Impairment of Gal-9 and Tim-3 crosstalk between Tregs and Th17 cells drives tobacco smoke-induced airway inflammation.

Overexpression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells has been observed in smokers. However, whether and how galectin-9 (Gal-9)/TIM-3 signal between T-regulatory cells (Tregs) and type 17 helper (Th17) cells contributes to tobacco smoke-induced airway inflammation remains unclear. Here, we aimed to explore the role of the Gal-9/TIM-3 signal between Tregs and Th17 cells during chronic tobacco smoke exposure. Tregs phenotype and the expression of TIM-3 on CD4+ T cells were detected in a mouse model of experimental emphysema. The role of TIM-3 in CD4+ T cells was explored in a HAVCR2-/- mouse model and in mice that received recombinant anti-TIM3. The crosstalk between Gal-9 and Tim-3 was evaluated by coculture Tregs with effector CD4+ T cells. We also invested the expression of Gal-9 in Tregs in patients with COPD. Our study revealed that chronic tobacco smoke exposure significantly reduces the frequency of Tregs in the lungs of mice and remarkably shapes the heterogeneity of Tregs by downregulating the expression of Gal-9. We observed a pro-inflammatory but restrained phenotypic transition of CD4+ T cells after tobacco smoke exposure, which was maintained by TIM-3. The restrained phenotype of CD4+ T cells was perturbed when TIM-3 was deleted or neutralised. Tregs from the lungs of mice with emphysema displayed a blunt ability to inhibit the differentiation and proliferation of Th17 cells. The inhibitory function of Tregs was partially restored by using recombinant Gal-9. The interaction between Gal-9 and TIM-3 inhibits the differentiation of Th17 cells and promotes apoptosis of CD4+ T cells, possibly by interfering with the expression of retinoic acid receptor-related orphan receptor gamma t. The expression of Gal-9 in Tregs was reduced in patients with COPD, which was associated with Th17 response and lung function. These findings present a new paradigm that impairment of Gal-9/Tim-3 crosstalk between Tregs and Th17 cells during chronic tobacco smoke exposure promotes tobacco smoke-induced airway/lung inflammation.

Irisin inhibits NLRP3 inflammasome activation in HG/HF incubated cardiac microvascular endothelial cells with H/R injury.

PURPOSE: NLRP3 inflammasome mediates myocardial ischemia/reperfusion (MI/R) injury and diabetic vascular endothelia dysfunction. However, the role of NLRP3 inflammasome in MI/R injury with diabetes has not been fully described. Irisin plays an important role in anti-inflammation and improves endothelial function in type 2 diabetes. The current study aimed to investigate the effect of irisin on regulating NLRP3 inflammasome activation in diabetic vascular endothelia dysfunction. METHODS: Cardiac microvascular endothelial cells (CMECs) were cultured and subjected to high glucose/high fat (HG/HF) receiving hypoxia/reoxygenation (H/R) with irisin incubation or not. Then, apoptosis, viability, migration, NO secretion, and inflammasome activation were examined. RESULTS: The hypoxic CMECs exhibited increased apoptosis, impaired viability, and migration, even decreased NO secretion and enhanced inflammasome activation. Moreover, irisin incubation decreased NLRP3 activation and attenuated cell injury in HG/HF cultured CMECs subjected to H/R injury, which was abolished by NLRP3 inflammasome activation. Meanwhile, NLRP3 inflammasome siRNA also attenuated H/R injury in CMECs under HG/HF condition. CONCLUSION: The current study demonstrated for the first time that irisin inhibits NLRP3 inflammasome activation in CMECs as a novel mechanism in myocardial ischemia/reperfusion injury in diabetes.

FNDC5/irisin reduces ferroptosis and improves mitochondrial dysfunction in hypoxic cardiomyocytes by Nrf2/HO-1 axis.

