BACKGROUND:Studies have confirmed that Pterocarya hupehensis skan total flavonoids(PHSTF)can improve the level of collagen-induced arthritis in rats,but there is still a lack of research on the regulation of Wnt/β-catenin signaling pathway in fibroblast-like synoviocytes and its effect on related cell functions.OBJECTIVE:To investigate the effect and mechanism of PHSTF on lipopolysaccharide-induced proliferation,migration and apoptosis of fibroblast-like synoviocytes based on the Wnt/β-catenin signaling pathwayMETHODS:Fibroblast-like synoviocytes were divided into control group,lipopolysaccharide group,lipopolysaccharide+low-,medium-,and high-dose PHSTF groups(10,20,and 40 μg/mL),lipopolysaccharide+Wnt pathway inhibitor DKK1 group,and lipopolysaccharide+Wnt pathway inhibitor DKK1+high-dose PHSTF group(40 μg/mL).The cell counting kit-8 method was used to detect the effect of PHSTF on the viability of fibroblast-like synoviocytes,and the final drug concentration and time were screened.Flow cytometry was used to detect the apoptosis of fibroblast-like synoviocytes.Cell scratch assay,EDU staining and cell cloning assay were used to detect the migration and proliferation of fibroblast-like synoviocytes.Western blot assay was used to detect the protein expression levels of Wnt3a,β-catenin,tumorigenic genes,matrix metalloproteinase 2,matrix metalloproteinase 9,Bax and Bcl-2 in fibroblast-like synoviocytes.RESULTS AND CONCLUSION:(1)Compared with the control group,the cell viability decreased significantly when the concentration of PHSTF was＞40 μg/mL(P＜0.01).Therefore,the drug concentration of≤40 μg/mL was selected for subsequent experiments.(2)Compared with the lipopolysaccharide group,the wound healing rate,cell clone formation rate and the number of EDU-positive cells in the low-,medium-and high-dose PHSTF groups were significantly reduced,while the apoptosis rate was significantly increased(P＜0.05-0.01).(3)Western blot results showed that compared with the lipopolysaccharide group,low-,medium-and high-dose PHSTF significantly inhibited cellular Wnt3a,β-catenin,cellular tumorigenic genes,matrix metalloproteinase 2,matrix metalloproteinase 9,and Bcl-2 protein expression,and promoted the expression of Bax protein(P＜0.01).(4)Compared with the DKK1 group,the combination of DKK1 and high-dose PHSTF significantly inhibited the protein expression of Wnt3a,β-catenin,matrix metalloproteinase 2,matrix metalloproteinase 9 and Bcl-2 protein expression and promoted the protein expression of Bax(P＜0.01).To conclude,PHSTF may inhibit the proliferation and migration of fibroblast-like synoviocytes and promote apoptosis by inhibiting the Wnt/β-catenin signaling pathway.

BACKGROUND:Studies have confirmed that Pterocarya hupehensis skan total flavonoids(PHSTF)can improve the level of collagen-induced arthritis in rats,but there is still a lack of research on the regulation of Wnt/β-catenin signaling pathway in fibroblast-like synoviocytes and its effect on related cell functions.OBJECTIVE:To investigate the effect and mechanism of PHSTF on lipopolysaccharide-induced proliferation,migration and apoptosis of fibroblast-like synoviocytes based on the Wnt/β-catenin signaling pathwayMETHODS:Fibroblast-like synoviocytes were divided into control group,lipopolysaccharide group,lipopolysaccharide+low-,medium-,and high-dose PHSTF groups(10,20,and 40 μg/mL),lipopolysaccharide+Wnt pathway inhibitor DKK1 group,and lipopolysaccharide+Wnt pathway inhibitor DKK1+high-dose PHSTF group(40 μg/mL).The cell counting kit-8 method was used to detect the effect of PHSTF on the viability of fibroblast-like synoviocytes,and the final drug concentration and time were screened.Flow cytometry was used to detect the apoptosis of fibroblast-like synoviocytes.Cell scratch assay,EDU staining and cell cloning assay were used to detect the migration and proliferation of fibroblast-like synoviocytes.Western blot assay was used to detect the protein expression levels of Wnt3a,β-catenin,tumorigenic genes,matrix metalloproteinase 2,matrix metalloproteinase 9,Bax and Bcl-2 in fibroblast-like synoviocytes.RESULTS AND CONCLUSION:(1)Compared with the control group,the cell viability decreased significantly when the concentration of PHSTF was＞40 μg/mL(P＜0.01).Therefore,the drug concentration of≤40 μg/mL was selected for subsequent experiments.(2)Compared with the lipopolysaccharide group,the wound healing rate,cell clone formation rate and the number of EDU-positive cells in the low-,medium-and high-dose PHSTF groups were significantly reduced,while the apoptosis rate was significantly increased(P＜0.05-0.01).(3)Western blot results showed that compared with the lipopolysaccharide group,low-,medium-and high-dose PHSTF significantly inhibited cellular Wnt3a,β-catenin,cellular tumorigenic genes,matrix metalloproteinase 2,matrix metalloproteinase 9,and Bcl-2 protein expression,and promoted the expression of Bax protein(P＜0.01).(4)Compared with the DKK1 group,the combination of DKK1 and high-dose PHSTF significantly inhibited the protein expression of Wnt3a,β-catenin,matrix metalloproteinase 2,matrix metalloproteinase 9 and Bcl-2 protein expression and promoted the protein expression of Bax(P＜0.01).To conclude,PHSTF may inhibit the proliferation and migration of fibroblast-like synoviocytes and promote apoptosis by inhibiting the Wnt/β-catenin signaling pathway.

