Conjugated linoleic acid (CLA) reduces HFD-induced obesity by enhancing BAT thermogenesis and iWAT browning via the CD36-AMPK pathway.

The antiobesity effect of conjugated linoleic acid (CLA) has been reported. However, the underlying mechanisms have not been fully clarified. Thus, this study aimed to investigate the effects of CLA on thermogenesis of interscapular brown adipose tissue (iBAT) and browning of inguinal subcutaneous white adipose tissue (iWAT) and explore the possible signaling pathway. The in vivo results showed that CLA enhanced the O2 consumption and heat production in HFD (high-fat diet)-fed female mice by roughly 38%. Meanwhile, CLA increased the average iBAT temperature by 2°C at the room temperature and cold exposure, respectively. Correspondingly, CLA caused 1.6- and 2.4-fold increases in the expression of UCP1 (uncoupling protein 1) of BAT and iWAT, respectively, suggesting the activated iBAT thermogenesis and iWAT browning in HFD-fed female mice. Meanwhile, CLA could promote the formation of brown and beige adipocytes in differentiated stromal vascular cells (SVCs) isolated from iBAT and iWAT (the expressions of UCP1 were promoted by about twofold changes). In possible mechanisms, CLA stimulated the expression of CD36 and the activation of the AMPK pathway in mice iBAT and iWAT as well as the differentiated SVCs. However, inhibition of CD36 and AMPK (adenosine 5'-monophosphate-activated protein kinase) abolished the promotive effects of CLA on brown and beige adipocytes formation. Hence, we showed that CLA reduced HFD-induced obesity through enhancing iBAT thermogenesis and iWAT browning via the  CD36-AMPK pathway.

Effect and molecular mechanism of Sulforaphane alleviates brain damage caused by acute carbon monoxide poisoning:Network pharmacology analysis, molecular docking, and experimental evidence.

Sulforaphane (SFN) has attracted much attention due to its ability on antioxidant, anti-inflammatory, and anti-apoptotic properties, while its functional targets and underlying mechanism of action on brain injury caused by acute carbon monoxide poisoning (ACOP) have not been fully elucidated. Herein, we used a systematic network pharmacology approach to explore the mechanism of SFN in the treatment of brain damage after ACOP. In this study, the results of network pharmacology demonstrated that there were a total of 81 effective target genes of SFN and 36 drug-disease targets, which were strongly in connection with autophagy-animal signaling pathway, drug metabolism, and transcription disorders in cancer. Upon the further biological function and KEGG signaling pathway enrichment analysis, a large number of them were involved in neuronal death, reactive oxygen metabolic processes and immune functions. Moreover, based on the results of bioinformatics prediction associated with multiple potential targets and pathways, the AMP-activated protein kinase (AMPK) signaling pathway was selected to elucidate the molecular mechanism of SFN in the treatment of brain injury caused by ACOP. The following molecular docking analysis also confirmed that SFN can bind to AMPKα well through chemical bonds. In addition, an animal model of ACOP was established by exposure to carbon monoxide in a hyperbaric oxygen chamber to verify the predicted results of network pharmacology. We found that the mitochondrial ultrastructure of neurons in rats with ACOP was seriously damaged, and apoptotic cells increased significantly. The histopathological changes were obviously alleviated, apoptosis of cortical neurons was inhibited, and the number of Nissl bodies was increased in the SFN group as compared with the ACOP group (p < .05). Besides, the administration of SFN could increase the expressions of phosphorylated P-AMPK and MFN2 proteins and decrease the levels of DRP1, Caspase3, and Casapase9 proteins in the brain tissue of ACOP rats. These findings suggest that network pharmacology is a useful tool for traditional Chinese medicine (TCM) research, SFN can effectively inhibit apoptosis, protect cortical neurons from the toxicity of carbon monoxide through activating the AMPK pathway and may become a potential therapeutic strategy for brain injury after ACOP.

Influences of two transport strategies on AMPK-mediated metabolism and flesh quality of shrimp (Litopenaeus vannamei).

