AMPKα1 overexpression improves postoperative cognitive dysfunction in aged rats through AMPK-Sirt1 and autophagy signaling.

Postoperative cognitive dysfunction (POCD) is a common complication in elderly patients who undergo surgery involving anesthesia. Its underlying mechanisms remain unclear. Autophagy plays an important role in the damage and repair of the nervous system and is associated with the development of POCD. Using a rat model, adenosine monophosphate-activated protein kinase α1 (AMPKα1), an important autophagy regulator, was found to be significantly downregulated in rats with POCD that was induced by sevoflurane anesthesia or by appendectomy. Overexpression of AMPKα1-ameliorated POCD, as indicated by decreased escape latencies and increased target quadrant swimming times, swimming distances, and platform crossing times during Morris water maze tests. AMPKα1 overexpression activated autophagy signals by increasing the expression of light chain 3 II (LC3-II) and Beclin1 and decreasing the expression of p62 in the hippocampus of rats with POCD. Moreover, blocking autophagy by 3-methyladenine partly attenuated AMPKα1-mediated POCD improvement. Furthermore, overexpression of AMPKα1 could upregulate the expression of p-AMPK and Sirt1 in the hippocampus of rats with POCD. Intriguingly, inhibiting AMPK signals via Compound C effectively attenuated AMPKα1-mediated POCD improvement, concomitant with the downregulation of p-AMPK, Sirt1, LC3-II, and Beclin1 and the upregulation of p62. We thus concluded that overexpression of AMPKα1 can improve POCD via the AMPK-Sirt1 and autophagy signaling pathway.

Sirt3-Mediated Autophagy Contributes to Resveratrol-Induced Protection against ER Stress in HT22 Cells.

Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in neuronal survival and death. Resveratrol is a non-flavonoid polyphenol that has neuroprotective effects against many neurological disorders. Here, we investigated the potential protective effects of resveratrol in an in vitro ER stress model mimicked by tunicamycin (TM) treatment in neuronal HT22 cells. We found that TM dose-dependently decreased cell viability and increased apoptosis, which were both significantly attenuated by resveratrol treatment. Resveratrol markedly reduced the expression or activation of ER stress-associated factors, including GRP78, CHOP, and caspase-12. The results of immunocytochemistry and western blot showed that resveratrol promoted autophagy in TM-treated cells, as evidenced by increased LC3II puncta number, bcelin1 expression and LC3II/LC3I ratio. Pretreatment with the autophagy inhibitor chloroquine could reduce the protective effects of resveratrol. In addition, the expression of Sirt3 protein and its downstream enzyme activities were significantly increased in resveratrol-treated HT22 cells. To confirm the involvement of Sirt3-mediated mechanisms, siRNA transfection was used to knockdown Sirt3 expression in vitro. The results showed that downregulation of Sirt3 could partially prevented the autophagy and protection induced by resveratrol after TM treatment. Our study demonstrates a pivotal role of Sirt3-mediated autophagy in mediating resveratrol-induced protection against ER stress in vitro, and suggests the therapeutic values of resveratrol in ER stress-associated neuronal injury conditions.

[Activation of sterol regulatory element binding protein and its involvement in endothelial cell migration].

OBJECTIVE: To study the activation of sterol regulatory element binding protein (SREBP) and its critical role in endothelial cell migration. METHODS: Bovine aortic endothelial cells (ECs) were cultured. The expression of SREBP and Cdc42 were determined by Western blot and quantitative real-time PCR. Moreover, outward growth migration model and transwell chamber assay were used to detect ECs migration. RESULTS: (1) SREBP was activated during ECs migration. Western blot analysis demonstrated increased active form SREBP in migrating as compared to non-migrating ECs population. SREBP activation decreased as ECs migration slowed;(2) Coincidental with SREBP activation, mRNA expression of its target genes such as low density lipoprotein receptor, HMG-CoA reductase, and fatty acid synthase also increased in migrating ECs population as detected by real-time PCR; (3) Migration induced SREBP activation in ECs was inhibited by SREBP-acting protein RNAi and pharmacologically by 25-hydroxycholesterol; (4) Inhibition of SREBP led to decreased ECs migration in various models; (5) Cells genetically deficient in SREBP-acting protein, S1P, or S2P, phenotypically exhibited impaired migration; (6) SREBP inhibition in ECs suppressed the activity of small GTPase Cdc42, a key molecule for ECs motility. CONCLUSIONS: SREBP is activated during and plays a critical role in ECs migration. Targeting SREBP could become a novel approach in fighting diseases involving abnormal ECs migration.

