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.

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.

Retinal vein occlusion(RVO)is often accompanied by macular edema(ME), which is the main cause of visual impairment in patients. From the perspective of traditional Chinese medicine theory, the key pathogenesis lies in Qi stagnation and blood stasis, as well as internal retention of water and dampness, which is closely related to the dysfunction of internal organs such as liver depression and qi stagnation, spleen failure to function properly, and kidney deficiency with water retention. Although anti-vascular endothelial growth factor(VEGF)therapy has become the first-line treatment option for RVO-ME, some patients show a low response or no response to this therapy, resulting in recurrent ME. According to traditional Chinese medicine, such difficult-to-treat cases are often caused by long-term illness entering the meridians and the interplay of phlegm and blood stasis, or by deficiency of the body's vital energy and the lingering of pathogenic factors. Intervention should be carried out through therapeutic methods such as promoting blood circulation and diuresis, resolving phlegm and unblocking meridians, and strengthening the body's vital energy and eliminating pathogenic factors. At present, the pathogenesis of RVO-ME is not yet fully understood. Modern medicine believes that it may involve multiple factors such as retinal microstructure damage, abnormal blood flow and systemic diseases throughout the body, while traditional Chinese medicine emphasizes the overall connection between local lesions and the imbalance of Qi, blood, Yin and Yang throughout the body. This article systematically reviews the existing research achievements of traditional Chinese and Western medicine on RVO-ME, analyzes its possible high-risk factors, and provides a theoretical basis for formulating individualized treatment plans integrating the advantages of traditional Chinese and Western medicine for such patients.

Nanoparticle delivery systems have good application prospects in the field of precision therapy, but the preparation process of nanomaterial has problems such as short in vivo circulation time, easy recognition, and clearance by the immune system in the body. In recent years, biomimetic nanoparticle delivery systems mediated by natural cell membranes have become a research hotspot to address these issues. The natural membrane biomimetic nanoparticle delivery system cleverly integrates the advantages of natural biofilm "autologous" and "artificial" functional carriers by using endogenous cell membranes to modify the surface of nanocarriers, endowing them with characteristics such as tumor targeting, low immunogenicity, and long blood circulation. Currently, biomimetic nanoparticle delivery systems have been used in the treatment of malignant tumors, cardiovascular diseases, bacterial infections, and other diseases. This paper analyzes the development status and current research hotspots of natural cell membrane camouflaged biomimetic nanoparticle delivery systems mainly reviews the latest research progress of red blood cell membrane camouflaged biomimetic nanoparticle delivery systems in the field of disease treatment in recent years. It focuses on exploring the advantages, future development prospects, and limitations of biomimetic nanoparticle delivery systems based on red blood cell membrane camouflage in improving drug delivery, to provide a reference for the in-depth research and development of this system.

Objective To analyze chemical constituents of compound Maxing Shigan decoction by ultra-high perfor-mance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). Methods The separation was performed on a UPLC BEH C₁₈ column (2.1 mm×100 mm, 2.5 µm)，with a gradient elution applying 0.1% aqueous formic acid solution and 0.1% formic acid acetonitrile as a mobile phase. The column temperature was 40 °C. The flow rate was 0.4 ml/min and the analysis time was 15 min. Mass spectrometry (MS) data were collected in both positive and negative ESI ion modes. Results Through UPLC-QTOF/MS analysis and reference validation, a total of 59 chemical components in Maxing Shigan decoction were identified. Conclusion An ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method was established to identify the chemical components of Maxing Shigan decoction. This method is simple, efficient, sensitive and accurate, and provides a basis for the elucidation of the pharmacodynamic material basis and mechanism of Maxing Shigan decoction. It can provide data reference for the optimization of the compatibility of traditional Chinese medicine in the treatment of COVID-19.