Objective To develop and validate a fast Monte Carlo(MC)-based patient-specific quality assurance(PSQA)tool using the treatment log files that is suitable to be used in the online adaptive radiotherapy for pencil beam scanning proton therapy(PBSPT-ART).Methods The proposed tool first used the delivery log file of a PBSPT plan to reversely reconstruct the PBSPT(rPBSPT)plan,and then used an in-house developed graphic processing unit(GPU)-accelerated virtual particle MC(VPMC)dose engine to calculate the dose distribution of the rPBSPT plan.The rPBSPT dose calculated by VPMC was then compared to the rPBSPT dose calculated by another independent MC dose engine(MCsquare),using 3D gamma analysis to verify the accuracy of VPMC calculation.As a demonstration of the feasibility of developed log file-based PSQA,the VPMC calculated dose of the rPBSPT plan was compared to the pre-delivery second check dose of the corresponding PBSPT plan calculated by MCsquare,using 3D gamma analysis.3D gamma analysis employes a criterion of 2 mm/2%/10%.Twenty patients with different disease sites were representatively selected to validate the efficiency and accuracy of the tool.Results The average calculation time of a rPBSPT plan by VPMC was(5.88±4.00)s in the accuracy verification.Compared to MCsquare,the passing rate of the 3D gamma analysis was 99.47%±0.72%.In the proposed PSQA tool demonstration,the passing rate of comparing the VPMC calculated rPBSPT dose to MCsquare calculated second check dose of the corresponding PBSPT plan was 98.91%±0.92%.Conclusion The accuracy and efficiency of the tool can meet the requirements of PSQA in the online PBSPT-ART workflow.

AIM:To investigate the mechanism of Qianggu-Kangshu formula(QGKSF)in the treatment of rheumatoid arthritis(RA)bone destruction based on network pharmacology,molecular docking,and cell experiments.METHODS:The main active ingredients and potential targets of QGKSF were obtained through TCMSP database and litera-ture search.OMIM database and GeneCards database were used to search the targets related to RA bone destruction,and Venny 2.1.0 was employed to screen the intersected targets of QGKSF and RA bone destruction.The protein-protein inter-action network of potential intersected targets was constructed by STRING database,and topological analysis was carried out by Cytoscape software to screen core targets.The screened targets of QGKSF related to RA bone destruction were evalu-ated through the Metascape system.Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology were applied to complete enrichment analysis.AutoDock and PyMOL software was used to carry out molecular docking between the core components and the core proteins.Mouse RAW264.7 macrophages were cultured in vitro,and the cell viability was de-tected by CCK-8 assay.The number of tartrate-resistant acid phosphatase(TRAP)positive multinucleated cells in each group was calculated by TRAP staining.The enzyme activity of the cells was evaluated by determination of TRAP activity.F-actin ring formation was detected by phalloidin staining.Western blot analysis was conducted to detect the protein levels of nuclear factor of activated T cells 1(NFATc1),TRAP,cathepsin K(CTSK),c-Fos,matrix metalloproteinase 9(MMP9),osteoprotegerin(OPG),receptor activator of nuclear factor κB(RANK),RANK ligand(RANKL)and phos-phorylated protein kinase B(p-AKT).RESULTS:A total of 136 active ingredients and 126 targets were selected corre-lated with QGKSF,whil 207 intersected targets of QGKSF and RA bone destruction were screened,175 of which were core targets.There were 199 pathways obtained by GO enrichment,and 20 pathways related to osteoclasts were screened out,including phosphatidylinoinosiol 3-kinase/AKT signaling pathway and osteoclast differentiation,etc.The results of TRAP staining,TRAP enzyme activity determination and phalloidin staining showed less positive cell formation,de-creased enzyme activity and decreased F-actin ring formation in QGKSF and methotrexate(MTX)groups compared with model group.Western blot results showed that compared with model group,the protein levels of NFATc1,TRAP,CTSK,c-Fos,MMP9,p-AKT,RANK and RANKL were decreased(P＜0.05),while the expression of OPG protein was in-creased(P＜0.05)in QGKSF and MTX groups.CONCLUSION:Treatment with QGKSF inhibits RANKL-induced dif-ferentiation of RAW264.7 cells into osteoclasts possibly by inhibiting the over-differentiation of osteoclasts via regulating RANKL/RANK/OPG signaling pathway.

