ObjectiveTo verify the association between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis by constructing an in vitro co-culture system of testis. MethodsTesticular tissue blocks from 20-25-day-old male rats were placed in an in vitro culture system， and the culture medium was replaced every 2 to 3 days. PCR was used to verify the expression of marker genes of various spermatogenic cells. RNA interference technology was employed to verify the correlation between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis. ResultsThe co-culture system could be continuously cultured for more than 2.5 months in vitro. RT-PCR showed that specific marker genes of spermatogonia， spermatocyte and spermoblast were expressed. The RNA and protein expression of Trib3 and Akt changed after the knocking down of Ddit3 and Trib3， respectively. It demonstrated the existence of Ddit3-Trib3-Akt signaling pathway in rat spermatogenesis. ConclusionThe culture time of more than 2.5 months indicates that the culture system can temporarily maintain the proliferation and differentiation of stem cells， and simultaneously maintain and stabilize spermatogenesis in a simple system. The successful validation of the Ddit3-Trib3-Akt signaling pathway also confirms that this culture system can be used to study possible molecular mechanisms of spermatogenesis in vitro.

ObjectiveThe Golgi apparatus serves as a central hub in the eukaryotic secretory pathway, responsible for the processing, sorting, and trafficking of proteins and lipids. In mammalian cells, the Golgi typically forms a perinuclear ribbon-like structure composed of laterally connected cisternae stacks.The maintenance of Golgi ribbon structure depends on the balance of membrane flux across multiple intracellular trafficking pathways, yet the specific contributions of distinct trafficking branches to Golgi macroscopic morphology remain elusive. In mammalian cells, the Golgi ribbon is typically organized as a perinuclear, laterally connected structure composed of stacked cisternae, and its integrity is highly dynamic and sensitive to perturbations in membrane trafficking. This study aims to elucidate the role of coat protein complex I (COPI)-mediated retrograde transport in maintaining the Golgi ribbon and to dissect the functional relationship between the transmembrane cargo receptors LEPROT/LEPROTL1 (LEPROTs) and the COPI adaptor GOLPH3. MethodsUsing siRNA interference and gene-deficient cell lines, we selectively perturbed COPI- or adaptor protein complex 1 (AP-1)-mediated trafficking pathways in HeLa cells. To quantitatively evaluate Golgi morphology, we employed a “Golgi Angle”-based measurement to assess its circumferential distribution around the nucleus. The spatial distribution of the Golgi ribbon was quantitatively analyzed using confocal microscopy, while Golgi ultrastructure and vesicle density were examined via transmission electron microscopy. Additionally, the subcellular distribution of COPI components was assessed by immunofluorescence co-localization. ResultsSelective inhibition of COPI retrograde transport significantly induced the circumferential extension of the Golgi ribbon around the nucleus, whereas blocking AP-1-mediated anterograde transport resulted in Golgi compaction, indicating opposing roles. These results suggest that different trafficking branches downstream of ARF1 exert distinct and even antagonistic effects on Golgi morphology. LEPROTs-deficient cells exhibited a Golgi extension phenotype highly consistent with COPI impairment. Furthermore, knockdown of GOLPH3 in a LEPROTs double-knockout background produced a significant additive effect on Golgi extension, suggesting that LEPROTs and GOLPH3 play non-redundant roles in regulating COPI-related trafficking processes. Mechanistically, loss of either LEPROTs or GOLPH3 led to the aberrant accumulation of COPI components at endoplasmic reticulum exit sites, accompanied by a reduction in COPI-like vesicles around the Golgi. This redistribution indicates a defect in COPI recycling between the ER-Golgi interface and the Golgi apparatus. Ultrastructural analysis revealed that Golgi cisternae in defective cells became shorter and thicker while maintaining a stable number of stacks. In parallel, the density of Golgi-associated vesicles was markedly decreased, further supporting an impairment in COPI vesicle formation or budding processes. ConclusionThis study demonstrates that active COPI retrograde transport is a critical factor in restricting the over-connection of the Golgi ribbon and maintaining its compactness. Rather than causing fragmentation, partial disruption of COPI function leads to a distinct morphological outcome characterized by Golgi ribbon extension at the light microscopy level and cisternal remodeling at the ultrastructural level. LEPROTs and GOLPH3 cooperatively promote the recycling and vesiculation of COPI components, thereby imposing a structural constraint on the Golgi periphery. Our findings support a model in which multiple adaptor proteins act in parallel to sustain efficient COPI cycling, thereby maintaining Golgi structural homeostasis. These findings provide new cell biological evidence for the membrane trafficking basis of Golgi morphological homeostasis.

