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Diabetes is closely related to hearing loss, but its specific pathogenesis is not clear. Cochlear stria vascularis and pericytes play an important role in hearing. The purpose of this study was to investigate whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes through oxidative stress, thus affecting hearing loss. The model of type Ⅱ diabetes was established in C57BL/6J mice. Compared with the control group, the hearing threshold of diabetic mice decreased continuously, the density of vascular stria decreased, the oxidative stress and apoptosis of various organs increased, and the level of antioxidation decreased. At the same time, after the pericytes were extracted from the stria vascularis and given in high glucose environment, the apoptosis level of pericytes increased, the content of ROS in pericytes increased, and the mitochondrial membrane potential decreased. AIF and cytochrome C in pericyte mitochondria were gradually released to the cytoplasm. On the basis of the intervention of high glucose, the release of mitochondrial AIF and cytochrome C from pericytes was alleviated and the level of pericyte apoptosis decreased by adding oxidative scavengers. It is clear that high glucose can pass through oxidative stress to induce mitochondrial apoptosis in cochlear vascular striated pericytes.

Increasing evidence suggests that transmembrane protein 16A (TMEM16A) in nociceptive neurons is an important molecular component contributing to peripheral pain transduction. The present study aimed to evaluate the role and mechanism of TMEM16A in chronic nociceptive responses elicited by spared nerve injury (SNI). In this study, SNI was used to induce neuropathic pain. Drugs were administered intrathecally. The expression and cellular localization of TMEM16A, the ERK pathway, and NK-1 in the dorsal root ganglion (DRG) were detected by western blot and immunofluorescence. Behavioral tests were used to evaluate the role of TMEM16A and p-ERK in SNI-induced persistent pain and hypersensitivity. The role of TMEM16A in the hyperexcitability of primary nociceptor neurons was assessed by electrophysiological recording. The results show that TMEM16A, p-ERK, and NK-1 are predominantly expressed in small neurons associated with nociceptive sensation. TMEM16A is colocalized with p-ERK/NK-1 in DRG. TMEM16A, the MEK/ERK pathway, and NK-1 are activated in DRG after SNI. ERK inhibitor or TMEM16A antagonist prevents SNI-induced allodynia. ERK and NK-1 are downstream of TMEM16A activation. Electrophysiological recording showed that CaCC current increases and intrathecal application of T16Ainh-A01, a selective TMEM16A inhibitor, reverses the hyperexcitability of DRG neurons harvested from rats after SNI. We conclude that TMEM16A activation in DRG leads to a positive interaction of the ERK pathway with activation of NK-1 production and is involved in the development of neuropathic pain after SNI. Also, the blockade of TMEM16A or inhibition of the downstream ERK pathway or NK-1 upregulation may prevent the development of neuropathic pain. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02520-9.

Based on the antibody typing classification, Helicobacter pylori infection can be divided into type I H. pylori infection and type II H. pylori infection. To observe the effects of different H. pylori infection types on the distribution of histopathological characteristics and the levels of three items of serum gastric function (PG I, PG II, G-17). 1175 cases from October 2018 to February 2020 were collected with ratio 1:2. All patients were performed with 14C-Urea breath test (14C-UBT), H. pylori antibody typing classification, three items of serum gastric function detection, painless gastroscopy, pathological examination, etc. According to H. pylori antibody typing classification, patients were divided into three groups: type I H. pylori infection group, type II H. pylori infection group and control group. Significant difference existed among type I H. pylori infection group, type II H. pylori infection group and control group in inflammation and activity (χ2 = 165.43, 354.88, P all χ2 = 67.99, P all H. pylori infection group and control group, the level of pepsinogen I, pepsinogen II, gastrin17 in type I H. pylori infection group increased, and PG I/PG II ratio (PG I/PG II ratio, PGR) decreased, which was statistically significant (χ2 = 35.08, 166.24, 134.21, 141.19; P all H. pylori infection worsened the severity of gastric mucosal inflammation and activity. H. pylori infection was prone to induce atrophy of gastric mucosa, while type I H. pylori infection played a key role in promoting the progress of atrophic gastritis and affected the level of serum gastric function. The study indicated that the eradication of H. pylori should be treated individually.

