Expression Of PI3K Research Articles

Mechanistic insights into Retama raetam's anti-proliferative and pro-apoptotic effects in A549 lung cancer cells: targeting PI3K/Akt pathway and ROS production.

Lung cancer, particularly non-small cell lung cancer (NSCLC), is a leading cause of cancer-related deaths worldwide. This study investigates the molecular mechanisms behind the anti-cancer effects of the tropical desert plant Retama raetam (R. raetam) on the A549 NSCLC cell line. The research examined R. raetam's anti-proliferative effects, cytotoxicity, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential, and cell morphology in NSCLC A549 and L-132 cells. In addition, the influence of R. raetam on DNA fragmentation, apoptotic signaling, and PI3K/Akt pathways for its anti-cancer mechanism was examined. Our results indicated that R. raetam's effects were dose- and time-dependent to exhibit anti-proliferative effects on A549 cells. R. raetam treatment promoted apoptotic cell death cycle arrest, increased apoptotic cells, depolarized the mitochondrial membrane, and induced morphological alterations in cells and nuclei. It also inhibited A549 cell migration (P < 0.05), colonization, and invasiveness. Moreover, the study demonstrated that R. raetam treatment resulted in the upregulation of Bax expression, downregulation of Bcl-2 expression, and apoptotic fragmented DNA in A549 cells. The top five bioactive compounds derived from R. raetam exhibited molecular interactions that inhibit PIK3CA and AKT1. This inhibition leads to an increased frequency of apoptosis and subsequent death of cancer cells. Additionally, R. raetam extract induced an increase in ROS formation and cytochrome c levels, indicating that its toxic effects on A549 cells involve both ROS-dependent cytotoxicity through the disruption of mitochondrial transmembrane potential ΔΨm and ROS-independent cell cycle arrest through downregulation BCL-2, PARP, E-Cadherin, PI3K, and Akt expressions pathways.

Characterization of Central and Nasal Orbital Adipose Stem Cells and their Neural Differentiation Footprints.

The unique potential of stem cells to restore vision and regenerate damaged ocular cells has led to the increased attraction of researchers and ophthalmologists to ocular regenerative medicine in recent decades. In addition, advantages such as easy access to ocular tissues, non-invasive follow-up, and ocular immunologic privilege have enhanced the desire to develop ocular regenerative medicine. This study aimed to characterize central and nasal orbital adipose stem cells (OASCs) and their neural differentiation potential. The central and nasal orbital adipose tissues extracted during an upper blepharoplasty surgery were explant-cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS). Cells from passage 3 were characterized morphologically by osteogenic and adipogenic differentiation potential and by flow cytometry for expression of mesenchymal (CD73, CD90, and CD105) and hematopoietic (CD34 and CD45) markers. The potential of OASCs for the expression of NGF, PI3K, and MAPK and to induce neurogenesis was assessed by real-time PCR. OASCs were spindle-shaped and positive for adipogenic and osteogenic induction. They were also positive for mesenchymal and negative for hematopoietic markers. They were positive for NGF expression in the absence of any significant alteration in the expression of PI3K and MAPK genes. Nasal OASCs had higher expression of CD90, higher potential for adipogenesis, a higher level of NGF expression under serum-free supplementation, and more potential for neuron-like morphology. We suggested the explant method of culture as an easy and suitable method for the expansion of OASCs. Our findings denote mesenchymal properties of both central and nasal OASCs, while mesenchymal and neural characteristics were expressed stronger in nasal OASCs when compared to central ones. These findings can be added to the literature when cell transplantation is targeted in the treatment of neuro-retinal degenerative disorders.

