p-Akt Protein Research Articles

Short and long-term blockades of adenosine 2A, 5-HT2A, and 5-HT7 receptors induce apoptosis, reduce proliferation, and show differential effects on miR-27b-3p expression in neuroblastoma cell lines.

The first of our aims in this study was to investigate the effects of 5-HT2AR, 5-HT7R, and A2AR blockades on miR-27b-3p expression in the short and long-term in neuroblastoma cells. Our second aim was to reduce the expression of pERK and suppress proliferation by blocking the 5-HT2AR with ketanserin. Our third aim was to reduce the expression of pAKT and induce apoptosis by blocking the A2AR and 5-HT7R with MSX3 and SB269970. Thus, we aimed to investigate the therapeutic efficacy of ketanserin, MSX3 and SB269970, individually or in combination, on neuroblastoma cells. We found that short and long-term blockades of A2A, 5-HT2A, and 5-HT7 receptors had different effects on miR-27b-3p expression. Blockade of A2AR and 5-HT7R with MSX3 and SB269970 decreased miR-27b-3p expression in the short term while increasing it in the long term. Ketanserin increased miR-27b-3p expression in both the short and long term. When 5-HT2AR was blocked with ketanserin, no significant difference was observed in pERK expression and proliferation in the short term. In contrast, a substantial decrease in pERK expression and proliferation was detected in the long term. Our findings show that the MSX3 + SB269970 dual combination and ketanserin + MSX3 + SB269970 triple combination are especially critical in suppressing pAKT expression in the long term. These findings showed that pAKT protein expression induced apoptosis due to decreased in neuroblastoma cells. Our study provides the first evidence for the relationships between ketanserin/miR-27b-3p/pERK, MSX3/miR-27b-3p/pAKT, and SB269970/miR-27b-3p/pAKT in neuroblastoma cells. Ketanserin, MSX3, and SB269970 drug combinations may be promising therapeutic agents in neuroblastoma cells.

Repurposing of sericin combined with dactolisib or vitamin D to combat non-small lung cancer cells through computational and biological investigations

This study aims to repurpose sericin in combating non-small lung cancer cells (A549 and H460) by combining it with dactolisib or vitamin D to reduce the dose of dactolisib and boost the anticancer effectiveness of dactolisib and vitamin D. Therefore, the binding affinities of individual and combined drugs were examined using in silico and protein-protein interaction studies, targeting NF-κB, Cyclin D1, p-AKT, and VEGF1 proteins. The findings manifested remarkable affinities for combinatorial drugs compared to individual compounds. To substantiate these findings, the combined IC50 for each combination (sericin + dactolisib and sericin + vitamin D) were determined, reporting 31.9 and 41.8 µg/ml, respectively, against A549 cells and 47.9 and 55.3 µg/ml, respectively, against H460 cells. Furthermore, combination indices were assessed to lower the doses of each drug. Interestingly, in vitro results exhibited marked diminutions in NF-κB, Cyclin D1, p-AKT, and VEGF1 after treatment with sericin + dactolisib and sericin + vitamin D compared to control lung cancer cells and those treated with a single drug. Moreover, A549 and H460 cells treated with both combinations demonstrated augmented caspase-3 levels, implying substantial apoptotic activity. Altogether, these results accentuated the prospective implementation of sericin in combination with dactolisib and vitamin D at low doses to preclude lung cancer cell proliferation.

RNF19A inhibits bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway

BackgroundThe role of the RING finger protein superfamily in carcinogenesis has been widely studied, but one member of this family, RNF19A, has not yet been thoroughly explored in bladder cancer (BCa).MethodsThe expression levels of RNF19A in BCa samples and cell lines were analysed through data mining of public resources and further experiments. BCa cells in which RNF19A was stably overexpressed or knocked down were generated through lentivirus infection. The effects of RNF19A on cell proliferation, migration, and invasion were explored by performing a series of in vitro experiments, including CCK-8, colony formation, wound healing, and Transwell invasion assays. Using bioinformatics methods and multiple experiments, including western blot, qRT‒PCR, immunoprecipitation, cycloheximide, ubiquitination, and rescue assays, the mechanism underlying the effect of RNF19A on the progression of BCa was investigated.ResultsHere, we found that RNF19A expression was reduced in BCa samples and cell lines and that lower RNF19A expression predicted shorter overall survival of BCa patients. Functionally, forced expression of RNF19A suppressed BCa cell proliferation, migration, and invasion by inactivating the AKT/mTOR signalling pathway, whereas silencing RNF19A had the opposite effects. Mechanistically, RNF19A could directly interact with ILK and promote its ubiquitination and degradation. Rescue experiments revealed that forced ILK expression partially rescued the decreased phosphorylation of AKT, mTOR, and S6K1 caused by RNF19A overexpression and that the increased levels of the p-AKT, p-mTOR, and p-S6K1 proteins induced by RNF19A knockdown were eliminated after silencing ILK. Similarly, the effects of RNF19A overexpression or knockdown on the phenotypes of cell proliferation, migration, and invasion could also be restored by forced or decreased ILK expression.ConclusionsRNF19A suppressed the proliferation, migration, and invasion abilities of BCa cells by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway. RNF19A might be a potential prognostic biomarker and promising therapeutic target for BCa.

