Rhein Protects Pancreatic β-Cells From Dynamin-Related Protein-1–Mediated Mitochondrial Fission and Cell Apoptosis Under Hyperglycemia

Rhein, an anthraquinone compound isolated from rhubarb, has been shown to improve glucose metabolism disorders in diabetic mice. The mechanism underlying the protective effect of rhein, however, remains unknown. Here, we demonstrate that rhein can protect the pancreatic β-cells against hyperglycemia-induced cell apoptosis through stabilizing mitochondrial morphology. Oral administration of rhein for 8 or 16 weeks in db/db mice significantly reduced fasting blood glucose (FBG) level and improved glucose tolerance. Cell apoptosis assay using both pancreatic sections and cultured pancreatic β-cells indicated that rhein strongly inhibited β-cell apoptosis. Morphological study showed that rhein was mainly localized at β-cell mitochondria and rhein could preserve mitochondrial ultrastructure by abolishing hyperglycemia-induced mitochondrial fission protein dynamin-related protein 1 (Drp1) expression. Western blot and functional analysis confirmed that rhein protected the pancreatic β-cells against hyperglycemia-induced apoptosis via suppressing mitochondrial Drp1 level. Finally, mechanistic study further suggested that decreased Drp1 level by rhein might be due to its effect on reducing cellular reactive oxygen species. Taken together, our study demonstrates for the first time that rhein can serve as a novel therapeutic agent for hyperglycemia treatment and rhein protects pancreatic β-cells from apoptosis by blocking the hyperglycemia-induced Drp1 expression.Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is an anthraquinone compound isolated from rhubarb that has been used for more than 2,000 years in China to treat constipation, gastrointestinal hemorrhage, and ulcers (1). In our previous work, we found that rhein could improve glucose metabolism disorders in diabetic mice, and its effect on reducing blood glucose level was even stronger than rosiglitazone and benazepril (2,3). Moreover, rhein also inhibited apoptosis of islet cells and protected islet function (4). Using mouse nonalcoholic fatty liver disease as an animal model associated with obesity, insulin resistance, and inflammatory disorders, Sheng et al. (5) reported that rhein could ameliorate fatty liver disease in diet-induced obese mice via negative energy balance, hepatic lipogenous regulation, and immunomodulation. Recent antihyperglycemic study by Chatterjee et al. (6) suggests that rhein, as well as other natural inhibitors such as aloins and capparisine, may be a foundation for a better antidiabetic therapy. However, the mechanism underlying these protective effects of rhein remains unclear.Increasing evidence suggests that β-cell failure is the mainstay of the pathogenesis of type 2 diabetes (7). Although the precise mechanisms underlying the β-cell dysfunction in type 2 diabetes are not fully understood, hyperglycemia has been shown as a major factor to cause the β-cell apoptosis. Once hyperglycemia develops, the pancreatic β-cell is exposed to increased metabolic flux and associated cellular stress, leading to impairment of β-cell function and survival, a process called glucotoxicity (8,9). In type 2 diabetes, hyperglycemia is commonly associated with deregulation of lipid metabolism and elevation of free fatty acids, which also contribute to β-cell dysfunction (8,10). Moreover, high levels of glucose can also amplify lipotoxicity (10). The thiazolidinedione peroxisome proliferator–activated receptor-γ activator drugs, rosiglitazone and pioglitazone, have been widely used to suppress insulin resistance in type 2 diabetic patients (11). Although rhein shows a similar or even better effect on reducing mouse blood glucose level than rosiglitazone, the underlying mechanism remains unclear. It has been known that mitochondrial fission and fusion modulators, dynamin-related protein 1 (Drp1) (12), optic atrophy protein 1 (Opa1) (13), prohibitin (14), and mitofusin (15), collectively control the dynamic balance of mitochondria fission and fusion processes and consequent mitochondria functions. Previous studies have demonstrated that Drp1 plays an important role in promoting hyperglycemia-induced apoptosis of β-cells and neurons (12,16,17). Drp1 expression was increased drastically in islet β-cells under hyperglycemia conditions. Estaquier and Arnoult (18) further demonstrated that inhibiting Drp1-mediated mitochondrial fission could selectively prevent the release of cytochrome c, a mediator of apoptosis, from mitochondria. In contrast to the mitochondria fission modulators, which are upregulated or activated by stress factors such as high concentration of glucose (HG), mitochondria fusion modulators are generally reduced when cells are challenged with proapoptotic insults. Recent studies by Kushnareva et al. (19) and Leboucher et al. (15) showed that stress-induced loss of Opa1 and mitofusin can facilitate mitochondrial fragmentation and cell apoptosis. However, it remains to be determined whether rhein executes its protective role in pancreatic β-cells through regulating the expression or activation of these mitochondria fission/fusion modulators.In the current study, we used db/db mice and a pancreatic β-cell line (NIT-1) to study the protective effect of rhein. Our results showed that rhein largely localized at mitochondria in the β-cells and that it strongly protected pancreatic β-cells from hyperglycemia-induced apoptosis through suppressing Drp1 activation and Drp1-mediated mitochondria fission.     

Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.     

High mobility group-box 3 overexpression is associated with poor prognosis of resected gastric adenocarcinoma

AIM:To elucidate high mobility group-box 3(HMGB3) protein expression in gastric adenocarcinoma,its potential prognostic relevance,and possible mechanism of action.METHODS:Ninety-two patients with gastric adenocarcinomas surgically removed entered the study.HMGB3 expression was determined by immunohistochemistry through a tissue microarray procedure.The clinicopathologic characteristics of all patients were recorded,and regular follow-up was made for all patients.The inter-relationship of HMGB3 expression with histological and clinical factors was analyzed using nonparametric tests.Survival analysis was carried out by Kaplan-Meier(log-rank) and multivariate Cox(Forward LR) analyses between the group with overexpression of HMGB3 and the group with low or no HMGB3 ex-pression to determine the prognosis value of HMGB3 expression on overall survival.Further,HMGB3 expression was knocked down by small hairpin RNAs(shRNAs) in the human gastric cancer cell line BGC823 to observe its influence on cell biological characteristics.The MTT method was utilized to detect gastric cancer cell proliferation changes,and cell cycle distribution was analyzed by flow cytometry.RESULTS:Among 92 patients with gastric adenocarcinomas surgically removed in this study,high HMGB3 protein expression was detected in the gastric adenocarcinoma tissues vs peritumoral tissues(P 0.001).Further correlation analysis with patients' clinical and histology variables revealed that HMGB3 overexpression was obviously associated with extensive wall penetration(P = 0.005),a positive nodal status(P = 0.004),and advanced tumor-node-metastasis(TNM) stage(P = 0.001).But there was no correlation between HMGB3 overexpression and the age and gender of the patient,tumor localization or histologic grade.Statistical Kaplan-Meier survival analysis disclosed significant differences in overall survival between the HMGB3 overexpression group and the HMGB3 no or low expression group(P = 0.006).The expected overall survival time was 31.00 ± 3.773 mo(95%CI = 23.605-38.395) for patients with HMGB3 overexpression and 49.074 ± 3.648 mo(95%CI = 41.925-57.311) for patients with HMGB3 no and low-level expression.Additionally,older age(P = 0.040),extensive wall penetration(P = 0.008),positive lymph node metastasis(P = 0.005),and advanced TNM tumor stage(P = 0.007) showed negative correlation with overall survival.Multivariate Cox regression analysis indicated that HMGB3 overexpression was an independent variable with respect to age,gender,histologic grade,extent of wall penetration,lymph nodal metastasis,and TNM stage for patients with resectable gastric adenocarcinomas with poor prognosis(hazard ratio = 2.791,95%CI = 1.233-6.319,P = 0.019).In the gene function study,after HMGB3 was knocked down in the gastric cell line BGC823 by shRNA,the cell proliferation rate was reduced at 24 h,48 h and 72 h.Compared to BGC823 shRNA-negative control(NC) cells,the cell proliferation rate in cells that had HMGB3 shRNA transfected was significantly decreased(P 0.01).Finally,cell cycle analysis by FACS showed that BGC823 cells that had HMGB3 knocked down were blocked in G1/G0 phase.The percentage of cells in G1/G0 phase in BGC823 cells with shRNA-NC and with shRNA-HMGB3 was 46.84% ± 1.7%,and 73.03% ± 3.51% respectively(P = 0.001),whereas G2/M cells percentage decreased from 26.51% ± 0.83% to 17.8% ± 2.26%.CONCLUSION:HMGB3 is likely to be a useful prognostic marker involved in gastric cancer disease onset and progression by regulating the cell cycle.     

以心血管系统病变为主要表现的双硫仑样反应32例报道

目的探讨使用头孢菌素类抗生素(头孢菌素)与饮酒相互作用导致的以心血管系统病变为主要表现的双硫仑样反应及其救治方法。方法对诊断为双硫仑样反应且主要表现为心血管系统病变的32例患者的临床资料进行回顾性分析,包括涉及药物、饮酒情况、致双硫仑样反应时间、临床表现、治疗及预后。结果引起双硫仑样反应的头孢菌素以头孢哌酮加舒巴坦最为常见。双硫仑样反应多为用药后饮酒所致,致双硫仑样反应时间多发生在饮酒后5 min～1 h。心血管系统主要表现为心悸、呼吸困难、低血压、胸闷、胸痛。心电图可表现为窦性心动过速、心房颤动、室性期前收缩、ST-T改变等;容易误诊为急性冠脉综合征、急性左心力衰竭、休克。给予积极有效治疗后,多数患者于治疗后30 min～3 h缓解。无1例死亡。结论应用头孢菌素与饮酒相互作用可引起双硫仑样反应,可主要以心血管系统表现为主,表现复杂且较危重,经积极治疗大多预后良好。