Myocardial infarction is characterized by cardiomyocyte death and mitochondrial dysfunction induced by ischemia. Ferroptosis, a novel form of cell death, has been found to play critical roles under ischemic conditions. Recently, several studies have shown that fibronectin type III domain-containing 5 (FNDC5) and its cleaved form, irisin, protect the heart against injury. However, its protective effect on ferroptosis and mitochondrial impairments is still unclear. Thus, our aim was to investigate the role of irisin in ferroptosis and mitochondrial dysfunction in cardiomyocytes under hypoxic conditions. Cardiomyocytes were treated with FNDC5 overexpression and/or irisin under normoxic and hypoxic conditions. Cell viability was assessed by Cell Counting Kit-8 assay. Reactive oxygen species production was evaluated by dihydroethidium staining. In addition, the intracellular ferrous iron level (Fe2+ ) and the relative concentration of malondialdehyde and ATP content were determined using an Iron Assay Kit, Lipid Peroxidation Assay Kit, and ATP Bioluminescent Assay Kit. The superoxide dismutase level in cells was measured using an Enzyme-Linked Immunosorbent Assay Kit. Furthermore, an immunoblotting assay was used to determine ferroptosis-related mitochondrial proteins. Hypoxia promoted cell death, increased ferroptosis, and caused mitochondrial dysfunction in cardiomyocytes. Interestingly, FNDC5 overexpression and/or irisin administration elevated cell viability, decreased ferroptosis, and reversed mitochondrial impairments induced by hypoxia. Mechanistically, FNDC5/irisin reduced ferroptosis and reversed mitochondrial impairments by Nrf2/HO-1 axis in hypoxic cardiomyocytes. Thus, we have demonstrated that FNDC5/irisin plays a protective role in ferroptosis and mitochondrial dysfunction in hypoxia-induced cardiomyocytes.

Autophagy inhibits high glucose induced cardiac microvascular endothelial cells apoptosis by mTOR signal pathway.

Cardiac microvascular endothelial cells (CMECs) dysfunction is an important pathophysiological event in the cardiovascular complications induced by diabetes. However, the underlying mechanism is not fully clarified. Autophagy is involved in programmed cell death. Here we investigated the potential role of autophagy on the CMECs injury induced by high glucose. CMECs were cultured in normal or high glucose medium for 6, 12 and 24 h respectively. The autophagy of CMECs was measured by green fluorescence protein (GFP)-LC3 plasmid transfection. Moreover, the apoptosis of CMEC was determined by flow cytometry. Furthermore, 3-Methyladenine (3MA), ATG7 siRNA and rapamycin were administrated to regulate the autophagy state. Moreover, Western blotting assay was performed to measure the expressions of Akt, mTOR, LC3 and p62. High glucose stress decreased the autophagy, whereas increased the apoptosis in CMECs time dependently. Meanwhile, high glucose stress activated the Akt/mTOR signal pathway. Furthermore, autophagy inhibitor, 3-MA and ATG7 siRNA impaired the autophagy and increased the apoptosis in CMECs induced by high glucose stress. Conversely, rapamycin up-regulated the autophagy and decreased the apoptosis in CMECs under high glucose condition. Our data provide evidence that high glucose directly inhibits autophagy, as a beneficial adaptive response to protect CMECs against apoptosis. Furthermore, the autophagy was mediated, at least in part, by mTOR signaling.

