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ObjectiveTo observe the intervention effect of Huaiqihuang granules （HQH） on immunoglobulin A vasculitis nephritis （IgAVN） mice and explore the underlying therapeutic mechanism. MethodFifty SPF-grade male Kunming mice were randomly divided into a normal group， an IgAVN model group， a dexamethasone group （2.5 mg·kg-1·d-1）， a low-dose HQH group （4 g·kg-1·d-1）， and a high-dose HQH group （8 g·kg-1·d-1）. The mouse model was established using oral administration of gliadin combined with intravenous injection of India ink. After successful modeling， the mice were euthanized after 4 weeks of gastric gavage according to groups. The 24 h urinary total protein （24 h UTP）， urine β2-microglobulin （β2-MG）， serum total protein， albumin， IgA， etc. were detected in each group. Flow cytometry was used to determine the proportion of T helper 17 （Th17） cells in spleen cell suspension. Western blot was employed to detect the expression of adenosine 5'-monophosphate-activated protein kinase α （AMPKα）， phosphorylated AMPKα （p-AMPKα）， acetyl-CoA carboxylase 1 （ACC1）， and phosphorylated ACC1 （p-ACC1） in Th17 cells. Pathological changes in the spleen and kidneys were observed. ResultCompared with the normal group， the IgAVN model group showed significant increases in 24 h UTP， urine β2-MG， total cholesterol （P<0.05）， serum interleukin-17 （IL-17）， IgA， Th17 proportion in the spleen cell suspension， and IL-17 expression in the spleen tissue （P<0.01）， and significantly decreased serum total protein， albumin， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.01）. Compared with the IgAVN model group， in the 4th week， the 24 h UTP， urine β2-MG， serum IL-17， IgA levels， and renal IgA deposition were significantly reduced in each treatment group （P<0.01）， and the Th17 proportion and IL-17 expression in spleen tissue were significantly decreased （P<0.05， P<0.01）. Serum albumin levels significantly increased （P<0.05）. Compared with the IgAVN model group， the dexamethasone group and the high-dose HQH group showed increases in serum total protein （P<0.01）， p-AMPKα/AMPKα， and p-ACC1/ACC1 expression of Th17 cells （P<0.05， P<0.01）. The high-dose HQH group showed a significant decrease in total cholesterol level （P<0.05）. Various treatment groups showed different degrees of improvement in spleen and kidney pathological changes. ConclusionHQH may affect Th17 cell differentiation by regulating the AMPK/ACC pathway， correcting immune inflammatory disorders， and exerting therapeutic effects on IgAVN.
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ObjectiveTo discuss the impact of Buzhong Yiqitang on lipid metabolism in skeletal muscle of exercise-induced fatigue （EIF） mice through adiponectin receptor 1 （Adipor1）/adenosine 5'-monophosphate（AMP）-activated protein kinase （AMPK）/peroxisome proliferator-activated receptor gamma coactivator-1α （PGC-1α）. MethodC57BL6J mice were randomly divided into the control group， model group， low， middle， and high dose groups of Buzhong Yiqitang， and vitamin C group. No intervention was given to the control group， while the other groups were subjected to exhaustive swimming training to establish the EIF model. One hour before exhaustion， 0.2 mL distilled water was given to the control group and the model group， while the mice in the low， middle， and high dose groups of Buzhong Yiqitang were given intragastrically Buzhong Yiqitang of 4.1， 8.2， and 16.4 g·kg-1， respectively， and the vitamin C group was given vitamin C of 0.04 g·kg-1 via gavage for a duration of six weeks. After six weeks of the experiment， the growth rate of body weight， organ index， and exhaustive swimming time were calculated. Enzyme colorimetry was utilized to detect the levels of blood urea nitrogen （BUN）， creatine kinase acid （CK）， lactate dehydrogenase acid （LDH）， and lactic acid （LD）. The pathological changes of skeletal muscle were observed using hematoxylin -eosin （HE） staining， while the ultrastructure of skeletal muscle was observed with transmission electron microscope （TEM）. The contents of free fatty acids （NEFA） and triglyceride acid （TG） in serum were also examined