Potential value of canonical and non-canonical roles of connexin 43 in disease treatment / 中国组织工程研究

BACKGROUND:Connexin 43(Cx43),which is thought to be engaged in the gap junction process and build the structural groundwork for the development of direct material signaling channels between cells,is crucial for maintaining the homeostatic balance of tissue metabolism.Recent research,however,has revealed fresh information about its distinct hemichannel function and highlighted the significance of its subcellular localization and self-fragmentation for cellular physiological activities and pathological processes. OBJECTIVE:To systematically summarize the molecular characteristics and expression of Cx43 in a variety of cells,concentrate on the pathological and physiological roles of channel-dependent Cx43 and channel-independent Cx43,and investigate the potential value in disease treatment by reviewing the pertinent literature in the database. METHODS:The Chinese and English keywords were"gap junction,connexin 43(Cx43),hemichannel,channel-dependent Cx43,channel-independent Cx43,extracellular vesicles(EVs),mitochondria,GJA1-20k",which were searched in PubMed and CNKI.Finally,81 articles were selected for review. RESULTS AND CONCLUSION:(1)The canonical role of Cx43 is to form a gap junction channel.Channel-dependent Cx43 has primarily involved in disease physiopathological processes by directly constituting gap junction channels,but full attention should be paid to the issue of its structural and functional integrity.Adhesion is a crucial characteristic of gap junctions,which are strongly associated with barrier-like diseases.(2)The non-canonical role of Cx43 is non-gap junction channel-dependent effect.In addition to being localized at the plasma membrane,inner mitochondrial membrane,extracellular vesicle surface,and other structures,Cx43 hexamer has also been found to play a role in positive pro-inflammatory mechanisms,mitochondrial functional metabolism,and targeted uptake of extracellular vesicles in inflammatory diseases.Selective shortened segments control mitochondrial homeostasis by encouraging the polymerization of peri-mitochondrial actin and are involved in the targeted translocation of full-length Cx43 to intracellular structural domains.(3)The development of targeted medicines and the solving of issues like the mechanism of seed cell transformation in tissue engineering-based therapies are both made possible by these two categories of impacts.The interactions of various types of Cx43,however,are frequently not fully taken into account in some of the existing original studies,which confuses the overall characteristics and skews the results.(4)It is necessary to systematically frame the physiological characteristics of Cx43 in different forms and its potential mechanisms in various diseases,so as to provide a reference for the exploration of the Cx43 integrity mechanism and the diagnosis and treatment of multiple diseases.

The Structure and Function of Glial Networks: Beyond the Neuronal Connections / 神经科学通报·英文版

Glial cells, consisting of astrocytes, oligodendrocyte lineage cells, and microglia, account for >50% of the total number of cells in the mammalian brain. They play key roles in the modulation of various brain activities under physiological and pathological conditions. Although the typical morphological features and characteristic functions of these cells are well described, the organization of interconnections of the different glial cell populations and their impact on the healthy and diseased brain is not completely understood. Understanding these processes remains a profound challenge. Accumulating evidence suggests that glial cells can form highly complex interconnections with each other. The astroglial network has been well described. Oligodendrocytes and microglia may also contribute to the formation of glial networks under various circumstances. In this review, we discuss the structure and function of glial networks and their pathological relevance to central nervous system diseases. We also highlight opportunities for future research on the glial connectome.

Research progresses on correlation between connexin subcellular distribution and tumorigenesis and development / 中国药理学通报

Connexin (Cx), a multigene-encoded transmembrane protein family, forms either gap junctions ( GJ) or hemichannels (HC) to mediate intercellular communication in plasma mem¬brane between adjacent cells or interacts with proteins by its car- boxyl terminal in the cytoplasm to participate in the process of tumor cell proliferation, apoptosis, necrosis, invasion, metasta¬sis, drug resistance and stem cell characteristics.However, mi- slocalization of Cx in cytoplasm or nucleus often occurs in many tumors, and involved in the occurrence and development of tumors.Subcellular localization of Cx is affected by post-transla- tional modifications, including phosphorylation, ubiquitination, and acetylation.In this paper the classification and function of Cx, the relationship between subcellular localization of Cx and tumorigenesis and the regulation of post-translational modifica¬tion on Cx are reviewed in order to provide new ideas for the study of Cx as a potential target for cancer therapy.

