ObjectiveTo investigate the therapeutic effect and mechanism of Xueshisanjia San against liver fibrosis by regulating PTEN-induced putative kinase （PINK1）/Parkin signaling pathway-mediated mitophagy. MethodsForty specific pathogen free （SPF）-grade male C57BL/6 mice were randomized into the control， model， silibinin （100 mg·kg^-1）， high-dose （15.16 g·kg^-1） Xueshisanjia San， and low-dose （7.58 g·kg^-1） Xueshisanjia San groups. The mouse model of liver fibrosis was constructed by intraperitoneal injection of 20% carbon tetrachloride solution. The treatment lasted for 6 weeks. Blood was collected from the abdominal aorta after intraperitoneal anesthesia， and the liver was separated. Liver pathology was examined by hematoxylin-eosin （HE） staining， Masson staining， and Sirius Red staining. Transmission electron microscopy （TEM） was employed to observe the mitochondrial morphology in the liver tissue. The levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， C-reactive protein （CRP）， total bilirubin （TBil）， transforming growth factor-β₁ （TGF-β₁）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） in the serum of mice were measured by enzyme-linked immunosorbent assay. Immunohistochemistry， immunofluorescence assay， and Western blot were employed to determine the protein levels of liver fibrosis markers α-smooth muscle actin （α-SMA） and collagen Ⅰ， as well as mitophagy markers microtubule-associated protein 1 light chain 3 （LC3）， p62， Beclin-1， PINK1， Parkin， and translocase of outer mitochondrial membrane 20 （TOM20）. ResultsCompared with the control group， the model group exhibited elevated levels of ALT， AST， CRP， TBil， IL-6， TGF-β₁， and TNF-α in the serum （P<0.05）， pathological changes such destroyed structure of hepatic lobules， disarrangement of hepatic cells， and collagen accumulation， swollen， vacuolated， and fragment mitochondria， down-regulated expression of p62 and TOM20， and up-regulated expression of LC3， Beclin-1， PINK1， and Parkin （P<0.05）. Compared with the model group， all the treatment groups exhibited declined levels of ALT， AST， CRP， TBil， IL-6， TGF-β₁， and TNF-α in the serum （P<0.05）， alleviated pathological damage of liver tissue and mitochondrial damage， up-regulated expression of p62 and TOM20， and down-regulated expression of α-SMA， COL1A1， LC3， Beclin1， PINK1， and Parkin （P<0.05）. ConclusionXueshisanjia San may prevent excessive mitophagy and improve mitochondrial quality by inhibiting PINK1/Parkin signaling pathway， thereby alleviating liver fibrosis.

OBJECTIVE To investigate the impact of vesicular stomatitis virus envelope glycopro-tein-G(VSV-G)modification on the mucosal immune efficacy of antigen-loaded engineered exosome vaccines.METHODS In vitro experiments:Dendritic cells(DCs)were divided into three groups:cell-control(treated with culture medium),receptor binding domain(RBD)(transfected with plasmid RBD),and RBD+VSV-G(co-transfected with plasmids RBD and VSV-G).Expression levels of RBD and VSV-G were assessed using Western blotting,flow cytometry,and immunofluorescence.Exosomes were extracted via ultracentrifugation,whose morphology,size distribution,and marker proteins were analyzed using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting that confirmed the expressions of RBD and VSV-G in the exosomes.In vivo experiments:① Female BALB/c mice were divided into the control group Mock exosomes(Mock-Exo)(derived from the supernatant of cell-control),RBD decorated exosomes(RBD-Exo)(derived from the RBD cell supernatant),and RBD and VSV-G decorated exosomes(RBD+VSV-G-Exo)(derived from RBD+VSV-G cell supernatant).Follow-ing intranasal immunization with the respective vaccines,the nasal retention effects were evaluated using in vivo imaging.Flow cytometry was used to assess the ability to recruit immune cells to the nasal tissue.Serum RBD-specific immunoglobulin G(IgG)and mucosal immunoglobulin A(IgA)(bronchoal-veolar lavage fluid/nasal wash)were quantified at 7 and 21 d post-immunization by enzyme-linked immuno-sorbent assay.Body weight changes were monitored and key serum biochemical parameters along with histopathological damage to major organs were analyzed following immunization.② Female BALB/c mice were divided into the Mock-Exo group(intranasally inoculated with Mock-Exo),RBD+VSV-G-Exo group(intranasally inoculated with RBD+VSV-G-Exo),and RBD+VSV-G-Exo(im)group(intramus-cularly injected with RBD+VSV-G-Exo).RESULTS In vitro experiments:RBD and VSV-G were successfully expressed in cells,with positive rates of RBD+and VSV-G+cells at 64.4%and 31.2%,respectively.The extracted exosomes exhibited regular morphology and qualified purity,with a particle size of approximately 138 nm and successfully loaded RBD and VSV-G proteins.In vivo experiments:Compared to Mock-Exo and RBD-Exo,RBD+VSV-G-Exo prolonged nasal retention time to 96 h and markedly increased the numbers of CD49B+natural killer cells,CD11c+dendritic cells,and F4/80+macrophages in nasal tissues.RBD+VSV-G-Exo induced robust RBD-specific immune responses,with serum IgG titers,BALF IgA titers,and nasal wash IgA titers reaching 1∶5 215,1∶2 560,1∶1 114,respec-tively.In contrast,no RBD-specific IgA antibody titers were detected in the BALF and nasal wash of mice treated with RBD+VSV-G-Exo(im).Mice showed stable body weight gain during 30 d post-immu-nization.Major serum biochemical indices were within normal reference ranges,and no obvious patho-logical changes were observed in major organs or olfactory bulbs 7 d after immunization.CONCLU-SION VSV-G modification extends the retention time of engineered exosome vaccines in nasal tissues,enhance their ability to recruit immune cells,and induce a high-level antigen-specific respiratory mucosal immune response.

