AKT,also known as Protein Kinase B(PKB),plays a critical role in cell proliferation and metabolism.There are three isoforms of AKT:AKT1,AKT2,and AKT3.The effects of these isoforms on the pluripotency and differentiation of mouse embryonic stem cells(mESCs)remain unclear.This study aims to explore the impact of three AKT isoform-specific knockouts on the self-renewal and differen-tiation of mouse embryonic stem cells.Using CRISPR/Cas9 gene-editing technology,AKT isoform-spe-cific knockout cell lines were established.The phenotypic and molecular changes were analyzed through Western blotting,flow cytometry,qRT-PCR,CCK-8 assays,Alkaline Phosphatase(AP)staining,and RNA-seq.The construction of AKT isoform-specific knockout cell lines was successful.The loss of AKT1 and AKT2 inhibited the proliferation of mESCs.The knockout of any single AKT isoform did not affect the expression of pluripotency genes at both mRNA or protein levels.However,during embryoid body forma-tion,the deletion of any of the three AKT isoforms affected the mRNA expression levels of genes in all three germ layers.Transcriptome analysis showed that compared to wild-type mESCs,995,547,and 429 differentially expressed genes(|log2FC|≧1,P＜0.05)were identified inAKT1,AKT2,and AKT3 isoform-specific knockout cells,respectively.There was some overlap in the differentially expressed genes regulated by these three isoforms.In conclusion,the independent knockout of AKT isoforms does not af-fect the maintenance of pluripotency in mouse embryonic stem cells,but they are crucial for differentia-tion.The three AKT isoforms can collectively regulate gene expression while retaining their own regulato-ry specificity.This study provides a foundation for understanding the unique and overlapping roles of AKT isoforms in stem cell biology,highlighting their importance in maintaining stem cell function and differen-tiation.

Aim To identify the underlying target of bullatine A(BA)against rheumatoid arthritis(RA)u-sing limited proteolysis-small molecule mapping(LiP-SMap)drug target proteomics and to provide a scientif-ic basis for clinical application of Aconiti brachypodi Radix in the treatment of RA.Methods LiP-SMap drug target proteomics was employed to perform bioin-formatics analysis for comparing and validating the dif-ferential protein expression after BA intervention.A collagen-induced arthritis(CIA)model was estab-lished in DBA/1 mice using bovine type Ⅱ collagen.The mice were then divided into the CIA model group,methotrexate-positive control group(MTX group),and BA groups(10 mg·kg-1 and 20 mg·kg-1)based on their clinical scores.After drug intervention,the thera-peutic efficacy against RA was assessed by joint index scores and foot thickness measurements.Histopatholog-ical changes in the arthritic joints of CIA mice were e-valuated using hematoxylin and eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was employed to detect inflammatory cytokines interleukin-17(IL-17)and total IgG and IgG3 anti-collagen-spe-cific antibodies levels from the serum of CIA mice.Flow cytometry was used to detect the expression levels of intracellular Th17 cells(IL-17+CD4+T cells)and Th1 cells(IFN-γ+CD4+T cells).Fluorescent quanti-tative PCR was performed to detect the expression of genes related to differential proteins.Results The proteomic analysis identified Serpinb1a as a protein with strong binding affinity to BA,and KEGG enrich-ment analysis indicated IL-17 signaling pathway was a crucial pathway of BA in against RA.BA treatment significantly reduced clinical scores and foot thickness,improved local arthritis symptoms in CIA mice,and al-leviated inflammatory cell infiltration into arthritic joints(P＜0.05).Differential protein validation re-sults showed that BA had strong affinity with Serpinb1a(-5.92 kJ·mol-1)and downregulated the expres-sion of Serpinb1a mRNA.Furthermore,the administra-tion of BA markedly reduced serum IL-17 A levels from CIA mice,inhibited the expression of intracellular IL-17 A and IFN-γ cytokines in splenic CD4+T cells(P＜0.05),and significantly downregulated the transcrip-tional expression of IL-17F(P＜0.05).Conclusion BA exhibits therapeutic effects on collagen-induced arthritis,and its mechanism of action may involve the regulation of Serpinb1a and the IL-17 signaling path-way.

