Regulatory Effects and Mechanism of Action of Green Tea Polyphenols on Osteogenesis and Adipogenesis in Human Adipose Tissue-Derived Stem Cells.

We previously showed that green tea polyphenols (GTPs) exert antiadipogenic effects on preadipocyte proliferation. Here, we investigated the regulatory effects of GTPs on osteogenesis and adipogenesis during early differentiation of human adipose tissue-derived stem cells (hADSC). Adipogenesis of hADSCs was determined by oil-red-O staining and triglycerides synthesis measurement. Osteoporosis of hADSC was measured using alkaline phosphatase assays and intracellular calcium levels. Immunofluorescence staining and qRT-PCR were used to detect PPARγ-CEBPA regulated adipogenic pathway regulated by PPAR-CEBPA and the osteogenic pathway mediated by RUNX2-BMP2. We found that GTPs treatment significantly decreased lipid accumulation and cellular triglyceride synthesis in mature adipocytes and attenuated pioglitazone-induced adipogenesis in a dose-dependent manner. GTPs downregulated protein and mRNA expression of Pparγ and attenuated pioglitazone-stimulated-Cebpa expression. GTPs treatment significantly enhanced hADSCs differentiation into osteoblasts compared to control and pioglitazone-treated cells. GTPs upregulated RunX2 and Bmp2 proteins and mRNA expression compared to control and significantly attenuated decreased RunX2 and Bmp2 mRNA expression by pioglitazone. In conclusion, our data demonstrates GTPs possesses great ability to facilitate osteogenesis and simultaneously inhibits hADSC differentiation into adipogenic lineage by upregulating the RUNX2-BMP2 mediated osteogenic pathway and suppressing PPARγ-induced signaling of adipogenesis. These findings highlight GTPs' potential to combat osteoporosis associated with obesity.

Laminaria japonica Peptides Suppress Liver Cancer by Inducing Apoptosis: Possible Signaling Pathways and Mechanism.

The anticancer properties of Laminaria japonica peptides (LJPs) have never been studied. Here, we extracted LJPs from fresh seaweed and explored their anti-liver cancer activity (in vivo and in vitro). LJPs were isolated/purified by HPLC-ESI-MS. HepG2 cell apoptosis and cell cycle were evaluated. MTT assays were used to examine the cytotoxicity of LJPs. Caspase activation of caspases 3 and 9, cleaved caspases 3 and 9, and cleaved PARP was examined by Western blotting. The PI3K/AKT pathway and the phosphorylation states of MAPKs (p38 and JNK) were examined. We found that the LJP-1 peptide had the most antiproliferative activity in H22 cells in vitro. LJP-1 blocked H22 cells in the G0/G1 phase, accompanied by inhibition of cyclin expression. LJP-1 induced apoptosis through caspase activation and regulation of the ASK1/MAPK pathway. Concurrent in vivo studies demonstrated that LJP-1 significantly inhibited tumor growth and induced tumor cell apoptosis/necrosis. In conclusion, LJPs, particularly LJP-1, exert strong inhibitory effects on liver cancer growth in vivo and in vitro. LJP-1 induces HCC cell apoptosis through the caspase-dependent pathway and G0/G1 arrest. LJP-1 induces caspase-dependent apoptosis, in part by inhibiting PI3K, MAPK signaling pathways, and cell cycle proteins. LJP-1 has the potential to be a novel candidate for human liver cancer therapeutics.

Paeoniflorin extract reverses dexamethasone-induced testosterone over-secretion through downregulation of cytochrome P450 17A1 expression in primary murine theca cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polycystic Ovarian Syndrome (PCOS) is a complex endocrine and reproductive disorder. A main hallmark includes increased androgen production. The root of Paeonia lactiflora Pall. (Bai Shao) is used in Chinese herbal medicine for reproductive disorders, however its effects and mechanisms on ovarian theca cells has not yet been fully elucidated. AIM OF THE STUDY: The aim of this study was to evaluate effect of paeoniflorin extract (PFE), the main constituents of Bai Shao, on androgen production in ovarian theca cells. MATERIALS AND METHODS: Primary murine theca cells were treated with concentrations of PFE (1-100 µg/mL) in the presence of dexamethasone (10 µM) with media-only treated cells used as the control. After 24 h, culture media was collected for biochemistry assays of testosterone and progesterone. Expression of key steroidogenic enzymes, cholesterol side-chain cleavage (CYP11A1) and 17α-hydroxylase (CYP17A1) was characterized using immunofluorescence staining, immunoblotting and qRT-PCR. RESULTS: Dexamethasone significantly enhanced testosterone secretion (P < 0.05 vs. the control cells). PFE reversed over-production of testosterone induced by dexamethasone in a dose-dependent manner. The treatment with PFE also normalized production of progesterone in dexamethasone-treated cells. Expression of CYP11A1 and CYP17A1 in the theca cells were visualised by immunofluorescence staining. All doses of PFE significantly inhibited CYP17A1 expression detected by immunoblotting, but only 100 µg/mL of PFE downregulated CYP11A1 expression and reduced CYP11A1 significantly in dexamethasone-treated theca cells. CONCLUSIONS: PFE may reduce over-secretion of testosterone in theca cells through downregulation of CYP17A1 and CYP11A1. These findings provide scientific evidence to treat ovarian hyperandrogenism with the root of Paeonia lactiflora Pall.

