Circ_0049979 ameliorates myocardial infarction through improving Cx43-mediated endothelial functions.

Endothelial injury is a fundamental pathogenesis of coronary atherosclerotic heart disease (CHD). Circular RNAs (circRNAs) are important post-transcriptional regulators in many human major diseases, including CHD. The aim of the present study was to explore the role of circ_0049979, a novel identified circRNA from ANO8 gene locus, in endothelial injury during CHD. We found that expression of circ_0049979 was reduced by ox-LDL treatment in HUVECs in a dose-dependent manner. Loss- and gain-of-function experiments demonstrated that knockdown of circ_0049979 decreased the capacities of proliferation, migration and tube formation in normal HUVECs. While, overexpression of circ_0049979 improved these capacities in both normal and ox-LDL-incubated HUVECs. Then, the online bioinformatic tool Circinteractome was used to predicted the target miRNAs of circ_0049979, and miR-653 was selected as the candidate. We demonstrated that miR-653 directly interacted with and was negatively regulated by circ_0049979, and played a negative role in regulating proliferation, migration and tube formation of HUVECs. In terms of the mechanism, miR-653 post-transcriptionally suppressed the expression of the gap junction protein 43 (Cx43), a key protein of endothelial tight junction. Finally, we verified that overexpression of circ_0049979 was able to alleviate plaque formation, lipid deposition, and endothelial cell apoptosis, as well as myocardial infarction, in coronary atherosclerotic mice in vivo. In conclusion, circ_0049979 plays a protective role in coronary atherosclerotic myocardial infarction by improving miR-653/Cx43-mediated endothelial functions.

Connexin 43 regulates intercellular mitochondrial transfer from human mesenchymal stromal cells to chondrocytes.

BACKGROUND: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. METHODS: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. RESULTS: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. CONCLUSIONS: Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.

Mutations of the Cx43 Gene in Non-Small Cell Lung Cancer: Association with Aberrant Localization of Cx43 Protein Expression and Tumor Progression.

Background and Objectives: The Connexin43 (Cx43) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Biomarker Profiles and Clinicopathological Features in Head and Neck Squamous Cell Carcinoma Patients.

Background and Objectives: Head and neck squamous cell carcinomas (HNSCCs) vary significantly in terms of invasiveness, growth rate, and metastatic potential. This study aimed to investigate the expression of several prognostic biomarkers (Ki67, p53, EGFR, COX-2, Cx43, and p16) in HNSCC from various anatomical regions and to correlate these expressions with clinicopathological parameters. Materials and Methods: We performed immunohistochemistry on 91 histologically verified HNSCC cases from the County Emergency Hospital, Targu Mures. Biomarker expression for Ki67, COX-2, and Cx43 was assessed using a standard immunoexpression scoring system: S1: 0-10%, S2: 11-25%, S3: 26-50%, S4 > 50%; EGFR was scored based on membrane staining intensity: 0, 1+, 2+, 3+; we classified p16 as positive or negative; p53 was grouped into mutant and wild-type; and we compared these across histopathological types, tumor grades, anatomical locations, gender, and different age groups. We performed a comparative analysis of Cx43 expression levels in relation to the expression of the rest of the markers. Statistical analysis was conducted using GraphPad InStat 3 software, version 3.06 (GraphPad Software Inc., San Diego, USA). Results: The majority of tumors were in males (95.6%) aged 51-60 years. Mutant p53 expression was prevalent in most cases. Elevated Ki67 and EGFR expression were associated with more aggressive tumors. COX-2 levels varied, with a higher proportion of moderate and high immunoexpression (S3 + S4) observed in patients under 70 years old. Cx43 expression was generally low, especially in extralaryngeal tumors. Conclusions: HNSCC primarily affects older males, with the larynx being the most common site. High levels of Ki-67 and EGFR suggest more aggressive tumors, while low COX-2 levels reflect varying prognoses. Women may develop more aggressive tumors, and extralaryngeal tumors often present with more challenging prognoses. Low Cx43 expression may be more likely to coincide with higher Ki67 and COX-2 levels, possibly indicating a link with more aggressive tumor behavior.

Blockage of p38MAPK in astrocytes alleviates brain damage in a mouse model of embolic stroke through the CX43/AQP4 axis.