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the

Nav1.7 is closely associated with neuropathic pain. Hydrogen sulfide (H2S) has recently been reported to be involved in numerous biological functions, and it has been shown that H2S can enhance the sodium current density, and inhibiting the endogenous production of H2S mediated by cystathionine β-synthetase (CBS) using O-(carboxymethyl) hydroxylamine hemihydrochloride (AOAA) can significantly reduce the expression of Nav1.7 and thus the sodium current density in rat dorsal root ganglion (DRG) neurons. In the present study, it was shown that the fluorescence intensity of H2S was increased in a spared nerve injury (SNI) model and AOAA inhibited this increase. Nav1.7 is expressed in DRG neurons, and the expression of CBS and Nav1.7 were increased in DRG neurons 7, 14 and 21 days post-operation. AOAA inhibited the increase in the expression of CBS, phosphorylated (p)-MEK1/2, p-ERK1/2 and Nav1.7 induced by SNI, and U0126 (a MEK blocker) was able to inhibit the increase in p-MEK1/2, p-ERK1/2 and Nav1.7 expression. However, PF-04856264 did not inhibit the increase in CBS, p-MEK1/2, p-ERK1/2 or Nav1.7 expression induced by SNI surgery. The current density of Nav1.7 was significantly increased in the SNI model and administration of AOAA and U0126 both significantly decreased the density. In addition, AOAA, U0126 and PF-04856264 inhibited the decrease in rheobase, and the increase in action potential induced by SNI in DRG neurons. There was no significant difference in thermal withdrawal latency among each group. However, the time the animals spent with their paw lifted increased significantly following SNI, and the time the animals spent with their paw lifted decreased significantly following the administration of AOAA, U0126 and PF-04856264. In conclusion, these data show that Nav1.7 expression in DRG neurons is upregulated by CBS-derived endogenous H2S in an SNI model, contributing to the maintenance of neuropathic pain.

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-β1)/Smads signaling pathway is involved in the neuroprotective effect of isoflurane postconditioning. TGF-β3 has a highly homologous sequence relative to that of TGF-β1. In this study, we explored the roles of the TGF-β3/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-β3, p-Smad3, and MEF2C. However, the neuroprotective effect was reversed by pirfenidone, a TGF-β3/Smad3 signaling pathway inhibitor. Also, pirfenidone treatment downregulated the expression of MEF2C. These results indicate that the TGF-β3/Smad3 signaling pathway contributes to the neuroprotection of isoflurane postconditioning after CIRI and is possibly related to MEF2C.

Angiotensin II (AngII) serves an important inflammatory role in cardiovascular disease; it can induce macrophages to differentiate into the M1‑type, produce inflammatory cytokines and resist pathogen invasion, and can cause a certain degree of damage to the body. Previous studies have reported that connexin 43 (Cx43) and NF‑κB (p65) are involved in the AngII‑induced inflammatory pathways of macrophages; however, the mechanisms underlying the effects of Cx43 and NF‑κB (p65) on AngII‑induced macrophage polarization have not been determined. Thus, the present study aimed to investigate the effects of Cx43 and NF‑κB (p65) on the polarization process of AngII‑induced macrophages. The macrophage polarization‑related proteins and mRNAs were examined by flow cytometry, western blotting, immunofluorescence, ELISA and reverse transcription‑quantitative PCR analyses. RAW264.7 macrophages were treated with AngII to simulate chronic inflammation and it was subsequently found that AngII promoted RAW 264.7 macrophage polarization towards the M1‑type by such effects as the release of inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)‑α, IL‑1β, the secretion of IL‑6, and the expression of M1‑type indicators, such as CD86. Simultaneously, compared with the control group, the protein expression levels of Cx43 and phosphorylated (p)‑p65 were significantly increased following AngII treatment. The M1‑related phenotypic indicators, iNOS, TNF‑α, IL‑1β, IL‑6 and CD86, were inhibited by the NF‑κB (p65) signalling pathway inhibitor BAY117082. Similarly, the Cx43 inhibitors, Gap26 and Gap19, also inhibited the expression of M1‑related factors, and the protein expression levels of p‑p65 in the Gap26/Gap19 groups were significantly decreased compared with the AngII group. Altogether, these findings suggested that AngII may induce the polarization of RAW264.7 macrophages to the M1‑type through the Cx43/NF‑κB (p65) signalling pathway.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl– (chloride ion) fluorescent probe technique was used to evaluate the change of Cl– concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl– fluorescent probe whose fluorescence intensity decreases as Cl– concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl– concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

