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ObjectiveTo explore the mechanism of Huangqisan （HQS） in regulating autophagy to alleviate hepatic steatosis and improve non-alcoholic fatty liver disease （NAFLD） based on adenosine 5'-monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodThe main chemical components and targets of HQS and NAFLD-related targets were collected from database and the intersection targets were used for Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis. The protein-protein interaction （PPI） network was constructed， and in vivo experimental verification was conducted. Sixty C57BL/6J male mice were randomly divided into normal control group （NCD）， model group high-fat diet （HFD）， metformin group （MET， 0.25 g·kg-1）， low-dose Huangqisan group （HQS-L， 0.5 g·kg-1）， and the high-dose Huangqisan group （HQS-H， 1 g·kg-1）， with 12 mice in each group after a one-week acclimatization period. NAFLD model was induced by HFD， and intragastric administration was performed at the same time， once a day for 13 weeks. Random blood glucose， serum total cholesterol （TC）， triglyceride （TG）， non-esterified fatty acid （NEFA）， low density lipoprotein-chdesterol （LDL-C） levels， and liver TG content were determined. The liver weight was weighed， and liver index was calculated. Hematoxylin-eosin （HE） staining， oil red O staining， transmission electron microscope （TEM）， real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， and Western blot were used to verify the effect and reveal the potential mechanism of C57BL/6J mice in vivo. ResultThrough network pharmacology analysis， combined with previous studies， it was predicted that HQS may improve NAFLD by regulating autophagy via the AMPK/mTOR signaling pathway. The result of in vivo experiment showed that， as compared with NCD group， random blood glucose， body weight， serum TC， LDL-C， NEFA， liver weight， liver index， and liver TG content of mice in the HFD groups were significantly increased （P<0.01）. HE staining showed massive lipid droplets （LDs） vacuolated， oil red O staining showed lipid accumulation in liver cells， and no obvious autophagosomes and autolysosome were observed under TEM. The relative mRNA expression of LC3A、LC3B、AMPKα1 and protein expression of AMPK， phosphory phosphorylated（p）-AMPK， and p-AMPK/AMPK were significantly down-regulated （P<0.01）， while the protein expression of microtubule-associated protein 1 light chain 3 （LC3）Ⅱ/Ⅰ and p-mTOR was significantly up-regulated （P<0.01）. As compared with HFD groups， liver weight， serum TG， and NEFA levels in HQS-L and HQS-H groups were significantly deceased （P<0.05， P<0.01）. HE staining and oil red O staining showed the improvement of liver pathological changes after HQS administration. Under TEM， a small amount of autophagosome and autolysosome were observed. Besides， liver index was significantly decreased in the HQS-L group （P<0.01）， and random blood glucose， serum TC level and liver TG content were significantly decreased in the HQS-H group （P<0.05）. The results of Western blot and Real-time PCR showed that the mRNA expression of LC3A and LC3B and the protein expression of LC3Ⅱ/Ⅰ， p-AMPK， and p-AMPK/AMPK were significantly up-regulated （P<0.01）， while the mRNA expressions of p62 and protein expression of p62 and p-mTOR were significantly down-regulated （P<0.05， P<0.01）. ConclusionHQS may promote autophagy and restore autophagy flux via the AMPK/mTOR signaling pathway to alleviate hepatic steatosis improving NAFLD.
