[Zuogui Pills improve testicular development of offspring rats exposed to prenatal stress via Cx43].

This study aims to reveal the mechanism of prenatal stress in affecting the testicular development of offspring rats and the intervention effects of Zuogui Pills via connexin 43(Cx43). Forty pregnant SD rats were randomized into a blank control group, a mo-del group, a high-dose(18.9 g·kg~(-1)) Zuogui Pills group, a low-dose(9.45 g·kg~(-1)) Zuogui Pills group, and a vitamin E(1.44 mg·kg~(-1)) group. The other groups except the blank control group was subjected to chronic unpredictable mild stress for the modeling of prenatal stress. The model was evaluated by sucrose preference test, open field test, and enzyme-linked immunosorbent assay(ELISA) of the glucocorticoid level. ELISA was employed to measure the thyroxine 4(T4), testosterone(T), and follicle-stimulating hormone(FSH) levels to assess kidney deficiency. Hematoxylin-eosin(HE) staining was employed to evaluate the status of testicular germ cells. An automatic sperm analyzer was used to measure the sperm quality. Immunofluorescence double staining was employed to detect the expression of Cx43 and follicle-stimulating hormone receptor(FSHR) in the testes of offspring rats. The mRNA and protein levels of Cx43, FSHR, phosphatidylinositol 3-kinase(PI3K), and protein kinase B(Akt) were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. Prenatal stress induced testicular development disorders in offspring rats. The HE staining results showed that on the day of birth, the model group had reduced seminiferous tubules in the testes, elevated FSH level in the serum, and lowered Cx43 level in the testicular tissue. Male offspring rats of 60 days old had reduced testicular spermatogenic function, decreased sperm quality, elevated FSH level and lowered T level in the serum, and down-regulated protein and mRNA levels of Cx43, FSHR, PI3K, and Akt in the testicular tissue. Zuogui Pills alleviated the abnormal development and dysfunction of testicles in the offspring rats caused by prenatal stress. In summary, Zuogui Pills may weaken the effects of prenatal stress on testicular development and spermatogenic function of offspring rats by activating the PI3K/Akt pathway to regulate Cx43 expression in the testicular tissue.

Pericytes modulate endothelial inflammatory response during bacterial infection.

Pericytes are located around blood vessels, in close contact with endothelial cells. We discovered that pericytes dampen pro-inflammatory endothelial cell responses. Endothelial cells co-cultured with pericytes had markedly reduced expression of adhesion molecules (PECAM-1 and ICAM-1) and proinflammatory cytokines (CCL-2 and IL-6) in response to bacterial stimuli (Brucella ovis, Listeria monocytogenes, or Escherichia coli lipopolysaccharide). Pericyte-depleted mice intraperitoneally inoculated with either B. ovis, a stealthy pathogen that does not trigger detectable inflammation, or Listeria monocytogenes, developed peritonitis. Further, during Citrobacter rodentium infection, pericyte-depleted mice developed severe intestinal inflammation, which was not evident in control mice. The anti-inflammatory effect of pericytes required connexin 43, as either chemical inhibition or silencing of connexin 43 abrogated pericyte-mediated suppression of endothelial inflammatory responses. Our results define a mechanism by which pericytes modulate inflammation during infection, which shifts our understanding of pericyte biology: from a structural cell to a pro-active player in modulating inflammation. IMPORTANCE: A previously unknown mechanism by which pericytes modulate inflammation was discovered. The absence of pericytes or blocking interaction between pericytes and endothelium through connexin 43 results in stronger inflammation, which shifts our understanding of pericyte biology, from a structural cell to a player in controlling inflammation.

Higenamine exerts antidepressant effect by improving the astrocytic gap junctions and inflammatory response.

