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

Background::Cardiovascular disease (CVD) has emerged as the leading cause of death from prostate cancer (PCa) in recent decades, bringing a great disease burden worldwide. Men with preexisting CVD have an increased risk for major adverse cardiovascular events when treated with androgen deprivation therapy (ADT). The present study aimed to explore the prevalence and risk evaluation of CVD among people with newly diagnosed PCa in China.Methods::Clinical data of newly diagnosed PCa patients were retrospectively collected from 34 centers in China from 2010 to 2022 through convenience sampling. CVD was defined as myocardial infarction, arrhythmia, heart failure, stroke, ischemic heart disease, and others. CVD risk was estimated by calculating Framingham risk scores (FRS). Patients were accordingly divided into low-, medium-, and high-risk groups. χ2 or Fisher’s exact test was used for comparison of categorical variables. Results::A total of 4253 patients were enrolled in the present study. A total of 27.0% (1147/4253) of patients had comorbid PCa and CVD, and 7.2% (307/4253) had two or more CVDs. The enrolled population was distributed in six regions of China, and approximately 71.0% (3019/4253) of patients lived in urban areas. With imaging and pathological evaluation, most PCa patients were diagnosed at an advanced stage, with 20.5% (871/4253) locally progressing and 20.5% (871/4253) showing metastasis. Most of them initiated prostatectomy (46.6%, 1983/4253) or regimens involving ADT therapy (45.7%, 1944/4253) for prostate cancer. In the present PCa cohort, 43.1% (1832/4253) of patients had hypertension, and half of them had poorly controlled blood pressure. With FRS stratification, as expected, a higher risk of CVD was related to aging and metabolic disturbance. However, we also found that patients with treatment involving ADT presented an originally higher risk of CVD than those without ADT. This was in accordance with clinical practice, i.e., aged patients or patients at advanced oncological stages were inclined to accept systematic integrative therapy instead of surgery. Among patients who underwent medical castration, only 4.0% (45/1118) received gonadotropin releasing hormone antagonists, in stark contrast to the grim situation of CVD prevalence and risk.Conclusions::PCa patients in China are diagnosed at an advanced stage. A heavy CVD burden was present at the initiation of treatment. Patients who accepted ADT-related therapy showed an original higher risk of CVD, but the awareness of cardiovascular protection was far from sufficient.

Objective To assess the effect of ascorbic acid/ferric chloride (AA/FeCl3) in attenuating cartilage damage in rats with osteoarthritis. Methods Thirty adult male Wistar rats with surgically induced osteoarthritis were randomized into 2 groups for treatment with intra-articular injection of saline (control group) or AA/FeCl3 mixture (AA group) once a week starting from the third week after the operation. At 6, 9, and 12 weeks after the operation, 5 rats from each group were sacrificed for observing subchondral bone changes on X-ray films and evaluation of cartilage degeneration in the right knee joints using safranin-O/Fast green staining and a modified OARSI scoring system. The degradation of the cartilage matrix was observed by immunohistochemical staining for type II collagen. Results X-ray examination in saline control group revealed the presence of osteophytes and narrowing of the joint space at 9 weeks, and the joint line disappeared at 12 weeks after the surgery; only slight irregularity of the articular surface was observed in the AA group at 9 and 12 weeks. OARSI scores were significantly lower in AA group than in the control group at 9 weeks (18.67 ± 0.67 vs 12.17 ± 2.75;P<0.05) and 12 weeks (20.11 ± 1.84 vs 13.77 ± 0.40;P<0.05) but not at 6 weeks after the surgery. The content of type 2 collagen in AA group was significantly higher than that in the control group at 6 weeks (0.36 ± 0.039 vs 0.49 ± 0.029;P<0.05) and 9 weeks after the surgery (0.25 ± 0.041 vs 0.38 ± 0.040;P<0.05). Conclusions Early intra-articular injection of AA/FeCl3 can effectively delay the progression of post-traumatic osteoarthritis in rats.

Objective To assess the effect of ascorbic acid/ferric chloride (AA/FeCl3) in attenuating cartilage damage in rats with osteoarthritis. Methods Thirty adult male Wistar rats with surgically induced osteoarthritis were randomized into 2 groups for treatment with intra-articular injection of saline (control group) or AA/FeCl3 mixture (AA group) once a week starting from the third week after the operation. At 6, 9, and 12 weeks after the operation, 5 rats from each group were sacrificed for observing subchondral bone changes on X-ray films and evaluation of cartilage degeneration in the right knee joints using safranin-O/Fast green staining and a modified OARSI scoring system. The degradation of the cartilage matrix was observed by immunohistochemical staining for type II collagen. Results X-ray examination in saline control group revealed the presence of osteophytes and narrowing of the joint space at 9 weeks, and the joint line disappeared at 12 weeks after the surgery; only slight irregularity of the articular surface was observed in the AA group at 9 and 12 weeks. OARSI scores were significantly lower in AA group than in the control group at 9 weeks (18.67 ± 0.67 vs 12.17 ± 2.75;P<0.05) and 12 weeks (20.11 ± 1.84 vs 13.77 ± 0.40;P<0.05) but not at 6 weeks after the surgery. The content of type 2 collagen in AA group was significantly higher than that in the control group at 6 weeks (0.36 ± 0.039 vs 0.49 ± 0.029;P<0.05) and 9 weeks after the surgery (0.25 ± 0.041 vs 0.38 ± 0.040;P<0.05). Conclusions Early intra-articular injection of AA/FeCl3 can effectively delay the progression of post-traumatic osteoarthritis in rats.