FAS and SREBP-1c Inhibition via AMPK Activation in HepG2 Cells by Biovip, Tox-off, and Traphanoside GO1 from Glinus oppositifolius.

Glinus oppositifolius is an endemic herbaceous plant found in tropical Asian countries and is native in Vietnam. It is used in traditional folk medicine because of its flavor and antiseptic and laxative effects. In the current research, the effects of Tox-off, Biovip, and the purified compounds isolated from G. oppositifolius in the previous study were evaluated on the activation of adenosine 5'-monophosphate-activated protein kinase (AMPK)-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) in C2C12 myoblasts. In addition, the most potent active compounds, traphanoside-GO1 (TRA-GO1) and TRA-GO5 have validated the reduction of fatty acid synthase (FAS) and sterol regulatory element binding protein (SREBP)-1c in HepG2 cells. We found that Tox-off and Biovip significantly increased the phosphorylation of AMPK and ACC in C2C12 myoblasts. Furthermore, TRA-GO1 and TRA-GO5 significantly increased the AMPK activation and phosphorylation of its downstream substrate ACC in a concentration-dependent way compared to the dimethyl sulfoxide (DMSO) control. Besides, the protein level of FAS and SREBP-1c decreased by TRA-GO1 and TRA-GO5 in a concentration-dependent manner. Taken together, our results showed that the increased AMPK and ACC phosphorylation by active components of G. oppositifolius may activate the AMPK signaling pathways, which are useful for the anti-obesity and its related metabolic disorders.

Paliurus spina-christi Mill fruit extracts improve glucose uptake and activate the insulin signaling pathways in HepG2 insulin-resistant cells.

BACKGROUND: Paliurus spina-christi Mill. (PSC) fruit is frequently used in the treatment of diabetes mellitus in Mediterranean regions. Here, we investigated the effects of various PSC fruit extracts (PSC-FEs) on glucose consumption and some key mediators of insulin signaling pathways in high glucose and high insulin-induced insulin-resistant HepG2 cells. METHODS: The effects of methanolic, chloroform and total extracts on cell proliferation were assessed by the MTT assay. The potential of non-toxic extracts on glucose utilization in insulin-resistant HepG2 cells was checked using a glucose oxidase assay. AKT and AMP-activated protein kinase (AMPK) pathway activation and mRNA expression levels of insulin receptor (INSR), glucose transporter 1 (GLUT1), and glucose transporters 4 (GLUT4) were determined by western blotting and real-time PCR, respectively. RESULTS: We found that high concentrations of methanolic and both low and high concentrations of total extracts were able to enhance glucose uptake in an insulin-resistant cell line model. Moreover, AKT and AMPK phosphorylation were significantly increased by the high strength of methanolic extract, while total extract raised AMPK activation at low and high concentrations. Also, GLUT 1, GLUT 4, and INSR were elevated by both methanolic and total extracts. CONCLUSIONS: Ultimately, our results shed new light on methanolic and total PSC-FEs as sources of potential anti-diabetic medications, restoring glucose consumption and uptake in insulin-resistant HepG2 cells. These could be at least in part due to re-activating AKT and AMPK signaling pathways and also increased expression of INSR, GLUT1, and GLUT4. Overall, active constituents present in methanolic and total extracts of PCS are appropriate anti-diabetic agents and explain the use of these PSC fruits in traditional medicine for the treatment of diabetes.

The protective effects of Esculentoside A through AMPK in the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear. PURPOSE: To investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model. METHODS: EsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins. RESULTS: EsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3ß activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3ß, and blocked autophagy. CONCLUSIONS: To our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

Prevention and Treatment of NAFLD with Traditional Chinese Medicine Monomers by Regulating AMPK Pathway： A Review / 中国实验方剂学杂志

