Differential cytokine expression in gastric tissues highlights helicobacter pylori's role in gastritis.

Helicobacter pylori (H. pylori), known for causing gastric inflammation, gastritis and gastric cancer, prompted our study to investigate the differential expression of cytokines in gastric tissues, which is crucial for understanding H. pylori infection and its potential progression to gastric cancer. Focusing on Il-1ß, IL-6, IL-8, IL-12, IL-18, and TNF-α, we analysed gene and protein levels to differentiate between H. pylori-infected and non-infected gastritis. We utilised real-time quantitative polymerase chain reaction (RT-qPCR) for gene quantification, immunohistochemical staining, and ELISA for protein measurement. Gastric samples from patients with gastritis were divided into three groups: (1) non-gastritis (N-group) group, (2) gastritis without H. pylori infection (G-group), and (3) gastritis with H. pylori infection (GH-group), each consisting of 8 samples. Our findings revealed a statistically significant variation in cytokine expression. Generally, cytokine levels were higher in gastritis, but in H. pylori-infected gastritis, IL-1ß, IL-6, and IL-8 levels were lower compared to H. pylori-independent gastritis, while IL-12, IL-18, and TNF-α levels were higher. This distinct cytokine expression pattern in H. pylori-infected gastritis underscores a unique inflammatory response, providing deeper insights into its pathogenesis.

Intermittent fasting attenuates lipopolysaccharide-induced acute lung injury in mice by modulating macrophage polarization.

Acute lung injury (ALI) is a spectrum of acute and life-threatening pulmonary inflammatory conditions. Treatment of ALI remains a clinical challenge. Recently, intermittent fasting (IF) has been shown to improve health and alleviate many diseases. In this study, we tested whether IF attenuated ALI and investigated the mechanism underlying this process. In vivo, the effects of IF on ALI were evaluated in a lipopolysaccharide (LPS)-induced murine ALI model. We found that two times of 24-h fasting in a week before ALI efficiently ameliorated LPS-induced lung injury in mice, characterized by alleviated lung lesions, wet-to-dry weight ratio, myeloperoxidase activity, malondialdehyde content, and lower levels of tumor necrosis factor-α, interleukin-6, and interleukin-1ß. In vitro, functional assays were conducted to assess IF on the inflammatory response and macrophage polarization of bone marrow-derived macrophages (BMDMs) treated with LPS or IL-4. And PPARγ antagonist GW9662 and AMPK siRNA were used to test the role of PPARγ and AMPK in the IF-mediated improvement of ALI. The results showed that IF (serum deprivation) suppressed macrophage M1 activation and promoted M2 activation in LPS-treated BMDMs. While, IF also augmented macrophage M2 polarization in IL-4-treated BMDMs. Further mechanistic studies showed that the promotive effect of IF on M2 polarization was related to the activation of the PPARγ and AMPK pathways. In conclusion, this study suggests that IF enhances M2 polarization by activating the AMPK and PPARγ pathways, thus facilitating anti-inflammatory response and ameliorating ALI.

Isolation of Novel Mycobacterium Species from Skin Infection in an Immunocompromised Person.

We investigated a case of cutaneous infection in an immunocompromised patient in China that was caused by a novel species within the Mycobacterium gordonae complex. Results of whole-genome sequencing indicated that some strains considered to be M. gordonae complex are actually polyphyletic and should be designated as closely related species.

Testosterone ameliorates vascular aging via the Gas6/Axl signaling pathway.

Low serum testosterone level is associated with aging-related vascular stiffness, but the underlying mechanism is unclear. The Growth arrest-specific protein 6 (Gas6) /Axl pathway has been proved to play important roles in cell senescence. In this study, we intend to explore whether Gas6/Axl is involved in the effect of testosterone on vascular aging amelioration. Vascular aging models of wild type and Axl-/- mice were established by natural aging. Mice of these two gene types were randomized into young group, aging group and testosterone undecanoate (TU) treatment group. Mice were treated with TU (37.9 mg/kg) in the TU group, which treated with solvent reagent served as control. The aging mice exhibited decreases in serum testosterone, Gas6 and Axl levels and an increase in cell senescence, manifested age-related vascular remodeling. Testosterone treatment induced testosterone and Gas6 levels in serum, and ameliorated cell senescence and vascular remodeling in aging mice. Furthermore, we uncover the underlying molecular mechanism and show that testosterone treatment restored the phosphorylation of Akt and FoxO1a. Axl knockout accelerated cell senescence and vascular remodeling, and resisted the anti-aging effect of testosterone. Testosterone might exert a protective effect on vascular aging by improving cell senescence and vascular remodeling through the Gas6/Axl pathway.

