Researchers commonly use cyclization recombination enzyme/locus of X-over P1 (Cre/loxP) technology-based conditional gene knockouts of model mice to investigate the functional roles of genes of interest in Sertoli and Leydig cells within the testis. However, the shortcomings of these genetic tools include high costs, lengthy experimental periods, and limited accessibility for researchers. Therefore, exploring alternative gene silencing techniques is of great practical value. In this study, we employed adeno-associated virus (AAV) as a vector for gene silencing in Sertoli and Leydig cells. Our findings demonstrated that AAV serotypes 1, 8, and 9 exhibited high infection efficiency in both types of testis cells. Importantly, we discovered that all three AAV serotypes exhibited exquisite specificity in targeting Sertoli cells via tubular injection while demonstrating remarkable selectivity in targeting Leydig cells via interstitial injection. We achieved cell-specific knockouts of the steroidogenic acute regulatory ( Star ) and luteinizing hormone/human chorionic gonadotropin receptor (Lhcgr) genes in Leydig cells, but not in Sertoli cells, using AAV9-single guide RNA (sgRNA)-mediated gene editing in Rosa26-LSL-Cas9 mice. Knockdown of androgen receptor ( Ar ) gene expression in Sertoli cells of wild-type mice was achieved via tubular injection of AAV9-short hairpin RNA (shRNA)-mediated targeting. Our findings offer technical approaches for investigating gene function in Sertoli and Leydig cells through AAV9-mediated gene silencing.
Animals ; Male ; Leydig Cells/metabolism* ; Mice ; Dependovirus/genetics* ; Sertoli Cells/metabolism* ; Gene Silencing ; Genetic Vectors ; Testis/cytology*

Prostate cancer is the second most common malignancy and the sixth leading cause of cancer-related death in men worldwide. Radical prostatectomy (RP) is the standard treatment for localized prostate cancer, but the procedure often results in postoperative erectile dysfunction (ED). The poor efficacy of phosphodiesterase 5 inhibitors after surgery highlights the need to develop new therapies to enhance cavernous nerve regeneration and improve the erectile function of these patients. In the present study, we aimed to examine the potential of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in preserving erectile function in cavernous nerve injury (CNI) mice. We found that HB-EGF expression was reduced significantly on the 1 st day after CNI in penile tissue. Ex vivo and in vitro studies showed that HB-EGF promotes major pelvic ganglion neurite sprouting and neuro-2a (N2a) cell migration. In vivo studies showed that exogenous HB-EGF treatment significantly restored the erectile function of CNI mice to 86.9% of sham levels. Immunofluorescence staining showed that mural and neuronal cells were preserved by inducing cell proliferation and reducing apoptosis and reactive oxygen species production. Western blot analysis showed that HB-EGF upregulated protein kinase B and extracellular signal-regulated kinase activation and neurotrophic factor expression. Overall, HB-EGF is a major promising therapeutic agent for treating ED in postoperative RP.
Animals ; Male ; Heparin-binding EGF-like Growth Factor/therapeutic use* ; Erectile Dysfunction/etiology* ; Mice ; Penis/drug effects* ; Nerve Regeneration/drug effects* ; Penile Erection/drug effects* ; Peripheral Nerve Injuries/drug therapy* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Cell Movement/drug effects* ; Prostatectomy/adverse effects* ; Mice, Inbred C57BL ; Reactive Oxygen Species/metabolism*

OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*

OBJECTIVES: To investigate the mechanism through which pinostrobin (PSB) alleviates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: C57BL/6 mice were randomized into control group, DSS model group, and PSB intervention (30, 60, and 120 mg/kg) groups. Colitis severity of the mice was assessed by examining body weight changes, disease activity index (DAI), colon length, and histopathology. The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining, and macrophage infiltration and polarization were analyzed with flow cytometry. ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors (TNF‑α and IL-6) and chemokines (CCL₂, CXCL₁₀, and CX₃CL₁) in the colon tissues, and PI3K/AKT phosphorylation levels were analyzed with Western blotting. In cultured Caco-2 and RAW264.7 cells, the effect of PSB on CCL₂-mediated macrophage migration was assessed using Transwell assay. Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB. RESULTS: In DSS-induced mouse models, PSB at 60 mg/kg optimally alleviated colitis, shown by reduced weight loss and DAI scores and increased colon length. PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues, inhibited colonic macrophage infiltration, and promoted the shift of macrophage polarization from M1 to M2 type. In cultured intestinal epithelial cells, PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL₂ expression. PSB treatment obviously blocked CCL₂-mediated macrophage migration of RAW264.7 cells, which could be reversed by exogenous CCL₂. Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL₂ signaling as the core targets of PSB. CONCLUSIONS PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling, reducing CCL₂ secretion, and blocking macrophage chemotaxis and migration, highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.
Animals ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Colitis/drug therapy* ; Dextran Sulfate ; Proto-Oncogene Proteins c-akt/metabolism* ; Macrophages ; Chemokine CCL2/metabolism* ; Humans ; Signal Transduction/drug effects* ; Caco-2 Cells ; RAW 264.7 Cells ; Epithelial Cells/drug effects* ; Intestinal Mucosa/metabolism*

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

High flow nasal cannula（HFNC）is a novel oxygen therapy developed in recent years，and has been successfully used in pediatric diseases such as bronchiolitis and pneumonia.Although there has been a lack of clinical application guidelines in pediatrics，it has been increasingly applied to the treatment of exacerbations of bronchial asthma.This review focused on efficacy，application timing，complications and parameters adjustment of HFNC in children with asthma exacerbation，so as to further guide the clinical use.