BACKGROUND: Depression is a refractory psychiatric disorder closely associated with dysfunction of the gap junctions (GJs) between astrocytes as well as neuroinflammation. Higenamine (Hig) is a potent cardiotonic ingredient in Fuzi (i.e., Aconitum carmichaeli Debx.) with anti-inflammatory and antioxidant effects, which has a significant protective effect on damaged nerve cells and has great potential for the treatment of neuropsychiatric diseases. METHODS: Rats were stimulated by chronic unpredictable stress (CUS) for 28 days while given Hig (5, 10, 20 mg/kg) and then analyzed behaviorally by the open field test, sucrose preference test, and forced swimming test. Changes in astrocyte GJs function and morphology were observed by dye transfer and transmission electron microscopy, respectively. Expression and phosphorylation of connexin 43 (Cx43) were analyzed by Western blot. Also, considering the close relationship between depression and neuroinflammation, we determined the inflammatory response in serum with ELISA kits and analyzed the expression of inflammation-related proteins with Western blot. RESULTS: Hig ameliorated CUS-induced depression-like behavior in rats. Hig administration improved gap junctional dysfunction in astrocytes, reduced gap junctional gaps and elevated the expression of Cx43 and decreased the phosphorylation of Cx43. Meanwhile, Hig administration was also able to attenuate the inflammatory response that occurs after CUS in rats. LIMITATIONS: For the role of Cx43 in depression, we did not validate it more deeply in animal models with knockout Cx43. In addition, GJs dysfunction might be associated with the inflammatory response seen in depression, but this needs to be further investigated. CONCLUSIONS: Hig ameliorates depression and exerts its antidepressant effect possibly by improving the dysfunctional GJs between astrocytes and the inflammatory response.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.

FGF21 protects against doxorubicin-induced cardiotoxicity by inhibiting connexin 43 ubiquitination.

BACKGROUND: Fibroblast growth factor 21 (FGF21) regulates glycolipid metabolism and insulin homeostasis and acts as a cardioprotective factor by protecting against myocardial ischemia/reperfusion injury, hypertension, and vascular dysfunction. FGF21 has been reported to prevent Doxorubicin (Dox)-induced cardiotoxicity, and the related signaling pathway is worthy of further study. Connexin43 (Cx43) protein was reduced by Dox treatment, especially low phosphorylated form of Cx43. Thus the aim of study is to explore the protection effect of FGF21 on Dox induced cardiotoxicity by improving the expression of Cx43 and the involved signaling pathway. METHODS AND RESULTS: FGF21 inhibited apoptosis in Dox-treated mice and cardiomyocytes. FGF21 increased the levels of connexin43 phosphorylated at serine (S) 282 (p-Cx43 S282) and total Cx43 to inhibit Dox-induced apoptosis. By RNA sequencing, we found that deubiquitinase monocyte chemoattractant protein-induced protein 1 (MCPIP1) expression was increased by FGF21. We further found that FGF21 induced the phosphorylation of fibroblast growth factor receptor 1 (FGFR1), extracellular signal-regulated kinase 1 and 2 (Erk1/2), and Elk. Phosphorylated Elk translocated to the nucleus and increased the expression of MCPIP1. Then, MCPIP1 bound neural precursor cell expressed developmentally downregulated protein 4 (Nedd4), an E3 ubiquitination ligase, as shown by co-immunoprecipitation (Co-IP), and suppressed Cx43 ubiquitination and degradation, competitively inhibiting the binding of Cx43 with Nedd4. Thus Nedd4 could not bind and ubiquitinate Cx43, leading to the up-regulation of Cx43 and phosphorylation of Cx43 at S282. CONCLUSIONS: FGF21 inhibited the effects of Dox on cardiomyocytes by elevating the phosphorylation of Cx43 at S282 and total Cx43 expression. This study suggests a previously unknown mechanism for the FGF21-mediated enhancement of cardiomyocyte survival and provides an effective approach to protect against the adverse cardiac effects of Dox.

Connexin 43 Phosphorylation: Implications in Multiple Diseases.

Connexin 43 (Cx43) is most widely distributed in mammals, especially in the cardiovascular and nervous systems. Its phosphorylation state has been found to be regulated by the action of more than ten kinases and phosphatases, including mitogen-activated protein kinase/extracellular signaling and regulating kinase signaling. In addition, the phosphorylation status of different phosphorylation sites affects its own synthesis and assembly and the function of the gap junctions (GJs) to varying degrees. The phosphorylation of Cx43 can affect the permeability, electrical conductivity, and gating properties of GJs, thereby having various effects on intercellular communication and affecting physiological or pathological processes in vitro and in vivo. Therefore, clarifying the relationship between Cx43 phosphorylation and specific disease processes will help us better understand the disease. Based on the above clinical and preclinical findings, we present in this review the functional significance of Cx43 phosphorylation in multiple diseases and discuss the potential of Cx43 as a drug target in Cx43-related disease pathophysiology, with an emphasis on the importance of connexin 43 as an emerging therapeutic target in cardiac and neuroprotection.