Fe/Mn/Gd polymetallic nanooxide(FMGN)were prepared by one-step solvent thermal reaction by using Fe(acac)3,Mn(acac)2 and Gd(acac)3 as reaction precursors.Next,hyaluronic acid(HA)was used to modify FMGN to fabricate tumor-targeting T 1-T 2 dual-mode magnetic resonance imaging(MRI)contrast agent(HA-FMGN)for accurate diagnosis of prostate cancer.The structure and morphology of FMGN were observed by transmission electron microscope(TEM).It was found that FMGN exhibited a uniform nanocluster spherical structure when the feeding ratio of iron acetylacetonate,manganese acetylacetonate,and gadolinium acetylacetonate was 3:2:1.X-ray diffraction(XRD)analysis showed that FMGN had a typical inverse spinel structure of Mn doped Fe 3O 4,with Gd existing in the form of amorphous gadolinium oxide.The longitudinal relaxivity(r 1)and transverse relaxivity(r 2)of FMGN were 13.395 and 428.535 L/(mmol·s),respectively,measured by 0.5 T MRI analyzer,which proved that FMGN had excellent T 1-T 2 dual-mode MRI contrast capability.The cytotoxicity and hemolysis test found that HA-FMGN didn't damage red cells and induce toxicity for normal cells,indicating that HA-FMGN had excellent cell biocompatibility.The internalization efficacy of HA-FMGN was observed by CLSM,and the results showed that HA-FMGN possessed excellent prostate tumor-targeting ability.In vivo MRI experiment showed that HA-FMGN significantly enhanced T 1 and T 2 weighted MRI signal to noise ratio(SNR)of prostate tumor,which promoted the accurate diagnosis of orthotopic prostate cancer.

The objective assessment of hearing loss is one of the critical components in forensic clini-cal research.Auditory brainstem response(ABR)is an important method for objectively assessing hearing levels.It is divided into various types based on different stimulus signals,each with its own characteris-tics and applications.Among them,narrow-band Chirp ABR,due to its frequency specificity,fulfills the basic requirements for objective assessment of forensic audiology,promising to be an important method of objective hearing assessment in forensic medicine.This article reviews the development history,charac-teristics and clinical applications of Chirp ABR,and envisions its application prospects in forensic audi-tory identification.

Abstract： ObjectiveThe study aims to explore the effects and regulatory roles of glucose transporter 1 （GLUT1） on the proliferation， migration， adhesion， and angiogenesis of human umbilical vein endothelial cells （HUVECs） under ischemia-hypoxic conditions. MethodsIn vitro experiments were conducted to subject HUVECs to an ischemia-hypoxic-mimicking environment （1% O₂， 5% CO₂， 94% N₂）. The biological characteristics of HUVECs under normoxic and ischemia-hypoxic conditions were compared by assessing cell viability， proliferation capacity， and examining the expression changes of GLUT1， HIF-1α， and VEGFA proteins under ischemia-hypoxia using Western blot technology. Further， GLUT1 was overexpressed using plasmid transfection and the proliferation， migration， adhesion， and angiogenic capabilities of HUVECs were evaluated through scratch assays， cell adhesion assays， and tube formation assays. Mitochondrial morphological changes were observed by transmission electron microscopy，and oxygen consumption rate （OCR） was detected by Seahorse metabolic analyzer to evaluate mitochondrial function. ResultsCompared with normoxic conditions， the ischemia-hypoxic environment significantly inhibited the proliferation， cell viability， migration， and adhesion capabilities of HUVECs and impaired their angiogenic potential. The expression levels of GLUT1， HIF-1α and VEGFA proteins were also markedly reduced. However， when GLUT1 expression was upregulated， the migration， adhesion， and angiogenic capabilities of HUVECs were significantly improved， and the protein expression levels of HIF-1α， VEGFA and VEGFR were increased. Transmission electron microscopy revealed that ischemic-hypoxia leads to mitochondrial swelling and matrix damage， while GLUT1 overexpression significantly alleviates mitochondrial morphology abnormalities. OCR results suggest that GLUT1 overexpression may enhance oxidative phosphorylation of endothelial cells in ischemic-hypoxic environments to improve energy metabolism. These results suggest that GLUT1 may influence the function and angiogenic potential of HUVECs by regulating glucose metabolism and energy supply. ConclusionsThis study reveals the significant regulatory role of GLUT1 in the function of HUVECs under ischemia-hypoxic conditions， potentially through modulating cellular energy metabolism and signal transduction pathways， thereby affecting cell proliferation， migration， adhesion， and angiogenesis. These findings provide a new perspective on the role of GLUT1 in cardiovascular diseases and may offer potential targets for the development of new therapeutic strategies.