Objective::To compare the analgesic effects of ibuprofen administered orally via two modes combined with a conventional, patient-controlled intravenous analgesia pump on maternal pain after cesarean section (CS).Methods::This prospective, randomized, controlled study enrolled females who underwent CS from August 2022 to August 2023 at Peking University First Hospital, Beijing, China. Participants were randomly assigned to either an as-needed ibuprofen group (300 mg orally upon request) or a scheduled ibuprofen group (300 mg every 12 hours for 48 hours). The primary outcomes assessed were postoperative pain levels using the Wong-Baker Faces Pain Scale-Revised and cumulative oxycodone consumption at multiple time points up to 48 hours post-delivery. Secondary outcomes included recovery parameters (time to first flatus, ambulation, and lactation initiation), patient satisfaction with pain control, and postpartum depression scores evaluated by the Edinburgh Postnatal Depression Scale on postoperative day 3. Normally distributed data analyzed with t-tests; non-normal data with Mann-Whitney U tests; categorical variables with chi-square or Fisher’s exact tests (SPSS 26.0, P < 0.05). Results::After excluding 61 non-eligible cases, 339 patients were included (171 as-needed vs. 168 scheduled). The scheduled group showed significantly better pain control at 12 hours (4.00 (2.00-5.50) vs. 4.00 (4.00-6.00), P < 0.001), 24 hours (4.00 (2.00-4.00) vs. 4.00 (2.00-6.00), P < 0.001), and 36 hours (2.00 (2.00-4.00) vs. 4.00 (2.00-4.00), P < 0.001), and 48 hours (2.00 (2.00-4.00) vs. 2.00 (2.00-4.00), P = 0.004) post-delivery and lower levels of oxycodone consumption at 36 hours (10.20 (8.20-13.35) vs. 11.00 (8.80-14.40), P = 0.042) and 48 hours (12.40 (10.40-15.95) vs. 13.80 (11.00-16.00), P = 0.020) postpartum compared with those in the as-needed group. Additionally, the time to the return of bowel movements was shorter in the scheduled group than in the as-needed group (23.50 (16.94, 31.47) vs. 27.00 (19.88, 35.97), P = 0.004). Differences in post-delivery ambulation, lactation initiation, satisfaction levels, and depression scores were not significantly different between the two groups. Conclusion::The results of this study promote the use of ibuprofen (scheduled oral administration) combined with a conventional, patient-controlled intravenous analgesia pump for achieving better post-CS pain control than an as-needed dosage regimen.Registration::Chinese Clinical Trial Registry, ChiCTR2400082474.

