The persistent non-healing of cutaneous wounds in diabetic patients represents a critical clinical challenge requiring urgent resolution. Excessive inflammation, impaired angiogenesis, and aberrant collagen remodeling profoundly hinder wound recovery. This study explores the potential therapeutic value of miR-221-3p in diabetic cutaneous wound healing using murine models. Subcutaneous administration of miR-221-3p was associated with accelerated wound closure; histological staining and Western blot analyses showed decreased expression of inflammatory markers (IL-1β, IL-6, MPO, CD68), increased levels of angiogenic markers (CD31, VEGFA), and enhanced collagen I/III deposition at wound margins. Complementary experiments using Mir221 knockout mice showed delayed healing, which was accompanied by upregulated MPO/CD68 expression, reduced CD31 levels, and decreased collagen fiber formation. These bidirectional observations suggest that miR-221-3p may contribute to diabetic wound healing, potentially through effects on inflammatory responses, neovascularization, and collagen synthesis. These findings suggest that miR-221-3p could serve as a potential therapeutic target for refractory wounds in diabetic patients, including diabetic foot ulcers and other chronic cutaneous lesions.

BACKGROUND:Identifying differentially expressed miRNAs in plasma-derived exosomes associated with type 2 diabetes mellitus (T2DM) complicated by atherosclerosis (AS) and elucidating their roles in high-glucose/high-lipid-induced vascular endothelial dysfunction. METHODS:Plasma-derived exosomal miRNAs were isolated from people with T2DM complicated by AS and healthy controls. Followed by miRNA sequencing to characterize differential expression profiles between groups. GO and KEGG pathway enrichment analyses were performed on differentially expressed miRNAs. Human umbilical vein endothelial cells (HUVECs) were exposed to combined hyperglycemia and hyperlipidemia to establish an in vitro model of diabetic endothelial injury. HUVECs were subsequently transfected with miR-887-5p mimics, inhibitors or negative controls, and assessed for proliferation, migration, apoptosis, oxidative stress, and nitric oxide (NO) content. RESULTS:Sequencing revealed globally reduced plasma exosomal miRNA expression in people with T2DM and AS relative to healthy controls, with 21 upregulated and 23 downregulated miRNAs identified. Among these, hsa-miR-887-5p exhibited the greatest fold change of all detected miRNAs, while hsa-miR-96-5p and hsa-miR-183-5p (upregulated) and hsa-miR-410-3p (downregulated) harbored the most target genes implicated in diabetic atherosclerosis. Functionally, miR-887-5p enhanced HUVEC proliferation and migration under high-glucose/high-lipid conditions, elevated superoxide dismutase (SOD) activity, reduced lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, suppressed intracellular iNOS/NO and attenuated apoptosis. CONCLUSION:Plasma exosomal miRNA expression is broadly reduced in people with T2DM complicated by AS. hsa-miR-887-5p, hsa-miR-96-5p, hsa-miR-183-5p, and hsa-miR-410-3p may emerge as candidates with diagnostic and therapeutic relevance in this context. Specifically, miR-887-5p mitigates high-glucose/high-lipid-induced vascular endothelial injury, warranting further investigation as a therapeutic target.

Cerebral stroke has a high mortality rate primarily driven by the pathophysiological processes of ischemia-reperfusion (I/R) injury. [D-Ala2, D-Leu5]-enkephalin (DADLE), the agonist of δ receptor, has raised interest as a tissue-protective agent for its ability to improve I/R injury, although its specific mechanism remains unclear. In this study, middle cerebral artery occlusion/reperfusion (MCAO/R) was induced in rats, while oxygen-glucose deprivation/reoxygenation (OGD/R) was applied to brain microvascular endothelial cells (BMECs). The effect of DADLE effect on autophagy regulation was assessed, and the interaction between β-arrestin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified. DADLE elevated autophagy levels in both in vivo and in vitro I/R models and improve BMEC viability under I/R conditions. After I/R injury, β-arrestin-GAPDH interaction tended to increase; however, DADLE treatment released GAPDH that was bound to β-arrestins. Simultaneously, DADLE reversed GAPDH activity caused by I/R injury. Quantitative real-time PCR detection revealed that, BMECs dominantly express a subtype of β-arrestin, β-arrestin1, which is necessary for DADLE-dependent cellular autophagy level and GAPDH activity enhancement. Therefore, this study provides evidence that DADLE may serve as a potential anti-ischemic stroke therapeutic agent.

