AGEs-RAGE-KCa3.1 pathway mediates palmitic acid-induced migration of PBMCs from patients with type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronic low-grade systemic inflammation. Tissue infiltration by monocyte migration contributes to the pathogenesis of vascular complications in T2DM. We studied the role of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels in the palmitic acid (PA)-induced migration of peripheral blood mononuclear cells (PBMCs) from T2DM patients and the influence of advanced glycation endproducts (AGEs). A total of 49 T2DM patients and 33 healthy subjects was recruited into this study. Using flow cytometry and Western blotting analysis as well as cell migration assay, we found that there was a significant decrease in frequency of T lymphocytes and monocytes in CD45+ leukocyte population. PA at 100 µM stimulated migration of PBMCs from T2DM individuals, which was inhibited by the specific KCa3.1 channel blocker TRAM-34 (1 µM). The PBMC migration was positively correlated with glycosylated hemoglobin A1 chain (HbA1c) level of T2DM patients, an indicator of AGEs, and PBMCs with higher level of HbA1c showed upregulated expression of toll-like receptor (TLR) 2/4 and KCa3.1 channels. In THP-1 cells, AGEs at 200 µg/ml increased protein expression of TLR 2/4 and KCa3.1 channels, and were synergistically involved in PA-induced migration through receptors of AGEs (RAGE)-mediated KCa3.1 upregulation. In conclusion, in PBMCs of T2DM patients, AGEs promotes PA-induced migration via upregulation of TLR2/4 and KCa3.1 channels.

Epidemiology of norovirus gastroenteritis in hospitalized children under five years old in western China, 2015-2019.

OBJECTIVES: Norovirus is associated with one-fifth of all gastroenteritis cases, but basic epidemiological data is lacking, especially in developing countries. As long-term surveillance on norovirus gastroenteritis is scarce in western China, this study aims to update the epidemiological knowledge of norovirus gastroenteritis and to characterize the genotypes of norovirus strains. METHODS: Stool samples were collected from hospitalized children under 5 years old with gastroenteritis in Chengdu, China. All samples were tested for norovirus as well as rotavirus, sapovirus, enteric adenovirus, and astrovirus by real-time RT-PCR. RdRp and VP1 genes were sequenced in norovirus-positive samples to investigate viral phylogenies. RESULTS: Of the 1181 samples collected from 2015 to 2019, 242 (20.5%) were positive for norovirus. Among norovirus-positive cases, 65 cases had co-infection with another virus; norovirus/enteric adenovirus was most frequently detected (50.8%, 33/65). The highest positive rate was observed in children aged 13-18 months (23.7%, 68/287). Norovirus infection peaked in autumn (36.6%, 91/249), followed by summer (20.3%, 70/345). Pearson correlation analysis showed significant correlation between the norovirus-positive rate and humidity (r = 0.773, P < 0.05). GII.4 Sydney 2012 [P31] (48.5%, 79/163) and GII.3 [P12] (35.6%, 58/163) were the dominant norovirus strains. CONCLUSIONS: Norovirus has become one of the most common causes of viral gastroenteritis in children under 5 years old in western China. Continuous monitoring is imperative for predicting the emergence of new epidemic strains and for current vaccine development.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

Chronic islet inflammation is associated with development of type 2 diabetes mellitus (T2DM). Intermediate-conductance calcium-activated K+ (KCa3.1) channel plays an important role in inflammatory diseases. However, the role and regulation of KCa3.1 in pancreatic ß cells in progression of T2DM remain unclarified. In the present study, we evaluated the effect of the specific KCa3.1 channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) on diabetic phenotype in the db/db model. In diabetic mice, blockade of KCa3.1 significantly improved glucose tolerance, enhanced secretion of postprandial insulin level, and reduced loss of ß-cell mass through attenuating the expression and secretion of inflammatory mediators. Furthermore, in cultured pancreatic ß cells, exposure to high levels of glucose or palmitic acid significantly increased expression and current density of the KCa3.1 channel as well as secretion of proinflammatory chemokines, and the effects were similarly reversed by preincubation with TRAM-34 or a NF-κB inhibitor pyrrolidinedithiocarbamate. Additionally, expression of KCa3.1 in pancreas islet cells was up-regulated by activation of NF-κB with IL-1ß stimulation. In summary, up-regulated KCa3.1 due to activation of NF-κB pathway leads to pancreatic inflammation via expression and secretion of chemokines and cytokines by pancreatic ß cells, thereby facilitating progression of T2DM.-Pang, Z.-D., Wang, Y., Wang, X.-J., She, G., Ma, X.-Z., Song, Z., Zhao, L.-M., Wang, H.-F., Lai, B.-C., Gou, W., Du, X.-J., Deng, X.-L. KCa3.1 channel mediates inflammatory signaling of pancreatic ß cells and progression of type 2 diabetes mellitus.

