Spinal cord injury (SCI) is a severe condition with an extremely high disability rate. It is mainly manifested as the loss of motor, sensory and autonomic nerve functions below the injury site. High-frequency transcranial magnetic stimulation, a recently developed neuromodulation method, can increase motor function in mice with spinal cord injury. This study aimed to explore the possible mechanism by which transcranial magnetic stimulation (TMS) restores motor function after SCI. A complete T8 transection model of the spinal cord was established in mice, and the mice were treated daily with 15 Hz high-frequency transcranial magnetic stimulation. The BMS was used to evaluate the motor function of the mice after SCI. Western blotting and immunofluorescence were used to detect the expression of Connexin43 (CX43) and autophagy-related proteins in vivo and in vitro, and correlation analysis was performed to study the relationships among autophagy, CX43 and motor function recovery after SCI in mice. Western blotting was used to observe the effect of magnetic stimulation on the expression of mTOR pathway members. In the control group, the expression of CX43 was significantly decreased, and the expression of microtubule-associated protein 1 A/1b light chain 3 (LC3II) and P62 was significantly increased after 4 weeks of spinal cord transection. After high-frequency magnetic stimulation, the level of CX43 decreased, and the levels of LC3II and P62 increased in primary astrocytes. The BMS of the magnetic stimulation group was greater than that of the control group. High-frequency magnetic stimulation can inhibit the expression of CX43, which negatively regulates autophagic flux. HF-rTMS increased the expression levels of mTOR, p-mTOR and p-S6. Our experiments showed that rTMS can restore hindlimb motor function in mice after spinal cord injury via regulation of the Cx43-autophagy loop and activation of the mTOR signalling pathway.

Cardiomyocyte apoptosis is a complex biological process involving the interaction of many factors and signaling pathways. In hypoxic environment, cardiomyocytes may trigger apoptosis due to insufficient energy supply, increased production of oxygen free radicals, and disturbance of intracellular calcium ion balance. The present research aimed to investigate the role of microRNA-29b1 (miR-29b1) in hypoxia-treated cardiomyocytes and its potential mechanism involved. We established an in vitro ischemia model using AC16 and H9C2 cardiomyocytes through hypoxia treatment (1% O2, 48 h). Cell apoptosis was evaluated by flow cytometry using Annexin V FITC-PI staining assay. Moreover, we used Western blot and immunofluorescence analysis to determine the expression of Bcl-2, Bax caspase-3 and Cx43 proteins. We found that miR-29b1 protected AC16 and H9C2 cells from hypoxia-induced injury as evidence that miR-29b1 attenuated the effects of hypoxia treatment on AC16 and H9C2 cell apoptosis after hypoxia treatment. In conclusion, our findings suggest that miR-29b1 may have potential cardiovascular protective effects during ischemia-related myocardial injury.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG\'s influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG\'s inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG\'s multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG\'s inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

Noncoding RNAs (ncRNAs) are a class of nucleotide sequences that cannot be translated into peptides. ncRNAs can function post-transcriptionally by splicing complementary sequences of mRNAs or other ncRNAs or by directly engaging in protein interactions. Over the past few decades, the pervasiveness of ncRNAs in cell physiology and their pivotal roles in various diseases have been identified. One target regulated by ncRNAs is connexin (Cx), a protein that forms gap junctions and hemichannels and facilitates intercellular molecule exchange. The aberrant expression and misdistribution of connexins have been implicated in central nervous system diseases, cardiovascular diseases, bone diseases, and cancer. Current databases and technologies have enabled researchers to identify the direct or indirect relationships between ncRNAs and connexins, thereby elucidating their correlation with diseases. In this review, we selected the literature published in the past five years concerning disorders regulated by ncRNAs via corresponding connexins. Among it, microRNAs that regulate the expression of Cx43 play a crucial role in disease development and are predominantly reviewed. The distinctive perspective of the ncRNA-Cx axis interprets pathology in an epigenetic manner and is expected to motivate research for the development of biomarkers and therapeutics.

BACKGROUND: Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated.
METHODS: We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation.
RESULTS: About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40.
CONCLUSIONS: The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy.
BACKGROUND: ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.

