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.