Expression and significance of stanniocalcin 2 in diabetic retinopathy

AIM To investigate the expression level of stanniocalcin 2 (STC2) in vitreous tissues of rats with diabetic retinopathy (DR), and to explore the relationship between STC2 and vascular endothelial growth factor (VEGF). METHODS Wistar rats were randomly divided into control group and DR group. The DR model was constructed by injection of streptozotocin. RT-qPCR, Western blot and ELISA were used to detect the expression levels of STC2 and VEGF in rat vitreous tissues. Rat retinal ganglion cells were treated with VEGFA, and the expression of STC2 was detected by RT-qPCR, Western blot and ELISA. The retinal ganglion cells were also treated with STC2 protein, and then the expression of VEGF was detected by RT-qPCR, Western blot and ELISA. Co-immunoprecipitation was used to detect the interaction between VEGF and STC2. RESULTS Compared with control group, the mRNA and protein levels of STC2 were significantly increased in vitreous tissues of the rats with DR, and the expression level of VEGF was also significantly increased in DR group (P<0.01). The expression level of STC2 was positively correlated with VEGF expression. VEGF induced the expression of STC2 in rat retinal ganglion cells and promoted its secretion. STC2 protein induced the expression and secretion of VEGF in rat retinal ganglion cells, and VEGF had certain interaction with STC2. CONCLUSION STC2 expression is significantly increased in vitreous tissues of the rats with DR, and is closely related to VEGF.     

Effect of hyperlipidemia on nitric oxide levels of blood and myocardial tissues in rats

AIM: To study the impact of hyperlipidemia on vasoactive substances and the mechanism of gemfibrozil in regulating the endothelial function. METHODS: The hyperlipidemic model was established with rats. The serum levels of lipid, NO, angiotensin Ⅱ (Ang Ⅱ), vascular endothelial growth factor(VEGF) and levels of NO, AngⅡ in myocardial tissues were measured. RESULTS: Hyperlipidemic rats had lower level of NO and higher level of VEGF than control subjects. Significant correlation had been shown between serum levels of lipid and NO. Administration of gemfibrozil significantly elevated serum VEGF, NO and myocardial tissue NO levels. CONCLUSIONS: Hyperlipidemia not only reduced serum NO level but also reduced myocardial NO content, which could cause endothelial dysfunction. Gemfibrozil reversed the above changes induced by hyperlipidemia, which might have relevance to VEGF.     

Effect of scutellarin on VEGF expression in human retinal pigment epithelial cells and retinas of diabetic rats

AIM: To evaluate the influence of scutellarin on the expression of vascular endothelial growth factor (VEGF) in high glucose-treated human retinal pigment epithelial cell line ARPE-19 and to observe the effects of scutellarin on the protein expression of VEGF, p-ERK and VEGFR2 in the retinas of type II diabetic rats. METHODS: Cultured ARPE-19 cells were divided into normal control group, scutellarin group, high glucose group and high glucose+scutellarin group. The protein levels of VEGF, p-ERK and VEGFR2 were measured by Western blot. The VEGF release in ARPE-19 cells was detected by ELISA. Normal rats were randomly divided into normal control group and scutellarin group. Diabetic rat model was established by feeding with high-fat diet and injecting with streptozocin, and randomly divided into diabetes group and diabetes treated with scutellarin group. After 16 weeks, the eyes were removed. The morphological changes of the retinas were observed under light microscope with HE staining, and histopathological score was recorded. The expression of VEGF in the retinas was observed by the method of immunohistochemistry. RESULTS: Compared with normal control group, the protein levels of VEGF, p-ERK and VEGFR2 in the ARPE-19 cells decreased in scutellarin group, but increased in high glucose group. The histopathological score of the retinas showed significant difference among diabetes group, diabetes treated with scutellarin group and normal control group, and no significant difference between normal control group and scutellarin group was observed. The expression of VEGF increased in diabetic group and was significantly higher than that in scutellarin treatment group (P<0.05). CONCLUSION: Scutellarin inhibits the increased protein le-vels of VEGF, p-ERK and VEGFR2 in ARPE-19 cells, and decreases the expression of VEGF in the retinas of diabetic rats. The suppression of the diabetic retinopathy development by scutellarin may be partly involved in the ERK/MAPK pathway.     

