AMP-activated Protein Kinase Expression Research Articles

Heme Oxygenase-1-Modified BMMSCs Activate AMPK–Nrf2–FTH1 to Reduce Severe Steatotic Liver Ischemia–Reperfusion Injury

BackgroundIschemia–reperfusion injury (IRI) is an important cause of graft dysfunction post-liver transplantation, where donor liver with severe steatosis is more sensitive to IRI. Liver IRI involves ferroptosis and can be alleviated by heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs).AimsTo explore the role and mechanism of HO-1/BMMSCs in severe steatotic liver IRI.MethodsA severe steatotic liver IRI rat model and a hypoxia/reoxygenation (H/R) of severe steatosis hepatocyte model were established. Liver and hepatocyte damage was evaluated via liver histopathology and cell activity. Ferroptosis was evaluated through ferroptosis indexes. Nuclear factor erythroid 2-related factor 2 (Nrf2) was knocked down in severe steatotic hepatocytes. The role of Nrf2 and AMPK in HO-1/BMMSC inhibition of ferroptosis was examined using the AMP-activated protein kinase (AMPK) pathway inhibitor Compound C.ResultsThe HO-1/BMMSCs alleviated severe steatotic liver IRI and ferroptosis. HO-1/BMMSCs promoted ferritin heavy chain 1(FTH1), Nrf2, and phosphorylated (p)-AMPK expression in the H/R severe steatotic hepatocytes. Nrf2 knockdown decreased FTH1 expression levels but did not significantly affect p-AMPK expression levels. The protective effect of HO-1/BMMSCs against H/R injury in severe steatotic hepatocytes and the inhibitory effect on ferroptosis were reduced. Compound C decreased p-AMPK, Nrf2, and FTH1 expression levels, weakened the HO-1/BMMSC protective effect against severe steatotic liver IRI and H/R-injured severe steatotic hepatocytes, and reduced the inhibition of ferroptosis.ConclusionsFerroptosis was involved in HO-1/BMMSC reduction of severe steatotic liver IRI. HO-1/BMMSCs protected against severe steatotic liver IRI by inhibiting ferroptosis through the AMPK–Nrf2–FTH1 pathway.Graphical HO-1/BMMSCs activate AMPK, which activates Nrf2, promotes its nuclear transcription, then promotes the expression of its downstream protein FTH1, thereby inhibiting ferroptosis and attenuating severe steatotic liver IRI in rats. Glu: glutamic acid; Cys: cystine; GSH: glutathione; GPX4: glutathione peroxidase 4; HO-1/BMMSCs: HO-1-modified BMMSCs; Fer-1: ferrostatin-1; DFO: deferoxamine; FTH1: ferritin heavy chain1; p-AMPK: phosphorylated AMP-activated protein kinase; Nrf2: nuclear factor erythroid 2-related factor 2; IRI: ischemia-reperfusion injury; MCD: methionine-choline deficiency

Differential G Protein-Coupled Estrogen Receptor-1 Regulation of Counter-Regulatory Transmitter Marker and 5′-AMP-Activated Protein Kinase Expression in Ventrolateral versus Dorsomedial Ventromedial Hypothalamic Nucleus

Introduction: The ventromedial hypothalamic nucleus (VMN) is an estrogen receptor (ER)-rich structure that regulates glucostasis. The role of nuclear but not membrane G protein-coupled ER-1 (GPER) in that function has been studied. Methods: Gene silencing and laser-catapult microdissection/immunoblot tools were used to examine whether GPER regulates transmitter and energy sensor function in dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN counter-regulatory nitrergic and γ-Aminobutyric acid (GABA) neurons. Results: Intra-VMN GPER siRNA administration to euglycemic animals did not affect VMNdm or -vl nitrergic neuron nitric oxide synthase (nNOS), but upregulated (VMNdm) or lacked influence on (VMNvl) GABA nerve cell glutamate decarboxylase_65/67 (GAD) protein. Insulin-induced hypoglycemia (IIH) caused GPER knockdown-reversible augmentation of nNOS, 5′-AMP-activated protein kinase (AMPK), and phospho-AMPK proteins in nitrergic neurons in both divisions. IIH had dissimilar effects on VMNvl (unchanged) versus VMNdm (increased) GABAergic neuron GAD levels, yet GPER knockdown affected these profiles. GPER siRNA prevented hypoglycemic upregulation of VMNvl and -dm GABA neuron AMPK without altering pAMPK expression. Conclusions: Outcomes infer that GPER exerts differential control of VMNdm versus -vl GABA transmission during glucostasis and is required for hypoglycemic upregulated nitrergic (VMNdm and -vl) and GABA (VMNdm) signaling. Glycogen metabolism is reported to regulate VMN nNOS and GAD proteins. Data show that GPER limits VMNvl glycogen phosphorylase (GP) protein expression and glycogen buildup during euglycemia but mediates hypoglycemic augmentation of VMNvl GP protein and glycogen content; VMNdm glycogen mass is refractory to GPER control. GPER regulation of VMNvl glycogen metabolism infers that this receptor may govern local counter-regulatory transmission in part by astrocyte metabolic coupling.

