Effect of Linggui Zhugantang on Mitochondrial Fusion-fission and Sirt3/ AMPK Signaling Pathway in Chronic Heart Failure Rats After Myocardial Infarction / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Linggui Zhugantang on mitochondrial fission and fusion and silencing information regulator 3（Sirt3）/adenosine monophosphate dependent protein kinase （AMPK） signaling pathway in chronic heart failure （CHF） rats after myocardial infarction （MI）. MethodSD rats randomly divide into sham operation group （normal saline ，thread only without ligature）， model group （normal saline， ligation of the left anterior descending coronary artery proximal to the heart）， Linggui Zhugantang group （4.8 g·kg-1） and Captopril group （0.002 57 g·kg-1）， with 10 rats in each group. Administere drug continuously for 28 days. Echocardiography detected cardiac function parameters. Hematoxylin eosin （HE） staining observed the pathological changes of the heart. Immunofluorescence detected the levels of reactive oxygen species （ROS）. JC-1 detect mitochondrial membrane potential. Colorimetry measure adenosine triphosphate （ATP）， superoxide dismutase （SOD）， malondialdehyde （MDA）， mitochondrial respiratory chain complex activity （Ⅰ-Ⅳ）. TdT-mediated dUTP nick end labeling （TUNEL） staining detected the apoptosis rate of myocardial tissue. Western blot detected protein expression levels of Sirt3， phosphorylated AMPK （p-AMPK）， phosphorylated dynamic-related protein 1（p-Drp1）， mitochondrial fission protein 1（Fis1）， mitochondrial fission factor （MFF）， optic atrophy protein 1（OPA1）. ResultCompared to the sham group， the left ventricular end diastolic diameter （LVIDd） and left ventricular end systolic diameter （LVIDs） were significantly increased in model group （P<0.01）， while the left ventricular short axis shortening rate （LVFS） and left ventricular ejection fraction （LVEF） were significantly decreased （P<0.01）. There were inflammatory cell infiltration and obvious pathological injury in myocardial tissue. ROS， MDA levels and myocardial cell apoptosis rate were significantly increased （P<0.01）， SOD level， ATP content， and membrane potential were significantly decreased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） was significantly decreased （P<0.01）. Levels of p-Drp1， Fis1， MFF proteins were significantly up-regulated （P<0.01）， while Sirt3， p-AMPK， OPA1 proteins level were significantly down-regulated （P<0.01）. Compared with model group， LVIDd and LVIDs were significantly decreased （P<0.01）， LVEF and LVFS were significantly increased （P<0.01）. Inflammatory cell infiltration and pathological damage of myocardial tissue were significantly relieved. ROS， MDA levels and myocardial cell apoptosis rate were significantly decreased in Linggui Zhugantang group and Captopril group （P<0.01）， SOD level， ATP content， and membrane potential significantly increased （P<0.01）. The activity of mitochondrial respiratory chain complexes （Ⅰ-Ⅳ） increased significantly （P<0.01），and p-Drp1， Fis1， MFF protein levels were significantly down-regulated （P<0.01）， Sirt3， p-AMPK， OPA1 protein were significantly up-regulated （P<0.01）. ConclusionLinggui Zhugantang can alleviate oxidative stress and apoptosis damage of myocardial cells， maintain mitochondrial function stability， and its effect may be related to mitochondrial mitosis fusion and Sirt3/AMPK signaling pathway.

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.

Huazhi Rougan Granules attenuates steatosis in cell model of nonalcoholic fatty liver disease by inducing autophagy / 中国中药杂志