BACKGROUND: Water-free transportation (WFT), as a novel strategy for express delivery of live shrimp (Litopenaeus vannamei), was developed recently. However, air exposure during this transportation arouses a series of abiotic stress to the shrimp. In the present study, the influences of WFT stress on glycolysis and lipolysis metabolism and meat quality (umami flavor and drip loss) were investigated in comparison with conventional water transportation (WT). RESULTS: The results showed that type II muscle fibers with the feature of anaerobic metabolism were dominated in shrimp flesh. In addition, the increments of intracellular Ca2+ was detected in WFT and WT, which then activated the AMP-activated protein kinase pathway and promoted the consumption of glycogen, as well as the accumulation of lactate and lipolysis, under the enzymolysis of hexokinase, pyruvate kinase, lactate dehydrogenase and adipose triglyceride lipase. Glycogen glycolyzed to latate. Meanwhile, ATP degraded along with glycolysis resulting in the generation of ATP-related adenosine phosphates such as inosine monophosphate with umami flavor and phosphoric acid. More remarkable (P < 0.05) physiological changes (except lactate dehydrogenase and lactate) were observed in WFT compared to WT. Additionally, the fatty acid profile also slightly changed. CONCLUSION: The transport stress induced significant energy metabolism changes of shrimp flesh and therefore effected the flesh quality. The intensifications of freshness (K-value) of shrimp flesh were detected as a result of ATP degradation, which were more pronounced after WFT. However, the drip loss of shrimp flesh was more significantly increased (P < 0.05) after WFT compared to WT. © 2023 Society of Chemical Industry.

LncRNA CEBPA-AS1 alleviates cerebral ischemia-reperfusion injury by sponging miR-340-5p regulating APPL1/LKB1/AMPK pathway.

Long non-coding RNAs (lncRNAs) regulate neurological damage in cerebral ischemia-reperfusion injury (CIRI). This study aimed to investigate the biological roles of lncRNA CEBPA-AS1 in CIRI. Middle cerebral artery occlusion and ischemia-reperfusion injury (MCAO/IR) rat model and oxygen-glucose deprivation and reoxygenation (OGD/R) cell lines were generated; the expression of CEBPA-AS1 was evaluated by qRT-PCR. The effects of CEBPA-AS1 on cell apoptosis and nerve damage were examined. The downstream microRNA (miRNA) and mRNA of CEBPA-AS1 were predicted and verified. We found that overexpression of CEBPA-AS1 could attenuate MCAO/IR-induced nerve damage and neuronal apoptosis in the rat model. Knockdown of CEBPA-AS1 aggravated cell apoptosis and enhanced the production of LDH and MDA in the OGD/R cells. Upon examining the molecular mechanisms, we found that CEBPA-AS1 stimulated APPL1 expression by combining with miR-340-5p, thereby regulating the APPL1/LKB1/AMPK pathway. In the rescue experiments, CEBPA-AS1 overexpression was found to attenuate OGD/R-induced cell apoptosis and MCAO/IR induced nerve damage, while miR-340-5p reversed these effects of CEBPA-AS1. In conclusion, CEBPA-AS1 could decrease CIRI by sponging miR-340-5, regulating the APPL1/LKB1/AMPK pathway.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 µM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

The protective effect of the mitochondrial-derived peptide MOTS-c on LPS-induced septic cardiomyopathy.

Septic cardiomyopathy is associated with mechanisms such as excessive inflammation, oxidative stress, regulation of calcium homeostasis, endothelial dysfunction, mitochondrial dysfunction, and cardiomyocyte death, and there is no effective treatment at present. MOTS-c is a mitochondria-derived peptide (MDP) encoded by mitochondrial DNA (mtDNA) that protects cells from stresses in an AMPK-dependent manner. In the present study, we aim to explore the protective effect of MOTS-c on lipopolysaccharide (LPS)-induced septic cardiomyopathy. LPS is used to establish a model of septic cardiomyopathy. Our results demonstrate that MOTS-c treatment reduces the mRNA levels of inflammatory cytokines ( IL-1ß, IL-4, IL-6, and TNFα) in cardiomyocytes and the levels of circulating myocardial injury markers, such as CK-MB and TnT, alleviates cardiomyocyte mitochondrial dysfunction and oxidative stress, reduces cardiomyocyte apoptosis, activates cardioprotection-related signaling pathways, including AMPK, AKT, and ERK, and inhibits the inflammation-related signaling pathways JNK and STAT3. However, treatment with the AMPK pathway inhibitor compound C (CC) abolishes the positive effect of MOTS-c on LPS stress. Collectively, our research suggests that MOTS-c may attenuate myocardial injury in septic cardiomyopathy by activating AMPK and provides a new idea for therapeutic strategies in septic cardiomyopathy.