Quercetin Attenuates Atherosclerosis via Modulating Apelin Signaling Pathway Based on Plasma Metabolomics.

To interpret the pharmacology of quercetin in treatment of atherosclerosis (AS). Fourteen apolipoprotein E-deficient (ApoE-/-) mice were divided into 2 groups by a random number table: an AS model (ApoE-/-) group and a quercetin treatment group (7 in each). Seven age-matched C57 mice were used as controls (n=7). Quercetin [20 mg/(kg·d)] was administered to the quercetin group intragastrically for 8 weeks for pharmacodynamic evaluation. Besides morphological observation, the distribution of CD11b, F4/80, sirtuin 1 (Sirt1) and P21 was assayed by immunohistochemistry and immunofluorescence to evaluate macrophage infiltration and tissue senescence. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MSC/MS) was performed to study the pharmacology of quercetin against AS. Then, simultaneous administration of an apelin receptor antagonist (ML221) with quercetin was conducted to verify the possible targets of quercetin. Key proteins in apelin signaling pathway, such as angiotensin domain type 1 receptor-associated proteins (APJ), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), tissue plasminogen activator (TPA), uncoupling protein 1 (UCP1) and angiotensin II receptor 1 (AT1R), were assayed by Western blot. Quercetin administration decreased lipid deposition in arterial lumen and improved the morphology of ApoE-/- aortas in vivo. Quercetin decreased the densities of CD11b, F4/80 and P21 in the aorta and increased the level of serum apelin and the densities of APJ and Sirt1 in the aorta in ApoE-/- mice (all P<0.05). Plasma metabolite profiling identified 118 differential metabolites and showed that quercetin affected mainly glycerophospholipids and fatty acyls. Bioinformatics analysis suggested that the apelin signaling pathway was one of the main pathways. Quercetin treatment increased the protein expressions of APJ, AMPK, PGC-1α, TPA and UCP1, while decreased the AT1R level (all P<0.05). After the apelin pathway was blocked by ML221, the effect of quercetin was abated significantly, confirming that quercetin attenuated AS by modulating the apelin signaling pathway (all P<0.05). Quercetin alleviated AS lesions by up-regulation the apelin signaling pathway.

Cornus officinalis Sieb. et Zucc. Alleviates Mitochondrial Abnormalities and Heart Failure through AMPK

Background: Traditional Chinese medicine (TCM) has clear advantages in stabilizing patients with heart failure (HF) and improving cardiac functions. Aims: This study aimed to investigate the role of Cornus officinalis Sieb. et Zucc. (Cor) in HF. Settings and Design: Rats with HF were treated with Cor or Cor with an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, dorsomorphin dihydrochloride (Dor). Materials and Methods: The weight and echocardiography findings of the rats were examined. AMPK and p-AMPK expression levels were assessed using Western blots, mitochondrial changes were evaluated using transmission electron microscopy (TEM), and adenosine triphosphate (ATP) levels were detected in each group. Finally, the targets of Cor were analyzed using HERB ( http://herb.ac.cn ). Statistical Analysis Used: A Student’s t-test was used to compare two groups, and a one-way analysis of variance was performed for intergroup comparison. A p &lt; 0.05 indicated that the difference was statistically significant. Results: Compared with that of the Sham group, the body weight of the Model group decreased; left ventricular end diastolic diameter (LVIDd) and left ventricular end systolic diameter (LVIDs) increased; and left ventricular end diastolic posterior wall thickness (LVPWd), left ventricular end systolic posterior wall thickness (LVPWs), left ventricular ejection fraction (EF), and stroke volume (SV) decreased. These effects were reversed by the Cor treatment. Western blot analysis indicated p-AMPK levels were higher in the Model group than those in the Sham group. After Cor treatment, p-AMPK levels increased but later decreased after treatment with Dor. In addition, myocardial mitochondrial abnormalities were reduced and ATP levels increased in the Cor group compared with the Model group, which were reversed by Dor. Lastly, Cor was found to target AMPK pathway-related genes. Conclusion: Cor exerts protective effects on HF by activating AMPK to improve mitochondrial function.

Asprosin contributes to nonalcoholic fatty liver disease through regulating lipid accumulation and inflammatory response via AMPK signaling.