Mechanism of Asperosaponin VI Related to EGFR/MMP9/AKT/PI3K Pathway in Treatment of Rheumtoid Arthritis.

To explore the mechanism of action of asperosaponin VI (AVI) in the treatment of rheumatoid arthritis (RA) and validate it in ex vivo experiments using network pharmacology and molecular docking methods. The predicted targets of AVI were obtained from PharmMaper, UniProt and SwissTarget Prediction platforms, the disease targets were collected from Online Mendelian Inheritance in Man, Therapeutic Target Database and GeneCards databases, the intersection targets of AVI and RA were obtained from Venny 2.1.0, and the protein-protein interaction (PPI) network was obtained from STRING database, which was analyzed by Cytoscape software and screened to obtain the core targets. Cytoscape software was used to analyze PPI network and screen the core targets. Based on the Database for Annotation, Visualization and Integrated Discovery database, Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed, and Cytoscape software was used to construct the "Disease-Pathway-Target-Drug" network, which was finally verified by molecular docking and animal experiments. Network pharmacological studies showed that AVI was able to modulate 289 targets, with 102 targets for the potential treatment of RA, with the core pathway being the AKT/PI3K signaling pathway, and the core targets being the epidermal growth factor receptor (EGFR) and matrix metalloproteinase 9 (MMP9). Molecular docking results showed that AVI could produce strong binding with both of the 2 core targets. In vitro cellular experiments showed that AVI reduced nitric oxide, prostaglandin E2, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1 β levels (P<0.05) and inhibited cyclooxygenase-2, nitric oxide synthase, EGFR, MMP9, phosphorylated phosphoinositide 3-kinase (p-PI3K), and phosphorylated serine-threonine kinase (p-AKT) proteins (P<0.05). The results of in vivo studies showed that AVI improved RA score and foot swelling thickness and decreased TNF-α, IL-6, p-PI3K and p-AKT levels in RA rats (P<0.05). AVI exerts anti-inflammatory and anti-RA effects which might be related to the EGFR/MMP9/AKT/PI3K pathway.

Effect of mesenchymal stem cell derived from umbilical cord blood on rabbit intrauterine adhesion model

Objective: To investigate the repair effect of human umbilical cord blood mesenchymal stem cells (hUCB-MSC) on endometrium of intrauterine adhesion (IUA) by establishing animal model. Methods: Eighteen healthy female New Zealand white rabbits were divided into a control group, an IUA group and an hUCB-MSC transplantation group according to random number table method. The control group underwent laparotomy only. The IUA group underwent IUA modeling surgery using curettage and lipopolysaccharide (LPS) cotton placed in uterine cavity for double injury. The hUCB-MSC transplantation group received the same IUA modeling surgery followed by multipoint injection of hUCB-MSC suspension (2×106 cells/ml, 500 μl) into the bilateral uterine myometrium one week after surgery. Four weeks after the IUA modeling surgery, the rabbits were euthanized and samples were collected. Hematoxylin and eosin (HE) staining and Masson staining were used to assess the number of endometrial glands and the fibrosis rate. RNA sequencing was performed on the endometrium of the IUA group and hUCB-MSC transplantation group.The mRNA and protein expression of the fibrosis markers [transforming growth factor β1 (TGF-β1) and tissue inhibitors of metalloproteinase 1 (TIMP1)] and RNA sequencing-related parameters were detected by quantitative real-time PCR (qRT-PCR) and Western blot. Results: Compared with the control group, the number of glands in IUA group decreased (26.33±1.53 vs 4.33±1.53, P<0.001), and the fibrosis rate increased (18.01%±2.21% vs 69.55%±2.42%, P<0.001), indicating successful modeling. HE and Masson staining revealed that the number of glands in the hUCB-MSC transplantation group was increased compared with that in IUA group (17.33±2.52 vs 4.33±1.53, P<0.001), and the fibrosis rate decreased (69.55%±2.42% vs 41.55%±1.99%,P=0.001). Western blot analyses of fibrosis-related proteins (TGF-β1 and TIMP1) showed elevated levels of TGF-β1 (0.91±0.05) and TIMP1 (0.99±0.01) proteins in the IUA group compared to control group (0.61±0.04, 0.68±0.07) (P=0.001, 0.015). The hUCB-MSC transplantation group exhibited reduced expression of TGF-β1 (0.69±0.04) and TIMP1 (0.62±0.08) proteins compared to the IUA group (P=0.005, 0.014). Western Blot results related to the PI3K/AKT signaling pathway [phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT)] showed that the expression of p-PI3K(1.05±0.05) and p-AKT(1.17±0.06) in the IUA group increased compared to the control group (0.78±0.03, 0.85±0.05) (P=0.002, 0.002). The expression of p-PI3K (0.74±0.02) and p-AKT (0.93±0.04) proteins in the hUCB-MSC transplantation group decreased compared to the IUA group (P=0.003, 0.005). Conclusion: hUCB-MSC can repair the damaged endometrium in rabbit intrauterine adhesion model by increasing the number of endometrial glands and improving its level of fibrosis.

