Pancreatic cancer cell-secreted exosomes promote apoptosis of β cells via activation of mitochondrial apoptotic pathways

AIM: To investigate the effects of exosomes secreted by pancreatic cancer cells on the viability and function of β cells and the possible mechanism. METHODS: ExoQuick-TC kit was used to extract exosomes in the supernatants of mouse pancreatic cancer Pan02 and MPC-83 cells, and the extracted exosomes were identified by transmission electron microscopy. Fluorescence-labeled exosomes were incubated with mouse insulinoma MIN6 cells for 48 h to detect whether exosomes secreted by pancreatic cancer cells were uptaken by MIN6 cells. MTT and glucose-stimulated insulin secretion (GSIS) assays were conducted to examine cell viability and insulin secretion of MIN6 cells after incubating with exosomes. The expression of miR-204 and Bcl-2 mRNA in MIN6 cells was detected by qPCR. The protein expression of Bcl-2, Bax, caspase-3 and cytochrome C (Cyt-C) in MIN6 cells was determined by Western blot. RESULTS: The results of transmission electron microscopy showed that both Pan02 cells and MPC-83 cells secreted exosomes, and Pan02 cells secreted more. The co-incubation results of fluorescence-labeled exosomes and MIN6 cells confirmed that MIN6 cells were able to ingest large amounts of exosomes secreted by pancreatic cancer cells. The results of MTT and GSIS assays showed that the viability and the level of high glucose-stimulated insulin secretion of MIN6 cells in exosome treatment group significantly decreased compared with nontreatment group (P<0.01). The results of qPCR showed that the exosomes secreted by pancreatic cancer cells were rich in miR-204, and the mRNA expression of Bcl-2 in MIN6 cells was significantly down-regulated by exosome incubation (P<0.01). The results of Western blot showed that the protein expression of Bcl-2 in the MIN6 cells treated with exosomes was significantly down-regulated (P<0.05), and the protein levels of Bax, cleaved caspase-3 and Cyt-C in exosomes treatment group were significantly up-regulated (P<0.01). CONCLUSION: Pancreatic cancer cells secrete exosomes. The exosomes secreted by pancreatic cancer cells are ingested by β cells, and reduce the viability and insulin secretion of β cells. The mechanism may be related to the increase in exosomal miR-204 in the β cells. Increasing miR-204 may inhibit the expression of Bcl-2 and promote the activation of mitochondrial apoptosis in β cells.     

Influence of amylin on apoptosis of human pancreatic islet β-cells and its molecular mechanism

AIM: To investigate the molecular mechanism of amylin in inducing apoptosis of human pancreatic islet β-cells. METHODS: Human pancreatic islet cells were isolated and cultured. The cells were treated with amylin or amylin and aminoguanidine (AG group) for 24 h, respectively. Apoptosis of pancreatic islet β-cells was studied by in situ TUNEL method combined with double staining for insulin and ELISA. The levels of insulin, NO2-/NO3- and glutathione (GSH), p53 mRNA and bcl-2 mRNA were also detected. RESULTS: (1) The enrichment factor and the apoptosis rate of pancreatic islet β-cells in amylin group were markedly higher than that in control group and AG group (P<001). (2) The insulin level in amylin group was significantly lower than that in control group and AG group (P<005). (3) The levels of NO2-/NO3- and p53 mRNA in amylin group were significantly higher than that in control group and AG group (P<001), while the levels of GSH and bcl-2 mRNA were markedly decreased as compared with control group and AG group. CONCLUSIONS: Amylin increases apoptosis of human pancreatic islets β cells, resulting in decrease in insulin secretion. This may be due to increased expression of p53 and decreased expression of bcl-2 during the of oxidative stress induced by amylin.     