The aim of the study was to clarify the effect of melatonin on neuropathic pain by N-type calcium channel (Cav2.2) inhibition in dorsal root ganglion (DRG) neurons after spared nerve injury (SNI) surgery. Immunofluorescence was used to identify the co-expression of Cav2.2 and the MT2 receptor and detect the changes in Cav2.2 expression in DRG neurons. Western-blot was also performed to detect the expression of Cav2.2 in DRG neurons. The action potential and current of Cav2.2 channels in DRG neurons were detected using whole-cell patch clamp analysis. Behavioral studies were conducted using thermal stimulation and acetone after melatonin was injected intraperitoneally. The results revealed that Cav2.2 and the MT2 receptor were co-expressed in medium and small sized DRG neurons, and the intensity of Cav2.2 increased after SNI. Injection of melatonin activated the MT2 receptor and relieved nociceptive pain through decreased the Cav2.2 expression and current in DRG neurons. Melatonin can significantly decrease the increase in Cav2.2 current density and excitability after SNI. In addition, the Cav2.2 activation curve shifted to the left after SNI, but there was no change in inactivation. 10 μM melatonin significantly inhibited the excitability of DRG neurons and Cav2.2 current, the inactivation curve of Cav2.2 current shifted significantly to the left. However, the MT2 receptor antagonist 4-P-PDOT reversed the inhibition of melatonin on Cav2.2 current. We conclude that melatonin inhibits the increased Cav2.2 expression and current; on the other hand, it reduces the excitability of DRG neurons after SNI surgery via the MT2 receptor pathway.

Type 1 and type 2 cannabinoid receptors (CB1 and CB2, respectively) mediate cannabinoid-induced analgesia. Loss of endogenous CB1 is associated with hyperalgesia. However, the downstream targets affected by ablation of CB1 in primary sensory neurons remain unknown. In the present study, we hypothesized that conditional knockout of CB1 in primary sensory neurons (CB1cKO) alters downstream gene expression in the dorsal root ganglion (DRG) and that targeting these pathways alleviates neuropathic pain. We found that CB1cKO in primary sensory neurons induced by tamoxifen in adult Advillin-Cre:CB1-floxed mice showed persistent hyperalgesia. Transcriptome/RNA sequencing analysis of the DRG indicated that differentially expressed genes were enriched in energy regulation and complement and coagulation cascades at the early phase of CB1cKO, whereas pain regulation and nerve conduction pathways were affected at the late phase of CB1cKO. Chronic constriction injury in mice induced neuropathic pain and changed transcriptome expression in the DRG of CB1cKO mice, and differentially expressed genes were mainly associated with inflammatory and immune-related pathways. Nerve injury caused a much larger increase in CB2 expression in the DRG in CB1cKO than in wildtype mice. Interfering with downstream target genes of CB1, such as antagonizing CB2, inhibited activation of astrocytes, reduced neuroinflammation, and alleviated neuropathic pain. Our results demonstrate that CB1 in primary sensory neurons functions as an endogenous analgesic mediator. CB2 expression is regulated by CB1 and may be targeted for the treatment of neuropathic pain.

It is widely accepted that the stria vascularis (SV) in cochlea plays a critical role in the generation of endocochlear potential (EP) and the secretion of the endolymph. 17β-estradiol (E2) is the most potent and abundant endogenous estrogen during the premenopausal period, thus, considered as the reference estrogen. This study aimd to investigate the protective effect of E2 by promoting the expression of vascular endothelial growth factor (VEGF) and thus promoting the vascular regeneration of the SV in elderly mice. After being treated with E2 either in vivo or in vitro, the hearing threshold changes of C57BL/6J elder mice continuously reduced, endothelial cell morphology improved, the number of endothelial cells (ECs) tubular nodes increased significantly, the ability of tubular formation enhanced significantly and the expression of VEGF increased. In vitro, cell model in conjunction with in vivo ovariectomized model was established to demonstrate for the first time that E2 promotes angiogenesis by promoting the secretion of VEGF through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT). In conclusion, E2 demonstrated potent angiogenesis properties with significant protection against Age-Related Hearing Loss (ARHL), which provides a new idea for the improvement of ARHL.