Effect of gap junction protein Cx43 inhibitor on cognitive function and its possible mechanism in epileptic rats / 中华神经医学杂志

Objective:To investigate the effect of gap junction protein Cx43 inhibitor carbenoxolone (CBX) on cognitive function and its possible mechanism in epileptic rats.Methods:One hundred and twenty Wistar rats were randomly divided into sham-operated group, epilepsy group, epilepsy+solvent group, and epilepsy+CBX group ( n=30). The models of temporal lobe epilepsy in the later three groups were prepared by injection of kainic acid in the hippocampus. Intraperitoneal injection of CBX (20 mg/kg) or equal amount of normal saline were given to the rats in the epilepsy+CBX group and epilepsy+solvent group 30 min before modeling. Western blotting was used to detect the protein expressions of phosphorylated (p)-Cx43 and microtubule associated protein light chain 3 (LC3) in the hippocampus 6, 12, and 24 h after modeling; the protein localization of p-Cx43 and LC3 in the hippocampus and optical density of their positive cells were detected by immunohistochemistry 24 h after modeling; the learning and memory abilities of rats were tested by Morris water maze experiment 30 d after modeling. Results:Western blotting results showed that as compared with those in the sham-operated group, p-CX43 and LC3 protein expressions in the hippocampal CA3 regions of epilepsy group and epilepsy+solvent group were significantly increased at 6, 12 and 24 h after modeling ( P<0.05); as compared with the epilepsy group and epilepsy+solvent group, the epilepsy+CBX group had statistically decreased p-CX43 and LC3 protein expressions in the hippocampal CA3 regions at each time point ( P<0.05). Immunohistochemical staining showed that p-CX43 was localized at the cell membrane and cytoplasm of hippocampal astrocytes; LC3 was located at the cytoplasm of hippocampal neurons. As compared with those in the sham-operated group, the optical density values of p-CX43 and LC3 positive cells in hippocampal CA3 regions of epilepsy group and epilepsy+solvent group were increased ( P<0.05). As compared with those in the epilepsy group and the epilepsy+solvent group, the optical density values of p-CX43 and LC3 positive cells in the hippocampal CA3 regions of the epilepsy+CBX group were significantly decreased ( P<0.05). Morris water maze test results showed that as compared with that in the sham-operated group, the escape latency in the epilepsy group and epilepsy+solvent group was significantly prolonged ( P<0.05); as compared with that in the epilepsy group and epilepsy+solvent group, the latency in the epilepsy+CBX group was significantly shortened ( P<0.05). Conclusion:CBX can weaken the neuronal autophagy and reduce the damage to cognitive function by inhibiting the p-Cx43 protein expression in the astrocytes of the hippocampal CA3 regions.

Astrocytic Gap Junctions Contribute to Aberrant Neuronal Synchronization in a Mouse Model of MeCP2 Duplication Syndrome / 神经科学通报·英文版

Abnormal synchronous neuronal activity has been widely detected by brain imaging of autistic patients, but its underlying neural mechanism remains unclear. Compared with wild-type mice, our in vivo two-photon imaging showed that transgenic (Tg1) mice over-expressing human autism risk gene MeCP2 exhibited higher neuronal synchrony in the young but lower synchrony in the adult stage. Whole-cell recording of neuronal pairs in brain slices revealed that higher neuronal synchrony in young postnatal Tg1 mice was attributed mainly to more prevalent giant slow inward currents (SICs). Both in vivo and slice imaging further demonstrated more dynamic activity and higher synchrony in astrocytes from young Tg1 mice. Blocking astrocytic gap junctions markedly decreased the generation of SICs and overall cell synchrony in the Tg1 brain. Furthermore, the expression level of Cx43 protein and the coupling efficiency of astrocyte gap junctions remained unchanged in Tg1 mice. Thus, astrocytic gap junctions facilitate but do not act as a direct trigger for the abnormal neuronal synchrony in young Tg1 mice, revealing the potential role of the astrocyte network in the pathogenesis of MeCP2 duplication syndrome.

