The effects of noise exposure on hippocampal cognition in C57BL/6 mice via transcriptomics.

BACKGROUND: Noise is an important environmental stressor in the industrialized world and has received increasing attention in recent years. Although epidemiological research has extensively demonstrated the relationship between noise and cognitive impairment, the specific molecular mechanisms and targets remain to be fully explored and understood. METHODS: To address this issue, 5-month-old C57BL/6 mice were divided into two groups, with one group exposed to white noise at 98 dB. The effects of noise on cognition in mice were investigated through molecular biology and behavioral experiments. Subsequently, transcriptomic sequencing of the hippocampus in both groups of mice was performed and enrichment analysis of differentially expressed genes (DEGs) was conducted using KEGG and GO databases. Furthermore, LASSO analysis was used to further narrow down the relevant DEGs, followed by enrichment analysis of these genes using KEGG and GO databases. The DEGs were further validated by rt-qPCR. RESULTS: Following noise exposure, the hippocampus levels of inflammation-related factors increased, the phosphorylation of Tau protein increased, the postsynaptic density protein decreased, the number of Nissl bodies decreased, and cell shrinkage in the hippocampus increased. Moreover, the behavioral experiments manifest characteristics indicative of a decline in cognitive.A total of 472 DEGs were identified through transcriptomic analysis, and seven relevant genes were screened by the LASSO algorithm, which were further validated by PCR to confirm their consistency with the omics results. CONCLUSION: In conclusion, noise exposure affects cognitive function in mice through multiple pathways, and the omics results provide new evidence for the cognitive impairment induced by noise exposure.

Activation of Ventral Tegmental Area Dopaminergic Neurons Projecting to the Parabrachial Nucleus Promotes Emergence from Propofol Anesthesia in Male Rats.

Since the clinical introduction of general anesthesia, its underlying mechanisms have not been fully elucidated. The ventral tegmental area (VTA) and parabrachial nucleus (PBN) play pivotal roles in the mechanisms underlying general anesthesia. However, whether dopaminergic (DA) projections from the VTA to the PBN play a role in mediating the effects of general anesthesia is unclear. We microinjected 6-hydroxydopamine into the PBN to damage tyrosine hydroxylase positive (TH+) neurons and found a prolonged recovery time from propofol anesthesia. We used calcium fiber photometry recording to explore the activity of TH + neurons in the PBN. Then, we used chemogenetic and optogenetic approaches either activate the VTADA-PBN pathway, shortening the propofol anesthesia emergence time, or inhibit this pathway, prolonging the emergence time. These data indicate the crucial involvement of TH + neurons in the PBN in regulating emergence from propofol anesthesia, while the activation of the VTADA-PBN pathway facilitates the emergence of propofol anesthesia.

A network pharmacology- and transcriptomics-based investigation reveals an inhibitory role of ß-sitosterol in glioma via the EGFR/MAPK signaling pathway.

Glioma is characterized by rapid cell proliferation, aggressive invasion, altered apoptosis and a poor prognosis. ß-Sitosterol, a kind of phytosterol, has been shown to possess anticancer activities. Our current study aims to investigate the effects of ß-sitosterol on gliomas and reveal the underlying mechanisms. Our results show that ß-sitosterol effectively inhibits the growth of U87 cells by inhibiting proliferation and inducing G2/M phase arrest and apoptosis. In addition, ß-sitosterol inhibits migration by downregulating markers of epithelial-mesenchymal transition (EMT). Mechanistically, network pharmacology and transcriptomics approaches illustrate that the EGFR/MAPK signaling pathway may be responsible for the inhibitory effect of ß-sitosterol on glioma. Afterward, the results show that ß-sitosterol effectively suppresses the EGFR/MAPK signaling pathway. Moreover, ß-sitosterol significantly inhibits tumor growth in a U87 xenograft nude mouse model. ß-Sitosterol inhibits U87 cell proliferation and migration and induces apoptosis and cell cycle arrest in U87 cells by blocking the EGFR/MAPK signaling pathway. These results suggest that ß-sitosterol may be a promising therapeutic agent for the treatment of glioma.

Noise exposure increase apoptosis in the hippocampus of AD mice through the upregulation of CTSS.

In recent years, it has become an acknowledged fact that noise exposure can lead to cognitive impairments, and researchers have shown increasing interest in this area. However, the detrimental impact of noise exposure on Alzheimer's disease (AD) animal models might be considerably greater than on ordinary model mice, yet the mechanisms by which noise exposure affects the hippocampus in these models have been scarcely investigated. This study we used 4D Label-free proteomics to identify distinctive differentially expressed proteins in the hippocampus of AD model mice following noise exposure. Among these proteins, the presence of Cathepsin S(CTSS) cannot be disregarded. Utilizing experimental techniques such as Western blot, immunofluorescence, and rt-qPCR, we confirmed the expression of CTSS in the hippocampus of APP/PS1 mice after noise exposure. Additionally, we examined downstream molecules including P53,BCL-2, BAX, and CASPASE3 using KEGG pathway analysis. The results indicated an elevation in CTSS expression, a reduction in the anti-apoptotic gene BCL-2, and an increase in the expression of BAX and cleaved CASPASE3. Based on these findings, we hypothesize that noise exposure potentially heightens apoptosis within the hippocampus through upregulating CTSS expression, subsequently posing a threat to AD model animals.

