ObjectiveThe malaria parasites remodel the host erythrocyte structure by exporting parasite proteins that interact with the membrane skeleton proteins of red blood cells (RBCs), facilitating their intracellular survival and pathogenicity. Skeleton-binding protein 1 (SBP1) is a conserved exported protein across Plasmodium species. In Plasmodium falciparum, SBP1 has been reported to interact with erythrocyte membrane skeleton proteins 4.1R and spectrin, while its contribution to erythrocyte remodeling and parasite virulence in Plasmodium berghei (Pb) remains unclear. This study aims to determine whether PbSBP1 associates with the host cytoskeletal protein 4.1R and to investigate its role in the remodeling of host RBCs and the pathogenicity of Plasmodium berghei. MethodsIn Plasmodium berghei, the relationship between PbSBP1 and the erythrocyte cytoskeletal protein 4.1R was examined using co-immunoprecipitation. A Pbsbp1 gene knockout mutant of Plasmodium berghei (Pbsbp1∆) was generated based on the principle of double crossover homologous recombination. The deformability of erythrocytes infected with Pbsbp1∆ parasites was assessed using microfluidic methods. Microchannels with an array of cylindrical pillars were used to detect modifications in infected RBC deformability. The infected RBCs were squashed between the rows and recovered between the columns and the transit velocity (μm/s) of infected RBCs travelling through the microchannel was recorded. The component of the erythrocyte membrane skeleton junctional complex, tropomodulin (TMOD), was fluorescently labeled, and the cytoskeletal network of infected erythrocytes was imaged using super-resolution stochastic optical reconstruction microscopy (STORM) to analyze ultrastructural changes in the cytoskeleton of wild-type (WT) and Pbsbp1∆-infected erythrocytes. Actin-based junctional complexes were displayed as individual clusters by the labeled TMOD in the STORM images, and the cluster densities and distances between adjacent clusters of infected RBCs were calculated. Additionally, rodent malaria models (BALB/c mice) and experimental cerebral malaria models (C57BL/6 mice) were employed to monitor the growth of Pbsbp1∆ and WT parasites during the intraerythrocytic stage and their capacity to induce cerebral malaria in mice. ResultsPbSBP1 may participate in the remodeling of infected erythrocytes through direct or indirect interaction with the erythrocyte cytoskeletal protein 4.1R. Microfluidic assays revealed that the deformability of erythrocytes infected with Pbsbp1∆ parasites was significantly enhanced compared to those infected with WT parasites. STORM imaging further demonstrated that the ultrastructure of the erythrocyte cytoskeleton in Pbsbp1∆-infected cells was altered relative to that in WT-infected erythrocytes. The distances between nearest neighbors of clusters had a tendency to increase while the cluster densities were decreased in Pbsbp1∆-infected RBCs compared to WT-infected RBCs. Subsequent phenotypic analysis indicated that the growth rate of Pbsbp1∆ parasites during the intraerythrocytic stage was significantly slower than that of WT parasites, and their ability to induce cerebral malaria in mice was also attenuated. These findings suggest that PbSBP1 is involved in the remodeling of the erythrocyte membrane skeleton, likely through its direct or indirect interaction with protein 4.1R, thereby regulating the deformability of infected erythrocytes and influencing the pathogenicity of the blood-stage parasites. ConclusionThis study establishes a role for PbSBP1 in host erythrocyte remodeling and parasite virulence, providing new research strategies for the prevention and treatment of malaria.