Protective effect of aFGF delivery by ultrasound-targeted microbubble destruction on left ventricular function in diabetic cardiomyopathy rats

AIM：To observe the protective effect of delivery of acidic fibroblast growth factor (aFGF) to myocardium by ultrasound-targeted microbubble destruction (UTMD) on left ventricular function in diabetic cardiomyopathy (DCM) rats and to investigate the possible mechanisms. METHODS：Twenty-four rats were intraperitoneally injected with streptozocin to induce DCM and were randomly divided into DCM group and aFGF treatment group. Twelve healthy rats served as normal controls. The rats in aFGF treatment group were infused with SonoVue-aFGF mixed fluid through tail vein and UTMD was simultaneously performed. Four weeks after intervention, all rats underwent cardiac catheterization to mea-sure left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the maximal increase/decrease rate of left ventricular pressure (LV±dp/dtmax). The microvessel density (MVD) of rat myocardial tissues was measured by immunohistochemical staining for CD31. The myocardial collagen volume fraction (CVF) was determined by improved Masson staining. The apoptotic index (AI) was detected by TUNEL method. RESULTS：Four weeks after intervention, the LVESP and LV±dp/dtmax in aFGF treatment group were significantly increased compared with DCM group (P<0.01), while the LVEDP in aFGF treatment group was significantly lower than that in DCM group (P<0.01). The MVD in aFGF treatment group was significantly increased compared with DCM group (P<0.01), but the CVF and AI in aFGF treatment group were significantly lower than those in DCM group (P<0.01). CONCLUSION: Delivery of aFGF to diabetic myocardium by UTMD could improve the left ventricular function of DCM rats and may be a new feasible therapeutic method for DCM.     

Expression of zinc-finger transcription factor Snail 1 in the kidney of diabetic rats

AIM: To explore the expression of Snail 1 in renal tissues of diabetic rats, and to investigate its contribution to the progression of diabetic nephropathy. METHODS: Streptozotocin-induced diabetic rats were randomly divided into 2, 4, 8, 12, 16, 20, 24 weeks groups and 16 week A, 20 week A and 24 week A groups. A groups were treated with insulin to control blood glucose to normal level from the 13th week. Control groups were set up in age-matched time points. Blood glucose, 24 h urine protein, serum creatinine (Scr) and kidney index of rats were measured. Periodic acid-silver (PAS) staining was used to observe the renal pathological changes. The mRNA and protein expressions of Snail 1 and FN in renal cortex were detected by RT-PCR and immunohistochemical staining, respectively. Western blotting was employed to detect the expression of Snail 1 protein in the renal cortex. RESULTS: The levels of blood glucose, Scr, kidney weight index were increased remarkably in diabetic rats as compared with those in control groups (P<0.05, P<0.01), and decreased remarkably in the insulin-treated rats as compared with those in the diabetic rats (P<0.05, P<0.01). The Snail 1 protein was not detected by immunohistochemical staining in normal renal tissues. However, strongly positive staining was observed in renal tubules of diabetic rats. A time-dependent loss of Snail 1 expression was detected in the kidney in insulin-treated rats. The Snail 1 protein and mRNA of Snail 1 and FN were significantly up-regulated in the diabetic rats as compared with those in controls (P<0.01), while down-regulated in the insulin-treated diabetic rats (P<0.01). A close positive relationship existed between the mRNA expression of Snail 1 and FN (r=0.800, P<0.01). The level of Snail 1 protein expression was positively correlated with blood glucose, urine protein, Scr, kidney index (r=0.877, 0.694, 0.522, 0.875, P<0.01).CONCLUSION: These findings suggest that Snail 1 gene and protein expression are up-regulated in the kidney of rats with diabetes and may be involved in the pathogenesis of diabetic nephropathy.     

