Main alkaloids of Rhizoma Coptidis improved palmitic acidinduced insulin resistance in HepG2 cells via AMPK and MAPK signaling pathway

Rhizoma Coptidis,a traditional Chinese herbal medicine,has been used for treating diabetes for thousands of years.However,the molecular basis for this action has not been elucidated.In the present study,the effects of seven main alkaloids of Rhizoma Coptidis on glycometabolism were investigated and the related molecular mechanism of five active compounds on insulin resistant(IR)cell model was explored for the first time.Results showed that berberine,palmatine,epiberberine,columbamine and groenlandicine enhanced glucose consumption in the palmitic acid(PA)-induced IR-HepG2 cells,indicating that these compounds could improve IR.In addition,we found that among these active alkaloids,berberine,columbamine,epiberberine and groenlandicine could inhibit the activation of ERK and p38 pathway,while berberine,columbamine,palmatine and epiberberine could activate AMPK pathway.Moreover,palmatine and columbamine regulated the mRNA expression of GLUT2 to ameliorate IR via activating AMPK and inactivating p38 MAPK signal pathway.To sum up,berberine,columbamine,palmatine,epiberberine and groenlandicine might be the active reagents,which contribute to the glucose lowering effects of Rhizoma Coptidis.

Liraglutide reduces oxidized LDL-induced oxidative stress and fatty degen- eration in Raw 264.7 cells involving the AMPK/SREBP1 pathway

Baekgound Recent studies have suggested a potential role for liraglutide in the prevention and stabilization ofatherosclerotic vascular disease. However, the molecular mechanisms underlying the effect of liraglutide on atherosclerosis have not been well elucidated. The pur- pose of this study was to examine whether liraglutide protects against oxidative stress and fatty degeneration via modulation of AMP-activated protein kinase （AMPK）/sterol regulatory element binding transcription factor 1 （SREBP1） signaling pathway in foam ceils. Methods Mouse macrophages Raw264.7 cells were exposed to oxidized low density lipoprotein （oxLDL） to induce the formation of foam cells. The cells were incubated with oxLDL （50 μg/mL）, liraglutide （0.1, 0.5, 1 and 2 nmol/L） or exendin-3 （9-39） （1, 10 and 100 nmol/L） alone, or in combination. Oil Red O staining was used to detect intracellular lipid droplets. The levels of TG and cholesterol were measured using the commercial kits. Oxidative stress was determined by measuring intracellular reactive oxygen species （ROS）, malondialdehyde （MDA） and superoxide dismutase 1 （SOD）. Western blot analysis was used to examine the expression of AMPKal, SREBP1, phosphory- lated AMPKal, phosphorylated SREBP1, glucagon-like peptide-1 （GLP-1） and GLP-1 receptor （GLP-1R）. Results Oil Red O staining showed that the cytoplasmic lipid droplet accumulation was visibly decreased in foam cells by treatment with liraglutide. The TG and cholesterol content in the liraglutide-treated foam cells was significantly decreased. In addition, foam ceils manifested an impaired oxidative stress following liraglutide treatment, as evidenced by increased SOD, and decreased ROS and MDA. However, these effects of liraglutide on foam cells were attenuated by the use of GLP-IR antagonist exendin-3 （9-39）. Furthermore, we found that the expression level of AMPKa 1 and phosphorylated AMPKct 1 was significantly increased while the expression level of SREBP 1 and phosphorylated SREBP 1 was significantly decreased in foam cells following treatment with liraglutide. Conclusions This study for the first time demonstrated that the effect of liraglutide on reducing oxidative stress and fatty degeneration in oxLDL-induced Raw264.7 cells is accompanied by the alteration of AMPK/SREBP1 pathway. This study provided a potential molecular mechanism for the effect of liraglutide on reducing oxidative stress and fatty degeneration.

