Objective To investigate the role of activating transcription factor 4 (ATF4) in benign prostatic hyperplasia (BPH) in order to elucidate the molecular mechanism underlying BPH progression. Methods Firstly, we analyzed the correlation between the expression of ATF4 and the volume of prostate transition zone in BPH tissues using publicly available datasets. Subsequently, we explored the potential association among ATF4, epithelial-mesenchymal transition (EMT) and BPH through single-cell RNA sequencing (scRNA-seq). Finally, we validated the findings by silencing ATF4 and treating BPH cells with ATF4 protein inhibitors, followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), CCK-8 assay, and Western blot. Results The expression of ATF4 in BPH tissues exhibited a significant positive correlation with the volume of prostate transition zone (r=0.64, P＜0.05). scRNA-seq analysis revealed that ATF4 was significantly upregulated in the prostate epithelial cells of BPH patients (P＜0.05), and its expression demonstrated a significant positive correlation with the Hallmark EMT singscore (r=0.14, P＜0.05). In vitro experiments further indicated that knockdown of ATF4 led to a significant reduction in the proliferative activity of BPH-1 cells (P＜0.05). Similarly, inhibition of ATF4 activity resulted in a marked decrease in the proliferative capacity and EMT of BPH-1 cells (P＜0.05). Conclusion ATF4 may exert a promoting effect on BPH by modulating the EMT signaling pathway;therefore, ATF4 may serve as a novel therapeutic target of BPH.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

Objective:To evaluate the role of peptidylarginine deiminase 4 (PAD4)-mediated development of neutrophil extracellular traps (NETs) in lung ischemia-reperfusion injury (LIRI) in mice.Methods:Ninety-six clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=24 each) using a table of random numbers: sham operation group (group S), sham operation + PAD4 specific inhibitor GSK484 group (group S+ G), lung ischemia-reperfusion group (group L), and lung ischemia-reperfusion + GSK484 group (group L+ G). After anesthesia and mechanical ventilation, mice were subjected to left hilum occlusion for 1 h followed by 2 h of reperfusion to establish the LIRI model in L and L+ G groups. Mice underwent thoracotomy for 3 h without left hilum occlusion in S and S+ G groups. In S+ G and L+ G groups, GSK484 4 mg/kg was intraperitoneally injected once a day for 3 days before developing the model. At the end of reperfusion, blood samples were collected from the abdominal aorta for blood gas analysis to record arterial partial pressure of oxygen (PaO 2). Mice were then sacrificed to collect bronchoalveolar lavage fluid (BALF) and to obtain lung tissues. The concentrations of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) in BALF were measured using enzyme-linked immunosorbent assay. The wet/dry lung weight (W/D) ratio was calculated. The lung tissues were obtained for microscopic examination of pathological changes (with a light microscope) which were scored after hematoxylin-eosin staining and for determination of the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) (by colorimetric assay) and expression of PAD4, neutrophil elastase (NE), high-mobility group box 1 (HMGB1), and citrullinated histone 3 (Cit-H3) (by Western blot). Results:Compared with group S, lung injury scores and W/D ratios were significantly increased, PaO 2 was decreased, the concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were increased, the content of SOD was decreased, the content of MDA was increased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was up-regulated in L and L+ G groups ( P<0.05), and no significant changes were observed in the aforementioned parameters in group S+ G ( P>0.05). Compared with group L, lung injury scores and W/D ratios were significantly decreased, PaO 2 was increased, concentrations of IL-1β, IL-6, TNF-α, and MPO in BALF were decreased, the content of SOD was increased, the content of MDA was decreased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was down-regulated in group L+ G ( P<0.05). Conclusions:Up-regulated PAD4 expression can promote the development of NETs and aggravate oxidative stress and inflammatory responses in lung tissues, thereby participating in LIRI in mice.