Aim To identify the underlying target of bullatine A(BA)against rheumatoid arthritis(RA)u-sing limited proteolysis-small molecule mapping(LiP-SMap)drug target proteomics and to provide a scientif-ic basis for clinical application of Aconiti brachypodi Radix in the treatment of RA.Methods LiP-SMap drug target proteomics was employed to perform bioin-formatics analysis for comparing and validating the dif-ferential protein expression after BA intervention.A collagen-induced arthritis(CIA)model was estab-lished in DBA/1 mice using bovine type Ⅱ collagen.The mice were then divided into the CIA model group,methotrexate-positive control group(MTX group),and BA groups(10 mg·kg-1 and 20 mg·kg-1)based on their clinical scores.After drug intervention,the thera-peutic efficacy against RA was assessed by joint index scores and foot thickness measurements.Histopatholog-ical changes in the arthritic joints of CIA mice were e-valuated using hematoxylin and eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was employed to detect inflammatory cytokines interleukin-17(IL-17)and total IgG and IgG3 anti-collagen-spe-cific antibodies levels from the serum of CIA mice.Flow cytometry was used to detect the expression levels of intracellular Th17 cells(IL-17+CD4+T cells)and Th1 cells(IFN-γ+CD4+T cells).Fluorescent quanti-tative PCR was performed to detect the expression of genes related to differential proteins.Results The proteomic analysis identified Serpinb1a as a protein with strong binding affinity to BA,and KEGG enrich-ment analysis indicated IL-17 signaling pathway was a crucial pathway of BA in against RA.BA treatment significantly reduced clinical scores and foot thickness,improved local arthritis symptoms in CIA mice,and al-leviated inflammatory cell infiltration into arthritic joints(P＜0.05).Differential protein validation re-sults showed that BA had strong affinity with Serpinb1a(-5.92 kJ·mol-1)and downregulated the expres-sion of Serpinb1a mRNA.Furthermore,the administra-tion of BA markedly reduced serum IL-17 A levels from CIA mice,inhibited the expression of intracellular IL-17 A and IFN-γ cytokines in splenic CD4+T cells(P＜0.05),and significantly downregulated the transcrip-tional expression of IL-17F(P＜0.05).Conclusion BA exhibits therapeutic effects on collagen-induced arthritis,and its mechanism of action may involve the regulation of Serpinb1a and the IL-17 signaling path-way.

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.

Background and Aims:Mucinous adenocarcinoma of the colorectum(MAC)is a distinct histologic subtype of colorectal cancer characterized by high malignancy and low diagnostic accuracy of preoperative biopsy,posing challenges for clinical decision-making.Given the critical role of the inflammatory microenvironment in tumor progression,this study aimed to develop and validate a nomogram model integrating preoperative systemic inflammatory indicators and clinical features to improve the preoperative diagnosis of MAC.Methods:Clinical data of 293 patients with colorectal cancer who underwent radical resection between June 2017 and June 2022 at the First Affiliated Hospital of the University of South China were retrospectively analyzed.Based on postoperative pathology,patients were classified into the mucinous adenocarcinoma(MAC)group and the non-specific adenocarcinoma(AC)group.Propensity score matching(PSM,1∶1)was used to balance age,T stage,and N stage.Differences in preoperative inflammatory indices were compared between groups.Univariate and multivariate logistic regression analyses were performed to identify independent predictors of MAC,which were incorporated into a diagnostic nomogram.The model's discrimination,calibration,and clinical utility were evaluated using the area under the receiver operating characteristic curve(AUC),calibration plots,and decision curve analysis(DCA).Results:Among the 293 patients,46 had MAC and 247 had AC,with a preoperative colonoscopic diagnostic rate of 54%for MAC.After PSM(43 pairs),platelet count,platelet lymphocyte ratio(PLR),systemic immune inflammation index(SII),inflammation related prognostic index(IPI),and systemic inflammation score(SIS)were significantly higher in the MAC group,while lymphocyte monocyte ratio(LMR)was lower(all P<0.05).Multivariate analysis identified tumor location,maximum tumor diameter,and preoperative IPI as independent predictors.The AUCs of the nomogram in the training(n=206)and validation(n=87)cohorts were 0.759(95%CI=0.662-0.856)and 0.776(95%CI=0.649-0.903),respectively.Calibration plots showed good agreement between predicted and observed probabilities,and DCA demonstrated satisfactory clinical applicability.Conclusion:A nomogram model integrating tumor location,tumor size,and preoperative IPI was successfully developed and validated for preoperative diagnosis of colorectal MAC.This model provides a practical,quantitative tool with good predictive performance to assist clinicians in individualized treatment planning,particularly for patients ineligible for surgical biopsy.