Objective To evaluate the efficacy and safety of recombinant staphylokinase in patients with acute ST-segment elevation myocardial infarction(STEMI)by a multi-center,randomized,position-controlled,parallel post-marketing clinical trial.Methods This study was a multi-center,randomized,positive drug parallel control,non-inferiority clinical trial.From July 2019 to June 2022,a total of 251 patients with STEMI were enrolled in 31 hospitals.Patients were randomly assigned to receive intravenous staphylokinase or alteplase in a ratio of 1∶1.Vascular recanalization was evaluated by clinical indicators 30 minutes,60 minutes and 120 minutes after the initiation of thrombolysis.Coronary angiography was performed 90 to 120 minutes after the initiation of thrombolysis.The proportion of infarct-related artery(IRA)with thrombolysis in myocardial infarction(TIMI)grade Ⅱ and Ⅲ,corrected TIMI frame count(CTFC)and TIMI myocardial perfusion grade(TMPG)were analyzed Major adverse cardiac events(MACE,including all-cause death,rehospitalization,reinfarction,urgent target vessel revascularization)and bleeding events were followed up at 30 days(±2 days)after thrombolysis.Results After excluding 7 subjects who did not use thrombolytic drugs,244 subjects were finally eligibled from 31 hospitals(117 in trial group and 127 in control group),and 232 subjects completed the follow-up(111 in trial group and 121 in control group).The vascular recanalization rate evaluated by clinical indicators at 120 minutes after thrombolysis was 85.6％ in trial group and 83.5％ in control group(P=0.657).The difference between the two groups was 2.11(95％CI-7.19-11.41).Given that the lower confidence limit of the 95％CI was greater than-12％,the non-inferiority of the vascular recanalization rate was established based on clinical judgment.Coronary angiography showed that the total patency rate of IRA(TIMIⅡ-Ⅲ)was 77.5％ in trial group and 77.7％ in control group(P=0.970).The difference between the two groups was-0.21(95％CI-10.95-10.54),with the lower bound of the 95％CI exceeding-12％.Therefore,the non-inferiority of the TIMI blood flow grade was confirmed,indicating that the total patency rate of IRA in the trial group was not inferior to that in the control group.The CTFC was(32.7±17.6)frames in trial group and(37.6±16.6)frames in control group,with no statistically significant difference between the two groups(P=0.054).The difference between the two groups was-4.9(95％CI-10.0-0.1).As the lower limit of the 95％CI exceeded-12％,the noninferiority of CTFC was successfully demonstrated.The proportions of TMPG 0-Ⅲ were 20.7％,6.3％,2.7％and 69.4％in trial group,and 22.3％,4.1％,6.6％ and 66.9％ in control group,respectively.There was no significant difference in TIMI myocardial perfusion grade between the two groups(P=0.086).The incidence of MACE was 7.7％ in trial group and 7.1％ in control group within 30 days after the initiation of thrombolysis,and there was no significant difference between the two groups(P=0.857).Further analysis showed that there was no significant difference in cardiovascular mortality(3.4％ vs.4.7％,P=0.751).All 244 subjects were included in the safety analysis set.There was no significant difference in the total incidence of bleeding events between the two groups(22.2％ vs.15.0％,P=0.144).There was no significant difference in the incidence of major bleeding(1.7％ vs.0.8％,P=0.609).Conclusions Recombinant staphylokinase is simple to use and has a rapid onset of action.The efficacy and safety of recombinant staphylokinase are not inferior to alteplase in the treatment of acute STEMI.

Objective:To investigate the risk factors and their diagnostic efficacy of perioperative lower limb deep vein thrombosis (DVT) in polytrauma patients with predominant severe limb trauma.Methods:A retrospective cohort study was conducted to analyze the clinical data of 155 polytrauma patients with predominant severe trauma who were admitted to Wuxi Ninth People′s Hospital from January 2021 to December 2024, including 64 males and 91 females, aged 13-95 years [(52.1±16.9)years]. Abbreviated injury scale (AIS) was 5-15 points [(7.4±2.1)points] and injury severity score (ISS) was 17-59 points [(21.3±6.5)points]. Based on the occurrence of DVT in the perioperative period, the patients were divided into preoperative DVT group with 17 patients (11.0%) and non-preoperative DVT group with 138 patients (89.0%) as well as postoperative DVT group with 24 patients (15.5%) and non-postoperative DVT group with 131 patients (84.5%). Basic clinical data were collected, including gender, age, body mass index (BMI), underlying diseases (hypertension, diabetes mellitus), hemoglobin level (Hb), platelet count (PLT), D-dimer, ISS, trauma site [cranial and brain trauma, thoracic and abdominal trauma, upper limb trauma, lower limb trauma (femoral fracture, patellar fracture, tibial or fibular fracture, foot fracture, vascular injury), and pelvic fracture], preoperative