Role of intercellular junctions in the biological behavior of SP cells of multiple myeloma / 药学实践杂志

Objective To observe the effects of the intercellular gap junction (GJIC) composed of connexin 43(Cx43) in mesenchymal stem cells (MSCs) from different sources and their signals on the biological behavior of multiple myeloma (MM) lateral population cells (SP cells), and to explore its possible mechanism. Methods Mesenchymal stem cells (MSCs) from different sources were isolated and cultured. SP cells of MM cell line RPMI 8266 were sorted by flow cytometry. RT-PCR and Western blot were used to detect the expression of Cx43 gene and protein in MSCs, RPMI 8266 and SP cells from different sources. The effects of MSCs from different sources on SP cell cycle, Cx43 protein expression, colony formation ability in vitro, stem cell related gene expression, cytokine secretion and drug resistance were observed. Results There was no significant difference in morphology and phenotype between MM-MSCs and ND-MSCs. Both MM-MSCs and RPMI 8266 cells expressed a higher level of Cx43. Co-culture with MM-MSCs induced more SP cells to enter G0 phase (P<0.001). The expressions of c-myc, Kif4 and Sox2 genes in SP cells were significantly up-regulated, while the expression of Oct-4 gene was down-regulated. After adding α-GA, c-myc, Kif4 and Sox2 were down-regulated in varying degrees, but there was no significant difference. The expression of Cx43 was up-regulated by (31.00±2)% and (39.00±2)%, respectively. The colony formation ability in vitro was up-regulated, and the addition of α-GA could partially inhibit this effect. A small amount of c-myc, Kif4, Sox2 and Oct-4 genes were expressed in RPMI 8266. These genes were significantly up-regulated in SP cell subpopulation. MM-MSCs secreted high levels of interleukin (IL)-6. After co-culture with SP cells, the expressions of IL-6, IL-10 and TGF-β in the supernatant of MM-MSCs were up-regulated (P=0.0072, P=0.037). bFGF and IL-17 had no significant change. After adding α-GA, the levels of IL-6, IL-10 and TGF-β in the supernatant decreased. MM cells were sensitive to bortezomib (BTZ) induced apoptosis, but SP cells were less sensitive. Co-culture with MM-MSCs significantly reduced BTZ-mediated apoptosis. The addition of α-GA partially restored the sensitivity of MM cells to bortezomib. Conclusion MM-MSCs and multiple myeloma SP cells up-regulate the expression of Cx43 protein, form more GJIC, and promote the proliferation and drug resistance of SP cells by changing the cytokine secretion profile of MSCs, which may be one of the reasons for the recurrence of MM.

Activating Connexin43 gap junctions primes adipose tissue for therapeutic intervention

Adipose tissue is a promising target for treating obesity and metabolic diseases. However, pharmacological agents usually fail to effectively engage adipocytes due to their extraordinarily large size and insufficient vascularization, especially in obese subjects. We have previously shown that during cold exposure, connexin43 (Cx43) gap junctions are induced and activated to connect neighboring adipocytes to share limited sympathetic neuronal input amongst multiple cells. We reason the same mechanism may be leveraged to improve the efficacy of various pharmacological agents that target adipose tissue. Using an adipose tissue-specific Cx43 overexpression mouse model, we demonstrate effectiveness in connecting adipocytes to augment metabolic efficacy of the β 3-adrenergic receptor agonist Mirabegron and FGF21. Additionally, combing those molecules with the Cx43 gap junction channel activator danegaptide shows a similar enhanced efficacy. In light of these findings, we propose a model in which connecting adipocytes via Cx43 gap junction channels primes adipose tissue to pharmacological agents designed to engage it. Thus, Cx43 gap junction activators hold great potential for combination with additional agents targeting adipose tissue.