The adaptive immune response mediated by T lymphocytes is a well-established factor in the pathogenesis of pulmonary inflammation. Changes in the expression of various connexins (Cxs) or disruption of connexin-mediated cellular communication in T lymphocytes contribute to inflammation or tissue remodeling. The aim of the present study was to investigate the potential therapeutic value of blocking Cxs in a monocrotaline (MCT)-induced pulmonary inflammation rat model. Carbenoxolone (CBX) was used to inhibit connexin-mediated cellular communication. An MCT rat model was established by intraperitoneal (i.p.) injection of a single dose of MCT (60 mg/kg), and CBX treatment (20 µg/kg/day, i.p.) was initiated on the day following MCT treatment for 28 days. Vehicle-treated male Sprague-Dawley rats were used as the negative control. The MCT rat model was evaluated by measuring the pulmonary artery flow acceleration time and right ventricular hypertrophy index (RVHI). Histopathological features of the lung tissues and pulmonary arteriolar remodeling were assessed. The proportions of T lymphocyte subtypes, Cx40/cx43 expression in the T cell subtypes and the cytokine levels in the plasma and the lung tissues were also analyzed. Pharmacological inhibition of Cxs using CBX attenuated MCT-induced right ventricular hypertrophy, pulmonary arteriolar remodeling, lung fibrosis and inflammatory cell infiltration by decreasing the RVHI, pulmonary arterial wall thickening, collagen deposition and pro-inflammatory cytokines production as well as CD3+ and CD4+ T cell accumulation in lung tissues of MCT-treated rats. Furthermore, flow cytometry analysis revealed that CBX may inhibit MCT-induced Cx40 and Cx43 expression in CD4+ and CD8+ T lymphocytes in lung tissues. The present study provides evidence that pharmacological inhibition of Cxs may attenuate MCT-induced pulmonary arteriolar remodeling and pulmonary inflammatory response, at least in part, by decreasing Cx expression. The results highlight the critical role of Cxs in T lymphocytes in the MCT-induced pulmonary inflammatory response and that targeting of Cxs may be a potential therapeutic method for treating pulmonary inflammatory diseases.

Aim The Wnt/β-catenin signaling pathway plays an important role in ischemia-reperfusion(I/R) injury, and the transforming growth factor(TGF)-β/Smad signaling pathway participates in the neuroprotection effect induced by isoflurane(ISO) postconditioning. In this study, we aimed to explore the role of the Wnt/|[beta]|-catenin β-catenin signaling pathway in the neuroprotection effect induced by ISO postconditioning, and investigate the interaction of Wnt/β-catenin and TGF-β/Smad signaling pathway in this neuroprotection effect. Methods Cerebral I/R injury was established in Sprague–Dawley rats by using the middle cerebral artery occlusion (MCAO) model for 90 min followed by 24 h reperfusion. Postconditioning by inhalation of ISO was performed for 60 min after ischemia at the onset of reperfusion. Neurological deficit scoring, 2,3,5-triphenyl tetrazolium chloride staining and Nissl staining were adopted to evaluate brain injury. Apoptosis of the hippocampus and cortex neurons was detected by TUNEL staining. The expression levels of Wnt3a, GSK-3β, β-catenin, Cyclin D1, VEGF, Caspase 3, TGF-β1, Smad3 and p-Smad3 were determined by immunofluorescence (IF) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Various targeted inhibitors were administered via intraperitoneal injection or lateral ventricle injection. Results In the cortex region, the neurological deficit score, infarct volumes and neuron apoptosis increased, and the expression level of the Wnt3a, GSK-3β, β-catenin, VEGF and Cyclin D1 decreased in the MCAO group compared with the Sham group. In the MCAO + ISO group, the neurological deficit score, infarct volumes and neuron apoptosis reduced significantly, the expression levels of Wnt3a, β-catenin, VEGF and Cyclin D1 increased, while the expression level of GSK-3β and Caspase 3 decreased relative to MCAO group. When Wnt inhibitor(DKK-1) was given in advance followed by ISO postconditioning, the neurological deficit score, infarct volumes, neuron apoptosis and the expression level of GSK-3β and Caspase 3 increased. qRT-PCR and IF showed similar changes in the protein levels of all groups. However, the expression level of β-catenin in nuclear and cytoplasm both decreased significantly after pre-injection with the TGF-β1 inhibitor(LY2157299) and Smad3 inhibitor(SIS3), whereas the expression levels of TGF-β1, Smad3 and p-Smad3 were almost unchanged. The expression levels of all the related proteins and morphological changes in the hippocampus region were consistent with that of the cortex. Conclusion ISO postconditioning can reduce cerebral I/R injury by activating the Wnt/β-catenin signaling pathway and may be related to the TGF-β/Smad3 signaling pathway.

