Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.     

Cardioprotection by St Thomas' solution is mediated by protein kinase C and tyrosine kinase

BACKGROUND: Intracellular signaling pathways, specifically the activation of protein kinase C and tyrosine kinase, are essential to the cardioprotection of ischemic preconditioning. We proposed that activation of PKC and TK contribute to the myocardial protection of St. Thomas' No. 2 cardioplegia solution (STC). MATERIALS AND METHODS: Isolated rat hearts were exposed to 40 min of global ischemia followed by 120 min of reperfusion. Before ischemia, hearts received no treatment (control; n = 7), STC (n = 7), phorbol 12-myristate 13-acetate (PMA; n = 6), PMA + chelerythrine (n = 6), anisomycin (n = 6), anisomycin + genistein (n = 7), STC + chelerythrine (n = 7), STC + genistein (n = 7), PMA + genistein (n = 7) or anisomycin + chelerythrine (n = 7). Left ventricular developed pressure (LVDP) recovery, myocardial infarct size, and lactate dehydrogenase release were measured. RESULTS: STC as well as PMA (protein kinase C activator) and anisomycin (tyrosine kinase activator) significantly reduced infarct size (6.9 +/- 2.9%, 9.6 +/- 2.1%, 14.0 +/- 4.4%) compared with controls (42.4 +/- 2.9%, P < 0.05). The infarct reduction of PMA and anisomycin were blocked by their inhibitors chelerythrine and genistein, respectively. Both chelerythrine (29.2 +/- 4.1%, P < 0.05) and genistein (40.4 +/- 4.3%, P < 0.05) attenuated the reduction of infarct size provided by STC. The recovery of LVDP improved with STC, PMA and anisomycin (72.6 +/- 1.4%, 60.4 +/- 4.7%, 57.2 +/- 4.6%) compared with control (33.8 +/- 3.6%, P < 0.05). Addition of chelerythrine or genistein to STC impaired recovery of LVDP (52.3 +/- 4.4%, 35.1 +/- 2.5%, P < 0.05) compared with STC treatment. CONCLUSION: Administration of the pharmacologic inhibitors chelerythrine and genistein blunts the cardioprotection caused by STC treatment.     

蛋白激酶C在异氟醚诱导原代培养大鼠心肌细胞分泌血管内皮生长因子中的作用

目的 探讨蛋白激酶C(PKC)在异氟醚诱导原代培养大鼠心肌细胞分泌血管内皮生长因子(VEGF)中的作用.方法 1～3 d SD新生大鼠,分离培养原代心肌细胞,随机分为6组(n=6):对照组(C组)培养后的细胞不经任何处理;不同浓度异氟醚组(Ⅰ1组～Ⅰ3组)细胞分别经0.7%、1.4%、2.1%异氟醚处理6 h;PKC抑制剂组(P组)细胞培养液中给予PKC抑制剂--calphostin C,终浓度50 nmol/L;PKC抑制剂+异氟醚组(PI组)心肌细胞培养液中加入calphostin C 50 nmol/L后,置入无菌密闭容器,持续输入1.4%异氟醚6 h.采用ELISA法测定细胞培养液VEGF浓度,Western blot法测定心肌细胞PKC亚型的表达.结果 与C组比较,Ⅰ2组和Ⅰ3组细胞培养液VEGF浓度升高,Ⅰ2组胞浆PKCε表达下调,胞膜PKCε表达上调(P＜0.01),胞浆和胞膜PKCα、PKCδ和PKCζ表达差异无统计学意义(P＞0.05),P组上述指标差异无统计学意义(P＞0.05).与胞膜比较,C组和Ⅰ2组胞浆PKCα、PKCδ和PKCζ表达上调(P＜0.05).随异氟醚浓度升高细胞培养液VEGF浓度升高(P＜0.05).与Ⅰ2组比较,PI组细胞培养液VEGF浓度降低(P＜0.05).结论 异氟醚可通过PKCε从胞浆转位到胞膜的途径诱导心肌细胞分泌VEGF,是异氟醚心肌保护作用的机制之一.     