Non-alcoholic fatty liver disease （NAFLD） is a chronic liver disease with complex and diverse pathogenesis， and there is no effective treatment or specific drugs for its clinical treatment. In recent years， its incidence has been on the rise， and it has become the earnest expectation of medical researchers in China and abroad that related patients could be treated. AMP-activated protein kinase （AMPK） functions to regulate cellular energy homeostasis and mitochondrial homeostasis. When activated， it has a good intervention effect on NAFLD progression with lipid metabolism disorders and mitochondrial homeostasis disorders. For NAFLD， the activation of AMPK can inhibit the production of new lipogenesis in the liver， promote the oxidation of fatty acids in the liver， and enhance the mitochondrial function of adipose tissues. As a key target of metabolic diseases， AMPK can also improve apoptosis， liver fibrosis， autophagy， and inflammation. Traditional Chinese medicine （TCM） is good at treating diseases from multiple targets and multiple pathways and is also commonly used in the treatment of chronic liver disease in clinical practice. A large number of in vitro and in vivo experimental studies on NAFLD have shown that TCM monomers have good prospects for the treatment of NAFLD through the AMPK signaling pathway， including glycosides， phenols， alkaloids， flavonoids， quinones， terpenoids， and lignans， which are natural activators of AMPK. This study reviewed the research progress on TCM monomers in regulating the AMPK pathway to prevent and treat NAFLD， providing a broader perspective for TCM treatment of NAFLD.

Traditional Chinese Medicine in Treatment of Myocardial Ischemia-Reperfusion Injury by Regulating Adenosine 5'-monophosphate （AMP）-activated Protein Kinase： A Review / 中国实验方剂学杂志

Due to its high incidence and mortality rate， acute myocardial infarction poses a serious threat to public health. Reperfusion therapy is the preferred treatment strategy for acute myocardial infarction， which can quickly restore blood circulation to the ischemic myocardium， rescue dying myocardial cells， reduce infarct size， and lower the mortality rate. However， reperfusion may lead to additional heart damage， known as myocardial ischemia-reperfusion injury （MIRI）. Therefore， how to alleviate MIRI has become one of the urgent issues in cardiovascular therapy. Traditional Chinese medicine （TCM） has the advantage of multiple components， multiple pathways， and multiple targets in the treatment of MIRI， providing new ideas for reducing MIRI. Adenosine 5'-monophosphate （AMP）-activated protein kinase （AMPK） is closely related to MIRI， and it plays an important role in alleviating MIRI by regulating inflammation， oxidative stress， autophagy， apoptosis， and ferroptosis. This article reviewed the basic structure of the AMPK signaling pathway and its role in MIRI， as well as the research progress of TCM in the treatment of MIRI by regulating the AMPK pathway， aiming to provide a theoretical basis for the prevention and treatment of MIRI.

Active Components in Chinese Medicinal Herbs Regulate AMPK/mTOR Pathway to Mediate Autophagy in Diabetic Kidney Disease： A Review / 中国实验方剂学杂志

Diabetic kidney disease （DKD） is one of the typical microvascular complications in patients with diabetes and a major cause of end-stage renal disease， with the pathogenesis remains to be elucidated. It may be associated with hemodynamic effects， genetic factors， kidney inflammatory injury， oxidative stress， autophagy dysregulation， metabolic disorders and so on. Because of its complex mechanism， there are no specific prevention and treatment measures in clinical practice. AMP-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway is a classical pathway involved in the regulation of autophagy. This pathway can be activated for treating DKD. Recent studies have demonstrated that the active components in Chinese medicinal herbs play a role in the prevention and treatment of DKD by directly acting on targeted cells and autophagy targets， which has attracted extensive attention. Researchers have extensively studied the occurrence and development of DKD and the mechanism of drug intervention in DKD， and the results prove that AMPK/mTOR pathway plays a role in the development of this disease. The active components in Chinese medicinal herbs regulate the AMPK/mTOR signaling pathway to affect autophagy， alleviate oxidative stress， inflammation， and extracellular matrix aggregation， and promote the generation of autophagosomes， thus mitigating kidney injury. This paper mainly reviews the relationship between AMPK/mTOR signaling pathway， autophagy， and DKD and the mechanism of active components in Chinese medicinal herbs in mediating autophagy via the AMPK/mTOR pathway， aiming to provide a theoretical basis for the clinical prevention and treatment of DKD.