Objective To investigate the effects of liraglutide on the gut microbiota of db/db diabetic mice and the predictive value of the microbial structure for the hypoglycemic efficacy.Methods The db/db mice were randomly divided into control group and liraglutide group(0.4 μg/g).Fasting blood glucose levels were measured in the mice,and fecal samples were collected to determine the structure of the gut microbiota.Results Compared with the control group,the liraglutide group exhibi-ted a significant increase in the average number of operational taxonomic unit(OTU)(P＜0.01).The number of observed species,Chao1 index and ACE index were all significantly higher in the liraglutide group than those in the control group(P＜0.05).Additionally,the liraglutide group showed a signifi-cant increase in the relative abundance of Firmicutes and a significant decrease in the abundance of Actinobacteria compared with the control group(P＜0.01).The percentage decrease in blood glucose levels in db/db mice was positively correlated with the abundance of Pseudomonadaceae and negatively correlated with the abundances of Clostridiaceae and Peptostreptococcaceae.Conclusion Liraglutide treatment can modulate the structure of the gut microbiota,increasing the number of OTU and diversity,particularly enhancing the abundance of beneficial bacteria such as Clostridium,Lachnospira and Oscillospira.The gut microbiota structure in mice serves as a predictive factor for the hypoglycemic efficacy of liraglutide,with diabetic mice exhibiting a higher abundance of Pseudomonas being more likely to benefit from liraglutide-induced hypoglycemic therapy.

Objective: To explore the longitudinal associations between dietary macronutrients and lung function in schoolaged children, so as to provide the nutritional research evidence for promoting children s lung health. Methods: In November 2021, two primary schools located in Chengdu, Sichuan Province were selected from the Southwest China Childhood Nutrition and Growth (SCCNG) cohort by a stratified cluster random sampling method, enrolling a total of 1 112 school aged children aged 8 to 13 years. At baseline, the dietary and sociodemographic characteristics of the children were assessed. One year later, the forced vital capacity (FVC) of the children was measured and converted into Z scores (FVC- Z ), while the vital capacity index (VCI) was also calculated. Generalized linear regression analysis was employed to examine the associations between dietary macronutrients and lung function, considering interactions with gender and age, followed by stratified analysis. Results: After adjusting for confounding factors, the analysis results of the generalized linear regression model showed that the carbohydrate energy ratio was negatively correlated with FVC- Z ( β =-0.02) and VCI ( β =-0.16), while the fat energy ratio showed a positive correlation with FVC- Z ( β =0.03) and VCI ( β =0.23) ( P <0.05). The protein energy ratio was positively correlated with FVC- Z ( β =0.09) and VCI ( β =0.60) specifically in girls ( P <0.05). Additionally, there was an interaction effect of age on the associations between macronutrients and lung function ( P <0.01); in children aged 8-9 and 10-11, the carbohydrate energy supply ratio was negatively correlated with FVC- Z ( β =-0.04, -0.03) and VCI ( β =-0.29, -0.21), and fat energy supply ratio was positively correlated with FVC- Z ( β =0.07, 0.05) and VCI ( β =0.46, 0.32) ( P <0.05). Conclusions There are age and sex differences in the association of dietary macronutrients with lung function, with a low carbohydrate, high fat diet promoting lung function in children. Additionally, protein intake appears to have a positive influence on the lung function of girls. The early school age period may represent a critical window for dietary interventions aimed at promoting lung health.

Objective:To evaluate the role of peptidylarginine deiminase 4 (PAD4)-mediated development of neutrophil extracellular traps (NETs) in lung ischemia-reperfusion injury (LIRI) in mice.Methods:Ninety-six clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=24 each) using a table of random numbers: sham operation group (group S), sham operation + PAD4 specific inhibitor GSK484 group (group S+ G), lung ischemia-reperfusion group (group L), and lung ischemia-reperfusion + GSK484 group (group L+ G). After anesthesia and mechanical ventilation, mice were subjected to left hilum occlusion for 1 h followed by 2 h of reperfusion to establish the LIRI model in L and L+ G groups. Mice underwent thoracotomy for 3 h without left hilum occlusion in S and S+ G groups. In S+ G and L+ G groups, GSK484 4 mg/kg was intraperitoneally injected once a day for 3 days before developing the model. At the end of reperfusion, blood samples were collected from the abdominal aorta for blood gas analysis to record arterial partial pressure of oxygen (PaO 2). Mice were then sacrificed to collect bronchoalveolar lavage fluid (BALF) and to obtain lung tissues. The concentrations of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) in BALF were measured using enzyme-linked immunosorbent assay. The wet/dry lung weight (W/D) ratio was calculated. The lung tissues were obtained for microscopic examination of pathological changes (with a light microscope) which were scored after hematoxylin-eosin staining and for determination of the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) (by colorimetric assay) and expression of PAD4, neutrophil elastase (NE), high-mobility group box 1 (HMGB1), and citrullinated histone 3 (Cit-H3) (by Western blot). Results:Compared with group S, lung injury scores and W/D ratios were significantly increased, PaO 2 was decreased, the concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were increased, the content of SOD was decreased, the content of MDA was increased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was up-regulated in L and L+ G groups ( P<0.05), and no significant changes were observed in the aforementioned parameters in group S+ G ( P>0.05). Compared with group L, lung injury scores and W/D ratios were significantly decreased, PaO 2 was increased, concentrations of IL-1β, IL-6, TNF-α, and MPO in BALF were decreased, the content of SOD was increased, the content of MDA was decreased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was down-regulated in group L+ G ( P<0.05). Conclusions:Up-regulated PAD4 expression can promote the development of NETs and aggravate oxidative stress and inflammatory responses in lung tissues, thereby participating in LIRI in mice.

