BACKGROUND:Bone tissue loss caused by tumors and trauma can have an adverse effect on postoperative rehabilitation.Therefore,scaffold materials are usually implanted during treatment.However,the existing implant materials are relatively simple and lack antibacterial properties.Early implantation may lead to iatrogenic autoinfection and have an adverse effect on osteogenesis.OBJECTIVE:To construct a KR-12-a5 polypeptide-nanohydroxyapatite-small intestinal submucosa composite scaffold and evaluate its feasibility as a material for promoting bone defect repair.METHODS:The small intestinal submucosa scaffold and the small intestinal submucosa scaffold containing 25,50,and 100 mg/mL nanohydroxyapatite(referred to as nHA-SIS scaffold)were prepared by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide cross-linking method.The appropriate scaffold was screened for subsequent experiments by mechanical property testing.The antibacterial properties of KR-12-a5 polypeptide solution against Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum were detected.The nHA-SIS scaffolds were immersed in 250,500,and 1 000 μg/mL KR-12-a5 peptide solutions for 24 hours,and then freeze-dried to obtain peptide-loaded nanohydroxyapatite-porcine small intestinal submucosa composite scaffolds(denoted as P-nHA-SIS scaffolds).The sustained-release properties of the three groups of scaffolds were characterized.The nHA-SIS scaffolds and the three groups of P-nHA-SIS scaffolds were co-cultured with Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum for 24 hours or 48 hours.The scaffolds with strong antibacterial ability were screened by live and dead bacteria staining and scanning electron microscopy for subsequent experiments.The degradation properties and water absorption rates of the uncross-linked small intestinal submucosa scaffolds,cross-linked small intestinal submucosa scaffolds,nHA-SIS scaffolds,and P-nHA-SIS scaffolds were characterized.The extracts of cross-linked small intestinal submucosal scaffolds,nHA-SIS scaffolds,and P-nHA-SIS scaffolds were co-cultured with MC3T3-E1 cells.CCK-8 assay and live-dead cell staining were performed.The effects of the extracts of the three scaffolds on the migration of MC3T3-E1 cells were detected by Transwell chamber assay.RESULTS AND CONCLUSION:(1)The elastic modulus and compressive strength of 25,50,and 100 mg/mL nHA-SIS scaffolds were higher than those of small intestinal submucosal scaffolds(P＜0.05),among which the elastic modulus and compressive strength of 25 mg/mL nHA-SIS scaffolds were the highest,and this group of scaffolds were selected for subsequent experiments to load peptides.(2)KR-12-a5 peptide had strong antibacterial activity against common bacteria in bone defects(Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum).The three groups of P-nHA-SIS scaffolds all had sustained release properties.With the increase of peptide mass concentration,the antibacterial property of P-nHA-SIS scaffold was enhanced.Among them,the P-nHA-SIS scaffold loaded with 500 μg/mL peptide had achieved a satisfactory antibacterial effect,and this group of scaffolds would be selected in the future.(3)The degradation rate of the three groups of cross-linked scaffolds was lower than that of the uncross-linked scaffolds,and the water absorption rate was greater than that of the uncross-linked scaffolds.P-nHA-SIS scaffolds could promote the proliferation and migration of MC3T3-E1 cells without affecting the activity of MC3T3-E1 cells.(4)The results show that P-nHA-SIS scaffolds have strong antibacterial properties and the ability to promote the proliferation and migration of MC3T3-E1 cells,and are expected to be used in bone defect repair.