Effects of grape seed proanthocyanidin extracts on aortic pulse wave velocity in streptozocin induced diabetic rats.

Grape seed proanthocyanidin extracts (GSPEs) have been reported to be effective in treating arteriosclerosis, while little is known about therapeutic agents against diabetic macrovascular complications. We used streptozocin to induce diabetic rats. GSPEs (250 mg/kg of body weight) were administrated to diabetic rats for 24 weeks. Aortic blood pressure and pulse wave velocity (PWV) were determined in anesthetized rats. Serum glycated hemoglobin and advanced glycation end products (AGEs) were determined. An electronic microscope was used to observe the changes in aortic ultrastructure. Immunohistochemistry was used to evaluate the receptor of advanced glycation end product (RAGE) protein expression in aortic tissue. GSPEs significantly decreased aortic PWV, blood pressure, and aortic medial thickness (P<0.05), and inhibited the migration of vascular smooth muscle cells. GSPEs significantly reduced the AGEs (P<0.05) and the expression of RAGE in aortas of diabetic rats. GSPEs play an important role against diabetic macrovascular complications. This study may provide a new recognition of natural medicine for the treatment of diabetic macrovascular complications.

Back-regulation of six oxidative stress proteins with grape seed proanthocyanidin extracts in rat diabetic nephropathy.

Diabetic nephropathy (DN) is a major cause of morbidity and mortality in diabetic patients. To prevent the development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE) have been reported to be effective in treating DN, while little is known about the functional protein changes. In this study, we used streptozotocin (STZ) to induce diabetic rats. GSPE (250 mg/kg body weight/day) were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin, and advanced glycation end products were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate kidney protein profiles among the control, untreated and GSPE treated diabetic rats. Twenty-five proteins were found either up-regulated or down-regulated in the kidneys of untreated diabetic rats. Only nine proteins in the kidneys of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in oxidative stress, glycosylation damage, and amino acids metabolism. Our findings might help to better understanding of the mechanism of DN, and provide novel targets for estimating the effects of GSPE therapy.

A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of visual impairment and blindness among the people of occupational age. To prevent the progress of retina injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts (GSPE) have been reported to be effective in treating diabetic complications, while little is discussed about the functional protein changes. METHODS: We used streptozotocin (STZ) to induce diabetes in rats. GSPE (250 mg/kg body weight per day) were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin and advanced glycation end products (AGEs) were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate retina protein profiles among control, STZ-induced diabetic rats, and GSPE treated diabetic rats. RESULTS: GSPE significantly reduced the AGEs of diabetic rats (P < 0.05). Moreover, GSPE significantly suppressed the vascular lesions of central regions, decreased capillary enlargements and neovascularization, similar to those of the control rats under light microscope. Eighteen proteins were found either up-regulated or down-regulated in the retina of STZ-induced diabetic rats. And seven proteins in the retina of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in many important biological processes such as heat shock, ubiquitin-proteasome system, cell proliferation, cell growth and glucose metabolism. CONCLUSIONS: These findings might promote a better understanding for the mechanism of DR, and provide novel targets for evaluating the effects of GSPE therapy.

Pulse wave velocity as a marker of arteriosclerosis and its comorbidities in Chinese patients.