Chinese Medicine Regulates mTOR Signaling Pathway to Prevent and Treat Osteoporosis： A Review / 中国实验方剂学杂志

Osteoporosis （OP）， a common systemic skeletal disease in the elderly， is characterised by bone loss and bone microstructural degeneration. Its clinical manifestations include increased bone fragility and bone pain. Furthermore， OP increases the risk of fracture due to the high bone fragility， which leads to lifelong disability or death， imposing a heavy economic and psychological burden on the patients and their families. The pathogenesis of OP is extremely complex and associated with a variety of factors such as proliferation and differentiation of osteoblasts， impairment of osteoclast activity and function， and abnormalities in autophagy activation. Recent studies have found that mammalian target of rapamycin （mTOR） signaing pathway is involved in the regulation of bone homeostasis， which can promote bone formation and improve bone metabolism and bone microstructure by regulating osteoblast proliferation and differentiation and osteoclast function and activating cellular autophagy， thus playing a crucial role in the prevention and treatment of OP. The prevention and treatment of OP with Chinese medicine has a long history， clear efficacy， multiple targets of action， low adverse effects， and wide medicine sources. Therefore， this paper briefly describes the role of mTOR signaling pathway in the development of OP by reviewing the latest research reports and summarizes in detail the latest research results on the treatment of OP with Chinese medicine extracts and prescriptions via the mTOR signaling pathway. This review aims to provide a basis for the in-depth research on the relationship between mTOR signaling pathway and OP and the clinical application of traditional Chinese medicine in the prevention and treatment of OP.

Paliperidone-Induced Acute Hyperglycemia Is Caused by Adrenaline Secretion via the Activation of Hypothalamic AMP-Activated Protein Kinase.

Although paliperidone-related hyperglycemia has been extensively examined, the underlying mechanisms have not yet been elucidated. We investigated the effects of a single intravenous injection of paliperidone (0.2, 0.4, or 0.6 mg/kg) on serum concentrations of glucose and other endogenous metabolites in rats. We also examined the effects of a single intravenous injection of paliperidone (0.4 mg/kg) on AMP-activated protein kinase (AMPK) activity in the hypothalamus and liver. To clarify the relationship between AMPK activity and adrenaline secretion, the effects of berberine, which inhibits hypothalamic AMPK, on paliperidone-induced hyperglycemia were assessed. Significant increases were observed in serum glucose, adrenaline, and insulin concentrations following intravenous injections of paliperidone at doses of 0.4 and 0.6 mg/kg. A propranolol pretreatment attenuated paliperidone-induced increases in serum concentrations of glucose, but not adrenaline. Significant increases were also noted in phosphorylated AMPK concentrations in the hypothalamus following the administration of paliperidone at a dose of 0.4 mg/kg. A berberine pretreatment attenuated paliperidone-induced increases in blood concentrations of glucose, adrenaline, and insulin and phosphorylated AMPK concentrations in the hypothalamus. Collectively, the present results demonstrated that an acute treatment with paliperidone induced hyperglycemia, which was associated with the effects of hypothalamic AMPK activation on the secretion of adrenaline.

Effect of Bombyx mori on the Liver Protection of Non-Alcoholic Fatty Liver Disease Based on In Vitro and In Vivo Models.

Edible insects, Bombyx mori (silkworm; SW), which feed on mulberry leaves, have been consumed by humans for a long time as supplements or traditional medication. Non-alcoholic fatty liver disease (NAFLD) is a liver metabolic disorder that affects many people worldwide. We examined the hepatoprotective effects of SW using in vitro and high-fat and high-fructose (HFHF) diet-induced obese in vivo model mice by real-time PCR, immunoblot analysis, and fecal microbiota analysis. SW significantly reduced lipid accumulation and expression of the lipogenic genes in HepG2 cells and the livers of HFHF-induced mice. SW caused significant reductions in triglycerides, and total cholesterol in serum and upregulation of fatty acid oxidation markers compared to the HFHF group. Besides, SW significantly induced phosphorylation of AMPK and ACC in both models, suggesting roles in AMPK activation and the ACC signaling pathway. Furthermore, the gut microbiota analysis demonstrated that SW treatment reduced Firmicutes to Bacteroidetes ratios and the relative abundance of the Lachnospiraceae family compared to HFHF-induced obese mice. These results provide a novel therapeutic agent of hepatoprotective effects of SW for non-alcoholic hepatic steatosis that targets hepatic AMPK and ACC-mediated lipid metabolism.