Green tea polyphenols ameliorate non-alcoholic fatty liver disease through upregulating AMPK activation in high fat fed Zucker fatty rats.

AIM: To investigate protective effects and molecular mechanisms of green tea polyphenols (GTP) on non-alcoholic fatty liver disease (NAFLD) in Zucker fatty (ZF) rats. METHODS: Male ZF rats were fed a high-fat diet (HFD) for 2 wk then treated with GTP (200 mg/kg) or saline (5 mL/kg) for 8 wk, with Zucker lean rat as their control. At the end of experiment, serum and liver tissue were collected for measurement of metabolic parameters, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), inflammatory cytokines and hepatic triglyceride and liver histology. Immunoblotting was used to detect phosphorylation of AMP-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein 1c (SREBP1c). RESULTS: Genetically obese ZF rats on a HFD presented with metabolic features of hepatic pathological changes comparable to human with NAFLD. GTP intervention decreased weight gain (10.1%, P = 0.052) and significantly lowered visceral fat (31.0%, P < 0.01). Compared with ZF-controls, GTP treatment significantly reduced fasting serum insulin, glucose and lipids levels. Reduction in serum ALT and AST levels (both P < 0.01) were observed in GTP-treated ZF rats. GTP treatment also attenuated the elevated TNFα and IL-6 in the circulation. The increased hepatic TG accumulation and cytoplasmic lipid droplet were attenuated by GTP treatment, associated with significantly increased expression of AMPK-Thr172 (P < 0.05) and phosphorylated ACC and SREBP1c (both P < 0.05), indicating diminished hepatic lipogenesis and triglycerides out flux from liver in GTP treated rats. CONCLUSION: The protective effects of GTP against HFD-induced NAFLD in genetically obese ZF rats are positively correlated to reduction in hepatic lipogenesis through upregulating the AMPK pathway.

Characterisation of Bone Beneficial Components from Australian Wallaby Bone.

Background: Osteoporosis is a condition in which the bones become brittle, increasing the risk of fractures. Complementary medicines have traditionally used animal bones for managing bone disorders, such as osteoporosis. This study aimed to discover new natural products for these types of conditions by determining mineral and protein content of bone extracts derived from the Australian wallaby. Methods: Inductively coupled plasma-mass spectrometry and Fourier transform infrared spectroscopic analysis were used for mineral tests, proteome analysis was using LC/MS/MS and the effects of wallaby bone extracts (WBE)s on calcium deposition and alkaline phosphatase activity were evaluated in osteogenic cells derived from adipose tissue-derived stem cells (ADSCs). Results: Concentrations of calcium and phosphorus were 26.21% and 14.72% in WBE respectively. Additionally, minerals found were wide in variety and high in concentration, while heavy metal concentrations of aluminium, iron, zinc and other elements were at safe levels for human consumption. Proteome analysis showed that extracts contained high amounts of bone remodelling proteins, such as osteomodulin, osteopontin and osteoglycin. Furthermore, in vitro evaluation of WBEs showed increased deposition of calcium in osteoblasts with enhanced alkaline phosphatase activity in differentiated adipose-derived stem cells. Conclusion: Our results demonstrate that wallaby bone extracts possess proteins and minerals beneficial for bone metabolism. WBEs may therefore be used for developing natural products for conditions such as osteoporosis and further investigation to understand biomolecular mechanism by which WBEs prevent osteoporosis is warranted.

Comparison of Cytotoxicity and the Anti-Adipogenic Effect of Green Tea Polyphenols with Epigallocatechin-3-Gallate in 3T3-L1 Preadipocytes.

Recent studies have demonstrated the effects of green tea polyphenols (GTP) and epigallocatechin-3-gallate (EGCG) on obesity. However, high doses of EGCG have also exhibited cytotoxicity. The aim of this study was to compare total GTP with purified EGCG on cytotoxicity, and to investigate the effects and the molecular mechanism of total GTP and EGCG on adipogenesis. Cytotoxicity was determined by cell viability assay. For the adipogenesis study, 3T3-L1 preadipocytes were incubated with three doses of GTP (1, 10, and 100 µg/ml) and the effect of EGCG (6.8 µg/ml) was compared with 10 µg/ml GTP containing 68% EGCG. Oil Red O staining and triglyceride content assay were carried out 10 days after differentiation and treatment. Adipogenic regulators CCAAT element binding protein α (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein-1c (SREBP-1c) were determined by qRT-PCR and immunoblotting. GTP at 1000 µg/ml and EGCG (68 and 680 µg/ml) significantly affected cell viability. Purified EGCG had greater cytotoxicity than corresponding doses of GTP. About 10 µg/ml of GTP showed stronger reduction in triglyceride accumulation than EGCG treatment. Transcriptional factors of C/EBPα, SREBP-1c and PPARγ were markedly decreased in both GTP and EGCG-treated cells and GTP exhibited stronger inhibitory effects on C/EBPα and PPARγ protein expression than EGCG (p < 0.05). In conclusion, total GTP exerted greater inhibitory effects than purified EGCG on adipogenesis through down-regulating the adipogenic factor C/EBPα, SREBP-1c and PPARγ expression. These findings support that a polyphenol mixture is safer and more effective than EGCG alone for preventing obesity and obesity-related chronic diseases.