Combination of cisplatin and ethyl acetate extract of Vernonia amygdalina Delile induces cell cycle arrest and apoptosis on PANC-1 cells via PI3K/mTOR

Context: Vernonia amygdalina Delile (VAD) is known as a potential plant with a wide variety of medicinal properties, including anticancer. Aims: To evaluate the combination effect of VAD extract with cisplatin (CIS) against PANC-1 cells, focusing on cell cycle arrest and apoptosis activities. Methods: This study is an experimental study using PANC-1 cells as objects. The phytochemical compounds were analyzed with LC-MS/MS. The cytotoxic activity of extracts and their combinations was determined using the MTT assay method in the PANC-1 cell line. Apoptosis, cell cycle arrest, and PI3K/mTOR profiles were analyzed with flow cytometry. Immunocytochemistry was used to determine Bcl-2, Cyclin D1, and p53 expression. Results: The phytochemicals found were five compounds with retention times of 8.96, 9.85, 10.52, 12.59, and 10.36. The results of the MTT assay showed that the IC50 values of extract and CIS were 21.83 ± 0.46 µg/mL and 3.02 ± 0.44 µg/mL, respectively. The combination index (CI) value of extract and CIS had a synergistic effect. Combination extract and CIS induced early and late apoptosis, inhibited cell cycle progression on the G1 phase, inhibited Bcl-2 and cyclin D1 expression, induced p53 expression, and inhibited PI3K and mTOR expression. Conclusions: The combination of extract and CIS showed anticancer activity against PANC-1 cells through induction of apoptosis and cell cycle arrest via inhibition of PI3K and mTOR expressions.

Electroacupuncture promotes angiogenesis by regulating miR-142-5p and activating ADAMTS1/PI3K/AKT pathway in ischemic stroke rats.

To observe the effect of electroacupuncture on miR-142-5p and ADAMTS1/PI3K/AKT pathway in rats with ischemic stroke, so as to explore the regulatory mechanism of electroacupuncture on angiogenesis after ischemic stroke. This study was divided into two parts. The first part of the experiment：SD rats were randomly divided into sham operation group, model group and electroacupuncture group. There were 20 rats in each group. The middle cerebral artery occlusion (MCAO) rat model was prepared using a modified Longa's method. In the electroacupuncture group, "Shuigou" (GV26) was selected for electroacupuncture intervention (4 Hz/20 Hz) for 30 min each time. The rats in the electroacupuncture group were given electroacupuncture immediately after successful modeling, once a day for 4 times. Hunter score and TTC staining were used to observe the neurological deficits and infarct volumes respectively；HE staining was used to observe the cortical pathological changes；immunohistochemistry was used to determine the changes of cerebral microvascular density. Real-time quantitative PCR and Western blot were used to observe the miR-142-5p expression, mRNA and protein expression levels of ADAMTS1, VEGF, PI3K, AKT, eNOS in ischemic cortex. The second part of the experiment：The rats were randomly divided into electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group with 8 rats in each group. MCAO model was established after injection. Electroacupuncture+control group was given 0.9% sodium chloride solution injected into the right ventricle.The rats in the electroacupuncture+miR-142-5p Antagomir group were injected with miR-142-5p inhibitor into the right ventricle 30 min before modeling. Rats in electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group were all given the same electroacupuncture treatment. Real-time fluorescence quantitative PCR was used to observe the effect of miR-142-5p Antagomir on the expression of miR-142-5p and ADAMTS1 mRNA. The effect of miR-142-5p Antagomir on ADAMTS1 protein was observed by Western blot. In the first part of the experiment, compared with the sham operation group, the Hunter score in the model group was significantly increased (P<0.01)；the volume of cerebral infarction in the model group was significantly increased (P<0.01)；the degree of brain edema and neuronal necrosis and the density of cerebral microvessels was increased；the cerebral microvascular density was significantly increased (P<0.01)；the expression levels of miR-142-5p and the mRNA expression levels of VEGF, AKT and eNOS were significantly decreased (P<0.01, P<0.05), and the protein expression levels of VEGF, p-AKT and eNOS were significantly down-regulated (P<0.01), while the mRNA expression levels of ADAMTS1 and PI3K, and the protein expression levels of ADAMTS1 and p-PI3K were all up-regulated (P<0.01, P<0.05) in the model group. Compared with the model group, after intervention, the Hunter score in the electroacupuncture group was decreased (P<0.01), the volume of cerebral infarction was significantly decreased (P<0.01)；the degree of brain edema and neuronal necrosis were alleviated；the cerebral microvascular density was significantly increased (P<0.01)；the expression of miR-142-5p and the mRNA expression of VEGF, PI3K, AKT and eNOS were increased (P<0.01), the protein expressions of VEGF, p-PI3K, p-AKT and eNOS were increased (P<0.01, P<0.05), while the mRNA and protein expression of ADAMTS1 were decreased (P<0.05, P<0.01). After injection of miR-142-5p inhibitor, compared with electroacupuncture+control group, the expression of miR-142-5p in electroacupuncture+miR-142-5p Antagomir group was decreased(P<0.05), while the mRNA and protein expression of ADAMTS1 were increased (P<0.01, P<0.05). Electroacupuncture at GV26 can improve the neurological damage of ischemic stroke rats, reduce the volume of cerebral infarction and promote angiogenesis. The mechanism may be associated with the function of electroacupuncture in promoting the expression of miR-142-5p, so as to inhibit the expression of its target gene ADAMTS1, mediate the up-regulation of VEGF expression, activate PI3K/AKT pathway, promote the release of eNOS, and participate in promoting angiogenesis in ischemic stroke rats.