Nonalcoholic fatty liver disease (NAFLD) is a primary contributor to liver-related morbidity and mortality. Asprosin has been reported to be implicated in NAFLD. This work is to illuminate the effects of Asprosin on NAFLD and the possible downstream mechanism. The weight of NAFLD mice induced by a high-fat diet was detected. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) examined serum Asprosin expression. RT-qPCR and western blot analysis examined Asprosin expression in mice liver tissues. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were implemented. Biochemical kits tested liver enzyme levels in mice serum and liver tissues. Hematoxylin and eosin staining evaluated liver histology. Liver weight was also tested and oil red O staining estimated lipid accumulation. RT-qPCR and western blot analysis analyzed the expression of gluconeogenesis-, fatty acid biosynthesis-, fatty acid oxidation-, and inflammation-associated factors. Besides, western blot analysis examined the expression of AMP-activated protein kinase (AMPK)/p38 signaling-associated factors. In palmitic acid (PA)-treated mice hepatocytes, RT-qPCR and western blot analysis examined Asprosin expression. Lipid accumulation, gluconeogenesis, fatty acid biosynthesis, fatty acid oxidation, and inflammation were appraised again. Asprosin was overexpressed in the serum and liver tissues of NAFLD mice and PA-treated mice hepatocytes. Asprosin interference reduced mice body and liver weight, improved glucose tolerance and diminished liver injury in vivo. Asprosin knockdown alleviated lipid accumulation and inflammatory infiltration both in vitro and in vivo. Additionally, Asprosin absence activated AMPK/p38 signaling and AMPK inhibitor Compound C reversed the impacts of Asprosin on lipid accumulation and inflammatory response. Collectively, Asprosin inhibition suppressed lipid accumulation and inflammation to obstruct NAFLD through AMPK/p38 signaling.

AMPK-mediated autophagy is involved in the protective effect of canagliflozin in the vitamin D3 plus nicotine calcification model in rats.

Vascular calcification (VC) is a major risk factor for cardiovascular events. A mutual interplay between inflammation, oxidative stress, apoptosis, and autophagy is implicated in its development.Herein, we aimed to evaluate the potential protective effects of canagliflozin in a vitamin D3 plus nicotine (VDN) model of VC, and to explore potential mechanisms. VC was induced by VDN in adult male Wistar rats on day one. Then, rats were randomly assigned into three groups to receive canagliflozin (10mg or 20mg/kg/day) or its vehicle for 4weeks. Age-matched normal rats served as a control group. After euthanization, aorta and kidneys were harvested for biochemical and histopathological evaluation of calcification. Aortic markers of oxidative stress, alkaline phosphatase (ALP) activity, runt-related transcription factor (Runx2) and bone morphogenic protein-2 (BMP-2) levels were determined. Additionally, the protein expression of autophagic markers, LC3 and p62, and adenosine monophosphate activated protein kinase (AMPK) were also assessed in aortic homogenates. Canagliflozin dose-dependently improved renal function, enhanced the antioxidant capacity of aortic tissues and reduced calcium deposition in rat aortas and kidneys. Both doses of canagliflozin attenuated ALP and osteogenic markers while augmented the expression of autophagic markers and AMPK. Histopathological examination of aortas and kidneys by H&E and Von Kossa stain further support the beneficial effect of canagliflozin. Canagliflozin could alleviate VDN-induced vascular calcification, in a dose dependent manner, via its antioxidant effect and modulation of autophagy. Further studies are needed to verify whether this effect is a member or a class effect.

Expression of enzymes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr heifers in a rotational grazing system supplemented with increasing protein levels

Studies evaluating the crude protein (CP) supplementation strategies across the year for grazing cattle and its association with the enzymes involved in the urea cycle and muscle and mammary gland developments are scarce. Thus, we aimed to evaluate the effect of supplementation with different levels of CP on the expression of genes involved in the urea cycle and muscle and mammary gland development of Holstein × Gyr crossbreed heifers grazing intensively managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial BW of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to 1 of 4 treatments: 3 protein supplements (SUP) fed at 5g/kg of body weight, plus a control group (CON, non-supplemented animals). The supplement CP levels evaluated were: 12, 24, and 36%. The study was divided into 4 seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). On the penultimate day of each season, ultrasound images of the carcass and mammary gland were taken. Five animals from each treatment were randomly chosen on the last day of each season, and liver and muscle tissue biopsies were performed. The target genes were the mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) in the muscle samples. Carbamoyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), arginosuccinate lyase (ASL), and arginase (ARG) were evaluated in the liver samples. Data were analyzed using PROC GLIMMIX of the SAS with repeated measures. We observed a greater rib eye area (cm2) and fat thickness (mm) in SUP animals than in non-supplemented animals. However, we did not observe differences among SUP levels for both variables. No effects of supplementation were detected on mammary gland development. Nevertheless, seasonal effects were observed, where the RDT and dry season had the most and least accumulated fat in the mammary gland. In muscle, we observed greater expression of AMPK in non-supplemented animals than SUP animals. On the other hand, no differences were observed in gene expression between SUP and non-supplemented animals and among SUP animals for mTOR. Season affected both AMPK and mTOR; heifers had a greater AMPK gene expression on rainy than RDT. For mTOR, we observed greater gene expression in RDT and DRT than in rainy. No differences were observed among RDT, dry, and DRT, and between dry and rainy seasons for mTOR. We observed greater CPS, ASL, and ARG gene expression in SUP animals than in non-supplemented animals. Among SUP animals, supplement CP linearly affected CPS. In conclusion, the supplementation strategy did not affect mammary gland development and mTOR expression in muscle tissue. However, we observed a seasonal effect on mammary gland development and AMPK and mTOR expression. The CP supplementation increased the rib eye area and fat thickness, directly affecting AMPK expression in the muscle. Moreover, the CP supplementation increased urea cycle enzyme expression, indicating greater urea production in the liver.