by microplate method. The protein expressions of Adipor1， p-AMPK/AMPK， PGC-1α， and HK2 in the skeletal muscle were measured by Western blot. ResultCompared with those of the control group， the growth rate of body weight and thymus index of the model group were decreased， and the serum levels of BUN， CK， LD， and LDH were increased （P<0.01）. The contents of NEFA and TG were decreased （P<0.01）， and the protein expression of Adipor1， p-AMPK/AMPK， PGC-1 α， and HK2 in the skeletal muscle decreased （P<0.05， P<0.01）. Compared with those in the model group， the growth rate of body weight， thymus index， and exhaustive swimming time were significantly increased （P<0.01）， and the levels of BUN， CK， LD， and LDH dropped in the high dose group of Buzhong Yiqitang （P<0.01）. The levels of NEFA and TG were greatly improved （P<0.01）. The protein expressions of Adipor1， p-AMPK/AMPK， PGC-1α， and HK2 in the skeletal muscle were significantly increased （P<0.05， P<0.01）. Compared with those in the model group， the thymus index and exhaustive swimming time were significantly increased in the vitamin C group， and the levels of BUN， CK， and LD dropped （P<0.05， P<0.01）. The levels of NEFA and TG were improved significantly （P<0.01）， and the protein expression of Adipor1 in skeletal muscle was increased greatly （P<0.01）. ConclusionBuzhong Yiqitang can delay the development of EIF， which may be connected with the regulation of the Adipor1/AMPK/PGC-1α signaling pathway and the improvement of the utilization rate of skeletal muscle to fat.
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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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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.
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Total flavonoids of Dracocephalum moldavica L. (TFDM) is an effective component extracted and isolated from the traditional Uighur medicinal herb Cymbidium fragrans. Cymbidium fragrans has the effects of tonifying the heart and brain, promoting blood circulation and resolving blood stasis, and has been widely used in the treatment of cardiovascular and cerebrovascular diseases for a long time. The purpose of this study was to determine the effect of total flavonoids from Cymbidium fragrans on hypoxia/re-oxygenation (H/R) injury in H9c2 (rat cardiomyocytes) cells and its mechanism. A model (H/R) of hypoxia/re-oxygenation injury in H9c2 cells was established using hypoxia and glucose deprivation for 9 h combined with re-oxygenation and rehydration for 2 h to simulate myocardial ischemia-reperfusion injury. The effects of total flavonoids from Cymbidium fragrans on cell viability, markers of myocardial cell damage, oxidative stress levels, and reactive oxygen radical (ROS) content were investigated, Western blot was used to detect the expression of vascular endothelial growth factor B (VEGF-B) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway related proteins. The results showed that the total flavonoids of Cymbidium fragrans significantly increased the viability of myocardial cells after H/R injury, and decreased the content of lactate dehydrogenase (LDH) and creatine kinase isozyme (CK-MB) in the cell supernatant. It significantly reduced malondialdehyde (MDA), increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and decreased intracellular ROS and nitric oxide (NO) content. Western blot analysis showed that the total flavonoids of Cymbidium fragrans decreased Bax levels in H9c2 cells damaged by H/R and increased Bcl-2 expression. Total flavones of Cymbidium fragrans upregulate VEGF-B/AMPK pathway related proteins VEGF-B, vascular endothelial growth factor receptor 1 (VEGFR-1), neuropilin 1 (NRP-1), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), phosphorylated adenosine monophosphate activated protein (p-AMPK) and phospho mechanistic target of rapamycin (p-MTOR) levels. The above research results indicate that the total flavonoids of Cymbidium can significantly reduce the H/R injury of myocardial cells, which may be related to the upregulation of VEGF-B/AMPK pathway and inhibition of oxidative stress response.