蛋白激酶C同工酶在糖尿病肾病肾小球的表达

目的：探讨糖尿病肾病（DN）发病过程中肾小球蛋白激酶C（PKC）同工酶的表达变化及其与DN发生，发展的关系。方法：将实验随机分为正常对照组及STZ－DM模型组，在模型成功后2周，4周和12周，采用免疫组织化学方法检测肾小球PKC同工酶的表达变化情况，结果：在肾小球内，PKCα在DM发病2周，4周，12周时表达显著上升；PKCβ1，PKCⅡ在DM发病2周时表达明显下降，4周，12周时逐渐上升，PKCε的表达无显著性变化。结论：在DN发病过程中的不同阶段，PKC各同工酶在肾小球内的表达变化不同，对DN的发生，发展也有着不同的调控效应。     

VEGF and podocytes in diabetic nephropathy

Vascular endothelial growth factor-A (VEGF-A) is a protein secreted by podocytes that is necessary for survival of endothelial cells, podocytes, and mesangial cells. VEGF-A regulates slit-diaphragm signaling and podocyte shape via VEGF-receptor 2-nephrin-nck-actin interactions. Chronic hyperglycemia-induced excess podocyte VEGF-A and low endothelial nitric oxide drive the development and the progression of diabetic nephropathy. The abnormal cross-talk between VEGF-A and nitric oxide pathways is fueled by the diabetic milieu, resulting in increased oxidative stress. Recent findings on these pathogenic molecular mechanisms provide new potential targets for therapy for diabetic renal disease.     

野黄芪甙原对糖尿病大鼠肾脏蛋白激酶C活性作用的研究

目的研究野黄芪甙原对实验性糖尿病大鼠肾脏蛋白激酶C(PKC)活性、肾功能及肾脏结构的影响.方法大鼠随机分为正常对照组(A组)、糖尿病组(B组)、野黄芪甙原治疗组(C组),分别于4、6周应用酶联免疫法测定肾脏PKC活性;同时测定尿蛋白、Ccr、Scr及肾脏肥大指数,以光镜及电镜观察肾脏组织.结果C组尿蛋白排泄率、Ccr、肾脏肥大指数及细胞膜PKC活性均低于B组(P均＜0.05);光镜、电镜病理改变C组较B组有所改善.结论野黄芪甙原对糖尿病大鼠肾脏病变有部分保护作用,其机制可能部分通过下调肾脏PKC活性.     

Metabolomics Reveals a Key Role for Fumarate in Mediating the Effects of NADPH Oxidase 4 in Diabetic Kidney Disease

The NADPH oxidase (NOX) isoform NOX4 has been linked with diabetic kidney disease (DKD). However, a mechanistic understanding of the downstream effects of NOX4 remains to be established. We report that podocyte-specific induction of NOX4 in vivo was sufficient to recapitulate the characteristic glomerular changes noted with DKD, including glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, albuminuria, and podocyte dropout. Intervention with a NOX1/NOX4 inhibitor reduced albuminuria, glomerular hypertrophy, and mesangial matrix accumulation in the F1 Akita model of DKD. Metabolomic analyses from these mouse studies revealed that tricarboxylic acid (TCA) cycle–related urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by the NOX1/NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (in mouse and human tissue), and administration of the NOX1/NOX4 inhibitor increased glomerular FH levels in diabetic mice. Induction of Nox4 in vitro and in the podocyte-specific NOX4 transgenic mouse led to reduced FH levels. In vitro, fumarate stimulated endoplasmic reticulum stress, matrix gene expression, and expression of hypoxia-inducible factor-1α (HIF-1α) and TGF-β. Similar upregulation of renal HIF-1α and TGF-β expression was observed in NOX4 transgenic mice and diabetic mice and was attenuated by NOX1/NOX4 inhibition in diabetic mice. In conclusion, NOX4 is a major mediator of diabetes-associated glomerular dysfunction through targeting of renal FH, which increases fumarate levels. Fumarate is therefore a key link connecting metabolic pathways to DKD pathogenesis, and measuring urinary fumarate levels may have application for monitoring renal NOX4 activity.     

Protein kinase C activation and its role in kidney disease

Protein kinase C (PKC) comprises a superfamily of isoenzymes, many of which are activated by cofactors such as diacylglycerol and phosphatidylserine. In order to be capable of activation, PKC must first undergo a series of phosphorylations. In turn, activated PKC phosphorylates a wide variety of intracellular target proteins and has multiple functions in signal transduced cellular regulation. A role for PKC activation had been noted in several renal diseases, but two that have had most investigation are diabetic nephropathy and kidney cancer. In diabetic nephropathy, an elevation in diacylglycerol and/or other cofactor stimulants leads to an increase in activity of certain PKC isoforms, changes that are linked to the development of dysfunctional vasculature. The ability of isoform-specific PKC inhibitors to antagonize diabetes-induced vascular disease is a new avenue for treatment of this disorder. In the development and progressive invasiveness of kidney cancer, increased activity of several specific isoforms of PKC has been noted. It is thought that this may promote the kidney cancer's inherent resistance to apoptosis, in natural regression or after treatments, or it may promote the invasiveness of renal cancers via cellular differentiation pathways. In general, however, a more complete understanding of the functions of individual PKC isoforms in the kidney, and development or recognition of specific inhibitors or promoters of their activation, will be necessary to apply this knowledge for treatment of cellular dysregulation in renal disease.     