The study aims to investigate the effects and potential mechanism of raw and processed Aconitum pendulum Busch on rheumatoid arthritis(RA) and analyze their toxicity attenuating and efficacy retaining effects. The bovine type Ⅱ collagen-induced arthritis(CIA) rat model was established. The weight, cardiac index, immune organ index, and arthritis index of the rats were recorded and calculated after administration. ELISA was used to measure the expressions of creatine kinase(CK), cardiac troponin T(cTnT), and multiple factors. The pathological morphological changes in heart tissue and ankle joint tissue were observed by hematoxylin-eosin staining. Connexin 43(Cx43) expression in the hearts of CIA rats was detected via immunohistochemical method. The levels of endogenous metabolites in the serum of CIA rats were detected by UPLC-Q-TOF-MS. Potential biomarkers were screened, and related metabolic pathways were analyzed. The results showed that raw A. pendulum could induce local myocardial fiber degeneration and necrosis, increase the cardiac index, decrease the average positive area of Cx43 expression significantly, and increase the expressions of CK and cTnT in cardiac tissue of rats. Meanwhile, raw A. pendulum could decrease the immune organ index, interleukin-6(IL-6), and other inflammatory cytokine contents in the serum and improve the damaged synovium and joint surface of CIA rats, with toxicity and efficacy coexisting. The Zanba stir-fired A. pendulum could reduce the index of arthritis, immune organ index, and content of IL-6 and inflammatory cytokines in serum and improve damaged synovium and joint surface of CIA rats with no obvious cardiac toxicity, showing significant toxicity attenuating and efficacy retaining effects. A total of 19 potential biomarkers of raw A. pendulum and Zanba stir-fired A. pendulum against RA were screened by serum metabolomics, including glycerophospholipid metabolism, glycine, serine, and threonine metabolism, arginine and proline metabolism, and steroid hormone synthesis. In conclusion, Xizang medicine A. pendulum is preventive and curative for RA. Raw A. pendulum has certain cardiotoxicity, and Zanba stir-fired A. pendulum has significant toxicity attenuating and efficacy retaining effects. The anti-RA mechanism may be related to the regulation of glycerophospholipid and amino acid metabolism.

Background and Objectives: Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods: Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results: The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein-protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions: GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.

The pathogenesis of ulcerative colitis (UC) involves chronic inflammation of the submucosal layer and disruption of epithelial barrier function within the gastrointestinal tract. Connexin 43 (Cx43) has been implicated in the pathogenesis of intestinal inflammation and its associated carcinogenic effects. However, a comprehensive analysis of Cx43\'s role in mucosal and peripheral immunity in patients with UC is lacking. In this study, the colon tissues of patients with UC exhibited severe damage to the intestinal mucosal barrier, resulting in a significant impairment of junctional communication as observed by transmission electron microscopy. The mRNA expression of Cx43 was found to be significantly elevated in the UC group compared to the control group, as determined using the Affymetrix expression profile chip and subsequently validated using qRT-PCR. The immunofluorescence analysis revealed a significantly higher mean fluorescence intensity of Cx43 in the UC group compared to the control group. Additionally, Cx43 was observed in both the cell membrane and nucleus, providing clear evidence of nuclear translocation. The proportion of Cx43 in the UC group for CD4+ and CD8+ T lymphocytes was increased in the control group, but only the proportion of Cx43 for CD8+ T lymphocytes showed significant difference by flow cytometry. The involvement of Cx43 in the pathogenesis of UC and its potential role in mucosal immunity warrants further investigation, as it holds promise as a prospective biomarker and therapeutic target for this condition. The proportion of Cx43 in the UC group for CD4+ and CD8+ T lymphocytes was increased in the control group, but only the proportion of Cx43 for CD8+ T lymphocytes showed a significant difference.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Ischemic heart disease is caused by coronary artery occlusion. Despite the increasing number and success of interventions for restoring coronary artery perfusion, myocardial ischemia-reperfusion (I/R) injury remains a significant cause of morbidity and mortality worldwide. Inspired by the impact of I/R on the Cx43 trafficking to the intercalated discs (ICDs), we aim to explore the potential mechanisms underlying the downregulation of Cx43 in ICDs after myocardial I/R. Gene set enrichment analysis (GSEA), Western blotting, and immunofluorescence experiments showed that Myocardial I/R activated the P38MAPK signaling pathway and promoted microtubule depolymerization. Inhibition of P38MAPK signaling pathway activation attenuated I/R-induced microtubule depolymerization. The ability of SB203580 to recover the distribution of Cx43 and electrophysiological parameters in I/R myocardium depended on microtubule stability. Our study suggests that microtubule depolymerization caused by the activation of the P38MAPK signaling pathway is an important mechanism underlying the downregulation of Cx43 in ICDs after myocardial I/R.