Muscone attenuates susceptibility to ventricular arrhythmia by inhibiting NLRP3 inflammasome activation in rats after myocardial infarction.

Fibrosis and abnormal expression of connexin 43 (Cx43) in the ventricle play vital roles in ventricular arrhythmias (VAs) after myocardial infarction (MI). Muscone, an active monomer of heart-protecting musk pill, has various biological activities, but its effect on susceptibility to VAs in rats with MI has not been determined. In the present study, we investigated the effects of muscone on ventricular inflammation, fibrosis, Cx43 expression, and the occurrence of VAs after MI. An MI model was established by ligating the proximal left anterior descending coronary artery. Then, the MI model rats were administered muscone (2 mg/kg/day) or vehicle (saline)via intragastric injection for 14 days. Cardiac function was evaluated by echocardiography, and an in vivo electrophysiological study was performed on Day 14. Cardiac inflammation, fibrosis, and Cx43 expression were determined by histochemical analysis and western blot analysis. Our results indicated that muscone treatment significantly improved cardiac function and inhibited ventricular inflammation, fibrosis, and nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome activation. Electrocardiogrphy and electrophysiology studies showed that muscone shortened the QRS interval, QT interval, QTc interval, and action potential duration; prolonged the effective refractory period; and reduced susceptibility to VAs in rats after MI. Furthermore, Cx43 expression in the BZ was increased by muscone treatment, and this change was coupled by inhibition of the NLRP3/IL-1ß/p38 MAPK pathway. Taken together, our results demonstrated that muscone reduces susceptibility to VA, mainly by decreasing ventricular inflammation and fibrosis, and attenuates abnormal Cx43 expression by inhibiting NLRP3 inflammasome activation after myocardial infarction in rats.

Sevoflurane Improves Ventricular Conduction by Exosomes Derived from Rat Cardiac Fibroblasts After Hypothermic Global Ischemia-Reperfusion Injury.

Purpose: This study investigated the effect of exosomes derived from sevoflurane-treated cardiac fibroblasts (Sev-CFs-Exo) on reperfusion arrhythmias (RA), ventricular conduction, and myocardial ischemia-reperfusion injury (MIRI). Methods: Primary cardiac fibroblasts (CFs) were isolated from the hearts of neonatal rats and identified by morphology and immunofluorescence. Exosomes were isolated from CFs at passages 2-3 after they had been treated with 2.5% sevoflurane for an hour and cultivated for 24-48 hours. The control group was CFs that did not receive any treatment. The hypothermic global ischemia-reperfusion injury model was established using the Langendorff perfusion technique following injection with exosomes through the caudal vein. Multi-electrode array (MEA) mapping was used to investigate the changes in RA and ventricular conduction in isolated hearts. Western blots and immunofluorescence were used to examine the relative expression and location of connexin 43 (Cx43). In addition, the MIRI was evaluated with triphenyl tetrazolium chloride and Hematoxylin-Eosin staining. Results: The primary CFs had a variety of morphologies, no spontaneous pulsation, and were vimentin-positive, which confirmed their successful isolation. Sev-CFs-Exo increased the heart rate (HR) at reperfusion for 15 minutes (T2) and 30 minutes (T3) and lowered the score and duration of RA and the time for restoration of heartbeat in reperfusion. Meanwhile, Sev-CFs-Exo increased conduction velocity (CV), decreased absolute inhomogeneity (P5-95) and inhomogeneity index (P5-95/P50) at T2 and T3, as well as promoted the recovery of HR, CV, P5-95 and P5-95/P50 after hypothermic global ischemia-reperfusion injury. Furthermore, Sev-CFs-Exo raised expression and reduced lateralization of Cx43, and improved myocardial infarct sizes and cellular necrosis. However, while cardiac fibroblast-derived exosomes (CFs-Exo) showed similar cardioprotective effects, the outcomes were not as significant. Conclusion: Sevoflurane reduces the risk of RA and improves ventricular conduction and MIRI by CFs-Exo, and this may be driven by the expression and location of Cx43.

Cx43-Delivered miR-181b Negatively Regulates Sirt1/FOXO3a Signalling Pathway-Mediated Apoptosis on Intestinal Injury in Sepsis.

