Quantification and Proteomic Characterization of ß-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice.

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid ß-oxidation, especially ß-hydroxybutyrate, are fatty energy-supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine ß-hydroxybutyrylation (Kbhb) is a ß-hydroxybutyrate-mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.

Exosomes from human umbilical cord mesenchymal stem cells protect aortas in Db/db mice characterized by combination of metabolomics and proteomics.

Diabetic cardiovascular complication is a common systemic disease with high morbidity and mortality worldwide. We hypothesise that exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs-exos) can rescue these disorders and alleviate vascular remodeling in diabetes. Morphological, non-targeted metabolomics and 4D label-free proteomics techniques were used to analyze the aortas of db/m mice as normal control group (NCA), saline treated db/db mice (DMA), and hUCMSCs-exos treated db/db mice (DMTA), and to clarify the molecular mechanism of the protection of hUCMSCs-exos in vascular remodeling from a new point of view. The results showed that 74 metabolites were changed significantly in diabetic aortas, of which 15 were almost restored by hUCMSCs-exos. In proteomics, 30 potential targets such as Stromal cell-derived factor 2-like protein 1, Leukemia inhibitory factor receptor, Peroxisomal membrane protein and E3 ubiquitin-protein ligase MYCBP2 were detected. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based analysis showed that Central carbon metabolism in cancer and Galactose metabolism pathway were up-regulated to near normal by hUCMSCs-exos in metabolomics, with janus associated kinase-signal transducer and activator of transcription (JAK-STAT) pathway displayed in proteomics. According to bioinformatics and integrated analysis, these targeted molecules of hUCMSCs-exos to attenuate the vascular remodeling were mainly associated with regulation of energy metabolism, oxidative stress, inflammation, and cellular communications. This study provided a reference for the therapy of diabetes-induced cardiovascular complications.

The protective effects of grape seed procyanidin B2 against asporin mediates glycated low-density lipoprotein induced-cardiomyocyte apoptosis and fibrosis.

The progression of diabetic cardiomyopathy is related to cardiomyocyte dysfunction and apoptosis. Our previous studies showed that asporin (ASPN) was significantly increased in the myocardium of db/db mice through proteomics, and grape seed procyanidin B2 (GSPB2) significantly inhibited the expression of ASPN in the heart of db/db mice. We report here that ASPN played a critical role in glycated low-density lipoproteins (gly-LDL) induced-cardiomyocyte apoptosis. We found that gly-LDL upregulated ASPN expression. ASPN increased H9C2 cardiomyocyte apoptosis with down-regulation of Bcl-2, upregulation of transforming growth factor-ß1, Bax, collagen III, fibronectin, and phosphorylation of smad2 and smad3. However, GSPB2 treatment reversed ASPN-induced impairments in H9C2 cardiomyocytes. These results provide evidence for the cardioprotective action of GSPB2 against ASPN injury, and thus suggest a new target for fighting against diabetic cardiomyopathy.

Low-dose phloretin alleviates diabetic atherosclerosis through endothelial KLF2 restoration.

We investigated whether low-dose phloretin served as daily dietary supplements could ameliorate diabetic atherosclerosis and the role of kruppel-like factor 2 (KLF2). HUVECs cultured in high glucose medium were treated with different concentrations of phloretin and KLF2 mRNA, and protein level was detected. Diabetes was induced using streptozotocin in Apoe-/- mice after which they were fed a high-cholesterol diet for 8 weeks. Diabetic mice injected with KLF2 shRNA-lentivirus or control virus were treated with 20 mg/kg phloretin. Glucose, lipid profile, aortic atheroma, and endothelial nitric oxide synthase (eNOS) expression were detected. Phloretin retained endothelial function by KLF2-eNOS activation under hyperglycemia. Low-dose phloretin helped with lipid metabolism, and blocked the acceleration of atherosclerosis in STZ-induced diabetic mice since the early stage, which was diminished by KLF2 knockdown. Low-dose phloretin exhibited athero-protective effect in diabetic Apoe-/- mice dependent on KLF2 activation. This finding makes phloretin for diabetic atherosclerosis.

Astaxanthin attenuated pressure overload-induced cardiac dysfunction and myocardial fibrosis: Partially by activating SIRT1.

