Expression Levels Of Akt Research Articles

Shenqu xiaoshi oral solution enhances digestive function and stabilizes the gastrointestinal microbiota of juvenile rats with infantile anorexia

Ethnopharmacological relevanceMassa Medicata Fermentata (“Shenqu”) has long been applied in the treatment of indigestion in China; in fact, it is the active ingredient in the medicine Shenqu xiaoshi oral solution (SQXSOS). Based on robust clinical evidence, SQXSOS has shown efficacy in treating infantile anorexia (IFA). Aim of the studyTo investigate the underlying mechanisms by which SQXSOS treats IFA. Material and methodsThe pharmacodynamic efficacy of SQXSOS was validated through a high-fat diet (HFD)-induced IFA model of juvenile rats, which share physiological similarities to two-year-old humans. Ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-TOF MS) was utilized to analyze the blood-dissolved components of SQXSOS in rats. After identification of the blood-dissolved components, the key components and target genes were predicted through network pharmacology analysis. To further validate the predicted key targets of the blood-dissolved components, RT-PCR and Western blotting were employed to measure the changes in their concentrations. Meanwhile, the efficacy of SQXSOS on the structure of gastrointestinal microbiota (GM) in IFA rats was investigated. ResultsSQXSOS, when administered to the IFA rats at a dosage equivalent to its clinical dose in humans (3.78 mL/kg/day), induced a significant increase in the gastric emptying rate (+1.9-fold) and small intestine advancement rate (+0.5-fold) compared to the IFA rats. Additionally, SQXSOS reversed the pathological changes observed in the serum levels of digestive functioning biochemicals (−32.4%～+250% compared to the model group, p < 0.05). A total of 40 blood-dissolved components were identified by UHPLC-TOF MS. Berberine, oleanolic acid, ganolucidic acid A, slicyluric acid, and glycyrrhetinic acid were identified as the key components of SQXSOS, while AKT1, STAT3, TP53, JUN, and MAPK1 were identified as the key targets enabling the therapeutic efficacy of SQXSOS in treating IFA. In a target validation study, the mRNA transcript levels of the abovementioned target genes were found to be significantly higher in the gastric antrum of IFA rats. However, SQXSOS administration (3.78 and 7.56 mL/kg/day) reduced the elevated mRNA transcript levels of the abovementioned target genes (41.1–77.3% compared to model group, p < 0.05). GM analysis revealed a significant increase in the Firmicutes/Bacteroidota ratio (F/B ratio, +214.2%) in the IFA fecal samples compared to normal rats, but the high dosage of SQXSOS induced a marked decrease in the F/B ratio (−44.1%) compared to IFA rats. Furthermore, the therapeutic efficacy of SQXSOS against IFA might be attributed to the increase in Muribaculaceae abundance and the decrease in Prevotellaceae_UCG_003 abundance. ConclusionMechanistic investigations indicated that the efficacy of SQXSOS in treating IFA could be manifested by regulating the transcription and expression levels of AKT1, MAPK1, STAT3, and TP53 genes in the gastric antrum as well as modulating the abundance of Muribaculaceae and Prevotellaceae_UCG_003 family. Furthermore, there are still some limitations: the contents of the key biochemicals remained to be determined, similar STAT3 transcription levels were observed in both normal rats and IFA rats, and it is crucial to further validate the potential target GM when transitioning from animal populations to humans.

Qibai Pingfei capsule induces apoptosis of pulmonary artery smooth muscle cells in hypoxic rats by regulating PI3K/AKT/mitochondrial signaling pathway

Purpose: To investigate the effect of Qibai Pingfei capsules (QBPF) medicated serum on the apoptosis of rat pulmonary artery smooth muscle cells (PASMC) in hypoxic rats, and to determine the relationship between that effect and PI3K/Akt/mitochondrial apoptosis pathway.Methods: Rat PASMCs were isolated, cultured, and the optimal hypoxic time and concentration of QBPF were determined by CCK-8 method. Hypoxic rats were treated with QBPF, QBPF + LY294002, or QBPF + SC79. Apoptosis and mitochondrial membrane potential were assessed using Annexin VFITC/ PI, Hoechst 33258, and Rho123 staining. The protein expression levels of AKT, P-AKT, and apoptosis-related proteins were evaluated via western blot.Results: CCK-8 studies showed that the optimal hypoxic time was 24 h, while the optimal concentration of QBPF was 20 %. Annexin V-FITC/PI double staining and Hoechst 33258 assay revealed that QBPF significantly promoted the apoptosis of PASMCs in hypoxic rats (p &lt; 0.05). Rho123 test results showed that QBPF inhibited mitochondrial membrane potential level in hypoxic rats' PASMCs, which was enhanced by PI3K inhibitor LY29002 and inhibited by AKT agonist SC79 (p &lt; 0.05). Western blot showed that QBPF reduced the protein level of P-AKT and Bcl-2, and raised the protein levels of Bad, Bax, CytC, casepase-9 and casepase-3, which was enhanced by LY29002 and blocked by SC79 (p &lt; 0.05). No major changes in AKT protein expression were seen between the groups.Conclusion: In hypoxic rats, QBPF blocks PI3K/AKT signaling pathway and regulates the activation of downstream Bcl-2 family members, thus activating mitochondrial apoptosis pathway and triggering PASMC death.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443.

