Astragalus Polysaccharide Injection Ameliorates the Apoptosis of Chondrocytes in the Ovariectomized Osteoporosis Mouse Model via Modulating Protein Kinase B/Glycogen Synthase Kinase 3 Beta Signal Transduction

The Akt/GSK3 signal pathway exerts an impact on the apoptosis of chondrocytes. We planned to elucidate the role and mechanism of Akt/GSK3 signal transduction in ovariectomized osteoporosis. 50 female mice in SPF-graded and healthy condition were processed to establish the ovariectomized osteoporosis model, while 12 of the healthy mice were set as blank controls. The successfully established ovariectomized osteoporosis models were divided into model group (12 mice) and Astragalus group (12 mice). The following measurements were conducted: the bone mineral density in the left humerus, serum estrogen level and Cx43 protein level in osteoblasts, proportion of apoptotic cells, as well as the protein and mRNA levels of Akt and GSK3β in murine bone tissues via Western blotting and RT-PCR detection, respectively. The bone mineral density of mice in Astragalus group was the highest (0.174±0.04) g/cm2. The positive rate of Cx43 protein expression, apoptosis rate, as well as the protein and mRNA levels of Akt in osteocytes were significantly decreased (P < 0.05), but still significantly higher than model group. Estrogen level in Astragalus group was (87.52 ±8.69) pmol/L. The positive rate of Cx43 expression, apoptosis rate, as well as the protein and mRNA levels of GSK3β in osteocytes were decreased in comparison to model group (P < 0.05). Astragalus polysaccharide injection could ameliorate the apoptosis of chondrocytes and downregulate Cx43 protein via modulating Akt/GSK3β signal transduction, thereby exerting a therapeutic effect on ovariectomized osteoporosis.

Upregulation of Cx43 Expression Under Stretch Condition is Mediated by TGF Beta1 and Cytoskeletal Network

Many physiological and pathophysiological processes in cells or tissues are involving mechanical stretch, which is inducing gap junction gene expression and cytokine TGF beta changes. However, the underlying mechanisms of gap junction gene expression changes remain unknown. Here, we showed that the expression of Cx43 at mRNA and protein level in Human umbilical-vein endothelial cells (HUVECs) is significantly increased after 24 h stretch stimulation, and TGF beta1, but not TGF beta2 expression is also upregulated. Administration of TGF betal into HUVECs without stretch also induced upregulation of Cx43 mRNA and protein expression. While simultaneously administration of TGF beta1 with SB431542, a specific inhibitor of TGF beta1 receptor, blocked the Cx43 protein upregulation by TGF beta1. Further, the increase of Cx43 protein expression under stretch condition can be partially blocked by SB431542; moreover, it also can be partially blocked by simultaneously administration of anti-TGF beta1 monoclonal neutralization antibody. Importantly, the stretch induced upregulation of Cx43 can be blocked by administration of actin and microtubule inhibitors, while NEDD4, a key element in mediating Cx43 protein ubiquitination and degradation, is not changed under stretch condition. Therefore, we conclude that upregulation of Cx43 expression under 24 h stretch condition is mediated by TGF beta1 via TGF beta1 receptor signaling pathway, and it also involves the actin and microtubule cytoskeletal network.

Effects of Shuwei Decoction on the Distribution of Connexin 43 Protein and the Repair and Regeneration of Interstitial Cells of Cajal in Rats with Functional Dyspepsia

