AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.

Objective: To analyze the consumption frequency of major foods among Chinese children aged 6-17 years old, and to provide a basis for optimizing the dietary structure of children in China. Methods: Using data from the China Nutrition and Health System Survey and Application Program for Children 0-18 years old, 56 734 children aged 6-17 years old from North, Norththeast East, Central, South, Southwest and Northwest seven regions in China were selected for the study using stratified cluster random sampling from 2019 to 2021. A food frequency questionnaire was used to investigate the intake frequency of eight food groups in a month, including fresh vegetables, fresh fruits, livestock and poultry meats, aquatic products, eggs, dairy products, legumes, and cereals and potatoes. The foods were grouped according to whether they met the recommended intake criteria outlined in the Dietary Guidelines for Chinese Residents 2022. The〖KG*2〗χ2 test was used to compare the differences in the proportion of childrens intake frequency of each food group meeting the standard in different regions and age groups. Results: The proportions of Chinese children aged 6-17 years who consumed fresh vegetables and cereals and potatoes ≥3 times/d were 12.1% and 67.2%, respectively. The proportions of children who consumed fresh fruits, livestock and poultry meats, eggs and dairy products ≥1 time/d were 50.8%, 58.8%, 36.0% and 54.3%, respectively. The proportion of legumes consumed ≥4 times/week was 37.4%, and the proportion of aquatic products consumed ≥2 times/week was 39.7%. Fresh vegetables (5.5%), fresh fruits (33.1%), and dairy products (36.4%) had the lowest frequency of meeting the recommended standards in South China, and aquatic products (27.4%) and eggs (21.1%) had the lowest frequency of meeting the recommended standards in Northwest (P<0.008 3). Conclusion The overall intake frequency of fresh vegetables, fresh fruits, legumes, and dairy products are insufficient among Chinese children, with significant regional variations.

OBJECTIVES: To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O₂) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. RESULTS: In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. CONCLUSIONS Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.
Humans ; Reactive Oxygen Species/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Human Umbilical Vein Endothelial Cells/drug effects* ; Membrane Proteins/metabolism* ; Proto-Oncogene Proteins/metabolism* ; Up-Regulation ; Cell Hypoxia ; Cells, Cultured ; Snake Venoms/chemistry* ; Beclin-1

BACKGROUND: Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown. METHODS: The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining. RESULTS: sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway. CONCLUSIONS sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals ; Mice ; Vascular Calcification/metabolism* ; Mice, Inbred C57BL ; Epoxide Hydrolases/metabolism* ; Diabetes Mellitus, Experimental/genetics* ; Male ; Gene Deletion ; MAP Kinase Signaling System/genetics* ; Cell Line ; Immunohistochemistry ; Muscle, Smooth, Vascular/metabolism* ; Signal Transduction/genetics* ; Mice, Knockout

Homo- or heterodimeric compounds that affect dimeric protein function through interaction between monomeric moieties and protein subunits can serve as valuable sources of potent and selective drug candidates. Here, we screened an in-house dimeric natural product collection, and panepocyclinol A (PecA) emerged as a selective and potent STAT3 inhibitor with profound anti-tumor efficacy. Through cross-linking C712/C718 residues in separate STAT3 monomers with two distinct Michael receptors, PecA inhibits STAT3 DNA binding affinity and transcription activity. Molecular dynamics simulation reveals the key conformation changes of STAT3 dimers upon the di-covalent binding with PecA that abolishes its DNA interactions. Furthermore, PecA exhibits high efficacy against anaplastic large T cell lymphoma in vitro and in vivo, especially those with constitutively activated STAT3 or STAT3^Y640F. In summary, our study describes a distinct and effective di-covalent modification for the dimeric compound PecA to disrupt STAT3 function.

Objective To explore the correlation between remnant cholesterol level and CI-AKI in elderly patients with ACS and concomitant T2DM following PCI.Methods A retrospective case-control study was conducted on 759 elderly T2DM patients with ACS undergoing PCI in the Affi-liated Hospital of Xuzhou Medical University between January 2020 and December 2023.The pa-tients were randomly allocated into a training group(n=608)and a validation group(n=151)in a ratio of 8∶2.Based on CI-AKI diagnostic criteria,the training group was further stratified into a CI-AKI subgroup(n=95)and a non-CI-AKI subgroup(n=513).Multivariate logistic regression analysis was used to identify the independent risk factors for CI-AKI.Results The remnant cho-lesterol level was significantly higher in the CI-AKI subgroup than the non-CI-AKI subgroup[0.78(0.51,1.07)mmol/L vs 0.57(0.40,0.81)mmol/L,P＜0.01].Red blood cell count,remnant cholesterol,age,uric acid,log-transformed systemic immune-inflammatory(SII)index,and cysta-tin C were identified as independent predictors for CI-AKI in T2DM patients with ACS after PCI(P＜0.01).ROC curve analysis demonstrated that the model combining remnant cholesterol with other independent predictors exhibited superior discriminative performance in both the training group(AUC=0.818,95％CI:0.773-0.863)and validation group(AUC=0.787,95％CI:0.686-0.889)when compared to the models excluding remnant cholesterol(P＜0.01).Conclusion Rem-nant cholesterol is an independent predictor of CI-AKI in elderly patients with ACS and T2DM af-ter PCI.A risk prediction model based on remnant cholesterol and other independent risk factors demonstrates enhanced predictive performance for postoperative CI-AKI in the patients.

