Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux.

The autophagy/apoptosis interaction has always been a focus of study in pathogenicity models. Neuritin is a neurotrophic factor that is highly expressed primarily in the central nervous system. Our previous study revealed that it protects against apoptosis in cortical neurons subjected to oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R), and later animal experiments revealed that it can increase the expression of the autophagy-related protein LC3. Whether this neuroprotective effect is closely related to autophagy is still unclear. In this study, we hypothesized that neuritin can promote autophagic flux to protect nerve cells after OGD/R. To verify this hypothesis, we induced OGD/R in primary cortical neurons and assessed cell viability by the CCK8 and LDH assays. Cell apoptosis was assessed by Annexin V-FITC/PI, staining, and the contents and mRNA abundances of the autophagy-related proteins LC3 and p62, the apoptotic protein Caspase3 were quantified by Western blotting and RT-PCR. Autophagic flux was assessed by immunofluorescence after RFP-GFP-LC3 virus transfection, and ultrastructural changes in autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability was decreased, apoptosis was increased and autophagy was enhanced after OGD/R. Neuritin significantly increased cell viability, decreased apoptosis, further increased the expression of the autophagic flux-related protein LC3, further decreased p62 expression, and significantly increased the autophagosome number and autophagosome to lysosome ratio. Bafilomycin A1 (BafA1) is a late autophagy inhibitor, aggravated cell damage and apoptosis and counteracted the enhancement of autophagy activation and protective effects of neuritin. In conclusion, neuritin may promote the completion of autophagic flux by ameliorating neuronal damage after OGD/R.

[Bioinformatics analysis of the microRNA expression profile in the peripheral blood lymphocytes of Kazakh patients with essential hypertension in Xinjiang].

This study aimed to investigate the differential expression profiles of microRNAs (miRNAs) in peripheral blood lymphocytes between patients with essential hypertension and healthy individuals in Xinjiang Kazakh and to provide insight into the mechanism involved in the pathogenesis of hypertension in this ethnic group. From April 2016 to May 2019, 30 Kazakh patients with essential hypertension in the inpatient and outpatient departments of Cardiology, First Affiliated Hospital of Shihezi University were used as the hypertension group; 30 healthy Kazakh patients were used as the control group. The miRNA expression profiles in peripheral blood lymphocytes of 6 Kazakh hypertensive patients and 6 matched healthy individuals were compared, and the differentially expressed miRNAs were analyzed by cluster analysis, GSEA enrichment analysis, target gene prediction, target gene annotation and other bioinformatics analyses. In addition, qRT-PCR was used to verify the differentially expressed miRNAs. The results showed that compared with the control group, 73 differentially expressed miRNAs were identified in the hypertension group, of which 39 miRNAs were up-regulated and 34 miRNAs were down-regulated. A total of 11 miRNAs related to hypertension were screened by GSEA enrichment analysis, including hsa-miR-100-5p, hsa-miR-150-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-296-5p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p, hsa-miR-874-3p, hsa-miR-543 and hsa-miR-940. qRT-PCR test found that the expression of hsa-miR-100-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p and hsa-miR-543 was up-regulated, while the expression of hsa-miR-150-5p, hsa-miR-296-5p, hsa-miR-874-3p and hsa-miR-940 was down-regulated in the hypertension group compared with the control group. The expression trend in the gene chip was consistent with the results verified by qRT-PCR. Using online database to predict target genes of 11 miRNAs related to hypertension, we found that a total of 1 647 target genes might be regulated by these 11 miRNAs. GO function enrichment showed that (a) in biological processes, the predicted hypertension related target genes are mainly relevant to nervous system development, cellular localization, regulation of cellular metabolic process, generation of neurons and positive regulation of biological process; (b) In terms of cellular components, they are mainly related to membrane-bounded organelle, cytoplasm, intracellular membrane-bounded organelle, synapse part, neuron part, and nucleoplasm; (c) In terms of molecular function, they are mainly related to protein binding, transcription regulatory region DNA binding, RNA polymerase II regulatory region DNA binding, transcription regulator activity, and ion binding. KEGG enrichment analysis showed that the p53 signaling pathway, adrenergic signaling in cardiomyocytes, cAMP signaling pathway, TGF-ß signaling pathway, endocrine and other factor-regulated calcium reabsorption, mTOR signaling pathway, and aldosterone-regulated sodium reabsorption may be related to the occurrence and development of hypertension. In conclusion, there are significant differences in the expression of miRNAs in peripheral blood lymphocytes between Kazakh patients with essential hypertension and healthy people. The differentially expressed miRNAs may be related to the occurrence and development of essential hypertension in Kazakh. However, the underlying mechanism needs to be further explored and verified.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension.