Neuregulin-1 induces expression of angiogenic factors in coronary artery smooth muscle cells

ATM: To investigate the effects of neuregulin-1 (NRG-1) on the expression of angiogenic factors in human coronary artery smooth muscle cells (HCASMCs). METHODS: HCASMCs were cultured in vitro, and the cells at the 3rd passage were collected to assess the expression and phosphorylation of ErbB by Western blot. After HCASMCs were cultured under normal condition, with hypoxia and serum deprivation, or with NRG-1 treatment, the expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) was determined by Western blot. RESULTS: The expression of ErbB2, ErbB3 and ErbB4 was observed in the HCASMCs, and the phosphorylation of these receptors was increased by NRG-1 treatment. Compared with control group, the expression of VEGF and Ang-1 in the HCASMCs was significantly increased in hypoxia and serum deprivation group (P<0.05﹚,while no difference in the expression of Ang-2 between the 2 groups was found.Compared with hypoxia and serum deprivation group,the expression of VEGF and Ang-1 in the H CASM Cs treated with NRG-1 was furtherincreased﹙P<0.05﹚,and no difference in the expression of Ang-2 between the 2 groups was observed.CONCLUSION: HCASMCs express ErbB2, ErbB3 and ErbB4, and the phosphorylation of the receptors is increased by NRG-1. Hypoxia, serum deprivation and NRG-1 treatment induce the increased expression of VEGF and Ang-1 significantly.     

Effect of Xuefuzhuyu decoction on cardiomyopathy in type 2 diabetic rats

AIM: To observe the pathologic changes of cardiomyopathy in type 2 diabetic rats and the therapeutic effect of Xuefuzhuyu decoction.METHODS: The diabetic model was established by feeding the animals with high-fat diet and injecting a middle dose of streptozotocin (50 mg/kg) intraperitoneally in 42 Wistar male rats. After 8 weeks, the damage of the heart in the model animals was detected by electrocardiogram and echocardiography, and the serum level of glucose, total cholesterol and triglyceride were determined by the methods of clinical chemistry. The content of collagen was quantified by Masson staining. The apoptosis of cardiomyocytes was measured by TUNEL apoptosis kit. The structures of myocardial damage were observed under light and electronic microscopes.RESULTS: (1) Compared with normal group at the same time points, the contents of serum glucose, triglyceride and cholesterol in model group increased (P<0.05). At the 11th and 14th weeks, the thickness of LVDS was significantly increased (P<0.01), the structure of myocardial tissues was severely damaged and collagen fiber content increased obviously (P<0.01). The cell apoptosis was also increased. (2) Compared with control group at the same time points, the contents of serum glucose, cholesterol and triglyceride in Xuefuzhuyu decoction group significantly decreased (P<0.05). The thickness of LVDS at the 11th and 14th weeks was decreased (P<0.05) and LVM at the 14th week became significantly thinner (P<0.01). The damage of the myocardium and subcellular structure was slighter and the content of collagen was lower than that in control group (P<0.05). The cell apoptosis was also attenuated.CONCLUSION: The levels of blood glucose, total cholesterol and triglyceride and the content of collagen fibers increase when diabetic cardiomyopathy develops, with more cell apoptosis and severe damage in the cardiac structure. Xuefuzhuyu decoction decreases the level of blood lipid in diabetic cardiomyopathy, alleviates the pathological changes of myocardial fibrosis and delays the progression of diabetic cardiomyopathy.     

Relationship between 3-nitrotyrosine and myocardial cell apoptosis in rat diabetic cardiomyopathy