To screen new antigens for novel tuberculosis(TB)vaccine research,we used bioinformatics to predict the B and T cell epitopes of Rv2318,and evaluated the immunogenicity of Rv2318 and its T/B epitope peptides(Rv2318p).The recombinant plas-mids pET32a-Rv2318 and pET32a-Rv2318p were constructed through gene synthesis methods.The recombinant proteins were ex-pressed in a prokaryotic system and purified with nickel affinity chromatography.Proteins were identified with SDS-PAGE and western blotting.BALB/c mice were immunized subcutaneously with the recombinant proteins to evaluate immunogenicity.Sera were collected,and antigen specific antibody titers were evaluated with ELISA.Splenocytes were isolated,and cytokines and T cell proliferation were analyzed with ELISA and flow cytometry,respectively.Rv2318 included two epitope fragments,aa10-130 and 350-410.SDS-PAGE and western blotting indicated that the target proteins were expressed and purified correctly,and their relative molecular weights were-approximately 68 kD and 42 kD,respectively.Rv2318 and Rv2318p induced stronger humoral immune responses than observed in the control groups(P<0.000 1,n=6).Compared with Rv2318,Rv2318p showed significantly greater enhancement of specific IgG and IgG subclass antibodies(P<0.000 1,n=6).In addition,Rv2318p increased the ratio of IgG2a/IgG1,thus indicating that it primarily induced a cellular immune response biased toward the Th1 type.Cytokine experiments revealed that IFN-γ,IL-2,IL-6,and IL-4 significantly increased after immunization with Rv2318p(P all<0.01,n=6),particularly Th1 type cytokines(IFN-γ and IL-2).Furthermore,Rv2318 increased the expression of only IL-2 and IL-6,particularly IL-6(P all<0.01,n=6).Although Rv2318 in-duced more IFN-γ,we observed no significant difference between Rv2318 and PBS immunized mice.Importantly,Rv2318p stimu-lated mice to express IFN-γ at 842 pg/mL,approximately 3 times the level elicited by Rv2318.Whereas both proteins increased the proportions of CD4+and CD8+T cells,Rv2318p promoted greater proliferation of T lymphocytes.These data indicated that both Rv2318 and its epitope peptides enhanced humoral and cellular immune responses,whereas the epitope peptides notably triggered a stronger Th1 type cellular response.In conclusion,the recombinant protein Rv2318 and its epitope peptides showed favorable immunogenicity,and the immunogenicity of Rv2318p was superior to that of Rv2318.This study provides a theoretical basis for TB vaccine development.