BACKGROUND: Sonic Hedgehog (SHH) signaling pathway controls cell proliferation, differentiation, and organ formation. The changes in Ca2+ and their regulatory role in the context of activated SHH signaling have not been fully examined. RESULTS: In this study, we induced activation of the SHH signaling pathway using activators, including Full-length Shh protein (Shh) and N-terminal Shh (ShhN) ligands or a Smoothened agonist (SAG), while concurrently manipulating calcium level by either increasing extracellular Ca2+ concentration or employing BAPTA to chelate intracellular Ca2+. We found that the activation of SHH target genes by the three activators was not affected by low intracellular Ca2+ but inhibited by excess Ca2+. Although both Shh and SAG activate Ca2+ influx via the store-operated calcium entry (SOCE), their effects differ in the presence of BAPTA: Shh directly increases intracellular Ca2+ concentration, whereas the effect of SAG is enhanced by high extracellular Ca2+. CONCLUSIONS: Excessive extracellular Ca2+ impairs SHH signal transduction, whereas intracellular Ca2+ levels have a negligible effect. At normal physiological extracellular Ca2+ concentrations, the depletion of intracellular Ca2+ does not affect SHH signal transduction.

Cyclophosphamide (CTX), a widely used chemotherapeutic agent, frequently causes ovarian toxicity and premature ovarian insufficiency, posing a serious threat to fertility in premenopausal women. There is growing interest in identifying safe, dietary-based strategies to mitigate such off-target effects. Here, we demonstrate the protective potential of aqueous extracts from two traditional botanical functional foods Lycium barbarum (LB) and Angelica sinensis (AS), which have long been used in East Asian medicine to support female reproductive health. In a murine model of CTX-induced ovarian injury, oral administration of LB or AS significantly preserved the ovarian function, as shown by increased numbers of DDX4+ oocytes and anti-Müllerian hormone (AMH)-positive follicles revealed through tissue-clearing–assisted whole-ovary immunofluorescence. Both extracts restored serum levels of AMH and estradiol (E2), reduced ovarian oxidative stress (evidenced by lower 8-OHdG) and inflammation (indicated by reduced IL-6), and activated the Foxo3a–PRDX4 antioxidant pathway—characterized by elevated total Foxo3a and PRDX4 with concomitant reduction in phosphorylated Foxo3a. Gut microbiota analysis revealed that each extract induced distinct, and beneficial shifts. LB exhibited more potent antioxidant effects, while AS showed superior efficacy in restoring hormone levels and suppressing inflammation, suggesting a potential link between gut microbiota modulation and ovarian protection. These findings support the potential of LB and AS as accessible, food-derived adjuvants to help preserve ovarian function during chemotherapy, offering a promising framework for safeguarding reproductive health and quality of life against chemotherapy-induced ovarian insufficiency.

Dysregulated mitochondrial Ca2+ influx is a unifying driver of neurodegeneration, compromising neuronal bioenergetics and survival. In Parkinson’s disease (PD), impaired mitochondrial Ca2+ uptake is a decisive trigger for dopaminergic (DA) neuron loss, yet the molecular identity of the responsible channel has remained unresolved. Although the mitochondrial Ca2+ uniporter (MCU) is regarded as the principal conduit, global MCU ablation is non-lethal and MCU-deficient neurons retain basal Ca2+ uptake, pointing to the existence of a vital alternative pathway. Here we identify prohibitin2 (PHB2) forms this long-sought MCU-independent Ca2+ channel. Conditional ablation of PHB2 in DA neurons of mice induces hallmark PD pathology, including >70% substantia nigra neuron loss and severe motor deficits. By integrating live-cell mitochondrial Ca2+ imaging, mitoplast patch-clamp, single-molecule photometry, and high-field solution NMR, we demonstrate that PHB2 oligomerizes into a hexameric Ca2+ channel (~30 Å lumen). Structure-guided mutagenesis of four pore-lining residues abolished Ca2+ conductance, and this disruption of PHB2 channel function in DA neurons drives degeneration. Crucially, rescue experiments by adeno-associated virus mediated re-expression of wild-type PHB2, but not channel-dead mutant, in PHB2-DA-knockout mice restores >80% of DA neurons and reverses motor deficits. These findings identify PHB2 as the essential mitochondrial Ca2+ channel sustaining dopaminergic survival and nigral integrity, resolve a central enigma in mitochondrial biology, and establish the PHB2 pore as a tractable therapeutic target in PD. More broadly, modulation of PHB2 channel activity may represent a general strategy to reinforce mitochondrial resilience across neurodegenerative diseases.