Antiresistin RNA Oligonucleotide Ameliorates Diet-Induced Nonalcoholic Fatty Liver Disease in Mice through Attenuating Proinflammatory Cytokines.

The aim of this study was to determine whether inhibition of resistin by a synthetic antiresistin RNA (oligonucleotide) oligo ameliorates metabolic and histological abnormalities in nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD) in mice. The antiresistin RNA oligo and a scrambled control oligo (25 mg/kg of body weight) were i.p. injected to HFD mice. Serum metabolic parameters and hepatic enzymes were measured after 4-week treatment. The treatment significantly reduced epididymal fat and attenuated the elevated serum resistin, cholesterol, triglycerides, glucose, and insulin with an improved glucose tolerance test. Antiresistin RNA oligo also normalized serum AST and ALT levels with improved pathohistology of NAFLD. Immunoblotting and qRT-PCR revealed that decreased protein and mRNA expression of resistin in fat and liver tissues of the treated mice were associated with reduction of adipose TNF-α and IL-6 expression and secretion into circulation. mRNA and protein expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) and sterol regulatory element-binding protein-1c (SREBP-1c) were also significantly decreased in the treated mice. Our results suggest that resistin may exacerbate NAFLD in metabolic syndrome through upregulating inflammatory cytokines and hepatic PEPCK and SREBP-1c. Antiresistin RNA oligo ameliorated metabolic abnormalities and histopathology of NAFLD through attenuating proinflammatory cytokines.

Managing the combination of nonalcoholic Fatty liver disease and metabolic syndrome with chinese herbal extracts in high-fat-diet fed rats.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome (MetS). The aim of the study was to evaluate the effects of Chinese herbal extracts from Salvia miltiorrhiza and Gardenia jasminoides (SGE) on the combination of NAFLD and MetS induced by a high-fat diet (HFD) in rats. After 6 weeks of HFD feeding, rats (n = 10 each group) were treated with saline, rosiglitazone (RSG), and SGE for 4 weeks. HFD rats were obese, hyperinsulinemic, hyperlipidemic and increased hepatic enzymes with the histological images of NAFLD. Treatment with SGE significantly reduced serum triglycerides (TG), nonesterified fatty acids and enhanced insulin sensitivity, and ameliorated the elevated serum hepatic enzymes compared with HFD-saline group. SGE treatment also attenuated hepatic TG by 18.5% (P < 0.05). Histological stains showed SGE decreased lipids droplets in hepatocytes (P < 0.05) and normalized macrovesicular steatosis in HFD rats. Significant reduction of TNF- α and IL6 in adipose tissue was detected in SGE treated rats. The anti-inflammatory action may be, at least in part, the mechanism of SGE on MetS associated with NAFLD. This study discovered that SGE is capable of managing metabolic and histological abnormalities of NAFLD and MetS. SGE may be an optimal treatment for the combination of NAFLD and MetS.

In vitro investigation of anti-diabetic effect of Taxus cuspidate extracts by ultrasound assisted method.

Extracting active components from Chinese medicinal herbs efficiently is a key step in the investigation of their pharmacological effects and modes of action. In this project, we compared the ultrasound-assisted method and the conventional solvent method for extracting the active compound of Taxus cuspidate (dong bei hong dou shan). Through the analysis of various extractions with a quadruple time-of-fight (Q-TOF) LC/MS, we demonstrated that the ultrasound-assisted method reduced solvent consumption and had shorter extraction time, while the extraction yields of the active compound (taxol) were equivalent to or even higher than those obtained with the conventional solvent extraction method. Through the comparison of Taxus cuspidate extracts (TCEs) with different concentrations of acetone and ethanol, we proved that 50% ethanol was an optimal solvent for extracting taxol from Taxus cuspidate. Based on traditional Chinese medicine (TCM) literature, we further determined whether TCEs possess antidiabetic effects by testing glucose uptake in 3T3-L1 adipocytes treated with TCEs from Taxus cuspidate bark and twigs under insulin stimulation (100 nM). The results showed that neither taxol (10 µg/ml) nor TCEs (1 and 0.1 mg/ml) changed glucose uptake significantly compared with insulin alone. This study demonstrated that the ultrasound-assisted method with 50% ethanol is a highly efficient approach for extracting Taxus cuspidate, which may be applicable for extraction of other Chinese medicinal herbs. Extracts of Taxus cuspidate bark and twigs had no effect on insulin stimulated-glucose uptake in vitro. This result conflicts with the description in TCM literature. Further in vivo study to clarify Taxus cuspidate's metabolic actions is necessary.