INTRODUCTION: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. METHODS: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. RESULTS: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. CONCLUSIONS: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.

Protective effect of wuzibushen recipe on follicular development via regulating androgen receptor in polycystic ovary syndrome model rats.

OBJECTIVES: The study aimed to explore the role and mechanism of WZBS recipe on PCOS. METHODS: PCOS model was established. After modeling, PCOS rats were intragastrically administered with Diane-35 or WZBS recipe (6.93 g/kg/d). Then, the ovarian and uterine morphology were observed, the estrous cycle was assessed. HE and oil red O staining were conducted for ovarian morphological analysis and counting ovarian follicle and corpora lutea number. Furthermore, the serum content of testosterone (T) and sex-hormone-binding globulin (SHBG) were assessed by ELISA kits. The androgen receptor (AR), CX43 mRNA and protein expression were measured by q-PCR and Western blot. RESULTS: WZBS recipe increased uterine implanted blastocysts, reduced cystic dilated follicles, and normalized estrous cycle in PCOS rats. Meanwhile, WZBS recipe alleviated ovarian injury, increased mature follicles and corpora lutea number in PCOS rats. Moreover, WZBS recipe decreased serum T content, AR expression and increased serum SHBG content, CX43 expression in PCOS rats. CONCLUSIONS: This study reveals that WZBS recipe may attenuate PCOS by protecting follicular development via down-regulating AR.

Experimental Investigations of Monomethyl and Dimethyl Fumarate in an Astrocyte-Microglia Co-Culture Model of Inflammation.

INTRODUCTION: Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the central nervous system. Dimethyl fumarate (DMF) and monomethyl fumarate (MMF) belong to the disease-modifying drugs in treatment of MS. There is evidence that astrocytes and microglia are involved in MS pathology, but few studies are available about MMF and DMF effects on astrocytes and microglia. The aim of this study was to investigate the effects of MMF and DMF on microglial activation and morphology as well as potential effects on glial viability, Cx43, and AQP4 expressions in different set-ups of an in vitro astrocyte-microglia co-culture model of inflammation. METHODS: Primary rat glial co-cultures of astrocytes containing 5% (M5, mimicking "physiological" conditions) or 30% (M30, mimicking "pathological, inflammatory" conditions) of microglia were treated with different concentrations of MMF (0.1, 0.5, and 2 µg/mL) or DMF (1.5, 5, and 15 µM) for 24 h. Viability, proliferation, and cytotoxicity of glial cells were examined using MTT assay. Immunocytochemistry was performed to analyze the microglial phenotypes. Connexin 43 (Cx43) and aquaporin 4 (AQP4) expressions were quantified by immunoblot analysis. RESULTS: Treatment with different concentrations of MMF or DMF for 24 h did not change the glial cell viability in M5 and M30 co-cultures. Microglial phenotypes were not altered by DMF under physiological M5 conditions, but treatment with higher concentration of DMF (15 µM) induced microglial activation under inflammatory M30 conditions. Incubation with different concentrations of MMF had no effects on microglial phenotypes. The Cx43 expression in M5 and M30 co-cultures was not changed significantly by immunoblot analysis after incubation with different concentrations of DMF or MMF for 24 h. The AQP4 expression was significantly increased in M5 co-cultures after incubation with 5 µm DMF. Under the other conditions, AQP4 expression was not affected by DMF or MMF. DISCUSSION: In different set-ups of the astrocyte-microglia co-culture model of inflammation, MMF has not shown significant effects. DMF had only limited effects on microglia phenotypes and AQP4 expression. In summary, mechanisms of action of fumarates probably do not involve direct effects on microglia phenotypes as well as Cx43 and AQP4 expression.

Connexin 43: An Interface Connecting Neuroinflammation to Depression.

Major depressive disorder (MDD) is a leading chronic mental illness worldwide, characterized by anhedonia, pessimism and even suicidal thoughts. Connexin 43 (Cx43), mainly distributed in astrocytes of the brain, is by far the most widely and ubiquitously expressed connexin in almost all vital organs. Cx43 forms gap junction channels in the brain, which mediate energy exchange and effectively maintain physiological homeostasis. Increasing evidence suggests the crucial role of Cx43 in the pathogenesis of MDD. Neuroinflammation is one of the most common pathological features of the central nervous system dysfunctions. Inflammatory factors are abnormally elevated in patients with depression and are closely related to nearly all links of depression. After activating the inflammatory pathway in the brain, the release and uptake of glutamate and adenosine triphosphate, through Cx43 in the synaptic cleft, would be affected. In this review, we have summarized the association between Cx43 and neuroinflammation, the cornerstones linking inflammation and depression, and Cx43 abnormalities in depression. We also discuss the significant association of Cx43 in inflammation and depression, which will help to explore new antidepressant drug targets.

