Predictive Value of Serum Inflammatory Factors and FT3 for Stroke-Associated Pneumonia in Patients With Acute Ischemic Stroke.

OBJECTIVE: The ability of serum inflammatory factors and free triiodothyronine (FT3) in predicting the occurrence of stroke-associated pneumonia (SAP) in patients with acute ischemic stroke (AIS) was assessed in this study. METHODS: A retrospective analysis was conducted on 285 consecutive patients with AIS initially diagnosed and admitted to our hospital from January to December 2022. Patients were categorized into SAP and non-SAP groups based on the presence of SAP. Both groups were compared in terms of baseline characteristics, including National Institute of Health Stroke Scale (NIHSS) score, SAP risk assessment (A2DS2), TOAST classification. Independent risk factors for SAP were identified using multivariate logistic regression analysis, and the predictive value of inflammatory markers was evaluated through ROC curves. RESULTS: Among 285 patients with AIS, 40 (14.03%) were found to have developed SAP. Higher NIHSS and A2DS2 scores, elevated serum IL-1ß, IL-8, and IL-33 levels, increased age, atrial fibrillation, swallowing difficulties, and a higher proportion of patients with low FT3 levels were observed in the SAP group compared with the non-SAP group (all P<0.05). Significant risk factors for SAP in patients with AIS were identified through multivariate logistic regression analysis, including age, swallowing difficulties, NIHSS, A2DS2 , IL-1ß , IL-8 , IL-33, and FT3 (P<0.05). The highest predictive values were observed for A2DS2, FT3, and IL-8 with AUC values of 0.854, 0.844, and 0.823, respectively. CONCLUSION: SAP can be highly predicted by A2DS2, FT3, and IL-8, enabling the early identification of patients with high-risk SAP and facilitating timely intervention and treatment.

[Mechanism of large-conductance calcium-activated potassium channel involved in inflammatory response in sepsis].

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 µg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1ß precursor (pro-IL-1ß) in cell, and the levels of caspase-1 p20, IL-1ß p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 µg/L LPS were significantly higher than that in the blank group (0 µg/L) [BKCa mRNA (2-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/ß-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1ß p17/pro-IL-1ß in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1ß p17/pro-IL-1ß: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1ß p17/pro-IL-1ß: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS: BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.

[Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity].

OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION: DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

[Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells].

OBJECTIVE: To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells. METHODS: DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/ß- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced. RESULTS: The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of ß- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05). CONCLUSION: Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/ß- catenin signaling pathway.

Bioinformatics analysis of high frequency mutations in myelodysplastic syndrome-related patients.

BACKGROUND: Myelodysplastic syndrome (MDS) is a group of hematological malignancies that may progress to acute myeloid leukemia (AML). Bioinformatics-based analysis of high-frequency mutation genes in MDS-related patients is still relatively rare, so we conducted our research to explore whether high-frequency mutation genes in MDS-related patients can play a reference role in clinical guidance and prognosis. METHODS: Next generation sequencing (NGS) technology was used to detect 32 mutations in 64 MDS-related patients. We classified the patients' genes and analyzed them by Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, protein-protein interaction (PPI) analysis, and then calculated the gene survival curve of high-frequency mutations. RESULTS: We discovered 32 mutant genes such as ASXL1, DNMT3A, KRAS, NRAS, TP53, SF3B1, and SRSF2. The overall survival (OS) of these genes decreased significantly after DNMT3A, ASXL1, RUNX1, and U2AF1 occurred mutation. These genes play a significant role in biological processes, not only in MDS but also in the occurrence and development of other diseases. Through retrospective analysis, genes associated with MDS-related diseases were identified, and their effects on the disease were predicted. CONCLUSIONS: Thirty-two mutant genes were determined in MDS and when mutations occur in DNMT3A, ASXL1, RUNX1, and U2AF1, their survival time decreases significantly. This results providing a theoretical basis for clinical and scientific research and broadening the scope of research on MDS.

MicroRNA-18a-5p regulates the Warburg effect by targeting hypoxia-inducible factor 1α in the K562/ADM cell line.

The Warburg effect is involved in drug resistance and recurrence of cancer, and poses a challenge for the treatment of chronic myelogenous leukemia (CML). Hypoxia-inducible factor 1α (HIF-1α) plays a key role in the Warburg effect. microRNAs (miRs) targeting HIF-1α have potential of regulating such aberrant metabolic process. The present study demonstrated that miR-18a-5p was expressed at a low level in K562/ADM cells via reverse transcription-quantitative PCR (RT-qPCR). The results of the luciferase reporter assay indicated that miR-18a-5p could specifically bind the 3'-untranslated region of HIF-1α. Through RT-qPCR and western blotting, it was revealed that miR-18a-5p downregulated the expression of HIF-1α. By inhibiting HIF-1α, miR-18a-5p suppressed aerobic glycolysis in K562/ADM cells, according to the results produced by glucose uptake, lactate production, pyruvate level and ATP synthesis measurement, along with the results obtained from extracellular acidification rate and oxygen consumption rate assays. These results provided new evidence that miR-18a-5p may suppress the Warburg effect by targeting HIF-1α. Furthermore, via CCK-8 and flow cytometry assays, cells transfected with miR-18a-5p mimics were more sensitive to Adriamycin (AMD) compared with AMD group. Reversing the Warburg effect by miR-30a-5p might provide a potential therapeutic strategy for CML.

[Effects of WNK1 on Human Chronic Myeloid Leukemia K562 Cells via MAPK7 Phosphorylation and Its Relative Mechanism].

OBJECTIVE: To investigate the biological effects and mechanism of WNK1 on K562 cells through regulating MAPK7. METHODS: Cells were routinely cultured in vitro, the expression of WNK1 and MAPK7 in different blood tumor cell lines was analyzed by RT-qPCR and Western blot analysis.K562 cells were transfected with siRNA-WNK1 lentivirus.The effect of WNK1 on K562 cell proliferation was analyzed by using CCK-8 reagent.And K562 cell apoptosis was analyzed by using flow cytometry. The expression level of phosphorylated MAPK7 protein and total MAPK7 protein in K562 cells was analyzed by Western blot. RESULTS: The mRNA and protein of WNK1 were highly expressed in HL60, THP-1, U266 and K562 cells, however, the expressions were the highest in K562 cells (P<0.05), while the changes of mRNA and protein expressions of MAPK7 were not significantly in HL60, THP-1, U266 and K562 cells (P>0.05), but the phosphorylated MAPK7 expression was the highest in K562 cells (P<0.05). Proliferation of K562 cells transfected by WNK siRNA was significantly suppressed, and apoptosis was obviously increased (P<0.05). And the pMAPK7 protein expression in K562 cells transfected by WNK1 siRNA significantly decreased (P<0.05), however, the total MAPK7 protein expression in K562 cells showed no obvious change (P>0.05). CONCLUSION: WNK1 is highly expressed in K562 cells, which can promote the proliferation of K562 cells and reduce apoptosis probably by promoting phosphorylation of its downstream MAPK7.