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-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β 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/β-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-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 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-1β p17/pro-IL-1β: 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.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases

BACKGROUND: Exposure to the ionizing radiation (IR) encountered outside the magnetic field of the Earth poses a persistent threat to the reproductive functions of astronauts. The potential effects of space IR on the circadian rhythms of male reproductive functions have not been well characterized so far. METHODS: Here, we investigated the circadian effects of IR exposure (3 Gy X-rays) on reproductive functional markers in mouse testicular tissue and epididymis at regular intervals over a 24-h day. For each animal, epididymis was tested for sperm motility, and the testis tissue was used for daily sperm production (DSP), testosterone levels, and activities of testicular enzymes (glucose-6-phosphate dehydrogenase (G6PDH), sorbitol dehydrogenase (SDH), lactic dehydrogenase (LDH), and acid phosphatase (ACP)), and the clock genes mRNA expression such as Clock, Bmal1, Ror-α, Ror-β, or Ror-γ. RESULTS: Mice exposed to IR exhibited a disruption in circadian rhythms of reproductive markers, as indicated by decreased sperm motility, increased daily sperm production (DSP), and reduced activities of testis enzymes such as G6PDH, SDH, LDH, and ACP. Moreover, IR exposure also decreased mRNA expression of five clock genes (Clock, Bmal1, Ror-α, Ror-β, or Ror-γ) in testis, with alteration in the rhythm parameters. CONCLUSION These findings suggested potential health effects of IR exposure on reproductive functions of male astronauts, in terms of both the daily overall level as well as the circadian rhythmicity.
ARNTL Transcription Factors/genetics* ; Acid Phosphatase ; Animals ; CLOCK Proteins/genetics* ; Circadian Rhythm/radiation effects* ; Epididymis/radiation effects* ; Gene Expression/radiation effects* ; Genitalia, Male/radiation effects* ; Glucosephosphate Dehydrogenase ; L-Iditol 2-Dehydrogenase ; L-Lactate Dehydrogenase ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Nuclear Receptor Subfamily 1, Group F, Member 1/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 2/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; RNA, Messenger/genetics* ; Radiation Exposure ; Radiation, Ionizing ; Reproductive Physiological Phenomena/radiation effects* ; Sperm Motility/radiation effects* ; Spermatozoa/radiation effects* ; Testis/radiation effects*

PURPOSE: Parkinson's disease (PD) is a common age-dependent neurodegenerative disease. MiR-212 has been demonstrated to exert protective effects in several neurological disorders. The present study aimed to investigate the role and underlying molecular mechanism of miR-212 in PD. MATERIALS AND METHODS: 1-methyl-4-phenylpyridinium (MPP+)-induced SH-SY5Y cells were applied as a PD model in vitro. RTqPCR was used to measure the expression of miR-212 and Kruppel-like factor 4 (KLF4) mRNA. Western blot analysis was performed to detect the protein levels of KLF4, Notch1 and Jagged1. Cell viability and apoptosis were determined by the Cell Counting Kit-8 and flow cytometry, respectively. Quantitative analysis of caspase-3 activity, lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1β) was conducted with corresponding ELISA kits. Dual-luciferase reporter assay was employed to evaluate the relationship between miR-212 and KLF4. RESULTS: MiR-212 was downregulated in MPP+-induced SH-SY5Y cells. Also, miR-212 alleviated MPP+-induced SH-SY5Y cell damage, embodied by increased cell viability, decreased caspase-3 activity, LDH release, ROS production, TNF-α, and IL-1β expression, as well as elevated SOD levels. KLF4 was a direct target of miR-212, and miR-212 repressed KLF4 expression in a post-transcriptional manner. Moreover, miR-212-mediated protection effects were abated following KLF4 expression restoration in MPP+-induced SH-SY5Y cells, represented as lowered cell viability and enhanced apoptotic rate. Furthermore, Notch signaling was involved in the regulation of miR-212/KLF4 axis in MPP+-induced SH-SY5Y cells. CONCLUSION: miR-212 might attenuate MPP+-induced neuronal damage by regulating KLF4/Notch signaling pathway in SH-SY5Y cells, a promising target for PD therapy.
1-Methyl-4-phenylpyridinium ; Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Count ; Cell Survival ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; In Vitro Techniques ; Interleukin-1beta ; L-Lactate Dehydrogenase ; Necrosis ; Nervous System Diseases ; Neurodegenerative Diseases ; Neurons* ; Parkinson Disease ; Reactive Oxygen Species ; RNA, Messenger ; Superoxide Dismutase