Background Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening zoonotic infection caused by SFTS bunyavirus (SFTSV), with no specific antiviral therapy or vaccine available. Thrombocytopenia is a hallmark feature of SFTS, but its quantitative prognostic value and optimal clinical cutoff for risk stratification remain undefined. This study aimed to identify the independent prognostic role of platelet count (PLT) in SFTS and define its optimal cutoff value for predicting severe progression and mortality, after adjusting for potential confounders including age, inflammatory markers, and organ injury indicators. Methods This single-center retrospective cohort study enrolled 223 hospitalized SFTS patients at the Second Affiliated Hospital of Anhui Medical University. Patients were stratified into severe (n = 42) and non-severe (n = 181) groups per clinical organ dysfunction criteria. LASSO regression followed by multivariate logistic regression identified independent risk factors for severe SFTS. Receiver operating characteristic (ROC) curve analysis determined the optimal PLT cutoff value. Kaplan–Meier analysis evaluated survival differences. All statistical analyses were performed using R software (v4.5.2), with P < 0.05 considered statistically significant. Results Severe SFTS patients had significantly older age, lower peripheral oxygen saturation (SpO₂), longer symptom-to-admission interval, higher rash incidence, greater oxygen therapy requirement, and markedly higher mortality (59.52% vs. 7.18%, P < 0.001) compared with non-severe patients. Laboratory findings revealed pronounced systemic inflammation, multi-organ dysfunction, metabolic disturbances, and higher SFTSV viral load in the severe group (all P < 0.05). LASSO-multivariate logistic regression identified six independent risk factors for severe SFTS: older age, lower SpO₂, higher neutrophil percentage, longer symptom-to-admission time, lower PLT, and elevated cardiac troponin T (cTnT) (all P < 0.05). PLT exhibited the highest predictive value for severe SFTS (AUC = 0.726, 95% CI: 0.658–0.794), with an optimal cutoff value of 51×10⁹/L (Youden index = 0.339, sensitivity = 0.723, specificity = 0.616). Kaplan–Meier analysis confirmed that patients with PLT < 51×10⁹/L had significantly higher mortality and shorter overall survival ( P < 0.001). Conclusions Baseline PLT < 51×10⁹/L is a simple, independent predictor of severe progression and mortality in SFTS, enabling early risk stratification and rational resource allocation, particularly in resource-limited settings.

Oral squamous cell carcinoma (OSCC) significantly impairs a person's physical and psychological health despite active treatment efforts. Thus, it is important to investigate the pathogenesis of OSCC to discover new therapeutic targets for clinical treatments. ORAI3 is a store-operated calcium (Ca2+) channel that has been associated with several cancers, including breast, prostate, and pancreas. Although the ORAI3 channel is recognized for its oncogenic potential, its pathological functions in OSCC are not well understood, especially concerning its involvement in cancer progression. In this study, we found that ORAI3 was upregulated in OSCC clinical samples and cell lines. When the expression of ORAI3 was knocked down in vitro in OSCC cells, store-operated calcium entry-mediated Ca2+ influx into the cells was reduced. Importantly, the proliferation, migration, and invasive capabilities of these OSCC cells were also markedly decreased. Application of calmodulin or calcineurin inhibitors (W-7 or CsA, respectively) further suppressed these cell functions. Transcriptomic analysis revealed that ORAI3 knockdown led to downregulation of ETV4, a member of the ETS transcription factor family that is involved in cell differentiation, proliferation, and apoptosis and has been shown to be upregulated in some types of cancer. Here, overexpression of ETV4 rescued the suppressive effects on the proliferation, migration, and invasive capabilities of OSCC cells caused by ORAI3 knockdown. In addition, treatment with the calcineurin inhibitor CsA markedly reduced ETV4 expression levels in OSCC cells. Taken together, these results indicated that ORAI3 drives cancer progression by activating the Ca2+/calmodulin/calcineurin/ETV4 signaling pathway. Overall, these findings suggest that ORAI3/ETV4 may be a therapeutic target for the treatment of OSCC.