Inhibition of the epidermal growth factor receptor preserves podocytes and attenuates albuminuria in experimental diabetic nephropathy

Aim: Early renal enlargement may predict the future development of nephropathy in patients with diabetes. The epidermal growth factor (EGF)‐EGF receptor (EGFR) system plays a pivotal role in mediating renal hypertrophy, where it may act to regulate cell growth and proliferation and also to mediate the actions of angiotensin II through transactivation of the EGFR. In the present study we sought to investigate the effects of long‐term inhibition of the EGFR tyrosine kinase in an experimental model of diabetes that is characterized by angiotensin II dependent hypertension. Methods: Female heterozygous streptozotocin‐diabetic TGR(mRen‐2)27 rats were treated with the EGFR inhibitor PKI 166 by daily oral dosing for 16 weeks. Results: Treatment of TGR(mRen‐2)27 rats with PKI 166 attenuated the increase in kidney size, glomerular hypertrophy and albuminuria that occurred with diabetes. The reduction in albuminuria, with EGFR inhibition in diabetic TGR(mRen‐2)27 rats, was associated with preservation of the number of glomerular cells staining positively for the podocyte nuclear marker, WT1. Immunostaining for WT1 inversely correlated with glomerular volume in diabetic rats. In contrast to agents that block the renin‐angiotensin system (RAS), EGFR inhibition had no effect on either the quantity of mesangial matrix or the magnitude of tubular injury in diabetic animals. Conclusion: These observations indicate that inhibition of the tyrosine kinase activity of the EGFR attenuates kidney and glomerular enlargement in association with podocyte preservation and reduction in albuminuria in diabetes. Accordingly, targeting the EGF‐EGFR pathway may represent a therapeutic strategy for patients who continue to progress despite RAS‐blockade.     

膀胱癌组织中蛋白激酶C的表达

目的　探讨蛋白激酶C(PKC)在膀胱癌发病机制中的作用及其意义。方法　应用免疫组化方法检测 82 例膀胱癌组织和12例正常膀胱组织中PKC和增殖细胞核抗原(PCNA)的表达。结果　膀胱癌组织中PKC阳性率为56.1%,显著高于正常膀胱组织的25.0%(P<0.05)。PKC表达与肿瘤分级、分期无显著相关性,复发者 PKC表达显著高于无复发者。膀胱癌 PKC阳性表达者的PCNA指数显著高于阴性表达者。结论　PKC异常表达促使细胞过度增殖,进而参与膀胱癌发病过程的调控。     

X-盒结合蛋白1、血管内皮生长因子C在肝癌中的表达及意义

目的 检测X-盒结合蛋白1(XBP1),血管内皮生长因子C(VEGF-C)在肝癌组织中的表达,探讨在肝癌发生发展过程中存在未折叠蛋白反应(UPR)的激活.方法 采用逆转录-聚合酶链反应(RT-PCR)法检测术中取的新鲜42例肝癌标本、15例同个体癌旁1.0cm肝组织和15例正常肝组织中XBP1 、VEGF-C的mRNA表达,应用Western blot法检测57例肝癌组织和正常肝组织标本中XBP1、VEGF-C在蛋白水平的表达.结果 肝癌组织、癌旁肝组织、正常肝组织中XBP1 mRNA相对表达量分别为0.4396±0.0241、0.4152±0.0252、0.4095±0.0149,XBP1 mRNA在3组之间的表达呈下降趋势(P＜0.05);肝癌组织、癌旁肝组织、正常肝组织中VEGF-C mRNA相对表达量分别为0.4447±0.0335、0.4195 ±0.0334、0.4019±0.0259,VEGF-C mRNA在3组之间的表达呈下降趋势(P＜0.05). 在肝癌组织和正常肝组织中均有两种蛋白的表达,但肝癌组织中的表达明显高于正常肝组织.结论 肝癌组织中XBP1、VEGF-C mRNA和蛋白的表达一致,均为高表达.     