ObjectiveMigraine is a leading neurological disorder and the fourth most common cause of years lived with disability worldwide, affecting nearly 116 million individuals. Although pharmacological treatments are available, their efficacy is often limited by side effects and variable response rates. Repetitive transcranial magnetic stimulation (rTMS) over the dorsolateral prefrontal cortex (DLPFC) offers a safe, non-invasive alternative for migraine management. However, the neurophysiological mechanisms, particularly how rTMS modulates local cortical excitability and distributed pain-related circuits, remain poorly understood. Elucidating these mechanisms is essential for optimizing treatment protocols and improving clinical outcomes. MethodsThis study employed concurrent transcranial magnetic stimulation and electroencephalography (TMS-EEG) to investigate neuroplastic and neurocircuitry mechanisms of DLPFC-rTMS in migraine. Study 1 compared 30 migraineurs and 28 healthy controls to identify abnormalities in TMS-evoked potentials (TEPs) and significant current density (SCD) within sensory-discriminative regions including the primary somatosensory cortex (S1) and posterior insula (pINS), cognitive-affective regions including the anterior insula (aINS) and midcingulate cortex (MCC), and a descending modulatory region, the periaqueductal gray (PAG). Study 2 used a single-blind, crossover, sham-controlled design in 34 healthy participants. Each participant received both active (10 Hz, 80% RMT, 1 500 pulses) and sham DLPFC-rTMS in counterbalanced order. TMS-EEG and cold pain tolerance were assessed before and after each session. ResultsIn Study 1, migraineurs showed a significantly less negative N120 amplitude compared to healthy controls (P=0.027, Cohen’s d=0.60), indicating local intracortical disinhibition. No group differences were observed for N40, P60, or P180 components. At the source level, migraineurs exhibited significantly higher SCD in the S1, pINS, aINS, and MCC (allQ<0.05), but not in the ventroposterior thalamus (vpTHAL), mediodorsal thalamus (mdTHAL), or PAG. In Study 2, active rTMS significantly reduced SCD from pre- to post-stimulation in the S1, aINS, and MCC (all Q<0.05). Sham stimulation also reduced SCD in the S1 (Q<0.05) but not in the aINS or MCC. Although no significant group-level analgesic effect was observed between active and sham conditions (P=0.107), correlation analyses revealed that greater SCD reductions in the S1 and MCC were significantly associated with higher post-rTMS pain tolerance (R=-0.487 and -0.495, both Q<0.01) and larger improvements in pain tolerance(R=-0.487 and -0.451, both Q<0.05). No such correlations were found following sham stimulation, suggesting that the behavioural relevance of neural changes is specific to active rTMS. ConclusionThis study provides novel evidence that migraineurs exhibit both local neuroplastic abnormalities (reduced N120 amplitude) and hyperactivity in key pain-processing regions (S1, pINS, aINS, MCC). A single session of DLPFC-rTMS reduced hyperactivity in the aINS, MCC, and S1. Notably, greater reductions in the S1 and MCC were associated with improved pain tolerance. These findings identify distinct cortical circuitries, particularly within the cognitive-affective pain network, that may serve as potential biomarkers for optimizing rTMS treatment in migraine and other chronic pain conditions. Future studies should validate these results in patient populations experiencing spontaneous migraine attacks and explore multi-session or accelerated rTMS protocols.

OBJECTIVE To screen a single matrix， prepare dissolving microneedles of Pterocarya hupehensis total flavonoids（PHTF-MN）， and characterize it while evaluating its analgesic effects.METHODS Using microneedle morphology and puncture rate as evaluation indicators， the matrix was screened from polyvinyl alcohol （PVA）， polyvinylpyrrolidone K30 （PVP K30）， PVP K90， and sodium hyaluronate with relative molecular weights of 15 000 and 60 000 （HA 1.5w and HA 6w ）. PHTF-MN was prepared by the layer-by-layer casting method with the selected matrix， and the appearance and morphology， total flavonoid content， mechanical properties， puncture performance， dissolution properties， and drug release behavior of the obtained PHTF-MN were characterized. Using male BALB/c mice as test subjects and aspirin （ASA） as a positive control， the analgesic effects of the microneedles were evaluated using a formalin-induced pain model.RESULTS Blank microneedles prepared with 30% HA 1.5w exhibited optimal surface flatness and the membrane puncture rate （through 4 layers of parafilm membrane） reaching 78%. The PHTF-MNs （with PHTF contents of 10， 20， 30， 40， and 50 mg/g in the matrix） exhibited uniform arrays and intact tips， with RSDs for total flavonoid contents all below 7% （ n ＝8）. The single-needle puncture forces were 1.46 and 1.07 N （with PHTF contents of 30 and 50 mg/g in the matrix， respectively）. The skin penetration rate approached 100%， with visible puncture marks in the stratum corneum and drug residue （with a PHTF content of 50 mg/g in the matrix）. After 30 min of fixation， the needles had almost completely dissolved， and the skin retention rates all exceeded 90% （with PHTF contents of 10， 30， and 50 mg/g in the matrix， respectively）. The cumulative release within 8 h was （134.64±2.03） μg/cm 2 ， and the fit to the first-order kinetic equation （ R 2 ＝0.950 1） was highest （with a PHTF content of 50 mg/g in the matrix）. Low- and high-dose PHTF-MN （containing 85.26 and 145.03 μg of total flavonoids per patch， respectively； 1 patch/day） significantly shortened the cumulative time of phase Ⅱ pain response in mice （ P ＜0.05）， with pain inhibition rates （43.67% and 59.78%） comparable to those of ASA （53.70%）.CONCLUSIONS PHTF-MN using HA 1.5w as a single matrix was successfully prepared. This formulation features a simplified preparation process， uniform drug loading， favorable mechanical properties， resistance to fracture， and rapid dissolution. Its drug release behavior conforms to a first-order kinetic model， and its analgesic effect is comparable to that of ASA.