SPAG4 enhances mitochondrial respiration and aerobic glycolysis in colorectal cancer cells by activating the PI3K/Akt signaling pathway.

Aerobic glycolysis plays a pivotal role in the progression of tumors. Previously, a glycolysis-associated prognostic model in CRC was constructed and the glycolysis-related gene SPAG4 was discovered to be upregulated in CRC and was correlated with adverse prognosis. To date, however, no study has elucidated the specific role of SPAG4 in the development of CRC. In our study, CRC cells were transfected with si-SPAG4 or OE-SPAG4 to evaluate the influence of SPAG4 silencing or overexpression on CRC cell malignant behaviors. CRC cell proliferation and metastasis were detected via CCK-8, colony formation, and Transwell assays. The oxygen consumption rate and extracellular acidification rate of CRC cells were determined by using an XF24 extracellular flux analyzer. The expression of SPAG4, key mitochondrial markers (NDUFA1, SDHB, ATP5A, and PGC-1α), key enzymes involved in glycolysis (GLUT1, HK2, LDHA, PKM2, and PFK1), and PI3K/Akt pathway-molecules and downstream transcription factor HIF-1α was assessed by RT-qPCR and western blot analysis. SPAG4 expression in CRC and normal tissue samples was tested through immunohistochemical staining. Finally, SPAG4-overexpressed CRC cells were treated with LY294002 to validate the inhibition of PI3K/Akt pathway on CRC cell malignant phenotypes. Our results showed that SPAG4 was upregulated in CRC cells and tissues, and high expression SPAG4 predicted shorter overall survival time. SPAG4 knockdown inhibited while SPAG4 overexpression enhanced CRC cell proliferation, migration, invasion, mitochondrial respiration, and aerobic glycolysis. Overexpressing SPAG4 elevated p-PI3K, p-Akt, p-mTOR, and HIF-1α protein levels, which were restored after LY294002 treatment. Furthermore, LY294002 abolished the promotion of SPAG4 overexpression on CRC malignant phenotypes. Collectively, SPAG4 plays an oncogenic role in CRC by promoting mitochondrial respiration and aerobic glycolysis through activating the PI3K/Akt signaling. These findings suggest that inhibition of SPAG4-mediated glucose metabolism may represent a potential strategy for the clinical treatment of CRC.

The Deubiquitinating Enzyme USP4 Promotes Trophoblast Dysfunction by Stabilizing RYBP.