To obtain reliable data on the epidemiology of arteriosclerosis and the comorbidities in patients with hypertension (HP), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM) and stroke, we evaluated the clinical significance of pulse wave velocity (PWV) as an indicator of arteriosclerosis and its comorbidities in Chinese patients. A total of 910 subjects, including 748 Chinese patients with one or more cardiovascular risk factors (80.2% male, mean age 73.69+/-5.03 years) and 162 healthy volunteers (78.4% male, mean age 73.60+/-5.32 years) were recruited into the study. PWV was measured in 910 subjects, and large artery arteriosclerosis was defined as PWV >or=12 m/s. Multivariable logistic regression analyses were performed to identify risk factors associated with arteriosclerosis. The prevalence of large artery arteriosclerosis in the patients overall was 67.4%, and the prevalence was higher in patients with than in those without HP (63.3% vs. 34.0%; odds ratio [OR]: 3.451), T2DM (24.8% vs. 11.1%; OR: 2.854), CHD (56.1% vs. 45.1%; OR: 1.246) and stroke (26.6% vs. 19.2%; OR: 1.236), but the OR values of CHD and stroke did not differ significantly (p>0.05). After multiple logistic regression analysis, female sex, older age, HP and T2DM were risk factors for large artery arteriosclerosis. In conclusion, PWV can be used as a routine measurement to scan arteriosclerosis in patients with HP or T2DM.

Grape seed proanthocyanidin extracts inhibit vascular cell adhesion molecule expression induced by advanced glycation end products through activation of peroxisome proliferators-activated receptor gamma.

Although evidence has shown that grape seed proanthocyanidin extracts (GSPE) can selectively inhibit cell adhesion molecule expression induced by advanced glycation end products (AGEs), the underlying molecular mechanism has not been extensively characterized. To study the antiinflammation mechanism of GSPE, we investigated the effect of GSPE on Von Willebrand factor (vWF) content and the expression of vascular cell adhesion molecule-1 (VCAM-1) induced by AGEs and the effect of GSPE on peroxisome proliferators-activated receptor gamma (PPAR gamma) and receptor for AGEs (RAGE) expression in human umbilical vein endothelial cells (HUVEC). HUVEC were preincubated with or without GSPE of different concentrations (10 mg/L, 50 mg/L, and 100 mg/L) for 4 hours before being treated with 200 mg/L AGEs or unmodified bovine serum albumin (BSA) for 24 hours. The expression of RAGE and PPAR gamma was investigated by Western blot. VCAM-1 expression was measured by flow cytometry and vWF content by enzyme-linked immunosorbent assay (ELISA). Results showed that GSPE significantly inhibited the expression of VCAM-1 in HUVEC and reduced the content of vWF in culture fluid induced by AGEs in a dose-dependent manner. AGEs activated the expression of RAGE and inhibited PPAR gamma expression in HUVEC, whereas GSPE inhibited the expression of RAGE through activation of PPAR gamma in HUVEC simultaneously. These findings indicated that GSPE inhibited the cell inflammatory factor expression and protected the function of endothelial cell through activation of PPAR gamma expression and inhibition of RAGE expression.

Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells.

The interaction of advanced glycation end products (AGE) with their cell surface receptors for AGEs (RAGE) has been causally implicated in the pathogenesis of diabetic vascular complications and has been shown to stimulate cell adhesion molecule expression in endothelial cells via induction of reactive oxygen species (ROS). Alternatively, grape seed proanthocyanidin extracts (GSPE), which are naturally occurring polyphenolic compounds, have been reported to possess potent radical scavenging and antioxidant properties and to display significant cardiovascular protective action. In this study, we investigated whether GSPE could inhibit AGE-induced cell adhesion molecule expression through interference with ROS generations in human umbilical vein endothelial cells. AGE-modified bovine serum albumin (AGE-BSA) was prepared by incubating BSA with a high concentration of glucose. Stimulation of cultured human umbilical vein endothelial cells with 200 microg/mL of AGE-BSA significantly enhanced intracellular ROS formation and subsequently upregulated the expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM-1), whereas both unmodified BSA and GSPE alone were without effect. However, preincubation of different concentrations of GSPE markedly downregulated AGE-BSA-induced VCAM-1 expression at the surface protein and mRNA level in a concentration-dependent manner, but the increased ICAM-1 expression was not affected by GSPE treatment. Meanwhile, the inhibition by GSPE of intracellular ROS generation was also observed at defined time periods. These results demonstrate that GSPE can inhibit the enhanced VCAM-1 expression but not ICAM-1 in AGE-exposed endothelial cells by suppressing ROS generation. Hence, GSPE may have therapeutic potential in the prevention and treatment of vascular complications in patients with diabetes.