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[Abstract] Objective To explore the role of purinergic ligand-gated ion chennel 7 receptor(P2X7R) in cognitive dysfunction in Parkinson’s disease rats. Methods Ninty rats was divided into 5 groups with 6 rats in each group with 3 repeats. The rat model of PD was established by 6-hydroxydopamine (6-OHDA). PD rats were injected with P2X7R agonists 2,3-adenosine 5-triethylammonium triphosphate (BzATP) and antagonists Comassie brilliant blue G (CBBG). The learning and memory ability and pain response of rats in the water maze were measured, and the expression of P2X7 in hippocampus was detected by Real-time PCR and Western blotting. Results Compared with the normal rats, PD rats had slow learning speed and weak memory ability. CBBG improved the learning and memory ability of PD rats, while BzATP decreased the learning and memory ability of PD rats. The result of Real-time PCR showed that compared with the control group, the expression of P2X7R mRNA was the highest in hippocampal tissue, the expression of P2X7R in CBBG group was down-regulated, and that of P2X7R in BzATP group was up-regulated. Compared with the PD group, Western blotting of P2X7R showed that the expression of P2X7 protein increased significantly in BzATP group, while the expression of P2X7 protein was lower in CBBG group. Conclusion Cognitive impairment exists in PD rats. CBBG can improve the learning and memory function of PD rats, and BzATP can inhibit the learning and memory function of PD rats.
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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.
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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 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 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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Protein neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of neddylation are cullin family proteins, which are the core component of Cullin-RING E3 ubiquitin ligases (CRLs). Given that cullin neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein neddylation from preclinical target validation, to drug screening, then to the clinical trials of neddylation inhibitors. In this review, we first briefly introduced the nature of protein neddylation and the regulation of neddylation cascade, followed by a summary of all reported chemical inhibitors of neddylation enzymes. We then discussed the structure-based targeting of protein-protein interaction in neddylation cascade, and finally the available approaches for the discovery of new neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of neddylation inhibitors.
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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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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.
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Objective To investigate the effect of Salvianolic Acid B(Sal B)on vascular function of db/db mice and reveal the potential mechanism. Methods 20 male db/db mice were divided into 2 groups,the con-trol group(n=10)and Sal B group(n=10). 10 age-matched male C57BL/KsJ mice were used as the wild type control. Mice in Sal B group were given Sal B ,100 mg/(kg · d)by tube. Mice in db/db control group and in wild type control were given the same volume of saline. Body weight,tail blood pressure,heart rate and fasting blood glucose level were measured every week. After 6-weeks treatment ,thoracic aorta was obtained and used to detect the levels ofsuperoxide anion and NO,vascular function,eNOS,p-eNOS,AMPK and p-AMPK. Results Sal B could reduce the body weight and fasting blood glucose level of db/db mice ,but had no effect on blood pressure. Sal B could decrease the level of superoxide inon,increased NO level,and improved endothelium-dependent but not endothelium- independent diastolic function. Sal B could increase phosphorylation levels of eNOS and AMPK. Conclusion Sal B can reduce the oxidative stress ,increases NO level in vasculature ,and improves the endo-thelium-dependent vasodilation in the diabetic mice ,which may be associated with the promotion of AMPK phos-phorylation.
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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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Objective To investigate the effect of Salvianolic Acid B(Sal B)on vascular function of db/db mice and reveal the potential mechanism. Methods 20 male db/db mice were divided into 2 groups,the con-trol group(n=10)and Sal B group(n=10). 10 age-matched male C57BL/KsJ mice were used as the wild type control. Mice in Sal B group were given Sal B ,100 mg/(kg · d)by tube. Mice in db/db control group and in wild type control were given the same volume of saline. Body weight,tail blood pressure,heart rate and fasting blood glucose level were measured every week. After 6-weeks treatment ,thoracic aorta was obtained and used to detect the levels ofsuperoxide anion and NO,vascular function,eNOS,p-eNOS,AMPK and p-AMPK. Results Sal B could reduce the body weight and fasting blood glucose level of db/db mice ,but had no effect on blood pressure. Sal B could decrease the level of superoxide inon,increased NO level,and improved endothelium-dependent but not endothelium- independent diastolic function. Sal B could increase phosphorylation levels of eNOS and AMPK. Conclusion Sal B can reduce the oxidative stress ,increases NO level in vasculature ,and improves the endo-thelium-dependent vasodilation in the diabetic mice ,which may be associated with the promotion of AMPK phos-phorylation.
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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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It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ± 1.04; Group B, 5.47 ± 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.