Objective:To identify the incidence and prevalence of inflammatory bowel disease (IBD) in the northern Chinese population of Jinan, Shangdong Province, along with its temporal trends from 2005 to 2022.Methods:By utilizing the data from the Jinan basic medical insurance system, a population-based IBD cohort was constructed. This facilitated the computation of both the incidence and prevalence rates of IBD, alongside their temporal trends throughout the 2005 to 2022 timeframe. The 95% confidence intervals were estimated using poisson regression.Results:The overall incidence rate of IBD showed a yearly increasing trend, with age-standardized incidence rates rising from 0.03/100 000 in 2005 to 5.39/100 000 in 2022. The age-standardized incidence rate of ulcerative colitis (UC) increased from 0.03/100 000 in 2005 to 4.97/100 000 in 2022. The age-standardized incidence rate of Crohn's disease (CD) rose from 0.05/100 000 in 2011 to 0.44/100 000 in 2022. The crude prevalence of IBD increased from 0.60/100 000 in 2005 to 32.39/100 000 in 2022. Specifically, the crude prevalence of UC increased from 0.60/100 000 in 2005 to 31.44/100 000 in 2022, while the crude prevalence of CD increased from 0.05/100 000 in 2011 to 1.19/100 000 in 2022.Conclusions:Analysis of recent medical insurance data reveals a continuous uptrend in both the incidence and prevalence of IBD in Jinan, a northern city in China. This underscores the urgent need for enhanced medical resources and healthcare guaruntee to ensure the well-being of individuals afflicted with IBD.

OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

Objective To investigate the correlation between glycemic variability metrics and the risk of diabetic foot(DF)in patients with type 2 diabetes mellitus(T2DM)utilizing flash glucose monitoring(FGM)tech-nology.Methods A retrospective analysis was conducted on 233 hospitalized patients with T2DM,with or without DF,who were treated in the Department of Endocrinology at Lianyungang First People's Hospital from January 2021 to May 2022 and monitored using FGM.Patients were categorized into a non-DF group(n=147)and a DF group(n=86)based on the presence of DF.The study compared general clinical characteristics,biochemical parameters,and glycemic variability metrics between the two groups and performed subgroup analyses.Binary logistic regression was employed to identify factors associated with the risk of DF,while receiver operating characteristic(ROC)curves were utilized to assess the predictive value of glycemic variability metrics for DF.Results Compared with the non-DF group,patients in the DF group exhibited significantly longer disease duration,higher body mass index(BMI),glycated hemoglobin(HbA1c),urinary albumin-to-creatinine ratio(UACR),alanine aminotransferase(ALT),serum uric acid(SUA),mean amplitude of glycemic excursions(MAGE),coefficient of variation(CV),mean of daily differences(MODD),and mean blood glucose(MBG),but lower fasting C-peptide(FCP),fasting insulin(FINS),high-density lipoprotein cholesterol(HDL-C),and time in range(TIR),with statistically signifi-cant differences(P<0.05).Subgroup analysis revealed that TIR was associated with the incidence of DF and diabetic retinopathy(DR).Binary logistic regression analysis identified HbA1c,MAGE,MODD,and MBG as risk factors for DF,while TIR was a protective factor(P<0.05).ROC curve analysis demonstrated that the area under the curve(AUC)for predicting DF using HbA1c,TIR,MAGE,MODD,MBG,and their combination were 0.646,0.850,0.868,0.764,0.619,and 0.967,respectively,indicating superior performance of the combined prediction model.Conclusions HbA1c,TIR,MAGE,MODD,and MBG are critical factors associated with the development of DF in patients with T2DM.Targeted early interventions aimed at optimizing these glycemic variability indicators may effectively reduce the incidence of DF.