BACKGROUND:Bone tissue loss caused by tumors and trauma can have an adverse effect on postoperative rehabilitation.Therefore,scaffold materials are usually implanted during treatment.However,the existing implant materials are relatively simple and lack antibacterial properties.Early implantation may lead to iatrogenic autoinfection and have an adverse effect on osteogenesis.OBJECTIVE:To construct a KR-12-a5 polypeptide-nanohydroxyapatite-small intestinal submucosa composite scaffold and evaluate its feasibility as a material for promoting bone defect repair.METHODS:The small intestinal submucosa scaffold and the small intestinal submucosa scaffold containing 25,50,and 100 mg/mL nanohydroxyapatite(referred to as nHA-SIS scaffold)were prepared by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide cross-linking method.The appropriate scaffold was screened for subsequent experiments by mechanical property testing.The antibacterial properties of KR-12-a5 polypeptide solution against Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum were detected.The nHA-SIS scaffolds were immersed in 250,500,and 1 000 μg/mL KR-12-a5 peptide solutions for 24 hours,and then freeze-dried to obtain peptide-loaded nanohydroxyapatite-porcine small intestinal submucosa composite scaffolds(denoted as P-nHA-SIS scaffolds).The sustained-release properties of the three groups of scaffolds were characterized.The nHA-SIS scaffolds and the three groups of P-nHA-SIS scaffolds were co-cultured with Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum for 24 hours or 48 hours.The scaffolds with strong antibacterial ability were screened by live and dead bacteria staining and scanning electron microscopy for subsequent experiments.The degradation properties and water absorption rates of the uncross-linked small intestinal submucosa scaffolds,cross-linked small intestinal submucosa scaffolds,nHA-SIS scaffolds,and P-nHA-SIS scaffolds were characterized.The extracts of cross-linked small intestinal submucosal scaffolds,nHA-SIS scaffolds,and P-nHA-SIS scaffolds were co-cultured with MC3T3-E1 cells.CCK-8 assay and live-dead cell staining were performed.The effects of the extracts of the three scaffolds on the migration of MC3T3-E1 cells were detected by Transwell chamber assay.RESULTS AND CONCLUSION:(1)The elastic modulus and compressive strength of 25,50,and 100 mg/mL nHA-SIS scaffolds were higher than those of small intestinal submucosal scaffolds(P＜0.05),among which the elastic modulus and compressive strength of 25 mg/mL nHA-SIS scaffolds were the highest,and this group of scaffolds were selected for subsequent experiments to load peptides.(2)KR-12-a5 peptide had strong antibacterial activity against common bacteria in bone defects(Staphylococcus aureus,Streptococcus gordonii,and Fusobacterium nucleatum).The three groups of P-nHA-SIS scaffolds all had sustained release properties.With the increase of peptide mass concentration,the antibacterial property of P-nHA-SIS scaffold was enhanced.Among them,the P-nHA-SIS scaffold loaded with 500 μg/mL peptide had achieved a satisfactory antibacterial effect,and this group of scaffolds would be selected in the future.(3)The degradation rate of the three groups of cross-linked scaffolds was lower than that of the uncross-linked scaffolds,and the water absorption rate was greater than that of the uncross-linked scaffolds.P-nHA-SIS scaffolds could promote the proliferation and migration of MC3T3-E1 cells without affecting the activity of MC3T3-E1 cells.(4)The results show that P-nHA-SIS scaffolds have strong antibacterial properties and the ability to promote the proliferation and migration of MC3T3-E1 cells,and are expected to be used in bone defect repair.

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.

AIM:To elucidate the mechanism by which Polygonatum sibiricum polysaccharides(PSP)miti-gate high-sugar diet-induced neuroinflammation through the gut microbiota-serum metabolome axis.METHODS:Fifty male ICR mice were divided into 5 groups:control group,model group,low-dose(250 mg/kg)PSP group,high-dose(500 mg/kg)PSP group,and donepezil hydrochloride(3 mg/kg)group.The neuroinflammation model was established through administration of high-sugar chow and 10%sucrose water for 12 weeks.Cognitive function assessment was per-formed utilizing the Morris water maze.Hippocampal histopathology was examined by hematoxylin-eosin(HE)staining,activated microglia were assessed via immunofluorescence,and neuronal apoptosis was evaluated by TUNEL assay.Serum and hippocampal levels of interleukin-6(IL-6),IL-1β,tumor necrosis factor-α(TNF-α)and nitric oxide(NO)were quantified using ELISA and Western blot.Gut microbiota diversity and serum untargeted metabolomics analyses were car-ried out employing 16S rRNA sequencing and LC-MS/GC-MS,respectively.Differential metabolites were screened,and key metabolic pathways were enriched using MetaboAnalyst 6.0.Spearman correlation analysis established relationships between gut microbiota,metabolites,and inflammatory factors.RESULTS:Model mice demonstrated increased escape latency(P<0.05 or P<0.01)and decreased platform crossings(P<0.01)compared with controls,which were reversed by PSP treatment(P<0.05 or P<0.01).Treatment with PSP substantially reduced IL-6,IL-1β,TNF-α and NO levels in se-rum and hippocampus(P<0.05 or P<0.01),diminished inflammatory infiltration,inhibited microglial activation,and re-duced neuronal damage.Gut microbiota analysis demonstrated that PSP increased Lactobacillus and Bacteroides abun-dance while reducing Alistipes(P<0.05).Metabolomics identified 15 differential metabolites(including betaine,kyotor-phin and ε-caprolactam)and highlighted the significance of alanine,aspartate and glutamate metabolism pathways.Spear-man analysis revealed that abundance of Alistipes and Bacteroides were positively correlated with IL-1β(P<0.05),while abundance of Lactobacillus was negatively correlated with inflammatory factors(P<0.05 or P<0.01).Key metabolites(be-taine,kyotorphin,ε-caprolactam,trans-cinnamate,cis-zeatin and galactitol)showed strong associations with inflamma-tion factors(P<0.05 or P<0.01).CONCLUSION:Treatment with PSP attenuates neuroinflammation through modula-tion of gut microbiota(Lactobacillus,Bacteroides and Alistipes),regulation of metabolites(betaine,kyotorphin and so on),and targeting amino acid metabolism pathways.