Role of the AMP-Activated Protein Kinase in the Pathogenesis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a complex disorder characterized by elevated androgen levels, menstrual irregularities, and polycystic morphology of the ovaries. Affecting 6-10% of women in childbearing age, PCOS is a leading cause of infertility worldwide. In recent years, there has been a growing acknowledgment of the involvement of adenosine monophosphate-activated protein kinase (AMPK) in the development of polycystic ovary syndrome (PCOS). The expression of AMPK is diminished in polycystic ovaries, and when AMPK is silenced in human granulosa cells, there is a rise in the expression of steroidogenic enzymes, resulting in increased production of estradiol and progesterone. Additionally, in mouse models, the inhibiting AMPK intensifies the polycystic appearance of ovaries and impairs the process of ovulation. Moreover, it has been shown that AMPK activators like metformin and resveratrol ameliorate PCOS associated signs and symptoms in experimental and clinical studies. These findings, collectively, indicate the key role of AMPK in the pathogenesis of PCOS. Understanding the role of AMPK in PCOS will offer rewarding information on details of PCOS pathogenesis and will provide novel more specific therapeutic approaches. The present review summarizes the latest findings regarding the role of AMPK in PCOS obtained in experimental and clinical studies.

Zoledronate Promotes Peri-Implant Osteogenesis in Diabetic Osteoporosis by the AMPK Pathway.

Together with diabetic osteoporosis (DOP), diabetes patients experience poor peri-implant osteogenesis following implantation for dentition defects. Zoledronate (ZOL) is widely used to treat osteoporosis clinically. To evaluate the mechanism of ZOL for the treatment of DOP, experiments with DOP rats and high glucose-grown MC3T3-E1 cells were used. The DOP rats treated with ZOL and/or ZOL implants underwent a 4-week implant-healing interval, and then microcomputed tomography, biomechanical testing, and immunohistochemical staining were performed to elucidate the mechanism. In addition, MC3T3-E1 cells were maintained in an osteogenic medium with or without ZOL to confirm the mechanism. The cell migration, cellular actin content, and osteogenic differentiation were evaluated by a cell activity assay, a cell migration assay, as well as alkaline phosphatase, alizarin red S, and immunofluorescence staining. The mRNA and protein expression of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphogenetic protein 2 (BMP2), and collagen type I (Col-I) were detected using real-time quantitative PCRs and western blot assays, respectively. In the DOP rats, ZOL markedly improved osteogenesis, enhanced bone strength and increased the expression of AMPK, p-AMPK, and Col-I in peri-implant bones. The in vitro findings showed that ZOL reversed the high glucose-induced inhibition of osteogenesis via the AMPK signaling pathway. In conclusion, the ability of ZOL to promote osteogenesis in DOP by targeting AMPK signaling suggests that therapy with ZOL, particularly simultaneous local and systemic administration, may be a unique approach for future implant repair in diabetes patients.

Effect of heavy metals on the energy metabolism in the brackish water flea Diaphanosoma celebensis

Heavy metals such as lead (Pb), cadmium (Cd), and arsenic (As) are of great concern in aquatic ecosystems because of their global distribution, persistence, and biomagnification via the food web. They can induce the expression of cellular protective systems (e.g., detoxification enzymes and antioxidant enzymes) to protect organisms from oxidative stress, which is a high-energy-consuming process. Thus, energy reserves (e.g., glycogen, lipids, and proteins) are utilized to maintain metabolic homeostasis. Although a few studies have suggested that heavy metal stress can modulate the metabolic cycle in crustaceans, information on changes in energy metabolism under metal pollution remains lacking in planktonic crustaceans. In the present study, the activity of digestive enzymes (amylase, trypsin, and lipase) and the contents of energy storage molecules (glycogen, lipid, and protein) were examined in the brackish water flea Diaphanosoma celebensis exposed to Cd, Pb, and As for 48 h. Transcriptional modulation of the three AMP-activated protein kinase (AMPK) and metabolic pathway-related genes was further investigated. Amylase activity was highly increased in all heavy metal-exposed groups, whereas trypsin activity was reduced in Cd- and As-exposed groups. While glycogen content was increased in all exposed groups in a concentration-dependent manner, lipid content was reduced at higher concentrations of heavy metals. The expression of AMPKs and metabolic pathway-related genes was distinct among heavy metals. In particular, Cd activated the transcription of AMPK-, glucose/lipid metabolism-, and protein synthesis-related genes. Our findings indicate that Cd can disrupt energy metabolism, and may be a potent metabolic toxicant in D. celebensis. This study provides insights into the molecular mode of action of heavy metal pollution on the energy metabolism in planktonic crustaceans.