Studies on the mechanism of inhibitory effect of lithium chloride on the proliferation of human Tenon capsule fibroblasts / 中华实验眼科杂志

Objective:To investigate the effect of lithium chloride (LiCl) on the gap junctional intercellular communication (GJIC) in human Tenon capsule fibroblasts (HTFs) and its underlying mechanism.Methods:The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mm×1 mm×1 mm.Primary culture and subculture were carried out, and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital (No.2019-023). Written informed consent was obtained from the subject.Results:The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike, and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53, which was significantly higher than 4.94±0.39 of the control group ( t=-18.79, P<0.01). Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group, and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1, the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23, showing a statistical significance between them ( t=-14.426, P<0.01). Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group, which was significantly higher than 0.446±0.028 in the control group ( t=-11.682, P<0.01). Conclusions:LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein, suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.

Effect of gap junction blockers on hippocampal ripple energy expression in rats with status epilepticus / 中国当代儿科杂志

OBJECTIVES@#To study the effect of gap junction blockers, quinine (QUIN) and carbenoxolone (CBX), on hippocampal ripple energy expression in rats with status epilepticus (SE).@*METHODS@#A total of 24 rats were randomly divided into four groups: model, QUIN, valproic acid (VPA), and CBX (@*RESULTS@#Ripple expression was observed in the hippocampal CA1, CA3, and dentate gyrus regions of normal rats. After 10 minutes of PILO injection, all groups had a gradual increase in mean ripple energy expression compared with 1 day before modeling, with the highest expression level before chloral hydrate injection in the model, VPA and CBX groups (@*CONCLUSIONS@#The change in ripple energy can be used as a quantitative indicator for early warning of seizures, while it cannot predict seizures in the interictal period. Gap junction blockers can reduce ripple energy during seizures.

Effect of gap junction blocker octanol on mitogen activated protein kinase signal pathway after cerebral ischemia reperfusion in rats / 中华神经医学杂志

Objective To investigate the effect of octanol,a gap junction blocker,on mitogen activated protein kinase (MAPK) signal pathway after ischemia reperfusion in rats.Methods Seventy-two male SD rats were randomly divided into sham-operated group,DMSO control group,saline control group and octanol group (n=1 8).The focal cerebral ischemia reperfusion models were established by suture method in the later three groups.Rats were injected 5 mmol/kg octanol solution (5％ octanol soluble in 5％ DMSO solution) into the abdominal cavity of rats in the octanol group 30 min before ischemia reperfusion;rats in the DMSO control group were injected with same amount of 5％ DMSO solution,and those in the sham-operated group and saline control group were injected with same amount of saline.At 24 h after reperfusion,Neurological Function Defect Scale was performed;water content in brain tissues was detected by dry-wet method;cerebral infarction volume percentage was detected by TTC staining;the total protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2),c-jun amino-terminal kinase (JNK),and p38,and protein expressions ofphosphorylased (p)-ERK1/2,p-JNK,and p-p38 were detected by Western blotting.Results The scores of Neurological Function Defect Scale,water content in brain tissues,and infarction volume percentage of the octanol group (1.583±0.651,78.363％±0.672％,and 24.34％±0.19％) were obviously reduced as compared with those in the DMSO control group (2.344±0.743,80.873％±0.831％,and 32.26％±0.21％) and saline control group (2.351±0.732,80.893％±0.734％,and 32.28％±0.24％),with significant differences (P＜0.05).The protein expressions ofp-ERKl/2,p-JNK,and p-p38 in the octanol group (0.201±0.009,0.211±0.011,and 0.191±0.009) were obviously reduced as compared with those in the DMSO control group (0.389±0.019,0.311±0.022,and 0.309±0.021) and saline control group (0.393±0.021,0.304±0.021,and 0.316±0.025),with significant differences (P＜0.05).Conclusion Octanol can reduce ischemic reperfusion injury,whose mechanism may be related to the regulation of MAPK signal pathway.