Microcystin leucine arginine induces human sperm damage: Involvement of the Ca2+/CaMKKß/AMPK pathway.

As a common pollutant in the water environment, microcystin leucine arginine (MC-LR) can enter semen and damage the sperm in animals. However, the mechanism by which MC-LR damages human sperm is unclear. Therefore, human sperm samples were obtained from the Henan Provincial Sperm Bank and exposed to different concentrations (0, 1, 10, and 100 µg/L) of MC-LR for 1, 2, 4, and 6 h, to invegest the effects and potential mechanism of MC-LR on sperm. The results showed that MC-LR mainly accumulated in the neck and flagellum of human sperm. Compared to the control group, the sperm capacitation rate and motility were significantly decreased in the 100 µg/L group. After exposure of 100 µg/L of MC-LR, the central microtubule and microtubule doublet of sperm flagellum were blurred, asymmetrical, or even lost. Furthermore, the expression levels of flagellin DNAH17, SPEF2, SPAG16, SPAG6, and CFAP44 in human sperm were reduced. Also, the phosphorylation levels of CaMKKß and AMPK can be inhibited by MC-LR. These findings revealed that MC-LR can induce functional and structural damage in human sperm, and the Ca2+/CaMKKß/AMPK pathway may be involved in this process. This study will provide a basis for prevention and treatment of male fertility declines caused by MC-LR.

Corilagin attenuates airway inflammation and collagen deposition in ovalbumin-induced asthmatic mice.

OBJECTIVE: To investigate the effects of corilagin on inflammation and collagen deposition in ovalbumin (OVA)-induced asthma mouse model and uncover the mechanism. METHODS: We constructed a mouse model of OVA-induced asthma. Enzyme-linked-immunosorbent serologic assays were conducted to detect the effects of corilagin on cytokines and Immunoglobulin E (IgE) production. Hematoxylin and eosin staining was used to show pathological features in lung tissues. Masson trichrome assay was used to examine collagen deposition. In addition, the lung function was detected by mouse lung function apparatus. Immunoblot was used to confirm the mechanism. RESULTS: Corilagin alleviates OVA-induced cytokine and IgE production. In addition, corilagin alleviates OVA-induced pathological changes and collagen deposition in lung tissues. Corilagin also suppressed airway resistance and lung function in mice. Mechanically, corilagin activated the adenosine monophosphate-activated protein kinase (AMPK) pathway in lung tissues. CONCLUSION: Corilagin attenuates airway inflammation and collagen deposition in OVA-induced asthmatic mice via AMPK pathway.

[Difference of lipid-lowering efficacy of "Xinjianqu" before and after fermentation and its mechanism based on LKB1-AMPK pathway and 16S rDNA sequencing technology].