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.

Pulmonary vascular remodelling is one of the most important factors for pulmonary hypertension (PH). Galectin-3 (Gal-3) is a β-galactoside-binding lectin. In the latest literature, Gal-3 has been reported to be involved in pulmonary vascular remodelling, and its underlying mechanism is unclear. Our research aims to prove the effect of Gal-3 on the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMC) induced by transforming growth factor β1 (TGF-β1) and to study its mechanism. In vivo experiment: In Sprague-Dawley (SD) rats, monocrotaline was injected intraperitoneally to establish a PH model, and the Gal-3 inhibitor (modified citrus pectin, MCP) 28 Ds was administered in the stomach. The results indicate that Gal-3 and TGF-β1 may be involved in the occurrence and development of PH, which may be related to the Smad2/3 signalling pathway. In vitro experiment: Human pulmonary artery smooth muscle cells were pretreated with the Gal-3 inhibitor (MCP) for 24 h, then TGF-β1 or Gal-3 was administered to the cells for 24 h. The results show that exogenous TGF-β1 and Gal-3 can activate the downstream Smad2/3 signalling pathway, and increase the proliferation and migration ability of HPASMC. However, the Gal-3 inhibitor (MCP) inhibited these effects. Further results display that TGF-β1 and Gal-3 could mutually regulate the protein and mRNA expression levels. In summary, the results of this study indicate that Gal-3 regulates the Smad2/3 signalling pathway through protein interaction with TGF-β1, in turn regulates the proliferation and migration of HPASMC, thereby regulating the occurrence and development of PH.

To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis.The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The CaThe current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency.The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.目的: 研究钙激活氯通道蛋白ANO1在心肌成纤维细胞(CFs)向成肌纤维细胞(MFs)分化过程中的表达规律及其电生理特性,探索ANO1在心肌纤维化进程中的作用。方法: 以刚分离贴壁的大鼠原代CFs及其分化的MFs为研究对象,利用全细胞膜片钳检测钙激活氯通道电流变化,采用免疫荧光标记技术和Western blot检测ANO1、α-SMA和vimentin在CFs和MFs细胞中的表达特征。结果: 刚贴壁培养的CFs钙激活氯通道电流密度远高于MFs;ANO1主要表达于CFs和MFs的细胞核周围及细胞核上,少量表达于细胞膜;ANO1在刚贴壁的CFs中表达较弱,而在有分裂增殖趋向的MFs细胞中表达较为明显。结论: ANO1的表达与MFs的分化过程密切相关,提示其可能参与调节心肌纤维化进程。.

Background: Currently, the specific mechanism of estrogen (E2) in protecting presbycusis is not clear. This study aimed to investigate whether E2 could affect the apoptosis of capillary pericytes (PCs) of cochlear stria vascularis (SV) in aged C57BL/6J mice by regulating transmembrane member 16A (TMEM16A), such that it plays a protective role in presbycusis.Methods: The model of C57BL/6J ovariectomized mice was established, and E2 was administered for 2 months. The hearing threshold was measured by auditory brainstem response (ABR). The changes in the cochlea were measured using hematoxylin-eosin (HE) and electron microscopy. qRT-PCR was used to examine the expression of TMEM16A and apoptosis-related protein mRNA. The PCs were cultured in vitro, and the cell senescence model was established by the continuous passage method. TMEM16A expression was assessed using immunofluorescence. Flow cytometry was performed to explore the apoptosis rate.Results: The results of animal experiments showed that E2 intervention could reduce hearing loss and improve the atrophy of cochlear SV, loss of PC chromatin organelles, cytoplasmic swelling and nuclear porosity in aged mice. E2 also decreased the mRNA expression of TMEM16A, Caspase-3 and Bax in cochlear SV of aged mice and upregulated the expression of Bcl-2 mRNA. Cellular experiments showed that E2 could downregulate the expression of TMEM16A, and E2 or T16Ainh-A01(a specific blocker of TMEM16A) could reduce the apoptosis rate of PCs in aged mice.Conclusions: E2 may inhibit the apoptosis of PCs through downregulating the expression of TMEM16A, which plays a protective role in presbycusis. This study may provide a novel potential treatment and prevention method for presbycusis.