Autophagy regulates the apoptosis of bone marrow-derived mesenchymal stem cells under hypoxic condition via AMP-activated protein kinase/mammalian target of rapamycin pathway.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been demonstrated as an ideal autologous stem cells source for cell-based therapy for myocardial infarction (MI). However, poor viability of donor stem cells after transplantation limits their therapeutic efficiency, whereas the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for maintaining homeostasis and normal function. Here, we investigated the potential role of autophagy on apoptosis in BM-MSCs induced by hypoxic injury. BM-MSCs, isolated from male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury for 6, 12, and 24 h, respectively. The autophagy state was regulated by 3-methyladenine (3MA) and rapamycin administration. Furthermore, compound C was administrated to inhibit AMPK. The apoptosis induced by H/SD was determined by TUNEL assays. Meanwhile, autophagy was measured by GFP-LC3 plasmids transfection and transmission electron microscope. Moreover, protein expressions were evaluated by Western blot assay. In the present study, we found that hypoxic stress increased autophagy and apoptosis in BM-MSCs time dependently. Meanwhile, hypoxia increased the activity of AMPK/mTOR signal pathway. Moreover, increased apoptosis in BM-MSCs under hypoxia was abolished by 3-MA, whereas was aggravated by rapamycin. Furthermore, the increased autophagy and apoptosis in BM-MSCs induced by hypoxia were abolished by AMPK inhibitor compound C. These data provide evidence that hypoxia induced AMPK/mTOR signal pathway activation which regulated the apoptosis and autophagy in BM-MSCs. Furthermore, the apoptosis of BM-MSCs under hypoxic condition was regulated by autophagy via AMPK/mTOR pathway.

Metformin inhibits thyroid cancer cell growth, migration, and EMT through the mTOR pathway.

Mammalian target of rapamycin (mTOR) signaling pathways have been shown to be activated in thyroid cancer. Recent evidences have demonstrated that the antidiabetic agent metformin, an activator of 5'-AMP-activated protein kinase, can impair the proliferation and migration of cancer cells via inhibition of mTOR. However, the underlying mechanisms remain unclear. In this study, we show that metformin can inhibit mTOR pathway to impair growth and migration of the thyroid cancer cell lines. Cyclin D1 and c-Myc are important regulators of cancer cell growth, and we observed that treatment of thyroid cancer cells with metformin reduced c-Myc and cyclin D1 expression through suppression of mTOR and subsequent inhibition of P70S6K1 and 4E-BP1 phosphorylation. Metformin reduced epithelial to mesenchymal transition (EMT) in thyroid carcinoma cells. Moreover, metformin regulated expression of the EMT-related markers E-cadherin, N-cadherin, and Snail. Additionally, knockdown of TSC2, the upstream regulatory molecule of mTOR pathway, or treatment of rapamycin, the mTOR inhibitor, could abolish the effects of metformin to regulate thyroid cancer cell proliferation, migration, EMT, and mTOR pathway molecules. These results indicate that metformin can suppress the proliferation, migration, and EMT of thyroid cancer cell lines by inhibiting mTOR signaling. These findings suggest that metformin and its molecular targets may be useful in thyroid carcinoma therapy.

Management of bleeding gastric varices in patients with sinistral portal hypertension.

BACKGROUND AND AIM: Sinistral portal hypertension (SPH) is a rare cause of upper gastrointestinal hemorrhage. Besides splenectomy, there is no consensus on the role of sclerotherapy and splenic embolization for bleeding gastric varices (GVs). This retrospective study summarizes our experience in managing GV bleeding from SPH in patients with pancreatic diseases. METHODS: Patients with pancreatic diseases who had bleeding GVs from SPH in two tertiary hospitals were reviewed from January 2001 to December 2011. The etiology, clinical manifestations, diagnostic and therapeutic modalities were analyzed. RESULTS: Twenty-one patients (15.2 %) complicating bleeding GVs among 139 patients with SPH secondary to pancreatic diseases were enrolled. The etiologies were acute pancreatitis in one patient, chronic pancreatitis in seven patients, and pancreatic tumors in 13 patients. Emergent endoscopic sclerotherapy was initially performed in five patients, and succeeded in two patients, while one patient died of massive hemorrhage. Initial transcatheter artery embolization using Gianturco coils was successfully performed in six patients. Splenectomy combined with other surgical procedures was undertaken for 15 patients. The patients undergoing artery embolization or splencetomy achieved hemostasis. The survivors had no recurrent bleeding during a median 72-month follow-up period. CONCLUSIONS: The incidence of bleeding GVs from SPH is relatively rare. Splenic artery embolization could be selected as a first-line choice for bleeding SPH, especially for patients in poor conditions, and sclerotherapy may not be preferentially recommended. Further studies are required to evaluate the optimum treatment algorithm for bleeding GVs from SPH.