Danggui-Shaoyao-San (DSS) ameliorating cognitive impairment in ischemia–reperfusion vascular dementia mice through miR-124 regulating PI3K/Akt signaling pathway

Vascular dementia (VD) is a disease characterized by cognitive impairment and memory loss due to brain cell damage caused by cerebral vascular ischemia. Danggui-Shaoyao-San (DSS) has been used clinically to treat diseases for centuries. The VD model was established by bilateral common carotid artery (BCCA) repeated ischemia–reperfusion (I/R) and caudal bleeding. Target prediction of DSS and miR-124 in PI3K/Akt signaling pathway by network pharmacology. The effect of DSS on cognitive dysfunction were evaluated through methods such as behavioral experiments, cerebral blood flow monitoring, HE and Nissl staining, western blot, and q-PCR. Prediction result showed that both DSS and miR-124 could target Akt1. DSS treatment significantly reduced hippocampal cell damage, improved learning and memory ability. Mechanically, DSS treatment up-regulated the expression levels of PI3K and Akt protein, and its gene. Bcl-2/Bax index is up-regulated and cell apoptosis reduced. LC3II/LC3I index decreased and autophagy of brain cells increased. Moreover, DSS down-regulated the expression level of miR-124. And inhibition of miR-124 up-regulate the expression of PI3K, Akt. These results suggested that DSS can reduce the content of miR-124 in the hippocampus of VD mice, thus regulating the PI3K/Akt signaling pathway and improving the learning and memory ability of VD mice.

Analysis of core functional components in Yinchenhao Decoction and their pathways for treating liver fibrosis