The CRISPR-Cas9 crATIC HeLa transcriptome: Characterization of a novel cellular model of ATIC deficiency and ZMP accumulation.

In de novo purine biosynthesis (DNPS), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (EC 2.1.2.3)/inosine monophosphate cyclohydrolase (EC 3.5.4.10) (ATIC) catalyzes the last two reactions of the pathway: conversion of 5-aminoimidazole-4-carboxamide ribonucleotide [aka Z-nucleotide monophosphate (ZMP)] to 5-formamido-4-imidazolecarboxamide ribonucleotide (FAICAR) then to inosine monophosphate (IMP). Mutations in ATIC cause an untreatable and devastating inborn error of metabolism in humans. ZMP is an adenosine monophosphate (AMP) mimetic and a known activator of AMP-activated protein kinase (AMPK). Recently, a HeLa cell line null mutant for ATIC was constructed via CRISPR-Cas9 mutagenesis. This mutant, crATIC, accumulates ZMP during purine starvation. Given that the mutant can accumulate ZMP in the absence of treatment with exogenous compounds, crATIC is likely an important cellular model of DNPS inactivation and ZMP accumulation. In the current study, we characterize the crATIC transcriptome versus the HeLa transcriptome in purine-supplemented and purine-depleted growth conditions. We report and discuss transcriptome changes with particular relevance to Alzheimer's disease and in genes relevant to lipid and fatty acid synthesis, neurodevelopment, embryogenesis, cell cycle maintenance and progression, extracellular matrix, immune function, TGFß and other cellular processes.

Ginsenoside Rg3 protects heart against isoproterenol-induced myocardial infarction by activating AMPK mediated autophagy.

BACKGROUND: Panax ginseng is a well-known medicinal herb that is widely used in traditional Chinese medicine for treating various diseases. Ginsenoside Rg3 (Rg3) is thought to be one of the most important active ingredients of Panax ginseng. However, the molecular mechanism underlying the beneficial effects of Rg3 has been elusive. METHODS: In the mouse heart injury model induced by isoproterenol (ISO), we used brain natriuretic peptide (BNP), lactate dehydrogenase (LDH) and caspase-3 ELISA kits to test myocardium injury. To test whether Rg3 protects myocardial injury through AMPK mediated autophagy, we used specific AMPK inhibitor in combination with Rg3. NLRP3 inflammasome related molecules such as NLRP3, ASC and caspase-1 were measured by western-blot following Rg3 treatment. RESULTS: We found that Rg3 significantly reduced ISO induced myocardial injury indicated by the downregulation of serum BNP and LDH. In addition, we showed that the improvement of myocardial injury by Rg3 was associated with enhanced expression of autophagy related protein and activation of AMPK downstream signaling pathway. CONCLUSIONS: We observed that inhibition of AMPK significantly reversed the myocardial protective effect of Rg3, which is associated with a decrease of Rg3 induced autophagy. These together suggested that Rg3 may improve myocardial injury during MI through AMPK mediated autophagy. Our study also provides important translational evidence for using Rg3 in treating myocardial infarction (MI).

Lemon Balm and Its Constituent, Rosmarinic Acid, Alleviate Liver Damage in an Animal Model of Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) ranges in severity from hepatic steatosis to cirrhosis. Lemon balm and its major constituent, rosmarinic acid (RA), effectively improve the liver injury and obesity; however, their therapeutic effects on nonalcoholic steatohepatitis (NASH) are unknown. In this study, we investigated the effects of RA and a lemon balm extract (LBE) on NAFLD and liver fibrosis and elucidated their mechanisms. Palmitic acid (PA)-exposed HepG2 cells and db/db mice fed a methionine- and choline-deficient (MCD) diet were utilized to exhibit symptoms of human NASH. LBE and RA treatments alleviated the oxidative stress by increasing antioxidant enzymes and modulated lipid metabolism-related gene expression by the activation of adenosine monophosphate-activated protein kinase (AMPK) in vitro and in vivo. LBE and RA treatments inhibited the expression of genes involved in hepatic fibrosis and inflammation in vitro and in vivo. Together, LBE and RA could improve liver damage by non-alcoholic lipid accumulation and may be promising medications to treat NASH.