To investigate the effect of Huazhi Rougan Granules(HZRG) on autophagy in a steatotic hepatocyte model of free fatty acid(FFA)-induced nonalcoholic fatty liver disease(NAFLD) and explore the possible mechanism. FFA solution prepared by mixing palmitic acid(PA) and oleic acid(OA) at the ratio of 1∶2 was used to induce hepatic steatosis in L02 cells after 24 h treatment, and an in vitro NAFLD cell model was established. After termination of incubation, cell counting kit-8(CCK-8) assay was performed to detect the cell viability; Oil red O staining was employed to detect the intracellular lipid accumulation; enzyme-linked immunosorbnent assay(ELISA) was performed to measure the level of triglyceride(TG); to monitor autophagy in L02 cells, transmission electron microscopy(TEM) was used to observe the autophagosomes; LysoBrite Red was used to detect the pH change in lysosome; transfection with mRFP-GFP-LC3 adenovirus was conducted to observe the autophagic flux; Western blot was performed to determine the expression of autophagy marker LC3B-Ⅰ/LC3B-Ⅱ, autophagy substrate p62 and silent information regulator 1(SIRT1)/adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway. NAFLD cell model was successfully induced by FFA at 0.2 mmol·L~(-1) PA and 0.4 mmol·L~(-1) OA. HZRG reduced the TG level(P<0.05, P<0.01) and the lipid accumulation of FFA-induced L02 cells, while elevated the number of autophagosomes and autophagolysosomes to generate autophagic flux. It also affected the functions of lysosomes by regulating their pH. Additionally, HZRG up-regulated the expression of LC3B-Ⅱ/LC3B-Ⅰ, SIRT1, p-AMPK and phospho-protein kinase A(p-PKA)(P<0.05, P<0.01), while down-regulated the expression of p62(P<0.01). Furthermore, 3-methyladenine(3-MA) or chloroquine(CQ) treatment obviously inhibited the above effects of HZRG. HZRG prevented FFA-induced steatosis in L02 cells, and its mechanism might be related to promoting autophagy and regulating SIRT1/AMPK signaling pathway.

Prevention and Treatment of Obesity Type 2 Diabetes Mellitus by Traditional Chinese Medicine Through AMPK Signaling Pathway： A Review / 中国实验方剂学杂志

Obesity type 2 diabetes mellitus （T2DM） strengthens insulin resistance （IR） and metabolic abnormalities and significantly increases the risk of heart disease， cancer， and other diseases， and it is characterized by IR and malnutrition. As a metabolic regulation center， adenosine phosphate activated protein kinase （AMPK） mainly responds to the changes in intracellular serine/threonine kinase adenosine monophosphate （AMP） levels. After its activation， AMPK converts the cell metabolism mode from synthesis to decomposition to improve energy metabolism and acts on pathological conditions such as inflammation， ischemia， obesity， and aging. In recent years， a large number of studies have found that AMPK is an important target for the treatment of obesity T2DM. Traditional Chinese medicine（TCM） monomers/extracts and TCM formulas mainly affect the mammalian target of rapamycin （mTOR）， recombinant sirtuin 1 （SIRT1）， nuclear factor erythroid 2-related factor 2 （Nrf2）， nuclear factor kappa-B （NF-κB）， and other key signaling factors by regulating the AMPK signaling pathway， so as to achieve a variety of effects such as regulating metabolism and autophagy， reducing oxidative stress and inflammatory response， and treating obesity T2DM. It also has advantages such as multiple targets， comprehensiveness， and low toxicity. The regulation of the AMPK pathway by TCM in the prevention and treatment of obesity T2DM has become an important research direction at the present and in the future， but there is no systematic summary and induction in this field. Therefore， this article attempts to summarize the composition and regulatory mechanisms of the AMPK signaling pathway in affecting obesity. It provides a review of the current research status of TCM in regulating the AMPK signaling pathway for the prevention and treatment of obesity T2DM， so as to provide a reference for the diagnosis and treatment of obesity T2DM in TCM and the development of new drugs.