AIM:To investigate the potential effect of Dabuyuan Jian(DBYJ)on peroxisome proliferator-acti-vated receptor γ(PPARγ)/nuclear factor-κB(NF-κB)signaling pathway and related inflammatory proteins in mice with mild cognitive impairment(MCI),and to explore the mechanism of DBYJ in improving the learning and memory ability of MCI mice.METHODS:Forty C57BL/6 male mice were randomly divided into 5 groups,including negative control(NC)group,D-galactose(D-Gal)group,D-Gal+DBYJ group,D-Gal+GW9662(PPARγ inhibitor)group and D-Gal+GW9662+DBYJ group,with 8 mice each.The mice in NC group were subcutaneously injected with 0.9%saline solution on the back of the neck for 8 weeks,while those in the remaining 4 groups were subcutaneously injected with D-Gal(100 mg·kg-1·d-1)on the back of the neck for 8 weeks to establish the MCI model.From week 5 to week 8,the mice in D-Gal+GW9662 and D-Gal+DBYJ+GW9662 groups were intraperitoneally injected with GW9662(1 mg·kg-1·d-1).From week 5,the mice in D-Gal+DBYJ and D-Gal+DBYJ+GW9662 groups were treated with DBYJ(13.2 g/kg)by gavage,while those in the re-maining 3 groups were administered an equal volume of purified water for 4 weeks.The Morris water maze(including posi-tioning navigation experiment and space exploration experiment)was used to assess the learning and memory ability of the mice.The structural integrity of the hippocampus of the mice was assessed by hematoxylin-eosin(HE)staining.Nissl staining was used to evaluate damage to hippocampal neurons in mice,and Western blot was applied to detect the protein levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),PPARγ,P65 and phosphorylated P65(p-P65)in the hippocampus of the mice.RESULTS:Compared with NC group,the escape latency of the mice in D-Gal+D-Gal and D-Gal+GW9662 groups significantly increased(P＜0.01),while the number of platform crossing and the duration of staying in the target quadrant within 60 s significantly decreased(P＜0.01).The number of neurons in the CA3 region remarkably decreased(P＜0.01),and pyramidal cell disarrangement and neuronal shrinkage were observed.The levels of TNF-α,IL-1β and p-P65 were up-regulated,while the expression of PPARγ was down-regulated(P＜0.05).Compared with D-Gal group,the escape latency of the mice in D-Gal+DBYJ group significantly decreased(P＜0.01),while the number of plat-form crossing and the duration of staying in the target quadrant within 60 s remarkably increased(P＜0.01).The number of neurons in the CA3 region increased(P＜0.01),the pyramidal cells were more neatly arranged,and the cytoarchitec-ture was intact.The levels of TNF-α,IL-1β and p-P65 were down-regulated,while the expression of PPARγ was up-regu-lated(P＜0.05).Compared with D-Gal+GW9662 group,significantly decreased escape latency was observed in D-Gal+DBYJ+GW9662 group(P＜0.01),and the number of platform crossing and the duration of staying in the target quadrant within 60 s remarkably increased(P＜0.05).The number of neurons in the CA3 region increased(P＜0.01),the pyrami-dal cells were arranged more neatly,and the cytoarchitecture was relatively intact.The levels of TNF-α,IL-1β and p-P65 were down-regulated,while the expression of PPARγ was up-regulated(P＜0.05).The effects shown in D-Gal+DBYJ+GW9662 group were inferior to those in D-Gal+DBYJ group,indicating that the therapeutic effect of DBYJ was inhibited after the addition of GW9662.CONCLUSION:Dabuyuan Jian improves the learning and memory ability of MCI mice,and the mechanism may be related to the expression of key proteins in the PPARγ/NF-κB signaling pathway.

Objective::To compare the analgesic effects of ibuprofen administered orally via two modes combined with a conventional, patient-controlled intravenous analgesia pump on maternal pain after cesarean section (CS).Methods::This prospective, randomized, controlled study enrolled females who underwent CS from August 2022 to August 2023 at Peking University First Hospital, Beijing, China. Participants were randomly assigned to either an as-needed ibuprofen group (300 mg orally upon request) or a scheduled ibuprofen group (300 mg every 12 hours for 48 hours). The primary outcomes assessed were postoperative pain levels using the Wong-Baker Faces Pain Scale-Revised and cumulative oxycodone consumption at multiple time points up to 48 hours post-delivery. Secondary outcomes included recovery parameters (time to first flatus, ambulation, and lactation initiation), patient satisfaction with pain control, and postpartum depression scores evaluated by the Edinburgh Postnatal Depression Scale on postoperative day 3. Normally distributed data analyzed with t-tests; non-normal data with Mann-Whitney U tests; categorical variables with chi-square or Fisher’s exact tests (SPSS 26.0, P < 0.05). Results::After excluding 61 non-eligible cases, 339 patients were included (171 as-needed vs. 168 scheduled). The scheduled group showed significantly better pain control at 12 hours (4.00 (2.00-5.50) vs. 4.00 (4.00-6.00), P < 0.001), 24 hours (4.00 (2.00-4.00) vs. 4.00 (2.00-6.00), P < 0.001), and 36 hours (2.00 (2.00-4.00) vs. 4.00 (2.00-4.00), P < 0.001), and 48 hours (2.00 (2.00-4.00) vs. 2.00 (2.00-4.00), P = 0.004) post-delivery and lower levels of oxycodone consumption at 36 hours (10.20 (8.20-13.35) vs. 11.00 (8.80-14.40), P = 0.042) and 48 hours (12.40 (10.40-15.95) vs. 13.80 (11.00-16.00), P = 0.020) postpartum compared with those in the as-needed group. Additionally, the time to the return of bowel movements was shorter in the scheduled group than in the as-needed group (23.50 (16.94, 31.47) vs. 27.00 (19.88, 35.97), P = 0.004). Differences in post-delivery ambulation, lactation initiation, satisfaction levels, and depression scores were not significantly different between the two groups. Conclusion::The results of this study promote the use of ibuprofen (scheduled oral administration) combined with a conventional, patient-controlled intravenous analgesia pump for achieving better post-CS pain control than an as-needed dosage regimen.Registration::Chinese Clinical Trial Registry, ChiCTR2400082474.

