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.

Mechanism of Salvianolate in Inducing Autophagy in Podocytes of Rats with Membranous Nephropathy via AMPK/SIRT1/PGC-1α Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of salvianolate on the protein expressions of adenosine monophosphate （AMP）-activated protein kinase （AMPK）， silent information regulator 1 （SIRT1） and peroxisome proliferator-activated receptor-γ coactivator-1α （PGC-1α）， autophagy and apoptosis in kidney tissue of rats with membranous nephropathy （MN）， and to explore its possible molecular mechanism against MN. MethodEighty male SD rats were randomly divided into normal group， model group， benazepril hydrochloride group （10 mg·kg-1）， and salvianolate low-， medium-， and high-dose groups （16.7， 33.3 and 66.7 mg·kg-1）. The rats were modeled by injection of cationized bovine serum albumin （C-BSA） into the tail vein. After successful modeling， rats in the administration groups were given corresponding doses of drugs for 4 consecutive weeks， and then 24-hour urine， serum and kidney tissue were collected for the detection of 24-hour urinary protein （UTP）， blood urea nitrogen （BUN）， serum creatinine （SCr）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， C reactive protein （CRP）， glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， and malondialdehyde （MDA）. The pathological changes of kidneys were observed by light microscope， electron microscope and immunofluorescence. Western blot was used to detect the protein expressions of phospho-AMPK （p-AMPK）， AMPK， phospho-SIRT1 （p-SIRT1）， SIRT1 and PGC-1α in rat kidney tissue. The protein expressions of autophagy-specific gene （Beclin-1）， microtubule-associated protein 1 light chain 3 （LC3） Ⅱ， ubiquitin-binding protein （p62）， B cell lymphoma （Bcl-2）， Bcl-2-associated X （Bax）， and cysteine aspartic protease-7 （Caspase-7） in rat kidney tissue were determined by immunohistochemistry （IHC）. ResultCompared with the conditions in the normal group， the levels of UTP， IL-6， TNF-α， CRP and MDA in the model group were increased （P<0.05） while the levels of SOD and GSH-Px were decreased （P<0.05）， and there was no difference in BUN and SCr. Compared with the model group， the administration groups had lowered UTP， IL-6， TNF-α， CRP and MDA （P<0.05） while elevated SOD and GSH-Px （P<0.05）. It could be seen from hematoxylin and eosin （HE） staining， Masson staining， immunofluorescence and electron microscopy that the pathological damage of rat kidney tissue in the model group was significant， but after treatment with benazepril hydrochloride and salvianolate， the pathological damage of kidney cells was gradually improved. The expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1α， Bcl-2， Beclin-1 and LC3Ⅱ in rat kidney in the model group were lower than those in the normal group （P<0.05） while the expressions of Bax， Caspase-7 and p62 were higher （P<0.05）. Compared with the model group， benazepril hydrochloride group and salvianolate groups had an up-regulation in the expressions of p-AMPK/AMPK， p-SIRT1/SIRT1， PGC-1α， Bcl-2， Beclin-1 and LC3Ⅱ in the kidney （P<0.05） while a down-regulation in the expressions of Bax， Caspase-7 and p62 （P<0.05）. ConclusionThe protective effect of salvianolate on the kidneys of MN rats may be related to the activation of AMPK/SIRT1/PGC-1α signaling pathway， the up-regulation of autophagy and the reduction of apoptosis.

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.

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.

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.

Effect of Qingzao Jiufeitang on Protein Expression Related to Autophagy Initiation in Lung Cancer Cells Through AMPK inhibition / 中国实验方剂学杂志

