The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy.

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE-/- mice were then assessed by performing hematoxylin-eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE-/- mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

MiR-92a expression profiling of exfoliated colonocytes isolated from feces for colorectal cancer screening.

OBJECTIVE: This study seeks to assess the efficacy of exfoliated colonocytes isolated from feces (ECIF) miR-92a as a clinical colorectal cancer diagnostic marker in a larger cohort. METHODS: Clinicopathologic data from colorectal cancer patients and health controls that underwent colonoscopy, as well as patients of other cancers diagnosed, were included. A total of 963 Chinese participants were enrolled, with 292 (27.4%) having colorectal cancer, 140 (14.5%) having other types of cancer, e.g., pancreatic, liver, oral, bile duct, esophagus, and stomach cancer, 171 (17.8%) having infection in the intestine, rectal, stomach, appendix, and gastrointestinal ulcer, and 360 (37.4%) of healthy controls. ECIF samples were gathered and miR-92a levels were detected using TaqMan probe-based miR-92a real-time quantitative PCR (RT-qPCR) kit developed by Shenzhen GeneBioHealth Co., Ltd. RESULTS: Through a series of experiments, we demonstrated that the Ep-LMB/Vi-LMB magnetic separation system is feasible, highly specific, and highly sensitive at a cutoff value of 1053 copies per 6 ng of ECIF RNA. ECIF miR-92a levels were significantly higher in colorectal cancer patients than in controls. Colorectal cancer detection sensitivity and specificity were 87.3% and 86.9% respectively. Furthermore, the performance of this miR-92a detection kit demonstrated that it is an effective tool for colorectal cancer, with a high sensitivity of 84.1%, even in early cancer stages (0, I, and II). Furthermore, tumor removal resulted in lower stool miR-92a levels (3.21±0.58 vs. 2.14±1.14, P < 0.0001, n = 65). CONCLUSION: Finally, the miR-92a RT-qPCR kit detects ECIF-increased miR-92a and could be used for colorectal cancer screening.

Empagliflozin inhibits macrophage inflammation through AMPK signaling pathway and plays an anti-atherosclerosis role.

OBJECTIVE: In recent years, some authoritative clinical studies have found that SGLT2 inhibitor can reduce cardiovascular risk in patients with diabetes, which may imply that SGLT2 inhibitor can play a role beyond lowering blood glucose. In this study, we explored the effect of empagliflozin on vascular atherosclerosis after removing the effect of diabetes. METHODS: The interaction between SGLT2 inhibitor and the AMPK(Adenosine 5'-monophosphate-activated protein kinase) signal pathway to attenuate atherosclerosis was studied in both spontaneously atherosclerotic mice in vivo and oxidized low-density lipoprotein(ox-LDL) induced macrophage inflammation model in vitro. In vivo experiment the aorta tree and aortic valve area were stained with oil red, and the level of inflammatory factors in the diseased tissue was evaluated by immunohistochemistry. Meanwhile, serum was collected to detect the levels of inflammatory factors. In vitro experiment, the RAW264.7 cell line was selected and ox-LDL was used to induce the release of proinflammatory factors, and different doses of empagliflozin were added. The phagocytosis of macrophages to ox-LDL density lipoprotein, and the expression of inflammatory factors at the protein and RNA levels were measured. RESULTS: Empagliflozin reduced the area of atherosclerotic plaque and macrophage infiltration in atherosclerotic plaques, decreased the expression of inflammatory factors in local plaque tissues and serum of APOE-/- mice fed with high-fat diet. Empagliflozin can improve the protein expression level of p-AMPK affected by ox-LDL in cell and reduce the gene expression level of inflammatory factors and protein expression level of NF-κB, thus playing an anti-atherosclerosis role. CONCLUSIONS: Empagliflozin improves energy metabolism and reduces the expression of inflammatory factors by activating AMPK. As empagliflozin inhibits atherosclerosis progression, it may be of use in prevention of cardiovascular diseases.

Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing.

AIMS: We focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. METHODS: Potential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. RESULTS: BMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. CONCLUSIONS: Exosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

Tolerogenic dendritic cells induced the enrichment of CD4+Foxp3+ regulatory T cells via TGF-ß in mesenteric lymph nodes of murine LPS-induced tolerance model.