Progranulin Improves Acute Lung Injury through Regulating the Differentiation of Regulatory T Cells and Interleukin-10 Immunomodulation to Promote Macrophage Polarization.

Progranulin (PGRN), which plays an anti-inflammatory role in acute lung injury (ALI), is promising as a potential drug. Studies have shown that regulatory T cells (Tregs) and interleukin- (IL-) 10 can repress inflammation and alleviate tissue damage during ALI. In this study, we built a lipopolysaccharide- (LPS-) induced ALI mouse model to illustrate the effect of PGRN on regulation of Treg differentiation and modulation of IL-10 promoting macrophage polarization. We found that the proportion of Tregs in splenic mononuclear cells and peripheral blood mononuclear cells was higher after treatment with PGRN. The increased proportion of Tregs after PGRN intratracheal instillation was consistent with the decreased severity of lung injury, the reduction of proinflammatory cytokines, and the increase of anti-inflammatory cytokines. In vitro, the percentages of CD4+CD25+FOXP3+ Tregs from splenic naïve CD4+ T cells increased after PGRN treatment. In further research, it was found that PGRN can regulate the anti-inflammatory factor IL-10 and affect the polarization of M1/M2 macrophages by upregulating IL-10. These findings show that PGRN likely plays a protective role in ALI by promoting Treg differentiation and activating IL-10 immunomodulation.

MicroRNA-23 suppresses osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting the MEF2C-mediated MAPK signaling pathway.

BACKGROUND: The present study aimed to determine the role and mechanism of miR-23 with respect to regulating the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). MATERIALS: The expression of miR-23 and MEF2C was measured in osteoporosis (OP) patients and healthy controls by a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The correlation between miR-23 and MEF2C was determined by the Pearson correlation coefficient. Moreover, bioinformatic analysis was performed using public databases. Target gene function and potential pathways were further examined. Then, we used a miR-23 mimic or inhibitor to further explore the potential mechanism of miR-23. RESULTS: miR-23 is found to be up-regulated and MEF2C is down-regulated in OP patients compared to healthy controls. miR-23 had a negative correlation with MEF2C (r = -0.937, p = 0.001). Bioinformatic analysis revealed that a total of 664 overlapping target genes were found in the TargetScan (http://www.targetscan.org), miRDB (http://mirdb.org) and miRanda (http://www.microrna.org/microrna/home.do) databases. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that miR-23 may regulate the mitogan-activated protein kinase (MAPK) signaling pathway. miR-23 is down-regulated and MEF2C is significantly up-regulated in the osteogenic differentiation of hBMSCs. MEF2C was significantly up-regulated in the osteogenic differentiation of hBMSCs. Overexpression of miR-23 significantly down-regulated alkaline phosphatase (ALP) activity and calcium deposition, whereas the miR-23 inhibitor had the opposite effects. Moreover, overexpression of miR-23 significantly decreased osteoblast-related markers (Runx2, Osx, ALP and OCN). Further experiments confirmed that MEF2C is a direct target of miR-23. Moreover, the miR-23 mimic enhanced the expression of p-p38 but had no effect on p-JNK. CONCLUSIONS: miR-23 decreases the osteogenic differentiation of hBMSCs through the MEF2C/MAPK signaling pathway.

Curcumin regulates the differentiation of naïve CD4+T cells and activates IL-10 immune modulation against acute lung injury in mice.