Objective:To explore the potential role of lipoprotein-associated phospholipase A2 (Lp-PLA2) and components of metabolic syndrome (MS) in the early progression of carotid arteriosclerosis.Methods:The study was a cross-sectional study. Urban participants undergoing routine health check-ups were enrolled from all 11 prefecture-level cities in Zhejiang Province between January and December 2022. General clinical information was obtained through interviews, and data on MS was collected from the clinical health examinations. Serum Lp-PLA? levels were measured in all participants. All participants were divided into 3 groups according to the results of the carotid ultrasound: the normal group, the intima thickening group with carotid intima thickening change and the plaque group. Multivariable logistic regression models were used to evaluate the associations of Lp-PLA2, MS, and the components of MS with early carotid atherosclerosis. Receiver operating characteristic (ROC) curve analyses were performed to assess the diagnostic performance of combining Lp-PLA2 with MS and the cumulative number of MS components for early carotid atherosclerosis.Results:A total of 4 009 urban adults undergoing routine health check-ups were enrolled (mean age (48.9±8.46) years, 2 665(66.5 %)male). Of these, 1 398 were in the normal group, 1 650 in the intima thickening group, and 961 in the plaque group. Multivariable logistic regression demonstrated that Lp-PLA2 was independently associated with early carotid atherosclerosis ( OR=1.34, 95% CI: 1.11-1.63, P=0.003). Lp-PLA2 also showed independent positive associations with both carotid intima thickening and carotid plaque formation, with the latter being more pronounced (both P<0.05). MS was independently and positively associated with early carotid atherosclerosis ( OR=1.48, 95 % CI: 1.20-1.84, P<0.001), as well as with intima thickening and carotid plaque formation, with the association being stronger for the latter (both P<0.05). Furthermore, the strength of the association increased progressively with the number of MS components ( P<0.001), especially for carotid plaques formation (both P<0.001). Multivariable logistic regression revealed that, compared with individuals without MS and low Lp-PLA2 levels, the risk of early carotid atherosclerosis was increased in those with high Lp-PLA2 alone, MS alone, or both conditions concurrently, with the highest risk observed when both were present (all P<0.05). ROC analyses demonstrated that the combination of elevated Lp-PLA2 with 3, 4, or 5 MS components yielded good diagnostic performance for early carotid atherosclerosis ( AUC=0.869, 0.888, and 0.889, respectively), intima thickening ( AUC=0.844, 0.860, and 0.845, respectively), and carotid plaque formation ( AUC=0.899, 0.924, and 0.968, respectively) in urban health-screening participants. Conclusions:Lp-PLA2, MS, and the number of MS components were independently and positively associated with early carotid atherosclerosis in urban health chek-up populations. The combination of MS components and Lp-PLA2 provided favorable diagnostic performance for the detection of early carotid atherosclerosis.

Objective: To understand the prevalence of elevated blood pressure and its association with dietary patterns in children and adolescents in China, providing evidence for developing dietary intervention of hypertension in children and adolescents. Methods: Data were derived from the China Children s Nutrition and Health System Survey and Application Project(2019-2021). A stratified cluster random sampling method was used to include 7 933 participants from 28 survey sites in seven major regions of Northeast, North, Northwest, East, Central, South and Southwest China. Multivariate Logistic regression models were used to analyze associations between demographic characteristics, nutritional status and elevated blood pressure. Exploratory factor analysis identified dietary patterns, which were divided into three quartile groups (T3, T2, T1) based on factor scores (compliance for dietary pattern) from high to low, and multivariate Logistic regression model assessed the correlation between elevated blood pressure and dietary patterns. Results: The prevalence of elevated blood pressure was 15.4% among Chinese children aged 7-17 years. Significant differences were observed across nutritional status (reference: underweight; normal weight: OR =1.57; overweight: OR = 2.61 ; obesity: OR =3.85), urban/rural residence (reference: rural; urban: OR =0.86), and paternal education (reference: junior high school and below; bachelor degree or above: OR =0.68) ( P <0.05). The detection rates of high blood pressure in T3 group children and adolescents with four dietary patterns (staple food, animal based food, snacks, vegetables and fruits) were 15.7%, 14.6%, 16.8%, and 15.8%, respectively. After adjusting for residence, paternal education, and nutritional status, the "snack dietary pattern" (mainly candy, sugar sweetened beverages, and processed snacks) showed positive associations with elevated blood pressure in T2 ( OR =1.21) and T3 ( OR =1.19) tertiles ( P <0.05). Conclusions The snack dietary pattern is a related factor for elevated blood pressure in children and adolescents. Restricting unhealthy snack intake may promote cardiovascular health.