Objective To explore the relationship between the serum levels of absent in melanoma 2(AIM2)and apolipoprotein J(Apo J)and the severity and prognosis of vascular dementia(VD).Methods From October 2020 to September 2022,128 patients with vascular dementia were collected as the study group in the Second People's Hospital of Binzhou.Based on the mini-mental state examination(MMSE)score,the severity of the condition was evaluated and the study group was separated into mild group(n=43),moderate group(n=54)and severe group(n=31).After 90 days of treatment,the prognosis of patients was evaluated based on the ability of daily living(ADL)scale score and grouped into grade Ⅰ group(n=66),grade Ⅱ group(n=40)and grade Ⅲgroup(n=22).Another 96 healthy individuals who underwent physical examination were selected as the control group.The expression levels of serum AIM2 and Apo J were detected by qRT-PCR and ELISA methods,and the differences between the groups were compared.Spearman method was applied to analyze the correlation between serum AIM2 and ApoJ expression levels and MMSE scores,and the relationship between serum AIM2 and Apo J expression levels and prognosis in patients with vascular dementia.Logistic regression was applied to analyze the prognostic factors of vascular dementia patients.Results The expression level of serum AIM2 mRNA(3.11±0.57 vs 1.06±0.23)in the study group was higher than those in the control group,while the expression level of serum ApoJ(68.83±12.24 ng/L vs 81.07±13.15 ng/L)was lower than that in the control group,with significant differences(t=32.054,7.174,all P＜0.001).The expression level of serum AIM2 mRNA in the severe group was higher than that in the mild and moderate groups(q=12.807,15.780),the expression level of serum Apo J and MMSE score in the severe group were lower than those in the mild and moderate groups(q=26.201,4.301;12.193,20.802),the expression level of serum AIM2 mRNA in the moderate group was higher than that in the mild group(q=14.688),and the expression level of serum Apo J and MMSE score in the moderate group were lower than those in the mild group(q=20.338,37.537),with significant differences(all P＜0.001).The expression level of serum AIM2 mRNA(5.27±0.60)in the grade Ⅲgroup of vascular dementia was higher than that in the grade Ⅱ(3.36±0.58)and grade Ⅰ groups(2.23±0.55),while the expression level of serum Apo J(51.22±12.21 ng/L)was lower than that in the grade Ⅱ(64.15±12.23 ng/L)and grade Ⅰgroups(77.53±12.25 ng/L).The expression level of serum AIM2 mRNA in the grade Ⅱ group was higher than that in the gradeⅠ group,while the expression level of serum Apo J in the grade Ⅱ group was lower than that in the grade Ⅰ group,with significant differences(q=5.630～30.740,all P＜0.001).The level of serum AIM2 in patients with vascular dementia was negatively correlated with MMSE score but positively correlated with prognosis(ADL grade)(r=-0.535,0.432,all P＜0.001),while the level of serum Apo J in patients with vascular dementia was positively correlated with MMSE score but negatively correlated with prognosis(ADL grade)(r=0.467,-0.496,all P＜0.001).Serum AIM2 mRNA[OR(95％CI):2.746(1.481～5.091)],Apo J[OR(95％CI):0.496(0.311～0.791)],and MMSE score[OR(95％CI):0.568(0.347～0.931)]were influencing factors for the prognosis of vascular dementia patients(all P＜0.05).Conclusion The increase of serum AIM2 mRNA expression level and the decrease of serum Apo J expression level in patients with vascular dementia were closely related to the clinical severity and prognosis of patients,which may effectively evaluate the prognosis of patients.

Objective: To investigate the prospective effects of intake of each food group on the development of lung function of pupils,so as to provide theoretical basis for promoting the healthy development of lung function and preventing chronic respiratory diseases in Chinese children. Methods: A cluster stratified sampling method was used to select a total of 893 pupils in grades 2-5 from Chengdu in November 2021. Dietary data of respondents were collected using a food frequency questionnaire within the past year,then the food group intake was categorized into T1, T2 and T3 from low to high by the trichotomous method, and anthropometric measurements including lung capacity were obtained in 2022. Logistic regression models and test for trend were used to analyze the prospective effects of intake of each food group on lung function development of pupils. Results: Among male students, consumption of vegetables [118.6(50.5, 188.2)g/d] and milk and dairy products [200.0(73.3, 250.0)g/d] were higher in the excellent lung capacity group than in the non excellent lung capacity group [90.0(37.1, 192.9), and 178.6(35.7, 250.0)g/d],with statistically significant