waiting time for surgery, surgical site (pelvis and lower limb, other areas), surgical protocols (pelvic and lower limb internal fixation, external fixation of lower limb, lower limb amputation), operation duration less or more than 2 hours, amount of intraoperative blood loss, intraoperative blood transfusion requirement, venous thromboembolism (VTE) prophylaxis (pharmacological and mechanical modalities) and length of hospital stay. Univariate analysis and multivariate binary Logistic regression analysis were conducted to investigate the correlation between the aforementioned indicators and incidence of perioperative lower limb DVT in polytrauma patients with predominant severe limb trauma and determine the independent risk factors. Receiver operating characteristic (ROC) curve and area under the curve (AUC) of the relevant risk factors were analyzed to evaluate and compare the diagnostic efficacy of the risk factors for perioperative lower limb DVT in polytrauma patients with predominant severe limb trauma.Results:Univariate analysis results showed that age, history of hypertension, D-dimer, thoracic and abdominal trauma, pelvic fracture, preoperative waiting time for surgery, and length of hospital stay were significantly correlated with preoperative of DVT of the lower limbs in the patients ( P<0.05). The results of multivariate binary Logistic regression analysis showed that age ( OR=1.05, 95% CI 1.00, 1.10, P<0.05), pelvic fracture ( OR=5.03, 95% CI 1.09, 23.20, P<0.05), preoperative waiting time for surgery ( OR=1.10, 95% CI 1.00, 1.22, P<0.05) and length of hospital stay ( OR=0.89,95% CI 0.81,0.98, P<0.05) were highly correlated with preoperative DVT of the lower limbs in the patients ( P<0.05). Univariate analysis results showed that age, D-dimer, ISS, foot fracture, and length of hospital stay were significantly correlated with postoperative DVT of the lower limbs in the patients ( P<0.05). The results of multivariate binary Logistic regression analysis showed that age ( OR=1.05, 95% CI 1.01, 1.08, P<0.01), D-dimer ( OR=1.05, 95% CI 1.00, 1.10, P<0.05), ISS ( OR=1.09, 95% CI 1.01, 1.17, P<0.05), and foot fracture ( OR=3.51 , 95% CI 1.25 , 9.87 , P<0.05) were significantly correlated with postoperative DVT of the lower limbs in the patients ( P<0.05). The results of the ROC curve analysis indicated that preoperative waiting time for surgery (AUC=0.83, 95% CI 0.75, 0.91) had the highest diagnostic efficacy for preoperative DVT of the lower limbs in the patients, with the diagnostic efficacies of pelvic fracture (AUC=0.75, 95% CI 0.65, 0.85) and age (AUC=0.70, 95% CI 0.59, 0.82) decreasing successively. For postoperative DVT of the lower limbs in the patients, D-dimer (AUC=0.71, 95% CI 0.61, 0.81) exhibited the highest diagnostic efficacy, followed by age (AUC=0.70, 95% CI 0.59, 0.81), ISS (AUC=0.64, 95% CI 0.51, 0.76) and foot fracture (AUC=0.62, 95% CI 0.49, 0.74), with diagnostic efficacy decreased successively. Conclusions:For polytrauma patients with predominant severe limb trauma, age, pelvic fracture and preoperative waiting time for surgery are independent risk factors for preoperative DVT, while age, D-dimer, ISS and foot fracture are independent risk factors for postoperative DVT. Additionally, preoperative waiting time for surgery has the best diagnostic efficacy for preoperative DVT, followed by pelvic fracture and age. D-dimer has the best diagnostic efficacy for postoperative DVT, followed by age, ISS and foot fracture.

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree with recurrent Joubert syndrome with negative results by whole-exome sequencing in the prior proband. METHODS: Chinese pedigree which opted elective abortion at the Women and Children's Hospital Affiliated to Chongqing Medical University in December 2024 was selected as the study subject. Whole-genome sequencing was carried out on fetal tissue after termination of pregnancy. Candidate variants were validated by Sanger sequencing and interpreted, while non-coding variant was analyzed using in silico prediction tools. RNA sequencing and cDNA sequencing were conducted on fetal brain tissue. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.2024YL045-02). RESULTS: Both the fetus and the affected child were found to harbor compound heterozygous variants of the CEP290 gene, namely c.7341dup (p.Leu2448fs*8) (pathogenic, maternally inherited) and c.1523-408G>A (likely pathogenic, paternally inherited). Both in silico analysis and fetal brain RNA sequencing confirmed aberrant RNA splicing caused by the intronic variant. CONCLUSION This case has highlighted the value of combining whole-genome sequencing with RNA functional validation. Above results not only enriched the spectrum of CEP290 gene mutations but also underscored its diagnostic value in resolving complex prenatal cases, providing critical clues for the prenatal diagnosis and recurrence risk assessment in genetic counseling.
