Effects of L-carnosine on NIT-1 islet cells

AIM: To investigate the protective effect of L-carnosine on insulin secretion, proliferation and apoptosis of β-cells impaired by high glucose. METHODS: NIT-1 cells were pre-treated with glucose at concentrations of 11.1 mmol/L (control level) and 33.3 mmol/L (high level) for 72 h, and then the cells were stimulated with various concentrations of glucose (0, 5 and 25 mmol/L) and/or L-carnosine (0, 1 and 20 mmol/L). The level of insulin in the medium was measured by radioimmunoassay. To detect the effect of L-carnosine on proliferation and apoptosis, NIT-1 cells were divided into 4 groups according to different culture conditions for 72 h: group C (with 11.1 mmol/L glucose), group H (with 33.3 mmol/L glucose), group H+A (with 33.3 mmol/L glucose+ 1 mmol/L L-carnosine) and group H+B (with 33.3 mmol/L glucose +20 mmol/L L-carnosine). Proliferous or apoptotic cells were identified by BrdU labeling and flow cytometry (labeling with annexin V-FITC/PI),respectively. Total RNA was extracted and the mRNA expression of caspase-3 and bcl-2 was measured by RT-PCR. The caspase-3 activity was also checked by fluorometric assay kit. RESULTS: The insulin in high-level glucose group was lower than that in control-level glucose group. L-carnosine at concentration of 20 mmol/L notably increased the insulin secretion of the cells pre-treated with glucose at control level or high level. The proliferation and apoptosis were both increased in group H compared with group C, but the total cell counts declined because the apoptotic rate was higher than the proliferation rate. L-carnosine at concentration of 1 mmol/L significantly increased the proliferation rate and decreased the apoptotic rate. The mRNA level of caspase-3 was decreased and the mRNA level of bcl-2 was increased after the cells were treated with L-carnosine at concentration of 1 mmol/L. L-carnosine at concentrations of both 1 mmol/L and 20 mmol/L significantly decreased the caspase-3 activity. CONCLUSION: L-carnosine at high level directly stimulates insulin secretion in NIT-1 cells, and L-carnosine at normal level promotes the cell proliferation and inhibits apoptosis induced by high concentration of glucose. Caspsase-3 and Bcl-2 may be partly involved in this process.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Effects and possible mechanism of recombinant human erythropoietin on proliferation of endothelial progenitor cells

AIM: To observe the effects of recombinant human erythropoietin on proliferation of endothelial progenitor cells (EPCs) from healthy volunteers and patients with renal failure,and tried to elucidate the possible mechanism.METHODS: Various concentrations of rhEPO were added to the culture system of EPCs from 15 cases of patients with renal failure (RF group) and 15 cases of healthy volunteers (control group).MTT assays were used to detect proliferative rates.Annexin-V/PI stains were used to measure the apoptotic rates.Western blotting was used to determine the expression of Akt protein kinase.RESULTS: Numbers and proliferative ability of EPCs from control group and RF group were improved in dose-dependent manner when concentrations of rhEPO were 100 U/L,600 U/L and 1 200 U/L.However,compared to the control group,numbers and function of EPCs from RF group were remarkably decreased.The apoptosis rate of EPCs was decreased and the activity of Akt protein kinase was improved in the presence of 1 200 U/L rhEPO.Wortmannin was able to block the effects.CONCLUSION: rhEPO improves the number and function of EPCs from both healthy volunteer and patients with renal failure.PI3K/Akt might play an important role in it.     