To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Exploring the role and mechanism of Astragalus membranaceus and radix paeoniae rubra in idiopathic pulmonary fibrosis through network pharmacology and experimental validation

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic disease with an unclear etiology and no effective treatment. This study aims to elucidate the pathogenic mechanism networks involving multiple targets and pathways in IPF. Extracts and metabolites of Astragalus membranaceus (AM) and Radix paeoniae rubra (RPR), two well-known traditional Chinese medicines, have demonstrated therapeutic effects on IPF. However, the underlying mechanisms of AM and RPR remain unclear. Utilizing network pharmacology analysis, differentially expressed genes (DEGs) associated with IPF were obtained from the GEO database. Targets of AM and RPR were identified using the TCM Systems Pharmacology Database and Analysis Platform and SwissTargetPrediction. A protein–protein interaction (PPI) network was subsequently constructed and analyzed using the STRING database and Cytoscape software. Gene ontology enrichment analysis and kyoto encyclopedia of genes and genomes analysis were conducted using Metascape. Additionally, a component-target-pathway network and a Sankey diagram were employed to identify the main active components, and molecular docking was performed between these components and proteins encoded by key targets. Finally, in vivo studies were conducted based on network pharmacology. A total of 117 common targets between DEGs of IPF and drug targets were identified and included in the PPI network, in which AKT1, MAPK3, HSP90AA1, VEGFA, CASP3, JUN, HIF1A, CCND1, PTGS2, and MDM2 were predicted as key targets. These 117 targets were enriched in the PI3K-AKT pathway, HIF-1 signaling pathway, apoptosis, and microRNAs in cancer. Astragaloside III, (R)-Isomucronulatol, Astragaloside I, Paeoniflorin, and β-sitosterol were selected as the main active components. Docking scores ranged from − 4.7 to − 10.7 kcal/mol, indicating a strong binding affinity between the main active compounds and key targets. In vivo studies have indeed shown that AM and RPR can alleviate the pathological lung fibrotic damage caused by bleomycin treatment. The treatment with AM and RPR resulted in a reduction of mRNA levels for key targets AKT1, HSP90AA1, CASP3, MAPK3, and VEGFA. Additionally, the protein expression levels of AKT1, HSP90AA1, and VEGFA were also reduced. These results support the therapeutic potential of AM and RPR in ameliorating pulmonary fibrosis and provide insight into the molecular mechanisms involved in their therapeutic effects.

Helicobacter pylori-derived outer membrane vesicles suppress liver autophagy: A novel mechanism for H. pylori-mediated hepatic disorder

BackgroundHelicobacter pylori outer membrane vesicles (OMVs) are nano-sized structures, which have been recently suggested to play a crucial role in H. pylori pathogenesis. There are growing evidence indicating the relationship of H. pylori infection with extra-gastroduodenal diseases, especially liver-related disorders. This study was aimed to investigate the effects of H. pylori-derived OMVs on autophagy in hepatic stellate cells (HSCs). Material and methodsA selection of five clinical strains of H. pylori with different virulence genotypes were included. The OMVs were isolated by ultracentrifugation and characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The protein concentration of OMVs was measured by BCA assay. MTT assay was used to determine the viability of LX-2 cells (human HSCs) treated with OMVs. The expression level of MTOR, AKT, PI3K, BECN1, ATG16 and LC3B genes was assessed in OMVs-treated LX-2 cells using quantitative real-time PCR. Moreover, immunocytochemistry was performed to evaluate the protein expression of MTOR and LC3B autophagy markers. ResultsH. pylori strains produced round shape nano-vesicles ranging from 50 to 500 nm. Treatment of HSCs with H. pylori-derived OMVs at concentration of 10 μg/mL for 24 h significantly elevated the expression of autophagy inhibitory markers (PI3K, AKT, and MTOR) and suppressed the mRNA expression level of autophagy core proteins (BECN1, ATG16 and LC3B). Immunocytochemistry also presented a substantial reduction in the concentration of LC3B autophagy core protein, and a marked elevation in the amount of MTOR autophagy inhibitory protein. ConclusionThis study revealed that H. pylori-derived OMVs could potentially suppress autophagy flux in HSCs as a novel mechanism for H. pylori-mediated liver autophagy impairment and liver disease development. Further studies are required to elucidate the exact role of OMV-carried contents in liver autophagy, and liver-associated disorders.