FNDC5/irisin improves the therapeutic efficacy of bone marrow-derived mesenchymal stem cells for myocardial infarction.

BACKGROUND: The beneficial functions of bone marrow mesenchymal stem cells (BM-MSCs) decline with decreased cell survival, limiting their therapeutic efficacy for myocardial infarction (MI). Irisin, a novel myokine which is cleaved from its precursor fibronectin type III domain-containing protein 5 (FNDC5), is believed to be involved in a cardioprotective effect, but little was known on injured BM-MSCs and MI repair yet. Here, we investigated whether FNDC5 or irisin could improve the low viability of transplanted BM-MSCs and increase their therapeutic efficacy after MI. METHODS: BM-MSCs, isolated from dual-reporter firefly luciferase and enhanced green fluorescent protein positive (Fluc+-eGFP+) transgenic mice, were exposed to normoxic condition and hypoxic stress for 12 h, 24 h, and 48 h, respectively. In addition, BM-MSCs were treated with irisin (20 nmol/L) and overexpression of FNDC5 (FNDC5-OV) in serum deprivation (H/SD) injury. Furthermore, BM-MSCs were engrafted into infarcted hearts with or without FNDC5-OV. RESULTS: Hypoxic stress contributed to increased apoptosis, decreased cell viability, and paracrine effects of BM-MSCs while irisin or FNDC5-OV alleviated these injuries. Longitudinal in vivo bioluminescence imaging and immunofluorescence results illustrated that BM-MSCs with overexpression of FNDC5 treatment (FNDC5-MSCs) improved the survival of transplanted BM-MSCs, which ameliorated the increased apoptosis and decreased angiogenesis of BM-MSCs in vivo. Interestingly, FNDC5-OV elevated the secretion of exosomes in BM-MSCs. Furthermore, FNDC5-MSC therapy significantly reduced fibrosis and alleviated injured heart function. CONCLUSIONS: The present study indicated that irisin or FNDC5 improved BM-MSC engraftment and paracrine effects in infarcted hearts, which might provide a potential therapeutic target for MI.

Irisin Attenuates Myocardial Ischemia/Reperfusion Injury and Improves Mitochondrial Function Through AMPK Pathway in Diabetic Mice.

AIMS: Several recent reports have shown irisin protects the heart against ischemia/reperfusion injury. However, the effect of irisin on I/R injury in diabetic mice has not been described. The present study was designed to investigate the role of irisin in myocardial ischemia-reperfusion (MI/R) injury in diabetic mice. METHODS: A mouse model of diabetes was established by feeding wild type or gene-manipulated adult male mice with a high-fat diet. All the mice received intraperitoneal injection of irisin or PBS. Thirty minutes after injection, mice were subjected to 30 min of myocardial ischemia followed by 3h (for cell apoptosis and protein determination), 24 h (for infarct size and cardiac function). RESULTS: Knock-out of gene FNDC5 augmented MI/R injury in diabetic mice, while irisin treatment attenuated MI/R injury, improved cardiac function, cellular ATP biogenetics, mitochondria potential, and impaired mitochondrion-related cell death. More severely impaired AMPK pathway was observed in diabetic FNDC5-/- mice received MI/R. Knock-out of gene AMPK blocks the beneficial effects of irisin on MI/R injury, cardiac function, cellular ATP biogenetics, mitochondria potential, and mitochondrion-related cell death. CONCLUSIONS: Our present study demonstrated that irisin improves the mitochondria function and attenuates MI/R injury in diabetic mice through AMPK pathway.

Autophagy mediates the beneficial effect of hypoxic preconditioning on bone marrow mesenchymal stem cells for the therapy of myocardial infarction.