On the basis of establishing the prescription of Xinjianqu and clarifying the increase of the lipid-lowering active ingredients of Xinjianqu by fermentation, this paper further compared the differences in the lipid-lowering effects of Xinjianqu before and after fermentation, and studied the mechanism of Xinjianqu in the treatment of hyperlipidemia. Seventy SD rats were randomly divided into seven groups, including normal group, model group, positive drug simvastatin group(0.02 g·kg~(-1)), and low-dose and high-dose Xinjianqu groups before and after fermentation(1.6 g·kg~(-1) and 8 g·kg~(-1)), with ten rats in each group. Rats in each group were given high-fat diet continuously for six weeks to establish the model of hyperlipidemia(HLP). After successful modeling, the rats were given high-fat diet and gavaged by the corresponding drugs for six weeks, once a day, to compare the effects of Xinjianqu on the body mass, liver coefficient, and small intestine propulsion rate of rats with HLP before and after fermentation. The effects of Xinjianqu before and after fermentation on total cholesterol(TC), triacylglyceride(TG), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol(LDL-C), alanine aminotransferase(ALT), aspartate aminotransferase(AST), blood urea nitrogen(BUN), creatinine(Cr), motilin(MTL), gastrin(GAS), and the Na~+-K~+-ATPase levels were determined by enzyme-linked immunosorbent assay(ELISA). The effects of Xinjianqu on liver morphology of rats with HLP were investigated by hematoxylin-eosin(HE) staining and oil red O fat staining. The effects of Xinjianqu on the protein expression of adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK), phosphorylated AMPK(p-AMPK), liver kinase B1(LKB1), and 3-hydroxy-3-methylglutarate monoacyl coenzyme A reductase(HMGCR) in liver tissues were investigated by immunohistochemistry. The effects of Xinjianqu on the regulation of intestinal flora structure of rats with HLP were studied based on 16S rDNA high-throughput sequencing technology. The results showed that compared with those in the normal group, rats in the model group had significantly higher body mass and liver coefficient(P<0.01), significantly lower small intestine propulsion rate(P<0.01), significantly higher serum levels of TC, TG, LDL-C, ALT, AST, BUN, Cr, and AQP2(P<0.01), and significantly lower serum levels of HDL-C, MTL, GAS, Na~+-K~+-ATP levels(P<0.01). The protein expression of AMPK, p-AMPK, and LKB1 in the livers of rats in the model group was significantly decreased(P<0.01), and that of HMGCR was significantly increased(P<0.01). In addition, the observed＿otus, Shannon, and Chao1 indices were significantly decreased(P<0.05 or P<0.01) in rat fecal flora in the model group. Besides, in the model group, the relative abundance of Firmicutes was reduced, while that of Verrucomicrobia and Proteobacteria was increased, and the relative abundance of beneficial genera such as Ligilactobacillus and Lachnospiraceae＿NK4A136＿group was reduced. Compared with the model group, all Xinjianqu groups regulated the body mass, liver coefficient, and small intestine index of rats with HLP(P<0.05 or P<0.01), reduced the serum levels of TC, TG, LDL-C, ALT, AST, BUN, Cr, and AQP2, increased the serum levels of HDL-C, MTL, GAS, and Na~+-K~+-ATP, improved the liver morphology, and increased the protein expression gray value of AMPK, p-AMPK, and LKB1 in the liver of rats with HLP and decreased that of LKB1. Xinjianqu groups could regulate the intestinal flora structure of rats with HLP, increased observed＿otus, Shannon, Chao1 indices, and increased the relative abundance of Firmicutes, Ligilactobacillus(genus), Lachnospiraceae＿NK4A136＿group(genus). Besides, the high-dose Xinjianqu-fermented group had significant effects on body mass, liver coefficient, small intestine propulsion rate, and serum index levels of rats with HLP(P<0.01), and the effects were better than those of Xinjianqu groups before fermentation. The above results show that Xinjianqu can improve the blood lipid level, liver and kidney function, and gastrointestinal motility of rats with HLP, and the improvement effect of Xinjianqu on hyperlipidemia is significantly enhanced by fermentation. The mechanism may be related to AMPK, p-AMPK, LKB1, and HMGCR protein in the LKB1-AMPK pathway and the regulation of intestinal flora structure.

G protein-coupled receptors that influence lifespan of human and animal models.

Humanity has always sought to live longer and for this, multiple strategies have been tried with varying results. In this sense, G protein-coupled receptors (GPCRs) may be a good option to try to prolong our life while maintaining good health since they have a substantial participation in a wide variety of processes of human pathophysiology and are one of the main therapeutic targets. In this way, we present the analysis of a series of GPCRs whose activity has been shown to affect the lifespan of animal and human models, and in which we put a special interest in describing the molecular mechanisms involved. Our compilation of data revealed that the mechanisms most involved in the role of GPCRs in lifespan are those that mimic dietary restriction, those related to insulin signaling and the AMPK and TOR pathways, and those that alter oxidative homeostasis and severe and/or chronic inflammation. We also discuss the possibility of using agonist or antagonist drugs, depending on the beneficial or harmful effects of each GPCR, in order to prolong people's lifespan and healthspan.

