OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins

Objective To explore the resistance mechanism of tigecycline insensitivity of multidrug-resistant Acine-tobacter baumannii,and provide reference for clinical rational antimicrobial use as well as prevention and control of healthcare-associated infection.Methods 22 strains of tigecycline insensitive multidrug-resistant Acinetobacter bau-mannii(TIS-MDR-AB)and 22 strains of tigecycline sensitive multidrug-resistant Acinetobacter baumannii(TS-MDR-AB)isolated clinically from the First Affiliated Hospital of Guangzhou Medical University from April 2022 to May 2023 were collected.Efflux pump phenotype inhibition assay was performed using efflux pump inhibitor car-bonyl cyanide m-chlorophenylhydrazone(CCCP).The main efflux pump genes(adeB,adeG,adeJ),as well as tigecycline-resistant gene tet(X),were screened by polymerase chain reaction(PCR)technique,and their mRNA expression levels were detected by real-time fluorescence quantitative PCR.Mutations in the efflux pump regulatory gene adeRS were analyzed by Sanger sequencing analysis.Results The detection rates of efflux pump genes adeB,adeG and adeJ were all above 95％in two MDR-AB groups,and tet(X)gene was not detected.Efflux pump inhibi-tor assay showed that the minimum inhibitory concentration(MIC)of TIS-MDR-AB strains decreased after adding CCCP,3 strains showed positive efflux pump phenotype.The mRNA expression level of MDR-AB adeB in the TIS-MDR-AB group was higher than that in the TS-MDR-AB group(P＜0.01),while the expression of adeG and adeJ genes was no statistically different.Multiple mutations were found in the adeR and adeS genes,the adeS of 2 strains was inserted ISAba1,and 3 strains were inserted ISAba13.Conclusion The overexpression of adeABC in the efflux pump system may play an important role in the mechanism of reduced sensitivity of MDR-AB to tigecycline,and its overexpression may be related to the insertion sequence or mutation in the efflux pump regulatory gene adeRS.

Objective To explore the resistance mechanism of tigecycline insensitivity of multidrug-resistant Acine-tobacter baumannii,and provide reference for clinical rational antimicrobial use as well as prevention and control of healthcare-associated infection.Methods 22 strains of tigecycline insensitive multidrug-resistant Acinetobacter bau-mannii(TIS-MDR-AB)and 22 strains of tigecycline sensitive multidrug-resistant Acinetobacter baumannii(TS-MDR-AB)isolated clinically from the First Affiliated Hospital of Guangzhou Medical University from April 2022 to May 2023 were collected.Efflux pump phenotype inhibition assay was performed using efflux pump inhibitor car-bonyl cyanide m-chlorophenylhydrazone(CCCP).The main efflux pump genes(adeB,adeG,adeJ),as well as tigecycline-resistant gene tet(X),were screened by polymerase chain reaction(PCR)technique,and their mRNA expression levels were detected by real-time fluorescence quantitative PCR.Mutations in the efflux pump regulatory gene adeRS were analyzed by Sanger sequencing analysis.Results The detection rates of efflux pump genes adeB,adeG and adeJ were all above 95％in two MDR-AB groups,and tet(X)gene was not detected.Efflux pump inhibi-tor assay showed that the minimum inhibitory concentration(MIC)of TIS-MDR-AB strains decreased after adding CCCP,3 strains showed positive efflux pump phenotype.The mRNA expression level of MDR-AB adeB in the TIS-MDR-AB group was higher than that in the TS-MDR-AB group(P＜0.01),while the expression of adeG and adeJ genes was no statistically different.Multiple mutations were found in the adeR and adeS genes,the adeS of 2 strains was inserted ISAba1,and 3 strains were inserted ISAba13.Conclusion The overexpression of adeABC in the efflux pump system may play an important role in the mechanism of reduced sensitivity of MDR-AB to tigecycline,and its overexpression may be related to the insertion sequence or mutation in the efflux pump regulatory gene adeRS.