Endotoxin tolerance is an important state for the prevention of lethal infection and inflammatory response, which is closely associated with the participation of innate immune cells. Moreover, mesenteric lymph nodes (MLNs)-resident immune cells, such as CD4+Foxp3+ regulatory T (Treg) cells and dendritic cells, play important roles in the maintenance of peripheral immune tolerance. However, the potential roles of these cells in MLNs in the development of endotoxin tolerance remain largely unknown. Recent research work showed that CD4+Foxp3+ Treg cells contributed to the development of endotoxin tolerance. Here, we further analyzed the possible change on CD4+Foxp3+Tregs population in MLNs in murine LPS-induced endotoxin tolerance model. Our data showed that the proportion and absolute number of CD4+Foxp3+Tregs, expressing altered levels of CTLA4 and GITR, significantly increased in MLNs of murine LPS-induced tolerance model. Moreover, the expression level of TGF-ß in MLNs also increased obviously. Furthermore, TGF-ß blockade could obviously reduce the proportion and absolute number of CD4+Foxp3+Tregs in MLNs and subsequently impair the protection effect against LPS rechallenge. Of note, we found that tolerogenic dendritic cell (Tol-DC), expressing lower levels of MHC-II and CD86 molecules, dominantly secreted TGF-ß in MLNs in murine LPS-induced tolerance model. In all, our data provided an unknown phenomenon that the total cell number of CD4+Foxp3+Tregs significantly increased in MLNs in endotoxin tolerance, which was related to MLN-resident TGF-ß secreting CD11c+DCs, providing a new fundamental basis for the understanding on the potential roles of MLN-resident immune cells in the development of endotoxin tolerance.

The prognostic value of miR-126 expression in non-small-cell lung cancer: a meta-analysis.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death. Growing evidence from recent studies have shown indicated that microRNA-126 (miR-126) played an important role in the progression of NSCLC. However, the potential value of miR-126 expression in prognosis of NSCLC remains to be fully elucidated. Here, we carried out a meta-analysis to assess the potential prognostic value of miR-126 for NSCLC. METHODS: PubMed, Embase, the Cochrane library, Web of Science, CNKI and WanFang database, as well as the reference of included studies, were searched to recognize pertinent studies until April 30, 2017. New castle-Ottawa scale was used to evaluate the quality of studies. Pooled hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS) was extracted by using a fixed-effects or a random-effects model on the basis of heterogeneity. Publication bias was evaluated by using Begg's tests. RESULTS: We identified four eligible trials involving 666 non-small-cell lung cancer patients in this meta-analysis. The results indicated that a high level of miR-126 played a favorable role in the overall survival (HR 0.73, 95% CI 0.61-0.86, fixed-effects model). There was no bias existed in this study. CONCLUSIONS: Our study showed that high expression level of miR-126 was a promising positive factor for OS for non-small cell lung cancer patients, and miR-126 might be a potential target for non-small-cell lung cancer therapy in the future.

Hepatitis B virus X protein activates Notch signaling by its effects on Notch1 and Notch4 in human hepatocellular carcinoma.

Deregulated expression of Notch receptors and abnormal activity of Notch signaling have been observed in a growing number of malignant tumors, however, the expression and activity of Notch in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and their relationship with HBV X protein (HBx) are still not fully elucidated. To address this, we examined the overall expression of Notch receptors in HBV-associated HCC tissues, analyzed their relationship with HBx, and further investigated the role of Notch signaling in HBx stable transfected HepG2 cells (HepG2X). The results showed that Notch signaling could be activated by HBx in HepG2 cells. The expression of cytoplasmic Notch1 or nuclear Notch4 was correlated with the expression of HBx in HBV-associated HCC tissues. The expression of cytoplasmic Notch1 or nuclear Notch4 could also be upregulated by HBx in HepG2X cells. The upregulation of Notch1 by HBx was through p38 MAPK pathway. Moreover, HBx was found to directly interact with Notch1, whereas, not with Notch4 in HepG2X cells. Suppression of Notch signaling by γ-secretase inhibitor (GSI) decreased cell growth, blocked cell cycle progression and induced cell apoptosis in HepG2X cells. The present study indicates that HBx activates Notch signaling by its effects on Notch1 and Notch4, and therefore, recruits Notch signaling as a downstream pathway contributing to its carcinogenic role in HBV-associated HCC.

[Endoscopic intranasal structure reconstruction surgery in treating rhinogenic headache].

OBJECTIVE: To study the efficacy of endoscopic intranasal structure reconstruction surgery for rhinogenic headache. METHOD: One hundred and seventy cases of rhinologic headache were examined by nasal endoscopy and CT scan. The various abnormal anatomy were found in the patients. The nasal abnormality were corrected by nasal endoscopic surgery. RESULT: Followed up survey 6-12 months postoperatively, 156 cases were cured, the cure rate of 183 cases was 85.2%,18 cases were obviously recovered (9.8%). 9 cases did not recover. The clinical cure rate was 95.0%. CONCLUSION: Rhinogenic headache was caused by anatomic abnormality or disease. Endoscopic sinus surgery can accurately and completely clear the ill tissue, reconstruct the nasal structure, which have good therapeutic effect and important clinical value.

Highly accurate two-gene signature for gastric cancer.