Cellular signaling crosstalk between Wnt signaling and gap junctions inbenzo[a]pyrene toxicity.

Gap junctional intercellular communication (GJIC) is considered a key biological mechanism to maintain homeostasis in cell differentiation and growth. In addition, as another major signaling pathway associated with cell proliferation and differentiation, Wnt/ß-catenin signaling appears to trigger several cellular responses against injury. The purpose of the present study was to investigate the effects of a known toxic agent, benzo[a]pyrene (BaP), on the regulation and interaction between GJIC and Wnt/ß-catenin signaling. BaP treatment resulted in GJIC inhibition and decreases the major GJIC protein connexin 43 (Cx43) in WB-F344 rat liver epithelial cells. We also found BaP-mediated downregulation of Wnt/ß-catenin signaling related to the PI3K-Akt pathway. To identify the relationship between GJIC and Wnt/ß-catenin signaling, we treated WB-F344 cells with the Wnt agonist CHIR99021 and found that it inhibited GJIC while causing a significant reduction in Cx43 expression at both the mRNA and protein levels, through the repression of promoter activity. This Wnt agonist-mediated GJIC inhibition was confirmed using a small interfering RNA directed against the Wnt antagonist Dact2, indicating that Wnt/ß-catenin signaling negatively regulates GJIC. Despite the inverse correlation between Wnt/ß-catenin signaling and Cx43 promoter activation as indicated by downregulation of ß-catenin nuclear translocation and upregulation of Cx43 promoter activation involving HNF3ß, BaP treatment decreased the Cx43 protein expression, which was associated with protein degradation, possibly through protein kinase C activation. In conclusion, our results revealed the mechanism of BaP-induced inhibition of GJIC and Wnt/ß-catenin signaling. More importantly, linking Wnt/ß-catenin signaling to Cx protein expression will have profound implications in understanding the relationships among different major signaling pathways associated with cell proliferation and differentiation in toxicity.

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform.

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.

Contraction Band Necrosis with Dephosphorylated Connexin 43 in Rat Myocardium after Daily Cocaine Administration.

Contraction band necrosis (CBN) is a common abnormality found in the myocardium of cocaine abusers, but is rarely reported in experimental models of cocaine abuse. Connexin 43 (Cx43) is essential for cardiac intercellular communication and the propagation of CBN. Under stress or injury, cardiac Cx43 is dephosphorylated, which is related to cardiomyocyte dysfunction and pathogenesis, whereas adiponectin exerts beneficial effects in the myocardium. In this study, we explore the effects of cocaine on cardiac Cx43 in vivo. Rats were administered cocaine via the tail vein at 20 mg/kg/day for 14 days, and showed widespread CBN, microfocal myocarditis and myocardial fibrosis, corresponding to a dysfunction of cardiac mitochondria under increased oxidative stress. The increase in dephosphorylated cardiac Cx43 and its negative correlation with the myocardial distribution of CBN after cocaine administration were determined. In addition, apoptosis and necroptosis, as well as increased adiponectin levels, were observed in the myocardium after cocaine exposure. Accordingly, we found altered profiles of cardiac Cx43, CBN and its negative correlation with dephosphorylated cardiac Cx43, and the possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. The latter might play a protective role in the cardiotoxicity of cocaine. The current findings would be beneficial for establishing novel therapeutic strategies in cocaine-induced cardiac consequences.

Estrogen Protects against Renal Ischemia-Reperfusion Injury by Regulating Th17/Treg Cell Immune Balance.