Objective:To investigate the incidence and influencing factors of pneumothorax in elderly patients(aged ≥65 years)undergoing CT-guided percutaneous lung biopsy(PLB).Methods:A prospective cohort study was conducted, enrolling elderly patients who underwent CT-guided PLB at Beijing Hospital from January 2017 to December 2023.Pneumothorax occurrence was assessed through two CT scans performed immediately post-procedure and at 48 hours post-PLB.Based on pneumothorax status, patients were categorized into a pneumothorax group and a non-pneumothorax group.Baseline clinical and procedural data were compared between the two groups, and multivariate logistic regression analyses were conducted to identify factors associated with pneumothorax.Results:Among the 647 elderly patients who underwent PLB, 152 cases(23.5%, 152/647)developed pneumothorax, with 101 cases(15.6%)presenting with minor pneumothorax and 51 cases(7.9%)with major pneumothorax.Compared with the non-pneumothorax group, patients in the pneumothorax group had a higher incidence of chronic obstructive pulmonary disease(COPD), higher frailty proportion, smaller lesions, lesions located further from the chest wall, more frequent bronchial involvement, and a higher rate of puncture depth ≥5 cm(all P<0.05).Multivariate logistic regression analysis revealed the following as significant risk factors for minor pneumothorax: COPD( OR=1.256, 95% CI: 1.014-1.558, P=0.033), lesion size ≤2 cm( OR=1.345, 95% CI: 1.075-1.659, P=0.022), lesion-to-chest-wall distance ≥2 cm( OR=1.372, 95% CI: 1.105-1.703, P=0.007), presence of pulmonary bullae or emphysema( OR=1.524, 95% CI: 1.223-1.899, P<0.001), ground-glass opacity density( OR=1.313, 95% CI: 1.072-1.839, P=0.014), bronchial involvement( OR=1.211, 95% CI: 1.047-1.694, P=0.038), and needle insertion depth ≥5 cm( OR=1.312, 95% CI: 1.024-1.749, P=0.038).For major pneumothorax, significant risk factors included age( OR=1.286, 95% CI: 1.021-1.819, P=0.041), COPD( OR=1.248, 95% CI: 1.098-1.753, P=0.035), heart failure( OR=1.312, 95% CI: 1.027-1.956, P=0.042), frailty( OR=1.301, 95% CI: 1.063-1.981, P=0.036), lesion size ≤2 cm( OR=1.345, 95% CI: 1.040-1.872, P=0.039), lesion-to-chest-wall distance ≥2 cm( OR=1.482, 95% CI: 1.116-1.738, P=0.032), presence of bullae or emphysema( OR=1.705, 95% CI: 1.316-2.431, P=0.024), and puncture depth ≥5 cm( OR=1.343, 95% CI: 1.058-1.763, P=0.037). Conclusions:Elderly patients undergoing CT-guided PLB have a high risk of pneumothorax.Personalized predictive strategies are necessary to reduce the risk of pneumothorax, particularly major pneumothorax.