Objective:To investigate the efficacy and safety of the combination therapy of venetoclax (VEN) and azacitidine (AZA) in treatment of newly diagnosed elderly patients with acute myeloid leukemia (AML).Methods:A retrospective cohort study was conducted. The clinical data of 17 newly diagnosed elderly AML patients who received VEN+AZA regimen at the First Hospital of Putian City from April 2021 to June 2023 were collected. Treatment outcomes and adverse events were analyzed. Survival curves were plotted by using the Kaplan-Meier method, and intergroup comparisons were performed by using the log-rank test.Results:Among the 17 patients, the median age [ M ( Q1, Q3)] was 70 (68, 74) years, with 11 males (64.7%) and 6 females (35.3%). The median number of treatment courses was 4.0 (2.5, 8.5). After the first course, the composite complete remission (cCR) rate was 41.2% (7/17), minimal residual disease (MRD) negativity rate was 5.9% (1/17), and overall response rate (ORR) was 82.4% (14/17). By the end of follow-up in September 2023, the cCR rate reached 64.7% (11/17), MRD negativity rate was 52.9% (9/17), and ORR was 88.2% (15/17). The median number of courses to achieve cCR was 1.0 (1.0, 2.0), and to achieve MRD negativity was 3.0 (2.0, 3.5). The follow-up rate was 88.2% (15/17), and the median follow-up time was 17.3 months (95% CI: 7.0-27.6 months). The median progression-free survival (PFS) time was 6.5 months (95% CI: 1.7-11.3 months), and median overall survival (OS) time was 12.0 months (95% CI: 0.3-23.7 months). The median OS time after progression was 1.5 months (95% CI: 1.0-2.0 months). All patients experienced hematological adverse events, with 94.1% (16/17) experiencing grade ≥ 3 hematological adverse events. The most common non-hematological adverse event was infection (88.2%, 15/17), with the lung being the most frequent site of infection (82.4%, 14/17), while 41.2% (7/17) of patients had pre-existing infections before treatment. Conclusions:The VEN+AZA regimen demonstrates high remission rates and significant efficacies in treating newly diagnosed elderly AML patients. Although adverse events occur in nearly all patients, most are able to tolerate the treatment.

Objective To investigate the distribution and antimicrobial resistance profiles of clinically isolated Haemophilus influenzae and Moraxella catarrhalis in hospitals across China from 2015 to 2021,and provide evidence for rational use of antimicrobial agents.Methods Data of H.influenzae and M.catarrhalis strains isolated from 2015 to 2021 in CHINET program were collected for analysis,and antimicrobial susceptibility testing was performed by disc diffusion method or automated systems according to the uniform protocol of CHINET.The results were interpreted according to the CLSI breakpoints in 2022.Beta-lactamases was detected by using nitrocefin disk.Results From 2015 to 2021,a total of 43 642 strains of Haemophilus species were isolated,accounting for 2.91％of the total clinical isolates and 4.07％of Gram-negative bacteria in CHINET program.Among the 40 437 strains of H.influenzae,66.89％were isolated from children and 33.11％were isolated from adults.More than 90％of the H.influenzae strains were isolated from respiratory tract specimens.The prevalence of β-lactamase was 53.79％in H.influenzae strains.The H.influenzae strains isolated from children showed higher resistance rate than the strains isolated from adults.Overall,779 strains of H.influenzae did not produce β-lactamase but were resistant to ampicillin(BLNAR).Beta-lactamase-producing strains showed significantly higher resistance rates to these antimicrobial agents than the β-lactamase-nonproducing strains.Of the 16 191 M.catarrhalis strains,80.06％were isolated from children and 19.94％isolated from adults.M.catarrhalis strains were mostly susceptible to both amoxicillin-clavulanic acid and cefuroxime,evidenced by resistance rate lower than 2.0％.Conclusions The emergence of antibiotic-resistant H.influenzae due to β-lactamase production poses a challenge for clinical anti-infective treatment.Therefore,it is very important to implement antibiotic resistance surveillance for H.influenzae and guide rational antibiotic use.All local clinical microbiology laboratories should actively improve antibiotic susceptibility testing and strengthen antibiotic resistance surveillance for H.influenzae.