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ObjectiveTo explore the effect and mechanism of Zuogui Jiangtang Tongmai prescription （ZGJTTMP） on astrocytes （ASs） injured by advanced glycation end products（AGEs） combined with oxygen-glucose deprivation （OGD）. MethodCell counting kit-8 （CCK-8） was used to determine the optimal concentration of AGEs and the action time of OGD， and the optimal blood concentration of ZGJTTMP was selected for follow-up experiments. ASs were divided into normal group， model group （AGEs + OGD）， ZGJTTMP group， an adenosine 5'-monophosphate-activated protein kinase （AMPK） inhibitor （Compound C） group， AMPK activator （AICAR） group， and combination group （ZGJTTMP + AICAR）. The morphological changes in ASs in each group were observed under an inverted microscope. The cell survival rate in each group was detected by CCK-8. The content of interleukin-1β（IL-1β）， interleukin-6（IL-6）， and tumor necrosis factor-α（TNF-α） was detected by enzyme-linked immunosorbent assay （ELISA）. The number of autophagosomes in each group was counted under an electron microscope. The expression of microtubule-associated protein light chain 3 （LC3） was observed by immunofluorescence. The protein expression of LC3， p62， p-AMPK， AMPK， p-mammalian target of rapamycin （mTOR）， mTOR， p-UNC-51 like kinase 1 （ULK1）， and ULK1 was detected by Western blot. ResultAccording to the results of cell survival rate， 200 mg·L-1 AGEs and OGD for 6 h were selected as the optimal modeling conditions for the model group， and 5% was selected as the optimal blood concentration of ZGJTTMP. Under the inverted microscope， the cells were severely damaged after modeling， but the cell injury in the ZGJTTMP group and the Compound C group was significantly improved. As revealed by ELISA results， the content of IL-1β， IL-6， and TNF-α in the model group increased （P<0.01）， and the content of inflammatory factors in the ZGJTTMP group and the Compound C group decreased （P<0.01）. Under the electron microscope， the number of autophagosomes in the model group increased significantly. The immunofluorescence results showed that the expression area of LC3 increased in the model group （P<0.01）， and the ZGJTTMP group and the Compound C group showed decreased number of autophagosomes and reduced expression area of LC3 （P<0.01）. As demonstrated by the results of Western blot， compared with the normal group， the model group showed increased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and decreased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. Compared with the model group， the ZGJTTMP group and the Compound C group showed decreased expression of LC3Ⅱ/LC3Ⅰ and p-AMPK/AMPK （P<0.01） and increased p62， p-mTOR/mTOR， and p-ULK1/ULK1 （P<0.01）. ConclusionZGJTTMP possesses a protective effect on ASs with inflammatory injury by AGEs combined with OGD， which may be achieved by inhibiting the activation of the AMPK/mTOR/ULK1 pathway related to autophagy， thus inhibiting the overexpression of autophagy.
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As one of the most lethal diseases, pancreatic cancer shows a dismal overall prognosis and high resistance to most treatment modalities. Furthermore, pancreatic cancer escapes early detection during the curable period because early symptoms rarely emerge and specific markers for this disease have not been found. Although combinations of new drugs, multimodal therapies, and adjuvants prolong survival, most patients still relapse after surgery and eventually die. Consequently, the search for more effective treatments for pancreatic cancer is highly relevant and justified. As a newly re-discovered mediator of gasotransmission, hydrogen sulfide (H
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Objective To observe the effect of rosiglitazone on the protein expression of AMPK and GLUT4 in peripheral tissue (liver, skeletal muscle and fat) of type 2 diabetic db/db mice and to prove that rosiglitazone can regulate the glucose metabolism in db/db mice partly through the AMPK pathway. Methods db/db mice were randomly divided into model group and rosiglitazone group according to their blood glucose.The db/m mice were normal control group.After 4 weeks of administration, fasting blood glucose was detected in each group.Western blot was used to detect the contents of AMPK, p-AMPK and GLUT4 in liver, skeletal muscle and adipose tissue. Results (1) Rosiglitazone significantly reduced the fasting blood glucose of db/db mice; (2)Rosiglitazone increased the level of AMPK phosphorylation in the liver, skeletal muscle and adipose tissue of db/db mice, and increased the content of GLUT4 protein in skeletal muscle and adipose tissue. Conclusion Rosiglitazone can increase the phosphorylation of AMPK and the expression of GLUT4 protein in the liver, muscle and fat tissue of db/db mice, and promote the uptake and utilization of glucose in peripheral tissue, suggesting that it can regulate glucose metabolism in db/db mice partly through the AMPK pathway.