Inhibitory effect of connexin43 protein on autophagy in cisplatin-resistant testicular cancer I-10 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of connexin43 (Cx43) protein on autophagy in cisplatin (DDP)-resistant testicular cancer I-10 cells.@*METHODS@#The expression of Cx43 proteins in testicular cancer I-10 cells and I-10/DDP cells were detected with Western blotting. I-10/DDP cells were transfected with a full- length mouse Cx43 vector (mCx43) Lipofectamine, the empty vector or Lipofectamine (blank control group), and the changes in the expressions of LC3 and p62 proteins were determined with Western blotting. mCherry-GFP-LC3B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression.@*RESULTS@#Cx43 was significantly decreased in I-10/DDP cells compared with I-10 cells ( < 0.01). Transfection of the I-10/DDP cells with mCx43 vector resulted in significantly increased Cx43 expression in the cells ( < 0.01) and caused significantly decreased expression of LC3-Ⅱ ( < 0.01) and increased expression of p62 ( < 0.05) as compared with the negative control cells. Both transmission electron microscopy and mCherry-GFP-LC3B transfection showed that the number of autophagosomes was obviously reduced in mCx43-transfected cells as compared with the negative control cells.@*CONCLUSIONS@#Cx43 inhibits autophagy in cisplatin-resistant testicular cancer I-10 /DDP cells.

Aspirin alleviates endothelial gap junction dysfunction through inhibition of NLRP3 inflammasome activation in LPS-induced vascular injury

The loss of endothelial connective integrity and endothelial barrier dysfunction can lead to increased vascular injury, which is related to the activation of endothelial inflammasomes. There are evidences that low concentrations of aspirin can effectively prevent cardiovascular diseases. We hypothesized that low-dose aspirin could ameliorate endothelial injury by inhibiting the activation of NLRP3 inflammasomes and ultimately prevent cardiovascular diseases. Microvascular endothelial cells were stimulated by lipopolysaccharide (2 μg/mL) and administrated by 0.1-2 mmol/L aspirin. The wild type mice were stimulated with LPS (100 μg/kg/day), and 1 h later treated with aspirin (12.5, 62.5, or 125 mg/kg/day) and dexamethasone (0.0182 mg/kg/day) for 7 days. Plasma and heart were harvested for measurement of ELISA and immunofluorescence analyses. We found that aspirin could inhibit NLRP3 inflammasome formation and activation in dose-dependent manner and has correlation between the NLRP3 inflammasome and the ROS/TXNIP pathway. We also found that low-concentration aspirin could inhibit the formation and activation of NLRP3 inflammasome and restore the expression of the endothelial tight junction protein zonula occludens-1/2 (ZO1/2). We assume that aspirin can ameliorate the endothelial layer dysfunction by suppressing the activation of NLRP3 inflammasome.

Effects of oxygen glucose deprivation and reperfusion on gap junction and glucose uptake of astrocytes / 中华行为医学与脑科学杂志

Objective@#To explore the relationship between gap junction and glucose uptake of astrocytes under oxygen-glucose deprivation(OGD) and reperfusion.@*Methods@#Cerebral cortical astrocyte from 1 day newborn SD rats were undergone the primary culture. The ischemia cell model was established by OGD. This experiment were divided into control group, OGD group and OGD+ CBX group.After different reperfusion time (0 h, 12 h 24 h and 48 h), the glucose uptake of astrocyte was measured by 2-NBDG through flow cytometry analysis and connexin 43(Cx43) gap junction plaques was detected using immunofluorescene.@*Results@#Compared with the control group, the glucose uptake of astrocyte was up-regulated induced by OGD following different reperfusion time.The glucose uptake of OGD group was (2.32±0.43)nmol/μgDNA in 24 hours reperfusion time and was (0.95±0.28)nmol/μgDNA in control group. The up-regulation was up to 2.63-fold increase (t=13.99, P=0.0024) in 24 hours after reperfusion.Compared with the control group, the Cx43 gap junction number was up to 2.5- fold increase(t=11.34, P=0.003) and the size was 1.85-fold increase (t=10.27, P=0.004) in 24 h reperfusion. The glucose uptake of astrocyte after OGD was reduced by CBX and the decrease was 42% in 48 h after reperfusion.@*Conclusion@#Those results urges us consider the clinical treatment for interfering with Cx43 gap junction.

