OBJECTIVETo investigate the dual role of daphnetin in suppressing high mobility group box-1 protein (HMGB1) release and blocking HMGB1-induced inflammatory response.

METHODSMurine macrophage RAW264.7 cells were cultured in the presence of daphnetin, lipopolysaccharide (LPS), or both. HMGB1 release from the cells was determined using ELISA, and phosphorylations of JAK1/2 and of STAT1 were detected by Western blotting. Human monocytic THP-1 cells exposed to daphnetin, rhHMGB1, or both were examined for NO production using a NO detection kit, for the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) using ELISA, and for expressions of iNOS, COX-2 and phosphorylated p38, ERK, and JNK with Western blotting.

RESULTSDaphnetin dose-dependently reduced the release of HMGB1 in RAW264.7 cells and suppressed rhHMGB1-induced iNOS and COX-2 expressions and release of TNF-α, IL-6, PGE2, and NO in THP-1 cells. Western blotting revealed that daphnetin significantly down-regulated the phosphorylations of JAK-STAT1 pathway in LPS-stimulated RAW264.7 cells but did not suppress the phosphorylations of MAPKs signaling pathway induced by rhHMGB1 in THP-1 cells.

CONCLUSIONDaphnetin can reduce the release of HMGB1 and suppress HMGB1-induced inflammatory response. In RAW264.7 cells, daphnetin inhibited LPS induced HMGB1 release is at least partly mediated by suppressing JAK-STAT1 signaling pathway activation.

Animals ; Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Inflammation ; metabolism ; Interleukin-6 ; metabolism ; Janus Kinase 1 ; metabolism ; Lipopolysaccharides ; Macrophages ; drug effects ; Mice ; Monocytes ; drug effects ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; RAW 264.7 Cells ; STAT1 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism ; Umbelliferones ; pharmacology

OBJECTIVETo evaluate the therapeutic effect of MHSP65-TCL on melanoma and its effect on the activity of the immunocytes.

METHODSMHSP65-TCL was prepared by mixing MHSP65 with TCL derived from B16 melanoma cell lysate by repeated freezing and thawing. The MHSP65-TCL vaccine was administered in mice bearing B16 melanoma, and the changes in melanoma growth was observed. To investigate the influence of TCL in MHSP65-TCL on the activity of the immunocytes, we co-cultured TCL and mouse spleen cells in vitro, and analyzed CD69 expression on the cells, cell apoptosis, and levels of IL-10 and IFN-γ in the cell culture supernatant.

RESULTSThe MHSP65-TCL vaccine showed an anti-melanoma effect in the tumor-bearing mice. In the in vitro experiment, TCL in MHSP65-TCL strongly stimulated the activation of mouse spleen cells while causing apoptosis in some of the immunocytes and promoting cellular IL-10 secretion, but not IFN-γ.

CONCLUSIONSMHSP65-TCL derived from B16 melanoma cells has an anti-melanoma effect mediated by the activation of immunocytes. TCL in MHSP65-TCL also has immunosuppressive effect on immunocytes possibly due to the presence of suppressive components in TCL, and identifying and eliminating these components may potentially improve the anti-tumor actovoty of MSHP65-TCL vaccine.

Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; metabolism ; Apoptosis ; Bacterial Proteins ; administration & dosage ; immunology ; Cancer Vaccines ; Cell Extracts ; administration & dosage ; immunology ; Cell Line, Tumor ; Chaperonin 60 ; administration & dosage ; immunology ; Female ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Lectins, C-Type ; metabolism ; Melanoma, Experimental ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Spleen ; cytology ; immunology ; metabolism ; Tumor Burden ; immunology