Objective. The study aims to systematically evaluate the clinical effect of gabapentin in the treatment of postherpetic neuralgia (PHN). Method. Data were retrieved electronically from PubMed, Embase, CNKI, the China Biomedical Database, and the Library of Clinical Database, beginning from the time of inception to April 2017, in order to collect data on randomized controlled trials (RCTs) of gabapentin and placebo in PHN treatment. Results. A total of 11 RCTs (2376 people) were retrieved. The gabapentin group reported significantly reduced pain intensity [MD=−0.91, 95% CI −1.32 to −0.51, P Conclusion. Gabapentin can be used to effectively and safely treat PHN.

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

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.

Aquaporins (AQPs) are water-channels that play important roles in brain water homeostasis and cerebral edema induced by brain injury. This study aimed to investigate the relationship between AQP4, bone morphogenetic protein 4 (BMP4)/Smad1/5/8 signaling pathway and isoflurane post-conditiong, which has effects on brain edema in rats with cerebral ischemia/reperfusion (I/R) injury.Cerebral I/R injury was induced in rats by using the middle cerebral artery occlusion (MCAO) model for 90min, followed by 24h of reperfusion. Isoflurane post-conditioning (ISO) group received 90min ischemia and underwent 1.5% isoflurane post-conditioning for 60min after initiating reperfusion. Neurobehavior, brain water content, thionine staining and 2, 3, 5-triphenyl tetrazolium chloride staining were evaluated to measure levels of brain edema and damage. Expressions of AQP4, BMP4, Smad1/5/8 and phosphorylated Smad1/5/8 were detected by using Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence (IF) staining.Compared with the Sham group, neurological behavior score, brain infarct volume and water content of MCAO model rats increased with reperfusion injury. However, in the ISO group, cell edema and damage of brain was significantly ameliorated (P0.01). qRT-PCR showed less AQP4 mRNA expression in the hippocampal tissue of the ISO group than in the I/R group (P0.01). Western blot and immunofluorescence results showed similar changes in protein levels of both groups. Related protein expressions showed expressions of BMP4 and Smad1/5/8 increased in the ISO group (P0.01), whereas total Smad1/5/8 expression didn't change in all groups. When BMP4 inhibitor (LDN193189) was injected, expression levels of AQP4 increased and neuronal density decreased (P0.05). By contrast, expression levels of BMP4 did not change significantly after pre-injection of AQP4 inhibitor (TGN020) (P0.05), but neuronal density increased (P0.05).Isoflurane post-conditioning may inhibit occurrence of brain edema and reduce cerebral I/R injury through down-regulating expression of AQP4, This process may be related to the activation of BMP4/Smad1/5/8 signaling pathway.

The aim of the present study was to examine whether single-nucleotide polymorphisms (SNPs) of β1 subunit of large-conductance Ca2+-activated K+ channel (KCNMB1) and inwardly rectifying K+ channel, subfamily J, member-11 (KCNJ11) are associated with essential hypertension (EH) in Xinjiang Kazak Chinese patients. A polymerase chain reaction-restriction fragment length polymorphism technique was applied to detect the distribution of selected alleles and genotype frequencies in a cohort of Xinjiang Kazak Chinese patients. Samples from 267 patients with EH and 259 normotensive (NT) controls were analyzed. An unconditional logistic regression analysis was used to estimate the odds ratio and 95% confidence interval of the risk factors that are associated with the development of EH. Genotype and allele frequency analyses revealed that the frequency of genotypes KCNJ11-rs2285676 and KCNMB1-rs11739136 was not significantly different between the EH and NT groups. Individuals carrying the GG genotype of KCNJ11-rs5219 had a 2.08 times higher risk of having EH than individuals carrying the GA+AA genotype of KCNJ11-rs5219. Furthermore, the G allele frequency of KCNJ11-rs5219 in the EH group was significantly higher than that of the NT group (P=0.048). Additionally, logistic regression analysis revealed that the body weight and GG genotype of KCNJ11-rs5219 were positively associated with EH in Xinjiang Kazak Chinese patients (P