BACKGROUND: Recent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. MATERIAL AND METHODS: Our research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. RESULTS: Chronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. CONCLUSION: Our research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

[Down-regulation of the Expression of Senescence Proteins P16 and P21 by Activating Connexin 43 in the Smooth Muscle of Spiral Modiolar Artery of Guinea Pigs].

OBJECTIVE: To analyze the correlation between connexin 43 (Cx43) and the expression of P16 and P21, aging-related proteins, and to investigate the possible role of Cx43 in the development of cell senescence with an aging model prepared by D-galactose (D-gal) intervention in the vascular smooth muscle cells (VSMCs) of guinea pig spiral modiolar artery (SMA). METHODS: The VSMCs of guinea pig SMA were cultured with the adhesion method, and the markers of VSMCs were detected with immunofluorescence technique. The experiment has a control group, a D-gal group, and a group that received D-gal and gap junction agonist AAP10 intervention, hereafter referred to as the AAP10 group. Cell Counting Kit-8 (CCK-8) was used to check VSMC activity and to determine the concentration and duration of D-gal intervention. The mRNA expression of Cx43 in each group was checked with qRT-PCR. The expression of Cx43, P16 and P21 proteins in each group was examined with the Western blot. The expression and distribution of P16 and P21 proteins were examined with immunofluorescence assay. RESULTS: Immunofluorescence results showed that the positive expression rate of cell actin (α-SM-actin) was over 90%. CCK-8 results showed that the optimal concentration of D-gal intervention was 30 mg/mL and the intervention duration was 48 h. qRT-PCR test showed that the mRNA expression of Cx43 in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01), while it is higher in the AAP10 group than that of the D-gal group ( P<0.01); Western blot assay showed that the Cx43 expression level in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01) and the expression of P16 and P21 was significantly higher than that in the control group ( P<0.01), the expression of Cx43 protein in AAP10 group was significantly up-regulated compared with that in the D-gal group ( P<0.01), while the expression of P16 and P21 was down-regulated significantly ( P<0.01); The results of immunofluorescence showed that P16 and P21 were mainly expressed in the cell nucleus. Semi-quantitative analysis of fluorescence intensity showed that the level of P16 and P21 protein in the D-gal group was significantly higher than that in the control group, and the fluorescence intensity of AAP10 group was significantly lower than that in the D-gal group ( P<0.01). CONCLUSION: Up-regulation of Cx43 expression can reverse the D-gal-induced abnormal expression of P16 and P21, two aging-related proteins, in SMA. It is suggested that Cx43 on SMA may be involved in D-gal-induced cell senescence, which provides a theoretical basis and possible intervention target for the delay of cell senescence.

[G protein-coupled estrogen receptor alleviates cerebral ischemia-reperfusion injury through inhibiting endoplasmic reticulum stress].

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.

[Effects of Ramipril on the expression of connexin 43 in cerebral arteries of spontaneously hypertensive rats].