Enpp1 deficiency caused chondrocyte apoptosis by inhibiting AMPK signaling pathway

Objective and backgroundThe deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) causes the phenotype similar to knee osteoarthritis (OA). However, the molecular mechanism is poorly understood.MethodThe global deletion of Enpp1 (Enpp1−/−) mice was created to analyze the role of Enpp1 in the progress of knee OA. The apoptosis, proliferation and chondrogenic differentiation ability of chondrocytes from wild-type (WT) and Enpp1−/− joints were compared. According to the results of high-throughput quantitative molecular measurements, the proteins of chondrocytes from WT and Enpp1−/− mice were used to explore the mechanism of Enpp1 deficiency-associated knee OA.ResultIn Enpp1−/− knee joints, we found significant chondrocyte apoptosis and proteomic results showed that abnormal expression of AMP-activated protein kinase (AMPK) signaling pathway may contribute to this phenotype. In primary chondrocyte cultures in vitro, Enpp1 deletion dramatically enhancing chondrocyte apoptosis. Meanwhile, we found Enpp1 deletion inhibits the phosphorylation of AMPK (P-AMPK). We also found that decreased level of P-AMPK and chondrocyte apoptosis, which are caused by Enpp1 deficiency, can be reversed by Acadesine (AICAR), the activator of AMPK.ConclusionConsequently, Enpp1 deficiency plays an essential role in knee OA by regulating AMPK signaling pathway.

Effects of Dietary Chenodeoxycholic Acid Supplementation in a Low Fishmeal Diet Containing Clostridium autoethanogenum Protein on Growth, Lipid and Cholesterol Metabolism, and Hepatopancreas Health of Litopenaeus vannamei.

The study aimed to assess the impact of adding chenodeoxycholic acid (CDCA) to the diet of Litopenaeus vannamei on their growth performance, lipid and cholesterol metabolism, and hepatopancreas health while being fed a low fishmeal diet. Five diets were formulated, one of which contained 25% fishmeal (PC); fishmeal was partially replaced with Clostridium autoethanogenum protein in the remaining four diets and supplemented with 0, 0.03, 0.06, and 0.09% CDCA (NC, BA1, BA2, and BA3, respectively). In this study, four replicates of each diet were assigned and each replicate consisted of 30 shrimp with an average weight of (0.25 ± 0.03 g). The shrimp were fed four times a day for a period of 56 days. The results of this study indicate that the inclusion of CDCA in the diet had a positive impact on the growth performance of the shrimp. The final body weight (FBW), weight gain (WG), and specific growth rate (SGR) of the shrimp in the PC group were similar to those in the BA2 group, and significantly higher than those in the other three groups. The survival rate (SR) was similar among all groups. In comparison to the PC group, the low fishmeal groups exhibited a significant decrease in the crude lipid content of the whole shrimp, as well as the Total cholesterol (T-CHOL), Low-density lipoprotein (LDL-C), and High-density lipoprotein (HDL-C) levels in the hemolymph. Regarding the sterol metabolism, the dietary supplementation of CDCA up-regulated the mRNA expression of intracellular cholesterol transporter 1-like (npc1), 7-dehydrocholesterol reductase (7dhcr), Delta (24) sterol reductase (Δ24), HMG-CoA reductase membrane form (hmgcr), and sterol carrier protein 2 (scp). In the lipid metabolism, the mRNA expression of sterol-regulatory element binding protein (srebp) was significantly down-regulated in the shrimp fed the BA1 diet and the expression of AMP-activated protein kinase (ampk) was significantly up-regulated in the shrimp fed the BA1 and BA3 diets compared to the PC group. The mRNA expression of triacylglycerol lipase (tgl) was significantly up-regulated in the shrimp fed the BA2 diet compared to the NC group. Compared with the shrimp fed the PC diets, the dietary supplementation of CDCA significantly down-regulated the protein expression of SREBP1. The lumen damage in the BA1 group was significantly less severe than those in the NC group. The addition of 0.06% CDCA to low fishmeal diets can improve the growth performance, lipid and cholesterol metabolism, and hepatopancreas health of L. vannamei.

Antitumor Activity of Ligustilide Against Ehrlich Solid Carcinoma in Rats via Inhibition of Proliferation and Activation of Autophagy.

Background Cancer is the second-leading cause of death worldwide. According to a 2018 WHO report, 9.6 million deaths occurred globally due to cancer. Ehrlich carcinoma is characterized by rapid proliferation and a short survival time. Ligustilide is a phthalide derivative and is one of the main compounds in Danggui essential oil and Rhizoma Chuanxiong. It has many protective effects, such as anticancer, anti-inflammatory, antioxidant, and neuroprotective effects. Aims We conducted this study to investigate the antitumor activity of ligustilide against Ehrlich solid carcinoma (ESC) in rats by affecting beclin 1, mammalian target of rapamycin (mTOR), B-cell lymphoma 2 (BCL2), and 5' AMP-activated protein kinase (AMPK). Materials and methods Twenty rats were intramuscularly implanted in the thigh of the left hind limb with a 200-µL tumor cell suspension in PBS containing 2 × 106 cells. After eight days of inoculation, 10 rats out of the 20 were treated with oral 20 mg/kg ligustilide daily. At the end of the experiment, samples of muscles with ESC were separated. Sections prepared from the muscle samples with ESC were immunohistochemically stained with anti-Ki67 antibodies. Another part of the muscle samples with ESC was used to assess gene expression and protein levels of beclin 1, mTOR, BCL2, and AMPK. Results Treatment of carcinoma rats with ligustilide elevated the mean survival time and reduced tumor volume and weight. Moreover, examination of tumor tissue stained with hematoxylin/eosin showed an infiltrative, highly cell-dense mass supported by a small to moderate amount of fibrovascular stroma and intersected with multifocal myofibril necrosis. Treatment with ligustilide ameliorated all these effects in the carcinoma group without affecting the control group. Finally, treatment with ligustilide significantly decreased the expression of beclin 1, mTOR, and AMPK associated with elevated expression of BCL2. Conclusions Our study aimed to explore the potential chemotherapeutic activity of ligustilide against ESC. We found that ligustilide effectively reduced tumor size and weight, indicating its antineoplastic activity against ESC. We further investigated that ligustilide inhibits cell proliferation by suppressing Ki67 and mTOR and activates autophagy through beclin 1 activation. Moreover, ligustilide inhibits apoptosis by upregulating BCL2. Finally, ligustilide reduced the expression of AMPK, preventing its ability to promote tumor cell growth.