OBJECTIVES: Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Curcumin has been reported to be an anti-inflammatory factor through enhancing the function of regulatory T cells (Tregs). This study aimed to explore the effect of curcumin on the differentiation of Tregs and the role of curcumin in ALI/ARDS. METHODS: A cecal ligation and puncture (CLP)-induced acute lung injury mouse model was used to explore the effect of curcumin in ALI/ARDS. The severity of lung injury was evaluated. Immunohistochemistry of IL-17A and MPO in lung tissue was examined. Treg-related cytokine levels in serum and bronchoalveolar lavage fluid (BALF) were tested. The expression of nuclear factor-kappa B (NF-κB) in lung tissue was detected. Macrophages in lung tissue were detected by immunofluorescence. Splenic CD4+CD25+FOXP3+ Tregs were quantified, and the differentiation of Tregs from naïve CD4 + T cell and STAT5 was evaluated. The expression of IL-10 during naïve CD4 + T cell differentiation in vitro was tested. RESULTS: Curcumin alleviated lung injury in the induced CLP mouse model and suppressed inflammation. IL-17A, MPO-producing neutrophils, and NF-κB p65 expression in lungs of CLP mice decreased significantly after pretreatment with curcumin. We found curcumin could regulate M1/M2 macrophage levels in lungs of CLP mice. This may have been through regulating the differentiation of Tregs and the production of Treg-derived IL-10. Treg-derived IL-10 is the main factor that could affect macrophage polarization. We found curcumin could increase Treg proportions in vivo and up-regulate IL-10 expression in serum and BALF of CLP mice. In our in vitro experiments, we found curcumin could promote Treg differentiation and increase the production of IL-10. CONCLUSIONS: Curcumin can reduce the degree of severity of ALI and uncontrolled inflammation through promoting the differentiation of naïve CD4 + T cells to CD4+ CD25+ FOXP3+ Tregs. Curcumin promotes the conversion of macrophages from M1 to M2. The differentiation of Tregs induced by curcumin may be one source of IL-10 immune modulation.

Exogenous testosterone alleviates cardiac fibrosis and apoptosis via Gas6/Axl pathway in the senescent mice.

BACKGROUND: Androgen has been implicated in aging-related cardiac remodeling, but its precise role in aging heart remains controversial. We aimed to investigate the role of testosterone in the development of aging-related cardiac remodeling and the mechanisms involved. METHODS: Wild type and Axl knockout mice (Axl-/-) were randomized into three groups: the young group (n = 30, 3 months old), the aging group (n = 30, 18 months old), the testosterone undecanoate treatment group (TU, n = 30, 18 months old). Mice in the TU group were given testosterone undecanoate (39 mg/kg) by subcutaneous injection on the back at fifteen-months-old, once a month, a total of three times. The old group received solvent reagent (corn oil) by the same method. RESULTS: The aging mice exhibited a decrease in serum testosterone, and Gas6 levels and an increase in apoptosis, and manifested cardiac fibrosis. Testosterone injection to wild type mice increased the levels of testosterone and Gas6 in serum and decreased cardiac apoptosis and fibrosis. Axl-/-mice receiving testosterone injection exhibited no obvious improvement in cardiac remodeling although the levels of testosterone and Gas6 in serum elevated. CONCLUSIONS: These data indicated that testosterone replacement therapy (TRT) alleviates cardiac fibrosis and apoptosis, at least in part by enhancing Gas6 expression. Moreover, deletion of Axl disables testosterone, which indicated that Axl is an important downstream regulator of testosterone. TRT would improve aging-related cardiac remolding via Gas6/Axl signaling pathway, implicating its therapeutic potential to treat aging-related heart disease.

Effect of parthanatos on ropivacaine-induced damage in SH-SY5Y cells.

Ropivacaine is one of the most common but toxic local anaesthetics, and the mechanisms underlying its neurotoxicity are still largely unknown. This study was conducted to prepare a ropivacaine-induced neuronal injury model and research the effects of ropivacaine on PARP-1 activation and nicotinamide adenine dinucleotide (NAD)+ depletion. The cell death and apoptosis of ropivacaine-induced SH-SY5Y cells were detected with flow cytometry. The lactate dehydrogenase cycling reaction measured the NAD+ level, and western blots were used to analyze the expression levels of PARP-1 and apoptosis-inducing factor (AIF) after ropivacaine treatments with different concentrations and durations. A PARP-1 inhibitor (PJ-34) was used to confirm the relationship between PARP-1 activation and NAD+ depletion. Hoechst 33258 nuclear staining and a mitochondrial membrane potential (Δψm) assay were used to detect the role of exogenous NAD+ in ropivacaine-induced neuronal injury. Ropivacaine-induced SH-SY5Y cell death and apoptosis, PARP-1 activation, and AIF increase as well as intracellular NAD+ depletion occurred in a time- and concentration-dependent manner (P<.05). PARP-1 activation led to NAD+ depletion (P<.05). Exogenous NAD+ impaired ropivacaine-induced nuclear injury (P<.05). Ropivacaine treatment induced PARP-1 activation and NAD+ depletion (P<.05). Parthanatos (PARP-1-dependent cell death) was definitely involved in ropivacaine-induced neuronal injury, and exogenous NAD+ may be a novel therapeutic method for parthanatos-dependent neuronal injury.