Jian-Gan-Xiao-Zhi decoction ameliorates high-fat high-carbohydrate diet-induced non-alcoholic fatty liver disease and insulin resistance by regulating the AMPK/JNK pathway

Background:Non-alcoholic fatty liver disease(NAFLD)can cause insulin resistance(IR)and diabetes.Our previous studies have demonstrated that Jian-Gan-Xiao-Zhi decoction(JGXZ)could be effective for the treatment of NAFLD and IR.However,the possible mechanism underlying the effects of JGXZ on NAFLD and IR remains unknown.Methods:Fifty rats received a high-fat high-carbohydrate(HFHC)diet for 12 weeks to induce NAFLD.After 4 weeks of HFHC treatment,rats were orally treated with JGXZ(8,16,and 32 g/kg weight)for 8 weeks.Ten rats in the control group received standard chow.In the positive control group,rats were orally treated with metformin(90 mg/kg weight)for 8 weeks.After JGXZ and metformin treatment,H&E staining was conducted on rat livers and serum biochemical markers,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),and total cholesterol(TC),were measured using test kits.Moreover,a fasting blood glucose test and an oral glucose tolerance test(OGTT)were conducted.Serum levels of insulin were determined using ELISA kit,and the homeostatic model assessment of insulin resistance(HOMA-IR)was calculated.The levels of total insulin receptor substrate-1(IRS1),AMP-activated protein kinase-α(AMPKα)and c-Jun N-terminal kinase(JNK)as well as the levels of phosphorylation of IRS1(p-IRS1),phosphorylation of AMPK(p-AMPK)and phosphorylation of JNK(p-JNK)were measured using western blotting.Results:The body weights in JGXZ low-,middle-,and high-dose groups were lower than those in the model group(P<0.05,P<0.01,P<0.01,respectively).The serum levels of AST(P<0.05 in JGXZ middle-and high-dose groups),ALT(P<0.01 in JGXZ middle-dose group and P<0.05 in JGXZ high-dose group),TG(P<0.01 in JGXZ middle-and high-dose groups),and TC(P<0.01)upon JGXZ treatment were lower those than in NAFLD model rats.H&E staining showed that JGXZ treatment reduced steatosis of the hepatocytes in NAFLD model rats.JGXZ decreased the levels of fasting blood glucose(P<0.01),HOMA-IR(P<0.01),AUC(area under the curve)of the OGTT(P<0.05)and p-IRS1(P<0.01 in JGXZ middle-and high-dose groups,P<0.05 in JGXZ low-dose groups).Moreover,JGXZ regulated the hepatic AMPKα/JNK pathway in NAFLD model rats,which reflected the induction of p-AMPKαand inhibition of p-JNK.Conclusion:This study showed that JGXZ improved liver function and reduced steatosis of the hepatocytes in NAFLD model rats.Moreover,JGXZ improved IR in NAFLD model rats.The possible mechanism underlying the effects of JGXZ on NAFLD and IR involves the modulation of the AMPK/JNK pathway.

MIR-448 Regulates MAGEA6/AMPK Signaling Pathway in Hepatocellular Carcinoma Tumor Stem Cells

Objective: To explore the role of miR-448 in regulating MAGEA6/AMPK signaling pathway in the biological study of hepatocellular carcinoma (HCC) tumor stem cells. Methods: Using the database, the hepatocellular carcinoma related expression chips were obtained and the regulatory mirnas of candidate genes were predicted, and the predicted results were analyzed. The effects of miR-448 and MAGEA6 on the pellet formation rate and clone formation rate of hepatocellular carcinoma stem cells were detected by immunofluorescence identification of stem cell markers and light microscope counting method. The effects of miR-448 and MAGEA6 on migration and invasion of hepatocellular carcinoma stem cells were detected by scratch and Transwell assay. Dual luciferase reporter assay to verify whether miR-448 targets MAGEA6. The expression and influence of miR-448 on MAGEA6 and AMPK pathway were detected by qRT-PCR and Western blot. Results: It was found that miR-448 may directly regulate the expression of MAGEA6. Overexpression of miR-448 inhibited the characteristics, proliferation, migration, and invasion of hepatocellular carcinoma stem cells in vitro, as well as the ability of xenograft tumor formation in vivo. However, inhibition of miR-448 showed opposite results. In addition, miR-448 directly targets MAGEA6 and regulates AMPK signaling. Silencing MAGEA6 and adding AMPK activator further verified that miR-448 activated AMPK signaling pathway by targeting MAGEA6, thus affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. Conclusions: Our results reveal that miR-448 activates AMPK signaling pathway by targeting MAGEA6, thereby affecting characteristics, proliferation, migration and invasion of hepatoma stem cells. It is suggested that overexpression of miR-448 may be a new therapeutic strategy for hepatocellular carcinoma.