De novo pyrimidine biosynthetic complexes support cancer cell proliferation and ferroptosis defence.

De novo pyrimidine biosynthesis is achieved by cytosolic carbamoyl-phosphate synthetase II, aspartate transcarbamylase and dihydroorotase (CAD) and uridine 5'-monophosphate synthase (UMPS), and mitochondrial dihydroorotate dehydrogenase (DHODH). However, how these enzymes are orchestrated remains enigmatical. Here we show that cytosolic glutamate oxaloacetate transaminase 1 clusters with CAD and UMPS, and this complex then connects with DHODH, which is mediated by the mitochondrial outer membrane protein voltage-dependent anion-selective channel protein 3. Therefore, these proteins form a multi-enzyme complex, named 'pyrimidinosome', involving AMP-activated protein kinase (AMPK) as a regulator. Activated AMPK dissociates from the complex to enhance pyrimidinosome assembly but inactivated UMPS, which promotes DHODH-mediated ferroptosis defence. Meanwhile, cancer cells with lower expression of AMPK are more reliant on pyrimidinosome-mediated UMP biosynthesis and more vulnerable to its inhibition. Our findings reveal the role of pyrimidinosome in regulating pyrimidine flux and ferroptosis, and suggest a pharmaceutical strategy of targeting pyrimidinosome in cancer treatment.

Metformin protects fibroblasts from patients with GNE myopathy by restoring autophagic flux via an AMPK/mTOR-independent pathway

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is an autosomal recessive disease characterized by rimmed vacuoles (RVs). Previous studies have shown that metformin protects against several neuromuscular disorders. In the present study, we summarize the clinical features of three GNE patients with the p.D207V mutation. The pathogenesis of GNE myopathy is described, and the significance of metformin in this disease is observed. Skin biopsy-derived fibroblasts from patients with GNE myopathy, carrying a D207V mutation in GNE, were cultured. GNE fibroblasts and control fibroblasts were treated under normal culture conditions, serum starvation conditions, or serum starvation + metformin conditions. Histopathological and immunohistochemical analyses of muscle samples showed that autophagy was involved in the formation of RVs in the muscle of patients. Starved GNE fibroblasts showed decreased autophagy-related proteins and impaired autophagic flow (p < 0.05). The mRFP-GFP-LC3 assay showed that the fusion of autophagosomes with lysosomes was partially blocked in GNE cells. Notably, metformin treatment upregulated the expression of autophagy proteins, increased the number of autolysosomes (p < 0.001), and influenced the viability of GNE cells (p < 0.001). Furthermore, adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and phosphorylated (p)-AMPK expression levels were upregulated in serum-starved GNE fibroblasts, while the mammalian target of rapamycin (mTOR) and p-mTOR expression levels were downregulated in both groups. Metformin treatment inhibited the AMPK–mTOR signaling pathway. Our results suggest that metformin plays a protective role in the GNE fibroblast by restoring autophagic flux and through the AMPK/mTOR-independent pathway.

Integrated network pharmacology, transcriptomics, and metabolomics analysis to reveal the mechanism of salt Eucommiae cortex in the treatment of chronic kidney disease mineral bone disorders via the PPARG/AMPK signaling pathway