BACKGROUND: Myocardial fibrosis contributes to cardiac dysfunction. Astaxanthin (AST), a member of the carotenoid family, is a well-known antioxidant, but its effect on and underlying mechanisms in myocardial fibrosis are poorly understood. METHODS: In vivo, myocardial fibrosis and cardiac dysfunction were induced using transverse aortic constriction (TAC). AST was administered to mice for 12weeks post-surgery. In vitro, transforming growth factor ß1 (TGF-ß1) was used to stimulate human cardiac fibroblasts (HCFs). EX-527 (6-chloro-2, 3, 4, 9-tetrahydro-1H-carbazole-1-carboxamide) and SIRT1 siRNA were used to inhibit SIRT1 in vivo and in vitro, respectively. The effects of AST on cardiac function and fibrosis were determined. SIRT1 expression and activity were measured to explore the mechanisms underlying its effects. RESULTS: AST improved cardiac function and attenuated fibrosis. Receptor activated-SMADs (R-SMADs), including SMAD2 and SMAD3, played important roles in these processes. The TAC surgery-induced increases in the expression of phosphorylated and acetylated R-SMADs were attenuated by treatment with AST, the translocation and transcriptional activity of R-SMADs were also restrained. These effects were accompanied by an increase in the expression and activity of SIRT1. Inhibiting SIRT1 attenuated the acetylation and transcriptional activity of R-SMADs, but not their phosphorylation and translocation. CONCLUSIONS: Our data demonstrate that AST improves cardiac function and attenuates fibrosis by decreasing phosphorylation and deacetylation of R-SMADs. SIRT1 contributes to AST's protective function by reducing acetylation of R-SMADs. GENERAL SIGNIFICANCE: These data suggest that AST may be useful as a preventive/therapeutic agent for cardiac dysfunction and myocardial fibrosis.

Kv1.3 potassium channel mediates macrophage migration in atherosclerosis by regulating ERK activity.

Ion channels expressed in macrophages have been tightly related to atherosclerosis by coupling cellular function. How the voltage-gated potassium channels (Kv) affect macrophage migration remain unknown. The aim of our study is to investigate whether Kv1.3-ERK signaling pathway plays an important role in the process. We explored the expression of Kv1.3 in coronary atherosclerotic heart disease and found Kv1.3 channel was increased in acute coronary syndrome patients. Treatment of RAW264.7 cells with Kv1.3 small interfering RNA, suppressed cell migration. The expression of phosphorylated ERK1/2 also decreased after knockdown of Kv1.3. On the other hand, overexpression of Kv1.3 channel promoted cell migration and ERK1/2 phosphorylation. U-0126, the mitogen-activated protein kinase inhibitors, could reverse macrophage migration induced by Kv1.3 channel overexpression. Downregulation of Kv1.3 channel by siRNA could not further inhibit cell migration when cells were treated with U-0126. It means that ERK is downstream signal of Kv1.3 channel. We concluded that Kv1.3 may stimulate macrophage migration through the activation of ERK.

Blockade of 2-arachidonoylglycerol hydrolysis produces antidepressant-like effects and enhances adult hippocampal neurogenesis and synaptic plasticity.

The endocannabinoid ligand 2-arachidonoylglycerol (2-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184.

Cofilin1 is involved in hypertension-induced renal damage via the regulation of NF-κB in renal tubular epithelial cells.

BACKGROUND: Inflammation mediated by nuclear factor-κB (NF-κB) plays a critical role in the pathogenesis of hypertensive nephropathy (HN). Cytoskeletal remodelling is necessary for the activation of NF-κB. An actin-binding protein, cofilin-1 promotes dynamic alterations to the cytoskeleton by severing actin filaments. However, whether cofilin1 modulates NF-κB activity via cytoskeletal remodelling in the setting of hypertensive renal damage and what mechanisms underlie this phenomenon, remain unknown. METHODS: Twenty-one-week old spontaneously hypertensive rats (SHRs) were treated with an antioxidant (100 or 250 mg kg(-1) day(-1)), grape seed proanthocyanidins extract (GSPE), for 22 weeks. Twenty-four-hour urinary protein, serum creatinine and urea nitrogen levels were measured. Haematoxylin and eosin (HE) staining was performed, and the expression levels of renal cortex cofilin1, monocyte chemotactic protein 1 (MCP1), interleukin-1ß (IL1ß) and NF-κB were evaluated via either Western blotting or immunohistochemistry. In vitro, human proximal renal tubular epithelial cells (HK-2 cells) were pre-incubated either with or without GSPE and subsequently treated with angiotensinII (AngII). Furthermore, a lentiviral shRNA-vector was utilized to knockdown cofilin1 expression in the HK-2 cells, which were stimulated with AngII. Actin filaments, NF-κB activity and several downstream inflammatory factors, including MCP1 and IL-1ß, were investigated. RESULTS: In addition to elevated blood pressure and 24 h urinary protein levels, NF-κB activity and the expression levels of MCP1 and IL-1ß were significantly increased, resulting in tubulointerstitial inflammatory infiltration in SHRs. The phosphorylation (inactivation) of cofilin1 was increased in the kidneys of the SHRs. In vitro, AngII stimulation resulted in the phosphorylation of cofilin1, the formation of actin stress fibres and nuclear translocation of NF-κB p65 in the HK2 cells. Both GSPE pretreatment and the shRNA knockdown of cofilin1 inhibited Rel/p65 nuclear translocation, as well as the expression of both MCP-1 and IL-1ß in the AngII-induced HK2 cells. CONCLUSION: These results demonstrate that cofilin1 is involved in hypertensive nephropathy by modulating the nuclear translocation of NF-κB and the expression of its downstream inflammatory factors in renal tubular epithelial cells.

