Heart failure (HF) is the end stage of various kinds of cardiovascular diseases and leads to a high mortality worldwide.Numerous studies have demonstrated that frequencies of CD4+CD25+Foxp3+ regulatory T cells (Tregs) are reduced in HF patients and properly expanding Tregs attenuates HF progression.Histone deacetylase (HDAC) 9 has been revealed to contribute to several cardiovascular and cerebrovascular diseases.Plenty of studies showed that HDAC9 negatively regulated the number and function of Tregs.Thus,we aim to investigate the expression of HDAC 9 in patients with chronic heart failure (CHF) and the relationship among HDAC9,Tregs and CHF.Our research showed a reduced number of Tregs and an increased expression of HDAC9 mRNA in CHF patients.Patients with CHF were divided into two groups by heart function grade of New York Heart Association (NYHA),we found that the HDAC9 mRNA expression level in NYHA grade Ⅱ-Ⅲ group were lower than that in NYHA grade Ⅳ group.More importantly,the correlation study suggested that the expression of HDAC9 mRNA was negatively correlated to Tregs frequency and left ventricular ejection fraction (LVEF),whereas positively correlated to larger left ventricular end-diastolic dimension (LVEDD) and B-type natriuretic peptide (BNP) in patients with CHF.The correlation studies also showed a positive correlation between HDAC9 and the severity of CHF.Our research suggests that HDAC9 may be a new indicator for assessing CHF and it may offer a new direction for research of CHF.

The inflammatory response is involved in the pathogenesis of the most common types of heart disease.Sanguinarine (SAN) has various pharmacological properties such as anti-inflammatory,antioxidant,antibacterial,antitumor,and immune-enhancing properties.However,few studies have investigated the effects of SAN on lipopolysaccharide (LPS)-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes.Therefore,in this study,H9c2 cells were co-treated with SAN and LPS,and the mRNA levels of pro-inflammation markers and the apoptosis rate were measured to clarify the effect of SAN on cardiac inflammation.The underlying mechanism was further investigated by detecting the activation of Toll-like receptor (TLR)4/nuclear factor-κB (NF-κB) signaling pathways.As a result,increased mRNA expression of interleukin (IL)-1 β,IL-6,and TNFα induced by LPS was attenuated after SAN treatment;LPS-induced apoptosis of H9c2 cardiomyocytes and cleaved-caspase 8,9,3 were all significantly reduced by SAN.Further experiments showed that the beneficial effect of SAN on blocking the inflammation and apoptosis of H9c2 cardiomyocytes induced by LPS was associated with suppression of the TLR4/NF-κB signaling pathway.It was suggested that SAN suppressed the LPS-induced inflammation and apoptosis of H9c2 cardiomyocytes,which may be mediated by inhibition of the TLR4/NF-κB signaling pathway.Thus,SAN may be a feasible therapy to treat sepsis patients with cardiac dysfunction.

The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer.After designing specific gRNA sequences targeting HPV 16 E6,generating hCas9-EGFP and E6-gRNA-RFP plasmids,and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids,we determined the titer of the pseudotype virus using the TCID50 method.We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells.Experimental subjects were divided into control group,empty virus group,E6-gRNA transfected group,Cas9 transfected group and Cas9+E6-gRNA transfected group.The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis,and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time.RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups;the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups.Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6.The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells,and there were two cutting zones in the Cas9+E6-gRNA transfected group.However,the empty virus group,E6-gRNA transfected group and Cas9 transfected group had no corresponding zone.Compared with those in the control group,the empty virus group,E6-gRNA transfected group and Cas9 transfected group,the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9+E6-gRNA transfected group (P＜0.01).In addition,the proliferation and migration abilities of SiHa cells were significantly inhibited (P＜0.01).There were no significant differences among the other groups.In contrast to the control group,the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model (P＜0.01).The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells,which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors.Taken together,these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease,particularly in HPV-related tumors.