Ethnopharmacological relevanceThe skeletal complications associated with chronic kidney diseases from stages 3–5 in individuals are called Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), which increases the incidence of cardiovascular diseases drastically and affects the quality of life of patients seriously. Eucommiae cortex has the effect of tonifying kidneys and strengthening bones, and salt Eucommiae cortex is one of the most commonly used traditional Chinese medicines in the clinical treatment of CKD-MBD instead of Eucommiae cortex. However, its mechanism still remains unexplored. Aim of the studyThe aim of this study was to investigate the effects and mechanisms of salt Eucommiae cortex on CKD-MBD by integrating network pharmacology, transcriptomics, and metabolomics. Materials and methodsThe CKD-MBD mice induced by 5/6 nephrectomy and low calcium/high phosphorus diet were treated with salt Eucommiae cortex. The renal functions and bone injuries were evaluated by serum biochemical detection, histopathological analyses, and femur Micro-CT examinations. Differentially expressed genes (DEGs) between the control group and model group, model group and high-dose Eucommiae cortex group, model group and high-dose salt Eucommiae cortex group were analyzed by transcriptomic analysis. The differentially expressed metabolites (DEMs) between the control group and model group, model group and high-dose Eucommiae cortex group, model group and high-dose salt Eucommiae cortex group were analyzed by metabolomics analysis. The common targets and pathways were obtained by integrating transcriptomics, metabolomics, and network pharmacology, which were identified and verified by in vivo experiments. ResultsThe negative impacts on the renal functions and bone injuries were alleviated with salt Eucommiae cortex treatment effectively. Compared with CKD-MBD model mice, the levels of serum BUN, Ca, and urine Upr were significantly decreased in the salt Eucommiae cortex group. And the Integrated network pharmacology, transcriptomics, and metabolomics analysis revealed that Peroxisome Proliferative Activated Receptor, Gamma (PPARG) was the only common target, mainly involved by AMP-activated Protein Kinase (AMPK) signaling pathways. The activation of PPARG in the kidney tissue was significantly decreased in CKD-MBD mice but increased in the salt Eucommiae cortex treatment. The AMPK signaling pathway was verified and the AMPK expression levels were found to decrease in CKD-MBD mice but increase given salt Eucommiae cortex treatment. ConclusionsOur study presented that salt Eucommiae cortex alleviated the negative impact of CKD-MBD on the renal injury and bone injury of mice induced by 5/6 nephrectomy with the low calcium/high phosphorus diet effectively, which is highly likely achieved through the PPARG/AMPK signaling pathway.

Effects of Gestational Hypoxia on PGC1α and Mitochondrial Acetylation in Fetal Guinea Pig Hearts

Chronic intrauterine hypoxia is a significant pregnancy complication impacting fetal heart growth, metabolism, and mitochondrial function, contributing to cardiovascular programming of the offspring. PGC1α (peroxisome proliferator-activated receptor γ co-activator 1α) is the master regulator of mitochondrial biogenesis. We investigated the effects of hypoxia on PGC1α expression following exposure at different gestational ages. Time-mated pregnant guinea pigs were exposed to normoxia (NMX, 21% O2) or hypoxia (HPX, 10.5% O2) at either 25-day (early-onset) or 50-day (late-onset) gestation, and all fetuses were extracted at term (term = ~65-day gestation). Expression of nuclear PGC1α, sirtuin 1 (SIRT1), AMP-activated protein kinase (AMPK), and mitochondrial sirtuin 3 (SIRT3) was measured, along with SIRT3 activity and mitochondrial acetylation of heart ventricles of male and female fetuses. Early-onset hypoxia increased (P<0.05) fetal cardiac nuclear PGC1α and had no effect on mitochondrial acetylation of either growth-restricted males or females. Late-onset hypoxia had either no effect or decreased (P<0.05) PCC1α expression in males and females, respectively, but increased (P<0.05) mitochondrial acetylation in both sexes. Hypoxia had variable effects on expression of SIRT1, AMPK, SIRT3, and SIRT3 activity depending on the sex. The capacity of the fetal heart to respond to hypoxia differs depending on the gestational age of exposure and sex of the fetus. Further, the effects of late-onset hypoxia on fetal heart function impose a greater risk to male than female fetuses, which has implications toward cardiovascular programming effects of the offspring.

Therapeutic exploration of polyherbal formulation against letrozole induced PCOS rats: A mechanistic approach