Dietary apple polyphenols promote fat browning in high-fat diet-induced obese mice through activation of adenosine monophosphate-activated protein kinase α.

BACKGROUND: Promoting brown and beige adipogenesis contributes to adaptive thermogenesis, which provides a defense against obesity and related disorders. Apple polyphenols (APs) play a significant role in treating variety of metabolic diseases. This study was conducted to determine the effects of APs on the development of brown and beige adipocytes and thermogenesis and investigate whether these effects are mediated by adenosine monophosphate-activated protein kinase (AMPK). High-fat diet (HFD)-induced obese mice and differentiated 3T3-L1 adipocytes were subjected to APs treatment. The thermogenic program and associated regulatory factors, and the involvement of AMPKα was assessed. RESULTS: Dietary APs supplementation reduced adiposity and improved insulin sensitivity in HFD-induced obese mice. Moreover, APs increased the oxygen consumption and heat production and decreased respiratory exchange ratio, which were accompanied by the upregulation of thermogenic genes expression and the activation of AMPKα in brown fat and inguinal white fat. Further, APs treatment directly increased expression of brown adipogenic markers and induced phosphorylation of AMPKα in differentiated 3T3-L1 adipocytes, whereas the beneficial effects of APs were reversed by AMPK inhibition. CONCLUSION: Our results provide new insights into the function of APs in regulating brown/beige adipogenesis and adaptive thermogenesis and suggest the potential application of APs in the prevention and therapeutics of obesity and associated metabolic diseases. © 2020 Society of Chemical Industry.

Protectin DX attenuates IL-1ß-induced inflammation via the AMPK/NF-κB pathway in chondrocytes and ameliorates osteoarthritis progression in a rat model.

Protectin DX (PDX) has been reported to have extensive anti-inflammatory effects. However, it is unknown whether PDX acts as an anti-inflammatory agent in the context of osteoarthritis (OA). This study aimed to evaluate the anti-inflammatory activity of PDX in vitro and in vivo in a model of OA. Primary rat chondrocytes were preincubated with PDX 1 h prior to IL-1ß treatment for 24 h. We found that PDX was nontoxic, and pretreatment with PDX increased cell viability in IL-1ß-induced chondrocytes. Preincubation with PDX also efficiently inhibited the degradation of type II collagen dose-dependently. Additionally, the expression of MMP-3, MMP-13, ADAMTS4, iNOS, COX-2, NO, and PGE2 decreased after IL-1ß stimulation when cells were preincubated with PDX. Moreover, PDX inhibited the increase in phosphorylated NF-κB p65 and IκBα upon IL-1ß stimulation, and the negative effects of IL-1ß on chondrocytes were partially blocked by treatment with pyrrolidine dithiocarbamate (PDTC), a selective NF-κB inhibitor. In addition, we found that PDX increased AMPK phosphorylation in IL-1ß-mediated chondrocytes. The phosphorylation of AMPK could be inhibited by compound C, a classic AMPK inhibitor. Compound C also remarkably reversed the decrease in p65 phosphorylation and MMP-13 expression caused by PDX. Furthermore, nuclear translocation of NF-κB was visible by immunofluorescence after PDX-induced AMPK activation. Additionally, we verified that PDX ameliorated cartilage degradation in monosodium iodoacetate (MIA)-induced OA rats through histological evaluation and ELISA of TNF-α in the serum and intra-articular lavage fluid. In conclusion, we have shown that PDX suppresses inflammation in chondrocytes in vitro and in vivo, likely through the AMPK/NF-κB signaling pathway. Our results suggest that PDX could be a useful novel therapeutic agent for OA treatment.

A Systematic Review on Anti-diabetic Properties of Chalcones.