The KCNQ1+KCNE1 potassium channel complex forms the slow delayed rectifier current (IKs) critical for cardiac repolarization. Loss-of-function variants in KCNQ1 and KCNE1 cause long QT syndrome types 1 and 5 (LQT1/LQT5), accounting for over one-third of clinical LQTS cases. Despite prior structural work on KCNQ1 and KCNQ1+KCNE3, the structural basis of KCNQ1+KCNE1 remains unresolved. Using cryo-EM and electrophysiology, we determined high-resolution (2.5-3.4 Å) structures of human KCNQ1+KCNE1 in both closed and open states. KCNE1 occupies a pivotal position at the interface of three KCNQ1 subunits, inducing seven “helix-to-loop” transitions in KCNQ1 transmembrane segments. These structural rearrangements: 1) stabilize the closed pore and the conformation of the intermediate voltage-sensing domain, thereby determining channel gating, ion permeation, and single channel conductance; 2) enable a dual-PIP2 modulation mechanism, where one PIP2 occupies the canonical site, while the second PIP2 bridges the S4-S5 linker, KCNE1, and the adjacent S6’, stabilizing channel opening; 3) create a fenestration capable of binding compounds specific for KCNQ1+KCNE1 (e.g., AC-1). Together, these findings reveal a previously unrecognized large-scale secondary structural transition during ion channel gating that fine-tunes IKs function and provides a foundation for targeted LQTS therapy development. ### Competing Interest Statement The authors have declared no competing interest. Joint Funding of the Macau Science and Technology Development Fund and the Ministry of Science and Technology of the People's Republic of China, 0006/2021/AMJ National Natural Science Foundation of China, 32171221, 32271260 Macau Science and Technology Development Fund, 002/2023/ALC, 006/2023/SKL, 0074/2022/A2, 0098/2023/RIA2 Macau University of Science and Technology, FRG-23-030-SKL the CAS "Light of West China" Program, xbzg-zdsys-202005 Jiangxi Province Natural Science Foundation, 20224ACB206046

Tubular injury triggered by hyperglycemia is an important pathological characteristic in diabetic nephropathy (DN). Accumulated advanced glycation end products and their precursor methylglyoxal (MGO), contribute to the development of DN. Carnosine has been shown to prevent the development of DN but the underlying mechanism still needs to be studied in depth. In this study, we explored the potential proteins influenced by MGO and carnosine in tubule epithelial cells. HK-2 cells were treated with MGO, carnosine, or a combination. Differentially expressed proteins (DEPs) between different groups were identified by isobaric tag for relative and absolute quantitation-based mass spectrometry. In the comparison between MGO and control, 29 DEPs were found to be associated with antioxidation and RNA methylation. In the comparison between carnosine and control, 10 DEPs were associated with ubiquitin protein ligase activity and RNA metabolism. In the comparison between MGO + carnosine and MGO, carnosine-induced DEPs in the presence of MGO were mainly related to RNA splicing and mRNA processing. MGO effects on OSTC expression was inversely correlated with that of carnosine. Some DEPs (OSTC, PRDX5, NEDD4L, NOP2, TRMT6, and GEMIN2) were validated by Western blotting. Additional experiments showed the 28 kD particle of Smith antigen was also influenced by MGO and carnosine. Carnosine can influence RNA processing and spliceosome-related proteins, and change MGO’s effect on HK-2 cells. This study helps to understand the mechanism by which MGO contributes to the development of DN and promotes further identification of carnosine downstream proteins as therapeutic targets for DN.