Background and Aims:Mucinous adenocarcinoma of the colorectum(MAC)is a distinct histologic subtype of colorectal cancer characterized by high malignancy and low diagnostic accuracy of preoperative biopsy,posing challenges for clinical decision-making.Given the critical role of the inflammatory microenvironment in tumor progression,this study aimed to develop and validate a nomogram model integrating preoperative systemic inflammatory indicators and clinical features to improve the preoperative diagnosis of MAC.Methods:Clinical data of 293 patients with colorectal cancer who underwent radical resection between June 2017 and June 2022 at the First Affiliated Hospital of the University of South China were retrospectively analyzed.Based on postoperative pathology,patients were classified into the mucinous adenocarcinoma(MAC)group and the non-specific adenocarcinoma(AC)group.Propensity score matching(PSM,1∶1)was used to balance age,T stage,and N stage.Differences in preoperative inflammatory indices were compared between groups.Univariate and multivariate logistic regression analyses were performed to identify independent predictors of MAC,which were incorporated into a diagnostic nomogram.The model's discrimination,calibration,and clinical utility were evaluated using the area under the receiver operating characteristic curve(AUC),calibration plots,and decision curve analysis(DCA).Results:Among the 293 patients,46 had MAC and 247 had AC,with a preoperative colonoscopic diagnostic rate of 54%for MAC.After PSM(43 pairs),platelet count,platelet lymphocyte ratio(PLR),systemic immune inflammation index(SII),inflammation related prognostic index(IPI),and systemic inflammation score(SIS)were significantly higher in the MAC group,while lymphocyte monocyte ratio(LMR)was lower(all P<0.05).Multivariate analysis identified tumor location,maximum tumor diameter,and preoperative IPI as independent predictors.The AUCs of the nomogram in the training(n=206)and validation(n=87)cohorts were 0.759(95%CI=0.662-0.856)and 0.776(95%CI=0.649-0.903),respectively.Calibration plots showed good agreement between predicted and observed probabilities,and DCA demonstrated satisfactory clinical applicability.Conclusion:A nomogram model integrating tumor location,tumor size,and preoperative IPI was successfully developed and validated for preoperative diagnosis of colorectal MAC.This model provides a practical,quantitative tool with good predictive performance to assist clinicians in individualized treatment planning,particularly for patients ineligible for surgical biopsy.

Objective:Based on a single-center retrospective study,to explore the efficacy of intervertebral fusion through different approaches combined with pedicle screw internal fixation in the treatment of single-segment low-grade lumbar spondylolisthesis.Methods:This study retrospectively analyzed the clinical data of 87 patients with single-segment low-grade lumbar spondylolisthesis who were treated in our hospital from January 2021 to August 2022,the cases were divided into Group A(41 cases)and Group B(46 cases)according to the differences in surgical methods.Group A received treatment with posterior lumbar interbody fusion(PLIF)combined with pedicle screw internal fixation,while Group B received treatment with percutaneous endoscopic posterior lumbar interbody fusion(PE-PLIF)combined with pedicle screw internal fixation.Both groups were followed up for two years.The Visual Analogue Scale(VAS)scores of the waist and legs,perioperative indicators,recovery of lumbar function[Japanese Orthopaedic Association(JOA)score,Oswestry Disability Index(ODI)score],and serum inflammatory mediators levels[interleukin-1α(IL-1α),transforming growth factor β1(TGF-β1),interleukin-6(IL-6)],the rate of lumbar interbody fusion,the rate of lumbar spondylolisthesis and the incidence of postoperative complications of the two groups were compared.Results:The operation time and the number of intraoperative X-ray fluoroscopy sessions in group B were both more than those in group A,while the hospital stay,the intraoperative blood loss and total incision length in Group B were all shorter than those in group A(P＜0.05).The VAS scores of the waist and legs in both groups decreased at 3 d after operation and the last follow-up(P＜0.05),and the VAS scores of the waist and legs in group B were lower than those in group A(P＜0.05).The ODI scores of both groups decreased at 3 d after operation and the last follow-up,and the ODI scores of group B were lower than those in group A;The JOA score increased,and the JOA scores of group B were all higher than those in group A(P＜0.05).Serum IL-1α,TGF-β1 and IL-6 levels in both groups decreased at 3 d and 7 d after operation,and serum IL-1α,TGF-β1 and IL-6 levels in group B were all lower than those in group A(P＜0.05).There was no statistically significant difference in the incidence of complications,the rate of lumbar interbody fusion,and the rate of lumbar spondylolisthesis between the two groups(P＞0.05).Conclusion:Compared with PLIF,PE-PLIF combined with pedicle screw internal fixation in the treatment of single-segment low-grade lumbar.spondylolisthesis,can better relieve the degree of pain of the waist and legs,improve lumbar dysfunction,regulate serum inflammatory mediators levels more effectively,and has higher safety and significant therapeutic efficacy.