Objective To analyze the related risk factors affecting the prognosis of elderly patients with severe community acquired pneumonia (SCAP). Methods A retrospective study method was conducted; the elderly (≥ 75 years old) patients with SCAP treated in the First Affiliated Hospital of Hainan Medical College from January 2015 to January 2019 were enrolled. The general data of patients were collected, including sex, age, oxygenation index (PaO2/FiO2), involved organs, presence or absence of following diseases or treatment: damage in multiple lung lobes, septic shock, basic diseases (cardiovascular disease, chronic lung disease, diabetes, hypertension, and cerebrovascular disease), invasive mechanical ventilation, ventilator-associated pneumonia (VAP), misinhalation event, hyponatremia, respiratory acidosis, hypoproteinemia, intubation times, total mechanical ventilation time, etc. According to the prognosis, the patients were divided into a death group and a survival group. The general data were compared between the two groups with different prognoses. Single factor analysis was carried out by selecting variables. The indicators with statistical significant differences in the results of univariate analysis were introduced into the multivariate Logistic regression analysis to analyze the related risk factors affecting the prognosis of elderly patients with SCAP. The receiver operating characteristic (ROC) curve was drawn to analyze the predictive values of risk factors in the patients with SCAP. Results A total of 112 patients were included, 33 died, and the mortality rate was 29.46%. Univariate analysis showed that the following factors were higher in the death group than those in the survival group: organ involvement >2 [69.70% (23/33) vs. 35.44% (28/79)], lung lobe damage ≥ 3 [75.76% (25/33) vs. 51.90% (41/79)], invasive mechanical ventilation [72.73% (24/33) vs. 32.91% (26/79)], diabetes [30.30% (10/33) vs. 12.66% (10/79)], intubation times ≥2 [57.58% (19/33) vs. 48.10% (38/79)], hypoproteinemia [75.76% (25/33) vs. 41.77% (33/79)], hyponatremia [72.73% (24/33) vs. 48.10% (38/79)], respiratory acidosis [66.67% (22/33) vs. 44.30 %(35/79)] and total mechanical ventilation time ≥ 15 days [69.70% (23/33) vs. 40.51 (32/79)]; the factors in the death group lower than those in the survival group were: septic shock [3.03% (1/33) vs. 17.72% (14/79)], chronic lung disease [6.06% (2/33) vs. 25.32% (20/79)] and PaO2/FiO2 [mmHg (1 mmHg = 0.133 kPa): 102.89±14.78 vs. 109.56±14.08],the differences were statistically significant (all P < 0.05); there were no significant differences in gender, age, cardiovascular disease, hypertension, VAP, misinhalation events and cerebrovascular disease between the two groups (all P > 0.05). Multivariate Logistic regression analysis showed that diabetes mellitus [odds ratio (OR) = 1.074, 95% confidence interval (95%CI) = 1.017-1.287, P =0.045], septic shock (OR = 2.765, 95%CI = 1.083-3.411, P = 0.047), hyponatremia (OR = 1.792, 95%CI = 1.128-1.417, P = 0.006), hypoalbuminemia (OR = 2.187, 95%CI = 1.872-5.462, P = 0.046), invasive mechanical ventilation (OR = 5.870, 95%CI = 2.324-23.796, P = 0.001), respiratory acid poisoning (OR = 2.934, 95%CI = 2.454-7.275, P = 0.043), time of mechanical ventilation (OR= 1.986, 95%CI = 2.467-3.483, P = 0.034), number of intubation (OR = 6.760, 95%CI = 2.116-24.696, P = 0.001), PaO2/FiO2 (OR = 1.981, 95%CI = 1.006-1.417, P = 0.007), organ involvement > 2 (OR = 2.924, 95%CI = 2.534-6.285, P = 0.048), chronic lung disease (OR = 2.887, 95%CI = 1.487-3.483, P = 0.039), and lung lobe damage≥3 (OR = 2.754, 95%CI = 1.131-1.798, P = 0.045) were independent risk factors affecting the prognosis of elderly patients with SCAP. ROC analysis showed that hyponatremia, hypoalbuminemia, invasive mechanical ventilation, total mechanical ventilation time, PaO2/FiO2, organ involvement > 2, damage of lung lobes ≥ 3, had predictive values for the prognosis of SCAP [the areas under ROC curve (AUC) were 0.377, 0.267, 0.301, 0.646, 0.650, 0.329, and 0.381, respectively, all P < 0.05]. Conclusions Underlying disease, invasive mechanical ventilation, respiratory acidosis, total mechanical ventilation time, PaO2/FiO2, intubation times ≥ 2, chronic lung disease and lung damage≥ 3 lobes are the independent risk factors for the prognosis of elderly patients with severe community acquired pneumonia. Clinical treatment should focus on the above aspects to minimize the mortality of patients.