Mu-Xiang-You-Fang protects PC12 cells against OGD/R-induced autophagy via AMPK/mTOR signaling pathway

OBJECTIVE Mu-Xiang-You-Fang(MXYF)is a classic prescription of Hui medicine,composed of five herbs,which has been used to treat ischemic stroke for many years.However,the potential pharmacological mecha⁃nisms of MXYF remain unclear.The present research is to investigate the neuroprotective effect of MXYF and its role in modulating autophagy via AMPK/mTOR signaling pathway in the PC12 oxygen-glucose deprivation and reperfusion(OGD/R)injury model.METHODS MXYF was extracted by supercritical CO2 fluid extraction apparatus.PC12 OGD/R injury model was established by oxygen-glucose deprivation for 2 h and reperfusion for 24 h.The effects of MXYF on the viability and cytotoxicity of PC12 cells were determined through cell counting kit(CCK-8)assay.Colorimetric method was performed to determine the LDH leakage rate.The calcium concentration was determined by chemical fluorescence method and the mitochondrial membrane potential was determined through flow cytometry.Monodansylcadaverine(MDC)staining was conducted to detect autophagosome formation.The expression of LC3,Beclin1,p62,p-AMPK,ULK1,p-mTOR and p-p70s6k proteins were determined by immunofluorescence and Western blotting analyses.RESULTS MXYF(1,2 and 4 mg·L^-1)could significantly increase the cell viability and mitochondrial membrane potential,while decreased the release of lactate dehydrogenase(LDH)and calcium concentration in PC12 cells.Mechanistic studies showed that MXYF reduced the LC3-II/LC3-I ratio and inhibited the expression of beclin1,p-AMPK and ULK1.In comparison,the expres⁃sion of p-mTOR,p-p70s6k and p62 were significantly enhanced.CONCLUSION MXYF inhibits autophagy after OGD/Rinduced PC12 cell injury through AMPK-mTOR pathway,thus MXYF might have therapeutic potential for treating the ischemic stroke.

Fanlian Huazhuo Formula alleviates high-fat diet-induced nonalcoholic fatty liver disease by modulating autophagy and lipid synthesis signaling pathway

BACKGROUND Fanlian Huazhuo Formula(FLHZF)has the functions of invigorating spleen and resolving phlegm,clearing heat and purging turbidity.It has been identified to have therapeutic effects on type 2 diabetes mellitus(T2DM)in clinical application.Non-alcoholic fatty liver disease(NAFLD)is frequently diagnosed in patients with T2DM.However,the therapeutic potential of FLHZF on NAFLD and the underlying mechanisms need further investigation.AIM To elucidate the effects of FLHZF on NAFLD and explore the underlying hepatoprotective mechanisms in vivo and in vitro.METHODS HepG2 cells were treated with free fatty acid for 24 hours to induce lipid accumulation cell model.Subsequently,experiments were conducted with the different concentrations of freeze-dried powder of FLHZF for 24 hours.C57BL/6 mice were fed a high-fat diet for 8-week to establish a mouse model of NAFLD,and then treated with the different concentrations of FLHZF for 10 weeks.RESULTS FLHZF had therapeutic potential against lipid accumulation and abnormal changes in biochemical indicators in vivo and in vitro.Further experiments verified that FLHZF alleviated abnormal lipid metabolism might by reducing oxidative stress,regulating the AMPKα/SREBP-1C signaling pathway,activating autophagy,and inhibiting hepatocyte apoptosis.CONCLUSION FLHZF alleviates abnormal lipid metabolism in NAFLD models by regulating reactive oxygen species,autophagy,apoptosis,and lipid synthesis signaling pathways,indicating its potential for clinical application in NAFLD.

Effect and Mechanism of Dicliptera chinensis Polysaccharide on miR-141/AMPK/SIRT1 Signaling Pathway in Rats with NAFLD