Jatrophone: a cytotoxic macrocylic diterpene targeting PI3K/AKT/NF-κB pathway, inducing apoptosis and autophagy in resistant breast cancer cells

BackgroundBreast cancer is a prevalent malignant tumor that affects women worldwide. The primary challenge in treating breast cancer is combating drug resistance, which contributes to relapse and metastasis. Jatrophone is a unique macrocyclic jatrophane diterpene found in various Jatropha and Euphorbia species. It possesses diverse biological and pharmacological activities, including anticancer activity. However, it is unclear whether jatrophone can overcome drug resistance in breast cancer.MethodsThis study includes the investigation of the cytotoxicity of jatrophone on doxorubicin-resistant breast cancer cells (MCF-7ADR) and the underlying molecular mechanisms. The effects of jatrophone on cell viability were determined using the sulforhodamine B (SRB) assay, while flow cytometry was used to evaluate cell cycle progression, apoptosis, and autophagy. A scratch assay was conducted to observe cell migration, and western blotting was used to measure downstream protein levels (PI3K, AKT, and NF-κB). Unpaired Student’s t-tests were used for comparison between the two groups and the results were analyzed by one-way ANOVA with Tukey- Kremer post hoc test.ResultsIt was shown that jatrophone exhibited potent cytotoxic activity on MCF-7ADR cells in a dose-dependent manner, with an IC50 value of 1.8 µM. It also significantly induced cell cycle S and G/M phase arrest. Interestingly, jatrophone induced both early and late apoptotic cell death, as well as autophagic cell death, with negligible necrosis. Furthermore, jatrophone treatment diminished the migration of MCF-7ADR cells. At the molecular level, jatrophone treatment significantly down-regulated the expression levels of PI3K, AKT, and NF-κB. β.ConclusionsThe results of the study suggest that jatrophone decreases the proliferation of MCF-7/ADR cells at a low micromolar concentration; induces cell cycle arrest; promotes apoptotic, and autophagic cell death; inhibits migration and EMT; and works on resistance by a mechanism involving the inhibition of the PI3K/Akt/ NF-κB pathway. These findings provide evidence of the potential of jatrophone to be a promising lead compound for targeting doxorubicin-resistant breast cancer cells and could be further investigated for its clinical application as a chemotherapy adjuvant.

Poly(A)-specific ribonuclease protein promotes the proliferation, invasion and migration of esophageal cancer cells.

Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease (PARN) gene in gastric cancer, head and neck squamous cell carcinoma, melanoma, cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis. The expression of the PARN gene in esophageal cancer (EC) tissue is also significantly higher than that in normal tissues, but the effect of PARN on the proliferation, migration and invasion of EC cells remains unclear. To investigate the relationship between PARN and the proliferation, migration and invasion of EC cells. The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected. PARN mRNA levels were measured using a tissue microarray, and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients. In addition, the effects of PARN gene knockout on tumor cell proliferation, invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1, and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model. The expression of PARN in EC tissues was higher than that in adjacent normal tissues, and the level of PARN expression was significantly positively correlated with lymphatic metastasis. Patients with high PARN levels had poor overall survival. BIM, IGFBP-5 and p21 levels were significantly increased in the PARN knockout group, while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data. In addition, the expression levels of Akt, p-Akt, PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased. The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased, the growth and proliferation of tumor cells were significantly inhibited, and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout. In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA (sh-NC) and PARN shRNA (sh-PARN) showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC, indicating that PARN knockdown significantly inhibited tumor growth in vivo. PARN has antiapoptotic effects on EC cells and promotes their proliferation, invasion and migration, which is associated with the development of EC and poor patient prognosis. PARN may become a potential target for the diagnosis, prognosis prediction and treatment of EC.

Chemical constituents from the flowers of Inula japonica and their anti-inflammatory activity