Shuwei Decoction itself has many functions, not only good for human body, but also has many biological functions. For functional dyspepsia rats, the distribution of connexin 43 protein and the repair and regeneration of interstitial cells of Cajal(RRICC) have different changes in different treatment methods. The purpose of this paper is to study the effect of Shuwei Decoction on the distribution of connexin 43 protein and the RRICC in rats with functional dyspepsia. In order to prove the specific effect of Shuwei Decoction on rats with functional dyspepsia, 50 rats were selected as the research object, and the size of distribution area of Cx43 protein and the cell regeneration were observed. The speed of birth and repair, the distribution of Cx43 protein and the level of Cx43 protein were detected, and the statistical data were studied. The results show that Shuwei decoction can promote the regeneration and formation of ICC, thus obtaining the structural integrity of ICC, improving the potential intestinal disorder and treating FD. Shuwei decoction has a strong effect on the RRICC, which is the result of the increase of the number of intercellular stem cells, and the speed of cell repair and regeneration is increased by nearly 30%. On the basis of the distribution of Cx43 protein, the distribution area increased by 20% compared with the original basis. Therefore, Shuwei decoction has a great influence on the distribution of connexin 43 proteins and the RRICC in rats with functional dyspepsia.

Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of connexins of only 1-5 hours, resulting in constant endocytosis and biosynthetic replacement of gap junction channels has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and a zebrafish model ( cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx 43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that Cx43 gap junction endocytosis is an essential aspect of gap junction function and when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Role of Connexin 43 in an Inflammatory Model for TMJ Hyperalgesia

Temporomandibular joint disorders (TMD) consist of a heterogeneous group of conditions that present with pain in the temporomandibular joint (TMJ) region and muscles of mastication. This project assessed the role of connexin 43 (Cx43), a gap junction protein, in the trigeminal ganglion (TG) in an animal model for persistent inflammatory TMJ hyperalgesia. Experiments were performed in male and female rats to determine if sex differences influence the expression and/or function of Cx43 in persistent TMJ hyperalgesia. Intra-TMJ injection of Complete Freund's Adjuvant (CFA) caused a significant increase in Cx43 expression in the TG at 4 days and 10 days post-injection in ovariectomized (OvX) female rats and OvX females treated with estradiol (OvXE), while TG samples in males revealed only marginal increases. Intra-TG injection of interference RNA for Cx43 (siRNA Cx43) 3 days prior to recording, markedly reduced TMJ-evoked masseter muscle electromyographic (MMemg) activity in all CFA-inflamed rats, while activity in sham animals was not affected. Western blot analysis revealed that at 3 days after intra-TG injection of siRNA Cx43 protein levels for Cx43 were significantly reduced in TG samples of all CFA-inflamed rats. Intra-TG injection of the mimetic peptide GAP19, which inhibits Cx43 hemichannel formation, greatly reduced TMJ-evoked MMemg activity in all CFA-inflamed groups, while activity in sham groups was not affected. These results revealed that TMJ inflammation caused a persistent increase in Cx43 protein in the TG in a sex-dependent manner. However, intra-TG blockade of Cx43 by siRNA or by GAP19 significantly reduced TMJ-evoked MMemg activity in both males and females following TMJ inflammation. These results indicated that Cx43 was necessary for enhanced jaw muscle activity after TMJ inflammation in males and females, a result that could not be predicted on the basis of TG expression of Cx43 alone.

Effects of Atypical Antipsychotics, Clozapine, Quetiapine and Brexpiprazole on Astroglial Transmission Associated with Connexin43