AIM:To elucidate the mechanism by which Polygonatum sibiricum polysaccharides(PSP)miti-gate high-sugar diet-induced neuroinflammation through the gut microbiota-serum metabolome axis.METHODS:Fifty male ICR mice were divided into 5 groups:control group,model group,low-dose(250 mg/kg)PSP group,high-dose(500 mg/kg)PSP group,and donepezil hydrochloride(3 mg/kg)group.The neuroinflammation model was established through administration of high-sugar chow and 10%sucrose water for 12 weeks.Cognitive function assessment was per-formed utilizing the Morris water maze.Hippocampal histopathology was examined by hematoxylin-eosin(HE)staining,activated microglia were assessed via immunofluorescence,and neuronal apoptosis was evaluated by TUNEL assay.Serum and hippocampal levels of interleukin-6(IL-6),IL-1β,tumor necrosis factor-α(TNF-α)and nitric oxide(NO)were quantified using ELISA and Western blot.Gut microbiota diversity and serum untargeted metabolomics analyses were car-ried out employing 16S rRNA sequencing and LC-MS/GC-MS,respectively.Differential metabolites were screened,and key metabolic pathways were enriched using MetaboAnalyst 6.0.Spearman correlation analysis established relationships between gut microbiota,metabolites,and inflammatory factors.RESULTS:Model mice demonstrated increased escape latency(P<0.05 or P<0.01)and decreased platform crossings(P<0.01)compared with controls,which were reversed by PSP treatment(P<0.05 or P<0.01).Treatment with PSP substantially reduced IL-6,IL-1β,TNF-α and NO levels in se-rum and hippocampus(P<0.05 or P<0.01),diminished inflammatory infiltration,inhibited microglial activation,and re-duced neuronal damage.Gut microbiota analysis demonstrated that PSP increased Lactobacillus and Bacteroides abun-dance while reducing Alistipes(P<0.05).Metabolomics identified 15 differential metabolites(including betaine,kyotor-phin and ε-caprolactam)and highlighted the significance of alanine,aspartate and glutamate metabolism pathways.Spear-man analysis revealed that abundance of Alistipes and Bacteroides were positively correlated with IL-1β(P<0.05),while abundance of Lactobacillus was negatively correlated with inflammatory factors(P<0.05 or P<0.01).Key metabolites(be-taine,kyotorphin,ε-caprolactam,trans-cinnamate,cis-zeatin and galactitol)showed strong associations with inflamma-tion factors(P<0.05 or P<0.01).CONCLUSION:Treatment with PSP attenuates neuroinflammation through modula-tion of gut microbiota(Lactobacillus,Bacteroides and Alistipes),regulation of metabolites(betaine,kyotorphin and so on),and targeting amino acid metabolism pathways.