BACKGROUND: Cerebral edema, a significant complication arising from acute ischemic stroke (IS), has a critical influence on morbidity and mortality. p38MAPK has been shown to promote neuronal apoptosis and brain damage. However, the role of the p38MAPK inhibitor SKF-86002 in protecting against ischemic injury and cerebral edema remains unclear. METHODS: Infarct area was examined by TTC staining in middle cerebral artery occlusion (MCAO) mice. Neurological score and brain water content were evaluated. TUNEL and NeuN staining were used to assess neuronal apoptosis and the survival of neurons. Blood-brain barrier (BBB) permeability was determined by Evans blue. Double immunofluorescence staining detected the colocalization of AQP4 and CX43 in astrocytes. IHC staining revealed CX43 and AQP4 expression. EDU staining detected the proliferation of Oxygen and glucose deprivation/reoxygenation (OGD/R)-treated astrocytes. Levels of oxidative stress markers were determined using commercial kits. ELISA was used to assess the secretion of pro-inflammatory factors. RT-qPCR measured the expression of CX43, AQP4 and pro-inflammatory factors. Western blot analyzed the levels of p-p38/p38, AQP4 and CX43. Co-immunoprecipitation (Co-IP) determined the interaction between CX43 and AQP4. RESULTS: SKF-86002 attenuated brain damage, edema, and neuronal apoptosis in MCAO mice. Astrocyte proliferation was suppressed, and oxidative stress and inflammation were alleviated by SKF-86002 treatment. SKF-86002 negatively regulated p38 signaling and the expression of AQP4 and CX43. Additionally, the expression of CX43/AQP4 within astrocytes was modulated by SKF-86002. CONCLUSION: In summary, SKF-86002 alleviated IS injury and cerebral edema by inhibiting astrocyte proliferation, oxidative stress and inflammation. This effect was associated with the suppression of CX43/AQP4, suggesting that SKF-86002 shows promise as a novel therapeutic approach for preventing IS.

Macrophages enhance sodium channel expression in cardiomyocytes.

Cardiac macrophages facilitate electrical conduction through the atrioventricular-node (AV) in mice. A possible role for cardiomyocyte-macrophage coupling on the effect of antiarrhythmic therapy has not been investigated yet. Holter monitoring was conducted in LysMCrexCsf1rLsL-DTR mice (MMDTR) under baseline conditions and after an elctrophysiological stress test by flecainide. In vivo effects were recapitulated in vitro by patch-clamp experiments. The underlying mechanism was characterized by expression and localization analysis of connexin43 (Cx43) and voltage-gated-sodium-channel-5 (Nav1.5). ECG monitoring in MMDTR mice did not show any significant conduction abnormalities but a significantly attenuated flecainide-induced extension of RR- and PP-intervals. Patch-clamp analysis revealed that the application of flecainide to neonatal rat ventricular cardiomyocytes (CMs) changed their resting-membrane-potential (RMP) to more negative potentials and decreased action-potential-duration (APD50). Coupling of macrophages to CMs significantly enhances the effects of flecainide, with a further reduction of the RMP and APD50, mediated by an upregulation of Cx43 and Nav1.5 surface expression. Macrophage depletion in mice does not correlate with cardiac electric conduction delay. Cardiac macrophages amplify the effects of flecainide on electrophysiological properties of cardiomyocytes in vivo and in vitro. Mechanistically, formation of macrophage-cardiomyocyte cell-cell-contacts via Cx43 facilitates the recruitment of Nav1.5 to the cell membrane increasing flecainide effects.

Chrysophanol-mediated trx-1 activation attenuates renal fibrosis through inhibition of the JNK/Cx43 signaling pathway.

PURPOSE: This study aimed to investigate the inhibitory effect of chrysophanol on renal fibrosis and its molecular mechanism. METHODS: Initially, potential targets of chrysophanol were predicted through network pharmacology analysis, and a protein-protein interaction network of these targets was constructed using Venn diagrams and the STRING database. GO enrichment analysis predicted the biological process of chrysophanol in treating renal fibrosis. Subsequently, both in vivo and in vitro experiments were conducted using unilateral ureteral obstruction (UUO) induced CKD mouse model and HK-2 cell model, respectively. In the mouse model, different doses of chrysophanol were administered to assess its renal protective effects through biochemical indicators, histological examination, and immunofluorescence staining. In the cell model, the regulatory effect of chrysophanol on the Trx-1/JNK/Cx43 pathway was evaluated using western blotting and flow cytometry. RESULTS: Chrysophanol treatment significantly ameliorated renal dysfunction and histopathological damage in the UUO mouse model, accompanied by a reduction in serum oxidative stress markers. Furthermore, chrysophanol markedly upregulated the expression of Trx-1 in renal tissues and inhibited the activation of the JNK/Cx43 signaling pathway. At the cellular level, chrysophanol enhanced the activity of Trx-1 and downregulated the JNK/Cx43 signaling pathway, thereby inhibiting TGF-ß induced oxidative stress and cell apoptosis. CONCLUSION: This study demonstrated a significant inhibitory effect of chrysophanol on renal fibrosis, mediated by the activation of Trx-1 to inhibit the JNK/Cx43 pathway. These findings provide experimental support for the potential use of chrysophanol as a therapeutic agent for renal fibrosis.