AKT,also known as Protein Kinase B(PKB),plays a critical role in cell proliferation and metabolism.There are three isoforms of AKT:AKT1,AKT2,and AKT3.The effects of these isoforms on the pluripotency and differentiation of mouse embryonic stem cells(mESCs)remain unclear.This study aims to explore the impact of three AKT isoform-specific knockouts on the self-renewal and differen-tiation of mouse embryonic stem cells.Using CRISPR/Cas9 gene-editing technology,AKT isoform-spe-cific knockout cell lines were established.The phenotypic and molecular changes were analyzed through Western blotting,flow cytometry,qRT-PCR,CCK-8 assays,Alkaline Phosphatase(AP)staining,and RNA-seq.The construction of AKT isoform-specific knockout cell lines was successful.The loss of AKT1 and AKT2 inhibited the proliferation of mESCs.The knockout of any single AKT isoform did not affect the expression of pluripotency genes at both mRNA or protein levels.However,during embryoid body forma-tion,the deletion of any of the three AKT isoforms affected the mRNA expression levels of genes in all three germ layers.Transcriptome analysis showed that compared to wild-type mESCs,995,547,and 429 differentially expressed genes(|log2FC|≧1,P＜0.05)were identified inAKT1,AKT2,and AKT3 isoform-specific knockout cells,respectively.There was some overlap in the differentially expressed genes regulated by these three isoforms.In conclusion,the independent knockout of AKT isoforms does not af-fect the maintenance of pluripotency in mouse embryonic stem cells,but they are crucial for differentia-tion.The three AKT isoforms can collectively regulate gene expression while retaining their own regulato-ry specificity.This study provides a foundation for understanding the unique and overlapping roles of AKT isoforms in stem cell biology,highlighting their importance in maintaining stem cell function and differen-tiation.

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.

Objective:Based on a single-center retrospective study,to explore the efficacy of intervertebral fusion through different approaches combined with pedicle screw internal fixation in the treatment of single-segment low-grade lumbar spondylolisthesis.Methods:This study retrospectively analyzed the clinical data of 87 patients with single-segment low-grade lumbar spondylolisthesis who were treated in our hospital from January 2021 to August 2022,the cases were divided into Group A(41 cases)and Group B(46 cases)according to the differences in surgical methods.Group A received treatment with posterior lumbar interbody fusion(PLIF)combined with pedicle screw internal fixation,while Group B received treatment with percutaneous endoscopic posterior lumbar interbody fusion(PE-PLIF)combined with pedicle screw internal fixation.Both groups were followed up for two years.The Visual Analogue Scale(VAS)scores of the waist and legs,perioperative indicators,recovery of lumbar function[Japanese Orthopaedic Association(JOA)score,Oswestry Disability Index(ODI)score],and serum inflammatory mediators levels[interleukin-1α(IL-1α),transforming growth factor β1(TGF-β1),interleukin-6(IL-6)],the rate of lumbar interbody fusion,the rate of lumbar spondylolisthesis and the incidence of postoperative complications of the two groups were compared.Results:The operation time and the number of intraoperative X-ray fluoroscopy sessions in group B were both more than those in group A,while the hospital stay,the intraoperative blood loss and total incision length in Group B were all shorter than those in group A(P＜0.05).The VAS scores of the waist and legs in both groups decreased at 3 d after operation and the last follow-up(P＜0.05),and the VAS scores of the waist and legs in group B were lower than those in group A(P＜0.05).The ODI scores of both groups decreased at 3 d after operation and the last follow-up,and the ODI scores of group B were lower than those in group A;The JOA score increased,and the JOA scores of group B were all higher than those in group A(P＜0.05).Serum IL-1α,TGF-β1 and IL-6 levels in both groups decreased at 3 d and 7 d after operation,and serum IL-1α,TGF-β1 and IL-6 levels in group B were all lower than those in group A(P＜0.05).There was no statistically significant difference in the incidence of complications,the rate of lumbar interbody fusion,and the rate of lumbar spondylolisthesis between the two groups(P＞0.05).Conclusion:Compared with PLIF,PE-PLIF combined with pedicle screw internal fixation in the treatment of single-segment low-grade lumbar.spondylolisthesis,can better relieve the degree of pain of the waist and legs,improve lumbar dysfunction,regulate serum inflammatory mediators levels more effectively,and has higher safety and significant therapeutic efficacy.