Objective: To investigate the situation of road traffic injuries (RTIs) among middle school students in Shaoxing City, Zhejiang Province, so as to provide the basis for implementation of interventions against RTIs among students. Methods: From 2021 to 2023, a multi-stage stratified cluster sampling method was used to select 82 junior high school classes and 89 senior high school classes in Shaoxing City as the survey population. Data on basic information, commuting travel, road safety behaviors and road safety knowledge awareness were collected through questionnaires, and the prevalence of RTIs in the past year was analyzed. Results: A total of 6 287 middle school students were surveyed, and 971 cases of RTIs were reported, with a reporting rate of 15.44%. The reporting rate of RTIs was higher in males than in females (17.68% vs. 13.34%, P<0.05). The reporting rate of RTIs was higher in high school students than in junior high school students (17.70% vs. 12.66%, P<0.05). The students who mainly walked to school (18.00%), walked 5 days a week (17.82%) and traveled with classmates (17.58%) had higher reporting rates of RTIs. Among those who walked for ≥20 minutes, the reporting rate of RTIs was higher in males than in females (P<0.05). Among different road safety behaviors, the reporting rate of RTIs was higher in males than in females who used electronic devices (P<0.05). The reporting rates of RTIs were relatively high among students who played for ≥10 minutes on the way (32.92%), crossed traffic lights directly when being late for school (41.54%) and crossed traffic barriers directly (30.67%). The reporting rate of RTIs among middle school students decreased with the increase of road safety knowledge scores (P<0.05). Conclusions Male students, high school students, students with road risky behaviors and with low awareness of road safety knowledge have higher reporting rates of RTIs. It is necessary to strengthen road safety knowledge education for students.

ObjectiveTo observe the intervention effect of Huaiqihuang granules （HQH） on immunoglobulin A vasculitis nephritis （IgAVN） mice and explore the underlying therapeutic mechanism. MethodFifty SPF-grade male Kunming mice were randomly divided into a normal group， an IgAVN model group， a dexamethasone group （2.5 mg·kg^-1·d^-1）， a low-dose HQH group （4 g·kg^-1·d^-1）， and a high-dose HQH group （8 g·kg^-1·d^-1）. The mouse model was established using oral administration of gliadin combined with intravenous injection of India ink. After successful modeling， the mice were euthanized after 4 weeks of gastric gavage according to groups. The 24 h urinary total protein （24 h UTP）， urine β₂-microglobulin （β₂-MG）， serum total protein， albumin， IgA， etc. were detected in each group. Flow cytometry was used to determine the proportion of T helper 17 （Th17） cells in spleen cell suspension. Western blot was employed to detect the expression of adenosine 5'-monophosphate-activated protein kinase α （AMPKα）， phosphorylated AMPKα （p-AMPKα）， acetyl-CoA carboxylase 1 （ACC1）， and phosphorylated ACC1 （p-ACC1） in Th17 cells. Pathological changes in the spleen and kidneys were observed. ResultCompared with the normal group， the IgAVN model group showed significant increases in 24 h UTP， urine β₂-MG， total cholesterol （P<0.05）， serum interleukin-17 （IL-17）， IgA， Th17 proportion in the spleen cell suspension， and IL-17 expression in the spleen tissue （P<0.01）， and significantly decreased serum total protein， albumin， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.01）. Compared with the IgAVN model group， in the 4th week， the 24 h UTP， urine β₂-MG， serum IL-17， IgA levels， and renal IgA deposition were significantly reduced in each treatment group （P<0.01）， and the Th17 proportion and IL-17 expression in spleen tissue were significantly decreased （P<0.05， P<0.01）. Serum albumin levels significantly increased （P<0.05）. Compared with the IgAVN model group， the dexamethasone group and the high-dose HQH group showed increases in serum total protein （P<0.01）， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.05， P<0.01）. The high-dose HQH group showed a significant decrease in total cholesterol level （P<0.05）. Various treatment groups showed different degrees of improvement in spleen and kidney pathological changes. ConclusionHQH may affect Th17 cell differentiation by regulating the AMPK/ACC pathway， correcting immune inflammatory disorders， and exerting therapeutic effects on IgAVN.