Background/Aims: Endoscopic radiofrequency ablation (ERFA) is a treatment option for superficial esophageal squamous cell neoplasia (ESCN), with a relatively low risk of stenosis; however, the long-term outcomes remain unclear. We aimed to compare the long-term outcomes of patients with widespread superficial ESCN who underwent endoscopic submucosal dissection (ESD) or ERFA. Methods: We retrospectively analyzed the clinical data of patients with superficial ESCN who underwent ESD or ERFA between January 2015 and December 2021. The primary outcome measure was recurrence-free survival. Results: Ninety-two and 33 patients with superficial ESCN underwent ESD and ERFA, respectively. The en bloc, R0, and curative resection rates for ESD were 100.0%, 90.2%, and 76.1%, respectively. At 12 months, the complete response rate was comparable between the two groups (94.6% vs 90.9%, p=0.748). During a median follow-up of 66 months, recurrence-free survival was significantly longer in the ESD group than in the ERFA group (p=0.004), while no significant differences in overall survival (p=0.845) and disease-specific survival (p=0.494) were observed.Preoperative diagnosis of intramucosal cancer (adjusted hazard ratio, 5.55; vs high-grade intraepithelial neoplasia) was an independent predictor of recurrence. Significantly fewer patients in the ERFA group experienced stenosis compare to ESD group (15.2% vs 38.0%, p=0.016). Conclusions The risk of recurrence was higher for ERFA than ESD for ESCN but overall survival was not affected. The risk of esophageal stenosis was significantly lower for patients who underwent ERFA.

Background/Aims: Endoscopic radiofrequency ablation (ERFA) is a treatment option for superficial esophageal squamous cell neoplasia (ESCN), with a relatively low risk of stenosis; however, the long-term outcomes remain unclear. We aimed to compare the long-term outcomes of patients with widespread superficial ESCN who underwent endoscopic submucosal dissection (ESD) or ERFA. Methods: We retrospectively analyzed the clinical data of patients with superficial ESCN who underwent ESD or ERFA between January 2015 and December 2021. The primary outcome measure was recurrence-free survival. Results: Ninety-two and 33 patients with superficial ESCN underwent ESD and ERFA, respectively. The en bloc, R0, and curative resection rates for ESD were 100.0%, 90.2%, and 76.1%, respectively. At 12 months, the complete response rate was comparable between the two groups (94.6% vs 90.9%, p=0.748). During a median follow-up of 66 months, recurrence-free survival was significantly longer in the ESD group than in the ERFA group (p=0.004), while no significant differences in overall survival (p=0.845) and disease-specific survival (p=0.494) were observed.Preoperative diagnosis of intramucosal cancer (adjusted hazard ratio, 5.55; vs high-grade intraepithelial neoplasia) was an independent predictor of recurrence. Significantly fewer patients in the ERFA group experienced stenosis compare to ESD group (15.2% vs 38.0%, p=0.016). Conclusions The risk of recurrence was higher for ERFA than ESD for ESCN but overall survival was not affected. The risk of esophageal stenosis was significantly lower for patients who underwent ERFA.