Cinnamtannin D1 ameliorates DSS-induced colitis by preventing Th17/Treg imbalance through activation of the AMPK/mTOR pathway.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic idiopathic gastrointestinal disease, including ulcerative colitis (UC) and Crohn's disease (CD), which is typically characterized by chronicity and relapse. Cinnamtannin D1 (CTD1), extracted from Cinnamomum tamala, has been found to exert good immunosuppressive activity. However, the role of CTD1 in IBD is unclear. METHODS: The colitis mice model was established by dextran sulfate sodium (DSS) treatment. Protein levels (p-STAT3/STAT3, ROR-γt, p-STAT5/STAT5, FOXP3, p-AMPK/AMPK, and p-mTOR/mTOR) were examined using Western blotting analysis. Changes in histopathology were detected through hematoxylin and eosin staining. The proportion of T helper 17 (Th17) cells and regulatory T (Treg) cells was measured by flow cytometry analysis. RESULTS: CTD1 improved body weight and colon length, and alleviated inflammation and histological damage in DSS-induced colitis mice model. DSS treatment also demonstrated a negative effect on Th17-Treg cells balance whereas CTD1 restored the balance of Th17- Treg cells in DSS-induced colitis mice model. Regulatory factors (such as STAT3, ROR-γt, STAT5, and FOXP3) that closely related to the balance of Th17-Treg cells were regulated by CTD1. In addition, AMPK phosphorylation was increased and mTOR phosphorylation was inhibited by CTD1 in DSS-induced colitis mice model. CONCLUSION: These findings established that CTD1 improved DSS-induced colitis by suppressing Th17-Treg cells balance by activating the AMPK/mTOR pathway. This study provided a new strategy for developing novel treatments for patients with IBD.

Antitumor activity of pachymic acid in cervical cancer through inducing endoplasmic reticulum stress, mitochondrial dysfunction, and activating the AMPK pathway.

Pachymic acid has various pharmacological effects, including anti-inflammatory, antioxidant, immunomodulatory, and antitumor. However, the role of pachymic acid in cervical cancer remains unclear. So, we investigated the effects of pachymic acid in cervical cancer and elucidated the underlying mechanisms. We treated HeLa cells and normal cervical epithelial cells (HUCECs) with pachymic acid (0, 10, 20, 40, 80, or 160 µM) for 72 h, and found the cell activity was decreased in cells treated with 160 µM pachymic acid for 48 h or 80 µM pachymic acid for 72 h, while HUCECs viability without effect. Next, we observed that endoplasmic reticulum (ER) related gene expression, mitochondrial membrane potential (MMP) changes, ATP depletion, reactive oxygen species (ROS) generation and apoptosis were increased. Moreover, we observed that cytochrome C (Cytc) expression was increased and apoptosis-inducing factor (AIF) was decreased in the cytoplasm of pachymic acid-treated HeLa cells. Tauroursodeoxycholic acid (TUDCA) of ER stress inhibitor reversed the effects of pachymic acid on HeLa cells. Phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) of the AMPK pathway key protein was upregulated in pachymic acid-induced HeLa cells. Finally, we subcutaneously implanted HeLa cells into female nude mice and treated them with pachymic acid (50 mg/kg) for 3 weeks (5 days/week), and observed in pachymic acid induced xenograft mice, tumor growth was suppressed, cell apoptosis, ER-related gene expression, and ROS levels in tumor tissues were increased. Therefore, these findings demonstrated that pachymic acid plays an anti-tumor activity in cervical cancer through inducing ER stress, mitochondrial dysfunction, and activating the AMPK pathway.

SLC45A4 promotes glycolysis and prevents AMPK/ULK1-induced autophagy in TP53 mutant pancreatic ductal adenocarcinoma.