The effect of intra spinal administration of cerium oxide nanoparticles on central pain mechanism: An experimental study.

This study investigated Cerium oxide nanoparticles (CeONPs) effect on central neuropathic pain (CNP). The compressive method of spinal cord injury (SCI) model was used for pain induction. Three groups were formed by a random allocation of 24 rats. In the treatment group, CeONPs were injected above and below the lesion site immediately after inducing SCI. pain symptoms were evaluated using acetone, Radian Heat, and Von Frey tests weekly for six weeks. Finally, we counted fibroblasts using H&E staining. We evaluated the expression of Cx43, GAD65 and HDAC2 proteins using the western blot method. The analysis of results was done by PRISM software. At the end of the study, we found that CeONPs reduced pain symptoms to levels similar to those observed in normal animals. CeONPs also increased the expression of GAD65 and Cx43 proteins but did not affect HDAC2 inhibition. CeONPs probably have a pain-relieving effect on chronic pain by potentially preserving GAD65 and Cx43 protein expression and hindering fibroblast infiltration.

GDNF's Role in Mitigating Intestinal Reactive Gliosis and Inflammation to Improve Constipation and Depressive Behavior in Rats with Parkinson's disease.

Constipation is a common symptom in patients with Parkinson's disease (PD) and is often associated with depression. Enteric glial cells (EGCs) are crucial for regulating intestinal inflammation and colon motility, and their activation can lead to the death of intestinal neurons. Glial cell line-derived neurotrophic factor (GDNF) has been recognized for its neuroprotective properties in various neurological disorders, including PD. This study explores the potential of GDNF in alleviating intestinal reactive gliosis and inflammation, thereby improving constipation and depressive behavior in a rat model of PD. A PD model was established via unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA). Five weeks post-injury, AAV5-GDNF (2 ~ 5 × 10^11) was intraperitoneally injected into experimental and control rats. Fecal moisture percentage (FMP) and colonic propulsion rate (CPPR) were used to evaluate colon motility. Colon-related inflammation and colonic epithelial morphology were assessed, and depressive behavior was analyzed one week before sampling. PD rats exhibited reduced colonic motility and GDNF expression, along with increased EGC reactivity and elevated levels of pro-inflammatory cytokines IL-1, IL-6, and TNF-α. Additionally, there was an up-regulation of CX43 and a decrease in PGP 9.5 expression. The intraperitoneal injection of AAV-GDNF significantly protected colonic neurons by inhibiting EGC activation and down-regulating CX43. This treatment also led to a notable reduction in depressive-like symptoms in PD rats with constipation. GDNF effectively reduces markers of reactive gliosis and inflammation, and promotes the survival of colonic neurons, and improves colonic motility in PD rats by regulating CX43 activity. Furthermore, GDNF treatment alleviates depressive behavior, suggesting that GDNF or its agonists could be promising therapeutic agents for managing gastrointestinal and neuropsychiatric symptoms associated with PD.

Tamoxifen-induced alterations in the expression of connexin 43 in the chicken ovary.