The role and mechanism of KCa3.1 channels in human monocyte migration induced by palmitic acid.

Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate-conductance Ca2+-activated K+ (KCa3.1) channels play an important role in cell migration. However, the role of KCa3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP-1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of KCa3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA-treated monocytes exhibited increment of the protein levels of KCa3.1 channel and monocyte chemoattractant protein-1 (MCP-1), and the effects were reversed by co-incubation of PA with anti-TLR2/4 antibodies or by specific inhibitors of p38-MAPK, or NF-κB. In addition, PA increased monocyte migration, which was abolished by a specific KCa3.1 channel blocker, TRAM-34, or KCa3.1 small interfering RNA (siRNA). The expression and secretion of MCP-1 induced by PA was also similarly prevented by TRAM-34 and KCa3.1 siRNA. These results demonstrate for the first time that PA upregulates KCa3.1 channels through TLR2/4, p38-MAPK and NF-κB pathway to promote the expression of MCP-1, and then induce the trans-endothelial migration of monocytes.

The role of KCa3.1 channels in cardiac fibrosis induced by pressure overload in rats.

The intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels play a pivotal role in the proliferation and collagen secretion of cardiac fibroblasts. However, their contribution in cardiac fibrosis remains unknown. This study was designed to investigate whether KCa3.1 channels mediate the development of cardiac fibrosis. Pressure-overloaded rats were induced by abdominal aortic constriction and treated without or with KCa3.1 blocker (TRAM-34) or angiotensin type 1 receptor blocker (losartan) for 2 weeks. Besides the increase of blood pressure, angiotensin (Ang) II level in the plasma and myocardium, left ventricle mass and hydroxyproline concentration, myocardial hypertrophy, as well as significant collagen deposition in the perivascular regions and interstitium of the myocardium were observed in pressure-overloaded rats. The expression of leukocyte differentiation antigens (CD45 and CD3), macrophage surface marker (F4/80), tumor necrosis factor alpha, and monocyte chemotactic protein-1 (MCP-1) also significantly increased. All these alterations were prevented by losartan and TRAM-34. TRAM-34 also reduced the increase of renin and angiotensinogen in the plasma and myocardium of pressure-overloaded rats. Ang II promoted the migration of monocytes through endothelial cells and the secretion of MCP-1 from human umbilical vein endothelial cells in vitro, which was inhibited by TRAM-34. In conclusion, the present study demonstrates that TRAM-34 alleviates cardiac fibrosis induced by pressure overload, which is related to its inhibitory action on KCa3.1 channels and Ang II level. Our findings indicate that the inhibition of KCa3.1 channels may represent a novel approach of preventing the progression of cardiac fibrosis, and also add to the already developing literature of promising targets for TRAM-34.

An outbreak of type π vaccine-derived poliovirus in Sichuan province, China: emergence and circulation in an under-immunized population.

BACKGROUND: During August 2011-February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China. METHODS: A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment. RESULTS: One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8-1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5-12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February-May, 2012, and the strain has not been isolated since. CONCLUSION: High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary.

Advanced glycation end products impair K(Ca)3.1- and K(Ca)2.3-mediated vasodilatation via oxidative stress in rat mesenteric arteries.

The present study was designed to investigate the role of advanced glycation end products (AGEs) in intermediate-conductance and small-conductance Ca(2+)-activated potassium channels (KCa3.1 and KCa2.3)-mediated relaxation in rat resistance arteries and the underlying mechanism. The endothelial function of mesenteric arteries was assessed with the use of wire myography. Expression levels of KCa3.1 and KCa2.3 were measured by using Western blot. Reactive oxygen species (ROS) were measured by using dihydroethidium and 2', 7'-dichlorofluorescein diacetate. KCa3.1 and KCa2.3-mediated vasodilatation responses to acetylcholine and NS309 (opener of KCa3.1 and KCa2.3) were impaired by incubation of the third-order mesenteric arteries from normal rats with AGEs (200 µg ml(-1) for 3 h). In cultured human umbilical vein endothelial cells (HUVECs), AGEs increased ROS level and decreased the protein expression of KCa3.1 and KCa2.3. Antioxidant alpha lipoic acid restored the impairment in both vasodilatation function and expression of KCa3.1 and KCa2.3. H2O2 could mimic the effect of AGEs on the protein expression of KCa3.1 and KCa2.3 in cultured HUVECs. These results demonstrate for the first time that AGEs impaired KCa3.1 and KCa2.3-mediated vasodilatation in rat mesenteric arteries via downregulation of both KCa3.1 and KCa2.3, in which the enhanced oxidative stress was involved.