BACKGROUND: Somatic mutations of the TP53 gene occur frequently in pancreatic ductal adenocarcinoma (PDA). Solute carrier family 45 member A4 (SLC45A4) is a H+ -dependent sugar cotransporter. The role of SLC45A4 in PDA, especially in TP53 mutant PDA, remains poorly understood. METHODS: We explored the TCGA datasets to identify oncogenes in TP53 mutant PDA. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], colony formation and 5-ethynyl-2'-deoxyuridine (Edu) assays were performed to investigate the function of SLC45A4 in vitro. Glucose consumption, lactate production and ATP production were detected to evaluate glucose utilization. Extracellular acidification rate and oxygen consumption rate assays were used to evaluate glycolysis and oxidative phosphorylation. The subcutaneous xenotransplantation models were conducted to explore the function of SLC45A4 in vivo. RNA-sequencing and gene set enrichment analysis were employed to explore the biological alteration caused by SLC45A4 knockdown. Western blotting was performed to evaluate the activation of glycolysis, as well as the AMPK pathway and autophagy. RESULTS: SLC45A4 was overexpressed in PDA for which the expression was significantly higher in TP53 mutant PDA than that in wild-type PDA tissues. Moreover, high level of SLC45A4 expression was tightly associated with poor clinical outcomes in PDA patients. Silencing SLC45A4 inhibited proliferation in TP53 mutant PDA cells. Knockdown of SLC45A4 reduced glucose uptake and ATP production, which led to activation of autophagy via AMPK/ULK1 pathway. Deleting SLC45A4 in TP53 mutant HPAF-II cells inhibited the growth of xenografts in nude mice. CONCLUSIONS: The present study found that SLC45A4 prevents autophagy via AMPK/ULK1 axis in TP53 mutant PDA, which may be a promising biomarker and therapeutic target in TP53 mutant PDA.

MUC16 C-terminal binding with ALDOC disrupts the ability of ALDOC to sense glucose and promotes gallbladder carcinoma growth.

The MUC16 C-terminal (MUC16c) level is associated with tumor serum CA-125 levels, however, the roles remain unclear in gallbladder carcinoma (GBC). In this study, we found that MUC16c promoted glucose uptake and glycolysis for GBC cell proliferation. Mass spectrometry analysis suggested that MUC16c could combine with aldolase. The ALDOC mRNA and protein are overexpressed in GBC tumors. The IHC results also showed the consistent up-regulation of. ALDOC and MUC16c level in GBC tumor tissues than in peritumor tissues. We determined that MUC16c combining with ALDOC promoted ALDOC protein stability and disrupted the ability of ALDOC sensing glucose deficiency, which activated AMPK pathway and increased GBC cell proliferation. ALDOC knockdown significantly inhibited the glucose uptake and glycolysis induced by MUC16c. Our study established important roles of MUC16c promoting GBC cell glycolysis and proliferation and revealed the underlying mechanism of CA-125-related heavy tumor metabolic burden in GBC.

Sulindac and vitamin D3 synergically inhibit proliferation of MCF-7 breast cancer cell through AMPK/Akt/ß-catenin axis in vitro.

Breast cancer is associated with a high rate of recurrence, resistance therapy and mortality worldwide. We aimed at investigating the inhibitory effects of Sulindac and vitamin D3 (VD) on MCF-7 human breast cancer cells. MCF-7 cells were cultured with different concentrations of Sulindac and VD over a period of 24, 48 and 72 hours for cell viability and IC50 experiments. Hochst staining was used to evaluate apoptosis, whereas quantitative PCR (qPCR) was performed to measure mRNA levels of BCL-2 and BAX genes. Immunofluorescence staining was used to monitor intracellular ß-catenin expression. The protein levels of AKT, AMPK and P65 were measured by western blotting. The result showed that cell viability decreased in treated cells dose/time dependently (P < .05). Hochst staining showed an increase in fragmented nuclei in treated cells. The expression of BCL-2 and BAX genes decreased and increased in treated cells, respectively (P < .05). Immunofluorescence staining indicated that the expression of ß-catenin significantly reduced in treated cells. The AKT-1/p-Akt-1 and AMPK/p-AMPK ratio increased in treated cells (P < .05), but the P65/p-P65 ratio did not change significantly (P > .05). Our results indicated that the combination of Sulindac and VD has a growth-inhibiting effect on MCF-7 cells through AMPK/Akt/ß-catenin axis.