Connexin 43 (Cx43) is a gap junction protein that participates in small molecule exchange between adjacent cells. It is a predominant Cx within the mammalian ovary, where is associated with proper follicle development. The expression and regulation of Cx43 in the chicken ovary is largely unknown. The aim of the present study was to examine the expression of the Cx43 gene (GJA1) and protein as well as the immunolocalization of Cx43 in the laying hen ovary in relation to follicle development, and to examine how tamoxifen (TMX; an estrogen receptor modulator) treatment affects these factors. qRT-PCR and western blotting demonstrated differences in Cx43 mRNA transcript and protein abundances in ovarian white follicles, yellowish follicles, small yellow follicles, and the largest yellow preovulatory follicles (F3-F1). In general, Cx43 was more abundant in hierarchical than prehierarchical follicles and in granulosa cells compared with theca cells. Further, the response to TMX treatment depended on the stage of follicle development and the layer of the follicular wall. Ovarian regression following TMX treatment was accompanied by an increase in Cx43 expression in most ovarian tissues, which may impact the formation and function of Cx43 hemichannels. Overall, our results showed, for the first time, the differences in Cx43 mRNA and protein levels between ovarian follicles, suggesting the potential involvement of this gap junction protein in the regulation of ovarian follicle development and function. In addition, the results indicate a possible role for estradiol in regulation of Cx43 transcription and/or translation in the chicken ovary. Understanding the contribution of Cx43 in mechanisms underlying ovarian follicle development may be of considerable importance for poultry egg production.

Exosomes from myoblasts induced by hypoxic preconditioning improved ventricular conduction by increasing Cx43 expression in hypothermia ischemia reperfusion hearts.

Myocardial ischemia-reperfusion arrhythmia after cardiac surgery is common and seriously affects quality of life. Remote ischemic preconditioning can reduce the myocardial damage caused by severe ischemia. However, the underlying mechanism is not well understood. This study aimed to investigate the effects of exosomes derived from C2C12 mouse myoblasts after hypoxic preconditioning (HP) on ventricular conduction in hypothermic ischemia-reperfusion hearts. Myocardial ischemia-reperfusion model rats were established using the Langendorff cardiac perfusion system. Exosomes derived from normoxic (ExoA) and hypoxia-preconditioned (ExoB) C2C12 cells were injected into the jugular vein of the model rats. The time to heartbeat restoration, arrhythmia type and duration, and heart rate were recorded after myocardial ischemia-reperfusion. Conduction velocity on the surface of left ventricle was measured using a microelectrode array after 30 min of balanced perfusion, 15 min of reperfusion, and 30 min of reperfusion. Immunohistochemistry and western blotting were performed to determine the distribution and relative expression of connexin 43 (Cx43). ExoB contained more exosomes than ExoA, showing that HP stimulated the release of exosomes. The IR + ExoB group showed faster recovery of ventricular myocardial activity, a lower arrhythmia score, faster conduction velocity, and better electrical conductivity than the IR group. ExoB increased the expression of Cx43 and reduced its lateralization in the ventricular muscle. Our study showed that exosomes induced by hypoxic preconditioning can improve ventricular myocardial conduction and reperfusion arrhythmia in isolated hearts after hypothermic ischemia-reperfusion.

Effects of exogenous hydrogen sulfide and honokiol intervention on the proliferation, apoptosis, and calcium signaling pathway of rat enteric glial cells.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that influences digestive and nervous system functions. Enteric glial cells (EGCs) are integral to the enteric nervous system and play a role in regulating gastrointestinal motility. This study explored the dual effects of exogenous H2S on EGCs and the influence of apoptosis-related pathways and ion channels in EGCs. We also administered honokiol for further interventional studies. The results revealed that low-concentration H2S increased the mitochondrial membrane potential (MMP) of EGCs, decreased the whole-cell membrane potential, downregulated BAX and caspase-3, upregulated Bcl2 expression, reduced apoptosis, and promoted cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also increased. A high concentration of H2S had the opposite effect. In addition, GFAP mRNA expression was upregulated in the test-low group, downregulated in the test-high group, and upregulated in the test-high + Hon group. Honokiol treatment increased MMP, reduced whole-cell membrane potential, inhibited BAX and caspase-3 expression, increased Bcl2 expression, decreased cell apoptosis, and increased cell proliferation. The Ca2+ concentration, Cx43 mRNA, and protein expression were also upregulated. In conclusion, our study showed that exogenous H2S can bidirectionally regulate EGC proliferation and apoptosis by affecting MMP and cell membrane potential via the Bcl2/BAX/caspase-3 pathway and modulate Cx43-mediated Ca2+ responses in EGCs to regulate colonic motility bidirectionally. Honokiol can ameliorate the damage to EGCs induced by high H2S concentrations through the Bcl2/BAX/caspase-3 pathway and improve colon motility by increasing Cx43 expression and Ca2+ concentration.

Relaxin suppresses atrial fibrillation, reverses fibrosis and reduces inflammation in aged hearts.