OBJECTIVE To establish the fingerprint of Bushen ningshen ointment， determine the contents of its major constituents， and investigate its in vitro antioxidant activity. METHODS High performance liquid chromatography （HPLC） fingerprints of 10 batches of Bushen ningshen ointment were established. Similarity evaluation and identification of common peaks were subsequently performed. The contents of 10 components such as salidroside were determined using the same HPLC method. Using the scavenging rates against 2，2′-azino bis（3-ethylbenzothiazoline-6-sulfonic acid） （ABTS） and 1，1-diphenyl-2- picrylhydrazyl （DPPH） radicals， as well as ferric ion reducing antioxidant power （FRAP） as indicators， the anti-oxidant activity of the ointment was evaluated； grey relational analysis and partial least squares regression were conducted using SIMCA 14.1 software to establish the spectrum-effect relationship. RESULTS The fingerprint chromatogram of 10 batches of Bushen ningshen ointment contained 24 common peaks， with similarity values all exceeding 0.96. Eleven peaks were identified as adenosine （peak 1）， salidroside （peak 4）， morroniside （peak 6）， catechin （peak 7）， paeoniflorin （peak 10）， spinosin （peak 11）， ferulic acid （peak 12）， isoquercitrin （peak 13）， E-mail：wli1743@163.com verbascoside （peak 14）， paeonol （peak 23）， and emodin （peak 24）. Content determination results showed that the average contents of salidroside， morroniside， catechin， paeoniflorin， spinosin， ferulic acid， isoquercitrin， verbascoside， paeonol， and emodin were 0.725， 1.962， 0.214， 3.395， 0.124， 0.107， 0.286， 0.019， 0.034 and 0.067 mg/g， respectively. The antioxidant potency composite index （APC） for the 10 batches ranged from 85.08% to 96.35%. Spectrum-effect relationship analysis indicated that all 24 common peaks were positively correlated with the antioxidant capacity. Seventeen peaks had variable importance in projection values ＞1， specitically peaks 2， 5， 6， 7， 9， 10， 13- 21， 23， and 24. CONCLUSIONS This study successfully established the HPLC fingerprint and content determination method for Bushen ningshen ointment. The compounds represented by the 17 common peaks such as morroniside may be the active components contributing to its antioxidant effects.

Objective : To prepare silk protein(SF) guided bone regeneration(GBR) membranes reinforced by aragonite sheets(AP) and to investigate their potential as novel barrier membranes for bone tissue regeneration by testing their tensile properties,biocompatibility,and their effect on osteogenic differentiation. Methods : AP was extracted from natural abalone shell(AS) by oxidation method,and AP-SF composite and pure SF membranes with different component ratios were prepared by solution casting and evaporative self-assembly techniques,and their tensile properties were tested using a universal mechanical machine under wet state conditions.The tensile properties were tested using a universal mechanical machine under wet conditions.The microstructure of the AP-SF membrane with the highest tensile strength was observed using scanning electron microscopy(SEM) and characterized using Fourier transform infrared spectroscopy(FTIR) and X-ray diffraction(XRD).Observation of the adhesion of rat bone marrow mesenchymal stem cells(rBMSCs) on the surface by scanning electron microscopy,and exploration of the biocompatibility of AP-SF membrane by CCK-8 and live cell staining.Alkaline phosphatase staining(ALP) and alizarin red staining(ARS) were used to detect the osteogenic differentiation of rBMSCs in each group. Results: AP was successfully extracted from AS,and the membrane prepared from a mixed solution of SF and AP with a solid content of 10:9 had the strongest wet tensile performance,reaching 8.46 MPa.The CCK-8 and live dead cell staining test results of the AP-SF membrane showed no significant difference compared to the blank group and SF group,indicating that both membranes had good biocompatibility.The ALP test and ARS results indicated that AP-SF membrane had a more significant ability to promote osteogenic differentiation of rBMSCs compared to SF membrane. Conclusion The prepared AP-SF membrane has both good mechanical and biological properties,and has certain clinical application potential.