The role of phospho-adenosine monophosphate-activated protein kinase and vascular endothelial growth factor in a model of chronic heart failure

We established a stable and reproducible animal model of chronic heart failure (CHF) in sheep to investigate biomolecular changes. Therefore, two biomarkers, adenosine monophosphate-activated protein kinase (AMPK) and vascular endothelial growth factor-A (VEGF-A) were examined to reveal their role during chronic ischemic conditions of the heart. AMPK was studied because it plays an important role in cellular energy homeostasis and its upregulation is associated with myocardial ischemia, whereas VEGF-A was studied because it acts as an important signaling protein for neoangiogenesis. We examined 15 juvenile sheep (mean weight, 78±4kg; control, n=3; ShamOP, n=2; coronary microembolization [CME], n=10). CHF was induced under fluoroscopic guidance by multiple sequential microembolizations (MEs) through bolus injection of polysterol microspheres (90μm, n=25.000) into the left main coronary artery. CME was repeated up to three times at 2- to 3-week intervals until animals started to develop stable signs of CHF. All animals were followed for 3 months. Phosphorylation of AMPK, marking the activated protein form, was detected by Western blotting. VEGF-A and vascular endothelial growth factor-receptor 2 (VEGF-R2) mRNA were detected by real-time polymerase chain reaction. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as a reference housekeeping gene. All 10 CHF animals developed clinical signs of CHF as indicated by a significant decrease of cardiac output, decreased ejection fraction, as well as occurrence of tachycardia and tachypnoea. Western blots showed significant phosphorylation of AMPK in CME animals compared to the control group (phospho-adenosine monophosphate-activated protein kinase α) (GAPDH control: 0.0, CME left ventricle [LV]: 0.39±0.20, CME right ventricle [RV]: 0.53±0.30; P<0.05). VEGF-A and VEGF-R2 expression in CME animal myocardium was within the range of the control group, but this data did not reach statistical significance due to the small size of this group. While microinjection was performed into the left main coronary artery, phosphorylation of AMPK and expression of VEGF-A and VEGF-R2 were significantly higher in the RV than in the LV. Multiple sequential intracoronary MEs can effectively induce myocardial dysfunction with clinical and biomolecular signs of chronic ischemic cardiomyopathy. Quantitative analysis of biomolecular markers showed a significantly higher phosphorylation of AMPK in CHF animals compared with control myocardium.     

Role of protein kinase C in cisplatin nephrotoxicity

BACKGROUND: Cisplatin is widely used in cancer treatment. The major disadvantage of this antitumor agent is its nephrotoxicity. The mechanism of cisplatin-induced nephrotoxicity has not been clarified. Recent evidence suggests protein kinase C (PKC)-related signal transduction pathways may modulate cisplatin-induced cytotoxicity. METHODS: The effect of cisplatin administration on PKC expression in the kidney and the effect of a PKC inhibitor on cisplatin-induced renal impairment were investigated in rats. RESULTS: A single intraperitoneal injection of 8 mg/kg cisplatin induced remarkable damage in the proximal tubules located in the outer medulla, which was associated with impaired renal function, within 48 h. An immunoblotting study revealed marked expression of alpha-PKC in membrane fractions of medullary tubules prepared from cisplatin-treated rats. In addition, pretreatment with the PKC inhibitor (H-7) protected kidneys from cisplatin-induced damage. CONCLUSIONS: These findings suggest that the nephrotoxic effects of cisplatin may, in part, be related to PKC activation in the renal tubules.     

紫草素对糖尿病小鼠中性粒细胞胞外诱捕网表达的影响

目的:探讨紫草素对佛波酯(PMA)诱导的糖尿病小鼠骨髓来源的中性粒细胞胞外诱捕网（NETs）的影响,为其治疗糖尿病足溃疡提供依据。方法:采用链脲佐菌素腹腔注射构建糖尿病小鼠模型,密度梯度离心法提取小鼠骨髓来源的中性粒细胞,并进行体外培养,PMA刺激其诱导NETs模型,采用CCK-8法筛选紫草素的安全剂量;采用Sytox Green核酸荧光染料法定量检测NETs水平;PicoGreen荧光染料法定量检测dsDNA/NETs含量;荧光探针定量检测中性粒细胞活性氧(ROS)水平;荧光免疫组化法检测NETs标志物瓜氨酸化组蛋白H3(Cit-H3)的表达水平。结果：紫草素在0.125～1μg/mL浓度范围内对中性粒细胞活性无影响。与模型组比较,紫草素1、0.5、0.25μg/mL浓度组中性粒细胞NETs产生的荧光强度显著降低（P <0.01）,细胞外dsDNA水平显著降低（P <0.01）,细胞内ROS水平显著降低（P<0.01）,细胞Cit-H3表达显著下调（P <0.01）。结论:紫草素通过抑制糖尿病小鼠骨髓来源的中性粒细胞ROS水平和Cit-H3表达,从而抑制NET...     