The use of anti-diabetic drugs has been increasing worldwide and the evolution of therapeutics has been enormous. Still, the currently available anti-diabetic drugs do not present the desired efficacy and are generally associated with serious adverse effects. Thus, entirely new interventions, addressing the underlying etiopathogenesis of type 2 diabetes mellitus, are required. Chalcones, secondary metabolites of terrestrial plants and precursors of the flavonoids biosynthesis, have been used for a long time in traditional medicine due to their wide-range of biological activities, from which the anti-diabetic activity stands out. This review systematizes the information found in literature about the anti-diabetic properties of chalcones, in vitro and in vivo. Chalcones are able to exert these properties by acting in different therapeutic targets: Dipeptidyl Peptidase 4 (DPP-4); Glucose Transporter Type 4 (GLUT4), Sodium Glucose Cotransporter 2 (SGLT2), α-amylase, α-glucosidase, Aldose Reductase (ALR), Protein Tyrosine Phosphatase 1B (PTP1B), Peroxisome Proliferator-activated Receptor-gamma (PPARγ) and Adenosine Monophosphate (AMP)-activated Protein Kinase (AMPK). Chalcones are, undoubtedly, promising anti-diabetic agents, and some crucial structural features have already been established. From the Structure-Activity Relationships analysis, it can generally be stated that the presence of hydroxyl, prenyl and geranyl groups in their skeleton improves their activity for the evaluated anti-diabetic targets.

Explore Protective Mechanism of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma Extract on Cardiac Aging in Diabetic Mice Through AMPK/mTOR Pathway / 中国实验方剂学杂志

Objective::To explore the protective mechanism of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma (GNC) extracts on cardiac aging in diabetic mice by observing the activation of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, changes of cardiac pathomorphological and related senescent proteins. Method::C57BL/6 male mice, SPF level, were randomly divided into normal control group and high-glucose group. The mice in high-glucose group were intraperitoneally injected with streptozotocin (STZ) and fed with high-fat diet. After successful modeling, they were randomly divided into model group, low-dose GNC group (0.819 g·kg-1), high-dose GNC group (1.638 g·kg-1) and metformin group (150 mg·kg-1). The drug was administered by gavage once a day for a continuous period of 9 weeks. 4-week-old male C57BL/6 mice were normally fed for 1 week as a youth group. General conditions of mice were observed. Hematoxylin-eosin (HE) staining combined with transmission electron microscope (TEM) was used to observe the cardiac pathomorphology in mice. Von Kossa staining was used to determine the degree of calcium salt deposition in cardiac micro vessels. Western blot was used to detect the activation of signaling pathways in myocardial tissue of mice, as well as the expression levels of matrix metalloproteinases-2 (MMP-2), tumor suppressor p53 (p53), and phospho-tumor suppressor p53 (p-p53). Result::As compared with the normal group, the blood glucose in the model group increased (P<0.01), as compared with the model group, the blood glucose in each administration group decreased significantly (P<0.05, P<0.01). The results of three pathological morphology experiments (HE, TEM, and Von Kossa) showed that as compared with the normal control group, the mice in model group showed cardiomyocytes hypertrophy, disordered arrangement of myocardial fibers, focal dissolving and necrosis, mitochondria swelling, degeneration, crest fracture, vacuolar alteration, disordered microvascular structure of the heart, uneven staining, and a large amount of calcium deposition in tunica media and intima. As compared with the model group, the pathomorphological changes of mice in each administration group were improved in varying degrees. Compared with the normal group, the expression levels of MMP-2, p53 and p-p53 protein in the model group were significantly increased (P<0.05, P<0.01), the protein ratios of p-liver kinase B2(LKB1)/LKB1, p-AMPK/AMPK were significantly decreased (P<0.05, P<0.01), and the average gray level of p-mTOR/mTOR and p-p70S6 kinase(p70S6k)/p70S6k protein was significantly increased (P<0.05, P<0.01), while the protein ratios of p-mTOR/mTOR, p-p70S6k/p70S6k were increased (P<0.01). As compared with the model group, the expression levels of MMP-2, p53 and p-p53 protein in each administration group were significantly decreased (P<0.05, P<0.01), the protein ratios of p-LKB1/ LKB1, p-AMPK/AMPK were significantly increased (P<0.05, P<0.01), while the protein ratios of p-mTOR/mTOR and p-p70S6k/p70S6k were decreased (P<0.05, P<0.01). Conclusion::STZ combined with high-fat diet can induce cardiac aging in mice, and GNC can improve cardiac aging in diabetic mice, which may be related to the inhibition of AMPK/mTOR pathway related protein expression.

A Modified Formulation of Sutaehwan Ameliorates Menopausal Anxiety, Depression and Heart Hypertrophy in the VCD-Induced Menopausal Mouse Model.