Ethnopharmacological relevanceThe flowers of Inula japonica (Inulae Flos) can be used to treat cough and asthma and remove phlegm in traditional Chinese medicine (TCM). Aim of the studyOur research aimed to obtain active components with the inhibition of inflammation and MUC5AC production to alleviate asthma symptoms from I. japonica. Materials and methodsThese compounds were separated from the MeOH extract of Inulae Flos by column chromatography over silica gel, AB-8 macroporous resin column, MPLC, and semipreparative HPLC. Their structures were elucidated by detailed spectroscopic data analysis, ECD calculations, and chemical methods. NO production was determined to evaluate anti-inflammatory activity in RAW 264.7 cells. The expression of MUC5AC, IL-1β, and IL-4 were measured in NCI–H292 cells by qRT-PCR. The anti-asthma activity assessments in vivo were performed through H & E and PAS staining, pulmonary function analysis, and cytokines determination by qRT-PCR or ELISA. The expression levels of PI3K, p-PI3K, AKT, p-AKT, MEK, p-MKE, ERK, p-MEK, and IL-1β were analyzed through western blotting. ResultsOne undescribed 1,10-seco-eudesmanolide derivative (1), two previously unreported 1,10-seco-eudesmanolide glycosides (2 and 3), and thirty-two known compounds (4–35) were obtained from Inulae Flos. Compound 11 had the most inhibitory effect against LPS-induced NO production in RAW 264.7 murine macrophages. Meanwhile, compound 11 also attenuated the increase in MUC5AC, IL-1β, and IL-4 mRNA expression in NCI–H292 cells. The results of the animal experiment confirmed that compound 11 significantly ameliorated OVA-induced asthma in a murine model of allergic asthma demonstrated by elevated pulmonary function, reduced inflammatory cell infiltration and mucus production. In addition, compound 11 significantly inhibited the levels of OVA-specific IgE in serum, of IL-4 and IL-6 in BALF, and of MUC5AC, IL-1β , IL-4, IL-5, IL-6 and IL-13 in lung tissue. Finally, compound 11 suppressed PI3K/AKT/MEK/ERK signaling pathway in lung tissue of mice. ConclusionThis study indicated that compound 11 might be a potential therapeutic candidate ameliorating airway inflammation and mucus hypersecretion via PI3K/AKT/MEK/ERK signaling pathway in allergic asthma.

Lithium prevents glucocorticoid‑induced chondrocyte autophagy: An in vitro study.

Glucocorticoids can induce chondrocyte autophagy. Lithium is a classical regulator of autophagy. The present study aimed to determine whether lithium can prevent glucocorticoid‑induced chondrocyte autophagy by regulating the PI3K/AKT/mTOR signaling pathway. For this purpose, rat and human chondrocytes were treated with dexamethasone (200µM) or dexamethasone (200µM) combined with lithium chloride at various concentrations (0.01, 0.1, 1 and 10mM). CYTO‑ID® autophagy fluorescence staining and transmission electron microscopy were used to detect the levels of autophagy in the chondrocytes. Reverse transcription‑quantitative PCR and western blot analysis were used to measure the expression levels of the autophagy marker, LC3B and the autophagy regulatory signaling pathway (PI3K/AKT/mTOR signaling pathways) markers, AKT and mTOR. The viability of chondrocytes was measured using the Cell Counting Kit‑8 assay. It was found that compared with that in the control group, dexamethasone induced the autophagy of chondrocytes, decreased the expression levels of AKT and mTOR, and reduced cell viability. Compared with the treatment with dexamethasone alone, lithium chloride (10mM) + dexamethasone reduced the autophagy levels, increased the expression level of AKT and mTOR, and increased cell viability. In conclusion, the present study demonstrated that lithium can prevent glucocorticoid‑induced autophagy by activating the PI3K/AKT/mTOR signaling pathway and preventing the glucocorticoid‑induced decrease in chondrocyte viability.

Relief of gouty arthritis in rats by total saponins from Dioscorea nipponica Makino through suppression of neutrophil extracellular trap formation via the PI3K/AKT/mTOR axis.

Gouty arthritis (GA) is an inflammatory disorder that is associated with elevated serum levels of uric acid. Total saponins from Dioscorea nipponica Makino (TSDN) are a natural component that ameliorates inflammation while also decreasing uric acid levels. The aim of the present study was to unravel the mechanism of TSDN in gouty rats in regard to regulation of the formation of neutrophil extracellular traps (NETs) via the PI3K/AKT/mTOR axis. A total of 40 Wistar rats were divided into 4 groups: normal, model, TSDN and rapamycin groups. Reverse-transcription-quantitative PCR (RT-qPCR) and western blot analysis were used to assess the mRNA and protein expression levels of the PI3K/AKT/mTOR axis. The formation of NETs was detected by immunohistochemical and immunofluorescent methods. ELISA was used to measure the levels of IL-1β and TNF-α. RT-qPCR and western blotting demonstrated that TSDN compromised the mRNA and protein expression levels of activated protein kinase (AMPK) and mTOR, as well as the mRNA expression levels of AKT and PTEN. Furthermore, it increased the protein expression levels of phosphorylated (p-) PI3K, p-AKT and p-AMPK. Immunohistochemical and immunofluorescent analyses revealed that TSDN decreased the protein expression levels of neutrophil elastase, proteinase 3, cathepsin G, lactoferrin and myeloperoxidase, as well as the number of citrullinated histone 3+ cells. TSDN also reduced the release of IL-1β and TNF-α. Overall, the anti-inflammatory action of TSDN in gouty rats may be realized by suppressing the formation of NETs by regulating the PI3K/AKT/mTOR axis.

HEATR3 involved in the cell proliferation, metastasis and cell cycle development of bladder cancer acts as a tumor suppressor.