Inflammation is a critical mediator of renal ischemia-reperfusion (I/R) injury (IRI), and T lymphocytes exert a key role in the renal IRI-induced inflammation. Connexin 43 (Cx43) is related to the maintenance of T lymphocyte homeostasis. Various preclinical researches have reported that estrogen is a renoprotective agent based on its anti-inflammatory potential. The present research is aimed at studying the role of T lymphocytes activated by Cx43 in 17ß-estradiol-mediated protection against renal IRI. Female rats were classified into six groups: control rats, I/R rats, ovariectomized rats, ovariectomized I/R rats, and ovariectomized rats treated with 17ß-estradiol or gap27. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) and Paller scoring were dramatically increased in I/R rats, especially in ovariectomized rats. By contrast, these indicators were markedly decreased by administering estradiol or gap27. Immunofluorescence staining revealed that CD4+ T cells infiltrated kidney tissues in the early stage of IRI. In both peripheral blood and renal tissue, the proportion of CD3+CD4+ T cells and ratio of CD4+ to CD8+ were high in I/R rats, especially in ovariectomized rats. The proportion of CD3+CD8+ T cells was low in peripheral blood but high in renal tissues. Administration of estrogen or Gap27 reversed these effects. IL-17 levels in both serum and tissue homogenate were significantly increased in ovariectomized rats subjected to I/R but significantly decreased in estrogen or gap 27 treated rats. The opposite trend was observed for IL-10 levels. Correlation analysis demonstrated that IL-17 was correlated positively with BUN, Scr, and Paller scores, while IL-10 was negatively correlated with these indicators. Western blot showed that Cx43 expression was markedly increased in the peripheral blood T lymphocytes of I/R rats, especially ovariectomized rats. After intervention with estrogen and gap27, Cx43 expression was significantly downregulated. These findings indicate that Cx43 may participate in the regulation of Th17/Treg balance by estrogen against renal IRI.

Role of Endothelin-1 in Right Atrial Arrhythmogenesis in Rabbits with Monocrotaline-Induced Pulmonary Arterial Hypertension.

Atrial arrhythmias are considered prominent phenomena in pulmonary arterial hypertension (PAH) resulting from atrial electrical and structural remodeling. Endothelin (ET)-1 levels correlate with PAH severity and are associated with atrial remodeling and arrhythmia. In this study, hemodynamic measurement, western blot analysis, and histopathology were performed in the control and monocrotaline (MCT, 60 mg/kg)-induced PAH rabbits. Conventional microelectrodes were used to simultaneously record the electrical activity in the isolated sinoatrial node (SAN) and right atrium (RA) tissue preparations before and after ET-1 (10 nM) or BQ-485 (an ET-A receptor antagonist, 100 nM) perfusion. MCT-treated rabbits showed an increased relative wall thickness in the pulmonary arterioles, mean cell width, cross-sectional area of RV myocytes, and higher right ventricular systolic pressure, which were deemed to have PAH. Compared to the control, the spontaneous beating rate of SAN-RA preparations was faster in the MCT-induced PAH group, which can be slowed down by ET-1. MCT-induced PAH rabbits had a higher incidence of sinoatrial conduction blocks, and ET-1 can induce atrial premature beats or short runs of intra-atrial reentrant tachycardia. BQ 485 administration can mitigate ET-1-induced RA arrhythmogenesis in MCT-induced PAH. The RA specimens from MCT-induced PAH rabbits had a smaller connexin 43 and larger ROCK1 and phosphorylated Akt than the control, and similar PKG and Akt to the control. In conclusion, ET-1 acts as a trigger factor to interact with the arrhythmogenic substrate to initiate and maintain atrial arrhythmias in PAH. ET-1/ET-A receptor/ROCK signaling may be a target for therapeutic interventions to treat PAH-induced atrial arrhythmias.

Connexin 43 contributes to the sensitization of colorectal cancer cells to photodynamic therapy through Akt inhibition.

BACKGROUND: Photodynamic therapy could be one approach to treat colorectal cancer though resistance leads to failure of therapy. Akt activation is a cellular survival response to photodynamic therapy and is also a reason for resistance. Thus, inhibition of Akt is a strategy to decrease resistance. Akt interacts with connexin 43, another protein involved in photodynamic therapy resistance. Connexin 43 is widely expressed in different human tissues and has a complex role in tumor development. However, the mechanism of inhibition of Akt by connexin 43 that sensitizes colorectal cancer cells to photodynamic therapy needs further investigation. METHODS: In this study, two colorectal cancer cells with low phosphorylated connexin 43 level were used to explore this mechanism. LY294002 was used as an Akt inhibitor, and connexin 43-pCMV3 was transfected into cells to increase connexin 43 expression. RESULTS: Akt and connexin 43 inhibit each other in both colorectal cancer cell lines. In vitro and in vivo experiments showed that LY294002 and connexin 43 transfection sensitized cells to hematoporphyrin-Photodynamic therapy. LY294002 increased the sensitivity of cells to photodynamic therapy with a pronounced effect in cells with high expression levels of connexin 43. CONCLUSIONS: Connexin 43 should be considered an important factor in increasing the phototoxicity of photodynamic therapy in colorectal cancer through Akt inhibition.