Finding the novel drug from the effective components of traditional Chinese herbal medicine is a hotspot of the modern pharmacological research.Hyperoside (HYP) belongs to flavonoid glycosides,and it has various properties,such as anti-inflammation,anti-spasm,anti-diuretic,antitussive,lowering blood pressure,and lowering cholesterol effects as well as protective effects for the cardiac and cerebral blood vessels.The purpose of this study was to investigate the effects of HYP on inflammatory and apoptotic responses in vascular endothelial cells stimulated by lipopolysaccharide (LPS) and further to identify the possible mechanisms underlying these effects.In our study,human umbilical vein endothelial cells (HUVECs) were stimulated with 1 μg/mL LPS in the presence or absence of HYP (10,20 and 50 μrnol/L).Our results indicated that HYP alone exerted no cytotoxicity on HUVECs,while it had an up-regulatory effect on the viability of HUVECs induced by LPS in a dose-dependent manner;increased mRNA expression of IL-1β,IL-6,TNFα and iNOS induced by LPS was attenuated after treatment with HYP both in a dose-and time-dependent manner;LPS-induced HUVECs apoptosis and cleaved-caspase 8,9,3 were all significantly reduced by HYP.Furthermore,the possible pathway involved in apoptosis and inflammation by HYP was detected,and the results showed that when treated with HYP,LPS-induced mitochondrial membrane instability was significantly inhibited through up-regulation of Bcl-2 and down-regulation of Bax.Furthermore,the expression of TLR4 and the phosphorylation of Iκ Bα and p65 in LPS-treated cells were blocked by HYP.Our results suggested that HYP treatment prevented HUVECs from LPS-induced inflammation and apoptosis responses,which might be mediated by inhibiting TLR4/NFκB pathway.

The poor survival of mesenchymal stem cells (MSCs) compromises the efficacy of stem cell therapy.Growth factor deprivation is one of the important factors that have challenged the survival of donor MSCs in cell therapy.In this study,the aim was to evaluate the effect of serum deprivation on the cell death of MSCs and to investigate the underlying mechanisms.Apoptosis of MSCs was evaluated with Hoechst 33342/PI staining.Signaling pathways involved in serum-deprivation induced apoptosis were analyzed using Western blotting.The results revealed that serum deprivation induced apoptosis in MSCs within 72 h of treatment.Serum deprivation was shown to lead to protein expression alterations in Bax,Bcl-2,casepase-3,casepase-8,GRP78,and CHOP during experiments.The data suggested that the mitochondria death pathway,the extrinsic apoptotic pathway and the endoplastic reticulum(ER) stress pathway were all involved in MSCs apoptosis.The increase in expression of CHOP and the simultaneous decrease in Bcl-2 expression suggest a synergistic effect in apoptosis induction in both the mitochondrion and the ER.

Melatonin has been reported to inhibit hepatic fibrosis and the mechanism may be correlated to its anti-oxidant effect.Nevertheless,the mechanism is not completely identified.This study was conducted to investigate the effects of melatonin on TGF-β1/Smad signaling pathway in liver fibrosis in rats.The liver fibrosis model was made by the subcutaneous injection of CCl4.The liver pathology changes were detected using hematoxylin and eosin (H&E) staining and Van Gieson (VG) staining.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with an autoanalyzer.Glutathione peroxidase (GPx) activities and levels of malondialdehyde (MDA) and hydroxyproline (Hyp) in liver were evaluated by spectrophotometry.Expression levels of TGF-β1,Smad2/3,phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in liver were detected by immunohistochemistry and Western blot analysis.Results showed that melatonin suppressed CC14-induced liver fibrosis,along with an improvement in histological changes,significant decreases in pathologic grading sores and obvious decreases in Hyp levels in liver.Melatonin improved liver function indicated by decreased serum ALT and AST activities.In addition,melatonin exerted its anti-oxidant effects,as supported by decreased MDA levels and increased GPx activities in liver.Furthermore,melatonin inhibited TGF-β1/Smad pathway,as evidenced by decreased TGF-β1,Smad2/3 and p-Smad2/3 expression and increased Smad7 expression in liver.In conclusion,melatonin may suppress CCl4-induced hepatic fibrosis in rats via inhibiting TGF-β1/Smad pathway.It is possible for melatonin to be a potential reagent to treat and cure liver fibrosis.

Anoikis is a form of apoptosis induced upon cell detachment from extracellular matrix.It has been determined that acquisition of resistance to anoikis is a critical step for tumor cell metastasis.MiR-21,the most prominent oncomiR,plays an important role in tumor progression.In this study,we revealed that up-regulation of miR-21 in human esophageal adenocarcinoma (EA) is associated with lymph node metastasis and poor survival rate.Because of the established anti-apoptosis effect of miR-21,it is tempting to speculate that miR-21 might contribute to tumor metastasis by regulating anoikis,qRT-PCR analysis demonstrated that miR-21 expression in OE33/AR cells (subpopulation of human EA OE33 cells that acquired resistance to anoikis) was significantly increased.Also,transfection of miR-21 mimics provided OE33 cells resisting to anoikis.By luciferase assays,we verified that PDCD4 and PTEN were the functional targets of miR-21.In mouse model,via tail vein injection experiment,we showed that the metastasis formation of OE33 cells in vivo could be mediated by changing the miR-21 expression pattern.Taken together,our findings suggested that miR-21 was involved in the regulation of anoikis in human EA cells.Targeting miR-21 may provide a novel strategy to prevent metastasis.