Objective:To identify the incidence and prevalence of inflammatory bowel disease (IBD) in the northern Chinese population of Jinan, Shangdong Province, along with its temporal trends from 2005 to 2022.Methods:By utilizing the data from the Jinan basic medical insurance system, a population-based IBD cohort was constructed. This facilitated the computation of both the incidence and prevalence rates of IBD, alongside their temporal trends throughout the 2005 to 2022 timeframe. The 95% confidence intervals were estimated using poisson regression.Results:The overall incidence rate of IBD showed a yearly increasing trend, with age-standardized incidence rates rising from 0.03/100 000 in 2005 to 5.39/100 000 in 2022. The age-standardized incidence rate of ulcerative colitis (UC) increased from 0.03/100 000 in 2005 to 4.97/100 000 in 2022. The age-standardized incidence rate of Crohn's disease (CD) rose from 0.05/100 000 in 2011 to 0.44/100 000 in 2022. The crude prevalence of IBD increased from 0.60/100 000 in 2005 to 32.39/100 000 in 2022. Specifically, the crude prevalence of UC increased from 0.60/100 000 in 2005 to 31.44/100 000 in 2022, while the crude prevalence of CD increased from 0.05/100 000 in 2011 to 1.19/100 000 in 2022.Conclusions:Analysis of recent medical insurance data reveals a continuous uptrend in both the incidence and prevalence of IBD in Jinan, a northern city in China. This underscores the urgent need for enhanced medical resources and healthcare guaruntee to ensure the well-being of individuals afflicted with IBD.

OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.

OBJECTIVE To investigate the impact of vesicular stomatitis virus envelope glycopro-tein-G(VSV-G)modification on the mucosal immune efficacy of antigen-loaded engineered exosome vaccines.METHODS In vitro experiments:Dendritic cells(DCs)were divided into three groups:cell-control(treated with culture medium),receptor binding domain(RBD)(transfected with plasmid RBD),and RBD+VSV-G(co-transfected with plasmids RBD and VSV-G).Expression levels of RBD and VSV-G were assessed using Western blotting,flow cytometry,and immunofluorescence.Exosomes were extracted via ultracentrifugation,whose morphology,size distribution,and marker proteins were analyzed using transmission electron microscopy,nanoparticle tracking analysis,and Western blotting that confirmed the expressions of RBD and VSV-G in the exosomes.In vivo experiments:① Female BALB/c mice were divided into the control group Mock exosomes(Mock-Exo)(derived from the supernatant of cell-control),RBD decorated exosomes(RBD-Exo)(derived from the RBD cell supernatant),and RBD and VSV-G decorated exosomes(RBD+VSV-G-Exo)(derived from RBD+VSV-G cell supernatant).Follow-ing intranasal immunization with the respective vaccines,the nasal retention effects were evaluated using in vivo imaging.Flow cytometry was used to assess the ability to recruit immune cells to the nasal tissue.Serum RBD-specific immunoglobulin G(IgG)and mucosal immunoglobulin A(IgA)(bronchoal-veolar lavage fluid/nasal wash)were quantified at 7 and 21 d post-immunization by enzyme-linked immuno-sorbent assay.Body weight changes were monitored and key serum biochemical parameters along with histopathological damage to major organs were analyzed following immunization.② Female BALB/c mice were divided into the Mock-Exo group(intranasally inoculated with Mock-Exo),RBD+VSV-G-Exo group(intranasally inoculated with RBD+VSV-G-Exo),and RBD+VSV-G-Exo(im)group(intramus-cularly injected with RBD+VSV-G-Exo).RESULTS In vitro experiments:RBD and VSV-G were successfully expressed in cells,with positive rates of RBD+and VSV-G+cells at 64.4%and 31.2%,respectively.The extracted exosomes exhibited regular morphology and qualified purity,with a particle size of approximately 138 nm and successfully loaded RBD and VSV-G proteins.In vivo experiments:Compared to Mock-Exo and RBD-Exo,RBD+VSV-G-Exo prolonged nasal retention time to 96 h and markedly increased the numbers of CD49B+natural killer cells,CD11c+dendritic cells,and F4/80+macrophages in nasal tissues.RBD+VSV-G-Exo induced robust RBD-specific immune responses,with serum IgG titers,BALF IgA titers,and nasal wash IgA titers reaching 1∶5 215,1∶2 560,1∶1 114,respec-tively.In contrast,no RBD-specific IgA antibody titers were detected in the BALF and nasal wash of mice treated with RBD+VSV-G-Exo(im).Mice showed stable body weight gain during 30 d post-immu-nization.Major serum biochemical indices were within normal reference ranges,and no obvious patho-logical changes were observed in major organs or olfactory bulbs 7 d after immunization.CONCLU-SION VSV-G modification extends the retention time of engineered exosome vaccines in nasal tissues,enhance their ability to recruit immune cells,and induce a high-level antigen-specific respiratory mucosal immune response.