The study was designed to detect the expression and clinical significance of the HEATR3 gene in bladder cancer (BCa) and to preliminarily explore whether this gene can affect the occurrence and development of BCa through the AKT/ERK signaling pathway. The expression and prognostic value of HEATR3 were explored based on The Cancer Genome Atlas (TCGA) and Genotypic Tissue Expression (GTEx) databases. Microarray immunohistochemical analysis was performed in 30 BCa cases to investigate the level of HEATR3 protein and to explore the relationship between HEATR3 and the clinicopathological features of BCa. Western Blot and qRT-PCR were used to detect HEATR3 protein and mRNA in BCa cell lines (5637, TCCSUP, SW780) and fallopian tube epithelial cell (SV-HUC-1). CCK8 method was employed to study the proliferation of BCa cells after heat treatment. Transwell assay was conducted to analyze the effect of HEATR3 on cell migration and invasion. And cell cycle and apoptosis were detected by flow cytometry. Furthermore, Western Blot assay was used to probe the effects of down-regulation of HEATR3 expression on the expression and phosphorylation levels of AKT and ERK proteins in BCa cells. Bioinformatics analysis showed that HEATR3 was significantly up-regulated in BCa, and high HEATR3 expression was associated with poor prognosis of BCa patients. In vitro experiments demonstrated that HEATR3 expression was up-regulated in BCa tissues compared with that in adjacent tissues. HEATR3 protein was also up-regulated in malignant cell lines. HEATR3 knockdown in BCa cells could inhibit cell proliferation, invasion and migration, block cell cycle and promote cell apoptosis. At the same time, HEATR3 knockdowns reduced the expression levels of p-AKT and p-ERK proteins. HEATR3 knockdown inhibits the development of BCa cells through the AKT/ERK signaling pathway. and it may become one of the most promising molecular targets for BCa treatment.

Dietary inositol improved glucose and lipid metabolism mediated by the Insulin-PI3K-Akt signaling pathway in Pacific white shrimp (Litopenaeus vannamei)

The present study aimed to evaluate the effects of dietary inositol level (0.86, 1.34, 1.40, 2.30, 3.88 and 7.50 g kg−1) on glucose and lipid homeostasis in Litopenaeus vannamei. The results indicated that the contents of inositol, phosphatidyl inositol (PI) and glycogen in hepatopancreas and muscle, very low-density lipoprotein (VLDL) in hepatopancreas and hemolymph, the activity of carnitine palmitoyl-transferase (CPT1) and the expression of hmit and inox were positively related to the dietary inositol levels, while the contents of glucose (GLU), crustacean hyperglycemic hormone (CHH), triglyceride (TG), cholesterol (CHO) and non-esterified fatty acid (NEFA) in hemolymph and the expression of imp, pepck, g6pc and fbp were negatively correlated with the dietary inositol level. TG content, phosphoenolpyruvate carboxykinase (PEPCK) activity and the expression levels of srebp, fas, acc1, g6pd and 6gpd in shrimp fed 3.88 and 7.50 g kg−1 inositol diets were significantly lower compared with shrimp fed the basal diet, while the expression of cpt1, aco, fabp, fatp and idir were showed the reverse change trend (P < 0.05). The activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK) and the expression levels of ir, irs1, pi3k and akt were significantly increased as dietary inositol levels ranged from 1.40 to 7.50 g kg−1, while the expression levels of gsk-3β and foxo1 significantly down-regulated. Hepatopancreas R cells number in shrimp fed 2.30, 3.88 and 7.50 g kg−1 inositol diets were significantly increased compared with shrimp fed 0.86 g kg−1 inositol diet, while the diameters and areas showed an opposite trend. In addition, dietary inositol supplementation significantly increased hemolymph insulin like peptide (ILP) content but had no significant effect on high density lipoprotein cholesterol (HLDC) content in hemolymph, hepatic lipase (HL) activity and the ratio of n-3/ n-6 PUFA and DHA/EPA in hepatopancreas. In conclusion, appropriate dietary inositol could positively regulate the Insulin-PI3K-Akt pathway to promote glucose utilization and maintain the normal accumulation of glycogen and lipids required for development, while high dietary inositol could promote lipid mobilization and prevent hepatopancreas lipids deposition by negatively regulating the Insulin-PI3K-Akt pathway, improving ILP sensitivity and enhancing VLDL synthesis, which may therefore be an important mechanism in the action of inositol on glucose and lipid metabolism. These findings provided further insight and understanding of mechanisms by which dietary inositol regulated glucose and lipid metabolism in L. vannamei.