ObjectiveTo observe the effects of Qingzao Jiufeitang on the expression of adenosine 5′-monophosphate （AMP）-activated protein kinase （AMPK）， mammalian target of rapamycin （mTOR）， and UNC-51-like kinase 1 （ULK1） in lung cancer cells after the application of AMPK inhibitor （compound C）. MethodMale C57BL/6J mice were randomly divided into a model group， a cyclophosphamide （CTX） group （50 mg·kg-1）， a Qingzao Jiufeitang group （11 g·kg-1）， an AMPK inhibitor group （10 mg·kg-1）， and a Qingzao Jiufeitang combined with AMPK inhibitor group （combination group） （11 g·kg-1+10 mg·kg-1）. Lewis lung cancer cells were subcutaneously injected into the right axilla to induce a tumor-bearing model. 24 hours after modeling， the mice in the CTX group were intraperitoneally injected once every other day for seven times in total. The mice in the AMPK inhibitor group and the combination group received intraperitoneal injection of compound C， once a day for 14 days. The mice in the Qingzao Jiufeitang group and the combination group were administered orally at the set dose for 14 days before and after modeling. At the end of the experiment， the mice in each group were sacrificed. The tumor-bearing tissues were collected， and the tumor weight of each group was counted. Transmission electron microscopy （TEM） was used to observe the formation of autolysosomes in lung cancer tissues of each group. Western blot was used to detect the protein expression of AMPK， phosphorylated AMPK （p-AMPK）， mTOR， phosphorylated mTOR （p-mTOR）， ULK1， phosphorylated ULK1 （p-ULK1）， microtubule-associated protein 1 light chain 3B （LC3B）， and p62. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of lung cancer in each group. ResultCompared with the model group， the Qingzao Jiufeitang group showed decreased tumor weight （P<0.01）， the formation of autolysosomes under the electron microscope， increased protein expression of p-AMPK， p-ULK1， LC3B， LC3B-Ⅱ， and p-AMPK/AMPK， p-ULK1/ULK1， and LC3B-Ⅱ/LC3B-Ⅰratios （P<0.01， P<0.05）， and reduced protein expression of p-mTOR， p62， and p-mTOR/mTOR ratio （P<0.05）. Compared with the Qingzao Jiufeitang group， the combination group showed no autolysosomes formation under the electron microscope， decreased protein expression of p-AMPK， p-ULK1， LC3B， LC3B-Ⅱ， and p-AMPK/AMPK， p-ULK1/ULK1， LC3B-Ⅱ/LC3B-Ⅰ ratios （P<0.05， P<0.01）， and increased p62 protein expression （P<0.05）. HE staining results showed that the pathological changes of lung cancer tissues in the groups with drug intervention were improved compared with those in the model group. ConclusionQingzao Jiufeitang can promote the elevation of LC3B-Ⅱ and decrease the expression of p62 protein， thus inducing autophagy. The mechanism of autophagy initiation may be achieved by the AMPK/ULK1 pathway instead of the mediation by the AMPK/mTOR/ULK1 pathway.

Effects of Incremental Load Training on Phosphorylation of AMPK in Skeletal Muscle Satellite Cells of Aged Mice / 医用生物力学

Objective To investigate the effect of incremental load training on AMP-activated protein kinase (AMPK) phosphorylation in skeletal muscle satellite cells of aged mice. Methods Experimental mice were divided into 3 groups: young control group (YC group, n=12), old control group (OC group, n=12) and old training group (OT group, n=12). The mice in OT group received incremental load training, and CD45-/CD31-/Sca1-/VCAM (CD106) + cells were isolated by flow cytometry sorting. Desmin, Myod myogenic staining and myogenic differentiation culture were used for identification of muscle satellite cells, and the p-AMPK level of muscle satellite cells was detected by immunohistochemistry combined with Western blotting method. Results The expression levels of AMPK and p-AMPK in skeletal muscle satellite cells in YC group were significantly higher than those in OC group (P0.05), while p-AMPK expression level in OT group was significant higher than that in OC group (P<0.05). Conclusions Incremental load training can promote AMPK phosphorylation of skeletal muscle satellite cells in aged mice, and improve energy metabolism of skeletal muscle in aged mice.

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.

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

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 (P0.05). 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.

Effect and mechanism of metformin on TGF-β1expression in pancreatic cancer / 西安交通大学学报(医学版)

Objective: To investigate the effect of metformin on TGF-β1 expression in pancreatic cancer and further analyze the underlying mechanism. Methods: After metformin intervention, cell proliferation and invasion ability were detected by MTT assay and Transwell method, respectively. The expression of TGF-β1 in pancreatic cancer cell lines Panc-1 and BxPC-3 was detected by qRT-PCR, Western blot and immunofluorescence. The expression of AMPK/p-AMPK was detected by Western blot; ELISA method was used to measure the content of TGF-β1 in cell supernatant. After silencing AMPK expression in pancreatic cancer cell lines, the cells were treated with metformin and the expression of TGF-β1 was detected by qRT-PCR and Western blot. Results: The phosphorylation level of AMPK in Panc-1 (P<0.001) and BxPC-3 (P<0.001) cells was significantly decreased after metformin intervention. In addition, the expression of TGF-β1 in tumor cells was significantly decreased. However, the reduced expression of TGF-β1 by metformin was reversed by AMPK silencing in cancer cells (P<0.05). Conclusion: Metformin can inhibit TGF-β1 expression in pancreatic cancer cells by inducing AMPK phosphorylation.

Extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma in Delaying High Glucose-induced Vascular Senescence in Mice / 中国实验方剂学杂志

Objective: To investigate the protective effect of extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on vascular senescence induced by high glucose in mice from adenosine 5'-monophosphate (AMP)-activated protein kinase/mechanistic target of rapamycin (AMPK/mTOR) pathway. Method: A total of 130 male C57BL/6 mice were randomly divided into control group and high glucose group. The high glucose group was intraperitoneally injected with streptozocin(STZ) and fed with a high-fat diet continuously for seven months. Mice were divided into 4 groups:model group, low-dose extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma(0.819 g·kg-1) group, high-dose extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma group (1.638 g·kg-1) and metformin group (150 mg·kg-1), and intragastrically administered once a day for nine weeks. The changes in body weight and blood glucose were measured. At the end of the administration, htoxylin eosin(HE) was performed for the detection of aortic morphology, and the expressions of cyclin-dependent kinase inhibitor 2A (p16), cyclin-dependent kinase inhibitor 1A (p21), AMPK, p-AMPK, mTOR, p-mTOR, liver kinase B1 (LKB1), p-LKB1, Ribosomal protein s6 kinase (p70s6k) and p-p70s6k proteins in mouse aorta were detected by Western blot. Result: Compared with blank group, mice in model group had lower body weight and higher blood glucose (PPPPPPPPPPPConclusion: High glucose can induce vascular senescence, and extracts from Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma can improve vascular aging induced by high glucose through AMPK/mTOR pathway.

Effect of Buyang Huanwu Tang on Myocardial Mitochondrial Energy Metabolism and AMPK/PPARα Signaling Pathway in Rats with Diastolic Heart Failure / 中国实验方剂学杂志

Objective: To study the effect of Buyang Huanwu Tang on myocardial energy metabolism in rats with diastolic heart failure (DHF) based on adenosine monophosphate (AMP)-activated protein kinase (AMPK)/peroxisome proliferators-activated receptors α (PPARα) signaling pathway,and investigate its mechanism of action.Method: The 48 SD rats were randomly divided into sham operation group and model group.DHF rat model was established by abdominal aorta constriction method.The successfully modeled rats were randomly divided into model group,Buyang Huanwu Tang group (12.72 g·kg-1·d-1),metoprolol tartrate group (0.004 5 g·kg-1·d-1),with corresponding drugs in each group by intragastric administration.The sham operation group and model group were given with equal amount of deionized water,once a day.After 8 weeks of continuous drug intervention,the contents of adenosine monophosphate (AMP),adenosine diphosphate (ADP) and adenosine triphophate (ATP) in peripheral blood of rats were determined by enzyme linked immunosorbent assay (ELISA).The changes of myocardial mitochondrial ultrastructure were detected by electron microscope.The protein expression levels of AMPK,PPARα and peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α) in rat myocardium were detected by Western blot.Result: As compared with sham operation group,the contents of AMP and ADP in model group were increased significantly,and ATP content was decreased significantly (PPPPα and PGC-1α protein in the model group were decreased significantly (Pα and PGC-1α protein in Buyang Huanwu Tang group and metoprolol tartrate group were increased significantly (PConclusion: Buyang Huanwu Tang may improve the energy metabolism of the failed heart and delay the progression of heart failure by improving the structure and function of mitochondria,activating AMPK and up-regulating the expression of AMPK/PPARα signaling pathway.

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.