Relationship of PI3K-Akt/mTOR/AMPK signaling pathway genetic mutation with efficacy and prognosis in nasopharyngeal carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Genetic mutation is one of the important causes for tumor genesis and development, but genetic mutation in nasopharyngeal carcinoma (NPC) has rarely been reported. This study explored the role of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), mammalian target of rapamycin (mTOR), and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in the efficacy and prognosis in patients with NPC.@*METHODS@#A total of 31 patients with advanced NPC, who came from the Affiliated Cancer Hospital of Xiangya School of Medicine of Central South University/Hunan Provincial Cancer Hospital, were enrolled. All of the exons of 288 genes, introns of 38 genes and promoters or fusion breakpoint regions from the nasopharyngeal biopsy tissues before treatment were detected by the gene sequencing platform Illumina NextSeq CN500. The coding regions of 728 genes were carried out a high-depth sequencing of target region capture, and the 4 variant types of tumor genes (including point mutations, insertion deletions of small fragments, copy number variations, and currently known fusion genes) were detected. All of 31 patients received platinum-based induction chemotherapy combined with concurrent chemoradiotherapy and were followed up for a long time.@*RESULTS@#The 3-year regional failure-free survival (RFFS) and disease-free survival (DFS) in patients with PI3K-Akt pathway mutation were significantly lower than those in unmutated patients (χ2=6.647, P<0.05). The 3-year RFFS and DFS in patients with mTOR pathway mutations were significantly lower than those in unmutated patients, and there was significant difference (χ2=5.570, P<0.05). The rate of complete response (CR) in patients with unmutated AMPK pathway was significantly higher than that in patients with mutation at 3 months after treatment (P<0.05), and the 3-year RFFS and DFS in patients with AMPK pathway mutation were significantly lower than those in unmutated patients (χ2=4.553, P<0.05). PI3K-Akt/mTOR/AMPK signaling pathway mutations and pre-treatment EB virus DNA copy numbers were independent prognostic factors for 3-year RFFS and DFS in patients with NPC (both P<0.05).@*CONCLUSIONS@#The NPC patients with PI3K-Akt/mTOR/AMPK signaling pathway mutation have poor prognosis, and the detection of PI3K-Akt, mTOR, AMPK driver genes and signaling pathways by next-generation sequencing is expected to provide new idea for basic research and targeted therapy of NPC.

Preliminary Study on Effects of Solamargine on the Growth and Apoptosis of Human Hepatocarcinoma Cells HepG 2 and Its Mechanism / 中国药房

OBJECTIVE：To ex plore the effects of solamargine on the growth and apoptosis of human hepatocarcinoma cells HepG2 and its underlying mechanism. METHODS ：The effects of 0（blank group ）-12 μmol/L solamargine treatment of 24，48 h on survival rate of HepG 2 cells were investigated. The effects of 0（blank group ），6 μmol/L solamargine treatment of 10 days on cell clone formation were also investigated. The effects of 0（blank group ），4，6，8 μmol/L solamargine for 24 h on the apoptotic rate of cells，mRNA expression of Bcl- 2，Bax and caspase- 3， protein expression of Bcl- 2 and cleaved caspase- 3 as well as ratio of p-AMPKα to AMPKα were all tested. The effects of AMPK inhibitor as compound C on the protein expression of AMPKα and Bcl- 2 in cells were investigated after treated with 6 μmol/L solamargine for 24 h. RESULTS ：Compared with 020-39318678。E-mail：wujingjing6028@gzucm.edu.cn blank group ，1-12 μ mol/L solamargine for 24，48 h could significantly decrease the survival rates of cells （P＜0.05）in a concentration-dependent manner ；IC50 of them were 8.310 and 7.996 μmol/L，respectively；the rate of cell clone formation was decreased significantly after treated with 6 μmol/L solamargine for 10 days（P＜0.05）. The apoptotic rate of HepG 2 cells，mRNA expression of Bax and caspase- 3，protein expression of cleaved caspase-3（except for 8 μmol/L）as well as ratio of p-AMPKα to AMPKα（except for 8 μmol/L）were all increased significantly after treated with 6，8 μmol/L solamargine（P＜0.05）；mRNA and protein expression of Bcl- 2 were decreased significantly （P＜ 0.05）；the changes of some indexes were in a concentration-dependent manner. The compound C could inhibit protein expression of AMPKα，and reverse the inhibitory effect of solamargine on Bcl- 2 protein. CONCLUSIONS ：Solamargine can inhibit the proliferation of HepG 2 cells and induce apoptosis ，the mechanism of which may be associated with activating AMPK signaling pathway.