Mechanism of Liuwei Dihuangwan in Enhancing Hepatocarcinoma Suicide Gene Therapy from Cx32 / 中国实验方剂学杂志

Objective: To investigate the effect of Liuwei Dihuangwan on connexin 32 (Cx32) in hepatoma cell line CBRH7919 and its gap junction intercellular communication (GJIC), and furthermore study its mechanism of enhancing the bystander killing effect of suicide gene therapy. Method: Liuwei Dihuangwan (32 g·kg·d-1) and the same volume of normal saline were given to the rats by intragastrical administration. Blood was taken to prepare the medicated serum of Liuwei Dihuangwan and blank control serum, respectively. The hepatoma cell line CBRH7919 were treated by control serum and medicated serum of Liuwei Dihuangwan in different concentrations. There were four groups in experiment:the blank control group (volume fraction of 10%), medicated serum high dose group of Liuwei Dihuangwan (the volume fraction of 10%), medicated serum middle dose group of Liuwei Dihuangwan (the volume fraction of 5%), and medicated serum low dose group of Liuwei Dihuangwan (the volume fraction of 2.5%). The expression levels of Cx32 protein and mRNA in hepatoma cell line CBRH7919 were detected by indirect immunofluorescence assay (ⅡA) and Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) assay. The fluorescence redistribution after photobleaching (FRAP) method was used to detect the function of GJIC of hepatoma cell line CBRH7919. Result: ① The indirect immunofluorescence assay (ⅡA) analysis indicated that as compared with the blank control group, the cx32 expression of CBRH7919 cells was up-regulated in a concentration-dependent manner in each dose group of the serum containing Liuwei Dihuangwan (PPPPConclusion: The mechanism of medicated serum of Liuwei Dihuangwan in enhancing the bystander killing effect of suicide geneis related to gap junction. Liuwei Dihuangwan may enhance the function of GJIC by increasing the localization of cx32 on the cell membrane of CBRH7919 cells and increasing the expression of cx32 mRNA and protein to achieve the synergistic action.

Effect of Gegensan on Expressions of NOS, α-SMA, Cx43, TGF-β1 mRNA and Protein in Penile Smooth Muscle Tissue of Alcoholic ED Rats / 中国实验方剂学杂志

Objective:To investigate the activity of nitric oxide synthase (NOS) and α-smooth actin (α-SMA) in rat penile smooth muscle tissue of rats with alcoholic erectile dysfunction (ED). The effects of protein gene 43 (connexin43, Cx43) and transforming growth factor β1 (TGF-β1) mRNA and protein expressions provide an experimental basis for the clinical application of Gegensan in the treatment of alcoholic ED. Method:SD rats were randomly divided into five groups:normal group, model group, and low,medium,high-dose Gegensan groups (5,10,20 g·kg-1). Except the normal group, the other groups were administered with drugs after alcohol intervention for 30 min at 15 mL·kg-1·d-1. Colorimetric assay was used to detect NOS activity in the penile smooth muscle tissue of alcoholic ED rats. Quantitative real-time fluorescence polymerase chain reaction(Real-time PCR) and Western blot were used to detect α-SMA, Cx43, TGF-β1 mRNA and protein expressions in smooth muscle tissue of alcoholic ED rats. Result:Compared with the normal group, the expressions of NOS, α-SMA and Cx43 mRNA and protein in the penile smooth muscle of the model group decreased significantly (Pβ1 mRNA and protein increased significantly (Pβ1 mRNA expression, and α-SMA mRNA and protein expressions in the penis tissue of rats with alcoholic ED were significantly up-regulated (PConclusion:Gegensan has an obvious protective effect on the structure of penile smooth muscle of alcoholic ED rats. The specific mechanism may be related to the regulation of NOS activity and a-SMA, Cx43 and TGF-β1 mRNA and protein expressions.