The present study was designed to examine whether Ramipril (an inhibitor of angiotensin-converting enzyme) affected spontaneous hypertension-induced injury of cerebral artery by regulating connexin 43 (Cx43) expression. Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were randomly divided into WKY, WKY + Ramipril, SHR, and SHR + Ramipril groups (n = 8). The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin-eosin (HE) staining was used to show vascular remodeling. The expression and distribution of Cx43 was determined by using immunofluorescence and immunohistochemistry analysis. The protein and mRNA levels of Cx43 were examined by Western blot and real-time PCR analysis, respectively. The results showed that chronic Ramipril treatment significantly attenuated blood pressure elevation (P < 0.01, n = 8) and blood vessel wall thickness in SHR (P < 0.01, n = 8). The cerebral artery contraction rate in the SHR group was higher than that in the WKY group (P < 0.05, n = 8). The cerebral artery contraction rate in the SHR + Ramipril group was lower than that in the SHR group (P < 0.05, n = 8). Pretreatment with 2-APB (Cx43 non-specific blocker) or Gap26 (Cx43 specific blocker) significantly decreased the vasoconstriction rate, while pretreatment with AAP10 (Cx43 non-specific agonist) significantly increased the vasoconstriction in the SHR + Ramipril group (P < 0.05, n = 8). In addition, the expression of Cx43 mRNA and protein in cerebral arteries of SHR group was higher than that of WKY group (P < 0.05, n = 8). The mRNA and protein expression of Cx43 in cerebral arteries of SHR + Ramipril group was significantly lower than that of SHR group (P < 0.05, n = 8). These results suggest that Ramipril can down-regulate the expression of Cx43 mRNA and protein in cerebral arterial cells of SHR, lower blood pressure, promote vasodilation, and improve arterial damage and vascular dysfunction caused by hypertension.

[Effects of calcium-sensitive receptors on 11ß-hydroxysteroid dehydrogenase type 2 and cortisol in neonatal mice with persistent pulmonary hypertension].

OBJECTIVE: To investigate the effects of calcium-sensitive receptors (CaSR) on the expression of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) and cortisol concentration in a neonatal mouse model of persistent pulmonary hypertension (PPH). METHODS: Fifty-six newborn C57BL/6 mice were randomly divided into a control group (n=14), a PPH group (n=14), an agonist group (n=14), and an inhibitor group (n=14). The mice in the PPH, agonist, and inhibitor groups were exposed to a 12% oxygen concentration, and the agonist group and inhibitor group were given CaSR agonist (GdCl3, 16 mg/kg) and CaSR antagonist (NPS2390, 1 mg/kg) intraperitoneally once a day, respectively. The mice in control group were exposed to air, and then injected with an equal volume of normal saline as those in the PPH group every day. All mice were treated for 14 days. Morphological examination of heart and lung tissues was performed using hematoxylin-eosin staining. The expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein in lung tissues were measured by qRT-PCR and Western blot respectively. Brain natriuretic peptide (BNP) and cortisol levels in lung tissues were determined using ELISA. RESULTS: Compared with the control group, the PPH group had significantly increased pulmonary artery wall thickness (WT%), ratio of right to left ventricular thickness (RV/LV), alveolar mean linear intercept, and BNP concentration and a significantly reduced radial alveolar count (P<0.05); compared with the PPH group, the agonist group showed significant increases in WT% and BNP concentration, while the inhibitor group showed significant reductions in the two indicators (P<0.05). Compared with the control group, the PPH group showed significant reductions in the expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein, but a significant increase in cortisol concentration (P<0.05); compared with the PPH group, the agonist group had significantly lower expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein, but a significant higher cortisol concentration, while the inhibitor group showed opposite changes in these indicators (P<0.05). CONCLUSIONS: CaSR may control the development and progression of PPH in newborn mice by regulating the expression of 11ß-HSD2 and cortisol concentration.

[Effect of calcium-sensitive receptors on endothelial nitric oxide synthase and nitric oxide in neonatal mice with persistent pulmonary hypertension].