Zinc depletion induces JNK/p38 phosphorylation and suppresses Akt/mTOR expression in acute promyelocytic NB4 cells

BackgroundMyeloid leukemia is associated with reduced serum zinc and increased intracellular zinc. Our previous studies found that zinc depletion by TPEN induced apoptosis with PML-RARα oncoprotein degradation in acute promyelocytic NB4 cells. The effect of zinc homeostasis on intracellular signaling pathways in myeloid leukemia cells remains unclear. ObjectiveThis study examined how zinc homeostasis affected MAPK and Akt/mTOR pathways in NB4 cells. MethodsWe used western blotting to detect the activation of p38 MAPK, JNK, ERK1/2, and Akt/mTOR pathways in NB4 cells stimulated with the zinc chelator TPEN. Whether the effects of TPEN on these pathways could be reversed by zinc or the nitric oxide donor sodium nitroprusside (SNP) was further explored by western blotting. We used Zinpyr-1 staining to assess the role of SNP on labile zinc levels in NB4 cells treated with TPEN. In additional, we evaluated expressional correlations between the zinc-binding protein Metallothionein-2A (MT2A) and genes related to MAPKs and Akt/mTOR pathways in acute myeloid leukemia (AML) based on the TCGA database. ResultsZinc depletion by TPEN activated p38 and JNK phosphorylation in NB4 cells, whereas ERK1/2 phosphorylation was increased first and then decreased. The protein expression levels of Akt and mTOR were downregulated by TPEN. The nitric oxide donor SNP promotes zinc release in NB4 cells under zinc depletion conditions. We further found that the effects of zinc depletion on MAPK and Akt/mTOR pathways in NB4 cells can be reversed by exogenous zinc supplementation or treatment with the nitric oxide donor SNP. By bioinformatics analyses based on the TCGA database, we demonstrated that MT2A expression was negatively correlated with the expression of JNK, and was positively correlated with the expression of ERK1 and Akt in AML. ConclusionOur findings indicate that zinc plays a critical role in leukemia cells and help understanding how zinc depletion induces apoptosis.

Exercise mitigates Dapagliflozin-induced skeletal muscle atrophy in STZ-induced diabetic rats

BackgroundSodium-glucose cotransporter 2 inhibitors (SGLT2i) are commonly used in the management of type 2 diabetes mellitus (T2DM) and have been found to worsen the reduction of skeletal muscle mass in individuals with T2DM. This study aims to examine the potential of exercise in mitigating the skeletal muscle atrophy induced by SGLT2i treatment.MethodsA rat model of T2DM (40 male Sprague-Dawley rats; T2DM induced by a combination of high-fat diet and streptozotocin) was used to examine the effects of six-week treatment with Dapagliflozin (DAPA, SGLT2i) in combination with either aerobic exercise (AE) or resistance training (RT) on skeletal muscle. T2DM-eligible rats were randomized into the T2DM control group (CON, n = 6), DAPA treatment group (DAPA, n = 6), DAPA combined with aerobic exercise intervention group (DAPA + AE, n = 6), and DAPA combined with resistance training intervention group (DAPA + RT, n = 6). To assess the morphological changes in skeletal muscle, myosin ATPase and HE staining were performed. mRNA expression levels of Atrogin-1, MuRF1, and Myostatin were determined using quantitative PCR. Furthermore, protein expression levels of AKT, p70S6K, mTOR, FoXO1/3A, NF-κB, and MuRF1 were examined through western blotting.ResultsBoth the administration of DAPA alone and the combined exercise intervention with DAPA resulted in significant reductions in blood glucose levels and body weight in rats. However, DAPA alone administration led to a decrease in skeletal muscle mass, whereas RT significantly increased skeletal muscle mass and muscle fiber cross-sectional area. The DAPA + RT group exhibited notable increases in both total protein levels and phosphorylation levels of AKT and p70S6K in skeletal muscle. Moreover, the DAPA, DAPA + AE, and DAPA + RT groups demonstrated downregulation of protein expression (FoXO1/3A) and mRNA levels (Atrogin-1, MuRF1, and Myostatin) associated with muscle atrophy.ConclusionsOur findings provide support for the notion that dapagliflozin may induce skeletal muscle atrophy through mechanisms unrelated to protein metabolism impairment in skeletal muscle, as it does not hinder protein metabolic pathways while reduces muscle atrophy-related genes. Additionally, our observations reveal that RT proves more effective than AE in enhancing skeletal muscle mass and muscle fiber cross-sectional area in rats with T2DM by stimulating protein anabolism within the skeletal muscle.