Involvement of protein kinase C in growth regulation of human meningioma cells

Summary In order to investigate the possible role of protein kinase C (PKC)-mediated signal pathways in growth regulation of meningiomas, we examined the effect of two PKC-activating phorbol esters, 12-O-tetradecanoyl-13-phorbol acetate (TPA) and phorbol 12, 13-dibutyrate (PDBu), and PKC inhibitor, staurosporine, on cell proliferation using low-passage human meningioma cells in culture. TPA (0.1 to 100 ng/ml) caused a dose-dependent stimulation of cell proliferation in six of eight meningioma cultures. At optimal concentrations of TPA, the cell growth ranged from 113% to 251% versus control. In contrast, PDBu (0.1 to 100 ng/ml) caused a significant inhibition of cell proliferation in three of five meningioma cultures. At optimal concentrations of PDBu, the cell growth ranged from 52% to 79% of control. Staurosporine exhibited a stimulation of cell proliferation (135% to 178%) in three of four meningioma cultures studied at a concentration of 10^–10 to 10^–9M, although a tendency of growth inhibition was observed at a lower concentration. A time course of DNA synthesis in response to TPA, assessed by [³H] thymidine incorporation studies, revealed a time- and dose-dependent stimulation and/or inhibition which further depended on the serum concentration of the growth medium used. The overall results indicate that PKC-mediated signal pathways are closely involved in growth regulation of human meningioma cells. The results further suggest that the signalling processes consist of complex mechanisms which await to be elucidated.     

蛋白激酶C激活在高糖诱导肾系膜细胞中的作用

目的：探讨高糖对系膜细胞蛋白激酶C（PKC）活性的影响及PKC在系膜细胞增殖、细胞外基质积聚中的作用。方法：采用大鼠系膜细胞进行体外培养,高糖作为激动剂,佛波酯（PMA）作为PKC抑制剂,甘露醇作为渗透压对照,用液闪仪测定PKC活性,^3H-TdR渗入法检测细胞增殖,ELISA法测定培养上清中纤维连接蛋白（FN）含量。结果：高糖可增加系膜细胞颗粒部分PKC活性、抑制细胞增殖、促进FN分泌,且与渗透压无关。抑制PKC后,可阻止高糖诱导的FN分泌。结论：高糖可激活系膜细胞PKC,促进细胞外基质积聚和糖尿病肾症的发生。     

来氟米特对糖尿病大鼠血清高敏C反应蛋白及肾脏病理的影响

目的：探讨糖尿病大鼠血清中高敏C反应蛋白（Hs-CRP）的变化和肾小球病理改变,观察来氟米特治疗后对Hs-CRP的变化和肾小球病理改变的影响。方法：将50只SD大鼠随机分为正常对照组（C组）、糖尿病对照组（DM组）和来氟米特干预组（LEF组）。链脲佐菌素（STZ）诱导糖尿病大鼠模型,来氟米特对LEF组进行治疗性灌胃。12周后留取尿液测量24h尿蛋白定量（24h U-TP）、留取血标本测血糖（Glu）、血清肌酐（Scr）、尿素氮（BUN）、Hs-CRP;肾脏标本测肾脏重量,部分肾组织行HE和PAS染色,在光镜下观察肾小球组织形态学的改变;显微图像摄影系统采图后应用Image J医学图像分析系统,测量肾小球平均面积及肾小球平均体积。结果：12周末DM组大鼠与C组大鼠比较,DM组大鼠体重（BW）增长缓慢或体重明显下降,LEF治疗组大鼠体重增长缓慢,但较DM组体重增加,肾重指数（KI）、GLU、Scr、BUN、Hs-CRP及24h U-TP均有统计学差异（P〈0.01）。LEF组大鼠与C组大鼠比较GLU、KI、Scr、BUN、Hs-CRP、24h U-TP虽然仍明显增高（P〈0.05或P〈0.01）,除GLU外其余指标与DM组相比明显降低,有统计学差异（P〈0.01或P〈0.05）,相关分析显示Hs-CRP与24h U-TP（r=0.838,P〈0.01）、Scr（r=0.732,P〈0.01）、BUN（r=0.772,P〈0.01）,KI（r=0.869,P〈0.01）,提示Hs-CRP与Scr、BUN、KI及24h U-TP呈显著正相关,图像分析显示糖尿病大鼠肾小球平均面积及平均体积较对照组明显增大,LEF组较DM组明显减轻。结论：血清Hs-CRP的水平与糖尿病大鼠肾脏损害的严重程度一致,来氟米特可以通过减低血清Hs-CRP的水平,降低炎症反应,减轻糖尿病大鼠肾脏损害。