OBJECTIVE: To study the effect of calcium-sensitive receptors (CaSR) on the expression of endothelial nitric oxide synthase (eNOS) and the concentration of nitric oxide (NO) in a neonatal mouse model of persistent pulmonary hypertension (PPH). METHODS: Eighty neonatal C57BL/6 mice were randomly divided into control, PPH, agonist and antagonist groups. The control group was exposed to air, and the other three groups were exposed to 12% oxygen. The agonist and antagonist groups were intraperitoneally injected with a CaSR agonist (GdCl3 16 mg/kg) and a CaSR antagonist (NPS2390, 1 mg/kg), respectively, while the PPH and control groups were intraperitoneally injected with normal saline instead. All mice were treated for 14 days. Alveolar development and pulmonary vessels were assessed by hematoxylin-eosin staining. The protein and mRNA expression of eNOS and its localization in lung tissues were determined by Western blot, qRT-PCR and immunohistochemistry. The levels of brain natriuretic peptide (BNP) and NO in lung homogenate were determined using ELISA. RESULTS: Compared with the control group, the PPH and agonist groups showed significant increases in alveolar mean linear intercept, the percent wall thickness of pulmonary arterioles, right to left ventricular wall thickness ratio (RV/LV) and BNP concentration, but a significant reduction in radial alveolar count (P<0.05). The antagonist group had significant improvements in all the above indices except RV/LV compared with the PPH and agonist groups (P<0.05). Compared with those in the control group, the protein and mRNA expression of eNOS and NO concentration were significantly increased in the PPH group and increased more significantly in the agonist group, but were significantly reduced in the antagonist group (P<0.05). CONCLUSIONS: CaSR plays an important role in the development of PPH in neonatal mice, possibly by increasing eNOS expression and NO concentration.

Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function.

BACKGROUND/AIMS: Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs). METHODS: Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca2+ ([Ca2+]i) and Cl- ([Cl-]i) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and α-smooth muscle actin (α-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively. RESULTS: ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl-]i in CFs. T16Ainh-A01 considerably decreased [Cl-]i in the nucleus, whereas CaCCinh-A01 reduced [Cl-]i in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca2+]i in CFs. ANO1 and α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs. CONCLUSION: Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future.

Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy.

Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining. This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride (NaBH4) and Sudan black B (SBB) alone (p < 0.05). Under maximum effective concentration, semi-quantitative analysis revealed that inhibitory effect of SBB on AF was superior to that of NaBH4 (P < 0.01). When the two chemicals were combined, we observed that background can be reduced, and specific staining can be optimized at optimum concentration. Intensity of renal tissue was examined by confocal λ scanning, which showed that peaks were located at the range of approximately 480 - 590 nm and similar to those of flavin and lipofuscin. These results indicated that combined use of NaBH4 and SBB, when targeted at different sources of AF in renal tissue, is the most effective means of reducing background and preserving specificity of fluorescent labels. In addition, this method does not interfere with various steps of immunofluorescence experiments.

The Protective Effect of Propofol Against Ischemia-Reperfusion Injury in the Interlobar Arteries: Reduction of Abnormal Cx43 Expression as a Possible Mechanism.