Testosterone delays vascular smooth muscle cell senescence and inhibits collagen synthesis via the Gas6/Axl signaling pathway.

Testosterone deficiency is associated with a higher incidence of cardiovascular diseases in men. However, its effect on cell senescence, which plays a causal role in vascular aging, remains unclear. Here, we tested the hypothesis that testosterone alleviated vascular smooth muscle cell (VSMC) senescence and collagen synthesis via growth arrest-specific protein 6 (Gas6)/Axl- and Akt/FoxO1a-dependent pathways. Testosterone significantly ameliorated angiotensin II-induced VSMC senescence and collagen overexpression. In addition, testosterone inhibited angiotensin II-induced matrix metalloproteinase-2 (MMP-2) activity, which played a pivotal role in facilitating age-related collagen deposition. Testosterone increased the expression of tissue inhibitor of metalloproteinase-2 but decreased the expression of MMP-2 and membrane type-1 metalloproteinase which contributed to increase MMP-2 activity. The effects on VSMCs senescence and collagen synthesis were mediated by restoration of angiotensin II-induced downregulation of Gas6 and Axl expression and a subsequent reduction of Akt and FoxO1a phosphorylation. The effects of testosterone were reversed by a Gas6 blocker, Axl-Fc, and a specific inhibitor of Axl, R428. Treatment of VSMCs with PI3K inhibitor LY294002 abrogated the downregulating effect of testosterone on MMP-2 activity. Furthermore, when FoxO1a expression was silenced by using a specific siRNA, the inhibitory effect of testosterone on MMP-2 activity was revered as well, that indicated this process was Akt/FoxO1a dependence. Taken together, Gas6/Axl and Akt/FoxO1a were involved in protective effects of testosterone on VSMCs senescence and collagen synthesis. Our results provide a novel mechanism underlying the protective effect of testosterone on vascular aging and may serve as a theoretical basis for testosterone replacement therapy.

Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

BACKGROUND/AIMS: Growth arrest-specific protein 6 (Gas6) is a cytokine that can be synthesized by a variety of cell types and secreted into the extracellular matrix. Previous studies have confirmed that Gas6 is involved in certain pathophysiological processes of the cardiovascular system through binding to its receptor, Axl. In the present study, we investigated the role of Gas6 in cellular senescence and explored the mechanisms underlying its activity. METHODS: We used vascular smooth muscle cells (VSMCs) to create two cellular senescence models, one for replicative senescence (RS) and one for induced senescence (IS), to test the hypothesis that Gas6 delays senescence. RESULTS: Gas6-treated cells appear relatively younger compared with non-Gas6-treated cells. In particular, Gas6-treated cells displayed decreased staining for SA-ß-Gal, fewer G1 phase cells, and decreased levels of p16(INK4a) and p21(Cip1) expression; conversely, Gas6-treated cells displayed more S phase cells and significantly increased proliferation indexes. Furthermore, in both the IS and RS models with Gas6 treatment, the levels of PI3K, p-Akt, and p-FoxO3a decreased following Axl inhibition by R428; similarly, the levels of p-Akt and p-FoxO3a also decreased following PI3K inhibition by LY294002. CONCLUSION: Gas6/Axl signaling is essential for delaying the cellular senescence process regulated by the PI3K/Akt/FoxO signaling pathway.

Prophylactic effectiveness of budesonide inhalation in reducing postoperative throat complaints.

Postoperative sore throat (POST) is one of the main postoperative complaints. This study was to evaluate the efficacy of budesonide inhalation suspension (BIS) in reducing the incidence and severity of POST. One hundred and twenty patients scheduled for thyroid surgery with general anesthesia were enrolled and randomized into three groups. Group A received 200 mcg BIS 10 min prior to the tracheal intubation and received the same treatment 6 and 24 h after extubation. Group B received 200 mcg BIS 6 and 24 h after extubation. Control group received the same scheduled treatment as Group A, but the BIS was replaced with 2 ml normal saline. The patients were evaluated for sore throat and hoarseness 1, 24 and 48 h after extubation. The status of laryngopharynx was also recorded. There was no statistically significant difference in the incidence of sore throat among three groups. However, hoarseness occurred significantly less frequently in Group A (P < 0.05). One hour after extubation, Group A exhibited significantly less severe sore throat and hoarseness compared to the other two groups (P < 0.05), which disappeared 24 h later. The mucositis scores of laryngopharynx at 1, 24 and 48 h post-extubation were significantly lower in Group A (P < 0.05). BIS can reduce the incidence and severity of the POST prophylactically.