[Inhibition connexin 43 by mimetic peptide Gap27 mediates protective effects on 6-hydroxydopamine induced Parkinson's disease mouse model].

OBJECTIVE: To explore whether the using of mimetic peptide Gap27, a selective inhibitor of connexin 43 (Cx43), could block the death of dopamine neurons and influence the expression of Cx43 in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease mouse models. METHODS: Eighteen C57BL/6 mice were randomly divided into control group, 6-OHDA group and 6-OHDA+Gap27 group, with 6 mice in each group. Bilateral substantia nigra stereotactic injection was performed. The control group was injected with ascorbate solution, 6-OHDA group was injected with 6-OHDA solution, and 6-OHDA+Gap27 group was injected with 6-OHDA and Gap27 mixed solution. Immuno-histochemical staining was used to detect the number of dopamine neurons, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of Cx43 messenger ribonucleic acid (mRNA), immuno-fluorescence staining was used to detect the distribution of Cx43 protein, the contents of Cx43 protein and Cx43 phosphorylation at serine 368 (Cx43-ps368) in mouse midbrain were detected by Western blot. RESULTS: After injection of 6-OHDA, numerous dopamine neurons in substantia nigra died as Cx43 content increased, Cx43-ps368 content decreased. Mixing Gap27 while injecting 6-OHDA could reduce the number of death dopamine neurons and weaken the changes of Cx43 and Cx43-ps368 content caused by 6-OHDA. The number of tyrosine hydroxylase (TH) immunoreactive positive neurons in 6-OHDA group decreased to 27.7% ± 0.02% of the control group (P < 0.01); The number of TH immunoreactive positive neurons in 6-OHDA+Gap27 group was (1.64±0.16) times higher than that in 6-OHDA group (P < 0.05); The content of total Cx43 protein in 6-OHDA group was (1.44±0.07) times higher than that in 6-OHDA+Gap27 group (P < 0.05) while (1.68±0.07) times higher than that in control group (P < 0.01). In 6-OHDA group, the content of Cx43-ps368 protein and its proportion in total Cx43 protein were significantly lower than that in 6-OHDA+Gap27 group (P < 0.05). CONCLUSION: In 6-OHDA mouse models, mimetic peptide Gap27 played a protective role in reducing the damage to substantia nigra dopamine neurons, which was induced by 6-OHDA. The overexpression of Cx43 protein might have neurotoxicity to dopamine neuron. Meanwhile, decreasing Cx43 protein level and keeping Cx43-ps368 protein level may be the protective mechanisms of Gap27.

Study on the Mechanism and Oxidative Stress of Compound Danshen on the Degradation of Cx43 and Improvement of Myocardial Apoptosis in ICM Rats.

In order to investigate the mechanism by which Salvia miltiorrhiza degrades connexin 43 (Cx43) and improves myocardial cell apoptosis and oxidative stress in rats with myocardial ischemia (ICM), male SD rats of pure grade are selected (32 rats), adaptively bred for 1 week, and then bred in SPF medium. They are randomly divided into a normal group, a model group, a low-dose Danshen group, and a high-dose group, with 8 rats in each group. HE staining and immunohistochemistry are performed. Serum samples are detected using kits according to instructions, including nitric oxide (NO) and endothelin-1 (ET-1); malondialdehyde (MDA), ELISA for superoxide dismutase (SOD), and vascular cell adhesion molecules (VCAM-1); western blot for detection of cardiac Cx43 protein expression; immunohistochemistry to detect Cx43 expression; and detection of myocardial ischemia. For ICM rats, the application of high-dose compound Salvia miltiorrhiza can effectively prevent the degradation of Cx43 protein, improve the apoptosis of myocardial cells in rats, and have a certain protective effect on ischemic myocardium in rats.