PURPOSE: To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress. METHODS: We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 µM hydrogen peroxide (H₂O₂) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction. RESULTS: VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 µM H₂O₂, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF. CONCLUSIONS: When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.
Bevacizumab ; Blotting, Western ; Endothelial Growth Factors* ; Hydrogen Peroxide ; In Vitro Techniques ; L-Lactate Dehydrogenase ; Oxidative Stress ; Real-Time Polymerase Chain Reaction ; Receptors, Vascular Endothelial Growth Factor ; Retinal Ganglion Cells* ; Retinaldehyde* ; Staurosporine ; Survival Rate ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factor Receptor-1 ; Vascular Endothelial Growth Factor Receptor-2

OBJECTIVETo investigate whether the cardioprotective effect of hemin against ischemia/reperfusion (I/R) injury is through the inhibition of calpain activity, and to explore its underlying mechanism.

METHODSSixty-four SD rats were randomly divided into eight groups (n = 8): sham, I/R, MDL+ I/R, MDL, hemin + I/R, hemin, and ZnPP + hemin+ I/R, ZnPP. Iangendorff isolated rat heart perfusion model was used. The rat hearts were suffered from 40 min of ischemia followed by 30 min of reperfusion. After that, left ventricular developed pressure (LVDP) was recorded. Infarct size and release of lactate dehydrogenase (LDH) were measured. Calpain, heme oxygenase (HO), and caspase 3 activities were evaluated. Expression of calpastatin protein was detected by Western blot.

RESULTS(1) After suffered from ischemia/reperfusion, the calpain activity and caspase 3 activity increased. MDL28170, an inhibitor of calpain, prevented ischemia/reperfusion induced increases in LDH and infarct size, improved the LVDP recovery. (2) Compared with ischema/reperfusion rat hearts, pretreatment of hemin enhanced the HO-1 activity, decreased the calpain and caspase 3 activities, declined LDH release and infarct size, and improved LVDP recovery. (3) Ischemia/reperfusion reduced the expression of calpastatin protein in rat hearts, which was inhibited by hemin pretreatment. And HO-1 inhibitor could abolish the cardioprotection of hemin.

CONCLUSIONCardioprotective effect of hemin against ischemia/reperfusion injury is through the inhibition of calpain activity, the mechanism might be involved in the increase in calpastatin protein expression.

Animals ; Calpain ; metabolism ; Cardiotonic Agents ; pharmacology ; Caspase 3 ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hemin ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Sprague-Dawley