Berberine Attenuates Vascular Remodeling and Inflammation in a Rat Model of Metabolic Syndrome.

Berberine is a natural product that shows benefits for metabolic syndrome (MS). However, the effects of berberine on the improvement of vascular inflammation and remodeling in MS remain unclear. This study aimed to investigate whether berberine could prevent vascular remodeling and inflammation in the MS condition. A rat model of MS was established, and MS rats were divided into two groups: MS group without berberine treatment, and MSB group with berberine treatment (each group n-10). Ten normal Wistar rats were used as controls (NC group). Vascular damage was examined by transmission electron microscopy and pathological staining. Compared to the NC group, the secretion of inflammatory factors was increased and the aortic wall thicker in the MS group. The MSB group exhibited decreased secretion of inflammatory factors and improved vascular remodeling, compared to the MS group. In addition, the levels of p38 mitogen-activated protein kinase (p38 MAPK), activating transcription factor 2 (ATF-2) and matrix metalloproteinase 2 (MMP-2) were significantly decreased in the MSB group compared to the MS group. In conclusion, our data show that berberine improves vascular inflammation and remodeling in the MS condition, and this is correlated with the ability of berberine to inhibit p38 MAPK activation, ATF-2 phosphorylation, and MMP-2 expression.

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy.

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), Akt and glycogen synthase kinase-3beta (GSK-3ß) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3ß in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3ß signaling pathway.

Quantitative proteomics study of protective effects of grape seed procyanidin B2 on diabetic cardiomyopathy in db/db mice.

Diabetic cardiomyopathy is one of the major complications of diabetes mellitus. Oxidative stress appears to play a substantial role in cardiomyopathy. Grape seed procyanidin B2 (GSPB2) has been known as an anti-oxidant in treating diabetes mellitus; however, little is known about its effects and underlying mechanisms on diabetic cardiomyopathy. The present study is to explore the molecular targets of GSPB2 responsible for the anti-oxidative effects in db/db mice by quantitative proteomics. GSPB2 (30 mg/kg body weight/day) were intragastric administrated to db/db mice for 10 weeks. Proteomics of the heart tissue extracts by isobaric tags for relative and absolute quantification analysis was obtained from db/db mice. Our study provides important evidence that GSPB2 protect against cardiomyopathy in diabetes mellitus, which are believed to result from regulating the expression of key proteins involving cardiac fibrosis and proliferation. GSPB2 could be expected to become novel clinical application in fighting against diabetic cardiomyopathy.

Clinical significance of automatic warning function of cardiac remote monitoring systems in preventing acute cardiac episodes.

OBJECTIVE: In addition to ambulatory Holter electrocardiographic recording and transtelephonic electrocardiographic monitoring (TTM), a cardiac remote monitoring system can provide an automatic warning function through the general packet radio service (GPRS) network, enabling earlier diagnosis, treatment and improved outcome of cardiac diseases. The purpose of this study was to estimate its clinical significance in preventing acute cardiac episodes. METHODS: Using 2 leads (V1 and V5 leads) and the automatic warning mode, 7160 patients were tested with a cardiac remote monitoring system from October 2004 to September 2007. If malignant arrhythmias or obvious ST-T changes appeared in the electrocardiogram records was automatically transferred to the monitoring center, the patient and his family members were informed, and the corresponding precautionary or therapeutic measures were implemented immediately. RESULTS: In our study, 274 cases of malignant arrhythmia, including sinus standstill and ventricular tachycardia, and 43 cases of obvious ST-segment elevation were detected and treated. Because of early detection, there was no death or deformity. CONCLUSIONS: A cardiac remote monitoring system providing an automatic warning function can play an important role in preventing acute cardiac episodes.