BACKGROUND: Stem cell therapy has emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the poor viability of transplanted stem cells hampers their therapeutic efficacy. Hypoxic preconditioning (HPC) can effectively promote the survival of stem cells. The aim of this study was to investigate whether HPC improved the functional survival of bone marrow mesenchymal stem cells (BM-MSCs) and increased their cardiac protective effect. METHODS: BM-MSCs, isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP), were preconditioned with HPC (1% O2) for 12 h, 24 h, 36 h, and 48 h, respectively, followed by 24 h of hypoxia and serum deprivation (H/SD) injury. RESULTS: HPC dose-dependently increased the autophagy in BM-MSCs. However, the protective effects of HPC for 24 h are most pronounced. Moreover, hypoxic preconditioned BM-MSCs (HPCMSCs) and nonhypoxic preconditioned BM-MSCs (NPCMSCs) were transplanted into infarcted hearts. Longitudinal in vivo bioluminescence imaging (BLI) and immunofluorescent staining revealed that HPC enhanced the survival of engrafted BM-MSCs. Furthermore, HPCMSCs significantly reduced fibrosis, decreased apoptotic cardiomyocytes, and preserved heart function. However, the beneficial effect of HPC was abolished by autophagy inhibition with 3-methyladenine (3-MA) and Atg7siRNA. CONCLUSION: This study demonstrates that HPC may improve the functional survival and the therapeutic efficiencies of engrafted BM-MSCs, at least in part through autophagy regulation. Hypoxic preconditioning may serve as a promising strategy for optimizing cell-based cardiac regenerative therapy.

[Study on preparation of the pH sensitive hydroxyethyl chitin/poly (acrylic acid) hydrogel and its drug release property].

Hydroxyethyl chitin (HECH) is a water soluble chitin derivative made by etherification of chitin, ethylene chlorohydrin was used as etherification reagent in this reaction. A novel interpenetrating polymer network (IPN) composed of HECH/PAA was prepared. The IR spectra confirmed that HECH/PAA was formed through chemical bond interaction. The sensitivity of this hydrogel to temperature and pH was studied. The swelling ratio of this hydrogel in artificial intestinal juice is much greater than that in artificial gastric juice. The IPN hydrogel exhibited a typical pH-sensitivity, and its degree of swelling ratio increased with the increase of temperature. The sustained-release drug system of Dichlofenac potassium was prepared by using HECH/PAA as the drug carrier. The release experiment showed a perfect release behavior in artificial intestinal juice. This IPN is expected to be used as a good drug delivery system of enteric medicine.

[Studies on the preparation and characterization of immobilized neutral protease by carboxymethyl chitosan microsphere].

AS1. 398 neutral protease was immobilized onto carboxymethyl chitosan with glutaraldehyde as cross-linking agent. The effects of pH, time of cross-linking, amount of cross-linking agent and the ratio of enzyme to carrier on the activity of the immobilized enzyme were demonstrated, and the optimum immobilization condition of AS1. 398 neutral protease was established. Also studied was the characterization of immobilized enzyme,including pH, temperature, Km and the stability of storage.

The diagnostic performance of coronary artery angiography with 64-MSCT and post 64-MSCT: systematic review and meta-analysis.