BACKGROUND/AIMS: This experimental study aims to observe whether the protective effect of propofol against renal ischemia-reperfusion injury (IRI) in the rat interlobar artery occurs through altered expression of the gap junction protein connexin 43 (Cx43). METHODS: This study randomly divided male Sprague Dawley (SD) rats into an untreated control group, a sham-operated control group (sham group), an ischemia-reperfusion group (IR group), a propofol group (propofol+IR group) and a fat emulsion group (Intralipid group). The ischemia/reperfusion model was prepared through resection of the right kidney and noninvasive arterial occlusion of the left kidney. Forty-five minutes after renal ischemia-reperfusion, an automatic biochemical analyzer was employed to measure blood urea nitrogen (BUN) and serum creatinine (SCr); changes in renal tissue pathology were observed using hematoxylin and eosin (HE) staining, and the vasomotor activity of the interlobar artery was detected using a pressure mechanogram technique. The protein expression of Cx43 in renal artery cross-sections was determined through western blotting. RESULTS: The experimental study confirmed that the BUN and SCr of rats markedly increased after ischemia-reperfusion injury; additionally, we observed some coagulation necrosis and shedding of cells, some solidification of nuclear chromatin, degeneration of cytoplasmic vacuoles, high renal interstitial vascular congestion and obvious inflammatory cell infiltration, characterized by focal hemorrhages. Furthermore, the contraction activity of the renal interlobar artery greatly decreased, and the tension of the arteries in the renal lobe increased remarkably. After the gap junction blocking agents 2-APB and Gap27 were applied, the systolic velocity of blood vessels and the vascular contraction rate both decreased. In addition, the expression of Cx43 in kidney tissues increased markedly. The damage was more severe after 24 h of ischemic reperfusion than after only 4 h. However, after pretreatment with propofol, regardless of whether ischemia-reperfusion was applied for 4 h or 24 h, the previously increased expression of Cx43 decreased obviously, and all forms of renal damage were reversed. CONCLUSION: Our research suggests new ways for propofol to relieve ischemia-reperfusion injury by decreasing the abnormal expression of the gap junction protein Cx43. This study reveals a novel mechanism for the action of propofol against IRI, and we hope this finding will lead to new treatments for IRI.

Increased expression and functionality of the gap junction in peripheral blood lymphocytes is associated with hypertension-mediated inflammation in spontaneously hypertensive rats.

BACKGROUND: Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs). METHODS: The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A). RESULTS: The percentage of CD4+ T cells and the CD4+/CD8+ ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8+ and CD4+CD25+ T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes. CONCLUSIONS: Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation.

Hydrogen Sulfide Attenuates Hypertensive Inflammation via Regulating Connexin Expression in Spontaneously Hypertensive Rats.

BACKGROUND Hydrogen sulfide (H2S) has anti-inflammatory and anti-hypertensive effects, and connexins (Cxs) are involved in regulation of immune homeostasis. In this study, we explored whether exogenous H2S prevents hypertensive inflammation by regulating Cxs expression of T lymphocytes in spontaneously hypertensive rats (SHR). MATERIAL AND METHODS We treated SHR with sodium hydrosulfide (NaHS) for 9 weeks. Vehicle-treated Wistar-Kyoto rats (WKYs) were used as a control. The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin and eosin staining was used to show vascular remodeling and renal injury. The percentage of T cell subtypes in peripheral blood, surface expressions of Cx40/Cx43 on T cell subtypes, and serum cytokines level were determined by flow cytometry or ELISA. Expression of Cx40/Cx43 proteins in peripheral blood lymphocytes was analyzed by Western blot. RESULTS Chronic NaHS treatment significantly attenuated blood pressure elevation, and inhibited inflammation of target organs, vascular remodeling, and renal injury in SHR. Exogenous NaHS also improved vascular function by attenuating KCl-stimulated vasoconstrictor response in basilar arteries of SHR. In addition, chronic NaHS administration significantly suppressed inflammation of peripheral blood in SHR, as evidenced by the decreased serum levels of IL-2, IL-6, and CD4/CD8 ratio and the increased IL-10 level and percentage of regulatory T cells. NaHS treatment decreased hypertension-induced Cx40/Cx43 expressions in T lymphocytes from SHR. CONCLUSIONS Our data demonstrate that H2S reduces hypertensive inflammation, at least partly due to regulation of T cell subsets balance by Cx40/Cx43 expressions inhibition.

Enhanced gap junctional channel activity between vascular smooth muscle cells in cerebral artery of spontaneously hypertensive rats.