Effects of phlorizin on diabetic retinopathy according to isobaric tags for relative and absolute quantification-based proteomics in db/db mice.

PURPOSE: Diabetic retinopathy (DR) is a leading cause of vision loss in working-age people. To retard the development and progression of retina lesions, effective therapeutic strategies directed toward key molecular targets are desired. Phlorizin is effective in treating diabetic complications, but little is known about functional protein changes that may mediate its actions. The aim of this study was to identify retinal proteomic alterations in db/db mice treated with phlorizin. METHODS: We used C57BLKS/J db/db mice as a type 2 diabetic animal model, while C57BLKS/J db/m mice were selected as the control. Phlorizin (20 mg/kg bodyweight /d) was administrated to db/db mice for ten weeks. Serum fasting blood glucose and advanced glycation end products were determined. Meanwhile, retina cell apoptosis was determined with terminal transferase dUTP nick end labeling. Isobaric tags for relative and absolute quantification and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify and profile retinal proteins among control, untreated diabetic, and phlorizin-treated db/db mice. The expression of glial fibrillary acidic protein was measured in retinas using western blotting analysis. RESULTS: Phlorizin treatment significantly reduced fasting blood glucose and levels of advanced glycation end products (p<0.05) and remarkably inhibited retina cell apoptosis and the expression of glial fibrillary acidic protein in the retinas of db/db mice. In addition, we identified 1,636 proteins from retina tissue in total, of which 348 proteins were differentially expressed in db/db mice compared with the controls. Only 60 proteins in the retinas of the db/db mice were found to be differentially changed following phlorizin treatment, including 33 proteins that were downregulated and 27 proteins that were upregulated. Most of these differentially changed proteins were involved in oxidative stress, apoptosis, energy metabolism, and signaling transduction. CONCLUSIONS: Our study revealed the expression of proteins differentially changed after phlorizin therapy. These proteins are most likely to participate in the development and recovery of DR. Our findings help expand understanding of the mechanism underlying the onset and progression of DR, and provide novel targets for evaluating the effects of phlorizin therapy.

Effect of ouabain on myocardial ultrastructure and cytoskeleton during the development of ventricular hypertrophy.

The aim of this work is to study cytoskeletal impairment during the development of ouabain-induced ventricular hypertrophy. Male Sprague-Dawley rats were treated with either ouabain or saline. Systolic blood pressure (SBP) was recorded weekly. At the end of the 3rd and 6th week, the rats were killed and cardiac mass index were measured. Hematoxylin-eosin and Sirius red staining were carried out and cardiac ultrastructure were studied using transmission electron microscopy. The mRNA level of Profilin-1, Desmin, PCNA, TGF-ß(1) and ET-1 in the left ventricle were measured using real-time quantitative PCR while their protein levels were examined by Western blot or immunohistochemistry. After 3 weeks, there was no significant difference in the mean SBP, cardiac mass index, mRNA and protein expression of PCNA, TGF-ß(1) and ET-1 between the two groups. However, ouabain-treated rats showed disorganized cardiac cytoskeleton with abnormal expression of Profilin-1 and Desmin. After 6 weeks, the cardiac mass index remained the same in the two groups while PCNA, TGF-ß(1), and ET-1 have been upregulated in ouabain-treated rats. The cardiac cytoskeletal impairment was more severe in ouabain-treated rats with further changes of Profilin-1 and Desmin. Cytoskeletal abnormality is an ultra-early change during ouabain-induced ventricular hypertrophy, before the release of hypertrophic factors. Therapy for prevention of ouabain-induced hypertrophy should start at the early stage by preventing the cytoskeleton from disorganization.

Prevalence and clinical implications of polypharmacy and potentially inappropriate medication in elderly patients with heart failure: results of six months' follow-up.