AIM: To explore the relationship between 3-nitrotyrosine (3-NT) level in hearts or blood and myocardial cell apoptosis in rat diabetic cardiomyopathy (DCM). METHODS: Sixty Sprague-Dawley (SD) rats (male, 8-week-old) were randomly divided into 4 groups: normal group, diabetic cardiomyopathy group (DCM group), diabetic rats treated with valsartan (40 mg·kg^-1·d^-1, D+V group) and DCM rats treated with valsartan (40 mg·kg^-1·d^-1, DCM+V group). Apoptotic index (AI) of rat cardiac myocytes was examined by TUNEL. The expression index (EI) of 3-NT in rat cardiac myocytes was examined by immunohistochemistry. The 3-NT concentration in rat serum was examined by ELISA. RESULTS: (1) Significant differences of the heart weight indexes among the 4 groups were observed (P<0.01). The heart weight indexes in DCM group and DCM+V group were higher than those in normal group and D+V group (P<0.01). (2) The EI of 3-NT in the cardiac myocytes was positively correlated with the AI of the cardiac myocytes in the same group (P<0.01), but the concentration of 3-NT in blood had no correlation with the AI of cardiac myocytes (P>0.05). (3) The difference of AI of cardiac myocytes among the 4 groups had statistical significance (P<0.01). The arrangement from high to low of AI was DCM group > D+V group and DCM+V group > N group (P<0.05). (4) The EI of 3-NT in DCM group was the highest as compared to other groups (P<0.05). (5) No statistical difference of 3-NT concentration in blood among the 4 groups was observed (P>0.05). CONCLUSION: (1) The expression of 3-NT in DCM myocardial tissues in SD rats is significantly increased and closely correlated with the apoptosis in myocardial cells. Valsartan inhibits 3-NT expression in DCM myocardial cells, thus inhibits the DCM myocardium apoptosis. (2) The 3-NT level in blood can not be true for reflection of 3-NT expression in DCM myocardial tissues and its effect on myocardial cell apoptosis.     

Effects of phillyrin on VEGF and endostatin expression in Lewis lung carcinoma

AIM: To investigate the effects of phillyrin on vascular endothelial growth factor (VEGF) and endostatin expression in lung tumor tissues isolated from Lewis lung carcinoma. METHODS: The expression of VEGF and endostatin in control individuals and the patients with lung cancer was determined by immunohistochemistry. In the animal experiment, 5 groups of animals were examined: control, tumor model, and tumor model with 3 different concentrations of phillyrin treatments. For preparation of transplanted tumor model, Lewis cells were subcutaneously injected into the right limb armpit of the nude mice. After that, phillyrin was administered via oral gavage once daily for 20 d at dose of 5 or 10 g/kg, or twice daily at 10 g/kg. Lung tumor tissues isolated from each group were observed by hematoxylin-eosin staining. VEGF and endostatin expression were examined by immunohistochemistry. RESULTS: VEGF expression was increased in lung tumor tissues as compared with normal and pericarcinous tissues, while endostatin expression was decreased. Phillyrin significantly inhibited the tumor size and tumor tissue density dose-dependently, which was accompanied with a decrease in VEGF expression and an increase in endostatin expression. CONCLUSION: Phillyrin inhibits the development of lung tumor through reducing VEGF expression and increasing endostatin expression.     

Alteration of p38 MAPK activity in lung tissue in diabetic rats

AIM: To observe the pathologic changes in lung and the role of p38 MAPKinase signal pathways in pulmonary alteration in diabetic rats. METHODS: Diabetic rats were induced by intraperitoneally injected streptozotozin (STZ). After 4 weeks, we observed the pathologic changes in lungs, tested protein kinase C (PKC) activities by isotope in lungs of model rats, tested transforming growth factor (TGF-β₁) by Western blotting and immunohistochemical analysis, and determined the expression of p38 MAPKinase mRNA using in situ hybridization.RESULTS: After STZ administration for 4 weeks, we observed thickened pulmonary capillary basal lamina and increased number of fibre in Diabetes mellitus (DM) rats. TGF-β₁ levels, PKC and p38 MAPK activities were also found increased. CONCLUSION: The increased activities of TGF-β₁ and p38 MAPK suggeste that TGF-β₁ may play an important role in diabetic lung, and hyperglycemia-PKC-p38 MAPK signal pathways may be involved in the pathogenesis of diabetes.     

Expression of Raf kinase inhibitor protein and p-ERK in renal tissues of diabetic rats

AIM: To investigate the expression of Raf kinase inhibitor protein (RKIP) and phosphorylated extracellular signal-regulated kinase (p-ERK) in the renal tissues of diabetic rats. METHODS: Forty SD rats were randomly divided into normal control group and diabetic nephropathy group. The pathological changes of the renal tissues were observed under microscope with HE and periodic acid-Schiff (PAS) staining. The expression of RKIP and p-ERK1/2 was detected by the methods of immunohistochemistry and Western blotting. RESULTS: Following the extension of duration, the blood sugar of the rats in diabetic nephropathy group increased, with the appearance of proteinuria, increase in kidney weight, increase in renal p-ERK1/2 expression and decrease in RKIP expression as compared with control group. CONCLUSION: The development of diabetic nephropathy may be accelerated by the decrease in RKIP and the increase in p-ERK.     