Background Local brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by acute I/R injury in brain microvascular endothelial cells (BMECs).Objective This study aims to explore the mechanism by which DADLE enhances the level of mitophagy in BMECs by upregulating the expression of transient receptor potential vanilloid subtype 4 (TRPV4).Methods BMECs were extracted and made to undergo oxygen-glucose deprivation/reoxygenation (OGD/R) accompanied by DADLE. RNA-seq analysis revealed that DADLE induced increased TRPV4 expression. The CCK-8 method was used to assess the cellular viability; quantitative PCR (qPCR) was used to determine the mRNA expression of Drp1; western blot was used to determine the expression of TRPV4 and autophagy-related proteins; and calcium imaging was used to detect the calcium influx. Autophagosomes in in the cells’ mitochondria were observed by using transmission electron microscopy. ELISA was used to measure ATP content, and a JC-1 fluorescent probe was used to detect mitochondrial membrane potential.Results When compared with the OGD/R group, OGD/R+DADLE group showed significantly enhanced cellular viability; increased expression of TRPV4, Beclin-1, LC3-II/I, PINK1 and Parkin; decreased p62 expression; a marked rise in calcium influx; further increases in mitophagy, an increase in ATP synthesis and an elevation of mitochondrial membrane potential. These protective effects of DADLE can be blocked by a TRPV4 inhibitor HC067047 or RNAi of TRPV4.Conclusion DADLE can promote mitophagy in BMECs through TRPV4, improving mitochondrial function and relieving I/R injury.

IntroductionBaoTaiyin (BTY) is a traditional Chinese medicine decoction. It has been used to treat recurrent miscarriage (RM). However, there are no comprehensive systematic studies to identify the chemical compositions of BTY and molecular mechanisms on RM. Finding the chemical components of BTY and clarifying the underlying processes in the treatment of RM were the goals of the study.MethodsWe used ultra-high-performance liquid chromatography coupled with triple quadruple time-of-flight tandem mass spectrometry to analyze the chemical components of BTY, network analysis to predict the pharmacological effects of the identified active ingredients, and cell experiments to identify potential molecular mechanisms.ResultsWe found 12 active ingredients among 61 components identified in BTY. These identified activities were linked to regulatory effects on 127 key signaling pathways, targeting 107 proteins. Through network analysis, we determined that insulin-like growth factor 1 receptor, matrix metalloproteinases, PI3K, and STAT3 may be the core targets of BTY’s therapeutic effects on RM. We further explored this mechanism to find that aqueous extracts of BTY significantly enhanced IGFBP2 and CaMKK2 expression and trophoblast proliferation, whereas inhibitors of IGF1R/PI3K/AKT pathway or CaMKK2 blocked the effect of BTY on trophoblast proliferation. In addition, IGFBP2 siRNA suppressed BTY-induced CaMKK2 expression. Caffeic acid, as one of components of BTY, increased intracellular Ca2+ concentration and proliferation in trophoblast.ConclusionOur research showed that BTY may have therapeutic benefits on RM through multiple targets and pathways, such as the IGF1R/PI3K/AKT and Ca2+/calmodulin signaling pathways.

The impact of a high-salt (HS) diet on metabolic disturbances in individuals with coronary heart disease remains unclear. The arachidonic acid (AA) metabolic pathway is closely linked to the development of cardiometabolic diseases and atherosclerotic cardiovascular diseases. Furthermore, endoplasmic reticulum stress (ERS) has emerged as a major contributor to cardiometabolic diseases. AA-related inflammation and ERS are hypothesized to play a role in HS diet-induced coronary remodeling. Rats were subjected to an HS diet for 4 weeks, and the serum concentration of AA was measured via enzyme-linked immunosorbent assay. Immunofluorescence staining and vascular tension measurements were conducted on coronary arteries. In addition, AA-stimulated coronary artery smooth muscle cells (CASMCs) were treated with ERS inhibitors to explore the underlying pathway involved. Increased susceptibility to myocardial infarction in the HS diet-fed rats was accompanied by increased serum AA concentrations and increased expression of the key AA metabolic enzyme cyclooxygenase-2 (COX-2). AA incubation weakened the contraction of denuded coronary arteries, reduced the expression of contraction markers, and increased the fluorescence intensity of synthetic and ERS response markers in coronary arteries. Further investigation of CASMCs revealed that AA-induced phenotypic transformation was mediated via the ERS pathway. ERS and AA were found to be stimulated in CASMCs following an HS diet. AA triggers an ERS response through COX-2 catalysis, and the downstream inositol requiring enzyme 1 - X-box binding protein-1 - osteopontin pathway may contribute to the AA-induced phenotypic transformation of CASMCs, resulting in dysfunctional coronary tension. This study may provide potential therapeutic targets for cardiovascular diseases associated with excessive AA-derived ERS.