The current study observed the effects and investigated the mechanism of remifentanil (RMF) on the isolated cerebral basilar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. A pressure myograph system was used to observe and compare the effects of different concentrations of RMF (10−10-10−5 mol/l) on the diameter changes of freshly isolated cerebral basilar arteries, which have been pre-shrunk by phenylephrine (PE), an endothelium-independent vasoconstrictor. Vascular smooth-muscle cells of the cerebral basilar artery (BASMCs) were freshly obtained via enzymolysis. BKCa (large-conductance calcium-activated potassium channels) current (IBKCa) and Kv (voltage-gated potassium channels) current (IKv) were recorded using a whole-cell patch-clamp technique. The changes in IBKCa and IKv produced by different concentrations of RMF (10−10 to 10−5 mol/l) on the two types of rats with the holding potential of −40 mV were observed and compared. The cerebral basilar arteries of the SHR and WKY rats were relaxed by RMF in a concentration-dependent manner (P0.05). Outward currents were increased by RMF in both BASMCs of SHR and WKY rats in a voltage- and dose-dependent manner (P

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 18β-glycyrrhetinic acid (18β-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.

Pirfenidone (PFD) is an anti-fibrotic agent that is clinically used in the treatment of idiopathic pulmonary fibrosis. PFD has been shown to exert protective effects against damage to orbital fibroblasts, endothelial cells, liver cells and renal proximal tubular cells; however, its effect on myocardial cell apoptosis remains unclear. The present study aimed to characterize the effects of PFD on homocysteine (Hcy)-induced cardiomyocyte apoptosis and investigated the underlying mechanisms. H9C2 rat cardiomyocytes were pre-treated with PFD for 30 min followed by Hcy exposure for 24 h. The effects of PFD on cell cytotoxicity were evaluated by CCK-8 assay. The apoptosis rate of each group was determined by flow cytometry. The protein and mRNA levels of connexin 43 (Cx43), Bax, B-cell lymphoma-2 (Bcl-2) and caspase-3 were measured by western blot analysis and reverse transcription-quantitative PCR, respectively. The present results demonstrated that the apoptotic rate increased following Hcy exposure, whereas the apoptotic rate significantly decreased following PFD pre-treatment. Furthermore, the ratio of Bax/Bcl2 was upregulated following Hcy exposure, and Hcy upregulated the expression levels of cleaved caspase-3 and Cx43. Notably, these effects were prevented by PFD. Additionally, the effects of PFD were inhibited by the Cx43 agonist, AAP10. In summary, the findings of the present study demonstrate that PFD protects H9C2 rat cardiomyocytes against Hcy-induced apoptosis by modulating the Cx43 signaling pathway.

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.

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 (P0.01, n = 8) and blood vessel wall thickness in SHR (P0.01, n = 8). The cerebral artery contraction rate in the SHR group was higher than that in the WKY group (P0.05, n = 8). The cerebral artery contraction rate in the SHR + Ramipril group was lower than that in the SHR group (P0.05, 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 (P0.05, n = 8). In addition, the expression of Cx43 mRNA and protein in cerebral arteries of SHR group was higher than that of WKY group (P0.05, n = 8). The mRNA and protein expression of Cx43 in cerebral arteries of SHR + Ramipril group was significantly lower than that of SHR group (P0.05, 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.