Sutaehwan (STH) has been used in Korean medicine for the treatment of abortus habitualis such as fetal restlessness in the uterus. Previously, we reported that a modified formulation of STH, Sutaehwan-Gami, has phytoestrogen-like properties in an ovariectomized menopausal rat model. However, the therapeutic effects of STH and the precise mechanisms by which STH affects various menopausal symptoms remain poorly understood. The current study was designed to explore the effects of a modified form of STH on menopausal anxiety, depression and heart hypertrophy and its mechanisms in 4-vinylcyclohexene diepoxide (VCD)-induced menopausal mouse models. VCD-induced menopausal model mice were fed a modified form of STH, which contained water extract of 3 herbs (called STH_KP17001) at a dose of 100 or 300 mg/kg/d or as a positive control, estradiol at a dose of 0.2 mg/kg/d with standard mouse pellets for 13 weeks. The results show that STH_KP17001 significantly restored the VCD-induced weight reduction of uterine and ovary through the phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) in the uterus and ovary. Moreover, STH_KP17001 showed slight proliferative effects and estrogen receptor α phosphorylation in MCF-7 cells. Treatment with STH_KP17001 reversed VCD-induced anxiety and depression through AMP-activated protein kinase (AMPK) activation and brain-derived neurotrophic factor (BDNF) expression in the cerebral cortex, while improving heart hypertrophy through inactivation of inhibitor of kappaB α (IκBα) in the heart. The results indicate that STH_KP17001 improves menopause-induced anxiety, depression and heart hypertrophy, implying its protective role for the management of menopausal symptoms.

[Electroacupuncture at "Zusanli"(ST36) promotes gastrointestinal motility possibly by suppres-sing excessive autophagy via AMPK/ULK1 signaling in rats with functional dyspepsia].

OBJECTIVE: To explore the effect of electroacupuncture (EA) at "Zusanli" (ST36) on gastrointestinal motility and expression of autophagy marker LC3 and autophagy signaling pathway molecule AMP-activated protein kinase (AMPK) in rats with functional dyspepsia (FD), so as to explore its mechanisms underlying improvement of FD. METHODS: A total of 40 male SD rats were randomly divided into blank control, model, EA, AMPK inhibitor and EA＋AMPK inhibitor groups, with 8 rats in each group. The FD model was established by tail-clip (30 min/time, twice daily) ＋ single day feeding, and gavage of normal saline (2 mL/time, twice a day) for 2 successive weeks. For rats of EA and EA＋AMPK inhibitor groups, EA (4 Hz, 1.0 mA) was applied to bilateral ST36 for 20 min, once daily for 7 successive days. For rats of the AMPK-inhibitor and EA＋AMPK inhibitor groups, Compound C (20 mg/kg) solution was administered by intraperitoneal injection before every EA administration. The gastric residual rate and small intestinal transit rate were calculated based on the weight of stomach and length of ink propelling and total small intestine, respectively. The expression levels of c-kit, microtubule-associated protein 1 light chain 3, Beclin 1, phosphorylated (p)-AMPK and p-unc-51 like autophagy activating kinase 1(ULK1) in the gastric antrum tissue were detected by using Western blot. RESULTS: Compared with the blank control group, the gastric residual rate and the expression levels of LC3-Ⅱ/LC3Ⅰ， Beclin 1, p-AMPK and p-ULK1 proteins were significantly increased, and the small intestinal transit rate and the expression of c-kit protein obviously decreased in the model group (P<0.01). After EA intervention, modeling-induced increase of gastric residual rate and the expression of LC3-Ⅱ/LC3Ⅰ， Beclin 1, p-AMPK and p-ULK1 proteins, and decrease of small intestinal transit rate and expression of c-kit protein were reversed in the EA, AMPK inhibitor and EA＋AMPK inhibitor groups (P<0.05, P<0.01). The therapeutic effect of EA and EA＋AMPK was significantly superior to that of AMPK inhibitor in down-regulating the expression of LC3Ⅱ/LC3Ⅰ， Beclin 1, p-AMPK and p-ULK1 proteins and in up-regulating the expression of c-kit protein (P<0.05, P<0.01). No significant differences were found among the EA, AMPK inhibitor and EA＋AMPK inhibitor groups in lowering gastric residual rate and elevating the small intestinal transit rate (P>0.05). CONCLUSION: EA at ST36 can promote gastrointestinal motility in FD rats, which is possibly mediated by inhibiting excessive autophagy of interstitial cells of Cajal via down-regulating AMPK/ULK1 signaling.