Beneficial biological effects of Flavokawain A, a chalcone constituent from kava, on surgically induced endometriosis rat model

Ethnopharmacological relevanceShrub kava has long been grown and utilized, primarily in the South Pacific region, for ceremonial, religious, and social occasions. It has been used as a pain reliever and muscle relaxant in medicinal practices from the eighteenth century. Interestingly, relatively low incidence of lung cancer may attribute to the high consumption of kava products in this region. Aim of the studyKava extracts were used to produce the kava chalcones Flavokawain A, B and C, which have a variety of bioactivities. In the present study, we show that Flavokawain A has positive effects on endometriosis. Materials and methodsThe endometriosis rat model was surgically induced by the autologous transplantation of endometrial tissue. Rats were evaluated for clinical ratings and lesion volume following a 6-week Flavokawain A therapy. Peritoneal fluid and blood samples were taken and ELISA assay was used to measure the cytokines and chemokines levels. Transcriptional and expression levels of Akt, PI3K, NF-kB, iNOS, Bcl-2, Bax and caspase-3 were evaluated by Western blotting and RT-qPCR. Implanted tissue sections of the rats were also analyzed by immunofluorescent and histopathological staining. ResultsLesion volumes and adhesion scores were successfully decreased. Blood and peritoneal fluid levels of associated cytokines and chemokines were markedly down-regulated. Besides, Flavokawain A also mediated cell apoptosis of endometrial implants. Additionally, VEGF expression was reduced, which inhibited the angiogenesis process. As for the expression of Akt, p-Akt, PI3K, p-PI3K, and NF-kB in endometriosis lesions, Flavokawain A significantly reduced them. ConclusionFlavokawain A has beneficial effects on the surgically induced endometriosis rat model, by reducing inflammation, promoting apoptosis, and decreasing angiogenesis. Our findings suggest that these effects may be mediated through the regulation of PI3K/Akt and NF-κB signaling pathways.

Nervonic acid reduces the cognitive and neurological disturbances induced by combined doses of D-galactose/AlCl3 in mice.

Nervonic acid (NA) is a kind of ultra-long-chain monounsaturated fatty acid, which can repair nerve cell damage caused by oxidative stress. Alzheimer's disease (AD) is a nervous system disease and often accompanied by the decline of learning and memory capacity. In this study, the combined dose of D-galactose/AlCl3 was used to establish a mouse model of AD. Meanwhile, the mice were treated with different doses of NA (10.95 and 43.93 mg/kg). The results showed that NA delayed the decline of locomotion and learning ability caused by D-galactose/AlCl3, increased the activity of total superoxide dismutase, catalase, glutathione peroxidase, and reduced the content of malondialdehyde in vivo. Besides, NA reduced the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), aspartate aminotransferase, alanine aminotransferase, increased the levels of 5-hydroxytryptamine, dopamine, γ-aminobutyric acid, alleviated the cell morphology damage induced by D-galactose/AlCl3 in hippocampus and liver tissue. Furthermore, the intervention of NA upregulated the expression levels of PI3K, AKT, and mTOR genes and downregulated the expression levels of TNF-α, IL-6, and IL-1β genes. Therefore, we speculate the intervention of NA could be an effective way in improving cognitive impairment through the activation of PI3K signaling pathway. These results suggest that NA has the potential to be developed as antioxidant drug for the prevention and early therapy of AD.

Investigating the synergistic potential of TRAIL and SAHA in inducing apoptosis in MOLT-4 cancer cells

IntroductionT-cell acute lymphoblastic leukemia is characterized by its fast progression rate and high complications. TRAIL can be used to trigger apoptosis in cancer cells with minimal effects on normal cells, but most of cancer cells develop resistance to this agent through various mechanisms. HDAC inhibitors like SAHA can be used to make cancer cells more susceptible to TRAIL-induced apoptosis. In this study, this hypothesis was tested on MOLT-4 cancer cell line. Materials and methodsThe cells were divided into six groups including the control group, TRAIL 50 nM, TRAIL 100 nM, SAHA 2 μM, SAHA 2 μM + TRAIL 50 nM, and SAHA 2 μM + TRAIL 100 nM. Apoptosis was evaluated by flowcytometry after 24, 48 and 72 h. The expression levels of c-flip, DR4, DR5, CHOP, NF-κB, STAT3, Akt, and PI3K genes were investigated by quantitative real-time PCR. Data were analyzed using two-way variance analysis with Tukey's and Dunnett's multiple comparisons tests, and statistical significance was defined as having a p-value less than 0.05. ResultsGroups exposed to the combination of SAHA with TRAIL demonstrated the maximum apoptosis in MOLT-4 cells by increasing the expression of DR4, DR5, and CHOP and decreasing the expression of c-flip, STAT3, PI3k, Akt, and NF-kB genes. ConclusionIt can be concluded that SAHA increases the sensitivity of MOLT-4 cells to TRAIL-mediated apoptosis, which can be used as a strategy to overcome resistance to TRAIL in leukemic patients.

Circ_0011373 promotes papillary thyroid carcinoma progression by regulating miR-1271/LRP6 axis.