Airway smooth muscle (ASM) hyperplasia is a hallmark of airway remodeling in asthma, which still lacks an effective treatment. Low-density lipoprotein receptor-related protein 1 (LRP1) is involved in regulating the proliferation of various cell types, and the intracellular domain of LRP1 (LRP1-ICD) also exhibits unique biological functions. However, the role of LRP1 in asthma airway remodeling remains unclear. In the present study, LRP1 was increased in ASM cells of mice with OVA-induced chronic asthma, with the elevation in LRP1-ICD protein levels being significantly greater than that of the LRP1 β chain. In vivo experiments demonstrated that inhibiting LRP1 reduced ASM proliferation in these mice. Mechanistically, LRP1 knockdown inhibited the FGF2/ERK signaling pathway, thereby arresting cell cycle progression and suppressing ASM cell proliferation. Additionally, in vitro experiments revealed that the inhibitory effect of LRP1-ICD overexpression on ASM cell proliferation was lost after adjusting the levels of LRP1. LRP1-ICD overexpression inhibited full-length LRP1 protein levels by promoting its protein degradation rather than by suppressing its transcription, thus preventing further exacerbation of asthma. In conclusion, this study clarifies the molecular biological mechanism by which LRP1 regulates ASM proliferation, suggesting targeting full-length LRP1 as a strategy for therapeutic intervention in asthma airway remodeling.

Manganese (Mn2+) serves as an inorganic activator of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. However, its activation efficiency remains lower than conventional organic STING agonists, hindering widespread applications in immune modulation and therapy. Herein, we report an intermediate-crystalline phase manganese layered double hydroxide/oxide (Mn-LDH/O150) nanocomposite, featuring both LDH and LDO structural phases, as a potent cGAS-STING activator. Surprisingly, Mn-LDH/O150 induced a type-I interferon level significantly higher than pure Mn-LDH or LDO phase nanocomposites, and comparable to organic STING agonists (cGAMP/diABZI). Mechanistically, conventional Mn nanocomposite impairs energy metabolism in dendritic cells and significantly reduces mitochondrial ATP production. In contrast, Mn-LDH/O150 modulates mitochondrial metabolism by normalizing the electron transport chain (ETC) process, which is termed "immunometabolism normalization", thereby promoting ATP production that in turn facilitates cGAMP synthesis and STING activation. In mice models, Mn-LDH/O150 acts as a potent immune adjuvant in inducing antibodies production and T cell responses. Using a model antigen (ovalbumin) and melanoma neoantigens, we further demonstrate the excellent activity of Mn-LDH/O150-based vaccine in inducing antitumor immunity to prevent tumor progression and metastasis. Our discoveries highlight the crucial involvement of energy metabolism in modulating STING activation, and present a simple yet translational material engineering approach for boosting metalloimmunotherapy.

The KCNQ1 + KCNE1 potassium channel complex produces the slow delayed rectifier current (IKs) critical for cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 cause long QT syndrome (LQTS) types 1 and 5 (LQT1/LQT5), accounting for over one-third of clinical LQTS cases. Despite prior structural work on KCNQ1 and KCNQ1 + KCNE3, the structural basis of KCNQ1 + KCNE1 remains unresolved. Using cryo-electron microscopy and electrophysiology, we determined high-resolution (2.5–3.4 Å) structures of human KCNQ1APO, and KCNQ1 + KCNE1 in both closed and open states. KCNE1 occupies a pivotal position at the interface of three KCNQ1 subunits, inducing six helix-to-loop transitions in KCNQ1 transmembrane segments. Three of them occur at both ends of the S4–S5 linker, maintaining a loop conformation during IKs gating, while the other three, in S6 and helix A, undergo dynamic helix-loop transitions during IKs gating. These structural rearrangements: (1) stabilize the closed pore and the conformation of the intermediate state voltage-sensing domain, thereby determining channel gating, ion permeation, and single-channel conductance; (2) enable a dual-PIP2 modulation mechanism, where one PIP2 occupies the canonical site, while the second PIP2 bridges the S4–S5 linker, KCNE1, and the adjacent S6’, stabilizing channel opening; (3) create a fenestration capable of binding compounds specific for KCNQ1 + KCNE1 (e.g., AC-1). Together, these findings reveal a previously unrecognized large-scale secondary structural transition during ion channel gating that fine-tunes IKs function and provides a foundation for developing targeted LQTS therapy.