differences ( Z =-1.98, -2.24); among girls, the group with excellent lung capacity consumed less staple food [391.1(273.6, 511.4)g/d] than the group with non excellent lung capacity [407.4(309.5, 594.3)g/d], and the group with excellent lung capacity consumed more aquatic products [31.2(14.6, 69.8)g/d] and milk and dairy products [215.0(107.1, 250.1) g/d ] than that of the non excellent lung capacity [19.4(10.7, 58.3), 114.3(35.7, 250.0)g/d] ( Z =-2.01, -3.33, -5.10)( P < 0.05 ). After adjusting for energy, body mass index Z score(BMI Z ), mother s education level, averge family income monthly, whether presence of smokers in the living environment, and whether participation in physical activities during the past week, among male students, T3 group of vegetable intake ( OR =0.48, 95% CI = 0.27-0.86), T2 group of bean and soy product intake ( OR = 0.52 , 95% CI =0.27-0.96)，T2 and T3 groups of milk and dairy products intake (T2： OR =0.54, 95% CI =0.31-0.93; T3： OR = 0.52 , 95% CI =0.30-0.90) were negatively associated with non excellent lung capacity ( P <0.05). Among girls, T3 group of aquatic product intake( OR =0.52, 95% CI =0.28-0.97), T2 and T3 groups of milk and dairy product (T2: OR =0.44, 95% CI =0.25- 0.76 ;T3: OR =0.33, 95% CI =0.19-0.59) were negatively associated with nonexcellent lung capacity, whereas the T2 group of red meat intake ( OR =2.51, 95% CI =1.37-4.67) was positively associated with non excellent lung capacity. Non excellent lung capacity was found to be negatively associated with vegetable and milk and dairy product intake in boys by test for trend; in girls, milk and dairy products intake was negatively associated with non excellent lung capacity, whereas red meat intake was positively associated with non excellent lung capacity ( t =-1.13，-0.44；-3.03，1.95, P trend <0.05). Conclusions Milk and dariy products intakes reduce the risk of non excellent lung capacity in pupils, vegetables intakes reduce the risk of non excellent lung capacity in boys, and the intake of red meat increases the risk of non excellent lung capacity in girls. Promoting rational food choices is necessary for children to improve healthy lung development.

Objective: To explore the differences in the gut microbiota of primary school students with different levels of sugar sweetened beverage intake, so as to provide scientific evidence for better identification of health risks in children and the development of targeted health policies. Methods: In June 2022, a total of 192 healthy primary school students from Chengdu were selected using a stratified cluster random sampling method. The sugar sweetened beverage intake was assessed through a dietary frequency questionnaire. Based on the median daily sugar sweetened beverage intake, primary school students were categorized into a low intake group ( n =96) and a high intake group ( n =96). The gut microbiota in fresh fecal samples from the two groups of primary school students was analyzed using 16S rRNA high throughput sequencing, and the diversity and community structure differences in the gut microbiota were compared. Results: Children in the low intake group had a sugar sweetened beverage intake of (21.3±1.6) mL/d, while the high intake group had an intake of (269.6±37.3) mL/d. Diversity analysis results showed that there were no statistically significant differences between the low intake and the high intake group in terms of α diversity metrics: Observed_otus index [298.50 (259.75, 342.25), 305.50 (244.25, 367.75)], Goods_coverage index [1.00 (1.00, 1.00), 1.00 (1.00, 1.00)], Chao index [304.18 (260.75, 348.78), 305.88 (245.68, 370.88)], Shannon index [5.88 (5.29, 6.45), 5.71 (4.89, 6.28)] and Simpson index [0.95 (0.91, 0.97), 0.94 (0.88, 0.97)] ( Z =-0.64, -0.76, -0.54, -1.76, -1.67, P >0.05). Furthermore, no statistically significant difference was observed in β diversity between the two groups ( R 2=0.006, P >0.05). At the genus level, the abundance of Blautia [0.033 (0.018, 0.055)] and Fusicatenibacter [0.009 (0.005, 0.015)] were higher in the low intake group compared to the high intake group [0.024 (0.013, 0.041),0.006 (0.003, 0.011)]and differences were statistically significant ( Z =-2.52, -2.81, P <0.05). LEfSe analysis highlighted intergroup differences primarily in Blautia, Fusicatenibacter and Sarcina( LDA= 3.56,3.12,3.53, P <0.05). Conclusions There is no significant difference in the diversity and overall structure of the gut microbiota in primary school students with different levels of sugar sweetened beverage intake. However, there are species variations at the genus level. The information can serve as a scientific basis for identifying health risks in primary school students and formulating targeted health strategies.