Decreased expression of gap junction delta-2 (GJD2) messenger RNA and connexin 36 protein in form-deprivation myopia of guinea pigs / 中华医学杂志(英文版)

Background@#More than ten genome-wide association studies have identified the significant association between the gap junction delta-2 (GJD2) gene and myopia. However, no functional studies have been performed to confirm that this gene is correlated with myopia. This study aimed to observe how this gene changed in mRNA and protein level in the form-deprivation myopia (FDM) animal model.@*Methods@#Four-week-old guinea pigs were randomly divided into two groups: control and FDM groups (n = 12 for each group). The right eyes of the FDM group were covered with opaque hemispherical plastic lenses for 3 weeks. For all the animals, refractive status, axial length (AL), and corneal radius of curvature were measured at baseline and 3 weeks later by streak retinoscope, A-scan ultrasonography, and keratometer, respectively. Retinal GJD2 mRNA expression and connexin 36 (Cx36) levels in FDM and control groups were measured by quantitative real-time PCR and Western blot analyses, respectively. Those results were compared using independent t test, Mann-Whitney U test, or paired t test. A significance level of P < 0.05 was used.@*Results@#Three weeks later, the FDM group (form-deprived eyes) showed about a myopic shift of approximately -6.75 (-7.94 to -6.31) D, while the control group remained hyperopic with only a shift of -0.50 (-0.75 to 0.25) D (Z=-3.38, P < 0.01). The AL increased by 0.74 (0.61–0.76) and 0.10 (0.05–0.21) mm in FDM and control groups, respectively (Z = -3.37, P < 0.01). The relative mRNA expression of GJD2 in the FDM group decreased 31.58% more than the control group (t = 11.44, P < 0.01). The relative protein expression of CX36 on the retina was lowered by 37.72% in form-deprivation eyes as compared to the controls (t = 17.74, P < 0.01).@*Conclusion@#Both the mRNA expression of GJD2 and Cx36 protein amount were significantly decreased in the retina of FDM guinea pigs. This indicates that Cx36 is involved in FDM development, providing compensating evidence for the results obtained from genome-wide association studies.

Experimental study of metformin in relieving bone cancer pain by inhibiting JNK and improving gap junction function / 中国药科大学学报

@#This study aims to explore the involvement of c-Jun N-terminal kinase(JNK)-Gap junction regulation in the rat model of bone cancer pain and figure out whether adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK )activator metformin could attenuate bone cancer pain through this mechanism. Tumor cell implantation(TCI)induced bone cancer pain model in rats was established. The rats were administered, respectively, with 20 μL of metformin(50, 100 μg), JNK inhibitor SP600125(10 μg), gap junction inhibitor(carbenoxolone, CBX)(10 μg)and AMPK inhibitor Compound C(CC)(10 μg). The Von Frey Assay was applied to test the mechanical pain threshold. The activity of Glial fibrillary acidic protein(GFAP), ionized calcium-binding adaptor molecule 1(IBA-1)and Connexin 43(Cx43)in spinal cord was evaluated by immunohistochemistry. Changes of p-JNK expression were detected by Western blot. JNK inhibitor SP600125 relieved TCI-induced bone cancer pain significantly in rats, while this analgesic effect was almost canceled by the blocker of gap junction carbenoxolone(CBX). Various concentration of metformin(50, 100 μg, i. t. )significantly inhibited TCI-induced mechanical allodynia and the changes of p-JNK and p-Cx43 expression were also reversed in spinal cord in rats. Together, these data suggested that activation of AMPK with metformin attenuated TCI-induced bone cancer pain via regulating the function of JNK-Gap junction in rats.