Effects of metoprolol on phosphorylated connexin 43 expression in myocardial tissues and apoptosis of myocardial cells in heart failure rats

AIM：To explore the in vivo effects of metoprolol on the expression of phosphorylated connexin 43 (p-Cx43) in myocardial tissues and the apoptosis of myocardial cells in rats with heart failure (HF).METHODS：One hundred Sprague-Dawley rats were randomly divided into 5 groups (each n=20): sham group, HF group, low-dose (1.25 mg·kg-1·d-1) metoprolol treatment (MetoA) group, middle-dose (5 mg·kg-1·d-1) metoprolol treatment (MetoB) group and high-dose (20 mg·kg-1·d-1) metoprolol treatment (MetoC) group.The rats in HF group and metoprolol treatment groups were subject to abdominal aortic ligation, and different doses of metoprolol were given 4 weeks later till 8 weeks after operation.Echocardiography was conducted to monitor the hemodynamic parameters at the 4th and 8th weeks, and the rat hearts were taken at the 8th week after operation.The morphological changes and the proliferation of collagen fibers in myocardial tissues were observed by HE and Masson staining, respectively.The expression level of p-Cx43 was detected by Western blotting and the apoptosis of myocardial cells was assessed by TUNEL method.The relationship between p-Cx43 expression level and apoptotic index was analyzed by Pearson’s correlation.RESULTS：(1) Echocardiography showed that metoprolol could effectively improved cardiac hemodynamics in HF rats, and pathological findings suggested that metoprolol could effectively reverse HF-induced cardiac remodeling in a dose-dependent manner within the therapeutic dose range.(2) Western blotting showed that p-Cx43 expression in HF group was significantly higher than that in sham group (P<001), and that in all metoprolol treatment groups was significantly decreased compared with HF group (P<005 or P<001), among which pairwise comparisons also showed significant differences (P<001).(3) The myocardial apoptotic index in HF group ［(51.17±6.94)%］ was significantly increased compared with sham group ［(4.62±160)%, P<001］.Compared with HF group, myocardial apoptotic indexes in MetoA group ［(40.60±4.15)%］, MetoB group ［(30.66±4.00)%］ and MetoC group ［(22.24±5.69)%］ were significantly decreased (P<001), among which pairwise comparisons also showed significant differences (P<001).(4) The expression level of p-Cx43 was positively correlated with the apoptotic index (r=0.905, P<001).CONCLUSION: The mechanism of metoprolol against HF-induced myocardial apoptosis may be related to inhibition of p-Cx43 expression.     

Metoprolol inhibits norepinephrine-induced rat cardiomyocyte apoptosis and connexin 43 phosphorylation

AIM: To study the effects of metoprolol (Meto) on the apoptosis of neonatal rat cardiomyocytes and the phosphorylation of connexin 43 (Cx43) induced by norepinephrine (NE). METHODS: Neonatal SD rat cardiomyocytes were divided into the following five groups (n=6 in each group): (1) control (Con) group: no treatment; (2) NE group: treatment with NE at 0.1 μmol/L for 24 h; (3) NE+Meto group: simultaneous treatment with NE and Meto both at 01 μmol/L for 24 h; (4) NE+Meto+PD98059 group: pretreatment with extracellular signal-regulated kinase (ERK) phosphorylation inhibitor PD98059 at 10 μmol/L for 30 min and then treatment with NE and Meto both at 01 μmol/L for 24 h; (5) NE+PD98059 group: pretreatment with PD98059 at 10 μmol/L for 30 min and then treatment with NE at 01 μmol/L for 24 h. The beating rates of cardiomyocytes in various groups were calculated, and the viability of cardiomyocytes was assayed by MTT method. The Cx43 mRNA expression was detected by RT-PCR, and the protein expression of phosphorylated Cx43 (p-Cx43), phosphorylated ERK1/2 (p-ERK1/2) and cleaved caspase-3 was detected by Western blotting. RESULTS: (1) Separate NE treatment could significantly increased the beating rate of cardiomyocytes and reduced cell viability, while Meto showed the opposite effects. PD98059 treatment had no significant effect on cardiomyocyte beating rate, but suppressed Meto to improve cell viability to some extent. (2) Compared with Con group, separate NE treatment significantly increased the Cx43 mRNA expression (P<001). Compared with NE group, Meto or PD98059 intervention could significantly inhibited Cx43 mRNA expression (both P<001), and simultaneous treatment with Meto and PD98059 could further suppress Cx43 mRNA expression up-regulated by NE (P<001). (3) Compared with NE group, Meto significantly inhibited the increased p-Cx43, p-ERK1/2 and cleaved caspase-3 expression induced by NE (P<001), and simultaneous treatment with Meto and PD98059 could further enhance the inhibition of p-Cx43, p-ERK1/2 and cleaved caspase-3 expression by Meto (P<001). PD98059 treatment had no significant effect on the increased p-Cx43 and cleaved caspase-3 expression induced by NE (P>005). CONCLUSION: The inhibitory effect of Meto on NE-induced cardiomyocyte apoptosis is related to the inhibition of Cx43 phosphorylation, which may be partly mediated via ERK1/2 pathway.     