Female ; Humans ; Pregnancy ; Abnormalities, Multiple/genetics* ; Antigens, Neoplasm/genetics* ; Cell Cycle Proteins/genetics* ; Cerebellum/abnormalities* ; Cytoskeletal Proteins/genetics* ; Eye Abnormalities/genetics* ; Introns/genetics* ; Kidney Diseases, Cystic/diagnosis* ; Pedigree ; Retina/abnormalities* ; Sequence Analysis, RNA/methods* ; Whole Genome Sequencing/methods* ; Child

Objective:To investigate the risk factors and their diagnostic efficacy of perioperative lower limb deep vein thrombosis (DVT) in polytrauma patients with predominant severe limb trauma.Methods:A retrospective cohort study was conducted to analyze the clinical data of 155 polytrauma patients with predominant severe trauma who were admitted to Wuxi Ninth People′s Hospital from January 2021 to December 2024, including 64 males and 91 females, aged 13-95 years [(52.1±16.9)years]. Abbreviated injury scale (AIS) was 5-15 points [(7.4±2.1)points] and injury severity score (ISS) was 17-59 points [(21.3±6.5)points]. Based on the occurrence of DVT in the perioperative period, the patients were divided into preoperative DVT group with 17 patients (11.0%) and non-preoperative DVT group with 138 patients (89.0%) as well as postoperative DVT group with 24 patients (15.5%) and non-postoperative DVT group with 131 patients (84.5%). Basic clinical data were collected, including gender, age, body mass index (BMI), underlying diseases (hypertension, diabetes mellitus), hemoglobin level (Hb), platelet count (PLT), D-dimer, ISS, trauma site [cranial and brain trauma, thoracic and abdominal trauma, upper limb trauma, lower limb trauma (femoral fracture, patellar fracture, tibial or fibular fracture, foot fracture, vascular injury), and pelvic fracture], preoperative waiting time for surgery, surgical site (pelvis and lower limb, other areas), surgical protocols (pelvic and lower limb internal fixation, external fixation of lower limb, lower limb amputation), operation duration less or more than 2 hours, amount of intraoperative blood loss, intraoperative blood transfusion requirement, venous thromboembolism (VTE) prophylaxis (pharmacological and mechanical modalities) and length of hospital stay. Univariate analysis and multivariate binary Logistic regression analysis were conducted to investigate the correlation between the aforementioned indicators and incidence of perioperative lower limb DVT in polytrauma patients with predominant severe limb trauma and determine the independent risk factors. Receiver operating characteristic (ROC) curve and area under the curve (AUC) of the relevant risk factors were analyzed to evaluate and compare the diagnostic efficacy of the risk factors for perioperative lower limb DVT in polytrauma patients with predominant severe limb trauma.Results:Univariate analysis results showed that age, history of hypertension, D-dimer, thoracic and abdominal trauma, pelvic fracture, preoperative waiting time for surgery, and length of hospital stay were significantly correlated with preoperative of DVT of the lower limbs in the patients ( P<0.05). The results of multivariate binary Logistic regression analysis showed that age ( OR=1.05, 95% CI 1.00, 1.10, P<0.05), pelvic fracture ( OR=5.03, 95% CI 1.09, 23.20, P<0.05), preoperative waiting time for surgery ( OR=1.10, 95% CI 1.00, 1.22, P<0.05) and length of hospital stay ( OR=0.89,95% CI 0.81,0.98, P<0.05) were highly correlated with preoperative DVT of the lower limbs in the patients ( P<0.05). Univariate analysis results showed that age, D-dimer, ISS, foot fracture, and length of hospital stay were significantly correlated with postoperative DVT of the lower limbs in the patients ( P<0.05). The results of multivariate binary Logistic regression analysis showed that age ( OR=1.05, 95% CI 1.01, 1.08, P<0.01), D-dimer ( OR=1.05, 95% CI 1.00, 1.10, P<0.05), ISS ( OR=1.09, 95% CI 1.01, 1.17, P<0.05), and foot fracture ( OR=3.51 , 95% CI 1.25 , 9.87 , P<0.05) were significantly correlated with postoperative DVT of the lower limbs in the patients ( P<0.05). The results of the ROC curve analysis indicated that preoperative waiting time for surgery (AUC=0.83, 95% CI 0.75, 0.91) had the highest diagnostic efficacy for preoperative DVT of