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.

The novel coronavirus(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2),classified as a single-stranded RNA virus,replicates and transcribes its genome through the action of an RNA-dependent RNA polymerase,which is itself comprised of numerous non-structural pro-teins(non-structural proteins,Nsps).The present study delineates the development of a detection system founded on a bicistronic reporter plasmid in conjunction with an array of Nsp plasmids,ai-ming to investigate the influence of SARS-CoV-2 Nsps on the virus's replication and expression profiles.Specifically,a bicistronic reporter gene plasmid along with twelve distinct Nsp plasmids(encompassing Nsp3C-Flag,Nsp4,Nsp6-Nsp10,Nsp12-Nsp16)were meticulously constructed via molecular cloning techniques.The successful expression of these Nsps was subsequently confirmed through Western blot analysis.Initially,the activity of the RNA-dependent RNA polymerase was assessed by co-transfection of the reporter plasmid with Nsp12,in the presence of its auxiliary fac-tors Nsp7 and Nsp8,with careful regulation of the co-transfection ratio,culturing temperature,and the timing of activity determination for the triad of Nsp plasmids.The normalized NLuc fluorescein value,in reference to the FLuc fluorescein value of the housekeeping gene,served as a metric for determining the polymerase activity.Building upon this foundation,the co-transfection concentra-tions of Nsp9-16 were fine-tuned,followed by the incremental addition of varying doses of Nsp3C,Nsp4,and Nsp6,to further elucidate the activity of RNA-dependent RNA polymerase(RdRp).The findings indicated that upon transfection with varying ratios of Nsp7,Nsp8,and Nsp12 at a propor-tion of 1∶8∶24,the polymerase activity was markedly elevated compared to the control group,with a statistical significance level(P＜0.001).Furthermore,in the absence of Nsp3,Nsp4,and Nsp6,the inclusion of Nsp10-16 substantially augmented the activity of the RdRp,particularly in scenarios where Nsp9 was not introduced,achieving statistical significance(P＜0.001).In the pres-ence of Nsp3 and Nsp4,the RdRp activity was augmented further upon the addition of Nsp9,reac-hing a level of significance(P＜0.05).The data imply that Nsp9 is capable of enhancing the RdRp activity of SARS-CoV-2 exclusively in the context of Nsp3 and Nsp4 coexistence,suggesting that the stimulatory influence of Nsp9 on viral replication may be contingent upon the formation of double-membrane vesicles.

Objective:To explore the therapeutic effect of CD5L targeted therapy on a rat model of collagen-induced arthritis (CIA).Methods:The CIA rat model was established and treated with anti-CD5L antibody. The clinical arthritis score and tissue swelling degree of rats were evaluated. Twenty-four SD rats were randomly divided into the control group, the model group, the isotype control group and the CD5L antibody group. HE staining and safranin fast green staining were used to observe the synovial inflammation and cartilage erosion in the ankle joint of CIA rats. Micro-CT was used to scan the feet of CIA rats to detect bone mineral density and bone destruction. The levels of TNF-α, IL-6 and IL-8 in serum were detected by enzyme-linked immunosorbent assay (ELISA). All experimental data conforming to normal distribution were compared between groups by one-way ANOVA, and Dunnet t test was used to compare the two groups. Results:CD5L antibody improved joint swelling and deformity in CIA rats. The statistics of arthritis scores in rats showed that there was a statistically significant difference in arthritis scores between the CD5L antibody group and the isotype antibody group of rats from Day 23[Day 23 (6.6±0.5) vs. (8.2±0.7), t=-7.07, P<0.05; Day 26 (7.0±0.6) vs. (8.4±0.5), t=-7.59, P<0.05; Day 29 (7.6±0.4) vs. (9.4±0.8), t=-6.96, P<0.05; Day 32 (7.8±0.5) vs.(9.6±1.1), t=-6.50, P<0.05; Day 35 (8.2±0.7) vs. (10.4±0.7), t=-5.88, P<0.05]. The HE staining results showed that there was a statistically significant difference in the HE staining score between the CD5L antibody group and the isotype antibody group of rats [(2.5±0.6) vs. (5.0±0.8), t=-6.73, P<0.05]. The results of safranin fixation green staining showed that there was a statistically significant difference in the safranin fixation green staining score between the CD5L antibody group and the isotype antibody group of rats [(1.8±0.8) vs. (3.5±0.6), t=-5.22, P<0.05]. The Micro-CT imaging results showed that there was a statistically significant difference in bone mineral density between the CD5L antibody group and the isotype antibody group of rats [(5 618±128)g/cm 3vs. (5 202±39)g/cm 3, t=8.06, P<0.01]. The ELISA results showed that there were statistically significant differences in the contents of TNF-α, IL-6 and IL-8 in the serum of rats between the CD5L antibody group and the isotype antibody group [TNF-α: (164±22)pg/ml vs. (213±17)pg/ml, t=5.05, P<0.05; IL-6: (186±17)pg/ml vs. (230.7±25.3)pg/ml, t=4.78, P<0.05; IL-8: (384±21)pg/ml vs. (456±44)pg/ml, t=-5.21, P<0.05]. Conclusion:Targeting CD5L can effectively alleviate synovial inflammation and bone destruction in CIA rats. CD5L may be used as a potential therapeutic target for rheumatoid arthritis.

Body weight abnormalities, including overweight, obesity, and underweight, have become a dual public health challenge in Chinese adults: overweight and obesity lead to a variety of chronic complications, while underweight increases the risks of malnutrition, sarcopenia, and organ dysfunction. To systematically address these issues, multidisciplinary experts in endocrinology, sports science, nutrition, and psychiatry from various regions have held multiple weight management seminars. Based on the latest epidemiological data and clinical evidence, they expanded the guideline to include assessment and intervention strategies for underweight, in addition to the core content of obesity management. This guideline outlines the etiological mechanisms, evaluation methods, and multidimensional management strategies for overweight and obesity, covering key areas such as diagnosis and assessment, medical nutrition therapy, exercise prescription, pharmacological intervention, and psychological support. It is intended to provide a scientific and standardized approach to weight management across the adult population, aiming to curb the rising prevalence of obesity, mitigate complications associated with abnormal body weight, and improve nutritional status and overall quality of life.

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.

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.

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.