Microarray analysis of tRNA-derived small RNA (tsRNA) in LPS-challenged macrophages treated with metformin.

Metformin, a widely used anti-diabetic drug, has demonstrated its efficacy in addressing various inflammatory conditions. tRNA-derived small RNA (tsRNA), a novel type of small non-coding RNA, exhibits diverse regulatory functions and holds promise as both a diagnostic biomarker and a therapeutic target for various diseases. The purpose of this study is to investigate whether the abundance of tsRNAs changed in LPS versus LPS + metformin-treated cells, utilizing microarray technology. Firstly, we established an in vitro lipopolysaccharide (LPS)-induced inflammation model using RAW264.7 macrophages and assessed the protective effects of metformin against inflammatory damage. Subsequently, we extracted total RNA from both LPS-treated and metformin + LPS-treated cell samples for microarray analysis to identify differentially abundant tsRNAs (DA-tsRNAs). Furthermore, we conducted bioinformatics analysis to predict target genes for validated DA-tsRNAs and explore the biological functions and signaling pathways associated with DA-tsRNAs. Notably, metformin was found to inhibit the inflammatory response in RAW264.7 macrophages. The microarray results revealed a total of 247 DA-tsRNAs, with 58 upregulated and 189 downregulated tsRNAs in the Met + LPS group compared to the LPS group. The tsRNA-mRNA network was visualized, shedding light on potential interactions. The results of bioinformatics analysis suggested that these potential targets of specific tsRNAs were mainly related to inflammation and immunity. Our study provides compelling evidence that metformin exerts anti-inflammatory effects and modulates the abundance of tsRNAs in LPS-treated RAW264.7 macrophages. These findings establish a valuable foundation for using tsRNAs as potential biomarkers for metformin in the treatment of inflammatory conditions.

Transcriptome and single-cell transcriptomics reveal prognostic value and potential mechanism of anoikis in skin cutaneous melanoma.

BACKGROUND: Skin cutaneous melanoma (SKCM) is a highly lethal cancer, ranking among the top four deadliest cancers. This underscores the urgent need for novel biomarkers for SKCM diagnosis and prognosis. Anoikis plays a vital role in cancer growth and metastasis, and this study aims to investigate its prognostic value and mechanism of action in SKCM. METHODS: Utilizing consensus clustering, the SKCM samples were categorized into two distinct clusters A and B based on anoikis-related genes (ANRGs), with the B group exhibiting lower disease-specific survival (DSS). Gene set enrichment between distinct clusters was examined using Gene Set Variation Analysis (GSVA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. RESULTS: We created a predictive model based on three anoikis-related differently expressed genes (DEGs), specifically, FASLG, IGF1, and PIK3R2. Moreover, the mechanism of these prognostic genes within the model was investigated at the cellular level using the single-cell sequencing dataset GSE115978. This analysis revealed that the FASLG gene was highly expressed on cluster 1 of Exhausted CD8( +) T (Tex) cells. CONCLUSIONS: In conclusion, we have established a novel classification system for SKCM based on anoikis, which carries substantial clinical implications for SKCM patients. Notably, the elevated expression of the FASLG gene on cluster 1 of Tex cells could significantly impact SKCM prognosis through anoikis, thus offering a promising target for the development of immunotherapy for SKCM.

Associated predictors of prolonged length of stay in patients surviving extensive burns: A large multicenter retrospective study.

BACKGROUND: Patients with extensive burns are critically ill and have long treatment periods. Length of stay (LOS) is a good measure for assessing treatment. This study sought to identify predictors of prolonged LOS in patients with extensive burns (≥50% TBSA). METHODS: This retrospective multicenter cohort study included adults aged ≥ 18 years who survived extensive burns in three burn centers in Eastern China between January 2016 and June 2022. Epidemiological, demographic and clinical outcomes data were extracted from electronic medical records and compared between patients with/without prolonged LOS, which was defined as LOS greater than the median. Logistic regression analysis was used to identify predictors of prolonged LOS. RESULTS: The study sample included 321 patients, of whom 156 (48.6%) had an LOS of 58 days (IQR 41.0-77.0). Univariate regression analysis showed that increased total burn area and increased full-thickness burn area; electrical, chemical and other burns; increased erythrocytes, leukocytes, platelets or serum creatinine within 24 h of admission; concomitant inhalation injury, pulmonary edema, sepsis, bloodstream infection, wound infection, pulmonary infection, urinary tract infection, or HB < 70 g/L during hospitalization were associated with prolonged LOS in patients with extensive burns. Increased number of surgical operations, mechanical ventilation and renal replacement therapy were also associated with prolonged LOS (P < 0.05 or P < 0.001). Multivariate regression analysis revealed that increased total burn area (ratio 1.032, 95%CI 1.01-1.055; P = 0.004), electrical and chemical or other burns (3.282, 1.335-8.073; P = 0.01), development of wound infection (2.653 1.285-5.481; P = 0.008) and increased number of operative procedures (1.714, 1.388-2.116, P < 0.001) were significant predictors. CONCLUSIONS: Increased area of full-thickness burn,occurrence of electrical and chemical or other burns,occurrence of wound infection and increased number of surgeries are the best predictors of prolonged LOS in patients with extensive burns. Clarifying relevant predictors of burn patients' LOS provides a reliable reference for clinical treatment.

Regulatory Effect of Ellagic Acid on Immune Function in Burned Rats.