Cordycepin promotes browning of white adipose tissue through an AMP-activated protein kinase (AMPK)-dependent pathway

Obesity is a worldwide epidemic. Promoting browning of white adipose tissue (WAT) contributes to increased energy expenditure and hence counteracts obesity. Here we show that cordycepin (Cpn), a natural derivative of adenosine, increases energy expenditure, inhibits weight gain, improves metabolic profile and glucose tolerance, decreases WAT mass and adipocyte size, and enhances cold tolerance in normal and high-fat diet-fed mice. Cpn markedly increases the surface temperature around the inguinal WAT and turns the inguinal fat browner. Further investigations show that Cpn induces the development of brown-like adipocytes in inguinal and, to a less degree, epididymal WAT depots. Cpn also increases the expression of uncoupling protein 1 (UCP1) and other thermogenic genes in WAT and 3T3-L1 differentiated adipocytes, in which AMP-activated protein kinase (AMPK) plays an important role. Our results provide novel insights into the function of Cpn in regulating energy balance, and suggest a potential utility of Cpn in the treatment of obesity.

Effect of gentiopicroside on AMPKα signaling pathway of rats with non-alcoholic fatty liver disease / 中草药

Objective To investigate the therapeutic effect of gentiopicroside has a on rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat and high-sucrose diet, and explore its mechanism. Methods After 10 d adaptive feeding, 60 male SD rats were randomly divided into the normal group, model group, gentiopicroside low, medium, high dose treatment groups, and positive drug polyeno phosphoryl choline (PPC) intervention group. Except for the normal group, the rats in other groups were received a high fat and glucose diet for 12 weeks to establish NAFLD model; After model successfully established, gentiopicroside low, medium, and high dosetreatment groups were given 50, 100, and 200 mg/(kg•d) gentiopicroside, PPC group was ig given 23 mg/(kg•d) PPC, and 500 μL/(kg•d) saline was given to the normal and model groups. After treated for eight weeks, the rats were sacrificed, and the serum was collected from rats to detect the liver function, blood lipid, serum oxidation, antioxidant capacity, and inflammatory factors. HE staining was used to observe pathological changes of liver. In addition, western blotting and qRT-PCR were used to detect the expression of AMPKα and p-AMPKα. Results HE staining showed that the size of liver cells in the normal group was uniform and the nuclei were evenly distributed, there were obvious vacuoles and a certain inflammatory reaction in the model group. Compared with the model group, gentiopicroside treatment group and PPC group (especially the gentiopicroside middle and high dose group) had a significant improvement, but there were still some differences compared with the normal group; Compared with the normal group, the AST, ALT, HDL-C, LDL-C, MDA, IL-1, and IL-6 in the model group were significantly increased (P 0.05); The results of Western blotting and qRT-PCR showed that compared with the normal group, the expression of p-AMPKα protein and AMPKα mRNA in the model group was significantly decreased (P<0.05). After gentiopicroside and PPC administration, they were significantly increased (P<0.05), and gentiopicroside groups showed a significant dose-dependent manner, and the middle dose and high dose of gentiopicroside groups were better than the PPC group (P<0.05), while the expression of AMPKα protein has no significant difference in each group (P<0.05). Conclusion The NAFLD rats showed a obvious hepatic fat infiltration and dyslipidemia, the liver function index and inflammatory factors levels were elevated, and the anti-oxidant capacity was decreased. Gentiopicroside significantly improved above symptoms, which may be associated with the increased expression of p-AMPKα in liver tissue of NAFLD rats by gentiopicroside.

Diarylbutane-type Lignans from Myristica fragrans (Nutmeg) show the Cytotoxicity against Breast Cancer Cells through Activation of AMP-activated Protein Kinase

In our program to search for new AMP-activated protein kinase (AMPK) activators from plants that exert potential anticancer property, we found that an EtOAc extract of Myristica fragrans (nutmeg) activated AMPK enzyme in human breast cancer MCF-7 cells. Two major diarylbutane-type lignans, macelignan and meso-dihydroguaiaretic acid (MDGA), were isolated as active principles from this extract. Treatment of breast cancer cells with two compounds induced cellular apoptosis, evidenced by cleavage of poly-(ADP-ribose) polymerase (PARP) and Ser 15 phosphorylation of p53. Moreover, macelignan and MDGA significantly inhibited the colony formation of MCF-7 breast cancer cells on soft agar. Intraperitoneal injection of macelignan and MDGA (20 mg/kg) suppressed the tumor growth of 4T1 mammary cancer cells. These results indicate that the chemopreventive effects of two major diarylbutane-type lignans from Myristica fragrans (nutmeg) may be associated with induction of apoptosis presumably through AMPK activation.