Effects of oxygen glucose deprivation and reperfusion on gap junction and glucose uptake of astrocytes / 中华行为医学与脑科学杂志

Objective To explore the relationship between gap junction and glucose uptake of astrocytes under oxygen-glucose deprivation(OGD) and reperfusion.Methods Cerebral cortical astrocyte from 1 day newborn SD rats were undergone the primary culture.The ischemia cell model was established by OGD.This experiment were divided into control group,OGD group and OGD + CBX group.After different reperfusion time (0 h,12 h 24 h and 48 h),the glucose uptake of astrocyte was measured by 2-NBDG through flow cytometry analysis and connexin 43(Cx43) gap junction plaques was detected using immunofluorescene.Results Compared with the control group,the glucose uptake of astrocyte was up-regulated induced by OGD following different reperfusion time.The glucose uptake of OGD group was (2.32 ± 0.43)nmol/μgDNA in 24 hours reperfusion time and was (0.95±0.28)nmol/μgDNA in control group.The upregtulation was up to 2.63-fold increase (t=13.99,P=0.0024) in 24 hours after reperfusion.Compared with the control group,the Cx43 gap junction number was up to 2.5-fold increase(t=11.34,P=0.003) and the size was 1.85-fold increase (t=10.27,P=0.004) in 24 h reperfusion.The glucose uptake of astrocyte after OGD was reduced by CBX and the decrease was 42％ in 48 h after reperfusion.Conclusion Those results urges us consider the clinical treatment for interfering with Cx43 gap junction.

Genetics of Hearing Loss in North Iran Population: An Update of Spectrum and Frequency of GJB2 Mutations

Diagnosis of pre-lingual hearing loss (HL) is difficult owing to the high number of genes responsible. The most frequent cause of HL is DFNB1 due to mutations in the GJB2 gene. It represents up to 40% of HL cases in some populations. In Iran, it has previously been shown that DFNB1 accounts for 16-18% of cases but varies among different ethnic groups. Here, we reviewed results from our three previous publications and data from other published mutation reports to provide a comprehensive collection of data for GJB2 mutations and HL in northern Iran. In total, 903 unrelated families from six different provinces, viz., Gilan, Mazandaran, Golestan, Ghazvin, Semnan, and Tehran, were included and analyzed for the type and prevalence of GJB2 mutations. A total of 23 different genetic variants were detected from which 18 GJB2 mutations were identified. GJB2 mutations were 20.7% in the studied northern provinces, which was significantly higher than that reported in southern populations of Iran. Moreover, a gradient in the frequency of GJB2 mutations from north to south Iran was observed. c.35delG was the most common mutation, accounting for 58.4% of the cases studied. This study suggests that c.35delG mutation in GJB2 is the most important cause of HL in northern Iran.

Mutations in GJB2 as Major Causes of Autosomal Recessive Non-Syndromic Hearing Loss: First Report of c.299-300delAT Mutation in Kurdish Population of Iran

BACKGROUND AND OBJECTIVES: Autosomal recessive non-syndromic hearing loss (ARNSHL) with genetic origin is common (1/2000 births). ARNSHL can be associated with mutations in gap junction protein beta 2 (GJB2). To this end, this cohort investigation aimed to find the contribution of GJB2 gene mutations with the genotype-phenotype correlations in 45 ARNSHL cases in the Kurdish population. SUBJECTS AND METHODS: Genomic DNA was extracted from a total of 45 ARNSHL families. The linkage analysis with 3 short tandem repeat markers linked to GJB2 was performed on 45 ARNSHL families. Only 9 of these families were linked to the DFNB1 locus. All the 45 families who took part were sequenced for confirmation linkage analysis (to perform a large project). RESULTS: A total of three different mutations were determined. Two of which [c.35delG and c.-23+1G>A (IVS1+1G>A)] were previously reported but (c.299-300delAT) mutation was novel in the Kurdish population. The homozygous pathogenic mutations of GJB2 gene was observed in nine out of the 45 families (20%), also heterozygous genotype (c.35delG/N)+(c.-23+1G>A/c.-23+1G>A) were observed in 4/45 families (8.8%). The degree of hearing loss (HL) in patients with other mutations was less severe than patients with c.35delG homozygous mutation (p < 0.001). CONCLUSIONS: Our data suggest that GJB2 mutations constitute 20% of the etiology of ARNSHL in Iran; moreover, the c.35delG mutation is the most common HL cause in the Kurdish population. Therefore, these mutations should be included in the molecular testing of HL in this population.