Expression of GLUT4 in endometrium of rats with polycystic ovary syndrome

AIM:To explore the expression of glucose transporter 4 (GLUT4) in the endometrium of rats with polycystic ovarian syndrom (PCOS) and evaluate the relationship between GLUT4 expression and insulin resistance (IR). METHODS:54 female SD rats of 85 days were randomized to control group (n=20), PCOS model group (n=17) and metformin treatment group (n=17). The rats in the latter two groups were induced by Poretsky’s method for PCOS model, followed by placebo or metformin, respectively. After 14 days of treatment, the rats were sacrificed and the expression of GLUT4 in endometrium was detected by Elivision^TM Plus two steps immunohistochemical staining. RESULTS:The expression of GLUT4 and insulin receptor(INS-R) proteins of endometrial glandulan epitheliu in PCOS rats were significantly lower (P<0.01,P<0.05) than those in control group, however, the expression of insulin(INS) protein in PCOS rats was higher than that in control group (P<0.01). The expression of GLUT4 in the treatment group increased (P<0.01), but was still lower than that in control group (P<0.01). However, compared with PCOS group, the expression of INS protein was decreased (P<0.05), but was still higher than that in control group (P<0.05). There was no GLUT4 expression in interstitial cells in endometrium, and the changes of the expressions of INS and INS-R proteins in those cells were similar with those in glandulan epitheliu. CONCLUSION:The decrease in GLUT4 expression of endometrium in PCOS rats is related with endometrial insulin resistance.     

Effects of stretch on transient outward potassium and inward rectifier potassium current in cultured neonatal rat atrial myocytes

AIM：To investigate the effects of mechanical stretch on transient outward potassium current (Ito), inward rectifier potassium current (IK1) and action potential duration(APD) of cultured neonatal rat atrial myocytes. METHODS：Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12% in stretched group. The cells without stretch served as control. Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp. RESULTS：Compared with control group, Ito density in stretched myocytes was significantly reduced ［(16±04) pA/pF vs (121±29) pA/pF, P<001］, whereas IK1 density was increased ［(-108±08) pA/pF vs (-88±09) pA/pF, P<001］. The APDs at 50% and 90% levels of repolarization (APD50 and APD90) in the stretched cells were obviously decreased than those in non-stretched cells ［(105±14) ms vs (155±24) ms, (300±28) ms vs (563±36) ms, P<001］. CONCLUSION：Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes, which may contribute to atrial electrical remodeling induced by pressure overload.     

Mutation of ATP-sensitive K+ channels and neonatal diabetes iDEND syndrome

Activating mutation in the KCNJ11 gene encoding Kir6.2 subunit of adenosine triphosphate (ATP)-sensitive potassium (K_ATP) channel gives rise to intermediate developmental delay, epilepsy and neonatal diabetes (iDEND) syndrome, a rare hereditary endocrine metabolic disorder characterized by neonatal diabetes accompanied by developmental delay and muscle weakness, but no epilepsy. The Kir6.2 Val⁵⁹→Met⁵⁹ (V59M) activating mutation is the common cause of iDEND syndrome (>50%). Activating mutation causes iDEND syndrome by inhibiting normal closure of ATP-sensitive K⁺ channel, which leads to reduce insulin secretion. Most of such patients are more sensitive to sulfonylurea. High blood-brain barrier permeability and sulfonylurea receptor 1 (SUR1)-specific drugs are expected to become a major therapy.     