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OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on the expression of liver protein kinase 1 (LKB1), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and 6-phosphofructo-2-kinase (PFK2) in cardiomyocytes of rats with acute myocardial ischemia (AMI), so as to explore its mechanisms underlying cardioprotective effect. METHODS: Thirty male Wistar rats were randomly divided into sham-operation, model and EA pretreatment groups (n＝10 rats per group). The AMI model was established by ligation of the left anterior descending branch of coronary artery. Before modeling, EA preconditioning (2 Hz/15 Hz, 1 mA) was applied to bilateral "Neiguan"(PC6) for 30 min, once daily for 14 days. Histopathological changes of myocardium was observed by microscope after H．E. staining. The level of lactate dehydrogenase (LDH) in serum was detected by ELISA. The expression of autophagy-associated proteins and mRNAs as LKB1, AMPKa1, AMPKa2 and PFK2 were detected by Western blot and real-time PCR, respectively. RESULTS: Compared with the sham-operation group, serum LDH content, and expression levels of myocardial AMPKa2 and PFK2 proteins and mRNAs were significantly up-regulated (P<0.01), and those of LKB1 and AMPKa1 proteins and mRNAs were increased in the model group (P<0.05). Following the intervention, serum LDH were apparently down-regulated (P<0.01), and expression levels of myocardial LKB1, AMPKa1 and PFK2 proteins and mRNAs were apparently up-regulated (P<0.01), but that of AMPKa2 protein and mRNA was remarkably down-regulated in the EA group (P<0.01). H．E. staining showed cell swelling, disordered arrangement of myocardial fibers with obvious rupture, interstitial bleeding and inflammatory infiltration, which was relatively milder in the EA preconditioning group. CONCLUSION: EA pretreatment can trigger LKB1/AMPK/PFK2 signaling pathway in AMI rats, which may contribute to its cardioprotective effect against ischemic myocardial injury by activating autophagy of cardiomyocytes. .
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OBJECTIVE: To observe the effect of electroacupuncture on the expression of phosphorylated adenosine 5'-monophosphate-activated protein kinase(p-AMPK), phosphorylated mammalian target of rapamycin(p-mTOR) and phosphorylated Ulk1(p-Ulk1) proteins in the cortex of traumatic brain injury (TBI) rats, so as to explore its mechanisms underlying treatment of TBI. METHODS: Male SD rats were randomly divided into sham operation (sham), model, electroacupuncture Ⅰ (EA Ⅰ), electroacupuncture Ⅱ (EA Ⅱ) groups (n＝10 in each group). TBI model was established by using a free fall brain injury striking device after exposing the local cranial bone (to induce the left parietal cerebral contusion). Rats in EA Ⅰ group were treated by electroacupuncture at "Neiguan" (SP6) and "Zusanli" (ST36) combined with acupuncture at "Shuigou" (GV26) and "Baihui"(GV20) on the 7thday after modeling, once a day for 7 consecutive days. Rats in EA Ⅱ group received the treatments as those in EA Ⅰ group on 24 h after modeling, once a day for 14 d. After the treatment, histopathological changes of the injured cerebral cortex were observed by HE staining and Nissl staining. Western blot was used to detect the expression of AMPK, p-AMPK, mTOR, p-mTOR, Ulk1, p-Ulk1 proteins in the injured cerebral cortex tissue. RESULTS: After modeling and compared with the sham group, a large number of tissue necrosis, scattered arrangement of nerve fibers, vacuolar changes of cells, nuclear fragmentation, consolidation and hyperplastic scar tissue were found in the brain trauma area of rats in the model group. Nissl corpuscles were obviously absent. The ratio of p-AMPK/AMPK was up-regulated in the cortex of the wound region (P<0.01), and the ratio of p-mTOR/mTOR, p-Ulk1/Ulk1 were down-regulated (P<0.01). Compared with the model group, the pathological changes in brain injury area of rats in both EA groups were alleviated, the number of Nissl corpuscles increased, the ratio of p-AMPK/ AMPK was down-regulated in cortex of the injury area (P<0.01), and the ratios of p-mTOR/mTOR and p-Ulk1/Ulk1 were up-regulated (P<0.01). Compared with EA Ⅰ group, the pathological changes in the brain injury area in EA Ⅱ group showed obvious improvement, with down-regulation of p-AMPK/AMPK (P<0.05), and up-regulation of p-mTOR/mTOR and p-Ulk1/Ulk1 (P<0.05). CONCLUSION: Electroacupuncture may inhibit the over-activation of autophagy of cranial neurons by regulating the activation of AMPK, mTOR and Ulk1, thus exerting brain protection effect on TBI rats, and early electroacupuncture intervention is more effective in acute phase of TBI.