The aim of the present study is to investigate the effects of hypertension on the gap junctions between vascular smooth muscle cells (VSMCs) in the cerebral arteries (CAs) of spontaneously hypertensive rats (SHRs). The functions of gap junctions in the CAs of VSMCs in SHRs and control normotensive Wistar-Kyoto (WKY) rats were studied using whole-cell patch clamp recordings and pressure myography, and the expression levels of connexins were analyzed using reverse transcription-quantitative polymerase chain reaction and Western blot analyses. Whole-cell patch clamp measurements revealed that the membrane capacitance and conductance of in situ VSMCs in the CAs were significantly greater in SHRs than in WKY rats, suggesting that gap junction coupling is enhanced between VSMCs in the CAs of SHRs. Application of the endothelium-independent vasoconstrictors KCl or phenylephrine (PE) stimulated a greater vasoconstriction in the CAs of SHRs than in those of WKY rats. The EC50 value of KCl was 24.9 mM (n = 14) and 36.9 mM (n=12) for SHRs and WKY rats, respectively. The EC50 value of PE was 0.9 µM (n = 7) and 2.2 µM (n = 7) for SHRs and WKY rats, respectively. Gap junction inhibitors 18ß-glycyrrhetinic acid (18ß-GA), niflumic acid (NFA), and 2-aminoethoxydiphenyl borate (2-APB) attenuated KCl-induced vasoconstriction in SHRs and WKY rats. The mRNA and protein expression levels of the gap junction protein connexin 45 (Cx45) were significantly higher in the CAs of SHRs than in those of WKY rats. Phosphorylated Cx43 protein expression was significantly higher in the CAs of SHRs than in those of WKY rats, despite the total Cx43 mRNA and protein expression levels in the cerebral artery (CA) exhibiting no significant difference between SHRs and WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may promote gap junction communication among VSMCs in the CAs of SHRs, which may enhance the contractile response of the CA to vasoconstrictors.

[Role of calcium-sensing receptor in neonatal mice with persistent pulmonary hypertension].

OBJECTIVE: To study the effect of calcium-sensing receptor (CaSR) agonists and antagonists on the expression of CaSR in neonatal mice with persistent pulmonary hypertension (PPHN), and to clarify the role of CaSR in neonatal mice with PPHN. METHODS: Forty-nine neonatal mice were randomly divided into four groups: control (n=10), hypoxia (PPHN; n=11), agonist (n=13), and antagonist (n=15). The mice in the PPHN, agonist, and antagonist groups were exposed to an oxygen concentration of 12%, and those in the control group were exposed to the air. The mice in the agonist and antagonist groups were intraperitoneally injected with gadolinium chloride (16 mg/kg) and NPS2390 (1 mg/kg) respectively once daily. Those in the PPHN and the control groups were given normal saline daily. All the mice were treated for 14 consecutive days. Hematoxylin and eosin staining and immunohistochemistry were used to observe the changes in pulmonary vessels. Laser confocal microscopy was used to observe the site of CaSR expression and measure its content in lung tissues. qRT-PCR and Western blot were used to measure the mRNA and protein expression of CaSR in lung tissues. RESULTS: Compared with the control group, the PPHN group had significant increases in the pulmonary small artery wall thickness and the ratio of right to left ventricular wall thickness (P<0.05), which suggested that the model was successfully prepared. Compared with the control group, the PPHN group had a significant increase in the mRNA and protein expression of CaSR (P<0.05), and the agonist group had a significantly greater increase (P<0.05); the antagonist group had a significant reduction in the mRNA and protein expression of CaSR (P<0.05). CONCLUSIONS: CaSR may play an important role in the development of PPHN induced by hypoxia in neonatal mice.

The enhancement of Cx45 expression and function in renal interlobar artery of spontaneously hypertensive rats at different age.