Background/Aims: Endoscopic radiofrequency ablation (ERFA) is a treatment option for superficial esophageal squamous cell neoplasia (ESCN), with a relatively low risk of stenosis; however, the long-term outcomes remain unclear. We aimed to compare the long-term outcomes of patients with widespread superficial ESCN who underwent endoscopic submucosal dissection (ESD) or ERFA. Methods: We retrospectively analyzed the clinical data of patients with superficial ESCN who underwent ESD or ERFA between January 2015 and December 2021. The primary outcome measure was recurrence-free survival. Results: Ninety-two and 33 patients with superficial ESCN underwent ESD and ERFA, respectively. The en bloc, R0, and curative resection rates for ESD were 100.0%, 90.2%, and 76.1%, respectively. At 12 months, the complete response rate was comparable between the two groups (94.6% vs 90.9%, p=0.748). During a median follow-up of 66 months, recurrence-free survival was significantly longer in the ESD group than in the ERFA group (p=0.004), while no significant differences in overall survival (p=0.845) and disease-specific survival (p=0.494) were observed.Preoperative diagnosis of intramucosal cancer (adjusted hazard ratio, 5.55; vs high-grade intraepithelial neoplasia) was an independent predictor of recurrence. Significantly fewer patients in the ERFA group experienced stenosis compare to ESD group (15.2% vs 38.0%, p=0.016). Conclusions The risk of recurrence was higher for ERFA than ESD for ESCN but overall survival was not affected. The risk of esophageal stenosis was significantly lower for patients who underwent ERFA.

Background/Aims: Endoscopic radiofrequency ablation (ERFA) is a treatment option for superficial esophageal squamous cell neoplasia (ESCN), with a relatively low risk of stenosis; however, the long-term outcomes remain unclear. We aimed to compare the long-term outcomes of patients with widespread superficial ESCN who underwent endoscopic submucosal dissection (ESD) or ERFA. Methods: We retrospectively analyzed the clinical data of patients with superficial ESCN who underwent ESD or ERFA between January 2015 and December 2021. The primary outcome measure was recurrence-free survival. Results: Ninety-two and 33 patients with superficial ESCN underwent ESD and ERFA, respectively. The en bloc, R0, and curative resection rates for ESD were 100.0%, 90.2%, and 76.1%, respectively. At 12 months, the complete response rate was comparable between the two groups (94.6% vs 90.9%, p=0.748). During a median follow-up of 66 months, recurrence-free survival was significantly longer in the ESD group than in the ERFA group (p=0.004), while no significant differences in overall survival (p=0.845) and disease-specific survival (p=0.494) were observed.Preoperative diagnosis of intramucosal cancer (adjusted hazard ratio, 5.55; vs high-grade intraepithelial neoplasia) was an independent predictor of recurrence. Significantly fewer patients in the ERFA group experienced stenosis compare to ESD group (15.2% vs 38.0%, p=0.016). Conclusions The risk of recurrence was higher for ERFA than ESD for ESCN but overall survival was not affected. The risk of esophageal stenosis was significantly lower for patients who underwent ERFA.

OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVE: To evaluate the efficacy and safety of Shexiang Tongxin Dropping Pill (STDP) in treating stable angina patients with phlegm-heat and blood-stasis syndrome by exercise duration and metabolic equivalents. METHODS: This multicenter, randomized, double-blind, placebo-controlled clinical trial enrolled stable angina patients with phlegm-heat and blood-stasis syndrome from 22 hospitals. They were randomized 1:1 to STDP (35 mg/pill, 6 pills per day) or placebo for 56 days. The primary outcome was the exercise duration and metabolic equivalents (METs) assessed by the standard Bruce exercise treadmill test after 56 days of treatment. The secondary outcomes included the total angina symptom score, Chinese medicine (CM) symptom scores, Seattle Angina Questionnaire (SAQ) scores, changes in ST-T on electrocardiogram and adverse events (AEs). RESULTS: This trial enrolled 309 patients, including 155 and 154 in the STDP and placebo groups, respectively. STDP significantly prolonged exercise duration with an increase of 51.0 s, compared to a decrease of 12.0 s with placebo (change rate: -11.1% vs. 3.2%, P<0.01). The increase in METs was significantly greater in the STDP group than in the placebo group (change: -0.4 vs. 0.0, change rate: -5.0% vs. 0.0%, P<0.01). The improvement of total angina symptom scores (25.0% vs. 0.0%), CM symptom scores (38.7% vs. 11.8%), reduction of nitroglycerin consumption (100.0% vs. 11.3%), and all domains of SAQ, were significantly greater with STDP than placebo (all P<0.01). The changes in Q-T intervals at 28 and 56 days from baseline were similar between the two groups (both P>0.05). Twenty-five participants (16.3%) with STDP and 16 (10.5%) with placebo experienced AEs (P=0.131), with no serious AEs observed. CONCLUSION STDP could improve exercise tolerance in patients with stable angina and phlegm-heat and blood stasis syndrome, with a favorable safety profile. (Registration No. ChiCTR-IPR-15006020).
Humans ; Double-Blind Method ; Drugs, Chinese Herbal/adverse effects* ; Male ; Female ; Middle Aged ; Angina, Stable/physiopathology* ; Aged ; Syndrome ; Treatment Outcome ; Placebos ; Tablets