the lower limbs in the patients, with the diagnostic efficacies of pelvic fracture (AUC=0.75, 95% CI 0.65, 0.85) and age (AUC=0.70, 95% CI 0.59, 0.82) decreasing successively. For postoperative DVT of the lower limbs in the patients, D-dimer (AUC=0.71, 95% CI 0.61, 0.81) exhibited the highest diagnostic efficacy, followed by age (AUC=0.70, 95% CI 0.59, 0.81), ISS (AUC=0.64, 95% CI 0.51, 0.76) and foot fracture (AUC=0.62, 95% CI 0.49, 0.74), with diagnostic efficacy decreased successively. Conclusions:For polytrauma patients with predominant severe limb trauma, age, pelvic fracture and preoperative waiting time for surgery are independent risk factors for preoperative DVT, while age, D-dimer, ISS and foot fracture are independent risk factors for postoperative DVT. Additionally, preoperative waiting time for surgery has the best diagnostic efficacy for preoperative DVT, followed by pelvic fracture and age. D-dimer has the best diagnostic efficacy for postoperative DVT, followed by age, ISS and foot fracture.

Aim To study the mechanism of adipose mesenchymal stem cell exosomes(ASC-exo)inhibition of fluorescein isothiocyanate(FITC)-induced atopic dermatitis(AD).Methods The mouse age,extrac-tion method,and the concentration of a solution of typeⅠ collagen enzyme and other conditions were compared to study the effects on the morphology and quantity of adipose mesenchymal stem cells(ASCs)after extrac-ted.FITC-induced mouse model in vivo was estab-lished and different doses of ASC-exo were given to measure ear thickness,ear weight and ear scratching times of mice.HE staining was used to observe the pathological changes of ear tissue of mice.The non-toxicity of ASC-exo was detected.IgE,IL-5,IL-13 and other cytokines were detected by ELISA.The gene ex-pressions of TSLP,IL-33,occludin,Claudin-1(CLDN-1)and E-cadherin were detected by RT-qPCR.The protein expression was detected by immunohistochemis-try.Results An efficient method for extracting ASCs was established.Compared with the blank group,mice in the model group showed obvious AD symptoms.Compared with the model group,ASC-exo administra-tion group significantly reduced the number of ear scratches,epidermal thickening,inflammatory cell infil-tration and the secretion of Th2 cytokines IL-5 and IL-13.Meanwhile,ASC-exo administration group signifi-cantly increased the expression of structural proteins CLDN-1 and occludin in epithelial cells and decreased the expression of TSLP and IL-33.Conclusions ASC-exo can significantly improve Th2 skin inflamma-tion in AD mice,and its mechanism may be through in-creasing the expression of tight junction proteins and adhesion link protein in epithelial cells,repairing the skin barrier,and inhibiting the key promoters of allergy TSLP and IL-33.

Objective To establish a fingerprint spectrum for Qiangshen Oral Liquid;To predict its quality markers combined with network pharmacology.Methods HPLC-ELSD method was used to establish fingerprint spectrum of Qiangshen Oral Liquid,and common peaks were identified.Its targets and related pathways were predicted through network pharmacology.A component-target-pathway network was constructed,and molecular docking verification was performed to analyze the quality markers of Qiangshen Oral Liquid.Results The HPLC fingerprint spectrum of Qiangshen Oral Liquid was established.20 common peaks were identified and belonged to all prescription drug tastes.13 chromatographic peaks were located using reference standards.The similarity evaluation showed that the similarity of 19 batches of Qiangshen Oral Liquid ranged from 0 to 0.995.Network pharmacology identified 45 key targets related to 13 components,and KEGG enrichment analysis obtained 151 pathways.A component-target-pathway network was constructed,predicted that calycosin-7-O-glucoside,ginsenoside Rg1,ginsenoside Re,ginsenoside Rf,ginsenoside Rb1,astragaloside Ⅳ,ginsenoside Rd,ginsenoside Rb2,astragaloside Ⅱ,astragaloside Ⅰ,schisandrin A and panaxatriol were quality markers for Qiangshen Oral Liquid.Conclusion The fingerprint spectrum of Qiangshen Oral Liquid was established,and quality markers were predicted preliminarily,providing a reference for its standard establishment and quality evaluation.