Previous studies have suggested that impaired spiral artery remodeling, placental dysfunction, and insufficient trophoblast infiltration are the etiology and pathogenesis of Preeclampsia (PE). Ring 1 and YY1 binding protein (RYBP) has been reported to be associated with trophoblast dysfunction. However, the molecular mechanism of RYBP involved in trophoblasts in the pathogenesis of PE is poorly defined. RYBP and Ubiquitin-specific peptidase 4 (USP4) mRNA levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). RYBP, USP4, p-PI3K, PI3K, p-AKT, and AKT protein levels were measured using western blot assay. Cell viability, proliferation, apoptosis, invasion, and migration were assessed using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell, and wound healing assays. After ubibrowser database analysis, the interaction between USP4 and RYBP was verified using Co-immunoprecipitation (CoIP) assay. RYBP and USP4 expression were upregulated in placental tissues from PE patients. By using JEG-3 and HTR-8/SVneo trophoblast cells, RYBP overexpression or USP4 upregulation could hinder cell viability, proliferation, invasion, migration, and promote apoptosis. Mechanistically, USP4 could trigger the deubiquitination of RYBP and prevent its degradation. In addition, USP4 repressed the PI3K/AKT signaling pathway by regulating RYBP. In total, Decreased USP4-mediated ubiquitination results in an adverse impact on trophoblast function by enhancing RYBP expression, providing a novel therapeutic target for PE.

Glycyrrhizic Acid Inhibits Hippocampal Neuron Apoptosis by Activating the PI3K/ AKT Signaling Pathway.

Glycyrrhizic acid (GA), one of the main active substances in Glycyrrhiza, has anti-inflammatory, anti-viral, and neuroprotective effects. GA can significantly reduce cerebral infarction size in middle cerebral artery occlusion (MCAO) rats and suppress inflammatory responses. However, the underlying mechanism by which GA protects the neuronal system remains poorly understood. Cell proliferation and viability were tested using CCK-8 and Edu assays. The effects of GA on apoptosis were detected using flow cytometry and Tunel assays. Western blotting was performed to assess protein expression. Behavioral experiments were conducted using the Morris water maze and rotation tests. Infarct size was observed using TTC staining. We report that GA protects neurons by inhibiting apoptosis, mainly through the PI3K/AKT pathway in oxygen-glucose deprivation/reoxygenation (OGD/R) and MCAO rat models. GA increases the viability and proliferation of oxygen- and glucose-deprived hippocampal neurons. Hippocampal neuron apoptosis decreased after GA treatment in vitro and in vivo. Furthermore, we determined that GA treatment increased the active state of PI3K and its downstream protein p-AKT, whereas when using a specific inhibitor of PI3K, Y294002, the levels of p-PI3K and p-AKT decreased. Finally, we showed that GA treatment improved spatial memory and motor coordination in MCAO rats, while TTC staining showed that GA decreased cerebral infarct size in MCAO rats. We reveal that GA protects hippocampal neurons by inhibiting their apoptosis, mainly through the PI3K/AKT signaling pathway.

7872 Adiponectin Enhances Hepatic Insulin Sensitivity in a Rat Model of Polycystic Ovary Syndrome: Role of PI3K/Akt/mTORC2 Pathway

Abstract Disclosure: A. Abdelhameed: None. S. Rezq: None. R.M. Vinson: None. J. Flaherty: None. B. Kim: None. D.G. Romero: None. L.L. Yanes Cardozo: None. Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity and insulin resistance (IR). Phosphoinositide-3-kinase (PI3K)/Akt/ the mammalian target of rapamycin complex 2 (mTORC2) pathway is one major insulin signaling pathway and its impairment is involved in multiple metabolic disorders by inducing IR. Phosphorylation of protein kinase B (Akt) at (Ser)-473 by mTORC2 leads to its full activation and subsequently enhances insulin sensitivity. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist that enhances insulin sensitivity in different models of diabetes. However, its potential to attenuate androgen-mediated hepatic IR by modulating PI3K/Akt/mTORC2 pathway remains unknown in PCOS. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates hepatic IR via modulating PI3K/Akt/mTORC2 pathway in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA while fasting plasma glucose was measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, was calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (Ser473), mTOR, p-mTOR (Ser2481) and mTORC2 regulatory protein Rictor were quantified in the liver by Western-blot. Results: DHT significantly (p&amp;lt;0.05) increased BW, fat mass and lean mass by 1.4-, 2.2-, 1.4- folds, respectively compared to PBO group. DHT significantly (p&amp;lt;0.05) increased HOMA-IR by 3.8- fold, compared to PBO group. DHT downregulated hepatic p-Akt, Akt and mTOR protein expression by 20% to 40% while upregulating IRS-1 by 1.6- fold with no impact on PI3 kinase, p-mTOR, Rictor and p-IRS1 compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR (34.7% reduction in HOMA-IR) in PCOS rats. Interestingly, PEG-BHD1028 significantly (p&amp;lt;0.05) increased PI3K, active Akt (pAKT), mTOR and Rictor levels by 1.6-, 2.2-, 1.5-, 1.8- folds, respectively with no effect on the other insulin signaling markers compared to vehicle-treated group. Conclusion: Our findings suggest that PEG-BHD1028 is a novel and effective therapeutic agent to attenuate hepatic IR in PCOS via activating PI3K/Akt/mTORC2 pathway. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Presentation: 6/2/2024