Effect of Yiqi Fuzheng Jiedu Decoction on Autophagy and Growth of A549 Cells： An Exploration Based on p53/AMPK Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of Yiqi Fuzheng Jiedu decoction （YQFZJDD） on autophagy and growth of A549 cells. Method:A549 cells were intervened with YQFZJDD-containing serum prepared in advance. The levels of microtubule-associated protein 1 light chain 3 （LC3）， homologue of yeast autophagy-related gene 6 （Beclin1）， p62， p53， adenosine 5'-monophosphate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， mammalian target of rapamycin （mTOR）， and phosphorylated mTOR （p-mTOR） were detected by Western blot assay， and microtubule-associated protein 1A/1B light chain 3B （MAP1LC3B） by immunofluorescence （IF） assay. The proliferation， invasion， and senescence of A549 cells were separately measured by 5-ethynyl-2'-deoxyuridine （EDU） staining， Transwell assay， and <italic>β</italic>-galactosidase staining. In the presence of autophagy inhibitor 3-methyladenine （3-MA， 5 mmol·L^-1）， the cells were divided into the 10% fetal bovine serum （blank） group， 10% control serum （control） group， low-， medium-， and high-dose 10% YQFZJDD-containing serum groups， and high-dose 10% YQFZJDD-containing serum + 3-MA group， followed by the measurement of A549 cell proliferation， invasion， and senescence. In the adoption of p53 inhibitor Pifithrin-<italic>α</italic> （PFT-<italic>α</italic>， 10 μmol·L^-1）， the cells were divided into the control group， PFT-<italic>α</italic> group， high-dose YQFZJDD-containing serum group， and high-dose YQFZJDD-containing serum + PFT-<italic>α</italic> group. Then the LC3-Ⅱ and Beclin1 expression， MAP1LC3B fluorescence intensity， as well as A549 cell proliferation， invasion and senescence were determined. Result:Compared with the blank group and control group， YQFZJDD-containing serum at the medium and high doses up-regulated the protein expression levels of LC3-Ⅱ and Beclin1 in A549 cells after 48-h intervention in a dose-dependent manner （<italic>P</italic><0.01）. Besides， YQFZJDD-containing serum at the low-， medium-， and high-doses down-regulated p62 and p-mTOR/mTOR expression （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated p53 and p-AMPK/AMPK （<italic>P</italic><0.01）， decreased the number of proliferative and invasive cells， increased the number of senescent cells （<italic>P</italic><0.01）， and enhanced the IF intensity of MAP1LC3B， with the optimal effect observed in the high-dose YQFZJDD-containing serum group （<italic>P</italic><0.01）. Compared with the high-dose YQFZJDD-containing serum group， the high-dose YQFZJDD-containing serum + 3-MA group and high-dose YQFZJDD-containing serum + PFT-<italic>α</italic> group exhibited significantly increased proliferative and invasive cells but decreased senescent cells （<italic>P</italic><0.05， <italic>P<</italic>0.01）. Meanwhile， the IF intensity of MAP1LC3B in the high-dose YQFZJDD-containing serum + PFT-<italic>α</italic> group was weakened and the LC3-Ⅱ and Beclin1 protein expression levels declined （<italic>P</italic><0.05， <italic>P<</italic>0.01）. Conclusion:YQFZJDD promotes the autophagy of A549 cells through the p53/AMPK signaling pathway to inhibit their proliferation， invasion and migration but accelerate senescence， thus playing a crucial role in inhibiting the progression of lung cancer.

Effects of Mangiferin on Glucose and Lipid Metabolism of Insulin-resistant HepG2 Cells / 中国药房