Healthy aging results in cardiac structural and electrical remodeling that increase susceptibility to cardiovascular diseases. Relaxin has shown broad cardioprotective effects including anti-fibrotic, anti-arrhythmic and anti-inflammatory outcomes in multiple models. This paper focuses on the cardioprotective effects of Relaxin in a rat model of aging. Sustained atrial or ventricular fibrillation are readily induced in the hearts of aged but not young control animals. Treatment with Relaxin suppressed this arrhythmogenic response by increasing conduction velocity, decreasing fibrosis and promoting substantial cardiac remodeling. Relaxin treatment resulted in a significant increase in the levels of: Nav1.5, Cx43, ßcatenin and Wnt1 in rat hearts. In isolated cardiomyocytes, Relaxin increased Nav1.5 expression. These effects were mimicked by CHIR 99021, a pharmacological activator of canonical Wnt signaling, but blocked by the canonical Wnt inhibitor Dickkopf1. Relaxin prevented TGF-ß-dependent differentiation of cardiac fibroblasts into myofibroblasts while increasing the expression of Wnt1; the effects of Relaxin on cardiac fibroblast differentiation were blocked by Dickkopf1. RNASeq studies demonstrated reduced expression of pro-inflammatory cytokines and an increase in the expression of α- and ß-globin in Relaxin-treated aged males. Relaxin reduces arrhythmogenicity in the hearts of aged rats by reduction of fibrosis and increased conduction velocity. These changes are accompanied by substantial remodeling of the cardiac tissue and appear to be mediated by increased canonical Wnt signaling. Relaxin also exerts significant anti-inflammatory and anti-oxidant effects in the hearts of aged rodents. The mechanisms by which Relaxin increases the expression of Wnt ligands, promotes Wnt signaling and reprograms gene expression remain to be determined.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Hydrogen mitigates brain injury by prompting NEDD4-CX43- mediated mitophagy in traumatic brain injury.

BACKGROUND: Hydrogen (H2) has emerged as a potential therapeutic intervention for traumatic brain injury (TBI). However, the precise mechanism underlying H2's neuroprotective effects in TBI remain incompletely understood. METHODS: TBI mouse model was induced using the controlled cortical impact (CCI) method, and a cell model was established by exposing astrocytes to lipopolysaccharide (LPS). Cell viability was detected by CCK-8 kits. Cell apoptosis was measured by flow cytometry. ELISA was used to detect cytokine quantification. Protein and gene expression was detected by western blot and RT-PCR analysis. Co-immunoprecipitation (CO-IP) were employed for protein-protein interactions. Morris water maze test and rotarod test were applied for TBI mice. RESULTS: H2 treatment effectively inhibited the LPS-induced cell injury and cell apoptosis in astrocytes. NEDD4 expression was increased following H2 treatment coupled with enhanced mitophagy in LPS-treated astrocytes. Overexpression of NEDD4 and down-regulation of connexin 43 (CX43) mirrored the protective effects of H2 treatment in LPS-exposed astrocytes. NEDD4 interacts CX43 to regulates the ubiquitinated degradation of CX43. While overexpression of CX43 reversed the protective effects of H2 treatment in LPS-exposed astrocytes. In addition, H2 treatment significantly alleviated brain injury in TBI mouse model. CONCLUSION: H2 promoted NEDD4-CX43 mediated mitophagy to protect brain injury induced by TBI, highlighting a novel pathway underlying the therapeutic effects of H2 in TBI.

Inhibition of autophagy prevents cardiac dysfunction at early stages of cardiomyopathy in Bag3-deficient hearts.

The HSP70 co-chaperone BAG3 targets unfolded proteins to degradation via chaperone assisted selective autophagy (CASA), thereby playing pivotal roles in the proteostasis of adult cardiomyocytes (CMs). However, the complex functions of BAG3 for regulating autophagy in cardiac disease are not completely understood. Here, we demonstrate that conditional inactivation of Bag3 in murine CMs leads to age-dependent dysregulation of autophagy, associated with progressive cardiomyopathy. Surprisingly, Bag3-deficient CMs show increased canonical and non-canonical autophagic flux in the juvenile period when first signs of cardiac dysfunction appear, but reduced autophagy during later stages of the disease. Juvenile Bag3-deficient CMs are characterized by decreased levels of soluble proteins involved in synchronous contraction of the heart, including the gap junction protein Connexin 43 (CX43). Reiterative administration of chloroquine (CQ), an inhibitor of canonical and non-canonical autophagy, but not inactivation of Atg5, restores normal concentrations of soluble cardiac proteins in juvenile Bag3-deficient CMs without an increase of detergent-insoluble proteins, leading to complete recovery of early-stage cardiac dysfunction in Bag3-deficient mice. We conclude that loss of Bag3 in CMs leads to age-dependent differences in autophagy and cardiac dysfunction. Increased non-canonical autophagic flux in the juvenile period removes soluble proteins involved in cardiac contraction, leading to early-stage cardiomyopathy, which is prevented by reiterative CQ treatment.

