IRS1/PI3K/AKT pathway signal involved in the regulation of glycolipid metabolic abnormalities by Mulberry (Morus alba L.) leaf extracts in 3T3-L1 adipocytes.

BACKGROUND: Mulberry (Morus alba L.) leaf tea benefits the control of diabetes in Asian nations. This study was aim to investigate if the flavonoids, which extracts from mulberry leaves, could regulate the metabolism of glycolipid, and to investigate if flavonoids could regulate IRS1/PI3K/AKT pathway signal to affect the expression of FAS and membrane transfer capacity GLUT4 in 3T3-L1 adipocytes. RESULTS: Results revealed that flavonoids decreased the levels of free fatty acid and increased the glucose consumption and the levels of adiponectin and leptin in a dose-dependent manner, and remarkably increased the protein expression levels of p-IRS1, p-PI3K, p-Akt, total GLUT4, and membrane GLUT4, and decreased the protein expression levels of PTEN and FAS in 3T3-L1 adipocytes IR model. On the other hand, wortmannin (2 nM), a selective and irreversible PI3K inhibitor, significantly decreased the glucose consumption and the adiponectin and leptin levels, and increased the free fatty acid level in flavonoids treated 3T3-L1 adipocytes IR model. Furthermore, wortmannin (2 nM) partly eliminated the activation of PI3K/AKT signaling, the suppression of FAS, and the up-regulated membrane transfer capacity of GLUT4 in flavonoids treated 3T3-L1 adipocytes IR model. CONCLUSION: In conclusion, our results illustrated that mulberry leaf extracts flavonoids alleviated the glycolipid metabolic abnormalities in 3T3-L1 adipocytes IR model, and the effect was associated with the activation of IRS1/PI3K/AKT pathway, the suppression of FAS, and the up-regulation of membrane transfer capacity of GLUT4.

Liuwei Dihuang, a traditional Chinese medicinal formula, inhibits proliferation and migration of vascular smooth muscle cells via modulation of estrogen receptors.

The phenotypic modulation of vascular smooth muscle cells (VSMCs) serves an important role in atherosclerosis­induced vascular alterations, including vascular remodeling. However, the precise mechanisms underlying VSMC phenotypic modulation remain to be elucidated. Our previous study demonstrated that Liuwei Dihuang formula (LWDHF) could improve menopausal atherosclerosis by upregulating the expression of estrogen receptors (ERs). The present study examined the role of ERs in the effects of LWDHF on VSMC phenotypic modulation. VSMC proliferation and cell cycle progression were examined by MTT assay and flow cytometry, respectively. The expression levels of α­smooth muscle actin, osteopontin and ERs were determined using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis. Cell ultrastructure was observed under an electron microscope. F­actin polymerization was detected by fluorescein isothiocyanate­phalloidin staining using fluorescence microscopy. A modified Boyden chamber assay was employed to assess VSMCs migration. Small interfering (si)RNA technology was used to examine the role of ERα in the effects of LWDHF on phenotypic modulation. The results indicated that LWDHF (3­12 µg/ml) inhibited proliferation and induced a cell cycle arrest in VSMCs treated with angiotensin II (Ang II; 100 nM) in a concentration­dependent manner. In addition, Ang II­stimulated migration of VSMCs and reorganization of actin were markedly inhibited by treatment with 12 µg/ml LWDHF. Results of RT­qPCR and western blotting demonstrated that LWDHF markedly stimulated transcription and expression of ERα and ERß, and inhibited VSMC synthetic phenotype. Furthermore, LWDHF­induced inhibition of phenotypic switching was partially suppressed by tamoxifen, and transfection with ERα siRNA markedly abolished the effects of LWDHF on VSMC phenotypic switching. In conclusion, these results revealed that ERα served an important role in LWDHF­induced regulation of the VSMC phenotype, including proliferation and migration.

Catalpol protects glucose-deprived rat embryonic cardiac cells by inducing mitophagy and modulating estrogen receptor.