[Objectives]Non-alcoholic fatty liver disease(NAFLD)rat model was established by feeding high-fat and high-sugar fodder to rats,and the protective effect of Dicliptera chinensis polysaccharide(DCP)on NAFLD rats was studied to explore its potential mechanism.[Methods]45 SD rats were randomly divided into 4 groups:normal control group,model control group and DCP treatment groups(100 and 300 mg/kg).The rats in the normal control group were fed with ordinary fodder,and the rats in other groups were fed with high-fat and high-sugar diet for 14 weeks to establish NAFLD model.From the 9^(th)week,the rats in the DCP treatment groups were given different doses of DCP by intragastric administration(5 mL/kg)for 6 weeks.After the last intragastric administration,the rats fasted for 16 h,and the serum and liver of rats were collected for detection.Hematoxylin-eosin(HE)staining was conducted to observe the histopathological changes of rat liver,and alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor(TNF-α)and micrornA-141(micro RNA-141)were detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression of SIRT1 and adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)in rat liver was detected by western blot.[Results]Compared with the model control group,the inflammatory damage and steatodegeneration of rats in the DCP groups were relieved to varying degrees,and the number of lipid vacuoles significantly reduced.The ALT,AST,TC,TG and LDL-C content in the serum and MDA content in the liver tissue decreased to varying degrees,while the HDL-C,SOD and GSH-Px content increased.The expression of SIRT1 and AMPK increased,while the expression of miR-141,TNF-α,IL-6 and IL-1βdeclined,and the DCP 300 mg/kg treatment group had better improvement effect.[Conclusions]DCP had a certain protective effect on NAFLD rats,which may be related to the regulation of miR-141/AMPK/SIRT1 signaling pathway.

Analysis of feasibility of application of Beishu point acupressure therapy in treating gastroesophageal reflux disease via AMPK / ULK1 mediated autophagy pathway based on "adjusting central axis via pivot" theory

Gastroesophageal reflux disease(GERD)is a digestive system disease characterized by uncomfortable symptoms caused by reflux of gastric contents.It has increased sharply with the development of my country’s society and economy.If there is no reasonable and effective Prevention and treatment measures will inevitably increase the financial burden of patients,and also pose a major threat to the quality of life and health of patients.Cell signal transduction mediated by various receptors participates in the regulation mechanism of the body's various levels of biological functions.By inhibiting or activating its functions,the purpose of curing diseases can be achieved,and cell signal transduction has been used in traditional Chinese medicine.Studying.The theory of"adjusting the central axis"was explored by Professor Xie Sheng through decades of clinical experience.It has been proven in practice to treat GERD.It starts from the model of TCM viscera and expounds that the pathogenesis of GERD involves multiple viscera.Multi-system and multi-factor,explain the correlation of the disease with a variety of zang-fu syndromes,and use this as a basis to guide the clinical use of hidden prescriptions.The back-shu pointer therapy can prevent GERD by correcting the unbalanced state of the viscera and qi machine,and promoting the junction of the two channels of Ren and Du.Based on the theory of"adjusting the hub by the pivot",this article expounds the pathogenesis of GERD from the perspective of traditional Chinese medicine.By consulting the literature and combining with the previous research,it proposes to analyze the methods and methods of Backshu pointer therapy to prevent and treat GERD from the AMPK/ULK1 mediated autophagy pathway.

Dynamic Imaging of Autophagy-lysosomal Pathway and Autophagy Function Following Pulmonary Hypoxia/Reoxygenation In Vitro

Alterations of the autophagy-lysosomal pathway（ALP） and autophagy have been involved in lung ischemia-reperfusion（I/R） injury. However, dynamic imaging of ALP function under lung I/R injury particularly is not fully understood. Here we depicted the live-cell fluorescence imaging of autophagosome to monitor ALP activation and autophagy function. The p As Red2-N1-LC3 vectors were transfected into CRL-2192 NR8383（an alveolar macrophage cell line） and CCL149（an alveolar epithelial cell line） successfully. 0-h, 2-h, 4-h, and 6-h hypoxia/0-h, 2-h, 4-h, and 6-h reoxygenation were then induced with an ALP inhibitor（3-MA） or activator（rapamycin） in the culture of transfected cells separately. ALP activation was conformed by up-regulating AMPK and beclin1 expression. Apoptosis was not obvious in 2-h hypoxia/2-h reoxygenation. p As Red2-N1-LC3 CCL149 and p As Red2-N1-LC3 NR8383 cells revealed gradually enhanced As Red2 from 2-h to 6-h hypoxia/reoxygenation. As Red2 varied sensitively to 3-MA and rapamycin interventions during 2-h hypoxia/reoxygenation. Our data provides a simple method of autophagosome imaging to monitor ALP activation and autophagy function in lung I/R injury.