Effect of puerarin on myocardial injury in KKAy diabetic mice

AIM:To observe the effect of puerarin on myocardial injury according to the time of occurrence of myocardial injury in the development of type 2 diabetic mice. METHODS: The serum levels of glucose (GLU), triglyceride (TG), total cholesterin (TC), low density lipoprotein-cholesterol (LDL-C) and high density lipoprotein-cholesterol (HDL-C) in 17-week, 20-week, 24-week, 28-week KKAy mice were detected by automatic biochemical methods. The apoptotic percentage of cardiomyocytes was examined by flow cytometry. The expressions of bax and bcl-2 mRNA in cardiomyocytes were detected by RT-PCR. Caspase-3 expression in cardiomyocytes was determined by immunohistochemical staining. RESULTS: Compared to normal control mice, not only GLU level increased, but also the levels of TG, TC, LDL-C, HDL-C in 20-week, 24-week and 28-week KKAy mice increased apparently (P<0.01). Apoptosis of cardiomyocytes in KKAy mice began to show up at 28-week. The expression of bax mRNA increased and expression of bcl-2 mRNA reduced. At the same time, the expression of caspase-3 increased. Puerarin obviously decreased the apoptotic percentage of cardiomyocytes, reduced the expression level of bax mRNA, improved the expression level of bcl-2 mRNA (P<0.01), inhibited the expression of caspase-3 (P<0.01). CONCLUSION:Myocardial injury exits in type 2 diabetic mice. Mitochondria apoptotic pathway might participate in the hurting course.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway, thus promoting the apoptosis of cardiomyocytes.     

Effect of L-arginine on expression of bcl-2 and bax mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits

AIM:To investigate the effect of L-arginine (L-Arg) on expression of bcl-2, bax mRNA during pulmonary ischemia and reperfusion injury (PIRI) in rabbits.METHODS:Single lung ischemia and reperfusion animal model was used in vivo. The rabbits were randomly divided into three groups: sham operated group (sham, n=12), ischemia-reperfusion group (I/R, n=12) and I/R+ L-arginine group (L-Arg, n=12). Changes of several parameters, which included apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA), were measured at 300 min after reperfusion in lung tissue. Meanwhile the location and expression of bcl-2, bax mRNA as well as the ratio of bcl-2 mRNA/bax mRNA were observed. The lung tissue was prepared for light microscopic and electron microscopic observation at 60, 180 and 300 min after reperfusion. RESULTS:As compared with I/R group, in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia, the expression of bcl-2 mRNA and the ratio of bcl-2 mRNA/bax mRNA were increased, and the expression of bax mRNA was decreased in L-Arg treatment group. The values of AI, W/D and IQA showed significantly lower than that in I/R group at 180 minutes after reperfusion in lung tissue (P<0.01 and P<0.05). Meanwhile, abnormal changes of the lung tissue in morphologically were markedly lessened in L-Arg treatment group.CONCLUSION:L-arginine produces a notable protective effect on PIRI in rabbits by up-regulating bcl-2 mRNA expression, down-regulating bax mRNA expression in lung tissue and regulating the balance of bcl-2 mRNA and bax mRNA to decrease apoptosis.     

Insulin protects endothelial progenitor cells against functional damage caused by high glucose

AIM: To investigate the impact of various levels of glucose on endothelial progenitor cells (EPCs) proliferation, senescence, and nitric oxide (NO) secretion,and the effect of insulin under high glucose conditions.METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199, and identified to be EPCs at 7th day by flk-1 and AC133 double staining. EPCss were harvested and incubated with glucose (5, 10, 20, 40 mmol/L) or insulin (0.1, 1, 10, 100 nmol/L) under high glucose conditions for 24 h or 7 days. Proliferative capacity, senescence level and NO secretion (after 24 h of incubation) were subsequently determined.RESULTS: High glucose (40 mmol/L) markedly inhibited EPCs proliferation, accelerated EPCs senescence, and decreased NO production (all P<0.05). Compared with high glucose (40 mmol/L) group, insulin intervention promoted EPCs proliferation, inhibited EPCs senescence (prominent at 1 nmol/L, P<0.05), and enhanced NO secretion (prominent at 10 nmol/L, P<0.05).CONCLUSION: High glucose harms EPCs proliferation and function, while insulin alleviates this jeopardy, indicating the protective role of insulin for the cardiovascular system.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Changes of bcl-2, bax expression and neuron apoptosis in lymphostatic encephalopathy of rats