OBJECTIVES: To investigate the prevalence of polypharmacy and potentially inappropriate medication (PIM) in elderly patients with heart failure (HF) and their impact on readmission and mortality. METHODS: We conducted a study of 274 participants aged 60 years or older with HF. The prevalence of polypharmacy (defined as the use of five or more medications) was calculated, and the 2019 American Geriatrics Society Beers criteria were applied to access PIMs. Medications and PIMs were characterized at admission and discharge, and changes in prescriptions during hospitalization were compared. The impact of polypharmacy and PIM on readmission and mortality were investigated. RESULTS: The median age of this study population was 68 years old. The median number of prescribed drugs was 7 at admission and 10 at discharge. At discharge, 99.27% of all patients were taking five or more drugs. The incidence of composite endpoint and cardiovascular readmission increased with the number of polypharmacy within 6 months. The use of guideline-directed medical therapy reduced the incidence of composite endpoint events and cardiovascular readmission, while the use of non-cardiovascular medications increased the composite endpoint events. The frequency of PIMs was 93.79% at discharge. The incidence of composite endpoint events increased with the number of PIMs. "PIMs in older adults with caution" increased cardiovascular readmission and "PIMs based on kidney function" increased cardiovascular mortality. Several comorbidities were associated with cardiovascular mortality or non-cardiovascular readmission. CONCLUSIONS: Polypharmacy and PIM were highly prevalent in elderly patients with HF, and their use was associated with an increased risk of composite endpoint events, readmission and mortality. Non-cardiovascular medications, "PIMs in older adults with caution", "PIMs based on kidney function" and several comorbidities were important factors associated with hospital readmission and mortality. Our findings highlight the importance of medication optimization in the management of HF in elderly patients.

Multi-omics reveals aging-related pathway in natural aging mouse liver.

Aging is associated with gradual changes in liver structure, altered metabolites and other physiological/pathological functions in hepatic cells. However, its characterized phenotypes based on altered metabolites and the underlying biological mechanism are unclear. Advancements in high-throughput omics technology provide new opportunities to understand the pathological process of aging. Here, in our present study, both metabolomics and phosphoproteomics were applied to identify the altered metabolites and phosphorylated proteins in liver of young (the WTY group) and naturally aged (the WTA group) mice, to find novel biomarkers and pathways, and uncover the biological mechanism. Analysis showed that the body weights, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) increased in the WTA group. The grips decreased with age, while the triglyceride (TG) and cholesterol (TC) did not change significantly. The increase of fibrosis, accumulation of inflammatory cells, hepatocytes degeneration, the deposition of lipid droplets and glycogen, the damaged mitochondria, and deduction of endoplasmic reticulum were observed in the aging liver under optical and electron microscopes. In addition, a network of metabolites and phosphorylated proteomes of the aging liver was established. Metabolomics detected 970 metabolites in the positive ion mode and 778 metabolites in the negative ion mode. A total of 150 pathways were pooled. Phosphoproteomics identified 2618 proteins which contained 16621 phosphosites. A total of 164 pathways were detected. 65 common pathways were detected in two omics. Phosphorylated protein heat shock protein HSP 90-alpha (HSP90A) and v-raf murine viral oncogene homolog B1(BRAF), related to cancer pathway, were significantly upregulated in aged mice liver. Western blot verified that protein expression of MEK and ERK, downstream of BRAF pathway were elevated in the liver of aging mice. However, the protein expression of BRAF was not a significant difference. Overall, these findings revealed a close link between aging and cancer and contributed to our understanding of the multi-omics changes in natural aging.

Simvastatin exerts cardioprotective effects and inhibits the activity of Rho-associated protein kinase in rats with metabolic syndrome.

1. Insulin resistance (IR) is crucially involved in the pathophysiology of metabolic syndrome (MS). The aim of the present study was to investigate the effects of simvastatin on IR in rats with MS. 2. A rat model of MS was established and myocardial damage was examined by transmission electron microscopy. Twenty-two MS rats were divided into two groups of 11 rats each: (i) an MS group; and (ii) a simvastatin-treated MS. Ten Wistar rats were used as controls. The phosphorylation of myosin phosphatase target subunit 1 (MYPT-1), insulin receptor substrate 1 (IRS-1) and Akt were analysed by immunohistochemistry and western blotting. 3. Insulin resistance-induced MS was associated with a significant increase in Rho kinase (ROCK) activity and inhibition of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Decreased levels of phosphorylated (p-) MYPT-1 and p-IRS-1 (Ser³°7) and increased levels of p-Akt were found in hearts from the MS + simvastatin compared with the MS group. These results suggest that simvastatin reduces ROCK activity and increases Akt activity. 4. Simvastatin exerts cardioprotective effects and improves IR, which can be attributed, at least in part, to the inhibition of ROCK and activation of PI3-K/Akt.