Introduction:Primary biliary cholangitis (PBC) is a chronic autoimmune-mediated cholestatic liver disease that can progress to cirrhosis and liver failure. Intrahepatic biliary epithelial cells (IBECs) are the primary targets of early injury in PBC. Our previous studies have shown that exosomes derived from hepatic stellate cells (HSCs) deliver miR-122-5p to regulate the expression of human IBEC inflammatory factors via the p38 MAPK signaling pathway. The purpose of this study was to investigate the therapeutic potential and molecular mechanism of HSC-derived exosomal miR-122-5p in PBC. Methods:The effects of exosomal miR-122-5p in inhibiting apoptosis, epithelial-mesenchymal transition (EMT), and fibrosis were evaluated in lipopolysaccharide (LPS)-induced human IBEC models, and its anti-inflammatory and anti-fibrotic effects were measured in dnTGF-βRII mouse models. A variety of analytical procedures, such as flow cytometry, Cell Counting Kit-8 (CCK-8), RT-qPCR, ELISA, co-culture, Western blotting, immunofluorescence, gene transfection, immunohistochemistry, and several staining methods (H&E and Masson), were used to evaluate the effectiveness and mechanisms of these methods. Results:The results from clinical data showed that exosomal miR-122-5p was correlated with liver function, and when combined with gp210 and sp100 antibodies, it could improve the sensitivity of PBC diagnosis. The results from in vitro experiments showed that exosomal miR-122-5p promoted the proliferation and inhibited the apoptosis, EMT, and fibrosis indicators of IBECs via the p38 MAPK signaling pathway. Dual luciferase reporter assay indicated that tumor necrosis factor receptor superfamily (TNFRSF) 19 is a specific target of miR-122-5p and reduces ASK1 levels. The co-immunoprecipitation (Co-IP) experiment further indicates the interaction between TNFRSF19 and ASK1. In vivo results indicated that the degrees of inflammatory infiltration and fibrosis in liver tissues of both PBC patients and model mice were more severe than those of normal controls and were then alleviated with exosomal miR-122-5p treatment. Conclusion:In conclusion, exosomal miR-122-5p alleviates liver pathology in PBC by targeting the TNFRSF19/ASK1/p38 MAPK axis, highlighting its potential as both a diagnostic biomarker and a therapeutic target for PBC.

Background Exercise training may counteract the detrimental effects of obesity on endothelial function by enhancing the reparative capabilities of endothelial progenitor cells (EPC); however, the underlying mechanisms of exercise-induced EPC-mediated endothelial repair are still unclear. The present study aimed to determine the mechanisms by which exercise-induced circulating exosomes protect against endothelial dysfunction induced by obesity. Methods An 8-week aerobic exercise intervention in both obese human participants and high-fat diet-induced obese rats was conducted. Circulating exosomes were isolated and characterized. microRNA sequencing, molecular biology techniques, and functional assays (including proliferation, migration, and luciferase reporter assays) were employed to identify key exosomal microRNAs and their downstream targets. A microRNA-214-3p (miR-214-3p) knockout rat model was used to validate its role in vivo. Results Exercise promoted EPC-mediated repair of endothelial damage and upregulated exosomal miR-214-3p in both obese humans and rats, without altering exosome quantity. miR-214-3p enhanced EPC proliferation and migration directly, by upregulating collagen type I alpha 2 chain (COL1A2) expression, and indirectly, through the phosphatase and tensin homolog, phosphatidylinositol 3-kinase, serine/threonine kinase (PTEN-PI3K-Akt) signaling pathway. Knockout of miR-214-3p abolished the exercise-induced improvements in endothelial and EPC functionalities. The myocardium was identified as an important source of the exercise-induced increase in circulating exosomal miR-214-3p. Conclusion Long-term aerobic exercise promotes endothelial repair in obesity by enriching circulating exosomes with miR-214-3p, which enhances EPC function via the PTEN-PI3K-Akt pathway and direct regulation of COL1A2. These findings reveal a novel exosome-mediated mechanism through which exercise improves vascular health and suggest potential therapeutic strategies for obesity-related endothelial dysfunction.