Clinical significance of Kir2.1/KCNJ2 and MRP1/ABCC1 protein expression in small-cell lung cancer

AIM: To investigate the expression of inward rectifying potassium channel 2.1 (Kir2.1/KCNJ2) and multidrug resistance-associated protein 1 (MRP1/ABCC1) in small-cell lung cancer (SCLC), and the correlation between them. METHODS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69 and H69AR cell lines was detected by real-time PCR and Western blotting. Immunohistochemistry (EnVision) method was used to detect the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 proteins in 44 cases of SCLC tissues. The interaction of Kir2.1/KCNJ2 with MRP1/ABCC1 was tested by co-immunoprecipitation (co-IP). RESULTS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69AR cells was all significantly higher than that in H69 cells (P<005). The positive expression rates of Kir2.1/KCNJ2 and MRP1/ABCC1 in the 44 cases of SCLC tissues were 47.7% (21/44) and 52.3% (23/44), respectively. A positive correlation was found between the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 in SCLC tissues (r=0.853, P<0.01). Co-IP results showed that Kir2.1/KCNJ2 could bind MRP1/ABCC1 in H69AR cells. CONCLUSION: Kir2.1/KCNJ2 is related to the multidrug resistance of SCLC. Kir2.1/KCNJ2 and MRP1/ABCC1 may have common function roles. Therefore, Kir2.1/KCNJ2 may promote the multidrug resistance of SCLC by regulating the expression of MRP1/ABCC1.     

Effects of miR-92a and miR-19b on insulin gene expression in mouse pancreatic β-cells

AIM To investigate the effect of microRNA-92a （miR-92a） and microRNA-19b （miR-19b） on the insulin expression in mouse pancreatic β-cells. METHODS The relative expression levels of endogenous miR-92a and miR-19b in mouse insulinoma MIN6 cells were detected by qPCR. The MIN6 cells were divided into control group， and experimental groups I and II， with 3 samples in each group， and transfected with negative control miRNA （NC）， miR-92a and miR-19b， respectively. The over-expression of the miRNAs was detected by qPCR. The morphological changes and viability of the cells were detected by optical microscopy and CCK8 assay， respectively. The expression of insulin was detected by qPCR， Western blot and immunofluorescence. The possible mechanisms of miR-92a and miR-19b regulating insulin expression were analyzed by bioinformatics prediction， dual-luciferase reporter assay， and Western blot. RESULTS Compared with the adult pancreatic progenitor cells， the expression of endogenous miR-92a and miR-19b in the MIN6 cells was decreased significantly （P<0.05）. Over-expression of miR-92a and miR-19b had no effect on the viability of MIN6 cells， but inhibited the expression of insulin at mRNA and protein levels. miR-19b significantly inhibited the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1 （P<0.05）. miR-92a had a fine-tuning effect on the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1. CONCLUSION miR-92a and miR-19b inhibit the insulin expression in mouse pancreatic β-cells.     

S100A6 promotes proliferation and migration of human osteosarcoma cell line 143B through PI3K/Akt signaling pathway

AIM: To investigate the effect of PI3K/Akt signaling pathway on S100A6-induced proliferation and migration of human osteosarcoma cell line 143B. METHODS: Recombinant human S100A6 protein (rhS100A6) was prepared. The 143B cells were treated with rhS100A6 in the presence or absence of PI3K inhibitor (LY294002 or wortmannin) exposure. The final concentrations of rhS100A6, LY294002 and wortmannin were 30 mg/L, 10 μmol/L and 0.5 μmol/L, respectively. The expression levels of total Akt (t-Akt) and phosphorylated Akt (p-Akt) in the 143B cells were analyzed by Western blotting. The cell proliferation and migration were determined by MTT and Transwell assays. RESULTS: rhS100A6 protein was successfully prepared, and significantly increased the proliferation and migration of 143B cells (P<005). rhS100A6 up-regulated the phosphorylation of Akt in 143B cells (P<005). Compared with rhS100A6 group, the level of p-Akt in 143B cells and the proliferation and migration of the cells were decreased in combined treatment group of rhS100A6 with LY294002 or wortmannin (P<005), where the proliferation rate at different time points dropped from 10.3% to 69.7% (P<005), and the migration rate dropped from 34.9% to 47.7% (P<005). CONCLUSION: To some extent, S100A6 promotes proliferation and migration of human ostersarcoma cell line 143B through PI3K/Akt signaling pathway.     