Dual anti-inflammatory effects of curcumin and berberine on acetaminophen-induced liver injury in mice by inhibiting NF-κB activation via PI3K/AKT and PPARγ signaling pathways

Acetaminophen (APAP) overdose is still a leading cause of drug-induced liver injury (DILI), accompanied with severe inflammatory response. However, the therapy for APAP-induced DILI is rather limited. The combined application of natural products to treat DILI induced by APAP may be a new direction of the research. This study was conducted to evaluate the dual anti-inflammatory activity of curcumin (CUR) combined with berberine (BBR) against APAP-mediated DILI. Network pharmacology found that PI3K-Akt and PPAR signaling pathways were primarily involved in anti-DILI of the combination of CUR and BBR. APAP injection enhanced the levels of ALT, AST, IL-1β, IL-6, and TNF-α in mice, while such phenomenon was significantly reversed by the cotreatment of CUR and BBR, which was more effective than either single treatment. The increase of p-NF-κB and p-IKKα/β protein expression and the decrease of p-PI3K, p-AKT, and PPARγ protein expression in APAP-treated mice were markedly inhibited by the coadministration of CUR and BBR. Molecular docking further demonstrated that both CUR and BBR could stably bind to PI3K, AKT, and PPARγ protein. In conclusion, the combination of CUR and BBR more effectively protected liver from APAP-triggered DILI than individual treatment. The mechanism is to alleviate hepatic inflammation by inhibiting NF-κB activation, which is possibly mediated by PI3K/Akt and PPARγ signaling pathways.

Inhibitory Effects of Propolis Flavonoids on Migration and Invasion of Laryngeal Cancer Cell and Analysis of Related Signal Pathways

Laryngeal cancer (LGC) is a malignant tumor that occurs in the larynx, and it is mainly treated through chemotherapy, radiotherapy, and surgery. Nevertheless, the five-year survival rate for patients is poor. Bee propolis contains various bioactive compounds and abundant anti-tumor active ingredients. Nevertheless, research on the use of propolis extracts for the treatment of LGC is relatively limited. This research aimed to demonstrate the inhibitory effects of ethanol extracts of propolis on migration (Mig) and invasion (Inv ) of LGC cells, as well as the related signaling pathways. The effects of graded ethanol extraction of propolis on the proliferation (Pro), Inv, Mig, apoptosis (Apo), and related signaling pathways of Hep-2 cells were analyzed. Propolis was extracted using ethanol (0%, 25%, 50%, 75%, and 100%) for the graded extraction of crude propolis. The flavonoid content and yield of the extracts were determined. The effects of various concentrations of propolis flavonoids on the clearance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, O2- radicals, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were evaluated, as well as their effects on the Pro inhibition of normal human pancreatic ductal epithelial (hTERT-HPNE) cells. Hep-2 cells of LGC were cultured using media containing 0, 25, 50, and 100 μmol/L propolis flavonoids. The cell Pro activity, Inv, Mig, Apo, and expression of PI3K/Akt pathway-related proteins were evaluated using CCK-8 assay, Transwell chamber assay, acridine orange/ethidium bromide (AO/EB) double staining method, and Western blotting, respectively. It was revealed that extraction with 50% ethanol solution yielded a higher content and yield of flavonoids, which were 51.20% and 7.42%, respectively. As the concentration of propolis flavonoids increased, the clearance rates of DPPH, O2-, and ABTS radicals, as well as the inhibition of hTERT-HPNE Pro, gradually increased. The maximum clearance rates were 84.1%, 26.6%, and 92.3%, respectively, while the maximum cell Pro inhibition rate was only 8.6%. Relative to the 0 μmol/L propolis flavonoid treatment group, the Hep-2 cells treated with 25, 50, and 100 μmol/L propolis flavonoids exhibited decreased cell Pro activity, reduced number of invasive and migratory cells, increased Apo rate, decreased PI3K and p-Akt proteins, and demonstrated a concentration-dependent effect (P &lt; 0.05). In summary, the extraction with 50% ethanol solution resulted in a higher yield of flavonoids. Propolis flavonoids demonstrated marked antioxidant activity and did not cause damage to normal hTERT-HPNE cells. They exhibited inhibitory effects on the Pro, Inv, and Mig of Hep-2 cells in LGC, and promoted cell Apo. These effects may be associated with PI3K/Akt signaling inhibition.