Objective: To investigate the correlation between the levels of calcitonin gene-related peptide α (CGRPα) and the poor responses to the treatment of phenylephrine in pregnant women with hypotension during caesarean section under intraspinal anesthesia. Methods : A total of 87 pregnant women who underwent intraspinal anesthesia cesarean section were recruited . They were grouped according to the presence or absence of hypotension after anesthesia and the response to ephedrine and Phe . As to Anesthesia-0 group , pregnant women had no hypo- tension , 35 cases (40. 2% ) ; Anesthesia-1 group , pregnant women had hypotension and good response to ephed- rine treatment , 12 cases (13 . 8% ) ; Anesthesia-2 group , pregnant women had hypotension and had poor response to ephedrine treatment , 6 cases (6. 9% ) ; Anesthesia-3 group , pregnant women had hypotension and good response to Phe treatment , 8 cases (9. 2% ) ; Anesthesia-4 group , pregnant women had hypotension and no response to Phe treatment , 26 cases (29. 9% ) . The levels of CGRPαin serum and placental tissues of pregnant women were deter- mined by ELISA . The rats pregnant normally. A hypotension rat model was established by intragastric administra- tion of risserpine . 50 rats were randomly divided into control group , model group , pregnancy group , model + preg- nancy group , model + pregnancy + phenylephrine group , with 10 rats in each group . Body weight of the rats was continuously monitored during the 2nd to 6th week of risserpine administration . Systolic blood pressure was meas- ured at the end of the 6th induction week . And the peripheral blood sample and placental tissues were collected and the levels of CGRPαwere determined . Results : The detection of CGRPαlevels in the pregnant women serum and placental tissues showed that compared with Anesthesia-0 group , CGRPαlevels in Anesthesia-1-4 groups serum were higher (P < 0. 05) . Compared with Anesthesia-3 group , serum CGRPαlevels in Anesthesia-4 group were higher (P < 0. 05) . Compared with Anesthesia-2 group , CGRPαlevels in Anesthesia-4 group serum were higher (P < 0. 05) . Compared with Anesthesia-0 group , tissues CGRPαlevels in Anesthesia-4 group were higher (P < 0. 05) . Compared with Anesthesia-3 group , CGRPαlevels in Anesthesia-4 group tissues were higher (P < 0. 05) . Compared with Anesthesia-2 group , CGRPαlevels in Anesthesia-4 group tissues were higher ( P < 0. 05) . From the 2nd to 6th week of hypotension modeling induction , compared with pregnancy group , body weight decreased in the model + pregnancy group and the model + pregnancy + phenylephrine group (P < 0. 05) . There was no signifi- cant difference in baseline systolic blood pressure among all groups (P > 0. 05) . At the end of the 6th week , com- pared with pregnancy group , systolic blood pressure decreased in model + pregnancy group and model + pregnancy + phenylephrine group(P < 0. 05) ; the levels of CGRPαin serum and placental tissues increased ( P < 0. 05) . Compared with model + pregnancy group , systolic blood pressure increased in model + pregnancy + phenylephrine group (P < 0. 05) . Conclusion The poor response of pregnant women with hypotension during caesarean section under intraspinal anesthesia to the treatment of phenylephrine may be related to the increased levels of CGRPαin placenta and peripheral blood .