To investigate the effect of ellagic acid (EA) treatment on immune function in burned rats. First, 30 Sprague-Dawley rats were established as a deep second-degree burn model. They were randomly divided into three groups: Model group, EA 50 mg/kg, and EA 100 mg/kg group. The wound area of rats at 0-7 days was measured and the wound healing rate was calculated. The levels of inflammatory factors tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin (IL)-1ß, IL-6, IL-10, and immunoglobulins IgA, IgG, and IgM in rat serum were evaluated by ELISA. Flow cytometry was used to detect the CD4 +/CD8 + T cell ratio, levels of Foxp3 + Treg cells, and CD4 + CD25 + regulatory T cells (Treg) cells levels in the peripheral blood of rats. On the fourth to seventh day of the burn, EA treatment could significantly promote the decrease of the wound area and the increase of the wound healing rate in burned rats. Further examination revealed that the levels of inflammatory factors in serum were remarkedly decreased and immunoglobulins levels were increased in the EA group, compared with the Model group. Meanwhile, the levels of CD4 + CD25 + Treg cells and Foxp3+ Treg cells were significantly decreased, whereas the CD4+/CD8 + T cell ratio was observably increased in a concentration-dependent manner. Altogether, EA effectively promotes the wound healing of burned rats by regulating the levels of inflammatory factors, immunoglobulin, and T cells in burned rats, and improves the symptoms of burn immunosuppression.

[Effects of extracellular heat shock protein 70 on intestinal immune function of rats with severe scald injury].

OBJECTIVE: To explore the change in the expression of extracellular heat shock protein 70 (eHSP70) and interleukin 2 (IL-2) and their correlation in intestine of rats with severe scald injury, and to observe the effects of eHSP70 on CD3(+) T lymphocytes in Peyer's patch of intestine in rats with severe scald injury in vitro. METHODS: (1) Sixty male SD rats were divided into normal control group (NC, n=10, only anesthetized) and scald group (S, n=50) according to the random number table. Rats in scald group were inflicted with 30% total body surface area full-thickness scald on the back. Ten rats from group NC immediately after anesthetization and 10 rats from group S at post injury hour (PIH) 3, 6, 12, 24, 48 were sacrificed to harvest their small intestines. The expressions of eHSP70 and IL-2 were determined with enzyme-linked immunosorbent assay (ELISA), and their correlation was analyzed. (2) Another 2 male SD rats were inflicted with the same injury as above. At PIH 12, CD3(+) T lymphocytes in Peyer's patch of small intestine were isolated and cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum. Cells were divided into blank control group (BC) and 5, 10, 20 µg/mL eHSP70 groups according to the random number table, with 6 wells in each group. Cells in group BC didn't receive any other treatment, while cells in the latter three groups were treated with corresponding mass concentration of recombinant rat eHSP70. After being cultured for 48 hours, the proportions of Th1 and Th2 in CD3(+) T lymphocytes, and the apoptosis rate of CD3(+) T lymphocytes were detected with flow cytometer, while the expressions of IL-2 and IL-10 in culture supernatant of cells were determined with ELISA. The cell experiments were repeated for 10 times. Data were processed with one-way analysis of variance, Kruskal-Wallis rank sum test, SNK-q test, and Pearson correlation analysis. RESULTS: (1) Compared with those in group NC [(1 278±135) and (48.6±4.9) ng/mg], the levels of eHSP70 [(728±93), (412±31), (314±21), (528±40), (1 028±97) ng/mg] and IL-2 [(38.6±2.3), (32.3±1.0), (25.3±3.6), (33.9±4.1), (44.3±2.6) ng/mg] in intestine of rats in group S obviously decreased at PIH 3, 6, 12, 24, 48 (with q values from 3.48 to 5.32, P values below 0.05), reaching the nadir both at PIH 12, with a significantly positive correlation between the level of IL-2 and the level of eHSP70 (r=0.920, P<0.01). (2) Compared with those in group BC [(8.6±1.1)% and (3.75±0.45)%], the proportion of Th1 obviously increased [(11.3±2.1)%, (15.7±1.8)%, (10.8±1.5)%, with q values from 2.97 to 4.57, P values below 0.05], while the proportion of Th2 obviously decreased [(2.39±0.38)%, (1.05±0.23)%, (2.67±0.26)%, with q values from 2.48 to 4.32, P values below 0.05] in CD3(+) T lymphocytes of rats in 5, 10, 20 µg/mL eHSP70 groups. Compared with those in group BC [(34.3±2.2)% and (254±16) pg/mL], the apoptosis rate of CD3(+) T lymphocytes obviously decreased [(26.1±2.6)%, (20.7±1.5)%, (31.5±2.4)%, with q values from 3.47 to 4.95, P values below 0.05], while the level of IL-2 obviously increased [(417±22), (587±19), (307±27) pg/mL, with q values from 3.02 to 4.98, P values below 0.05] in culture supernatant of CD3(+) T lymphocytes of rats in 5, 10, 20 µg/mL eHSP70 groups. There was no significant difference in the level of IL-10 in culture supernatant of CD3(+) T lymphocytes of rats among the four groups (F=2.12, P>0.05). CONCLUSIONS: The expressions of eHSP70 and IL-2 in intestine of rats are decreased after severe scald, with a obviously positive correlation between them. eHSP70 can promote the differentiation of CD3(+) T lymphocytes in Th1 orientation, decrease the apoptosis rate of the cells, and promote the release of IL-2 of cells in Peyer's patch of intestine in rats with severe scald injury in vitro.