Statins are associated with reduced risk of gastric cancer: a meta-analysis.

BACKGROUND: Statins are widely prescribed to reduce cholesterol levels in the prevention of atherosclerotic cardiovascular disease. However, the debate about the effect of statins on cancer risk remains unsettled. The aim of this study was to investigate the association of utilization of statins with the risk of gastric cancer by carrying out a meta-analysis. METHODS: A literature search was performed on PubMed and EMBASE up to March 2013 to identify the cohort or case-control studies or randomized controlled trials (RCTs) that examined the relationship between statins use and the risk of gastric cancer. The bibliographies of the retrieved articles were also reviewed to identify additional studies. A random-effects model was used to calculate the summary relative risks (RRs) with 95 % confidence intervals (CIs). RESULTS: Three post-hoc analyses of 26 RCTs involving 290 gastric cancers and eight observational studies totaling 7,321 gastric cancers were included. Statins use was shown to be significantly associated with a 27 % reduction in the risk of gastric cancer (RR = 0.73, 95 % CI = 0.58-0.93), with considerable heterogeneity among studies (I (2) = 88.9 %). Excluding one study in which all subjects are diabetic patients obtained an attenuated, but homogeneous result (RR = 0.85, 95 % CI = 0.80-0.91, I (2) = 0.0 %). These findings were consistent in the subgroup analysis. CONCLUSION: A meta-analysis of existing evidence, primarily from observational studies, indicates that use of statins reduces the risk of gastric cancer.

The effects and underlying mechanisms of S-allyl l-cysteine treatment of the retina after ischemia/reperfusion.

PURPOSE: Retinal ischemia-associated ocular disorders are vision-threatening. The aim of the present study was to examine whether S-allyl l-cysteine (SAC) is able to protect against retina ischemia/reperfusion injury. METHODS: In vivo, retinal ischemia in the rat was induced by raising intraocular pressure (IOP) to 120 mmHg for 60 min. In vitro, an ischemic-like insult, namely oxidative stress, was established by incubating retinal ganglion cell-5 (RGC-5) with 500 µM H(2)O(2) for 24 h. The mechanisms involved in these processes were evaluated by electrophysiology, immunohistochemistry, and molecular biological approaches. RESULTS: The retinal changes caused by the high IOP were characterized by a decrease in electroretinogram b-wave amplitudes, a loss of choline acetyltransferase immunolabeling amacrine cell bodies/neuronal processes, and an upregulation of the mRNA levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelium growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9). The increased protein levels of HIF-1α, VEGF, and MMP-9 were also seen in RGC-5 cells subjected to defined oxidative stress. Of clinical importance, the ischemic/ischemic-like detrimental effects were concentration-dependently (least effect at 25 µM) and/or significantly (50 and/or 100 µM) blunted when SAC was applied 15 min before retinal ischemia or ischemic-like insult, respectively. CONCLUSION: SAC would seem to protect against retinal ischemia by acting as an antioxidant and inhibiting the upregulation of HIF-1α, VEGF, and MMP-9.

Detection of rib metastases in patients with lung cancer: a comparative study of MRI, CT and bone scintigraphy.

We retrospectively investigated the imaging findings of bone scintigraphy, chest CT and chest MRI in 55 cases of lung cancer. The sensitivity, specificity and accuracy of the detection of rib metastases were compared between imaging modalities on both a per-lesion and a per-patient basis. On a per-lesion basis, MRI sensitivity and accuracy were significantly higher than that of bone scintigraphy and CT (P<0.05). The sensitivities, specificities, and accuracy levels between CT and bone scintigraphy did not differ on either a per-lesion or per-patient basis (P>0.05). MRI appears to be superior for the detection of ribs metastases in lung cancer.

[Akt involved in diazoxide preconditioning against rat hippocampal neuronal apoptosis induced by anoxia-reoxygenation injury].