Ginger prevents obesity through regulation of energy metabolism and activation of browning in high-fat diet-induced obese mice.

Numerous natural herbs have been proven as safe anti-obesity resources. Ginger, one of the most widely consumed spices, has shown beneficial effects against obesity and related metabolic disorders. The present study aimed to examine whether the antiobesity effect of ginger is associated with energy metabolism. Mice were maintained on either a normal control diet or a high-fat diet (HFD) with or without 500 mg/kg (w/w) ginger supplementation. After 16 weeks, ginger supplementation alleviated the HFD-induced increases in body weight, fat accumulation, and levels of serum glucose, triglyceride and cholesterol. Indirect calorimetry showed that ginger administration significantly increased the respiratory exchange ratio (RER) and heat production in both diet models. Furthermore, ginger administration corrected the HFD-induced changes in concentrations of intermediates in glycolysis and the TCA cycle. Moreover, ginger enhanced brown adipose tissue function and activated white adipose tissue browning by altering the gene expression and protein levels of some brown and beige adipocyte-selective markers. Additionally, stimulation of the browning program by ginger may be partly regulated by the sirtuin-1 (SIRT1)/AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway. Taken together, these results indicate that dietary ginger prevents body weight gain by remodeling whole-body energy metabolism and inducing browning of white adipose tissue (WAT). Thus, ginger is an edible plant that plays a role in the therapeutic treatment of obesity and related disorders.

Effect of Gandou Decoction on Autophagy of SH-SY5Y Cell Induced by High Copper and Its Mechanism / 中国实验方剂学杂志

Objective: To explore the effect of Gandou decoction on autophagy of SH-SY5Y cells induced by high copper and its mechanism, in order to provide new therapeutic targets and research ideas for the prevention and treatment of brain-type Wilson disease (WD) with traditional Chinese medicine. Method: CuSO4 model showed a certain dose-effect and time-effect relationship according to methyl thiazolyl tetrazolium(MTT); lactate dehydrogenase(LDH) leakage rate was detected by LDH release assay; flow cytometry method was used to detect intracellular reactive oxygen species (ROS) content. The fluorescent dye JC-1 was used to detect the mitochondrial membrane potential of the cells. Flow cytometry was used to quantify autophagy. The expressions of liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK), microtubule-associated protein 1 light chain 3 (LC3A/B), mammalian target of rapamycin (mTOR) and UNC-51-like kinase-1 (ULK1), phosphorylation-ULK (p-ULK), phosphorylation-AMPK (p-AMPK) were detected by Western blot. Result: According to MTT results, CuSO4 showed a dose-effect and time-effect relationship with cells (P4, the survival rate of cells showed a downward trend (P4-induced cell death (P4 compared with the normal group (P4-injured cells (P4 significantly increased the production of ROS in cells (P4-induced intracellular ROS production (P4 induced a significant decrease in mitochondrial membrane potential in cells (P4-induced mitochondrial membrane potential in a dose-dependent manner (P1, AMPK, LC3A/B, ULK, p-AMPK in the model group were significantly increased, while the protein expressions of mTOR and p-ULK were significantly decreased (P1, AMPK, LC3A/B, p-AMPK and ULK were significantly decreased, whereas the protein expressions of mTOR and p-ULK were significantly increased in the rabbit serum group containing Gandou decoction (PConclusion: High copper can induce autophagic apoptosis in SH-SY5Y cells by inducing intracellular mitochondrial oxidative stress, up-regulating the expressions of autophagy-related proteins LKB1, AMPK, LC3A/B, ULK, p-AMPK and down-regulating the expressions of mTOR and p-ULK. However, Gandou decoction can inhibit the occurrence of autophagy, and cut off high copper-induced neuronal damage by down-regulating the expressions of autophagy-related proteins LKB1, AMPK, LC3A/B, ULK, p-AMPK, and up-regulating the expression of mTOR and p-ULK, so as to exert a neuroprotective effect.