Large amount of expression data were generated by high-throughput experimental techniques such as microarray. Single algorithm cannot be widely accepted as suitable method for mining of gene expression data. Therefore, integration of different algorithms and extraction of more useful information from the expression data are the key problems for identification of biomarkers. Here, we used three machine learning algorithms to select feature genes based on gene profiling data of gastric cancer (GC). Then, a common divisor was extracted as candidate feature genes aggregation for Tree Building and Tree Pruning analysis by Decision Tree (DT) algorithm. Real-time quantitative PCR and immunohistochemistry (IHC) staining were used to validate the relative expression levels of the candidate feature genes. Receiver operating characteristic curves were used to analyse the classification sensitivity and specificity of the feature genes. A total of 174, 202, 149 feature genes were selected by Class Information Index, Information Gain Index and Relief algorithms, with a common divisor consisting of 32 genes. Using a DT algorithm to contribute to the classification rule sets, we identified COL2A1 and ATP4B as candidate biomarkers of GC. The expression levels of these two genes were validated by real-time PCR and IHC with high sensitivity (>90 %) and specificity (>90 %) in both training and test samples. We first introduced an integral and systematic data-mining model for identification of biomarkers based on gene expression data. The two-gene signature obtained by our predictive model could be used for recognizing the biological characteristic of GC.

Identification of recurrence-related genes by integrating microRNA and gene expression profiling of gastric cancer.

We previously analyzed the microRNA (miRNA) expression pattern in gastric cancer with and without recurrence and obtained 17 differentially expressed miRNAs with potential to predict recurrence risk for GC patients. In the present study, we aimed to investigate recurrence-related genes which may be regulated by the differentially expressed miRNAs identified in our prior research. Three different miRNA target gene databases (miRanda, TargetScan and PicTar) were used for searching the potential genes regulated by miRNAs. A combination was performed between miRNA target genes and recurrence-related gene expression profiling. Three bioinformatics algorithms (PAM, SVM and RF) were used to feature recurrence-related gene selection. In addition, we validated the expression levels of the genes in GC patients using real-time PCR. A total of 3,263 genes were identified as potential targets of 17 miRNAs. We identified 2,736 differential expressed genes using the SAM method based on 22K oligo microarray data which included 7 recurrence and 4 without recurrence GC samples. Combining the target genes regulated by miRNAs and the differentially expressed genes between recurrence and non-recurrence groups, we identified 228 differential genes for further study. Finally, we identified HNRPA0 and PRDM4 as risk biomarkers of GC patients, which were regulated by hsa-miR­194 and hsa-miR-373, respectively. Our data indicated that HNRPA0 and PRDM4 may be involved in the recurrence process of GC and have potential to act as new prognostic biomarkers in predicting recurrence risk for gastric cancer patients.

Notch1 activation contributes to tumor cell growth and proliferation in human hepatocellular carcinoma HepG2 and SMMC7721 cells.

Notch signaling controls cellular differentiation and proliferation. Recent studies have shown that Notch signaling plays an important role in the carcinogenesis and progression of a growing number of malignant tumors. We investigated the effect of Notch1 activation on human hepatocellular carcinoma (HCC). In five human HCC cell lines, it was found that SMMC7721 had relatively high while HepG2 relatively low expression of Notch1 and the activity of Notch signaling. Notch1 activation by transfection of active intracellular region of Notch1 (ICN1) into HCC HepG2 cells enhanced cell growth and proliferation, including in vitro single cell colony formation, anchorage-independent proliferation, and in vivo tumorigenicity. Notch1 activation also promoted HepG2 cell cycle progression. Suppression of Notch1 activation by RNAi of Notch1 or by γ-secretase inhibitor (GSI) in HCC SMMC7721 cells decreased cell growth capability and blocked cell cycle progression. Moreover, it was found that suppression of Notch1 activation induced SMMC7721 cell apoptosis, as demonstrated by apoptosis assays. These findings indicate that Notch1 activation promotes human HCC cell growth and proliferation, which may contribute to the progression of this type of malignant carcinoma.

RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells.

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-J kappa (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.

Construction and expression of a eukaryotic expression vector containing a fusion gene of the Hantaan virus S gene and hsp70 gene.