This research aimed to explore the regulatory molecular mechanism among circular RNA (circ)_0011373, microRNA (miR)-1271, and lipoprotein receptor-related protein 6 (LRP6) in papillary thyroid carcinoma (PTC). Quantitative real-time PCR (qRT-PCR) assay was adopted to measure the expression of circ_0011373, miR-1271, and LRP6 mRNA. Furthermore, cell cycle distribution, apoptosis, migration and invasion were investigated by flow cytometry and transwell assay, respectively. The target relationship between miR-1271 and circ_0011373 or LRP6 was predicted by using the Starbase website and DIANA TOOL and verified by dual-luciferase reporter and RIP assay. Protein expression levels of LRP6, p-mTOR, mTOR, p-AKT, AKT, p-PI3K, and PI3K were tested by Western blot. The function of circ_0011373 on PTC tumor growth was validated by the xenograft tumor model in vivo. Circ_0011373 and LRP6 were upregulated, while miR-1271 was downregulated in PTC tissues and cell lines. Moreover, knockdown of circ_0011373 inhibited cell cycle, migration, and invasion and promoted apoptosis. Of particular importance was the fact that circ_0011373 directly interacted with miR-1271 and miR-1271 inhibitor was able to reverse the effect of circ_0011373 knockdown on PTC cell progression. Meanwhile, LRP6 was directly targeted by miR-1271, and its expression was positively regulated by circ_0011373. We further confirmed that miR-1271 overexpression suppressed cell cycle, migration, and invasion and enhanced apoptosis by regulating LRP6. In addition, circ_0011373 knockdown restrained PTC tumor growth in vivo. Circ_0011373 might be able to regulate PTC cell cycle, migration, invasion, and apoptosis by regulating the miR-1271/LRP6 axis.

Fenugreek Seed Extract Regulates Human Umbilical Vein Endothelial Cell Angiogenesis and Proliferation via the PI3K/Akt/Cyclin D1 Pathway.

The significance of angiogenesis in tumour progression has been widely documented. Hence, the identification of anti-angiogenic agents with fewer common side effects would be valuable in cancer therapy. In this study, we evaluated the anti-angiogenic and anti-proliferative effects of a hydro-alcoholic extract of fenugreek seed (HAEF) on human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cells were treated with various concentrations of HAEF and the half-maximal inhibitory concentration (IC50) value was estimated by using the MTT assay. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and matrix metalloproteinase enzyme (MMP-2 and MMP-9) gene expression profiles were evaluated by using quantitative RT-PCR (qRT-PCR). Moreover, MMP activities and PI3K, Akt and cyclin D1 protein expression levels were evaluated by gel zymography and Western blotting, respectively. HAEF reduced HUVEC viability, with an IC50 value of 200μg/ml. The qRT-PCR results demonstrated that treatment with HAEF markedly reduced MMP-2/MMP-9, VEGF and bFGF gene expression, as compared to the control group. We also found that MMP-2/MMP-9 enzyme activity and PI3K/Akt/cyclin D1 protein expression were notably decreased in cells treated with HAEF. Our results suggest that HAEF can potentially inhibit angiogenesis, and also affect cellular proliferation by targeting the PI3K/Akt/cyclin D1 pathway. Thus, fenugreek seed extract merits further investigation as a source of compounds with anti-cancer properties.

Curcumin inhibits lipopolysaccharide-induced proximal renal tubular epithelial cell death by regulating ADAMTS18 methylation and AKT/Notch pathways.

This study aimed to explore the effect of curcumin on the activity of lipopolysaccharide (LPS)-induced proximal renal tubular epithelial cells (PRTECs) and to analyze the molecular mechanism by which curcumin regulates their occurrence. LPS-induced PRTECs were used to construct an inflammatory cell model. RT-qPCR and western blot (WB) were used to detect ADAMTS18 expression. Methylation-specific PCR was used to detect ADAMTS18 methylation levels. After curcumin treatment, MTT assay was used to analyze cell viability, flow cytometry was used to analyze apoptosis, and ADAMTS18 expression and methylation levels were detected again. After transfection with siADAMTS18, cell viability and apoptosis were analyzed again. The levels of inflammatory factors were measured by enzyme-linked immunosorbent assay, and the expression levels of AKT, Notch-1 and Notch-2 were analyzed by WB. Curcumin strongly inhibited LPS-induced PRTEC inflammatory lesions, restored normal cell proliferation, and reduced the apoptosis rate by downregulating ADAMTS18 methylation and restoring ADAMTS18 expression. After siADAMTS18, the ability of curcumin to improve cell viability was reduced, and the ability of curcumin to downregulate inflammatory factors was significantly reduced. Curcumin could also inhibit the expression of AKT, Notch-1 and Notch-2 simultaneously. siADAMTS18 attenuated the abovementioned effects of curcumin. Curcumin inhibits LPS-induced PRTEC death by regulating ADAMTS18 methylation and AKT/Notch pathways.