Loganin regulates glycolipid metabolism by influencing intestinal microbiota and AMPK signaling in obese mice

Objective: We aimed to observe the effects of loganin(Log) on serum glycolipid levels and probe the mechanisms focusing on intestinal flora and AMP-activated protein kinase(AMPK) signaling in obese mice.Methods: A high-fat diet was given for 12 consecutive weeks to generate the obesity model in institute of cancer research(ICR) mice. Body weight was measured weekly and fasting blood glucose(FBG) was determined every 2 weeks. Both the oral glucose tolerance test and the intraperitoneal insulin tolerance test were performed. The serum levels of total cholesterol(TC), triglyceride, high-density lipoproteincholesterol, low-density lipoprotein-cholesterol(LDL-C), and free fatty acids(FFA) were measured. The expression of key proteins in the AMPK signaling pathway in skeletal muscle tissue was detected by immunoblotting, and gut microbiota were characterized using 16S rDNA sequencing.Results: Log significantly decreased the body weight and the FBG in obese mice(P <.05), and it could restore FBG to normal levels. The total cholesterol, LDL-C, and FFA levels were significantly reduced by Log compared with the obese controls(TC: P =.0020;LDL-C: P =.0233;FFA: P =.0127), and the glucose tolerance of animals was significantly improved(P =.0477). The western blot results showed that Log could upregulate the protein expression of Adenosine 5‘-monophosphate(AMP)-activated protein kinase(AMPKa), Sirtuin 1(SIRT1), and peroxisome proliferator-activated receptor-gamma coactivator-1 alpha(PGC1a) in skeletal muscle tissue of obese mice. 16S rDNA sequencing indicated that Log reduced the diversity of the gut flora in feces and altered the floral composition of obese mice.Conclusions: Log was effective in reducing body weight and improving glucolipid metabolism in obese mice, probably through activating AMPK signaling and regulating intestinal microbial diversity.

Dietary L-arginine supplementation influences the muscle fiber characteristics and meat quality of Mongolian sheep through the NO/AMPK/PGC-1α pathway

L-arginine serves as a substrate for the production of nitric oxide(NO)in animals,and it can also impact muscle fiber characteristics and meat quality in these animals.The present study was designed to explore the effects of adding 1%L-arginine to a basal diet regimen on the muscle fiber characteristics and meat quality of Mongolian sheep.Dietary L-arginine supplementation reduced shear force in the longissimus thoracis(LT)and increased a*in biceps femoris(BF)muscles(p<0.05).L-arginine supplementation also increased the proportion of type IIA muscle fiber in the LT(p<0.05)and type I muscle fiber in the BF(p<0.05)while reducing both the diameter and CSA of type IIB muscle fiber in both the LT and BF(CSA in LT,p<0.01;all others,p<0.05).L-arginine treatment was also associated with the upregulation of MyHC IIa(LT),MyHC I(BF),nNOS(LT&BF),AMPKα1(BF),PGC-1α(LT&BF)(PGC-1αin BF,p<0.01;all others,p<0.05),together with an increase in nNOS content(LT,p<0.01;BF,p<0.05).Dietary L-arginine supplementation was associated with a significant increase in the post-slaughter tenderness of lamb meat,which is related to transitions in muscle fiber types.The gene expression and nNOS analysis results generated herein further indicate that this effect is mediated by the NO/AMPK/PGC-1αpathway.Further studies are thus warranted to provide further insight into the role that NO signaling plays in controlling the associations between L-arginine,muscle fiber characteristics,and meat quality.

Effect of Platelet-rich Plasma in Stimulating Macrophage Transformation into M2 Type under AMPK Signaling Pathway

Objective:To explore the effect of platelet-rich plasma(RPR)in stimulating the transformation of pro-inflammatory(M1)macrophages into antiinflammatory(M2)under the adenosine-monophosphate-dependent protein kinase(AMPK)signaling pathway.Methods:Rat peritoneal macrophages(RAW264.7)were cultured and randomly divided into 8 groups:blank control group,LPS group,RPR group A,RPR group B,LPS+RPR(12 h)group,LPS+RPR(24 h)group A,LPS+RPR(24 h)group B,LPS+RPR(24 h)group C.RPR was prepared based on blood donors.The expressions of AMPK signaling pathway-related proteins(AMPK,ULK1,m TOR)and macrophage markers(i NOS,Arg-1)in the blank control group,LPS group,LPS+RPR(12 h)group and LPS+RPR(24 h)group were observed and compared.The expressions of macrophage markers in LPS+RPR(24 h)B and C groups were compared,and the expressions of AMPK and TGF-βin RPR A and B groups were compared.Results:The gray values of AMPK and ULK1 in LPS cells decreased significantly,while those in LPS+RPR(12 h)and LPS+RPR(24 h)A cells increased significantly.The gray values of AMPK and ULK1 in LPS+RPR(24 h)A cells were higher than those in LPS+RPR(12 h)cells(P<0.05).The m TOR gray value of LPS cells was significantly higher than that of LPS+RPR(24 h)A cells,and the m TOR gray value of LPS+RPR(24 h)A cells was significantly lower than that of LPS+RPR(12 h)cells(P<0.05).The expression of i NOS in LPS cells was significantly decreased,the expression of i NOS in LPS+RPR(12 h)and LPS+RPR(24 h)cells was significantly increased,and the expression of i NOS in LPS+RPR(24 h)cells was higher than that in LPS+RPR(12 h)cells(P<0.05).The expression of Arg-1 in LPS cells was significantly decreased,the expression of Arg-1 in LPS+RPR(12 h)and LPS+RPR(24 h)A cells was significantly increased,and the expression of Arg-1 in LPS+RPR(24 h)A cells was higher than that in LPS+RPR(12 h)cells(P<0.05).The i NOS expression level of LPS+PRP(24 h)C cells was significantly higher than that of LPS+PRP(24 h)B cells,and the Arg-1 expression level was significantly lower than that of LPS+PRP(24 h)B cells(P<0.05).The gray values of AMPK and TGF-βin PRP B cells were significantly lower than those in PRP A cells(P<0.05).Conclusion:RPR can stimulate macrophage transformation from M1 to M2 by up-regulating AMPK signaling pathway.

A soy glycinin derived octapeptide protects against MCD diet induced non-alcoholic fatty liver disease in mice

Soy glycinin derived octapeptide(SGP8)is a peptide obtained from degradation of the soy glycinin,whose amino acid sequence is IAVPGEVA.To determine the effect of SGP8 on non-alcoholic fatty liver disease(NAFLD),steatosis Hep G2 cells were induced by 1 mmol/L free fatty acid(FFA)and C57 BL/6 J mice were fed with methionine-choline defi cient(MCD)diet for 3 weeks to establish NAFLD model.The results of oil red O staining and total cholesterol(TC)/triglyceride(TG)contents showed that SGP8 could signifi cantly reduce the lipid content of steatosis Hep G2 cells.In vivo,SGP8 lowered plasma alanine aminotransferase(ALT)and low density lipoprotein(LDL)content,normalized hepatic superoxide dismutase(SOD)and malondialdehyde(MDA)production,and reduced the severity of liver infl ammation.The results of Western blotting showed that SGP8 increased expression of Sirtuin-1(SIRT1)and phosphorylation level of AMP activated protein kinase(AMPK)in hepatocytes.Through activation of SIRT1/AMPK pathway,SGP8 downregulated the expression of sterol regulatory element binding protein 1 c(SREBP-1 c)and its target genes ACC and FAS expression levels,and increased the phosphorylation level of acetyl Co A carboxylase(ACC).Furthermore,SGP8 also upregulated the expression of transcription factor peroxisome proliferator activated receptorα(PPARα),which was regulated by SIRT1/AMPK pathway,and its target gene CPT1 level.In conclusion,SGP8 might improve NAFLD by activating the SIRT1/AMPK pathway.Our data suggest that SGP8 may act as a novel and potent therapeutic agent against NAFLD.

Qili Qiangxin, a compound herbal medicine formula, alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro

Objective: Qili Qiangxin(QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxiareoxygenation(H/R)-induced myocardial injury model.Methods: The main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia–reperfusion injury. Apoptosis, autophagy,and generation of reactive oxygen species(ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.Results: Apoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3Ⅱ and p62 degradation(P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C(at 0.5 μmol/L),and QLQX(250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis(P < 0.01), while AICAR(an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger,N-Acetyl-L-cysteine(NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy(P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK(P < 0.01), which showed a similar effect to QLQX.Conclusion: QLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.

Ephedra Herb extract ameliorates adriamycin-induced nephrotic syndrome in rats via the CAMKK2/AMPK/mTOR signaling pathway

This study aimed to investigate the effect and mechanisms of Ephedra Herb(EH)extract on adriamycin-induced nephrotic syndrome(NS),providing an experimental basis for the clinical treatment of NS.Hematoxylin and eosin staining,creatinine,urea nitrogen,and kidn injury molecule-1 were used to evaluate the activities of EH extract on renal function.The levels of inflammatory factors and oxidative stress were detected by kits.The levels of reactive oxygen species,immune cells,and apoptosis were measured by flow cytometry.A network pharmacological approach was used to predict the potential targets and mechanisms of EH extract in the treatment of NS.The protein levels of apoptosis-related proteins and CAMKK2,p-CAMKK2,AMPK,p-AMPK,mTOR and p-mTOR in the kidneys were detected by Western blot.The effective material basis of EH extract was screened by MTT assay.The AMPK pathway inhibitor(compound C,CC)was added to investigate the effect of the potent material basis on adriamycin-induced cell injury.EH extract significantly improved renal injury and relieve inflammation,oxidative stress,and apoptosis in rats.Network pharmacology and Western blot results showed that the effect of EH extract on NS may be associated with the CAMKK2/AMPK/mTOR signaling pathway.Moreover,methylephedrine significantly ameliorated adriamycin-induced NRK-52e cell injury.Methylephedrine also significantly improved the phosphorylation of AMPK and mTOR,which were blocked by CC.In sum,EH extract may ameliorate renal injury via the CAMKK2/AMPK/mTOR signaling pathway.Moreover,methylephedrine may be one of the material bases of EH extract.

cPKCγ Deficiency Exacerbates Autophagy Impairment and Hyperphosphorylated Tau Buildup through the AMPK/mTOR Pathway in Mice with Type 1 Diabetes Mellitus

Type 1 diabetes mellitus(T1DM)-induced cognitive dysfunction is common,but its underlying mechanisms are still poorly understood.In this study,we found that knockout of conventional protein kinase C(cPKC)γsignificantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles,but did not affect the activities of GSK-3βand PP2A in the hippocampal neurons of T1DM mice.cPKCγdeficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice.Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγdeficiency in T1DM mice.Moreover,cPKCγdeficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro.The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor.In conclusion,these results indicated that cPKCγpromotes autophagy through the AMPK/mTOR signaling pathway,thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.

Anti-hyperglycemic effects of dihydromyricetin in streptozotocin-induced diabetic rats

Dihydromyricetin(DHM),as a bioactive flavanonol compound,is mainly found in“Tengcha”(Ampelopsis grossedentata)cultivated in south of China.This study aimed to investigate the anti-hyperglycemic and antidyslipidemic activities of DHM using type 2 diabetes mellitus(T2D)rats,which was induced by feeding with high fat and fructose diet for 42 days and intraperitoneal administration of streptozocin.Forty-eight freshlyweaned rats were randomly assigned into the negative control(Blank),low dose(100 mg/kg),medium dose(200 mg/kg),high dose(400 mg/kg),and positive(40 mg/kg,met)groups.Fasting blood glucose and body weight were measured at weekly interval.Oral glucose tolerance tests were performed on days 42.The results revealed that DHM possessed significant antihyperglycaemic and antihyperinsulinemic effects.Moreover,after the DHM treatment,p-Akt and p-AMPK expression was upregulated,and glycogen synthase kinase-3β(GSK-3β)expression was downregulated,indicating that the potential anti-diabetic mechanism of DHM might be due to the regulation of the AMPK/Akt/GSK-3βsignaling pathway.

Dietary choline activates the Ampk/Srebp signaling pathway and decreases lipid levels in Pacific white shrimp(Litopenaeus vannamei)