Isoflurane (ISO) post-conditioning attenuates cerebral ischemia/reperfusion (I/R) injury, but the underlying mechanism is incompletely elucidated. Transforming growth factor beta (TGF-β) and hedgehog (Hh) signaling pathways govern a wide range of mechanisms in the central nervous system. We aimed to investigate the effect of the TGF-β2/Smad3 and sonic hedgehog (Shh)/Glioblastoma (Gli) signaling pathway and their crosstalk in the hippocampus of rats with ISO post-conditioning after cerebral I/R injury. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), 1.5 h occlusion and 24 h reperfusion (MCAO/R). To assess the effect of ISO after I/R injury, various approaches were used, including neurobehavioral tests, TTC staining, HE staining, Nissl staining, TUNEL staining, immunofluorescence (IF), qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot. The ISO post-conditioning group (ISO group) received 1 h ISO post-conditioning when reperfusion was initiated, leading to lower infarct volumes and neurologic deficit scores, more surviving neurons, and less damaged and apoptotic neurons. IF staining, qRT-PCR and Western blot showed high expression levels of TGF-β2, Shh and Gli1 in the hippocampal CA1 of the ISO group. Phosphorylated Smad3 (p-Smad3), Patched (Ptch), and Smoothed (Smo) were also increased at protein level in the ISO group, whereas total Smad3 expression did not change in all groups. When TGF-β2 inhibitor, pirfenidone, or Smad3 inhibitor, SIS3 HCl, were administered, the expression levels of p-Smad3 and Gli1 were reduced, and surviving pyramidal neurons decreased. By contrast, the expression levels of TGF-β2 and p-Smad3 did not change significantly after pre-injection of Smo inhibitor cyclopamine, but reduced the expression levels of Shh, Ptch, and Gli1. Moreover, Gli showed the lowest expression levels with pirfenidone combined with cyclopamine. These findings indicate that the TGF-β and hedgehog signaling pathways mediate the neuroprotection of ISO post-conditioning after cerebral I/R injury, and crosstalk between two pathways at the Gli1 level.

Background: Stroke is the second leading cause of death worldwide. Angiogenesis facilitates the formation of microvascular networks and promotes recovery after stroke. The Shh/Gli signaling pathway is implicated in angiogenesis and cerebral ischemia-reperfusion (I/R) injury. This study aimed at investigating the influence of isoflurane (ISO) post-conditioning on brain lesions and angiogenesis after I/R injury. Methods: Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), 1.5 h occlusion and 24 h reperfusion (MCAO/R). The ISO post-conditioning group (ISO group) received 1 h ISO post-conditioning when reperfusion was initiated. Neurobehavioral tests, TTC staining, HE staining, Nissl staining, TUNEL staining, immunofluorescence (IF), immunohistochemistry (IH) and Western blot were performed to assess the effect of ISO after I/R injury. Results: ISO post-conditioning resulted in lower infarct volumes and neurologic deficit scores, higher rate of neurons survival, and less damaged and apoptotic cells after cerebral I/R injury in rats. Meanwhile, ISO post-conditioning significantly increased the expression levels of vascular endothelial growth factor (VEGF) and CD34 in the ischemic penumbra, relative to that in the Sham and I/R groups. However, cyclopamine, the specific inhibitor of the Sonic hedgehog (Shh) signaling pathway, decreased the expression levels of VEGF and CD34, and counteracted the protective effects of ISO post-conditioning against I/R injury in rats. Conclusions: ISO post-conditioning enhances angiogenesis in vivo partly via the Shh/Gli signaling pathway. Thus, Shh/Gli may represent new therapeutic targets for aiding recovery from stroke.

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion (I/R) injury. In this study, three key proteins in the endoplasmic reticulum stress pathway (glucose-regulated protein 78, caspase-12, and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor. Female Sprague-Dawley rats received ovariectomy (OVX), and then cerebral I/R rat models (OVX + I/R) were established by middle cerebral artery occlusion. Immediately after I/R, rat models were injected with 100 μg/kg E2 (OVX + I/R + E2), or 100 μg/kg G protein-coupled estrogen receptor agonist G1 (OVX + I/R + G1) in the lateral ventricle. Longa scoring was used to detect neurobehavioral changes in each group. Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining. Morphological changes in neurons were observed by Nissl staining. Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group, neurological function was remarkably improved, infarct volume was reduced, number of normal Nissl bodies was dramatically increased, and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention. To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum, caspase-12 distribution and expression were detected by immunofluorescence, and mRNA and protein levels of glucose-regulated protein 78, caspase-12, and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay. The results showed that compared with the OVX + I/R group, E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78, C/EBP homologous protein, and caspase-12. However, the G protein-coupled estrogen receptor antagonist G15 (OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury. These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus, thereby improving dysfunction caused by cerebral I/R injury. Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. SHZ A2017-171) on February 27, 2017.