OBJECTIVES: To describe the clinical course and treatment response of a case of thrombotic thrombocytopenic purpura (TTP) after a month of therapeutic plasma exchange (TPE) and immunosuppressive agents and to review related published literature regarding TTP and its response to TPE and immunosuppressive agents.
CASE AND DISCUSSION: A 64-year-old female presented with fever, bicytopenia, change in sensorium, and seizure of one day duration. Metabolic panel showed normal electrolytes with normal brain imaging. Hematologic work-up showed anemia, thrombocytopenia and leukocytosis with normal differential count. Reticulocyte was elevated. Peripheral smear showed significant schistocytes with nucleated red blood cells and marked thrombocytopenia. Thrombotic thrombocytopenic purpura was the initial consideration with the fulfilment of four out of the pentad of TTP, namely: microangiopathichaemolytic anemia, thrombocytopenia, fever and neurologic symptoms. Therapeutic plasma exchange was immediately initiated with daily platelet count and Lactate dehydrogenase (LDH) determination to assess response. However, despite daily plasma exchanges and continuous plasma infusion, there was inadequate response. In this light, immunosuppressive agents were started in the following order: high dose methylprednisolone, weekly rituximab and intravenous cyclophosphamide. On the 31st hospital day, after daily TPE and combined immunosuppression, she achieved complete response with a platelet count of 170 X 109/L to 250 X 109/L (baseline-14 X 109/L) and LDH 227U/L (baseline-1,191 U/L).
CONCLUSION: This study presented a challenging case of TTP which was successfully treated with the standard of care together with the available adjunctive treatment options.

Human ; Female ; Middle Aged ; Plasma Exchange ; Purpura, Thrombotic Thrombocytopenic ; Rituximab ; Thymidine 5'-triphosphate ; Lactate Dehydrogenase 1 ; Isoenzymes ; L-lactate Dehydrogenase ; Anemia

Retaining or improving periodontal ligament (PDL) function is crucial for restoring periodontal defects. The aim of this study was to evaluate the physiological effects of low-power laser irradiation (LPLI) on the proliferation and osteogenic differentiation of human PDL (hPDL) cells. Cultured hPDL cells were irradiated (660 nm) daily with doses of 0, 1, 2 or 4 J⋅cm(-2). Cell proliferation was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the effect of LPLI on osteogenic differentiation was assessed by Alizarin Red S staining and alkaline phosphatase (ALP) activity. Additionally, osteogenic marker gene expression was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Our data showed that LPLI at a dose of 2 J⋅cm(-2) significantly promoted hPDL cell proliferation at days 3 and 5. In addition, LPLI at energy doses of 2 and 4 J⋅cm(-2) showed potential osteogenic capacity, as it stimulated ALP activity, calcium deposition, and osteogenic gene expression. We also showed that cyclic adenosine monophosphate (cAMP) is a critical regulator of the LPLI-mediated effects on hPDL cells. This study shows that LPLI can promote the proliferation and osteogenic differentiation of hPDL cells. These results suggest the potential use of LPLI in clinical applications for periodontal tissue regeneration.
Adenine ; analogs & derivatives ; pharmacology ; Adenylyl Cyclase Inhibitors ; Alkaline Phosphatase ; analysis ; genetics ; radiation effects ; Anthraquinones ; Bone Morphogenetic Protein 2 ; genetics ; Calcium ; metabolism ; radiation effects ; Cell Culture Techniques ; Cell Differentiation ; radiation effects ; Cell Line ; Cell Proliferation ; radiation effects ; Coloring Agents ; Core Binding Factor Alpha 1 Subunit ; genetics ; Cyclic AMP ; antagonists & inhibitors ; radiation effects ; Gene Expression ; radiation effects ; Humans ; L-Lactate Dehydrogenase ; analysis ; Lasers, Semiconductor ; Low-Level Light Therapy ; instrumentation ; Osteocalcin ; genetics ; Osteogenesis ; genetics ; radiation effects ; Periodontal Ligament ; cytology ; radiation effects ; Radiation Dosage ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Tetrazolium Salts ; Thiazoles

OBJECTIVETo explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology.

METHODSThirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α.

RESULTSCounts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively).

CONCLUSIONAA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; complications ; pathology ; physiopathology ; Bone Marrow ; blood supply ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; blood ; L-Lactate Dehydrogenase ; blood ; Male ; Middle Aged ; Neovascularization, Pathologic ; Vascular Endothelial Growth Factor A ; blood ; Yang Deficiency ; complications ; pathology ; physiopathology ; Yin Deficiency ; complications ; pathology ; physiopathology ; Young Adult

OBJECTIVETo explore whether coal tar pitch smoke extract (CTP) induced pyroptosis in human bronchial epithelial cells (BEAS-2B).