This study was aimed at exploring the interaction between the nucleoprotein(NP)of influenza A virus(IAV)and TRIM25.The physicochemical properties and protein structure of IAV NP protein were analyzed through bioinformatics methods.The interaction between IAV NP and TRIM25 proteins was simulated with molecular docking techniques,and the in-teraction sites were predicted.With the cDNA of the A/Puerto Rico/8/1934(H1N1)PR8 strain as the template,the NP pro-tein was cloned into the eukaryotic expression vector pCMV-C-Flag through PCR amplification,the eukaryotic expression re-combinant plasmid pCMV-Flag-NP was constructed,and the expression was further verified.The protein expression levels of pCMV-Flag-NP and pCMV-HA-TRIM25 were detected at various time periods.The interaction between NP protein and TRIM25 protein was verified by co-immunoprecipitation.The co-localization of NP protein and TRIM25 protein in cells was ob-served with laser confocal microscopy.Bioinformatics analysis revealed that the NP protein consists of 498 amino acids and 20 amino acids,and is an unstable hydrophilic protein.The NP protein has multiple phosphorylation sites,as well as N-glycosyla-tion and O-glycosylation sites,but no transmembrane domain or signal peptide domain.Additionally,the NP protein's second-ary structure consists of a high proportion of alpha-helices and random coils.The molecular docking prediction results indicated that IAV NP interacts with TRIM25 protein and has multiple potential interaction sites,including the 233rd alanine,234th ala-nine,236th lysine,and 440th alanine of the NP protein.After successfully constructing and expressing the IAV NP protein,we verified the interaction between IAV NP and TRIM25 protein by immunoprecipitation and laser confocal microscopy obser-vations.Our results together suggested that the structure of the IAV NP protein is closely related to its function,and its im-portance to the virus is clear.In addition,the interaction between IAV NP and TRIM25 protein may be associated with TRIM25's anti-influenza virus mechanism.Further in-depth research may provide new ideas for anti-influenza virus strategies.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

Aim To investigate the regulatory mecha-nisms of donepezil on the expression and enzymatic ac-tivity of cytochrome P450 3A4(CYP3A4),elucidate the synergistic impact of hypoxia on CYP3A4 function,and reveal its potential association with drug-induced cardiotoxicity,particularly QT interval prolongation.Methods Western blot,co-immunoprecipitation,and gene knockdown techniques were employed to evaluate the effects of donepezil and hypoxia on CYP3A4 pro-tein expression.CYP3A4 enzymatic activity was as-sessed using an in vitro incubation system with rat liver microsomes combined with high-performance liquid chromatography(HPLC),and the half-maximal inhib-itory concentration(IC50)was determined.Results Donepezil(10 μmol·L-1)and hypoxia reduced CYP3A4 protein expression to 31.75％and 45.90％of the control levels,respectively.Both interventions activated the gp78-mediated ubiquitin-proteasome path-way,significantly increasing CYP3A4 ubiquitination levels by 2.1-fold compared to the control group,thereby promoting proteasomal degradation.Donepezil inhibited CYP3A4 enzyme activity with an IC50 of 83.4μmol·L-1,and hypoxia synergistically enhanced this inhibitory effect,reducing the IC50 to 20.79 μmol·L-1.Conclusion Donepezil downregulates CYP3A4 function through dual mechanisms involving ubiquitin-mediated proteasomal degradation and direct enzymatic inhibition.Hypoxia potentiates this effect,leading to impaired metabolism of CYP3A4 substrate drugs,ele-vated plasma drug concentrations(1.6-2.3-fold in-crease compared to normal metabolic conditions),and an increased risk of QT interval prolongation and other forms of cardiotoxicity.