AIM: To investigate the changes of bcl-2, bax expression and neuron apoptosis of cerebral cortex in lymphostatic encephalopathy of rats. METHODS: The model of lymphostatic encephalopathy was established by occluding and removing both the shallow and deep cervical lymph nodes in rats. The animals were sacrificed at 1, 2, 3, 5, 7 and 14 days after operation. HE staining was used to observe the structure of brain tissues and TUNEL staining was used to detect in situ cell apoptosis. The expressions of bcl-2 and bax were examined by RT-PCR. RESULTS: Cerebroedema appeared at the second day and was the most serious at the 5th day after blockage of cervical lymphatics. The number of TUNEL positive cells and the expression of bax began to increase at the 2nd day, reached a peak at the 5th day and dropped to control level at the 14th day. The expression of bcl-2 began to increase at the 1st day, reached a peak at the 5th day and dropped to control level at the 7th day. The increasing extent of bax was higher than that of bcl-2. CONCLUSION: The blockage of cervical lymphatics can lead to lymphostatic encephalopathy. Apoptosis is the main form of neuron death in the cortex and has relation to the increasing expression of bcl-2 and bax.     

Effect of blood glucose control on expression of Smad7 and renal fibrosis in diabetic rats

AIM： To verify the hypothesis that treatment with insulin to control the blood glucose (BG) may relieve or slow down the development of diabetic nephropathy (DN) in diabetic rats by increasing the expression of Smad7. METHODS：The diabetic rat model was established by tail-vein injection of streptozotocin. Sixteen rats were divided into 2 groups. Eight of these animals in diabetes mellitus (DM) group had no treatment. The remaining eight of them in insulin treatment (INS) group were injected with insulin. After 13 weeks, the rats in INS group were given individual treatment with insulin to let the blood glucose level keep within 4 to 7 mmol/L. Meanwhile, 8 rats were used for normal control (NC group). After 16 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and to observe the histophathological changes of the kidney and pancreas. In addition, immunohistochemical staining and Western blotting were employed to detect the protein expression of transforming growth factor β1 (TGF-β1), Smad ubiquitin regulatory factor 2 (Smurf2), Smad7, E-cadherin, α-sooth muscle actin (α-SMA), fibronectin (FN) and collagen I. RESULTS：Compared with NC group, the body weight was significantly reduced in DM group, whereas the body weight in INS group increased gradually. Compared with NC group, the levels of 24 h urine protein (24 h UP), BG and triglyceride (TG) were remarkably increased in DM group. Pathological detection on pancreas indicated that the islet was destroyed. The levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in the kidneys were increased in DM group, and the expression of Smad7 and E-cadherin, which were mainly located in renal tubular epithelial cells, was significantly reduced. Compared with DM group, the levels of 24 h UP and BG were significantly reduced in INS group, and the alleviated renal fibrosis was observed under light microscope. In addition, the protein levels of TGF-β1, Smurf2, α-SMA, FN and collagenⅠ in INS group were decreased compared with DM group, and the expression of Smad7 and E-cadherin was increased significantly. CONCLUSION：Target glucose control with insulin treatment restores the protein expression of Smad7 in the kidney of diabetic rats, reduces the accumulation of extracellular matrix and slows down DN progress. The decrease in TGF-β1 and Smurf2 expression, and the attenuation of Smad7 ubiquitination in renal tissues are the crucial parts in this process.     

Up-regulation of mRNA expressions of fas,bcl-2, bim,bax, caspase-3, caspase-9, and bcl2l12 in K562 treated with bortezomib

AIM: To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas, bcl-2, bcl2l12, bim, bax, caspase-9 and caspase-3.METHODS: MTT assay was used to detect the inhibition of proliferation. Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential (Δψm). RT-PCR was used to analyze the mRNA expressions of fas, bcl-2, bcl2l12, bim, bax, caspase-3 and caspase-9.RESULTS: Bortezomib caused a time- and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L, respectively. At the concentration of 104 nmol/L, bortezomib induced apoptosis in a time-dependent manner, including increasing annexin-V positivity and decreasing the Δψm. RT-PCR showed that bortezomib up-regulated the mRNA expression of fas, bcl2l12, caspase-9 and caspase-3, but mRNA expressions of bcl-2, bim and bax did not changed obviously.CONCLUSION: Bortezomib inhibits the proliferation of K562 and induces apoptosis, in which fas, bcl2l12, caspase-9 or caspase-3 gene is one of the main genes taking part in.     