OBJECTIVE：To analyze the effects of mangiferin （MGF）on glucose and lipid metabolism in insulin resistance （IR）HepG2 cells，and to explore the potential mechanism. METHODS ：Using human hepatoma HepG 2 cells as research objects ， 1 mmol/L palmitic acid and 2 mmol/L oleic acid were used to establish the IR-HepG 2 cell model. Using metformin hydrochloride as positive control ，the effects of low-concentration ，medium-concentration and high-concentration MGF （125，250，500 μmol/L）on the corrected glucose consumption ，the contents of triglyceride （TG）and total cholesterol （TC）in IR-HepG 2 cells were detected. The mRNA expression of APN ，AdipoR2，APPL1，AMPK in the upstream of AMPK signaling pathway and IRS- 1，Akt and GLUT4 in the downstream insulin signaling pathway were detected by RT-PCR. The phosphorylation level of AMPK protein was detected by Western blot assay. RESULTS ：Compared with control group ，corrected glucose consumption ，mRNA expression of APN，AdipoR2，APPL1，AMPK，IRS-1 and GLUT 4，as well as the phosphorylation level of AMPK protein were decreased significantly in model group ，while the contents of TG and TC were increased significantly （P＜0.05 or P＜0.01）. Compared with model group ， corrected glucose consumption ， mRNA expression of APN （except for MGF medium-concentration and high-concentration groups ），AdipoR2，APPL1，AMPK（except for MGF medium-concentration and high-concentration groups ）， IRS-1（except for MGF medium-concentration and high-concentration groups ），Akt（except for positive control group ），GLUT4 （except for MGF high-concentration group ）were increased significantly in administration groups ，while the contents of TG and TC were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Mangiferin may activate APN ，which is the upstream target of pathway ，and then regulate AMPK signaling pathway ，so as to promote glucose uptake of IR-HepG 2 cells，reduce TG and TC contents，and improve IR and abnormal glucose and lipid metabolism.

Study on the Mechanism of Huanglian Jiedu Decoction Regulating Macrophage Polarization Based on Network Pharmacology / 中国药房

OBJECTIVE：To study the mechanism of H uanglian jiedu decoction （HJD）regulating macrophage polarization in order to explore its anti-inflammatory mechanism. METHODS ：The active components and predicted targets of HJD were screened through TCMSP and Swiss Target Prediction database ；the related targets of macrophage polarization were obtained by GeneCards and OMIM database ，and the network diagram of active ingredient-macrophage polarization target of HJD was drawn by using Cytoscape 3.6.0 software；protein interaction network was constructed by String database and core targets were extracted. Gene ontology（GO）enrichment analysis and Kyoto Encyclopedia of genes and genomes （KEGG）pathway enrichment analysis were carried out by using Cytoscape 3.6.0 software and DAVID website. Combined with the results of network pharmacology analysis ， RAW264.7 macrophage cells were divided into blank control group ，model group ，simvastatin group （10 μmol/L）and serum containing HJD group （obtained from the blood after the rats were given HJD at the dose of 10 g/kg）. Except the blank control group and model group were added culture medium ， the other groups were added with 100 μ L of relevant drug solution or serum containing drug. After 2 h of culture ，except for the blank control group ，LPS solution （100 μg/L）was added to the other groups for 24 h to induce inflammation. Western blot assay was used to detect the expression of AMPK. mRNA expression of M 1 type polarization factor （IL-1 β，iNOS）and M 2 type polarization factor （IL-10，Fizz1）were detected by RT-PCR. RESULTS ：A total of 50 active components of HJD （such as acacetin ，wogonin，quercetin，β-sitosterol）were screened ， which could regulate macrophage polarization through 12 GO items （such as anoikis ，astrocyte activation ），and 20 KEGG pathways（such as estrogen signaling pathway ，bladder cancer pathway ，AMPK signaling pathway ）. The results of cell test showed that compared with blank control group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in model group were significantly decreased （P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly increased（P＜0.01）； compared with model group ，the expression of AMPK protein ，Fizz1 and IL- 10 mRNA in serum containing HJD group and simvastatin group were significantly increased （P＜0.05 or P＜0.01），while the expression of IL- 1β and iNOS mRNA were significantly decreased （P＜0.01）. CONCLUSIONS ：HJD can regulate macrophage polarization through multiple targets and pathways；it can up regulate the expression of M 2-type polarization factors and down-regulate the expression of M 1-type polarization factors through AMPK signaling pathway ，regulate macrophage polarization and play an anti-inflammatory role.