Oxymatrine ameliorates high fat-induced insulin resistance in ApoE-/- mice

ATM: To investigate the effect of oxymatrine (OXY) on high fat-induced insulin resistance in mice, and to investigate the mechanism. METHODS: ApoE^-/-mice with high-fat diet for 16 weeks were divided into insulin resistance group, and OXY groups at concentrations of 25, 50 and 100 mg/kg. C57BL/6J mice served as normal control group. The mice in OXY groups were gavaged with OXY for 8 weeks. Glucose tolerance test in the mice was performed. Fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), fatty acid (FFA) and fasting insulin (FINS) in the plasma were measured. The mRNA expression of insulin receptor (INSR), insulin receptor substrate-2 (IRS-2), glucose transporter 2 (GLUT2) in the liver tissues was examined by RT-qPCR. The protein levels of GLUT2, INSR, IRS-2, p-INSR, p-IRS-2, PI3K, p-PI3K, serine/threonine protein kinase (AKT) and p-AKT were examined by Western blot.RESULTS: OXY reduced the levels of FBG, TC, TG, FFA and FINS, and attenuated insulin resistance. Compared with insulin resistance group, the mRNA expression of INSR, IRS-2 and GLUT2 significantly increased in OXY groups (P<0.05). The protein levels of p-INSR/INSR, p-IRS-2/IRS-2, p-PI3K/PI3K, p-AKT/AKT and GLUT2 also increased in OXY groups (P<0.05).CONCLUSION: OXY ameliorates high fat-induced insulin resistance in mice via PI3K/AKT pathway.     

Establishment and verification of insulin resistance model in mouse skeletal muscle cells

AIM:To establish a stable and repeatable insulin resistance model of skeletal muscle cells in vitro, so as to promote the exploration of the pathological mechanism of insulin resistance and the development and screening of related drugs. METHODS:C2C12 mouse myoblasts were used to induce differentiation in normal differentiation medium and differentiation medium containing glucose at 40 and 60 mmol/L, respectively. The effects of glucose at different concentrations on cell convergence, fusion and formation of multinucleated myotubes were observed under phase contrast microscope every day. After 1, 3, 5 and 7 d of differentiation, 2-NBDG assay was used to detect the effects of different interventions on C2C12 basal glucose uptake and insulin-stimulated glucose uptake. The effects of different interventions on the protein expression of glucose transporter 4 (GLUT4) after 5 d and 7 d of differentiation were determined by Western blot. The effects of different interventions on the distribution of GLUT4 protein after 5 d of differentiation were detected by immunofluorescence staining. RESULTS:After treated with glucose at 60 mmol/L, the morphological observation showed that high glucose treatment significantly inhibited the growth and differentiation of C2C12 cells after 3 d. High glucose treatment significantly inhibited basal glucose uptake and insulin-stimulated glucose uptake of the C2C12 cells after 5 d and 7 d (P<0.01). No difference between insulin-stimulated GLUT4 expression and basal GLUT4 expression after 5 d and 7 d of high glucose treatment was observed (P>0.05), but there was significant difference between control group and 60 mmol/L group (P<0.05) determined by Western blot. Immunofluorescence staining observation showed that the distribution of GLUT4 protein in the C2C12 cell membrane was significantly decreased after 5 d of high glucose treatment (P<0.01). Glucose treatment (40 mmol/L) also played a role to some extent, but the effect was not as obvious and stable as 60 mmol/L glucose. CONCLUSION:A stable insulin resistance model of mouse skeletal muscle cells in vitro was successfully established by high glucose stimulation. The treatment of glucose at 60 mmol/L for 5 d was the best. Morphological observation and detection of basic and insulin-stimulated glucose uptake and GLUT4 protein expression and distribution evaluates the insulin resistance level of skeletal muscle cells in vitro.     