Background The prevalence of malnutrition in older adults is high. Early use of appropriate screening scales for malnutrition risk and early intervention can effectively improve life quality of the elderly in communities. Objective To evaluate the risk of malnutrition among the community-dwelling elderly in a district of Shanghai and explore its influencing factors. Methods From October to December 2021, a total of 960 seniors aged 65 years and above in community committees (villages) of Minhang District were selected by stratified random sampling. Trained investigators conducted one-to-one interviews with included seniors using questionnaires. The questionnaires included the Geriatric Depression Scale (GDS), the Activity of Daily Living Scale (ADL), and the malnutrition risk assessment for elderly adults. Height, weight, waist circumference, and calf circumference were measured. Chi-square test and logistic regression were used to analyze potential influencing factors of malnutrition in the elderly. Results Among the 960 community-dwelling seniors of Minhang District, 13 (1.35%) were malnourished and 311 (32.40%) were at the risk of malnutrition. There were statistically significant differences in nutritional status across different categories of age, sex, monthly family income, education level, marital status, waist circumference, dental health status, activity of daily living, nutrition knowledge, suffering from chronic diseases, having > 3 chronic diseases, taking > 3 long-term prescriptions, depression symptoms, sleeping duration, daily outdoor activity time, number of daily food species (milk/soy products/fish/meat/poultry/eggs), daily intake of vegetables and fruits, daily consumption of cooking oil, frequency of physical exercise, frequency of smoking, and living alone (P < 0.05). The logistic regression analysis results showed that poor dental conditions, insufficient daily intake of milk/soy products/fish/meat/poultry/eggs (<3 kinds), insufficient daily intake of vegetables and fruits (<500 g), excessive daily consumption of cooking oil (>25 g), insufficient daily outdoor activities (<1 h·d⁻¹), living alone, low educational level (primary school and below), suffering from chronic diseases, having > 3 chronic diseases, taking > 3 long-term prescriptions, and being single/widowed/divorced were the main risk factors for nutritional abnormalities in the elderly (P< 0.05). Conclusion The elderly in Minhang District of Shanghai have a high malnutrition risk, and their nutritional status is affected by multiple factors, including poor dental status, irrational dietary structure, insufficient time for outdoor activities, suffering from chronic diseases, having > 3 chronic diseases, taking > 3 long-term prescriptions, low educational level, living alone, and being single/widowed/divorced.

Objective : To explore the feasibility of applying the Malocclusion Impact Questionnaire (MIQ) of Chinese version among Chinese teenagers, through the verification and evaluation of the Chinese version. Methods: According to the standard procedures of the international quality of life assessment program, the MIQ was translated, back translated, adapted and updated culturally, and the Chinese version was established. The 161 teenagers with the first orthodontic treatment were included. This patient group was assessed for oral health-related quality of life by the Chinese version of the MIQ and Children's Perception Questionnaire (11-14 years old). The reliability and validity of the Chinese version of the MIQ were evaluated statistically by Spearman and factor analysis. Results : A total of 161 valid questionnaires were collected. The internal consistency Cronbach's α of the Chinese scale was 0.887. The correlation coefficient between items and the scale ranged from 0.000 1 to 0.824. A significant positive correlation of the scores was noted between the translated scale and the Children's Perception Questionnaire (11-14 years old), and the correlation coefficient was 0.444 (P<0.001). Conclusion The reliability and validity of the Chinese version of the MIQ are reliable and can be applied for clinical orthodontic treatment.

Objectives To compare the differences in intestinal phylum firmicutes between elderly patients with type 2 diabetes (T2DM)and the healthy elder people.Methods 37 elderly patients with T2DM and 69 healthy controls in Shaoxin city were recruited.DNA of phylum firmicutes from fecal samples was extracted.The real-time quantitative PCR was used with special primers for bacterial genus including Faecalibacterium prausnitzii,Eubacterium rectale,Clostridium leptum,and Peptostrepyococc.The differences in content of different bacteria between two groups were analyzed and compared.Results In healthy elderly group versus the elder patient with T2DM,the contents of intestinal phylum firmicutes were[(6.22±1.41) × 107 versus(5.41± 1.40) × 107,t=2.83,P=0.006] in Eubacterium rectale,[(7.46 ± 0.98) × 107 versus (6.96 ± 1.40) × 107),t =2.13,P =0.036] in Faecali bacterium prausnitzii,[(7.89±0.89) × 107 versus(7.46±1.11) × 107,t=2.15,P=0.034]in Clostridium leptum,and[(4.86 ± 1.33) × 107 versus (4.21 ± 1.24) × 107,t=2.45,P =0.016] in Peptostrepyococc,which showed that the contents of intestinal phylum firmicutes were less in T2DM group than in healthy elderly group.Conclusions There are some differences in intestinal flora between the elderly patients with T2DM and healthy people.These intestinal flora may play an important role in the development of T2DM.This study may provide new evidences for probiotic treatment of T2DM.