Action mechanism of Sijunzi Decoction in treatment of type 2 diabetes mellitus based on network pharmacology / 中草药

Objective: The main active components and targets of Sijunzi Decoction for type 2 diabetes mellitus (T2DM) treatment were predicted by network pharmacology, and the potential mechanism of multi-component-multi-target-multi-pathway was discussed, and INSR and PI3K in the AMPK signal pathway were verified in animal experiment. Methods: Based on TCM system pharmacology database and analysis platform (TCMSP), literature mining and previous studies of our research group, the chemical components of Sijunzi decoction and its four herbs Codonopsis pilosula, Atractylodes macrocephala, Poria cocos, Glycyrrhiza uralensis were screened. Three conditions of oral absorption rate (OB), drug-like (DL), half-life (HL) were used to screen the active components of the drug; The targets of active components were predicted through the Swiss Target Prediction database; TTD, Drupe and DisGNET databases were used to screen the targets of T2DM. After mapping the component target and disease target, the interaction network of the target proteins of the active components was constructed by using the software of Cytoscape 3.2.1. Through topological analysis of the network, the nodes with degree of freedom ≥ average degree of freedom were screened out, and the core target network of core active ingredient was obtained, while using the String database to draw core-target protein-protein interaction (PPI) networks, using DAVID database for GO analysis and KEGG analysis of core target genes to construct core active components-core target-metabolism Path network diagram to explore the potential mechanism of Sijunzi Decoction against T2DM. The system docking site was used for molecular docking of the top 5 components and the top 3 proteins. Further, animal experiments were used to verify. SD rats were fed with high-sugar high-fat diet combined with low-dose streptozotocin (STZ) to induce T2DM model, and administrated with 5.65 g/kg Sijunzi Decoction for 28 d, and the levels of blood glucose (FBG) and oral glucose tolerance (OGTT) of each group of mice were determinated. The InsR, PI3K mRNA expression in AMPK signaling pathway was detected by RT-PCR. Results: A total of 113 chemical components were selected from Sijunzi Decoction, involving 47 targets for the treatment of T2DM. Twenty-two core components and 27 core targets were screened according to node degree of freedom ≥ average degree of freedom. The GO analysis results showed that it involved seven biological processes such as blood glucose homeostasis, positive regulation of adipose tissue development, four molecular functions including steroid hormone receptor activation and drug binding, and cell composition including the plasma membrane and nuclear chromatin. The results of KEGG analysis showed that it might treat diabetes through 14 signaling pathways, such as AMPK signaling pathway, PPAR signaling pathway and insulin resistance. A total of 60% of the molecules had intense binding activity and 40% had stronger binding activity. The results of animal experiments showed that Sijunzi Decoction could significantly improve OGTT (P < 0.001), and decrease FBG (P < 0.01) in T2DM rats, and significantly increase the expression of INSR mRNA (P < 0.001) and PI3K mRNA (P < 0.001). Some of the predicted results of network pharmacology were verified. Conclusion: This study reflects that Sijunzi Decoction can treat type 2 diabetes mellitus through multi-target and multi-channel, which lays a foundation for the future study of molecular mechanism.

Role of mitochondrial fusion and fission in protective effects of dexmedetomidine against cerebral ischemia/reperfusion injury in mice / 南方医科大学学报

OBJECTIVE@#To investigate the protective effects of dexmedetomidine (DEX) against cerebral ischemia/reperfusion (I/R) injury in mice and its relation with mitochondrial fusion and fission.@*METHODS@#Male ICR mice were randomly divided into sham-operated group, I/R group, I/R+DEX group and I/R+DEX+dorsomorphin group. Mouse models of cerebral I/R injury were established by modified thread occlusion of the middle cerebral artery. DEX (50 μg/kg) was injected intraperitoneally at 30 min before cerebral ischemia, which lasted for 1 h followed by reperfusion for 24 h. The neurobehavioral deficits of the mice were evaluated based on Longa's scores. The volume of cerebral infarction was detected by TTC staining. The changes in mitochondrial morphology of the brain cells were observed with transmission electron microscopy. Western blotting was performed to detect the expressions of phosphorylated AMP-activated protein kinase (p-AMPK), mitochondrial fusion protein (Mfn2) and mitochondrial fission protein (p-Drp1) in the brain tissues.@*RESULTS@#DEX pretreatment significantly reduced the neurobehavioral score and the percent volume of cerebral infarction in mice with cerebral I/R injury. Treatment with dorsomorphin (an AMPK inhibitor) in addition to DEX significantly increased the neurobehavioral score and the percent volume of cerebral infarction in the mouse models. Transmission electron microscopy showed that DEX obviously reduced mitochondrial damage caused by cerebral I/R injury and restored mitochondrial morphology of the brain cells, and such effects were abolished by dorsomorphin treatment. Western blotting showed that DEX pretreatment significantly increased the expressions of p-AMPK and Mfn2 protein and decreased the expression of p-Drp1 protein in the brain tissue of the mice, and these changes were also reversed by dorsomorphin treatment.@*CONCLUSIONS@#Preconditioning with DEX produces protective effects against cerebral I/R injury in mice possibly by activating AMPK signaling to regulate mitochondrial fusion and fission in the brain cells.