Objective To analyze the clinical values of super early enteral nutrition combined with microecopharmaceutics and delayed enteral nutrition on patients with severe acute pancreatitis. Methods Clinical data of thirty patients diagnosed as severe acute pancreatitis in our emergency department during January 2013 and December 2017 were reviewed retrospectively. Patients were divided into the treatment group (n=15, patients given enteral nutrition combined with microecopharmaceutics within 24 h after admission) and the control group (n=15, patients given delayed enteral nutrition after 48 h of admission). Two weeks after the treatment, the serum variables of C-reactive protein, total protein, albumin, recovery time of urine and blood amylase, length of hospital stay and APACHE Ⅱ score were compared between the two groups by using paired samples t test. Results The C-reactive protein [(46.7±13.1) mg/L vs. (190.72±19.3) mg/L, t=10.4, P<0.01] and APACHE Ⅱ score [(7.2±1.9) vs.(9.3±2.4),t=2.7,P<0.05] of the treatment group were significantly lower than those in the control group. The total protein [(58.1±6.3)g/L vs.(52.6±5.4)g/L, t=2.5, P<0.05] and albumin [(29.9±3.2)g/L vs.(22.0±2.8)g/L, t=7.12, P<0.01] of the treatment group were significantly higher than those in the control group. The recovery time of urine amylase [(13.2±2.1)d vs.(18.7±3.9)d, t=4.9, P<0.01] and blood amylase [(7.5±3.0)d vs.(11.1±3.4)d, t=3.1, P<0.01], and length of hospital stay[(14.9±4.5)d vs.(27.1±5.3)d, t=6.9, P<0.01] were significantly shorter in the treatment group compared with those in the control group. Conclusions Ultra-early enteral nutrition combined with microecopharmaceutics can shorten the length of hospital stay of patients with severe acute pancreatitis, and is safe and effective.

OBJECTIVE: To investigate the inhibitory effects of dihydromyricetin on the proliferation and migration of gastric cancer BGC-823 cells and explore the molecular mechanisms. METHODS: BGC-823 cells in routine culture were treated with different concentrations of dihydromyricetin (0, 40, 60, 80, 100, and 120 μg/mL) for 24 h, and the changes in cell viability were detected using CCK-8 assay; colony forming assay and Transwell assay were performed to assess the changes in colonyforming and migration abilities of the cells, respectively. The levels of MMP-2 and MMP-9 in the treated cells were determined using ELISA, and Western blotting was used to detect the expressions of E-cadherin, N-cadherin, cyclin D1, cyclin E1, HSP70 and HMGB1 and the phosphorylation levels of Akt and Stat3. RESULTS: CCK-8 assay showed that dihydromyricetin treatment dose-dependently inhibited the viability of BGC-823 cells ( CONCLUSIONS Dihydromyricetin inhibits the proliferation and migration of BGC-823 cells through suppressing the activation of Akt/stat3 signaling pathways and HMGB1 expression.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Flavonols ; HMGB1 Protein/metabolism* ; Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; STAT3 Transcription Factor ; Stomach Neoplasms