Objective:To explore the risk factors of septic acute kidney injury (sAKI) in patients with sepsis, construct a predictive model for sAKI, verify the predictive value of the model, and develop a dynamic nomogram to help clinical doctors identify patients with high-risk sAKI earlier and more easily.Methods:A cross-sectional study was conducted. A total of 245 patients with sepsis admitted to intensive care unit (ICU) of the Affiliated Hospital of Jining Medical University from May 2013 to November 2023 were enrolled as the research subjects. The patients were divided into sAKI group and non-sAKI group based on whether they suffered from sAKI during ICU hospitalization. The differences of the demographic, clinical and laboratory indicators of patients between the two groups were compared. Logistic ordinal regression analysis was performed to analyze the imbalanced variables between the two groups, and to construct a sAKI predictive model. The predictive value of the sAKI predictive model was evaluated through 5-fold cross validation, calibration curve, and decision curve analysis (DCA), and to develop an online dynamic nomogram for the predictive model.Results:A total of 245 patients were enrolled in the final analysis. 110 (44.9%) patients developed sAKI during ICU hospitalization and 135 (55.1%) patients did not develop sAKI. Compared with the non-sAKI group, the patients in the sAKI group had higher ratios of female, hypertension, invasive mechanical ventilation (IMV), renal replacement therapy (RRT), vasopressin usage, and neutrophil count (NEU), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (SCr), uric acid (UA), Na +, K +, procalcitonin (PCT), acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score, and sequential organ failure assessment (SOFA) score. Multivariate Logistic ordinal regression analysis showed that female [odd ratio ( OR) = 2.208, 95% confidence interval (95% CI) was 1.073-4.323, P = 0.020], hypertension ( OR = 2.422, 95% CI was 1.255-5.073, P = 0.012), vasopressin usage ( OR = 2.888, 95% CI was 1.380-6.679, P = 0.002), and SCr ( OR = 1.015, 95% CI was 1.009-1.024, P < 0.001) were independent risk factors for sAKI in septic patients, and a sAKI predictive model was constructed: ln[ P/(1+ P)] = -4.665+0.792×female+0.885×hypertension+1.060×vasopressin usage+0.015×SCr. The 5-fold cross validation showed that the average area under the receiver operator characteristic curve (AUC) was 0.860, indicating the sAKI predictive model had a good performance. The calibration curve analysis showed that the calibration degree of the sAKI predictive model was good. DCA showed that the net profit of the sAKI predictive model was relatively high. A static nomogram and an online dynamic nomogram were constructed for the sAKI predictive model. Compared with the static nomogram, the dynamic nomogram allowed for manual selection of corresponding patient characteristics and viewing the corresponding sAKI risk directly. Conclusions:Female, hypertension, vasopressin usage, and SCr are the main risk factors for sAKI in patients with sepsis. The sAKI predictive model constructed based on these factors can help clinical doctors identifying high-risk patients as early as possible, and intervene in a timely manner to provide preventive effects. Compared with the common static nomogram, online dynamic nomogram can make predictive models clearer, more intuitive, and easier.

Objective:To investigate the incidence and influencing factors of pneumothorax in elderly patients(aged ≥65 years)undergoing CT-guided percutaneous lung biopsy(PLB).Methods:A prospective cohort study was conducted, enrolling elderly patients who underwent CT-guided PLB at Beijing Hospital from January 2017 to December 2023.Pneumothorax occurrence was assessed through two CT scans performed immediately post-procedure and at 48 hours post-PLB.Based on pneumothorax status, patients were categorized into a pneumothorax group and a non-pneumothorax group.Baseline clinical and procedural data were compared between the two groups, and multivariate logistic regression analyses were conducted to identify factors associated with pneumothorax.Results:Among the 647 elderly patients who underwent PLB, 152 cases(23.5%, 152/647)developed pneumothorax, with 101 cases(15.6%)presenting with minor pneumothorax and 51 cases(7.9%)with major pneumothorax.Compared with the non-pneumothorax group, patients in the pneumothorax group had a higher incidence of chronic obstructive pulmonary disease(COPD), higher frailty proportion, smaller lesions, lesions located further from the chest wall, more frequent bronchial involvement, and a higher rate of puncture depth ≥5 cm(all P<0.05).Multivariate logistic regression analysis revealed the following as significant risk factors for minor pneumothorax: COPD( OR=1.256, 95% CI: 1.014-1.558, P=0.033), lesion size ≤2 cm( OR=1.345, 95% CI: 1.075-1.659, P=0.022), lesion-to-chest-wall distance ≥2 cm( OR=1.372, 95% CI: 1.105-1.703, P=0.007), presence of pulmonary bullae or emphysema( OR=1.524, 95% CI: 1.223-1.899, P<0.001), ground-glass opacity density( OR=1.313, 95% CI: 1.072-1.839, P=0.014), bronchial involvement( OR=1.211, 95% CI: 1.047-1.694, P=0.038), and needle insertion depth ≥5 cm( OR=1.312, 95% CI: 1.024-1.749, P=0.038).For major pneumothorax, significant risk factors included age( OR=1.286, 95% CI: 1.021-1.819, P=0.041), COPD( OR=1.248, 95% CI: 1.098-1.753, P=0.035), heart failure( OR=1.312, 95% CI: 1.027-1.956, P=0.042), frailty( OR=1.301, 95% CI: 1.063-1.981, P=0.036), lesion size ≤2 cm( OR=1.345, 95% CI: 1.040-1.872, P=0.039), lesion-to-chest-wall distance ≥2 cm( OR=1.482, 95% CI: 1.116-1.738, P=0.032), presence of bullae or emphysema( OR=1.705, 95% CI: 1.316-2.431, P=0.024), and puncture depth ≥5 cm( OR=1.343, 95% CI: 1.058-1.763, P=0.037). Conclusions:Elderly patients undergoing CT-guided PLB have a high risk of pneumothorax.Personalized predictive strategies are necessary to reduce the risk of pneumothorax, particularly major pneumothorax.