Antibody-activation of connexin hemichannels in bone osteocytes with ATP release suppresses breast cancer and osteosarcoma malignancy.

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.

Wound Healing Efficacy of Cucurbitaceae Seed Oils in Rats: Comprehensive Phytochemical, Pharmacological, and Histological Studies Tackling AGE/RAGE and Nrf2/Ho-1 Cue.

The Cucurbitaceae family includes several edible species that are consumed globally as fruits and vegetables. These species produce high volumes of seeds that are often discarded as waste. In this study, we investigate the chemical composition and biological activity of three seed oils from Cucurbitaceae plants, namely, cantaloupe, honeydew, and zucchini, in comparison to the widely used pumpkin seed oil for their ability to enhance and accelerate wound healing in rats. Our results showed that honeydew seed oil (HSO) was effective in accelerating wound closure and enhancing tissue repair, as indicated by macroscopic, histological, and biochemical analyses, as compared with pumpkin seed oil (PSO). This effect was mediated by down-regulation of the advanced glycation end products (AGE) and its receptor (RAGE) cue, activating the cytoprotective enzymes nuclear factor erythroid 2 (Nrf2) and heme oxygenase-1 (HO-1), suppressing the inflammatory mediators tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-κB), and nod-like receptor protein 3 (NLRP3), and reducing the levels of the skin integral signaling protein connexin (CX)-43. Furthermore, immunohistochemical staining for epidermal growth factor (EGF) showed the lowest expression in the skin after treatment with HSO, indicating a well-organized and complete healing process. Other seed oils from cantaloupe and zucchini exhibited favorable activity when compared with untreated rats; however, their efficacy was comparatively lower than that of PSO and HSO. Gas chromatographic analysis of the derivatized oils warranted the superior activity of HSO to its high nutraceutical content of linoleic acid, which represented 65.9% of the fatty acid content. This study's findings validate the use of honeydew seeds as a wound-healing fixed oil and encourage further investigation into the potential of Cucurbitaceae seeds as sources of medicinally valuable plant oils.

Pegylated polymeric micelles of boswellic acid-selenium mitigates repetitive mild traumatic brain injury: Regulation of miR-155 and miR-146a/BDNF/ Klotho/Foxo3a cue.

This study aims to explore the protective machinery of pegylated polymeric micelles of boswellic acid-selenium (PMBS) against secondary neuronal damage triggered by mild repetitive traumatic brain injury (RTBI). After PMBS characterization in terms of particle size, size distribution, zeta potential, and transmission electronic microscopy, the selected formula was used to investigate its potency against experimental RTBI. Five groups of rats were used; group 1 (control) and the other four groups were subjected to RTBI. Groups 2 was RTBI positive control, while 3, 4, and 5 received boswellic acid (BSA), selenium (SEL), and PMBS, respectively. The open-field behavioral test was used for behavioral assessment. Subsequently, brain tissues were utilized for hematoxylin and eosin staining, Nissl staining, Western blotting, and ELISA in addition to evaluating microRNA expression (miR-155 and miR-146a). The behavioral changes, oxidative stress, and neuroinflammation triggered by RTBI were all improved by PMBS. Moreover, PMBS mitigated excessive glutamate-induced excitotoxicity and the dysregulation in miR-155 and miR-146a expression. Besides, connexin43 (Cx43) expression as well as klotho and brain-derived neurotrophic factor (BDNF) were upregulated with diminished neuronal cell death and apoptosis because of reduced Forkhead Box class O3a(Foxo3a) expression in the PMBS-treated group. The current study has provided evidence of the benefits produced by incorporating BSA and SEL in PEGylated polymeric micelles formula. PMBS is a promising therapy for RTBI. Its beneficial effects are attributed to the manipulation of many pathways, including the regulation of miR-155 and miR-146a expression, as well as the BDNF /Klotho/Foxo3a signaling pathway.

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.