Cyanidin-3-O-glucoside attenuates high glucose-induced podocyte dysfunction by inhibiting apoptosis and promoting autophagy via activation of SIRT1/AMPK pathway.

Diabetic nephropathy (DN) is a common and complicated chronic kidney disease around the world. To elucidate and find effective therapies of DN is of vital importance. In this paper, we have discovered that cyanidin-3-O-glucoside (C3G), which is one of the anthocyanins, could alleviate high glucose-induced podocyte dysfunction. MTT, flow cytometry assay, and Western blot analysis showed that C3G could reverse the increase of cell apoptosis under high glucose treatment in MPC5 cells by upregulation of Bcl2 and downregulation of Bax and cleaved caspase-3. Moreover, C3G improved the autophagy decrease that was induced by high glucose through regulating the expression level of LC3-II/LC3-I, Beclin1, and p62. In addition, C3G inhibited epithelial-mesenchymal transition (EMT) by increasing E-cadherin and reducing Vimentin. By further study of the mechanisms, we found C3G activated the SIRT1 and AMPK which were inhibited in high glucose condition. Silencing SIRT1 blocked the effect of C3G on regulating cell apoptosis, autophagy, and EMT. In summary, our current findings suggest the protective effect of C3G against high glucose-induced podocyte dysfunction is by improving autophagy and reducing apoptosis and EMT via activating SIRT1/AMPK pathway. It might be a new insight for the treatment of DN.

Ghrelin Affects Gastric Cancer Progression by Activating AMPK Signaling Pathway.

As the endogenous ligand for the GH secretagogue receptor (GHSR), Ghrelin is aberrant expressed in multiple malignant carcinoma, and involved in regulating a number of progression of cancer, especially in metastasis and proliferation. However, the precise role of Ghrelin in tumorigenesis of gastric cancer (GC) is still poorly understood. In this study, we extensively investigated the roles and mechanisms of Ghrelin in human gastric cancer. Ghrelin levels in cancer tissues and cell lines were analyzed by immunohistochemistry, qRT-PCR, and Western blot. Functional studies were performed after Ghrelin overexpressed or knockdown in AGS cell line. Cell proliferation was evaluated in by MTT and clone formation assays. The wound healing and Transwell system were used to assess the cell migration and invasive ability of GC cells. Cell apoptosis was detected by flow cytometry, and metabolic assays were performed to reveal the function of Warburg effect in the process. Ghrelin was lowly expressed in gastric cancer tissues and cell lines. Overexpression of Ghrelin inhibited gastric cancer cell proliferation, migration, invasion, and promoted apoptosis by activating the AMPK pathway, while D-[lys3]-GHRP-6 (a GHSR agonist) treatment relieved the effect, promoting tumorigenesis. Ghrelin knockdown increased the glucose uptake and lactic acid release, suggesting that Ghrelin elicited an anti-Warburg effect via AMPK pathway to inhibit gastric tumorigenesis. Ghrelin inhibits cell proliferation, migration, and invasion by eliciting an anti-Warburg effect via AMPK signaling pathway in gastric cancer cells.

Isoleucilactucin Ameliorates Coal Fly Ash-Induced Inflammation through the NF-κB and MAPK Pathways in MH-S Cells.