PURPOSE: To comprehensively investigate the diagnostic performance of coronary artery angiography with 64-MDCT and post 64-MDCT. MATERIALS AND METHODS: PubMed was searched for all published studies that evaluated coronary arteries with 64-MDCT and post 64-MDCT. The clinical diagnostic role was evaluated by applying the likelihood ratios (LRs) to calculate the post-test probability based on Bayes' theorem. RESULTS: 91 studies that met our inclusion criteria were ultimately included in the analysis. The pooled positive and negative LRs at patient level were 8.91 (95% CI, 7.53, 10.54) and 0.02 (CI, 0.01, 0.03), respectively. For studies that did not claim that non-evaluable segments were included, the pooled positive and negative LRs were 11.16 (CI, 8.90, 14.00) and 0.01 (CI, 0.01, 0.03), respectively. For studies including uninterruptable results, the diagnostic performance decreased, with the pooled positive LR 7.40 (CI, 6.00, 9.13) and negative LR 0.02 (CI, 0.01, 0.03). The areas under the summary ROC curve were 0.98 (CI, 0.97 to 0.99) for 64-MDCT and 0.96 (CI, 0.94 to 0.98) for post 64-MDCT, respectively. For references explicitly stating that the non-assessable segments were included during analysis, a post-test probability of negative results >95% and a positive post-test probability <95% could be obtained for patients with a pre-test probability of <73% for coronary artery disease (CAD). On the other hand, when the pre-test probability of CAD was >73%, the diagnostic role was reversed, with a positive post-test probability of CAD >95% and a negative post-test probability of CAD <95%. CONCLUSION: The diagnostic performance of post 64-MDCT does not increase as compared with 64-MDCT. CTA, overall, is a test of exclusion for patients with a pre-test probability of CAD<73%, while for patients with a pre-test probability of CAD>73%, CTA is a test used to confirm the presence of CAD.

Nebivolol protects against myocardial infarction injury via stimulation of beta 3-adrenergic receptors and nitric oxide signaling.

Nebivolol, third-generation ß-blocker, may activate ß3-adrenergic receptor (AR), which has been emerged as a novel and potential therapeutic targets for cardiovascular diseases. However, it is not known whether nebivolol administration plays a cardioprotective effect against myocardial infarction (MI) injury. Therefore, the present study was designed to clarify the effects of nebivolol on MI injury and to elucidate the underlying mechanism. MI model was constructed by left anterior descending (LAD) artery ligation. Nebivolol, ß3-AR antagonist (SR59230A), Nitro-L-arginine methylester (L-NAME) or vehicle was administered for 4 weeks after MI operation. Cardiac function was monitored by echocardiography. Moreover, the fibrosis and the apoptosis of myocardium were assessed by Masson's trichrome stain and TUNEL assay respectively 4 weeks after MI. Nebivolol administration reduced scar area by 68% compared with MI group (p<0.05). Meanwhile, nebivolol also decreased the myocardial apoptosis and improved the heart function after MI (p<0.05 vs. MI). These effects were associated with increased ß3-AR expression. Moreover, nebivolol treatment significantly increased the phosphorylation of endothelial NOS (eNOS) and the expression of neuronal NOS (nNOS). Conversely, the cardiac protective effects of nebivolol were abolished by SR and L-NAME. These results indicate that nebivolol protects against MI injury. Furthermore, the cardioprotective effects of nebivolol may be mediated by ß3-AR-eNOS/nNOS pathway.

Diagnostic accuracy of dual-source CT coronary angiography in patients with atrial fibrillation: meta analysis.

RATIONALE AND OBJECTIVE: To synthesize the available data to underscore the diagnostic accuracy of dual-source CT (DSCT) coronary angiography in patients with atrial fibrillation (AF). MATERIALS AND METHODS: We searched in the electronic databases of PubMed for all published studies that examined patients with AF using DSCT. We used an exact binomial rendition of the bivariate mixed-effects regression model to synthesize the diagnostic data. RESULTS: The positive and negative likelihood ratios (LRs) at the patient level were 6.0 (CI, 3.6-10.1) and 0.03(CI, 0.004-0.2), respectively. The negative predictive values higher than 90% were available for a CAD prevalence <78%. The pooled vessel- and segment-level estimates showed higher positive and negative LRs than the patient-level estimates (15.3 [CI, 9.8-23.9] and 0.1 [CI, 0.07-0.3]; 25.1 [CI, 10.8-58.5] and 0.2 [CI, 0.2-0.3], respectively). No statistically significant heterogeneity between studies and publication bias were found at the patient level estimate. A sensitivity analysis showed that no study influenced the pooled results larger than 0.02. CONCLUSIONS: Cardiac angiography with DSCT can be applied as an imaging test for ruling out CAD in patient with AF. However, DSCT angiography may be not an effective tool for risk stratification for the high negative LR at the artery and segment levels.