BACKGROUND/AIMS: This study was designed to investigate the expression and function of gap junction protein connexin 45 (Cx45) in renal interlobar artery (RIA) of spontaneously hypertensive rats (SHR), and the association between hypertension and enhanced vasoconstrictive response in SHR. METHODS: Western blot analysis and pressure myography were used to examine the differences in expression and function of Cx45 in vascular smooth muscle cells (VSMCs) of RIA between SHR and normotensive Wistar-Kyoto (WKY) rats. RESULTS: Our results demonstrated that 1) whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ RIA VSMCs of SHR were significantly greater than those of WKY rats (p<0.05, n=6), suggesting that the coupling of gap junction between VSMCs of RIA was enhanced in SHR; 2) the KCl or phenylephrine (PE)-stimulated RIA constriction was more pronounced in SHR than that in WKY rats (p<0.05, n=10). After applying a gap junction inhibitor 18ß-glycyrrhetintic acid (18ß-GA), the inhibitory effect of 18ß-GA on KCl or PE-induced vasoconstriction was greater in SHR (p<0.05, n=10); and 3) the expression of Cx45 in RIA of SHR was greater than that in WKY rats (p<0.05, n=3) at 4, 12 and 48 wks of age. CONCLUSIONS: The hypertension-induced elevation of Cx45 may affect communication between VSMCs and coupling between VSMCs and endothelium, which results in an increased vasoconstrictive response in renal artery and might contribute to the development of hypertension.

[Isoliquiritigenin relaxes the cerebral basilar artery by enhancing BKCa current in spontaneously hypertensive rat: role of sGC/cGMP].

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.

PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 µmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 µmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 µmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 µmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCß. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²âº-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

[Effects of calcium-activated chloride channels on vascular activity of rat cerebral basilar artery].

This study investigated the role of calcium-activated Cl⁻ channels (CaCCs) in mediating vasomotor activity of cerebral basilar artery (BA) of Wistar rat. Pressure myograph was used to examine the changes in diameter of isolated BA to vasoactive reagents. The results showed that (1) The rate of pressure-induced vasomotor activity was 78.6% (n = 28) in BA from 0 to 100 mmHg working pressure. The contractile phase of the response was faster than the relaxation phase; (2) The amplitude of contraction was (62.6 ± 6.4) µm (n = 22), the frequency of contraction was variable and the highest value was 8.0 ± 2.3 per 5 min at 60 mmHg working pressure (n = 22); (3) The pressure-induced vasomotor activity of BA was markedly attenuated when Ca²âº was removed from medium; (4) The pressure-induced vasomotor activity was blocked by voltage dependent Ca²âº channel blocker nimodipine; (5) The pressure-induced vasomotor was inhibited by CaCC antagonists NFA and NPPB. These results suggest that the pressure-induced vasomotor activity of isolated BA is associated with Ca²âº influx that activates CaCCs.

[Inhibitory effect of 18ß-glycyrrhetinic acid on KCl- and PE-induced constriction of rat renal interlobar artery in vitro].

The aim of the present study is to investigate the effect of 18ß-glycyrrhetinic acid (18ß-GA) on KCl- and PE-induced constriction of rat renal interlobar artery (RIA). Pressure myograph system was used to observe the constriction induced by KCl and PE (endothelial independent vasoconstrictor) in acutely separated RIA of Wistar rats with or without 18ß-GA pretreatment. Whole-cell patch clamp recordings were used to observe the effect of 18ß-GA on membrane input capacitance (C(input)), membrane input conductance (G(input)) or membrane input resistance (R(input)) of smooth muscle cells embedded in arteriole segment. The results showed that both KCl (30-100 mmol/L) and PE (0.1-30 µmol/L) induced contraction of RIA in a concentration-dependent way. After pretreatment with 18ß-GA (100 µmol/L), KCl- or PE-induced constriction of RIA was significantly decreased. After application of 18ß-GA (100 µmol/L), the C(input), G(input) and R(input) of the in situ smooth muscle cells were very close to those of dispersed single smooth muscle cells. These results suggest 18ß-GA inhibits the contraction induced by KCl and PE, and the underlying mechanism may involve the inhibitory effect of 18ß-GA on gap junction.