Tujia medicine Toddalia asiatica improves synovial pannus in rats with collagen-induced arthritis through the PI3K/Akt signaling pathway

To investigate the therapeutic mechanism of Tujia medicine Toddalia asiatica alcohol extract (TAAE) for synovial pannus formation in rats with college-induced arthritis (CIA). Sixty male SD rats were randomized into normal control group, CIA model group, TGT group, 3 TAAE treatment groups at low, medium and high doses (n=10). Except for those in the normal control group, all the rats were subjected to CIA modeling using a secondary immunization method and treatment with saline, TGT or TAAE by gavage once daily for 35 days. The severity of arthritis was assessed using arthritis index (AI) score, and knee joint synovium pathologies were examined with HE staining. Serum levels of TNF-α, IL-6, and IL-1β were detected with ELISA; the protein expressions of PI3K, Akt, p-PI3K, p-Akt, VEGF, endostatin, HIF-1α, MMP1, MMP3, and MMP9 in knee joint synovial tissues were determined using Western blotting, and the mRNA expressions of TNF‑α, IL-6, IL-1β, VEGF, HIF-1α, PI3K, and Akt were detected with RT-PCR. Treatment of CIA rat models with TAAE and TGT significantly alleviated paw swelling, lowered AI scores, and reduced knee joint pathology, neoangiogenesis, and serum levels of inflammatory factors. TAAE treatment obviously increased endostatin protein expression, downregulated p-PI3K, p-Akt, MMP1, MMP3, MMP9, VEGF, and HIF-1α proteins, and reduced TNF‑α, IL-6, IL-1β, PI3K, Akt, VEGF, and HIF-1α mRNA levels in the synovial tissues, and these changes were comparable between high-dose TAAE group and TGT group. TAAE can improve joint symptoms and inhibit synovial pannus formation in CIA rats by regulating the expressions of HIF-1α, VEGF, endostatin, MMP1, MMP3, and MMP9 via the PI3K/Akt signalling pathway.

Eucommia ulmoidesOliv. leaves flavonoids attenuate methylglyoxal-induced endothelial cell apoptosis invitro and invivo by upregulating AKT-Nrf2 signaling and downregulating oxidative stress.

Methylglyoxal (MGO) triggers oxidative stress responses in vascular endothelial cells, leading to apoptosis linked to diabetic vascular complications. Total flavonoids of Eucommia ulmoides leaves (TFEL) display antioxidant activity, yet its prevention of MGO-induced apoptosis and mechanisms are unclear. Our study used western blotting and ELISA to evaluate protein levels and enzyme activities. Cell viability and apoptosis were evaluated using CCK8 assay and PE Annexin V/7-AAD double staining. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured using fluorescence probes. Vascular pathological changes and apoptosis were analyzed through H&E and TUNEL staining. Invitro, MGO-stimulated human umbilical vein endothelial cells (HUVECs) were treated with varying TFEL concentrations. Our results demonstrated that TFEL significantly enhanced cell viability, reduced apoptosis, downregulated caspase-3 activity, and Bax/Bcl-2 ratio. Moreover, TFEL markedly suppressed MGO-induced ROS and malondialdehyde (MDA) production while restoring antioxidant enzyme activity and MMP. TFEL pretreatment promoted the expression of p-Akt, Nrf2, and HO-1 proteins. Pharmacological inhibition of p-Akt significantly suppressed the upregulation of Nrf2 and HO-1 protein levels mediated by TFEL. Consistently, pharmacological inhibition of Nrf2 or p-Akt partially abrogated the protective effects of TFEL against MGO-induced damage in HUVECs. Invivo studies revealed that TFEL (100 and 200 mg/kg) partially restored antioxidant capacity and reduced aortic thickness and apoptosis in MGO-injured mice. In conclusion, the findings indicate that TFEL mitigates MGO-induced apoptosis via activation of p-Akt/Nrf2/HO-1 and scavenging of oxidative stress, highlighting its potential in diabetic vascular complication management.

Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway

BackgroundTo explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism.MethodsAn obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee’s index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, β-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis.ResultsHFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, β-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs.ConclusionsNobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

LINC01370 suppresses hepatocellular carcinoma proliferation and metastasis by regulating the PI3K/AKT pathway

BackgroundHepatocellular carcinoma (HCC) poses a serious threat to human health worldwide. lncRNA dysregulation is frequently observed in various cancers, including HCC. However, the function of LINC01370 in HCC progression and its underlying mechanisms remain unclear.MethodsLINC01370 expression in HCC tissues with cells was analyzed by applying the GEO and GEPIA databases and qRT-PCR. CCK-8 and Transwell assays were used to assess HCC cell proliferation, migration, and invasion. The PI3K, AKT, with p-AKT protein expression were analyzed by western blotting.ResultsGene Expression Omnibus (GEO) and Gene Expression Profiling Interactive Analysis (GEPIA) showed that LINC01370 expression was significantly lower in HCC tissues than in normal tissues. LINC01370 overexpression markedly repressed HepG2 SMMC-7721 cells proliferation, migration, and invasion. To understand the downstream mechanism of LINC01370 regulation, we further analyzed the genes co-expressed with LINC01370 in GSE136247 and GSE132037 and then performed KEGG analysis. The PA pathway was found to be a downstream pathway regulated by LINC01370 in GSE136247 and GSE132037 via gene co-expression and KEGG analysis. Furthermore, PI3K and p-AKT protein levels decreased after LINC01370 overexpression. Importantly, rescue experiments showed that activation of the PI3K/AKT pathway disrupted the repressive effect of LINC01370 overexpression on the proliferation, migration, and invasion of HepG2 of SMMC-7721 cells.ConclusionsThis study verified that LINC01370 suppresses HCC proliferation with metastasis by regulating the PI3K/AKT pathway.

Sitagliptin synergizes 5-fluorouracil efficacy in colon cancer cells through MDR1-mediated flux impairment and down regulation of NFκB2 and p-AKT survival proteins.

5-fluorouracil (5-FU) is an inexpensive treatment for colon cancer; however, its efficacy is limited by chemoresistance. This study investigates the combination therapy approach of 5-FU with Sitagliptin (Sita), a diabetic drug with potential cancer-modulating effects. The combination was evaluated in vitro and in silico, focusing on the effects of Sita and 5-FU on colon cancer cells. The results showed that the addition of Sita significantly decreased the IC50 of 5-FU compared to 5-Fu monotherapy. The study also found that Sita and 5-FU interact synergistically, with a combination index below 1. Sita successfully lowered the 5-FU dosage reduction index, decreasing the expression of MDR1 mRNA and p-AKT and NFκB2 subunits p100/p52 protein. Molecular docking analyses confirmed Sita's antagonistic action on MDR1 and thymidylate synthase proteins. The study concludes that sitagliptin can target MDR1, increase apoptosis, and significantly reduce the expression of p-AKT and NFκB2 cell-survival proteins. These effects sensitize colon cancer cells to 5-FU. Repurposing sitagliptin may enhance the anticancer effects of 5-FU at lower dosages.

RNF135 Promotes Human Osteosarcoma Cell Growth and Inhibits Apoptosis by Upregulating the PI3K/AKT Pathway.

Ring finger protein 135 (RNF135) is an E3 ubiquitin ligase that has been implicated in the tumorigenesis of multiple human malignancies. However, whether RNF135 plays a role in the development of human osteosarcoma (OS) remains unknown. RNF135 expression in 20 human OS and 20 human osteochondroma specimens were evaluated by means of immunohistochemistry staining. The effects of shRNA-mediated RNF135 knockdown on human OS cell growth and apoptosis were evaluated through a panel of in vitro studies on cell proliferation, colony formation, exposure of phosphatidylserine on the cell surface, and caspase 3/7 activation. The protein levels of PI3K, AKT, and p-AKT were determined by western blot analysis. We detected significantly higher RNF135 levels in human OS tissues than human osteochondroma tissues. In in vitro studies, shRNA-mediated RNF135 knockdown in human OS cells inhibited proliferation and induced apoptosis. In addition, RNF135 knockdown reduced PI3K and p-AKT protein levels and activated caspase 3 and 7. These results supported that RNF135 contributes to human OS development through PI3K/AKT-dependent mechanisms. Targeting RNF135 may provide a new therapeutic approach for treating this human malignancy.