MicroRNA-23a knockdown attenuates angiotensin Ⅱ induced hypertrophy in rat H9c2 cells via activating PTEN and AMPK pathway / 中华心血管病杂志

Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK 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.

Effect of Lycium barbarum polysaccharide on atherosclerosis in mice by up-regulating adiponectin expression / 药物评价研究

Objective To explore the effect of Lycium barbarum polysaccharide (LBP) on adiponectin (APN) expression and the mechanism of lowering blood-lipid and anti-inflammation in atherosclerotic (AS) mice.Methods C57BL/6J mice with normal feed were chosen as control group.Thirty-two ApoE-/-mice with high cholesterol diet were successfully established as AS models,and then the mice were randomly divided into model group and three LBP groups,which were feed with high,medium,and low dose of LBP.After feeding for four weeks,aortic blood and tissues were collected.Blood-lipid,inflammatory factors,endothelin-1 (ET-1),APN,AdipoR1,and AMPK pathway related protein expression were detected.Differentiated 3T3-L1 cells were divided into control group,LBP group,and LBP + BML-275 group.Triglyceride (TG),inflammatory factors,APN,AdipoR1,and AMPK pathway related protein expression was investigated.Results In mice,compared with the control group,typical AS pathomorphologic changes were found in aorta and the levels of TG,total cholesterol (TC),nitric oxide (NO),ET-1,interleukin-6 (IL-6),and tumor necrosis factor (TNF-α) in the model group were significantly increased,while the protein expression of HDL-C,APN,AdipoR1,PPARα,AMPKα,and p-AMPK-α and Acyl-CoA oxidase (ACO) mRNA expression was reduced.Compared with the model group,AS pathomorphologic state was obviously improved in aorta and the amount of TG,TC,NO,ET-1,IL-6,and TNF-α in LBP groups were markedly decreased,while the protein expression of HDL-C,APN,AdipoR1,PPARα,AMPKα,p-AMPKα,and ACO mRNA expression was up-regulated.These changes were all in a dose-dependent manner.In differentiated 3T3-L1 fat cells,compared with control group,LBP enhanced the expression of APN,AdipoR1,PPARα,AMPKα,p-AMPKα,and ACO,but decreased the amount of TG,IL-6,and TNF-α.Compared with LBP group,the levels of TG,IL-6 and TNF-α was notably increased in BML-275 group.Conclusion LBP up-regulates the expression of APN and AdipoR1,activates APN/AMPK pathway,plays a role in lowering blood-lipid and anti-inflammation,and thus relieves AS in mice.

Piperine regulates glucose metabolism disorder in HepG2 cells of insulin resistance models via targeting upstream target of AMPK signaling pathway / 中国中药杂志

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.

Research progression of AMPK in adjuvant treatment strategy of breast cancer / 医学研究生学报

Activated protein kinase ( AMPK) , an important energy receptor , plays a very important role in regulating organ-ism and the energy metabolism of the cell .AMPK has complex relationship with survival of different types of breast cancer cells .Ac-cording to different conditions , AMPK may exist both anti or promoting effect on tumor .In this review , we summarize the relationship between AMPK and breast cancer related signal pathways , AMP and breast cancer endocrine therapy , breast cancer chemotherapy , ra-diotherapy for breast cancer , treatment of triple negative breast cancer and multi drug resistance of the relationship , we also expound some drugs related to AMPK and used in clinical setting .