OBJECTIVE: Investigate whether preconditioning with diazoxide (DZ), a mitochondrial ATP-sensitive potassium channel opener, could enhance Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis. METHODS: Cultured for 9 - 10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control (Group A), Anoxia (Group B), Anoxia + DZ100 micromol/L (Group C), Anoxia + DZ100 micromol/L + 5-hydroxydecanoate (Group D), Anoxia + DZ100 micromol/L + LY294002 50 micromol/L (Group E). Prior to oxygen deprivation, the hippocampal neurons were treated with DZ for 1 h per day and this treatment was persisted for 3 d. Each experiment was repeated for three times and each group contained 16 wells or 2 dishes of neurons for each time. Thereafter, neurons were derived of oxygen for 4 h. At 48 h of reoxygenation the neuronal optical density (A) were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Akt, Bcl-2 and Bax proteins were detected and evaluated by Western blot. RESULTS: Compared with other pretreatment, DZ 100 micromol/L led to the elevation of A, whereas promoted the decrease of apoptotic rate (P < 0.05). Compared with those in other anoxic groups, the expressions of Akt protein and Bcl-2 protein in Group C were increased significantly (P < 0.05), whereas the Bax density were reduced significantly (P < 0.05). Preceding administration of 100 micromol/L DZ took protective effects on the neurons induced by anoxia-reoxygenation. CONCLUSIONS: DZ 100 micromol/L increased Akt protein to up-regulate Bcl-2/Bax protein ratio against apoptosis induced by anoxia-reoxygenation.

[Epidemiological survey of rheumatic heart disease in schoolchildren in Guangdong and Xinjiang].

OBJECTIVE: To investigate the prevalence of rheumatic heart disease (RHD) among schoolchildren in Guangdong Province and Xinjiang Uygur Autonomous Region. METHODS: Using a cluster sampling method, an epidemiological survey of RHD was conducted in 16 682 primary and high school students by auscultation in Guangdong Province and Xinjiang Uygur Autonomous Region from 2005 to 2006. Review of the clinical records, RHD survey in adults, and examination of the positivity rate of group A beta-hemolytic Streptococcus (GAS) by throat swab cultures in the students aged between 9 and 12 years in the sampled schools were also carried out. RESULTS: No RHD patient was found in the sampled population. In Xinjiang, the prevalence of RHD was 12.9/1000 among adults, higher than that (2.2/1000) in Guangdong Province. The GAS-positive rate in the schoolchildren in Xinjiang ranged from 9.8% to 12.6%, higher than that in Guangdong (2.3%-3.9%). CONCLUSION: The GAS-positive rate among children and incidence of RHD in adults are higher in Xinjiang than in Guangdong. The prevalence of RHD among the schoolchildren shows a reduction compared with that in 1994.

[Effects of genistein on expression of TGF-beta1 and intracellular free Ca2+ concentration in fibroblasts derived from human hypertrophic scar].

OBJECTIVE: To observe the effects of Genistein on TGF-beta1 expression and the intracellular free Ca2+ concentration in human hypertrophic scar fibroblasts, and to discuss the mechanism of the anti-fibrosis effect. METHODS: Fibroblasts were derived from human hypertrophic scar tissue and cultured in vitro. Genistein in different concentrations (25, 50, 100 micromol/L) was administrated to the fibroblasts, respectively. After 48 hours of co-culture, the expression of TGF-beta1 mRNA and protein were examined by RT-PCR and Western-Blot assay respectively. The intracellular free Ca2+ concentration in hypertrophic scar fibroblasts pretreated by Genistein was determined by laser confocal scanning microscopy with or without the stimulation of bFGF. RESULTS: Genistein inhibited the expression of TGF-beta1 in hypertrophic scar fibroblasts on a concentration-dependent manner. bFGF significantly elevated the intracellular free Ca2+ concentration, however its stimulating effect was remarkably alleviated when the fibroblasts were pre-treated by Genistein. CONCLUSIONS: Genistein can reduce the expression of TGF-beta1 and block the accumulation of intracellular free calcium induced by growth factors. It maybe one of the possible mechanisms of Genistein's antifibrosis effect.

Diazoxide preconditioning alleviates apoptosis of hippocampal neurons induced by anoxia-reoxygenation in vitro through up-regulation of Bcl-2/Bax protein ratio.