Protective effect of pyrrolidine dithiocarbamate on cardiac muscles in diabetic rats

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC) on reducing blood glucose level and its protective effect on cardiac muscles in diabetic rats.METHODS: Thirty-seven male Wistar rats were randomly divided into normal control (NC) group and the high-fat diet (HFD) group. After 8 weeks of feeding, the rats in high-fat diet group were given a single dose of streptozotocin (STZ, 27 mg/kg) by intraperitoneal injection to induce type 2 diabetes. The diabetic rats were randomly divided into diabetes mellitus (DM) group and PDTC treatment(PDTC) group. The rats in PDTC group were intraperitoneally injected with PDTC (50 mg/kg) once daily. The rats in NC group and DM group were injected with equivalent volume of saline in the same way. After 1-week treatment, the level of blood glucose was measured, and all animals were killed. The concentration of malondialdehyde (MDA) and the activity of superoxide dismutases (SOD) and glutathione peroxidase (GSH-Px) were determined using commercial kits. The ultrastructural changes of the cardiac tissues were observed under transmission electron microscope. The expression of inducible nitric oxide synthase(iNOS) and content of nitrotyrosine was examined by the method of immunohistochemistry.RESULTS: The levels of blood glucose and MDA were significantly higher, while the activity of SOD and GSH-Px was lower in DM group than those in NC group (P<0.01). Treatment with PDTC markedly decreased the blood glucose and MDA content, and increased the activity of SOD and GSH-Px. Severe degeneration, necrosis, mitochondrial damage and inflammatory cell infiltration were found in the cardiac tissues in DM group. Treatment with PDTC markedly attenuated mitochondrial damage. The expression of iNOS and content of nitrotyrosine in cardiac tissues were significantly higher in DM group than those in NC group, and those were reduced after administration of PDTC.CONCLUSION: High glucose induces oxidative stress, increases the expression of iNOS and content of nitrotyrosine, and impairs the structure and function of myocardium. PDTC reduces blood glucose level, decreases the expression of iNOS and content of nitrotyrosine, and delays or attenuates the development of diabetic cardiomyopathy in diabetic rats.     

Effect of erlotinib on renal injury in rats with STZ-induced diabetic nephropathy

AIM: To investigate the effect of epidermal growth factor receptor (EGFR) inhibitor erlotinib on kidney injury in diabetic nephropathy (DN) rat and the underlying mechanism. METHODS: The rat model of DN was induced by intraperitoneal injection of streptozotocin (STZ) at dose of 55 mg/kg. One week after STZ injection, the rats with blood glucose level exceeding 16.7 mmol/L were identified as diabetic. Diabetic rats were randomly divided into 2 groups:STZ group and STZ+erlotinib group. In addition, the normal rats were used as control group. The rats in STZ+erlotinib group were treated with erlotinib at 100 mg·kg^-1·d^-1 for 4 weeks(5th~8th week). The fasting blood glucose (FBG), serum creatinine (SCr) and 24 h urine protein were measured. The pathological changes of the kidney were observed by HE staining and Masson staining. The protein levels of EGFR, p-EGFR, transforming growth factor β1 (TGFβ1), Smad2/3, p-Smad2/3, collagen Ⅳ (ColⅣ) and fibronectin in the kidney tissues were determined by Western blot. The reactive oxygen species (ROS) level and malondialdehyde (MDA) content in the renal tissues were futher analyzed. RESULTS: Compared with control group, the levels of FBG, 24 h urine protein and Scr were significantly increased in STZ group (P<0.01). Compared with STZ group, the levels of FBG, 24 h urine protein and SCr in STZ+erlotinib group were markedly decreased (P<0.05). In additon, the glomerular structure was restored to normal, the proliferative degree of mesangial cells markedly attenuated, and the epithelial cells were in alignment in STZ+erlotinib group. Moreover, erlotinib significantly inhibited the protein levels of p-EGFR, TGFβ1, p-Smad2/3, ColⅣ and fibronectin in the kidney tissues of STZ rats. In addition, erlotinib also significantly inhibited the levels of ROS and MDA in the kidney tissues of STZ rats. CONCLUSION: Erlotinib ameliorates STZ-induced diabetic nephropathy possibly through inhibiting the activation of EGFR/TGFβ1-Smad2/3 signaling pathway in association with suppression of fibrosis and oxidative stress.     