Ulcerative colitis (UC) is a chronic inflammatory disease and its involvement area in colon is influenced by a complex network of gene interactions.We analyzed the weighted gene co-expression networks in microarray dataset from colonic mucosa of patients with UC and identified one gene co-expression module that was highly associated with the progression of involved area in UC colon (Pearson coefficient=0.81,P＜0.0001).In total,523 hub genes in this module were found to be involved in immune system process after enrichment analysis in Gene Ontology.By the STRING and Cytoscape analysis,we observed that interleukin-8 (IL-8) and matrix metalloproteinase-9 (MMP-9) were centered in the network of hub genes.We then detected the expression of IL-8 and MMP-9 in mucosa from left-sided colon of patients using quantitative PCR and immunofluorescence assay respectively.Both quantitative PCR and immunofluorescence assay revealed the expression levels of IL-8 and MMP-9 were significantly different among the healthy controls,left-sided colitis group and pancolitis group (P＜0.05).IL-8 and MMP-9 were detected with an enhanced expression in pancolitis as compared with left-sided colitis and healthy controls,respectively (P＜0.05).This study demonstrates that immune system process is indispensable in the progression of disease in colon,and identifies that IL-8 and MMP-9 play potential critical roles for the progression.

Variant pulmonary vein anatomy (PVA) has been reported to influence the recurrence of atrial fibrillation (AF) after radiofrequency ablation.However,the effects of PVA on AF in patients undergoing cryoballoon ablation (CBA) remain unknown.The present study aimed to examine the impact of PVA on the long-term outcome of CBA for AF.A total of 78 patients (mean age 60.7±10.9 years,64.1％ males) with symptomatic and drug-refractory paroxysmal AF were enrolled in the study.Left atrium (LA) and PVA acquired at computed tomography angiography (CTA) were reconstructed with CARTO(R) 3 SYSTEM.Patients were routinely evaluated by 24-hour Holter monitoring following CBA.Cox regression was used to detect the predictors of AF recurrence after CBA.The results showed abnormal PVA in 30 patients (38.5％) and 18 patients (23.1％) had left common PV (LCPV).Electrical pulmonary vein isolation was achieved in all patients.After a mean follow-up of 689.5±103.8 days,it was found that patients with abnormal PVA had similar AF recurrence rate to those with normal PVA (26.7％ vs.25.0％,P=0.54),and there was no significant difference in AF recurrence rate between LCPV patients and non-LCPV patients (33.7％ vs.23.3％,P=0.29).Cox regression analysis showed that AF duration (72.9±9.0 vs.42.3±43.2 months,HR 1.001;95％CI 1.003-1.014;P＜0.001) and cryo-applications of right-side PVs (3.0±1.6 vs.4.7±1.7,HR 0.661;95％ CI 0.473-0.925;P=0.016) were independent predictors of freedom from AF,but PVA was not identified as a predictor of long-term success.In conclusion,the variant PVA cannot significantly influence the long-term outcome of AF patients undergoing CBA;longer AF duration and less cryo-applications of right-side PVs are associated with higher AF recurrent rate.

The relationship between the levels of renalase and changes in proteinuria,hypertension,renal function,renal tubular epithelial cell apoptosis and B-cell lymphoma-2 (Bcl-2) expression was investigated in patients (chronic nephritis,primary nephrotic syndrome or other kidney disease) that underwent renal biopsy.The study group comprised 72 patients undergoing renal biopsy.Patient profiles and renal function were collected.Concentrations of renalase and Bcl-2 were measured by immunohistochemistry.Tubular injury was detected by periodic acid Schiff staining (PAS) and renal tubular epithelial cell apoptosis was assessed by TUNEL assay.The expression of renalase was significantly lower in renal biopsy specimens than in normal kidney tissues.There was a positive linear relationship between renalase and some serum and cardiac indices;a negative correlation was found between age,eGFR,Ccr and 24-h urinary protein.Renal tubule injury index and tubular epithelial cell apoptosis index showed a negative linear correlation with renalase.The results showed that renalase probably increased the expression of Bcl-2.By two independent samples t-test,renalase levels were significantly increased in the non-hypertension group than in the hypertension group.One-way ANOVA showed that renalase expression was higher in samples with Lee's grade Ⅲ than in those with Lee's grade V.The expression of renalase was significantly decreased in patients who underwent renal biopsy,and was also associated with blood and renal function.The research proved that renalase may reduce renal tubular injury and apoptosis of renal tubular epithelial cells through the mitochondrial apoptosis pathway,finally achieving the purpose of delaying the progress of renal failure.