METHODSBEAS-2B cells were treated with different concentrations of CTP (1, 3 µg/ml) for 8h and 24 h, respectively. Lactic dehydrogenase (LDH) activity and interleukin-1 beta (IL-1β) levels in the supernatants of cell culture media were measured with LDH activity or human IL-1β ELISA kit, respectively. The activity of Caspase-1 was measured with Caspase-1 colorimetric assay kit.

RESULTSThe activity of caspase-1 in 1 and 3 µg/ml CTP groups were (9.29 ± 0.30) and (8.67 ± 0.59) µmol/ml respectively which were both significantly increased compared to that (7.42 ± 0.59) µmol/ml in the control group (P < 0.05) after 8 h exposure, but there was no significant difference in the activity of LDH and levels of IL-1β in the cell culture media among the CTP and control groups. 24 h after exposure, the activity of LDH in the CTP (1, 3 µg/ml) groups were (1323.03 ± 28.53) and (1148.45 ± 16.42) U/dl respectively which were significantly higher than that (1091.93 ± 26.64) U/dl in the control group (P < 0.05), and the levels of IL-1β in the CTP (1 and 3 µg/ml) groups were (125.37 ± 25.00) pg/ml and (92.04 ± 19.09) pg/ml respectively which were significantly higher than that (46.20 ± 14.43) pg/ml in the control group (P < 0.05), but there was no significant difference in the activity of Caspase-1 among CTP and control groups (P < 0.05).

CONCLUSIONCTP treatment induced early increase in caspase-1 activity followed by the increase in LDH activity and IL-1 levels, indicative of pyroptosis in human bronchial epithelial cells.

Apoptosis ; Bronchi ; cytology ; Caspase 1 ; metabolism ; Cell Line ; Coal Tar ; adverse effects ; Epithelial Cells ; cytology ; Humans ; Interleukin-1beta ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Smoke ; adverse effects

OBJECTIVETo investigate the effect of genistein on osteoblast proliferation, cellular cycle, apoptosis and differentiation of osteoblasts cultivated under hypoxia conditions.

METHODRat osteoblasts were isolated from calvarias by enzyme digestion and a hypoxic model was established by in a triple-gas incubator. Rat osteoblasts were grouped into the normoxic control group, the hypoxia control group and the hypoxia administration group which was subdivided into Ge-6 group, Ge-5 group and Ge-4 group, to which genistein was administered at doses of 1 x 10(-6), 1 x 10(-5), 1 x 10(-4) mol x L(-1). The cell survival rate, lactic dehydrogenase leakage rate, apoptosis and differentiation of osteoblasts were observed for each group at 3 h after hypoxia, and the gene expression of HIF-1alpha, Bcl-2, Caspase-3 was detected by Real time RT-PCR. Forty-eight hours after hypoxia, osteogenic differentiation markers including alkaline phosphatase activity and nodules were detected.

RESULTCompared with the hypoxia control group, the hypoxia administration group displays a significant increase in the survival rate and a decreased in LDH leakage rate, apoptosis rate and percentage of S + G2 phases. Besides, the mRNA level of HIF-1alpha and Bcl-2 were enhanced, the mRNA level of Caspase-3 was inhibited.

CONCLUSIONGenistein has an effect on protecting osteoblasts from hypoxia.

Alkaline Phosphatase ; metabolism ; Animals ; Apoptosis ; drug effects ; Calcification, Physiologic ; drug effects ; Caspase 3 ; genetics ; Cell Cycle ; drug effects ; Cell Hypoxia ; Cell Survival ; drug effects ; Cells, Cultured ; Genes, bcl-2 ; Genistein ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; L-Lactate Dehydrogenase ; metabolism ; Osteoblasts ; cytology ; drug effects ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley