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.

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.

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.

Mechanism of Profilin-1 in regulating eNOS/NO signaling pathway and its role in hypertensive myocardial hypertension

Objective:To explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy.Methods:Spontaneously hypertensive rats(SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1expression knockdown(SHR-I) or over express(SHR-H) or to use as control(SHR-C).All these treatment were compared with Wistar-Kyoto rats(SKY) treated with control adenovirus vectors(WKY-C).The same injection was executed at the sixth week during the experiment of 12 weeks.After experiment,the left ventricular weight-to-heart weight ratio(LVW/HW)and left ventricular long axis(LVLA) were measured.Meanwhile.NO contents in blood and myocardium,Profilin-1,eNOS and Caveolin-3 niRNA and protein levels and phosphorylated eNOS(P-eNOS) protein level in myocardium were determined.Results:Compared with WKY-C group,the SHR-C group was statistically higher in LVW/HW(0.79±0.03).LVLA(11.82±0.58 mm) and Profilin-1 niRNA and prolein level(P<0.05).but lower in NO content[(18.63±6.23) μmol/L| in blood and[(2.71±0.17) μmol/L]in myocardium).eNOS activity and Caveolin-3 expression(P<0.05).The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW[(0.93±0.03) mm and LVLA(14.17±0.69) mm]in comparison with SHR-C group(P<0.05).and to a lower value of NO content(in myocardium).eNOS activity and Caveolin-3 expression(P<0.05):however,this phenomenon was reversed by the knockdown Profilin-1 expression(SHR-I group).Conclusions:Profilin-1 expression,being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity,promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mT OR-ULK1 signaling pathway

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

麻元通便止痛汤调控AMPK/eNOS信号通路改善大鼠慢传输型便秘的作用机制

目的研究麻元通便止痛汤调控AMP活化蛋白激酶(AMPK)/内皮型一氧化氮合酶(eNOS)信号通路改善大鼠慢传输型便秘(STC)的作用机制。方法将大鼠随机分为空白组、模型组和麻元通便止痛汤低、中、高剂量组(6、12、18 mg/kg),每组10只。除空白组外,其余各组大鼠灌胃复方地芬诺酯混悬液复制STC模型。造模成功后,空白组和模型组大鼠灌胃生理盐水,麻元通便止痛汤各剂量组大鼠灌胃相应药物,每天1次,连续2周。观察大鼠治疗前后的粪便数量及含水率;计算大鼠的炭末推进率;观察大鼠结肠组织的病理学变化;检测大鼠结肠组织中一氧化氮(NO)、一氧化氮合酶(NOS)水平及AMPK、eNOS、哺乳动物雷帕霉素靶蛋白(mTOR)、结节性硬化复合物1(Tsc-1)、Tsc-2、真核启动因子4E结合蛋白(4ebp)的表达水平;筛选麻元通便止痛汤君药火麻仁、枳壳、生地的活性成分,选择Degree值最高的活性成分与AMPK、eNOS进行分子对接,以验证相互作用。结果与治疗前比较,麻元通便止痛汤各剂量组大鼠粪便数量及含水率均显著增加(P<0.05)。与空白组比较,模型组大鼠炭末推进率显著降低(P<0.05);结肠组织结构紊乱,黏膜下层见大量炎症细胞浸润;结肠组织中NO、NOS水平和AMPK、eNOS、mTOR、Tsc-1、Tsc-2、4ebp蛋白表达水平均显著升高(P<0.05)。与模型组比较,麻元通便止痛汤各剂量组上述指标(低剂量组NOS除外)均显著逆转(P<0.05)。分子对接结果显示,火麻仁、枳壳、生地中Degree值最高的活性成分分别为(Z)-3-(4-hydroxy-3-methoxy-phenyl)-N-[2-(4-hydroxyphenyl)ethyl]acrylamide、nobiletin、stigmasterol,这3种成分与AMPK的结合能分别为-5.15、-4.61、-4.83 kJ/mol,与eNOS的结合能分别为-6.11、-5.40、-5.91 kJ/mol,且结合构象稳定、结合活性较高。结论麻元通便止痛汤可改善STC模型大鼠的便秘症状和肠道功能,其作用机制可能与抑制AMPK/eNOS信号通路有关。

基于AMPK/eNOS/NF-κB信号通路探讨虾青素对脑梗死大鼠的影响及作用机制

目的基于AMPK/eNOS/NF-κB信号通路探讨虾青素对脑梗死大鼠的影响及作用机制。方法采用改良的Longa法建立脑梗死大鼠模型,取50只造模成功的SD雄性大鼠随机分为模型对照组、阳性对照组、虾青素低、中、高剂量组,每组10只,另外未造模的10只作为空白对照组。空白对照组与模型对照组均给予等体积生理盐水,阳性对照组给予20mg/(kg·d)尼莫地平,虾青素低、中、高剂量组分别给予10、20、30mg/(kg·d)虾青素,连续给药14天。检测大鼠神经功能变化;氯化三苯基四氮唑染色法检测脑梗死面积;酶联免疫吸附法检测白细胞介素10(IL-10)、IL-1β、IL-6和肿瘤坏死因子α(TNF-α)水平;TUNEL法检测脑皮质缺血半影区的细胞凋亡情况;反转录-聚合酶链反应检测腺苷酸活化蛋白激酶(AMPK)、内皮型一氧化氮合酶(eNOS)、核因子κB(NF-κB)mRNA表达水平;Westernblot检测p-AMPK、AMPK、p-eNOS、eNOS、NF-κB蛋白表达。结果虾青素可明显改善脑梗死大鼠的神经功能及脑梗死面积(P<0.01);抑制促炎因子IL-1β、IL-6和TNF-α表达,促进抗炎因子IL-10表达(P<0.05);降低皮质缺血半影区细胞凋亡率(P<0.01);下调NF-κB mRNA和蛋白表达,促进AMPK和eNOS磷酸化(P<0.05),但AMPK、eNOSmRNA和蛋白表达无显著差异(P>0.05)。结论虾青素能明显改善脑梗死大鼠神经功能、脑梗死面积、皮质缺血半影区细胞凋亡率及血清细胞因子,其作用机制可能与AMPK/eNOS/NF-κB信号通路密切相关。

LncRNAs are involved in regulating ageing and age-related disease through the adenosine monophosphate-activated protein kinase signalling pathway

A long noncoding RNA(lncRNA)is longer than 200 bp.It regulates various biological processes mainly by interacting with DNA,RNA,or protein in multiple kinds of biological processes.Adenosine monophosphate-activated protein kinase(AMPK)is activated during nutrient starvation,especially glucose starvation and oxygen deficiency(hypoxia),and exposure to toxins that inhibit mitochondrial respiratory chain complex function.AMPK is an energy switch in organisms that controls cell growth and multiple cellular processes,including lipid and glucose metabolism,thereby maintaining intracellular energy homeostasis by activating catabolism and inhibiting anabolism.The AMPK signalling pathway consists of AMPK and its upstream and downstream targets.AMPK upstream targets include proteins such as the transforming growth factor b-activated kinase 1(TAK1),liver kinase B1(LKB1),and calcium/calmodulindependent protein kinase b(CaMKKb),and its downstream targets include proteins such as the mechanistic/mammalian target of rapamycin(mTOR)complex 1(mTORC1),hepatocyte nuclear factor 4a(HNF4a),and silencing information regulatory 1(SIRT1).In general,proteins function relatively independently and cooperate.In this article,a review of the currently known lncRNAs involved in the AMPK signalling pathway is presented and insights into the regulatory mechanisms involved in human ageing and age-related diseases are provided.

基于AMPK/eNOS/NF-κB信号通路观察丁苯酞对脑梗死大鼠的保护作用

目的基于腺苷酸活化蛋白激酶(AMPK)/内皮型一氧化氮合酶(eNOS)/核转录因子κB(NF-κB)(AMPK/eNOS/NF-κB信号通路)探讨丁苯酞(NBP)对脑梗死大鼠的保护机制。方法将80只12~15周龄的SPF级Wistar雄性大鼠随机分为4组(n=20):假手术组(sham组)、模型组、丁苯酞组(NBP组)和阳性对照药物尼莫地平组(NMDP组),采用线栓法构建大鼠永久性中动脉栓塞模型,造模手术结束3h后,NBP组、NMDP组分别腹腔注射20 mg/kg NBP和NMDP,注射计量10 mL,每日定点注射1次,sham和模型组腹腔给予等量生理盐水,连续给药14 d。第14天给药后,对大鼠进行神经功能缺损程度(NDS)评分,依次采用激光散斑成像、TTC染色、尼氏染色、ELISA法,免疫荧光法检测各组大鼠大脑皮层血流变化、脑梗死体积、脑组织内神经元的损伤、脑组织中IL-1β、IL-6和TNF-α表达水平、NF-κB的核移位,Western blot检测p-AMPK、p-eNOS和NF-κB蛋白的表达水平。结果与sham组相比,模型组大鼠神经功能损伤评分明显升高,大脑皮层血流灌注量明显减少,脑梗死体积明显增大,神经细胞凋亡率,IL-1β、IL-6和TNF-α表达水平,NF-κB入核的免疫荧光强度,p-AMPK、p-eNOS、NF-κB蛋白表达水平均升高,差异有统计学意义(P<0.05);与模型组相比,NBP组、NMDP组神经功能损伤评分明显下降,大脑皮层血流灌注量明显增加,脑梗死体积明显减小,神经细胞凋亡率,IL-1β、IL-6和TNF-α表达水平,NF-κB入核的免疫荧光强度,NF-κB蛋白表达水平均降低,p-AMPK、p-eNOS表达水平均升高,差异有统计学意义(P<0.05);NBP组和NMDP组相比,各指标相比组间差异无统计学意义(P>0.05)。结论丁苯酞对脑梗死大鼠的保护作用,与AMPK/eNOS/NF-κB信号通路有关,其机制可能是AMPK/eNOS被激活,抑制NF-κB的活化,从而减轻炎症反应,发挥对脑梗死后的脑组织的保护作用。

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.

Yiqi Yangyin and Huatan Quyu granule can improve skeletal muscle energy metabolism in a type 2 diabetic rat model by promoting the AMPK/SIRT/PGC-1α signalling pathway

Objective:To investigate how Yiqi Yangyin and Huatan Quyu granule (YYHO) improves skeletal muscle insulin resistance in a type 2 diabetic rat model and to discover whether the molecular mechanism is related to the promotion of the AMPK/SIRT/PGC-1α signalling pathway.Methods:Rats were randomly divided into 4 groups:the normal group,the model group,the YYHQ granule group,and the pioglitazone group.The type 2 diabetic rat model was established by feeding a high-fat diet for 5 weeks along with a single intraperitoneal injection of 30 mg/kg streptozotocin (STZ).After modelling successfully,the appropriate drug was intragastrically administered to diabetic rats for 2 weeks,once per day.The YYHQ granule group was given a dose of 4.8 g/kg body weight per day,the pioglitazone group was given a dose of 1.35 mg/kg body weight per day.The doses for both groups were equivalent to the clinical equivalent dose based on a previous study.Other groups were gavaged with the same amount of saline water.Body weight,food intake,water intake,urine volume and grip strength were recorded weekly.The fasting blood glucose(FBG) was determined weekly using blood glucose test strips.The related glucose and lipid metabolism indexes,e.g.,fasting insulin (Fins),glycated haemoglobin (GHb),HOMA-IR,ISI,triglycerides (TG),total cholesterol (TC),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C) and free fatty acid (FFA),were determined using biochemical method.The mRNA expression levels of adenosine monophosphate-activated protein kinase (AMPK),peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α),carnitine palmitoyl transterase-1 (CPT-1),Sirtuin 1 (SIRT1),and Sirtuin 3 (SIRT3) were assessed using quantitative real-time PCR (qRT-PCR).The protein expression levels of creatine kinase (CK),Ca2+ ATPase,α-Actin,AMPK,PGC-1α and CPT-1 were determined using enzyme-linked immunosorbent assay method (ELISA).Results:Body weight decreased significantly (P <.01),food intake,water intake and urine volume increased significantly (P <.01),and grip strength decreased significantly (P <.01) in the model group compared with the normal group.The levels of FBG,Fins,GHb and HOMA-IR increased significantly (P <.01),and the ISI decreased significantly (P <.01) in the model group.The levels of TG,TC,LDL-C and FFA increased significantly (P <.05 or P <.01),and the level of HDL-C decreased significantly (P <.05) in the model group.These changes were reversed after treatment with YYHQ granule or pioglitazone.Compared with the model group,the YYHQ granule and pioglitazone groups significantly improve body weight,water intake and urine volume (P <.05 or P <.01),however,both treatments had no significant effect on food intake (P >.05).The levels of FBG,Fins,GHb,HOMA-IR and ISI were improved significantly (P <.01) and the levels of TG,TC and LDL-C were improved significantly (P <.05 or P <.01),however,both treatments had no significant effect on the levels of HDL-C and FFA (P >.05).Further results indicated that YYHQ granule significantly decreased the mRNA expression of AMPK,PGC-1α,CPT-1,SIRT1 and SIRT3 in skeletal muscle (P <.01) and the pioglitazone group showed similar effects;moreover,the protein expression levels of CK,Ca2+ATPase,α-Actin,AMPK,PGC-1α and CPT-1 in skeletal muscle significantly decreased (P <.01),however,pioglitazone had no significant effect on CK and α-Actin (P >.05).Conclusion:The possible molecular mechanism of YYHQ granule improving skeletal muscle insulin resistance in a type 2 diabetic rat model may be related to the stimulation of energy metabolism in skeletal muscle via the AMPK/SIRT/PGC-1α signalling 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.

Hypoglycemic effect and the mechanism of action of a polysaccharide from sweet corncob in a high-fat diet and streptozotocin-induced diabetic mice

Type 2 diabetes mellitus(T2DM)is a metabolic disease caused by a glycolipid metabolism disorder and isletβ-cell dysfunction.SCP-80-I is a biologically active water-soluble polysaccharide isolated from sweet corncob,an agricultural byproduct.The hypoglycemic effects of SCP-80-I on T2DM mice and its mechanisms were investigated in this study.SCP-80-I was found to significantly reduce blood glucose and lipid deposition levels in T2DM mice,as well as decrease serum leptin and increase adiponectin secretion.Interestingly,real time-polymerase chain reaction(RT-PCR)and Western blotting results revealed that SCP-80-I could regulate the expression of several glycolipid metabolisms and insulin secretion genes and proteins,including 5'-AMP-activated protein kinase(AMPK),carnitine palmitoyltransferase I(CPTI),and acetyl coenzyme A carboxylase(ACC)in the liver and AMPK,sirtuin1(Sirtl),peroxisome proliferator-activated receptorycoactivator-1(PGC-1α),and uncoupling protein 2(UCP2)in the pancreas.To have a hypoglycemic effect,SCP-80-1 regulated glycolipid metabolism and islet cell function in the liver by regulating the AMPK/AC C/CPT1 signaling pathway and the AMPK/Sirt1/PGC-1αand AMPK/Sirtl/UCP2 signaling pathways.These findings improve our understanding of polysaccharides derived from sweet corncob and the use of SCP-80-I in the production of hypoglycemic foods.

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.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats

Objective To investigate the influence of electroacupuncture(EA)on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase(PI3K/Akt/eNOS)signaling pathway in spontaneously hypertensive rats(SHRs).Methods Eight Wistar-Kyoto rats were used as the healthy blood pressure(BP)control(normal group),and 32 SHRs were randomized into model group,EA group,EA plus ghrelin group(EA+G group),and EA plus PF04628935 group(a potent ghrelin receptor blocker;EA+P group)using a random number table.Rats in the normal group and model group did not receive treatment,but were immobilized for 20 min per day,5 times a week,for 4 continuous weeks.SHRs in the EA group,EA+G group and EA+P group were immobilized and given EA treatment in 20 min sessions,5 times per week,for 4 weeks.Additionally,1 h before EA,SHRs in the EA+G group and EA+P group were intraperitoneally injected with ghrelin or PF04628935,respectively,for 4 weeks.The tail-cuff method was used to measure BP.After the 4-week intervention,the rats were sacrificed by cervical dislocation,and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin(HE)staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of ghrelin,nitric oxide(NO),endothelin-1(ET-1)and thromboxane A2(TXA2)in the serum.Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation.Western blot was used to measure the expression of PI3K,Akt,phosphorylated Akt(p-Akt)and eNOS proteins in the abdominal aorta.Further,quantitative real-time polymerase chain reaction(qRT-PCR)was conducted to measure the relative levels of mRNA expression for PI3K,Akt and eNOS in the abdominal aorta.Results EA significantly reduced the systolic BP(SBP)and diastolic BP(DBP)(P<0.05).HE staining showed that EA improved the morphology of the vascular endothelium to some extent.Results of ELISA indicated that higher concentrations of ghrelin and NO,and lower concentrations of ET-1 and TXA2 were present in the EA group(P<0.05).The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group.Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K,p-Akt/Akt and eNOS proteins,as well as expression levels of PI3K,Akt and eNOS mRNAs(P<0.05).In the EA+G group,SBP and DBP decreased(P<0.05),ghrelin concentrations increased(P<0.05),and the concentrations of ET-1 and TXA2 decreased(P<0.05),relative to the EA group.In addition,the levels of PI3K and eNOS proteins,the p-Akt/Akt ratio,and the expression of PI3K,Akt and eNOS mRNAs increased significantly in the EA+G group(P<0.05),while PF04628935 reversed these effects.Conclusion EA effectively reduced BP and protected the vascular endothelium,and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

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.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

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.

四味黄芪散降低低氧性肺动脉高压的机制研究

目的探讨低氧不同时相内皮型一氧化氮合酶(eNOS)信号通路参与藏药四味黄芪散降低低氧性肺动脉高压的机制。方法将110只SD大鼠随机分为常氧组、低氧对照组和低氧药物组,后两组根据暴露低氧时间又各分为5个组。常氧组大鼠暴露于西宁地区(海拔2260 m)大气环境,正常喂养;10个低氧组置于模拟海拔5000 m的低压氧舱,其中,5个低氧药物组灌胃给予四味黄芪散混悬液,5个低氧对照组灌胃给予生理盐水,每日1次,持续30 d。分别于暴露低氧第1、3、7、15、30天时测定肺动脉压(PAP)、左右心室比值;并采集肺组织标本,用WB法测定肺组织中腺苷酸活化蛋白激酶(AMPK)、eNOS、内皮素1(ET-1)蛋白的含量。结果与常氧组比较,低氧对照组大鼠的PAP和左右心室比值逐渐增高,于暴露低氧第7天起显著升高,并随暴露低氧时间的进一步延长而持续增高;低氧药物组大鼠的PAP也随暴露低氧时间的延长而增高,但其增高幅度明显低于低氧对照组。低氧药物组大鼠肺组织eNOS蛋白的水平在急性低氧15 d内均低于常氧组和低氧对照组;于低氧第15天后,低氧药物组eNOS蛋白的水平逐渐升高,超过低氧对照组,于低氧30 d时显著增高,甚至超过常氧组水平。低氧下,eNOS蛋白水平的表达趋势与肺组织中AMPK蛋白及Beclin-1的水平呈高度相关。低氧对照组大鼠肺组织中ET-1蛋白的水平随暴露低氧时间的延长而增高,于低氧第7天时达峰值;低氧药物组大鼠肺组织中ET-1的水平无论在急性低氧期或慢性低氧期均显著低于低氧对照组。结论eNOS蛋白在不同低氧时相呈现不同的水平,四味黄芪散降低低氧性肺动脉高压的机制也不同。急性低氧期主要通过下调ET-1的水平来降低PAP;慢性低氧期主要通过上调eNOS的水平及调控AMPK-eNOS信号通路,抑制平滑肌细胞增殖,降低PAP。