We investigated whether isoleucilactucin, an active constituent of Ixeridium dentatum, reduces inflammation caused by coal fly ash (CFA) in alveolar macrophages (MH-S). The anti-inflammatory effects of isoleucilactucin were assessed by measuring the concentration of nitric oxide (NO) and the expression of pro-inflammatory mediators in MH-S cells exposed to CFA-induced inflammation. We found that isoleucilactucin reduced CFA-induced NO generation dose-dependently in MH-S cells. Moreover, isoleucilactucin suppressed CFA-activated proinflammatory mediators, including cyclooxygenase-2 (COX2) and inducible NO synthase (iNOS), and the proinflammatory cytokines such as interleukin-(IL)-1ß, IL-6, and tumor necrosis factor (TNF-α). The inhibiting properties of isoleucilactucin on the nuclear translocation of phosphorylated nuclear factor-kappa B (p-NF-κB) were observed. The effects of isoleucilactucin on the NF-κB and mitogen-activated protein kinase (MAPK) pathways were also measured in CFA-stimulated MH-S cells. These results indicate that isoleucilactucin suppressed CFA-stimulated inflammation in MH-S cells by inhibiting the NF-κB and MAPK pathways, which suggest it might exert anti-inflammatory properties in the lung.

Saikosaponin A and D Inhibit Adipogenesis via the AMPK and MAPK Signaling Pathways in 3T3-L1 Adipocytes.

Obesity is a lipid metabolism disorder caused by genetic, medicinal, nutritional, and other environmental factors. It is characterized by a complex condition of excess lipid accumulation in adipocytes. Adipogenesis is a differentiation process that converts preadipocytes into mature adipocytes and contributes to excessive fat deposition. Saikosaponin A (SSA) and saikosaponin D (SSD) are triterpenoid saponins separated from the root of the Bupleurum chinensis, which has long been used to treat inflammation, fever, and liver diseases. However, the effects of these constituents on lipid accumulation and obesity are poorly understood. We investigated the anti-obesity effects of SSA and SSD in mouse 3T3-L1 adipocytes. The MTT assay was performed to measure cell viability, and Oil Red O staining was conducted to determine lipid accumulation. Various adipogenic transcription factors were evaluated at the protein and mRNA levels by Western blot assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Here, we showed that SSA and SSD significantly inhibited lipid accumulation without affecting cell viability within the range of the tested concentrations (0.938-15 µM). SSA and SSD also dose-dependently suppressed the expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c (SREBP-1c), and adiponectin. Furthermore, the decrease of these transcriptional factors resulted in the repressed expression of several lipogenic genes including fatty acid binding protein (FABP4), fatty acid synthase (FAS), and lipoprotein lipase (LPL). In addition, SSA and SSD enhanced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylase (ACC), and inhibited the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) and p38, but not c-Jun-N-terminal kinase (JNK). These results suggest that SSA and SSD inhibit adipogenesis through the AMPK or mitogen-activated protein kinase (MAPK) pathways in the early stages of adipocyte differentiation. This is the first study on the anti-adipogenic effects of SSA and SSD, and further research in animals and humans is necessary to confirm the potential of saikosaponins as therapeutic agents for obesity.

Linagliptin Protects against Endotoxin-Induced Acute Kidney Injury in Rats by Decreasing Inflammatory Cytokines and Reactive Oxygen Species.

Septic shock can increase pro-inflammatory cytokines, reactive oxygen species (ROS), and multiple organ dysfunction syndrome (MODs) and even lead to death. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been proven to exert potential antioxidant and anti-inflammatory effects. We investigated the effects of linagliptin on endotoxic shock and acute kidney injury (AKI) in animal and cell models. In the cell model, linagliptin attenuated ROS by activating the AMP-activated protein kinase (AMPK) pathway, restoring nuclear-factor-erythroid-2-related factor (Nrf2) and heme oxygenase 1 (HO-1) protein, and decreasing pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß)). In the animal model, 14-week-old conscious Wistar-Kyoto rats were randomly divided into three groups (n = 8 in each group). Endotoxin shock with MODs was induced by the intravenous injection of Klebsiella pneumoniae lipopolysaccharide (LPS, 20 mg/kg). Linagliptin improved animal survival without affecting hemodynamic profiles. In the histopathology and immunohistochemistry examinations of the rat kidneys, linagliptin (10 mg/kg) suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducible nitric oxide synthase (iNOS), decreased injury scores, and preserved E-cadherin expression from LPS damage. In conclusion, linagliptin ameliorated endotoxin-shock-induced AKI by reducing ROS via AMPK pathway activation and suppressing the release of TNF-α and IL-1ß in conscious rats.