Changes of DNA methylation at CpG sites in promoter region of TNF-α gene in DENV2-infected peripheral blood mononuclear cells

AIM：To examine DNA methylation at CpG sites in the promoter region of tumor necrosis factor-alpha (TNF-α) gene in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMC).
METHODS：DNA methylation in the promoter region of TNF-α gene was measured by bisulfite sequencing PCR.
RESULTS：The promoter region of TNF-α gene was from -294 bp to +58 bp, including 11 CpG sites. The PCR products identified by aga-rose gel electrophoresis were consistent with the theoretical size. Two sites were methylated at 0 h and 6 h and 6 sites were methylated at 12 h in TNF-α gene promoter region in DENV2-infected PBMC. The average methylation rates were 103%, 121% and 255% at 0 h, 6 h and 12 h, respectively. Significant differences between 0 h and 12 h and between 6 h and 12 h were observed.
CONCLUSION：The DNA methylation in the promoter region of TNF-α gene is increased in DENV2-infected PBMCs.     

Expression of GATA3 in human breast cancer tissues and its clinical significance

AIM: To investigate the expression of GATA3 in human breast carcinoma and its clinical significance. METHODS: The expression level of GATA3 in breast cancer tissues from 124 patients was detected by the method of immunohistochemistry and the relationships between GATA3 expression and other clinicopathological factors were analyzed. RESULTS: Low expression of GATA3 in breast cancer tissues was associated with estrogen receptor (ER)/progesterone receptor (PR) negative, high histological tumor grade, p53 mutation and vascular invasion (P<005), but not with age, tumor size,human epidermal growth factor receptor 2 (HER-2) expression and lymph node metastasis (P>005). In all breast cancer tissues, the positive expression rate of GATA3 was 56.4%. The positive expression rate of GATA3 in luminal breast cancer is 684%, higher than that in non-luminal breast cancer (326%, P<005). In all breast cancer tissues, the expression of GATA3 in middle recurrence risk group was higher than that in high recurrence risk group (P<005). CONCLUSION: GATA3 expression in breast cancer is related to differentiation and biological characteristics of the tumor, which can be a factor for evaluation of the treatment and prognosis.     

High-fat diet affects plasma membrane GLUT4 content in skeletal muscle from Sprague-Dawley rats

AIM: To explore the change of the amount of GLUT4 protein at the plasma membrane of the rat skeletal muscle after high-fat feeding. METHODS: The animals were divided into three groups (ten for each): group I: control; group II: high-fat feeding; group III: high-fat feeding + dietary treatment. The rat model of insulin resistance (IR) was made by feeding high-fat diet for eight weeks. And then insulin-resistant rats were fed with chow diet for 4 weeks. Fasting plasma glucose and fasting serum insulin levels were measured before and after dietary treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (10 unit insulin per kg body weight) 15 minutes before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western bloting. RESULTS: The GLUT4 content at the plasma membrane in high-fat-fed rat skeletal muscle was significantly lower (about 31%) than that in controls (P<0.01). Dietary treatment partly corrected fasting blood glucose [from(6.20±0.39)mmol/L to(5.78±0.74)mmol/L]and fasting serum insulin levels [from(17.19±1.93)mU/L to(11.68±1.28)mU/L] and increased the GLUT4 content at the plasma membrane by 1.14-fold in insulin-resistant rat skeletal muscle. CONCLUSION: High-fat feeding induces IR in Sprague-Dawley rats. The mechanism may be involved in decreased cell-surface level of GLUT4 through affecting intracellular insulin signaling and then decreasing GLUT4 trafficking.     

Effects of taurine on expression of glucose transporters in rat brain with diffused brain injury