Objective:To investigate the clinical manifestation, therapy, and prognosis of Susac syndrome and enhance the understanding of this disease.Methods:A case summary was made.The clinical data of two siblings with Susac syndrome treated at Children′s Medical Center, Peking University First Hospital in January 2024 were summarized.Reported cases of pediatric Susac syndrome were reviewed.Results:The onset of the disease in the two siblings was at the age of 3.00 and 6.75 years, with recurrent headaches, tinnitus, hearing loss and encephalopathy symptoms.Cranial magnetic resonance imaging showed multiple cerebral microbleeding and microinfarction lesions, " snowball like" in the corpus callosum and diffuse white matter edema in the brain.Audiometry revealed sensorineural hearing loss.In one case, ophthalmic fluorescein angiography revealed ischemic changes due to branch retinal artery occlusions.No pathogenic variants were detected in gene testing.This child was diagnosed with Susac syndrome, and the symptoms were improved after treatment with Corticosteroids and Rituximab.No relapse was observed during the 9-month follow-up.A total of 20 pediatric cases of Susac syndrome were retrieved, including 18 reported previously and 2 cases from this study.There were 2 boys and 18 girls, with the age of onset ranging from 2.5 to 17.0 years.The common initial symptoms included headache (19 cases), vertigo and tinnitus or hearing loss (9 cases), and vision impairment or visual field defect (4 cases). The symptoms were improved after immunotherapy.Conclusions:With a low incidence, Susac syndrome is rare in children and difficult to diagnose.There may be a genetic predisposition in such disease.Early diagnosis and immunotherapy can low the relapse and improve the prognosis.

[Objective] To compare next generation sequencing (NGS) library construction technology between probe hybridization capture and amplicon methods, and analyze the influencing factors of HLA genotyping resolution level and its prospects in clinical applications. [Methods] A total of 207 clinical samples with known typing results and samples from the proficiency testing plan were selected. The conformity rate of HLA genotyping results, allele coverage and typing data analysis indicators were confirmed, and the effects of two library construction methods on the level of HLA genotyping discrimination were compared. [Results] The concordance rate of 207 samples with the feedback results of PT or prior well-characterized HLA genotypes was 100%. Among them, 91 samples were captured using hybridization probe capture method. Compared with the original amplicon method, the hybridization probe capture method can distinguish the alleles of DRB1 and DPB1 that cannot be determined in 13 samples. The allelic imbalance of DRB1, DPA1, and DQB1 loci in 6 samples was resolved. Three samples were found to have missed detection of alleles at the DQA1 and DQB1 loci. [Conclusion] The performance indicators of hybridization probe capture and amplicon performance confirmation meet the requirements of clinical detection of HLA genotyping, which provides an experimental method and basis for clinical application.

Objective:To investigate the clinical manifestation, therapy, and prognosis of Susac syndrome and enhance the understanding of this disease.Methods:A case summary was made.The clinical data of two siblings with Susac syndrome treated at Children′s Medical Center, Peking University First Hospital in January 2024 were summarized.Reported cases of pediatric Susac syndrome were reviewed.Results:The onset of the disease in the two siblings was at the age of 3.00 and 6.75 years, with recurrent headaches, tinnitus, hearing loss and encephalopathy symptoms.Cranial magnetic resonance imaging showed multiple cerebral microbleeding and microinfarction lesions, " snowball like" in the corpus callosum and diffuse white matter edema in the brain.Audiometry revealed sensorineural hearing loss.In one case, ophthalmic fluorescein angiography revealed ischemic changes due to branch retinal artery occlusions.No pathogenic variants were detected in gene testing.This child was diagnosed with Susac syndrome, and the symptoms were improved after treatment with Corticosteroids and Rituximab.No relapse was observed during the 9-month follow-up.A total of 20 pediatric cases of Susac syndrome were retrieved, including 18 reported previously and 2 cases from this study.There were 2 boys and 18 girls, with the age of onset ranging from 2.5 to 17.0 years.The common initial symptoms included headache (19 cases), vertigo and tinnitus or hearing loss (9 cases), and vision impairment or visual field defect (4 cases). The symptoms were improved after immunotherapy.Conclusions:With a low incidence, Susac syndrome is rare in children and difficult to diagnose.There may be a genetic predisposition in such disease.Early diagnosis and immunotherapy can low the relapse and improve the prognosis.