Recently, accumulating preclinical findings suggest the possibility that functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of schizophrenia and affective disorder. Therefore, to explore the novel mechanisms of mood-stabilizing effects associated with tripartite synaptic transmission, the present study determined the effects of mood-stabilizing antipsychotics, clozapine (CLZ), quetiapine (QTP) and brexpiprazole (BPZ), on the astroglial l-glutamate release and expression of connexin43 (Cx43) in the astroglial plasma membrane using cortical primary cultured astrocytes. Neither acute (for 120 min) nor subchronic (for 7 days) administrations of CLZ, QTP and BPZ affected basal astroglial l-glutamate release, whereas both acute and subchronic administration of CLZ, QTP and BPZ concentration-dependently enhanced astroglial l-glutamate release through activated hemichannels. Subchronic administration of therapeutic-relevant concentration of valproate (VPA), a histone deacetylase inhibiting mood-stabilizing antiepileptic drug, enhanced the stimulatory effects of therapeutic-relevant concentration of CLZ, QTP and BPZ on astroglial l-glutamate release through activated hemichannel. Subchronic administration of therapeutic-relevant concentration of CLZ, QTP and BPZ did not affect Cx43 protein expression in the plasma membrane during resting stage. After subchronic administration of VPA, acute and subchronic administration of therapeutic-relevant concentrations of CLZ increased Cx43 protein expression in the plasma membrane. Both acute administrations of therapeutic-relevant concentrations of QTP and BPZ did not affect, but subchronic administrations enhanced Cx43 protein expression in the astroglial plasma membrane. Furthermore, protein kinase B (Akt) inhibitor suppressed the stimulatory effects of CLZ and QTP, but did not affect Cx43 protein expression in the astroglial plasma membrane. These results suggest that three mood-stabilizing atypical antipsychotics, CLZ, QTP and BPZ enhance tripartite synaptic glutamatergic transmission due to enhancement of astroglial Cx43 containing hemichannel activities; however, the Cx43 activating mechanisms of these three mood-stabilizing antipsychotics were not identical. The enhanced astroglial glutamatergic transmission induced by CLZ, QTP and BPZ is, at least partially, involved in the actions of these three mood-stabilizing antipsychotics.

Impaired Cx43 gap junction endocytosis causes cardiovascular defects in zebrafish

Gap junction proteins, termed connexins (Cx), mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting exchange of molecules, ions, and electrical impulses. The most ubiquitously expressed gap junction protein, connexin43 (Cx43) has been implicated in cardiovascular diseases including arrhythmias, cardiomyopathies, hypertension and diabetes. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43Δ256-289) and zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired cardiovascular development. These findings suggest that normal, unimpaired Cx43 gap junction endocytosis and turnover is an essential aspect of gap junction function as demonstrated here for cardiovascular development that when impaired can give rise to arrhythmias, heart malformations and aberrant vasculature structure and function.

The Protective Effect of Cx43 Protein-Mediated Phosphocreatine on Myocardial Ischemia/Reperfusion Injury

Objectives. To verify the protective effect of phosphocreatine on myocardium in an ischemic model and the possible mechanism of action. Methods. The model of myocardial ischemia/reperfusion (I/R) was established by the ligation balloon method. 30 SD rats were randomly divided into three groups, n = 10 in each group. Sham operation group: the coronary artery was not blocked and observed for 120 minutes. The ischemia/reperfusion (I/R) group was given ischemia for 30 minutes and ischemia reperfusion for 90 minutes. Phosphocreatine (PCr) group: after 30 minutes of ischemia, the rats were intraperitoneally injected with PCr (200 mg/kg) for 90 minutes. The animal groups of myocardial ischemia/reperfusion model in vitro were the same as those in vivo. The heart was removed by thoracotomy and washed immediately in H-K buffer solution. Then, the heart was installed on the Langendorff instrument. The concentration of PCr perfusion fluid in the PCr group was 10 mmol/L. The changes in coronary blood flow in isolated myocardium were recorded. The heart rate and electrocardiogram were recorded by RM6240BT. At the end of the experiment, myocardial pathological sections and Cx43 immunofluorescence staining were made, and the contents of malondialdehyde (MDA) in myocardial tissue were detected. Results. Phosphocreatinine treatment improved the myocardial ischemia model, performance in electrocardiogram (ECG) changes (ST segment apparent), and histological changes (decrease in necrotic myocardial cells, inflammatory cell infiltration, and a reduction in myocardial edema). At the same time, MDA decreased, while coronary blood flow and Cx43 expression significantly improved. Conclusions. Phosphocreatine can improve the electrocardiogram and restore histologic changes in ischemic myocardium and coronary blood flow. The postulated mechanism is by inhibiting the generation of free oxygen radicals and restoring the expression of Cx43 protein.

Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia

Pulmonary hypertension (PH) is a disease associated with vasoconstriction and remodelling of the pulmonary vasculature. Pulmonary artery fibroblasts (PAFs) play an important role in hypoxic-induced remodelling. Connexin 43 (Cx43) is involved in cellular communication and regulation of the pulmonary vasculature. Using both in vitro and in vivo models of PH, the aims of this study were to (i) investigate the role of Cx43 in hypoxic-induced proliferation and migration of rat PAFs (rPAFs) and rat pulmonary artery smooth muscle cells (rPASMCs) and (ii) determine whether Cx43 expression is dysregulated in the rat sugen5416/hypoxic model of PH. The role of Cx43 in hypoxic-induced proliferation and migration was investigated using Gap27 (a pharmacological inhibitor of Cx43) or genetic knockdown of Cx43 using siRNA. Cx43 protein expression was increased by hypoxia in rPAFs but not rPASMCs. Hypoxic exposure, in the presence of serum, resulted in an increase in proliferation of rPAFs but not rPASMCs. Hypoxic exposure caused migration of rPAFs but not rPASMCs. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and ERK1/2 were increased by hypoxia in rPAFs. The effects of hypoxia on proliferation, migration and MAPK phosphorylation in rPAFs were attenuated in the presence of Gap27 or Cx43 siRNA. Cx43 protein expression was increased in sugen5416/hypoxic rat lung; this increased expression was not observed in sugen5416/hypoxic rats treated with the MAPK pathway inhibitor GS-444217. In conclusion, Cx43 is involved in the proliferation and migration of rPAFs in response to hypoxia via the MAPK signalling pathway.

Mechanism of antrocytic connexin-43 autophagy degradation in oxygen-glucose deprivation and its effect on neuroinflammation

Background : Connexin 43 (Cx43) are the most widely distributed gap junction proteins in the nervous system. Cx43 enables cell-to-cell communication and plays an important role in ion transport, substrate exchange and delivery of information , which have been implicated in cerebral ischemia injury. Our previous work revealed the relationships between Cx43 and glia-mediated neuroinflammation through the release of ATP in oxygen-glucose deprivation (OGD), which means degradation of Cx43 may improve neuroinflammatory damage during OGD injury . However, the roles of Cx43 degradation and neuroinflammation caused by OGD remain unclear. Methods: We used primary cultured astrocytes treated with OGD as an in vitro model of cerebral ischemia injury and we used middle cerebral artery occlusion (MCAO) model as an in vivo model of cerebral ischemia. HeLa cells were used in overexpression experiments. Cx43 protein levels were determined by western blotting. The interaction between Cx43 and related autophagy receptors was determined by co-immunoprecipitation and immunofluorescence. The gene knockdown (KD) of ATG5, OPTN, NDP52, PINK1 and Cx43 was applied by siRNA transfection. Related cytokines were detected by cytometric bead assay. Results: We found that Cx43 protein levels increased after ischemia in gene KD of ATG5, OPTN, NDP52 and PINK1 primary astrocytes. The interaction of Cx43 with OPTN, NDP52 and PINK1 was increased after cerebral ischemia injury in vitro and vivo. While the interaction was weakened after point mutation of Cx43 at Ser368, Tyr265 and Tyr247. Meanwhile, IL-10 upregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes , while TNF downregulated during OGD after KD of ATG5, OPTN, NDP52 and PINK1 in astrocytes. Conclusions: Our results suggest that degradation of Cx43 is caused by selective autophagy during ischemia injury and the autophagy degradation of Cx43 plays important roles in neuroinflammation mediated by OGD injury. Treatment targeting Cx43 degradation pathway can improve neuroinflammation responses induced by OGD injury , which provide novel therapeutic strategies and crosstalk between autophagy and neuroinflammation.