Expression of FBXW7 in diabetic cardiomyopathy

AIM:To investigate the expression of F-box/WD repeat-containing protein 7 (FBXW7) in the myocardium of type 1 diabetic rats and to clarify its role in the development of diabetic cardiomyopathy. METHODS:All rats were randomly divided into non-diabetic (ND) group, 4-week diabetes mellitus (DM) model group, 8-week DM mo-del group and 12-week DM model group. The DM model was prepared by intraperitoneal injection of streptozotocin (60 mg/kg). The change of myocardial pathological structure was investigated by HE staining. The FBXW7 expression level in the myocardium was determined by Western blot and immunohistochemistry methods. RESULTS:DM induced cardiomyocyte degeneration and necrosis as shown by cardiac histological analysis. Both Western blot and immunohistochemistry results showed that the expression level of FBXW7 was significantly increased in 4-week, 8-week and 12-week DM groups compared with control group (P<0.01). However, The FBXW7 expression level in 12-week DM group was decreased compared with 4-week and 8-week DM groups. CONCLUSION:With the development of diabetes, the expression of FBXW7 in the myocardium of the rats with diabetic cardiomyopathy shows a tendency to increase first and then decrease, suggesting that it plays some roles in the development of diabetic cardiomyopathy.     

Expression of vascular endothelial growth factor in chronic inflammatory mucosa of lacrimal sac

AIM: To study the expression of vascular endothelial growth factor (VEGF) in inflammatory mucosa of lacrimal sac. METHODS: Immunohistochemical S-P method was used to examine the expression of VEGF in the mocusa from 12 patients with chronic dacryocystitis and 8 volunteers. RESULTS: The positive rates of VEGF expression in different parts of the mocusa were: basal lamina: 44.3%±7.6％; surface epithelium: 16.9%±4.6％; connective tissue: 15.2%±4.9％, all normal mocusa of 8 cases were negative. There was a significant difference between the two groups (P<0.01), a significant difference among each part of the chronic inflammatory mocusa of lacrimal sac. CONCLUSION: VEGF may play an important role in hyperplasia of inflammatory mucosa of lacrimal sac.     

Role of p38 mitogen-activated protein kinase in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is debilitating, often fatal, expensive to treat and common. The intracellular signals following diabetes that lead to diminished contractility, apoptosis, fibrosis and ultimately heart failure are not fully understood but probably involve p38 mitogen-activated protein kinase (p38), one of serine/threonine kinases which, when activated, cause cardiomyocyte contractile dysfunction and death. Pharmacological inhibitors of p38 suppress inflammation and are undergoing clinical trials of rheumatoid arthritis, chronic obstructive pulmonary disease, psoriasis and acute coronary syndrome. In this review, we discuss the mechanisms, circumstances and consequences of p38 activation in DCM. The purpose is to evaluate p38 inhibition as a potential therapy for DCM.     

Expression of VEGF and bFGF in rat model of pressure ulcer

AIM:To study the effects of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) on the molecular pathogenesis of pressure ulcer.METHODS:SD rats were randomly divided into control group and experiment group. The pressure ulcer model was established by magnetic disk circulating compression method. HE staining was used to observe the pathological changes of the skin in the rats. The expression of VEGF and bFGF in the tissues was detected by immunohistochemical method. RESULTS:The expression of VEGF and bFGF in the tissues of rat Ⅲ-degree pressure ulcer was lower than that in the surrounding tissues and normal skin(P<0.01). The changes of VEGF and bFGF were consistent(κ=0.58). CONCLUSION:The expression levels of VEGF and bFGF are decreased in the tissues of rat pressure ulcer, suggesting that they may be the potential key factors in the difficult healing of pressure ulcer.