyroid tumors, suggesting their potential use as biomarkers. ROC curve analysis showed that among these four proteins, VEGF-C and bFGF were the best diagnostic biomarkers. A VEGF-C and bFGF cluster was the most useful factor for the differential diagnosis between metastatic and non-metastatic papillary thyroid cancers. Thus, the combined use of VEGF-C and bFGF as biomarkers may improve the diagnostic accuracy of papillary thyroid carcinoma and may be useful in multimodal screening programs for the clinical diagnosis of papillary thyroid carcinoma and early detection of papillary thyroid carcinoma with distant metastasis.