Catalpol, a bioactive component from Rehmannia glutinosa (Di Huang), has been widely used to protect cardiomyocytes against myocardial ischemia. The aim of the present study was to investigate the anti-apoptotic and anti-oxidative effects of Catalpol on glucose-starved H9c2 cells for cardio-protection and to elucidate the underlying mechanisms. Here, we showed that Catalpol protected the glucose-starved H9c2 cells through reducing apoptosis and attenuating oxidative damage. Moreover, the increases of autophagic lysosomes, LC3, autophagic flux and autophagic vacuole were observed in Catalpol-treated cells using flow cytometer and fluorescence microscope. Western blotting analyses showed that the autophagy-related proteins (LC3, Beclin1 and ULK) were markedly increased in Catalpol-treated cells, suggesting that Catalpol up-regulated autophagy in glucose starved H9c2 cells. Mechanistic investigations revealed that the autophagy inhibitor 3-MA markedly abrogated Catalpol's anti-apoptotic and anti-oxidative effects and prevented Catalpol-induced mitophagy. Furthermore, the estrogen receptor inhibitor tamoxifen significantly abolished Catalpol up-regulation of mitophagic related proteins (LC3, Beclin 1, p62, ATG5). Collectively, these data revealed that Catalpol inhibited apoptosis and oxidative stress in glucose-deprived H9c2 cell through promoting cell mitophagy and modulating estrogen receptor, supporting the notion that Catalpol could be a novel drug candidate against myocardial ischemia for the treatment of cardiovascular diseases.

A novel formula Sang-Tong-Jian improves glycometabolism and ameliorates insulin resistance by activating PI3K/AKT pathway in type 2 diabetic KKAy mice.

AIMS: Sang-Tong-Jian (STJ), a novel formula composed of flavonoids and alkaloids derived from mulberry leaf, has been found to reduce blood glucose levels in rats with type 2 diabetes mellitus (T2DM) in our previous studies. However, the precise mechanisms remain unknown. Insulin resistance is the main characteristic of T2DM, which may be due to impairment of the PI3K/AKT signaling pathway. In this study, we investigated the effects of STJ on glycometabolism and insulin resistance in KKAy mice. METHODS: A total of 50 KKAy male mice were randomly divided into five groups: model, metformin at 260mg/kg, and STJ at 105, 210 and 420mg/kg. C57BL/6J mice were used as the control group. Random blood glucose levels in KKAy mice were determined every 10days after treatments. At the 10th and 13th week, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were conducted after a 12h overnight fast, respectively. After 13-week treatments, glycosylated hemoglobin (GHb) and serum insulin were measured using a colorimetric method and ELISA kits. Liver glycogen and muscle glycogen levels were analyzed using a colorimetric method. The morphology of pancreas, liver, skeletal muscle and epididymal fat were visualized by haematoxylin and eosin staining. The gene level of GLUT2 (liver) and GLUT4 (skeletal muscle, epididymal fat) were detected by real-time PCR. The proteins of GLUT2, GLUT4, IRS1, PI3K, AKT and their phosphorylation were assayed by Western blot analyses. RESULTS: STJ significantly decreased the random blood glucose and GHb levels, and increased liver and muscle glycogen levels. The results of OGTT and ITT and measurement of serum insulin indicated that STJ ameliorated insulin resistance in KKAy mice. STJ treatments also ameliorated the histopathological alterations in pancreas, liver, skeletal muscle and epididymal fat in KKAy mice. Furthermore, STJ upregulated the gene and protein expression of GLUT2 (liver) and GLUT4 (skeletal muscle, epididymal fat). Meanwhile, GLUT4 translocation and phosphorylation of IRS1, p85-PI3K and AKT were significantly increased by STJ treatments. CONCLUSIONS: Our results indicated that STJ ameliorated glycometabolism and insulin resistance in KKAy mice, which might be due to activation of PI3K/AKT pathway.

Cardioprotective effect of Salvianolic acid B on acute myocardial infarction by promoting autophagy and neovascularization and inhibiting apoptosis.

OBJECTIVES: The aim of this study was to investigate the cardioprotective effect of salvianolic acid B (Sal B) on acute myocardial infarction (AMI) in rats and its potential mechanisms. METHODS: The AMI model was established in rats to study the effect of Sal B on AMI. Haematoxylin-eosin (HE) staining was used to evaluate the pathological change in AMI rats. Immunofluorescence and TUNEL staining were used to detect autophagy and apoptosis of myocardial cells in hearts of AMI rats, respectively. Protein expression of apoptosis-related, autophagy-related and angiogenesis-related proteins were examined by Western blot. KEY FINDINGS: Sal B attenuated myocardial infarction significantly compared with that of the model group. Rats administered with Sal B showed higher inhibition rate of infarction and lower infarct size than those of the model group. Moreover, Sal B decreased the serum levels of creatine kinase, lactate dehydrogenase and malondialdehyde, while increased such level of superoxide dismutase significantly compared with those of the model group. Sal B inhibited the expression of Bax, cleaved caspase-9 and cleaved PARP, while promoted the expression of Bcl-2, LC3-II, Beclin1 and VEGF. CONCLUSIONS: Sal B has cardioprotective effect on AMI and Sal B may be a promising candidate for AMI treatment.