An 8-week feeding trial was conducted in Pacific white shrimp(Litopenaeus vannamei)to evaluate the effects of dietary choline supplementation on choline transport and metabolism,hepatopancreas histological structure and fatty acid profile,and regulation of lipid metabolism.Six isonitrogenous and isolipidic diets were formulated to contain different choline levels of 2.91(basal diet),3.85,4.67,6.55,10.70 and 18.90 g/kg,respectively.A total of 960 shrimp(initial weight,1.38±0.01 g)were distributed randomly into twenty-four 250-L cylindrical fiber-glass tanks,with each diet assigned randomly to 4replicate tanks.The results indicated that dietary choline significantly promoted the deposition of choline,betaine and carnitine(P<0.05).The diameters and areas of R cells,total lipid and triglyceride contents in hepatopancreas,and triglyceride and non-esterified fatty acid contents in hemolymph were negatively correlated with dietary choline level.The contents of functional fatty acids in hepatopancreas,the activity of acetyl-CoA carboxylase(Acc),and the mRNA expression of fas,srebp and acc were highest in shrimp fed the diet containing 4.67 g/kg choline,and significantly higher than those fed the diet containing 2.91 g/kg,the lowest level of choline(P<0.05).The number of R cells,content of very lowdensity lipoprotein(VLDL),activities of carnitine palmitoyl-transferase(Cpt1),lipoprotein lipase and hepatic lipase,and the mRNA expression levels of cpt1,fabp,fatp,ldlr,and ampk in hepatopancreas increased significantly as dietary choline increased(P<0.05).In addition,hepatopancreas m RNA expression levels of ctl1,ctl2,oct1,badh,bhmt,ck,cept,and cct were generally up-regulated as dietary choline level increased(P<0.01).In conclusion,dietary choline promoted the deposition of choline and its metabolites by up-regulating genes related to choline transport and metabolism.Moreover,appropriate dietary choline level promoted the development of hepatopancreas R cells and maintained the normal accumulation of lipids required for development,while high dietary choline not only promoted hepatopancreas lipid export by enhancing VLDL synthesis,but also promoted fatty acidβ-oxidation and inhibited de novo fatty acid synthesis by activating the Ampk/Srebp signaling pathway.These findings provided further insight and understanding of the mechanisms by which dietary choline regulated lipid metabolism in L.vannamei.

A new peptide,VD11,promotes structural and functional recovery after spinal cord injury

The regenerative capacity of the central nervous system is very limited and few effective treatments are currently available for spinal cord injury.It is therefore a priority to develop new drugs that can promote structural and functional recovery after spinal cord injury.Previous studies have shown that peptides can promote substantial repair and regeneration of injured tissue.While amphibians have a pronounced ability to regenerate the spinal cord,few studies have investigated the effect of amphibian spinal cord-derived peptides on spinal cord injury.Here we report for the first time the successful identification and isolation of a new polypeptide,VD11(amino acid sequence:VDELWPPWLPC),from the spinal cord of an endemic Chinese amphibian(Odorrana schmackeri).In vitro experiments showed that VD11 promoted the secretion of nerve growth factor and brain-derived neurotrophic factor in BV2 cells stimulated with lipopolysaccharide,as well as the proliferation and synaptic elongation of PC12 cells subjected to hypoxia.In vivo experiments showed that intravertebral injection of VD11 markedly promoted recovery of motor function in rats with spinal cord injury,alleviated pathological damage,and promoted axonal regeneration.Furthermore,RNA sequencing and western blotting showed that VD11 may affect spinal cord injury through activation of the AMPK and AKT signaling pathways.In summary,we discovered a novel amphibian-derived peptide that promotes structural and functional recovery after spinal cord injury.

Salicylic acid sensitizes cervical cancer cells to radiotherapy by activating AMPK/TSC2/mTOR pathway

Objective:To investigate the radio-sensitizing effect of salicylic acid(SA)on human cervical cancer cells and its potential molecular mechanism.Methods:Cervical cancer cells were treated with SA and ionizing radiation.The expression ofγ-H2AX was evaluated by immunofluorescence(IF)assay.Cell cycle and apoptosis were analyzed by flow cytometry.Western blot was performed to detect the protein level of AMPK/TSC2/mTOR pathway.Results:SA inhibited basal proliferation of cervical cancer cells in a dose and time dependent manner.In addition,SA increased radiation-induced DNA damage,promoted apoptosis,triggered a redistribution of cell cycle from G2-M phase to G1-S phase of cervical cancer cells,and hence increased cell sensitivity to radiation.Moreover,SA treatment elevated the expression levels of p-AMPKα(t=3.996,P<0.05)and p-TSC2(t=5.308,P<0.05),whereas the level of p-mTOR(t=10.160,P<0.05)was significantly decreased.Conclusion:SA enhances the radiosensitivity of cervical cancer cells by targeting AMPK/TSC2/mTOR signaling pathway,and might serve as a promising therapeutic strategy to improve the efficacy of radiotherapy for cervical cancer.