Effects of phytochemicals sulforaphane on uridine diphosphate-glucuronosyltransferase expression as well as cell-cycle arrest and apoptosis in human colon cancer Caco-2 cells.

Our study investigated the effects of the chemopreventive agent sulforaphane (SFN) on human colon cancer Caco-2 cells and potential underlying mechanisms of the effects. When treated with SFN at hypotoxic levels, UDP-glucuronosyltransferase 1A (UGT1A) was induced in a dose-dependent manner. SFN at 25 µM showed the highest UGT1A-induction activity, inducing UGT1A1, UGT1A8, and UGT1A10 mRNA expression, and increasing UGT-mediated N-hydroxy-PhIP glucuronidation. SFN- induced UGT1A expression may have resulted from Nrf2 nuclear translocation or activation. At higher concentrations, e.g. 75 µM, SFN caused G1/G2 cell cycle arrest and induced apoptosis possibly through reducing anti-apoptotic bcl-2 expression and increasing apoptosis-inducing bax expression in Caco-2 cells. Taken together, these results demonstrated the chemopreventive effects of SFN on human colon cancer Caco-2 cells may have been partly attributed to Nrf2-mediated UGT1A induction and apoptosis induction, and our studies provided theoretic and experimental basis for clinical application of SFN to human colon cancer prevention.

Induction of lactadherin mediates the apoptosis of endothelial cells in response to advanced glycation end products and protective effects of grape seed procyanidin B2 and resveratrol.

One of characteristics of diabetes mellitus (DM) is endothelial cell (EC) dysfunction and apoptosis which contributes to the development of vasculopathy. Advanced glycation end products (AGEs) continuously produced in the setting of DM play an important role in causing EC dysfunction and apoptosis. However, the underlying molecular mechanism remains largely elusive. Lactadherin, a secreted glycoprotein of milk-fat globule, is expressed by multiple cell types of arterial wall including ECs. Our previous proteomic studies showed that the expression of lactadherin was significantly increased in the aorta of diabetic rats as compared with control rats and treatment with grape seed procyanidin extracts significantly inhibited the lactadherin expression in diabetic rats. We hypothesized that lactadherin plays a critical role in AGEs-induced EC apoptosis; grape seed procyanidin B2 (GSPB2) and resveratrol protect against AGEs-induced EC apoptosis through lactadherin regulation. Our results showed that AGEs upregulated lactadherin expression and lactadherin RNA interference significantly attenuated AGEs-induced EC apoptosis. Overexpression of lactadherin increased EC apoptosis with up-regulation of Bax/Bcl-2 ratio, cytochrome c release, caspase-9 and caspase-3 activation suggesting the involvement of mitochondria apoptosis pathway. Mechanistically, overexpression of lactadherin reduced the phosphorylation of GSK3beta at baseline. Our study also revealed nine proteins interacting with lactadherin in HUVEC and study of these candidate proteins could unveil further underlying molecular mechanisms. In summary, our study identified lactadherin as a key player responsible for AGEs-induced EC apoptosis and antioxidants GSPB2 and resveratrol protect against AGEs-induced EC apoptosis by inhibiting lactadherin. Targeting lactadherin with antioxidant could be translated into clinical application in the fighting against DM complications.

Grape seed procyanidin B2 inhibits advanced glycation end product-induced endothelial cell apoptosis through regulating GSK3ß phosphorylation.

To investigate the effects of GSPB2 (grape seed procyanidin B2) on the apoptosis of HUVECs (human umbilical endothelial cells) induced by AGEs (advanced glycation end products), HUVECs were treated with AGEs (200 µg/ml) in the presence or absence of GSPB2 (2.5, 5.0 and 10.0 µmol/l). Our findings showed that (i) AGEs induced HUVEC apoptosis and up-regulated the expression of caspase-3 activation and lactadherin and reduced the phosphorylation of GSK3ß (glycogen synthase kinase 3ß) at baseline. (ii) Treatment of HUVEC with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs. Moreover, GSPB2 inhibited intracellular reactive oxygen species in a dose-dependent manner in AGEs-treated cells as determined by flow cytometry. (iii) GSPB2 increased the phosphorylation of GSK3ß of HUVEC in response to AGEs. These findings suggest that the signalling pathway involving phosphorylation of GSK3ß and lactadherin might play a key role in the endothelial apoptosis. GSPB2 therapy could become an effective approach to battling AGEs-induced endothelial apoptosis.