Anti-aging traditional Chinese medicine： potential mechanisms involving AMPK pathway and calorie restriction based on "medicine-food homology" theory / 中国中药杂志

There have been many reports about the anti-aging effect of traditional Chinese medicine (TCM), but the material basis and mechanism of action have not been clearly elucidated. AMP-activated protein kinase (AMPK) is the receptor of energy metabolism and its life extending effect has been confirmed in different experiments. Over expression or activation with metform in of AAK-2/AMPK has been shown to extend life expectancy in nematodes and Drosophila. The possible downstream pathways of AMPK against aging include TOR/S6k pathway, FOXOs pathway and CRTC pathway. One of the core concepts of traditional Chinese medicine is disease prevention, for which one of manifestations is to improve the body with the same source of medicine and food to achieve longevity. It is possible to activate AMPK to achieve the goal of health preservation and prolonging the life by some of the "medicine-food harmony" treatments. Our survey finds that in "medicine-food harmony" compound TCM, "invigorating the kidney deficiency and promoting blood circulation" class dominates and Salviae Miltiorrhizae Radix Rhizoma, Astragali Radix, Coptidis Rhizoma, Poria, Atractylodes Macrocephalae Rhizoma, Radix et Rhizoma Rhei, and Ginseng Radix et Rhizoma are used in high frequency. Network pharmacology analysis using ingenuity pathway analysis (IPA) software revealed that TCM-derived drugs interacting with AMPK target proteins included berberine, emodin, curcumin, resveratrol, alcohol, cordyceps, arctiin, suggesting in a certain extent the feasibility of "medicine -food homology" drugs to extend the lifespan through the AMPK pathway. Our study combines a comprehensive database query and an IPA network pharmacology analysis to identify Chinese medicine monomer and components that may activate AMPK pathway to delay aging and to discuss the potential of these medicine by improving energy metabolism to delay the aging process, based on the concept of traditional Chinese medicine "medicine-food homology".

Cellular and molecular mechanisms of metformin for clinical application / 中国糖尿病杂志

[Summary] Considerable efforts have been made to understand the cellular and molecular mechanisms of metformin ,a potent antihyperglycemic agent now recommended as the first‐line treatment of patients with T2DM. The main effect of this drug is to reducethe hepatic glucose output ,primarily through inhibition of the mitochondrial respiratory chain complex Ⅰ and then activating AM P‐activated protein kinase (AMPK) ,which provide a generally acceptable mechanism for the action of metformin on hepatic gluconeogenesis. Beyond its effect on glucose metabolism ,metformin has been reported to improve ovarian function of patients with polycystic ovary syndrome (PCOS) and to reduce the risk of microvascular and macrovascular complications in patients with T2DM. In addition ,metfomin has also recently been suggested as an adjuvant treatment of cancer. Here we reviewed the progress of mechanism research and clinical application of metformin.

Losartan Inhibits Vascular Smooth Muscle Cell Proliferation through Activation of AMP-Activated Protein Kinase

Losartan is a selective angiotensin II (Ang II) type 1 (AT1) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the G0/G1 cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.

Overexpression of AMPKalpha1 Ameliorates Fatty Liver in Hyperlipidemic Diabetic Rats

5'-AMP-activated protein kinase (AMPK) is a heterotrimeric complex consisting of a catalytic (alpha) and two regulatory (beta and gamma) subunits. Two isoforms are known for catalytic subunit (alpha1, alpha2) and are encoded by different genes. To assess the metabolic effects of AMPKalpha1, we examined the effects of overexpression of adenoviral-mediated AMPKalpha1 in hyperlipidemic type 2 diabetic rats. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an established animal model of type 2 diabetes that exhibits chronic and slowly progressive hyperglycemia and hyperlipidemia. Thirty five-week-old overt type 2 diabetic rats (n=10) were administered intravenously with Ad.AMPKalpha1. AMPK activity was measured by phosphorylation of acetyl CoA carboxlyase (ACC). To investigate the changes of gene expression related glucose and lipid metabolism, quantitative real-time PCR was performed with liver tissues. Overexpression of AMPKalpha1 showed that blood glucose concentration was decreased but that glucose tolerance was not completely recovered on 7th day after treatment. Plasma triglyceride concentration was decreased slightly, and hepatic triglyceride content was markedly reduced by decreasing expression of hepatic lipogenic genes. Overexpression of AMPKalpha1 markedly improved hepatic steatosis and it may have effective role for improving hepatic lipid metabolism in hyperlipidemic state.