The oral and gastrointestinal opportunistic pathogen contamination was compared between tooth mugs placed upward and down-ward(n=9)for 1 4 days.Selective cultivation of the pathogens was uesd to measure the extent of contamination.The colony forming units (CFU)of colibacillus in group up and group down were 4.25 ±0.71 and 2.84 ±1 .40(P=0.046),S.mutans 89 ±0.31 and 2.84 ±1 .40 (P<0.001 ),Candida 2.28 ±1 .36 and 2.53 ±1 .92(P=0.002),fungus 2.44 ±0.99 and 0,respecitvely.Thus,tooth mug placed open-ing down is superior for health.

OBJECTIVETo investigate the effect of mTOR signal transduction pathway and down-regulating anti-oncogene PTEN on the growth of breast cancer MCF-7 cells.

METHODSMCF-7 cells were transfected with the eukaryotic expression plasmid pcDNA3.1-mTOR and non-loaded plasmid, and the expression of mTOR in the cells was detected using Western blotting. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells, and the expression of PTEN was detected after transfection.

RESULTSThe cells transfected with pcDNA3.1-mTOR showed a increased growth rate than those transfected with the non-loaded plasmid and those without transfection. The expression of the protein PTEN decreased obviously in the cells after mTOR trasnfection.

CONCLUSIONmTOR can regulate the expression of PTEN via PI3K/AKT/PTEN pathways through a negative feedback mechanism. Increased mTOR expression promotes MCF-7 cell growth, suggesting the potential value of mTOR specific inhibitor in the treatment of breast cancer.

Apoptosis ; Breast Neoplasms ; pathology ; Cell Cycle ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; PTEN Phosphohydrolase ; metabolism ; Plasmids ; Signal Transduction ; TOR Serine-Threonine Kinases ; genetics ; Transfection

Objective To investigate the effect of mTOR signal transduction pathway and down-regulating anti-oncogene PTEN on the growth of breast cancer MCF-7 cells. Methods MCF-7 cells were transfected with the eukaryotic expression plasmid pcDNA3.1-mTOR and non-loaded plasmid, and the expression of mTOR in the cells was detected using Western blotting. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells, and the expression of PTEN was detected after transfection. Results The cells transfected with pcDNA3.1-mTOR showed a increased growth rate than those transfected with the non-loaded plasmid and those without transfection. The expression of the protein PTEN decreased obviously in the cells after mTOR trasnfection. Conclusion mTOR can regulate the expression of PTEN via PI3K/AKT/PTEN pathways through a negative feedback mechanism. Increased mTOR expression promotes MCF-7 cell growth, suggesting the potential value of mTOR specific inhibitor in the treatment of breast cancer.

Objective To investigate the effect of mTOR signal transduction pathway and down-regulating anti-oncogene PTEN on the growth of breast cancer MCF-7 cells. Methods MCF-7 cells were transfected with the eukaryotic expression plasmid pcDNA3.1-mTOR and non-loaded plasmid, and the expression of mTOR in the cells was detected using Western blotting. Flow cytometry was used to analyze apoptosis and cell cycle of the transfected cells, and the expression of PTEN was detected after transfection. Results The cells transfected with pcDNA3.1-mTOR showed a increased growth rate than those transfected with the non-loaded plasmid and those without transfection. The expression of the protein PTEN decreased obviously in the cells after mTOR trasnfection. Conclusion mTOR can regulate the expression of PTEN via PI3K/AKT/PTEN pathways through a negative feedback mechanism. Increased mTOR expression promotes MCF-7 cell growth, suggesting the potential value of mTOR specific inhibitor in the treatment of breast cancer.