Activating mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels is a critical event of pharmacological preconditioning, which can enhance neuronal ability against various insults. mitoK(ATP) channels are abundant in neurons and can be selectively opened by diazoxide (DZ). The aim of this study was to determine whether DZ could restrain neuronal apoptosis induced by anoxia-reoxygenation and to reveal the effect of DZ preconditioning on the expressions of Bcl-2 and Bax proteins in cultured hippocampal neurons. Cultured for 9~10 d in vitro, the hippocampal neurons of Sprague-Dawley rats were assigned to the following 5 groups randomly: Control, DZ 0 mumol/L, DZ 30 mumol/L, DZ 100 mumol/L, DZ 100 mumol/L+5-hydroxydecanoate (5-HD, a selective mitoK(ATP) channel blocker) 100 mumol/L. Prior to oxygen deprivation, the hippocampal neurons except those in the control group were treated with DZ or DZ+5-HD for 1 h per day and this treatment persisted for 3 d. Thereafter, neurons were subjected to anoxia for 4 h and followed by reoxygenation. At 24 h of reoxygenation the neuronal survival rates were measured by MTT method, while the apoptotic rates were assayed by annexin V-FITC staining. The expressions of Bcl-2 and Bax proteins were detected with immunocytochemistry and evaluated by Western blot. Anoxia-reoxygenation injury reduced the survival rates and increased apoptotic rates significantly. In comparison with those in other groups, the survival rate in DZ 100 mumol/L group was increased by about 15%, whereas the apoptotic rate was decreased by almost 12% simultaneously. 5-HD could abolish the neuroprotection afforded by 100 mumol/L DZ. Bcl-2 and Bax proteins in the control normoxic neurons were both expressed slightly, while anoxia-reoxygenation led to high expression of Bax protein. The administration of 100 mumol/L DZ enhanced the expression of Bcl-2 protein by nearly 60%, whereas Bax protein was reduced by approximately 30%. Lower concentrations of DZ had no detectable effects on the expressions of Bcl-2 and Bax proteins. However, beneficial effects of DZ on the expressions of Bcl-2 and Bax proteins were reversed after the co-treatment with 5-HD. In conclusion, 100 mumol/L DZ prevented cultured hippocampal neurons from apoptosis induced by anoxia-reoxygenation possibly through up-regulating the expression of Bcl-2 protein and down-regulating the expression of Bax protein.

Diazoxide preconditioning plus subsequent hypothermia increased resistance of rat cultured hippocampal neurons against hypoxia-reoxygenation injury.

BACKGROUND: Cerebral ischemia-reperfusion/hypoxia-reoxygenation insult triggers lots of pathophysiological and biochemical events that separately affect the evolution of cerebral damage. Accordingly, all known effective neuroprotective measures should be taken to get the optimal efficacy of therapy. This study was undertaken to investigate whether diazoxide (DZ) preconditioning combined with the following hypothermia could contribute to synergistic neuroprotection compared with either hypothermia or DZ preconditioning alone. METHODS: Cultured for 9-10 days in vitro, the hippocampal neurons of SD rats were preconditioned with DZ 0 micromol/L or DZ 250 micromol/L for 1 hour per day and this treatment lasted for 3 days. Subsequently, neurons were subjected to deprivation of oxygen for 4 hours at 37 degrees C, 34 degrees C, 30 degrees C and 22 degrees C, respectively. This experiment consisted of 8 groups (4 temperature groups and 4 combination groups) and each group contained 12-well or 2-dish cells. Survival rate, expression of Bcl-2, fluorescence magnitude of intracellular calcium, and concentration of malondialdehyde (MDA) were determined at 24 hours after reoxygenation. RESULTS: The survival rate and expression of Bcl-2 were both increased in individually hypothermic conditions compared with those at 37 degrees C (P < 0.05), whereas intracellular calcium and MDA did the opposite exhibition simultaneously (P < 0.05). 22 degrees C contributed to a higher survival rate and greater expression of Bcl-2 in comparison with other hypothermia (P < 0.05). Preceding administration of 250 micromol/L DZ took the similar effects on the neurons like hypothermia. Moreover, compared with individual hypothermia or DZ preconditioning, the neuronal survival rate and expression of Bcl-2 in the combination group were increased significantly (P < 0.05), whereas the calcium fluorescence density and concentration of MDA were reduced further (P < 0.05). 250 micromol/L DZ preconditioning combined with 22 degrees C provided a maximal neuroprotection. CONCLUSIONS: Compared with either individual hypothermia or DZ preconditioning, the combination of both treatments conferred synergistic protection for cultured hippocampal neurons in vitro against hypoxia-reoxygenation insult.