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OBJECTIVE@#To investigate whether ginsenoside-Rb1 (Gs-Rb1) improves the CoCl-induced autophagy of cardiomyocytes via upregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway.@*METHODS@#Ventricles from 1- to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group (normal level oxygen), hypoxia group (500 μmol/L CoCl), Gs-Rb1 group (200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), Ara A group (500 μmol/L Ara A + 500 μmol/L CoCl), Ara A+ Gs-Rb1 group (500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) + 500 μmol/L CoCl], and AICAR+Gs-Rb1 group (1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl). Cells were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cardiac troponin I (cTnI) levels were detected by enzyme-linked immunosorbent assay (ELISA). AMPK activity was assessed by 2',7'-dichlorofluorescein diacetate (DCFH-DA) ELISA assay. The protein expressions of Atg4B, Atg5, Atg6, Atg7, microtubule-associated protein 1A/1B-light chain 3 (LC3), P62, and active-cathepsin B were measured by Western blot.@*RESULTS@#Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes (P0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes (P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A (P<0.05) and were not affected by AICAR (P=0.871).@*CONCLUSION@#Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.
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AIM:To investigate the effects of losartan on lipopolysaccharide (LPS)-induced glial fibrillary acidic protein (GFAP) expression,and to determine whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation is involved in the mechanism.METHODS:Adult male KM mice were divided into control group,LPS model group,losartan treatment group,and losartan and Compound C co-treatment group.To establish a model of central nervous system inflammation,the mice received daily intracerebroventricular injection of LPS (24 μg/d) for 2 d.Daily losartan administration (0.5,1 or 5 mg · kg-1 · d-1,ip) initiated at 14 d prior to LPS injection.Compound C (10 mg/kg,ip),a selective AMPK inhibitor,started to be injected daily at 2 d prior to LPS injection.The hippocampal tissues in each group were isolated at 3 d after the last LPS injection,and then the protein levels of GFAP,AMPK,p-AMPK,mammalian target of rapamycin (mTOR) and p-mTOR were determined by Western blot.RESULTS:Twice LPS injections significantly increased the expression of GFAP in the hippocampus (P ＜ 0.01).Losartan inhibited LPS-induced GFAP expression in a concentration-dependent way,and losartan at 5 mg· kg-1 · d-1 significantly inhibited GFAP expression and AMPK activation (P ＜ 0.05),but it had no obvious effect on mTOR activation.Furthermore,Compound C significantly reversed the effect of losartan treatment on LPS-induced GFAP expression and AMPK phosphorylation (P ＜ 0.05).CONCLUSION:Losartan inhibits LPS-induced GFAP expression in the mouse hippocampus,and AMPK activation but not mTOR,is involved in the mechanism.
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AIM:To investigate the effects of losartan on lipopolysaccharide (LPS)-induced glial fibrillary acidic protein (GFAP) expression,and to determine whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) activation is involved in the mechanism.METHODS:Adult male KM mice were divided into control group,LPS model group,losartan treatment group,and losartan and Compound C co-treatment group.To establish a model of central nervous system inflammation,the mice received daily intracerebroventricular injection of LPS (24 μg/d) for 2 d.Daily losartan administration (0.5,1 or 5 mg · kg-1 · d-1,ip) initiated at 14 d prior to LPS injection.Compound C (10 mg/kg,ip),a selective AMPK inhibitor,started to be injected daily at 2 d prior to LPS injection.The hippocampal tissues in each group were isolated at 3 d after the last LPS injection,and then the protein levels of GFAP,AMPK,p-AMPK,mammalian target of rapamycin (mTOR) and p-mTOR were determined by Western blot.RESULTS:Twice LPS injections significantly increased the expression of GFAP in the hippocampus (P ＜ 0.01).Losartan inhibited LPS-induced GFAP expression in a concentration-dependent way,and losartan at 5 mg· kg-1 · d-1 significantly inhibited GFAP expression and AMPK activation (P ＜ 0.05),but it had no obvious effect on mTOR activation.Furthermore,Compound C significantly reversed the effect of losartan treatment on LPS-induced GFAP expression and AMPK phosphorylation (P ＜ 0.05).CONCLUSION:Losartan inhibits LPS-induced GFAP expression in the mouse hippocampus,and AMPK activation but not mTOR,is involved in the mechanism.
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With the social development,the number of obese people is increasing.Recent studies have shown that obese women to have breast cancer disease's risk is higher than normal body weight,and etabolic disorders growing threat to human health.Estrogen,aromatase have contributed to the occurrence and development of breast cancer after menopause.AMPK is a negative regulator of aromatase,it can inhibit proliferation of cancer cells by metabolic pathways.What's more metformin,resveratrol inhibited proliferation by this route has been confirmed in breast cancer cells.Here,the author do an overview of the role of obesity-related factor for postmenopausal breast tissue estrogen synthesis and metabolic abnormalities affect aromatase expression in breast.