Objective: To explore the molecular mechanism of resveratrol (RES) in the treatment of oral squamous cell carcinoma (OSCC) through the use of biological information methods such as network pharmacology and molecular docking and to provide a theoretical reference for the clinical application of RES in the treatment of OSCC. Methods: The Swiss Target Prediction(http://www.swisstargetprediction.ch), SEA (http://sea.bkslab.org)database, and Pharm mapper database(http://lilab-ecust.cn) were used to retrieve RES-related targets, and the DISGENET (www.disgenet.org), OMIM (https://omim.org) and GeneCards (https://www.genecards.org) databases were used to screen OSCC disease targets. The intersection of drugs and disease targets was determined, and Cytoscape 3.7.2 software was used to construct a "drug-diseasetarget pathway" network. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a target protein interaction network, and the DAVID database was used for enrichment analysis of key proteins. Finally, molecular docking validation of key proteins was performed using AutoDock and PyMOL. The enrichment analysis and molecular docking results were integrated to predict the possible molecular mechanisms of RES treatment in OSCC; western blot was used to determine the effect of resveratrol at different concentrations (50, 100) μmol/L on the expression of Src tyrosine kinase (SRC), epidermal growth factor receptor (EGFR), estrogen receptor gene 1 (ESR1), and phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway proteins in OSCC HSC-3 cells. Results: A total of 243 targets of RES drugs and 6 094 targets of OSCC were identified. A total of 116 potential common targets were obtained by intersecting drugs with disease targets. These potential targets mainly participate in biological processes such as in vivo protein self-phosphorylation, peptide tyrosine phosphorylation, transmembrane receptor protein tyrosine kinase signaling pathway, and positive regulation of RNA polymerase Ⅱ promoter transcription, and they interfere with the PI3K/AKT signaling pathway to exert anti-OSCC effects. The docking results of resveratrol with OSCC molecules indicated that key targets, such as EGFR, ESR1, and SRC, have good binding activity. The results of cell-based experiments showed that resveratrol inhibited the protein expression of SRC, EGFR, ESR1, p-PI3K, and p-AKT in HSC-3 cells in a dose-dependent manner. Conclusion RES can inhibit the expression of its targets EGFR, ESR1, SRC, p-PI3K, and p-AKT in OSCC cells.

OBJECTIVE: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism. METHODS: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 μg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 μg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy. RESULTS: Calenduloside E below 20 μg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1β, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells. CONCLUSIONS Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.
Animals ; Lipopolysaccharides ; Mice ; NF-kappa B ; Oleanolic Acid ; analogs & derivatives ; RAW 264.7 Cells ; Reactive Oxygen Species ; Saponins ; Signal Transduction

OBJECTIVETo study the effect of chrysin in inducing apoptosis of human hepatic carcinoma cells and explore the possible mechanism.

METHODSHuman hepatic carcinoma SMMC-7721 cells treated with DMSO or chrysin at different concentrations (5-200 μg/mL) were examined for changes in the cell proliferation using CCK-8 assay. The morphological changes of SMMC-7721 cells were observed in response to treatment with 5, 10, or 20 μg/mL chrysin, and the changes in the cell nuclei were observed using DAPI nuclear staining. Annexin Ⅴ-FITC/PI flow cytometry was used to determine the cell apoptosis rate. The changes in the apoptosis-related proteins (PARP and caspase-3) and MAPKs signal pathway were detected with Western blotting.

RESULTSChrysin treatment obviously suppressed the proliferation of SMMC-7721 cells in a dose-dependent manner below the concentration of 60 μg/mL. Chrysin (20 μg/mL) also caused significantly increased cell apoptosis and significant cleavage of PARP and caspase-3. Chrysin significantly activated MAPKs signaling pathway in a time-and dose-dependent manner, with the peak activation level occurring at 15 min. Pretreatment of the cells with specific inhibitors of the MAPKs pathway obviously inhibited the effect of chrysin in inducing cell apoptosis.

CONCLUSIONSChrysin inhibits the proliferation and promotes apoptosis of SMMC-7721 cells by regulating the activation of MAPKs signaling.

OBJECTIVE: To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells. METHODS: Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining. RESULTS: Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01). CONCLUSION Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.
Humans ; Animals ; Mice ; Stomach Neoplasms ; Cyclin B1 ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; HMGB1 Protein ; Signal Transduction ; Cell Proliferation ; STAT3 Transcription Factor

OBJECTIVETo investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism.

METHODSGastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells.

RESULTSAloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells.

CONCLUSIONSAloin induces apoptosis of human gastric cancer cells by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.