Hantavirus (HV) infection leads to a kind of severe systematic syndrome, hemorrhagic fever with renal syndrome (HFRS). Heat shock proteins (HSPs) can be used as adjuvants assisting soluble antigens to produce specific targets which can be attacked by cytotoxic T lymphocytes. For further research on HFRS vaccine, this study aimed to express Hantaan virus nucleocapsid protein (HTNV NP)-HSP70 fusion protein in COS-7 cells. First, an HTNV S gene encoding NP was amplified by PCR with a mutated termination code and cloned into eukaryotic expression vector pCDNA3.1(+), into which the full-length hsp70 gene had already been inserted, to form the S-hsp70 fusion expression vector pCDNA3.1(+)/S-hsp70. Then this recombinant plasmid was transfected into COS-7 cells by liposome, and eukaryotic expression of NP-HSP70 fusion protein was detected by immunocytochemistry and western blot. The results show that the eukaryotic expression vector pCDNA3.1(+)/S-hsp70 was successfully constructed and the NP-HSP70 fusion protein was effectively expressed in COS-7 cells. This study demonstrates that the NP-HSP70 fusion protein was expressed effectively from the pCDNA3.1(+)/S-hsp70 vector in a eukaryotic system and thus provides a basis for using this plasmid as a new DNA vaccine against HV infection.

Risk factors for ulcerative colitis in a Chinese population: an age-matched and sex-matched case-control study.

BACKGROUND AND AIMS: Cigarette smoking, alcohol use, appendectomy, and family history of inflammatory bowel disease (IBD) have all been shown to be associated with IBD, but there were no reports of risk factors for IBD in a Chinese population in which the incidence of IBD is increasing during the past decade. We conducted a case-control study to examine associations between previously reported environmental risk factors and development of ulcerative colitis (UC) in Wuhan city, central China. METHODS: A total of 177 patients with UC and 177 age-matched and sex-matched controls were prospectively studied in Wuhan city from January 2004 to December 2004. An age-matched and sex-matched case-control study was conducted to assess the role of smoking, alcohol use, appendectomy, and other potential risk factors in the development of UC by a detailed questionnaire. RESULTS: Smoking was a protective factor and exsmoking is a risk factor for UC [compared with nonsmokers, smokers: odds ratios (OR)=0.28, 95% confidence intervals (CI): 0.16-0.48, P=0.0001; exsmokers: OR=4.36, 95%CI: 1.46-13.04, P=0.008]. Positive family history of IBD was a risk factor (OR=4.35, 95%CI: 1.21-15.71, P=0.025) whereas appendectomy was a protective factor (OR=0.24, 95%CI: 0.07-0.86, P=0.028) for UC. There were no significant associations between UC and other factors examined. CONCLUSIONS: Although the incidence of UC in Chinese is relatively lower than that in white, the same risk factors for UC that were reported in white populations were associated with Chinese UC patients. Specifically, smoking was a protective factor for UC and exsmoking was associated with an increase risk of UC in a Chinese population. Family history of IBD was shown to be a risk for UC, whereas appendectomy was associated with a low risk for UC.

Retrospective survey of 452 patients with inflammatory bowel disease in Wuhan city, central China.

BACKGROUND: Inflammatory bowel disease (IBD) had been uncommon in China until about 1990, but since then, it has been seen in the clinical setting more and more. The prevalence and phenotype of IBD in the Chinese population is not well known. The present study investigates the trend of prevalence in ulcerative colitis (UC) and Crohn's disease (CD) in Wuhan City, central China, and evaluates clinical features, extraintestinal manifestations, and the treatment of IBD in the last 14 years. METHODS: Three hundred and eighty-nine patients with UC and 63 patients with CD were retrospectively collected from 5 central hospitals in Wuhan City, in which high-quality endoscopic and histological diagnoses were available from 1990 to 2003. UC and CD were diagnosed based on clinical, experimental, radiological, endoscopic, and histological examinations according to the internationally accepted Lennard-Jones criteria. RESULTS: The trend toward prevalence of UC and CD increased between 1990 and 2003 in Wuhan City. There was no change in the sex and age distribution comparing 1990 to 1996 with 1997 to 2003 both in UC and CD. However, the number of individuals with higher education and a professional occupation during 1997 to 2003 was significantly higher than that during the period 1990 to 1996 in patients with UC (OR 2.1, 95% CI 1.27-3.35, P = 0.004; OR 2.2, 95% CI 1.31-3.61, P = 0.003). The mean age of patients with CD was significantly younger than that of UC at the time of diagnosis (32.6 +/- 12.5 vs. 42 +/- 14.5, P < 0.0001). The ratio of male to female patients was 1.53:1 in UC and 2.32:1 in CD, respectively. The mean duration of onset of the disease to diagnosis was 1.4 years in UC and 1.1 years in CD. The extra intestinal manifestations of UC and CD were 5.7% and 19%, respectively, and complications of UC and CD were 6.4% and 50.8%, respectively. Only 3% of UC patients required surgery, whereas 27% of CD patients underwent surgical procedures (P < 0.001). CONCLUSION: The prevalence of IBD has increased in Wuhan City, central China, but is not as high as in Western countries. The disease in Wuhan City has often been associated with young adult professional males with a high level of education. The clinical presentation of UC was often mild and had few extra intestinal manifestations.