ObjectiveThis study aimed to develop an effective alternative medicine with multi potential herbs against polycystic ovarian syndrome (PCOS) in rats induced by letrozole treatment. Materials and methodPolyherbal syrup was prepared with a combination of S. asoca bark, G. sylvestre leaves, P. daemia aerial parts, C. zeylanium stem bark, C. bonduc seeds, and W. somnifera roots ethanolic extract. In vitro cell viability study, adenosine monophosphate-activated protein kinase (AMPK), and glucose transporter 4 (GLUT4) gene expression assay were carried out on the Chinese Hamster Ovarian (CHO) cell line. For the PCOS induction letrozole (1 mg/kg p. o.) was given for 21 consecutive days. The PCOS induction was confirmed by measuring estrus irregularity, insulin resistance by oral glucose tolerance test (OGTT), and hyperandrogenism by measuring serum total testosterone level 21 days after completion of letrozole treatment. After induction of PCOS, metformin (155 mg/kg p. o.), and polyherbal syrup (100 mg/kg, 200 mg/kg, and 400 mg/kg p. o.) were administered for further 28 days. The treatment efficacy was measured by measuring serum lipid profile, fasting insulin level, sex hormones level, ovarian steroidogenic enzymes, ovarian tissue insulin receptor, AMPK, and GLUT4 protein expression levels, and histomorphological studies. The post-treatment effect was confirmed by reproductive performance studies. ResultsLetrozole-induced PCOS rats showed significant estrus irregularity, abnormal sex hormones levels, and hyperandrogenism indicated by showing increased free androgenic index and decreased sex hormones binding globulin (SHBG) level. The insulin resistance in PCOS rats was indicated by increased fasting glucose levels with impaired glucose clearance in the OGT test. Homeostasis Model Assessment Index of Insulin Resistance (HOMA-IR) increased level, also decreases INSR, GLUT4, and AMPK mRNA expression in ovarian cells confirming the insulin resistance in PCOS rats. Ovarian histology in PCOS rats also showed many follicular cysts, atretic follicles, and the absence of corpus luteum. The administration of polyherbal syrup, in a dose-dependent manner, effectively restored these alterations. The treatment of polyherbal formulation 400 mg/kg possesses highly significant efficacy over the treatment of metformin in PCOS rats. It mainly acts by reducing peripheral and ovarian hyperandrogenism and improves insulin sensitivity via activating the insulin receptor and AMP-activated kinase-mediated transcription and translation of GLUT4 from the cytoplasm to the ovarian membrane improves glucose uptake and promotes the follicular development and ovulation. The higher fertility rate, delivery index, and survival of delivered pups confirm the broader and superior efficacy of PCOS. These beneficial actions are mainly attributable to the formulation's inclusion of the key secondary metabolites flavonoids and phytosterols. In conclusion, the prepared polyherbal syrup was found to be the safest and most effective alternative medicine for both endocrinal and metabolic complications of PCOS women.

AdipoRon Engages Microglia to Antinociception through the AdipoR1/AMPK Pathway in SNI Mice.

Microglia-associated neuroinflammation plays a crucial role in the initiation and development of neuropathic pain (NeuP). AdipoRon is an analog of adiponectin that exerts an anti-inflammatory effect in various diseases through the adiponectin receptor 1 (AdipoR1) signaling mechanism. Adenosine monophosphate-activated protein kinase (AMPK) is a downstream target of AdipoR1, and the AdipoR1/AMPK pathway is involved in the regulation of inflammation. This study is aimed at investigating whether AdipoRon could alleviate NeuP by inhibiting the expression of microglia-derived tumor necrosis factor-alpha (TNF-α) through the AdipoR1/AMPK pathway. In vivo, the NeuP model was established in mice through the spared nerve injury. The von Frey test was used to detect the effect of AdipoRon on the mechanical paw withdrawal threshold. Western Blot was performed to detect the effects of AdipoRon on the expression of TNF-α, AdipoR1, AMPK, and p-AMPK. Immunofluorescence was performed to observe the effects of AdipoRon on spinal microglia. In vitro, lipopolysaccharide (LPS) was used to induce inflammatory responses in BV2 cells. The effect of AdipoRon on cell proliferation was detected by CCK-8. qPCR was used to examine the effects of AdipoRon on the expression of TNF-α and polarization markers. And the effect of AdipoRon on the AdipoR1/AMPK pathway was confirmed by Western Blot. Intraperitoneal injection of AdipoRon alleviated mechanical nociception in SNI mice, and the application of AdipoRon reduced the expression of TNF-α and the number of microglia in the ipsilateral spinal cord. Additionally, AdipoRon decreased the protein level of AdipoR1 and increased the protein level of p-AMPK in the ipsilateral spinal cord. In vitro, AdipoRon inhibited BV2 cell proliferation and reversed LPS-induced TNF-α expression and polarization imbalance. Furthermore, AdipoRon reversed the LPS-induced increase in AdipoR1 expression and decrease in p-AMPK expression in BV2 cells. AdipoRon may alleviate NeuP by reducing microglia-derived TNF-α through the AdipoR1/AMPK pathway.

Effect and mechanism of Magnolia officinalis pharmacopuncture for treating localized fat via network pharmacology and experimental study

BackgroundRecently, for various reasons, the need for non-invasive treatment for localized fat has emerged. This study confirmed whether Magnolia officinalis (MO) pharmacopuncture reduces localized fat by promoting lipolysis and inhibiting adipogenesis. MethodsThe network was built using genes related to the active compound of MO and the mode of action of MO was predicted by the functional enrichment analysis. Based on the result from network analysis, 100 µL of 2 mg/mL MO pharmacopuncture was injected into the inguinal fat pad for 6 weeks in obese C57BL/6J mice. Normal saline was injected into the right-side inguinal fat pad as a self-control. ResultsIt was expected that the AMP-activated protein kinase (AMPK) signaling pathway would be affected by the MO Network. MO pharmacopuncture reduced the weight and size of inguinal fat in HFD-induced obese mice. The phosphorylation of AMPK along with the increases of lipases was significantly increased by MO injection. Also, the expression levels of fatty acid synthesize-related mediators were suppressed by MO injection. ConclusionOur results demonstrated that MO pharmacopuncture promoted the expression of AMPK, which has beneficial effects on activation of lipolysis and inhibition of lipogenesis. Pharmacopuncture of MO can be a non-surgical alternative therapy in the treatment of local fat tissue.