Expression of perilipin and ADRP in rat liver tissues with abnormal glucose metabolism

AIM: To observe the changes of perilipin and adipose differentiation-related protein (ADRP) du-ring the development of diabetes mellitus and to explore the effect of perilipin and ADRP on abnormal glucose metabolism with non-alcoholic fatty liver disease (NAFLD). METHODS: The rat model of impaired glucose tolerance (IGT) was induced by feeding high-fat diet, and the type 2 diabetes mellitus (T2DM) model was induced by feeding high-fat diet for 4 weeks and intraperitoneally injecting streptozotocin. The morphological change of the liver tissue was observed under optical microscope. The serum contents of perilipin and ADRP were measured by ELISA. The mRNA expression of perilipin and ADRP in the liver tissues was detected by real-time PCR. The protein expression of ADRP in the liver tissues was determined by Western blotting. RESULTS: HE staining showed steatosis in the liver of the rats in IGT group was more serious than that in T2DM group. The biochemical and the pathological processes of rat models were consistent with the clinical feature of related diseases. The serum content of perilipin had no difference among various groups. The mRNA expression of perilipin in IGT group and T2DM group was significantly higher than that in control group. Compared with IGT group, the mRNA expression of perilipin in T2DM group was significantly increased. The serum content of ADRP in T2DM group was significantly lower than that in control group. The mRNA and protein expression of ADRP in model groups was significantly lower than that in control group. Compared with IGT group, the mRNA expression of ADRP in T2DM group was significantly reduced. CONCLUSION: The serum content of ADRP plays a role in the development and progression of T2DM. It is negatively correlated with HOMA-IR. NAFLD occurs during progression of abnormal glucose metabolism induced by feeding high-fat diet. The development of abnormal glucose metabolism with NAFLD is probably related to the increased expression of perilipin and the reduced expression of ADRP.     

The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Growth-inhibitory effects of selective cyclooxygenase-2 inhibitor on colon cancer cells and its possible mechanisms

AIM: To evaluate the growth-inhibitory effects of NS-398, a selective cyclooxygenase-2 inhibitor, in human colon cancer HT-29 cells and its possible mechanisms. METHODS: MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect apoptosis rate and cell cycle. RT-PCR was used to detect the expression of bcl-2 mRNA and bax mRNA. Alteration of cytoskeleton component F-actin was observed by confocal laser scanning microscope. RESULTS: NS-398 could inhibit growth of HT-29 cells in dose-and time-dependent manners. Flow cytometry revealed that NS-398 could induce apoptosis and cause G0/G1 arrest of HT-29 cells in a dose-dependent manner. After 72 h incubation with NS-398 at different concentrations, the expression level of bcl-2 mRNA was lowered and the ratio of bcl-2 to bax was decreased in HT-29 cells. F-actin was mainly distributed around nuclei forming annular structure in HT-29 cells. After exposure to NS-398, the annular structure around nuclei disappeared and fluorescence intensity of F-actin decreased obviously. CONCLUSION: NS-398 can inhibit the growth effectively and induce apoptosis in HT-29 cells in vitro, which is associated with the down-regulation of bcl-2 to bax ratio and the disruption of cytoskeleton.     

Effect of shock lymph on apoptosis relative gene expression in pulmonary micro-vascular endothelial cells

AIM:To observe the effects of shock lymph on apoptosis relative gene expressions of pulmonary micro-vascular endothelial cells (PMVECs), and explore its mechanism.METHODS:The model of severe hemorrhagic shock was established by maintaining the blood pressure of rats in the condition of sepsis, mesentery lymph and shock portal vein blood was taken out. As control, mesentery lymph, portal vein blood of normal rats was taken out. The primary PMVECs of passages 3 were treated by different treatment factors, respectively. The apoptosis rate was analyzed by flow cytometry, and the expressions of relative genes of apoptosis such as fas, fas L, bcl-2 and bax were detected by RT-PCR. RESULTS:The apoptosis rate of PMVECs was 9.86%±3.24% after exposed to shock lymph at the final concentration of 4% for 4 hours and significantly higher than that in control (P<0.01). The expression levels of fas, fas L and bax mRNA were higher and bcl-2 mRNA was lower in shock lymph group than those in control group.CONCLUSION:The results demonstrated that the apoptosis of PMVECs of rats was induced by shock lymph, and its mechanism relate to high expression of apoptosis accelerative genes such as fas, fas L, bax mRNA and low expression of apoptosis inhibitory gene bcl-2.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.