PGC-1α affects cochlear pericytes migration in noise-exposed mice.

OBJECTIVE: The study aimed to observe the effects of noise exposure on the pericytes of the cochlear stria vascularis (SV) in mice and to investigate its molecular mechanism. METHOD: Male C57BL/6J mice aged 6-8 weeks were used as the subjects. Auditory Brainstem Response (ABR) was used to assess hearing loss. Hematoxylin and Eosin (HE) staining was conducted to observe morphological alterations in the SV. Immunofluorescence combined with transmission electron microscopy (TEM) was used to scrutinize changes in pericytes following acoustic injury. Western blotting (WB) was used to assess the expression variations of the migration-related protein Osteopontin (OPN). Evans Blue assay was performed to evaluate the permeability of the blood labyrinth barrier (BLB). 4-Hydroxynonenal (4-HNE) staining, in conjunction with measurements of Superoxide Dismutase (SOD), Malondialdehyde (MDA), and Catalase (CAT) content, was used to ascertain whether oxidative stress injury occurred in the SV. WB, combined with immunofluorescence, was used to examine alterations in the expression of proliferator-activated receptor-gamma coactivator 1α (PGC-1α) in the SV and pericytes. RESULTS: Noise exposure resulted in permanent hearing loss in C57BL/6J mice, accompanied by SV swelling, migration of pericytes from their vascular attachments, BLB leakage, elevated oxidative stress levels in the SV, and reduced expression of PGC-1α on both the SV and migrating pericytes. CONCLUSION: Noise exposure may potentially increase oxidative stress levels in the SV, downregulate the expression levels of PGC-1α, promote pericytes migration, and subsequently lead to an elevation in BLB permeability.

Activation of Kir2.1 improves myocardial fibrosis by inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

The inwardly rectifying potassium channel Kir2.1 is closely associated with many cardiovascular diseases. However, the effect and mechanism of Kir2.1 in diabetic cardiomyopathy remain unclear. In vivo, we use STZ to establish the model, and ventricular structural changes, myocardial inflammatory infiltration, and myocardial fibrosis severity are detected by echocardiography, histological staining, immunohistochemistry, and western blot analysis, respectively. In vitro, a myocardial fibrosis model is established with high glucose. The Kir2.1 current amplitude, intracellular calcium concentration, fibrosis-related proteins, and TGF-ß1/Smad pathway proteins are detected by whole-cell patch clamp, calcium probes, western blot analysis, and immunofluorescence, respectively. The in vivo results show that compared to diabetic cardiomyopathy, zacopride (a Kir2.1 selective agonist) significantly reduces the left ventricular systolic diameter and diastolic diameter, increases the left ventricular ejection fraction and left ventricular short-axis shortening, improves the degree of cell necrosis, and reduces the expression of myocardial interstitial fibrosis protein and collagen fibre deposition area. The in vitro results show that the current amplitude and protein expression of Kir2.1 are both decreased in the high glucose-induced myocardial fibrosis model. Additionally, zacopride significantly upregulates the expression of Kir2.1 and inhibits the expressions of the fibrosis-related proteins α-SMA, collagen I, and collagen III. Activation of Kir2.1 reduces the intracellular calcium concentration and inhibits the protein expressions of TGF-ß1 and p-Smad 2/3. Activation of Kir2.1 can improve myocardial fibrosis induced by diabetic cardiomyopathy, and the possible mechanism may be related to inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

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.

TGF-ß3/Smad3 Contributes to Isoflurane Postconditioning Against Cerebral Ischemia-Reperfusion Injury by Upregulating MEF2C.

Isoflurane postconditioning alleviates cerebral ischemic-reperfusion injury (CIRI), but the underlying mechanism has not been fully clarified. We previously demonstrated that the transforming growth factor beta-1 (TGF-ß1)/Smads signaling pathway is involved in the neuroprotective effect of isoflurane postconditioning. TGF-ß3 has a highly homologous sequence relative to that of TGF-ß1. In this study, we explored the roles of the TGF-ß3/Smad3 signaling pathway and myocyte enhancer factor 2C (MEF2C) in neuroprotection induced by isoflurane postconditioning. A CIRI rat model was established by middle cerebral artery occlusion for 1.5 h, followed by 24 h of reperfusion. Isoflurane postconditioning led to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons as compared with those of CIRI rats. Moreover, isoflurane postconditioning upregulated the expressions of TGF-ß3, p-Smad3, and MEF2C. However, the neuroprotective effect was reversed by pirfenidone, a TGF-ß3/Smad3 signaling pathway inhibitor. Also, pirfenidone treatment downregulated the expression of MEF2C. These results indicate that the TGF-ß3/Smad3 signaling pathway contributes to the neuroprotection of isoflurane postconditioning after CIRI and is possibly related to MEF2C.

[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 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.

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.