AIM: To investigate the effect of taurine on the expression of glucose transporter 1 (GLUT1) and transporter 3 (GLUT3) in rat brain with diffused brain injury (DBI).METHODS: Sixty-four male Sprague-Dawley rats were randomly divided into 4 groups: sham-operated group, DBI group, low-dose taurine group (200 mg/kg, ig) and high-dose taurine group (300 mg/kg, ig).After fed with the corresponding drugs for 7 days, the animal model of DBI was made, and the rats were executed 24 h after DBI.The expression of GLUT1 and GLUT3 in the brain was detected by the methods of immunohistochemistry and Western blotting.The pathomorphological changes of the cerebral cortex were observed under electron microscope.RESULTS: The expression of GLUT1 was detected in capillary vascular endothelial cells in each group, and cytoplasm-positive cells or the cells with buffy membrane were observed.No significant difference of the GLUT1 expression in brain tissues between DBI group and sham-operated group was detected.Compared with DBI group, the expression of GLUT1 in the brain tissues were significantly increased in low-and high-dose taurine groups (P<0.01).The expression of GLUT1 in the brain tissues in low-dose taurine group were significantly higher than that in high-dose taurine group (P<0.05).The positive staining of GLUT3 only appeared in the periphery of the third ventricle in each group in the cells with buffy membrane or positive cytoplasm.The expression of GLUT3 in the brain tissues in DBI group was significantly higher than that in sham-operated group (P<0.01).The expression of GLUT3 in the brain tissues in low-and high-dose taurine groups was significantly higher than that in DBI group (P<0.01).Compared with low dose taurine group, the expression of GLUT3 in the brain tissues were significantly increased in high-dose taurine group (P<0.01).The pathological damage of cerebral cortex in low-dose taurine group was obviously alleviated.CONCLUSION: Taurine may take part in the neuroprotective mechanisms in DBI by increasing the expression of GLUT1 and GLUT3 at protein level to maintain the energy supply in brain tissues.     

Effects of simvastatin on cigarette smoke extract-induced sEPCR and EPCR expression in human umbilical vein endothelial cells

AIM: To investigate the effects of simvastatin on cigarette smoke extract (CSE)-induced expression levels of soluble endothelial cell protein C receptor (sEPCR) and membrane-associated endothelial cell protein C receptor (mEPCR ) in human umbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs at passage 4 to 6 were randomly divided into control group, 5% CSE group, simvastatin groups and simvastatin+CSE groups. In simvastatin groups, HUVECs were incubated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 24 h. In simvastatin+CSE groups, the cells were treated with simvastatin at the concentrations of 50, 100 and 200 μmol/L for 2 h, and then exposed to CSE for 24 h. The protein level of sEPCR in the culture supernatants was measured by ELISA. The cells were collected for determining the mRNA expression of mEPCR by real-time PCR. RESULTS: Compared with control group, the protein level of sEPCR was significantly increased, and the mRNA expression of mEPCR was significantly decreased in 5% CSE group (both P<0.05). The protein levels of sEPCR were significantly increased, and the mRNA expression of mEPCR was significantly decreased in 100 μmol/L and 200 μmol/L simvastatin groups. However, the protein levels of sEPCR were lower, and the mRNA expression of mEPCR was significantly higher in 100 μmol/L and 200 μmol/L simvastatin groups than those in 5% CSE group. Compared with 5% CSE group, the protein levels of sEPCR in simvastatin+CSE groups were significantly decreased, but higher than those in control group and simvastatin group with corresponding concentration. On the contrary, the mRNA expression of mEPCR in simvastatin+CSE groups was significantly increased, but lower than that in control group and simvastatin group with corresponding concentration (all P<0.05). CONCLUSION: Simvastatin obviously increases the mRNA expression of mEPCR, decreases the protein level of sEPCR, and attenuates the CSE-induced endothelial injury in vitro .     

Effects of bradykinin on proliferation of porcine pulmonary artery smooth muscle cells induced by TGF-β1

AIM: To investigate the effects of bradykinin (BK) on the proliferation of pulmonary artery smooth muscle cells (PASMCs) induced by transforming growth factor beta 1 (TGF-β1) and its possible mechanisms. METHODS: Primary porcine PASMCs were isolated, cultured and identified, and the cells at passages 2~6 were used in this study. The viability of PASMCs was determined by Cell Counting Kit-8 assay. The protein expression of phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was detected by Western blotting. RESULTS: TGF-β1 promoted the proliferation of PASMCs in a dose-dependent manner (P<005). BK significantly inhibited the proliferation of PASMCs induced by TGF-β1 (P<005), and attenuated the elevated expression of PI3K, p-Akt and p-ERK1/2 proteins (P<005). HOE-140, a BK type 2 receptor (B2R) inhibitor, reversed the effects of BK (P<005). CONCLUSION: BK inhibits TGF-β1-induced proliferation of PASMCs, which may be associated with inactivation of PI3K/Akt and ERK1/2 signaling pathways.