TSP50 facilitates breast cancer stem cell-like properties maintenance and epithelial-mesenchymal transition via PI3K p110α mediated activation of AKT signaling pathway

BackgroundStudies have confirmed that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties are conducive to cancer metastasis. In recent years, testes-specific protease 50 (TSP50) has been identified as a prognostic factor and is involved in tumorigenesis regulation. However, the role and molecular mechanisms of TSP50 in EMT and CSC-like properties maintenance remain unclear.MethodsThe expression and prognostic value of TSP50 in breast cancer were excavated from public databases and explored using bioinformatics analysis. Then the expression of TSP50 and related genes was further validated by quantitative RT-PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). In order to investigate the function of TSP50 in breast cancer, loss- and gain-of-function experiments were conducted, both in vitro and in vivo. Furthermore, immunofluorescence (IF) and immunoprecipitation (IP) assays were performed to explore the potential molecular mechanisms of TSP50. Finally, the correlation between the expression of TSP50 and related genes in breast cancer tissue microarray and clinicopathological characteristics was analyzed by IHC.ResultsTSP50 was negatively correlated with the prognosis of patients with breast cancer. TSP50 promoted CSC-like traits and EMT in both breast cancer cells and mouse xenograft tumor tissues. Additionally, inhibition of PI3K/AKT partly reversed TSP50-induced activation of CSC-like properties, EMT and tumorigenesis. Mechanistically, TSP50 and PI3K p85α regulatory subunit could competitively interact with the PI3K p110α catalytic subunit to promote p110α enzymatic activity, thereby activating the PI3K/AKT signaling pathway for CSC-like phenotypes maintenance and EMT promotion. Moreover, IHC analysis of human breast cancer specimens revealed that TSP50 expression was positively correlated with p-AKT and ALDH1 protein levels. Notably, breast cancer clinicopathological characteristics, such as patient survival time, tumor size, Ki67, pathologic stage, N stage, estrogen receptor (ER) and progesterone receptor (PR) levels, correlated well with TSP50/p-AKT/ALDH1 expression status.ConclusionThe effects of TSP50 on EMT and CSC-like properties promotion were verified to be dependent on PI3K p110α. Together, our study revealed a novel mechanism by which TSP50 facilitates the progression of breast cancer, which can provide new insights into TSP50-based breast cancer treatment strategies.

The 3,3'-dimethoxy-4,4'-dihydroxy-stilbene Triazole (STT) Inhibits Liver Cancer Cell Growth by Targeting Akt/mTOR Pathway.

The present study was aimed to investigate the proliferation inhibitory ability of 3,3'-dimethoxy-4,4'-dihydroxy-stilbene triazole (STT) on SNU449 and Huh7 cells. Moreover, the mechanism associated with the suppression of liver cancer cell proliferation by STT was also studied. The results revealed that STT suppresses proliferation of SNU449 and Huh7 cells to 28 and 21%, respectively treatment with 20 µM. The clonogenic survival of SNU449 and Huh7 cells was also significantly reduced after incubation with STT compared to the control cultures. In comparison to the control, STT treatment significantly decreased the invasive potential of SNU449 cells. Treatment with STT led to a prominent suppression in p62 and increase in LC3B protein expression in SNU449 cells compared to the control cells. The STT treatment dramatically decreased p-Akt and p-mTOR protein expression in SNU449 cells. Docking study revealed that STT interacts via traditional hydrogen bonding with the glutamine, phenylalanine, leucine, serine, arginine, aspartic acid, and lysine residues of Akt protein. In summary, the current study demonstrates that STT effectively suppresses the viability of SNU449 and Huh7 liver cancer cells. Moreover, STT treatment of the liver cancer cells also significantly reduces the clonogenic survival and invasive potential of SNU449 cells. Treatment of liver cancer cells with STT increases the expression of autophagic, targets anti-autophagic protein expression and down-regulates Akt/mTOR pathway to inhibit cancer growth and proliferation. Thus, STT exhibits prominent anticancer effect and needs to be investigated further as a potential candidate for the treatment of liver cancer.

Discovery of sesquiterpene lactones with anti-inflammatory effect from Youngia japonica

The preliminary study revealed that the ethyl acetate eluate of Youngia japonica (YJ-E) could inhibit the expression of key proteins of p-p65, p-IκBα, p-IKKα/β, and p-AKT in LPS stimulated BV2 cell. Further phytochemical study led to the isolation of eight compounds from YJ-E, including one new sesquiterpene lactone. Their structures were elucidated by several spectroscopic data, and comparing the NMR data of known compound. In addition, all of the isolates were evaluated for the anti-inflammatory effect. As a result, compounds 3 and 4 distinctly attenuated the expressions of p-IκBα, p-p65, and p-AKT in LPS stimulated BV2 cell, respectively.