Objective:To analyze the clinical characteristics of eosinophilic lung diseases(ELD) in children to enhance pediatricians′ understanding of ELD.Methods:In this retrospective cross-sectional study, a total of 149 children with ELD were recruited from Beijing Children′s Hospital, Capital Medical University between April 1, 2007 and March 31, 2022.Chi-square test, Fisher′s exact test, Mann-Whitney U test and Kruskal-Wallis test were used to analyze data and conclude clinical characteristics.Spearman correlation was used to analyze the correlation between eosinophils in peripheral blood and bronchoalveolar lavage fluid.Chi-square test and Kappa consistency test were used to compare the differences and consistency in diagnostic results between bronchoalveolar lavage fluid or lung biopsy and eosinophil elevation with chest imaging abnormalities. Results:(1)The isolated lung involvement was mostly caused by allergic bronchopulmonary aspergillosis(9 patients), and other system involvement by idiopathic hypereosinophilic syndrome(89 patients).(2)The main respiratory manifestations included coughing(90 cases, 60.4%) and expectoration(41 cases, 27.5%), while 23.5%(35 cases) of patients had no respiratory symptoms; 50.3% had digestive system involvement, and 40.9% had skin involvement.These were the two most commonly affected organs.(3)Spearman correlation was performed between eosinophils in peripheral blood and bronchoalveolar lavage fluid( r=0.3, P<0.05).Chi-square test was performed to compare ELD diagnosed by bronchoalveolar lavage fluid or lung biopsy with peripheral blood eosinophilia accompanied by abnormal chest imaging( P<0.05).Kappa consistency test(Kappa<0.2) showed poor consistency between the two diagnostic methods. Conclusions:ELD are present in children, and multiple etiologies may be pathogenic.Among children with ELD, the isolated lung involvement is mainly caused by allergic bronchopulmonary aspergillosis.The digestive system and skin are the most commonly affected organs, except for lungs.The correlation between eosinophil levels in peripheral blood and bronchoalveolar lavage fluid is poor.

Objective To systematically evaluate the incidence of healthcare-associated infection(HAI)in adult cases with carbapenem-resistant Enterobacterales(CRE)colonization in intestine,and provide referential basis for the prevention and control of HAI in cases colonized with CRE intestinally.Methods Literatures on the incidence of HAI in cases with intestinal CRE colonization were retrieved from 8 databases,including Embase,Cochrane,PubMed,Web of Science,CNKI,Wanfang,VIP,and China Biomedical Literature Database(CBM),dating back from the establishment of the databases to June 2023.Meta-analysis was conducted by Stata 17.0 software.Stabili-ty of the research results was evaluated by sensitivity analysis,and publication bias was evaluated by Egger's test.Results A total of 16 articles were included in the study,with in total 2 151 cases from 5 Chinese articles and 11 English articles.Meta-analysis results showed that the incidence of HAI in adult cases with intestinal CRE coloniza-tion was 23.1％(95％CI:14.8％-32.5％).Subgroup analysis was conducted based on grouping factors,such as different research design types,publication years,as well as research regions,departments,and infection sites.The differences in the combined effects among subgroups were not statistically significant(all P＞0.05).Among the CRE developed from colonization to HAI,the proportion of carpabenem-resistant Klebsiella pneumoniae(CRKP)was 96.0％(95％CI:86.8％-100％),and the incidence of bloodstream infection in colonized cases was 18.2％(95％CI:10.3％-27.6％).The 30-day mortality of CRE colonized cases was 32.6％(95％CI:20.5％-45.9％),and the 30-day mortality of CRE infected cases was 36.9％(95％CI:16.0％-60.2％).Conclusion In recent years,the incidence of HAI in cases with CRE colonization is high,it is necessary to actively screen and focus on intervention in high-risk departments,so as to decrease the incidence of HAI in CRE colonized cases.

BACKGROUND:Osteoporosis is a common comorbidity in patients with knee osteoarthritis.The impact of osteoporosis on the prognosis of unicompartmental knee arthroplasty is a trending topic of current research. OBJECTIVE:To investigate the effect of different bone densities on the stress value and stress distribution of each structure in the joint after unicompartmental knee arthroplasty using finite element analysis,and to evaluate the correlation between osteoporosis and complications. METHODS:CT and MRI were adopted to obtain the lower limb image data of a volunteer.Geomagic Studio,Ansys workbench,and Mimics were used to establish a finite element model of the knee joint with normal sclerotin condition(T-value≥-1.0).The finite element model of the knee joint with osteopenia(-2.5

Objective:To explore the application effect of the modified sandwich teaching method based on constructivism theory in clinical teaching of neurology nursing interns.Methods:A total of 29 nursing interns who practiced in the Department of Neurology of the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) from June 2020 to May 2021 were selected as the control group, using the conventional clinical nursing teaching method, and 28 nursing interns who practiced from June 2021 to May 2022 were selected as the observation group, using the modified sandwich teaching method based on constructivism theory. Before graduating from the Department of Neurology, nursing interns in the two groups were compared in terms of the teaching effects, such as the score of learning initiative, theoretical examination, operation examination, and nursing round report. SPSS 25.0 was used for t test and chi-square test. Results:The score for each dimension (learning driving force, learning objectives, in-depth learning, controlled learning, and solid learning) and total score in the observation group were significantly higher than those in the control group ( P<0.05). There was no significant difference between the two groups in the scores of theoretical examination and operation examination (89.11±3.58 vs. 88.97±2.74, 93.79±2.48 vs. 93.86±2.20; P>0.05); the scores of nursing rounds in the observation group were significantly higher than those in the control group (88.61±2.60 vs. 83.38±3.97, P<0.05). Conclusions:The modified sandwich teaching method based on constructivism theory can enhance the learning initiative and comprehensive analysis ability of nursing interns in the Department of Neurology. It is suitable for clinical nursing teaching in the Department of Neurology and is worth popularizing.