To analyze the core functional component groups (CFCG) in Yinchenhao Decoction (YCHD) and their possible pathways for treating hepatic fibrosis based on network pharmacology. PPI data were extracted from DisGeNET, Genecards, CMGRN and PTHGRN to construct a weighted network using Cytoscape 3.9.1. The data of the chemical components in YCHD were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the potential active components and targets were selected using PreADMET Web server and SwissTargetPrediction. A fusion model was constructed to obtain the functional effect space and evaluate the effective proteins to identify the CFCG followed by GO and KEGG pathway enrichment analyses for all the targets. In cultured human hepatic stellate cells (LX-2 cells), the cytotoxicity of different compounds in YCHD was tested using CCK-8 assay; the effects of these compounds on collagen α1 (Col1a1) mRNA expression and the pathways in 20 ng/mL TGF-β1-stimulated cells were analyzed using RT-qPCR and Western blotting. A total of 1005 pathogenic genes, 226 potential active components and 1529 potential targets in YCHD and 52 potential targets of CFCG were obtained. Benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid were selected for CCK-8 verification, and they all showed minimal cytotoxicity below the concentration of 200 μmol/L. Clorius, polydatin, lauric acid and ferulic acid all effectively inhibited TGF-β1-induced LX-2 cell activation. At the concentration of 200 μmol/L, all these 4 components inhibited PI3K, p-PI3K, AKT, p-AKT, ERK, p-ERK, P38 MAPK and p-P38 MAPK expressions in TGF-β1-induced LX-2 cells. The therapeutic effect of YCHD on hepatic fibrosis is probably mediated by its core functional components including benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid, which inhibit the PI3K-AKT and MAPK pathways in hepatic stellate cells.

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.

Neuroprotective effects of resveratrol through modulation of PI3K/Akt/GSK-3β pathway and metalloproteases.

To analyze the expressional changes in the PI3K/Akt/GSK-3β pathway and metalloprotease in the cellular Alzheimer's Disease (AD) model with the effect of antioxidant resveratrol. Neuron-like cells were obtained by a two-step method of neuronal differentiation by using a combination of retinoic acid (RA) and brain-derived factor (BDNF) exposure. Then, the application of the amyloid beta peptide 25-35 (Aβ25-35) to the cell culture mimicked the environmental toxicity observed in AD. Afterward, cell viability and apoptosis assays were performed to determine whether the resveratrol exerts a cytotoxic and apoptotic effect. Finally, the expressional changes in genes in the cellular AD model with the effect of resveratrol were analyzed by Real-Time PCR. The analysis in silico was assessed using the STRING V12.0 database in each group. Apoptosis data findings were decreased by 1.5-fold and 2.5-fold respectively by Differentiated+Resveratrol (RES) and RES when compared to control but no significant difference was observed between RES and AD model groups. Real-time PCR analysis results revealed PI3K (3.38-fold), AKT (3.95-fold), and RELN (1.99-fold) expressions were significantly higher (p < .001), and also GSK-3β, TAU, ADAMTS-4, ADAMTS-5, and TIMP-3 gene expression levels were significantly downregulated (2.53-, 1.79-, 2.85-, 4.09-, and 6.62-fold, respectively) in the Differentiated+Aβ + RES groups compared to the Differentiated+Aβ group (p < .001). Network analysis shows the functional enrichment of 23 Alzheimer-related GO terms in the Wnt signaling, proteolysis, and extracellular matrix organization pathways. Resveratrol has inhibited GSK-3β by activating the PI3K/Akt insulin pathway in a neurotoxic environment. In addition, TAU, RELN, metalloproteases, and their inhibitors associated with Alzheimer's pathology have been regulated supporting the neuroprotective effect of resveratrol.

Eriodictyol attenuates osteoarthritis progression through inhibiting inflammation via the PI3K/AKT/NF-κB signaling pathway

Eriodictyol, a flavonoid distributed in citrus fruits, has been known to exhibit anti-inflammatory activity. In this study, destabilized medial meniscus (DMM)-induced OA model was used to investigate the protective role of eriodictyol on OA. Meanwhile, we used an IL-1β-stimulated human osteoarthritis chondrocytes model to investigate the anti-inflammatory mechanism of eriodictyol on OA. The production of nitric oxide was detected by Griess reaction. The productions of MMP1, MMP3, and PGE2 were detected by ELISA. The expression of LXRα, ABCA1, PI3K, AKT, and NF-κB were measured by western blot analysis. The results demonstrated that eriodictyol could alleviate DMM-induced OA in mice. In vitro, eriodictyol inhibited IL-1β-induced NO, PGE2, MMP1, and MMP3 production in human osteoarthritis chondrocytes. Eriodictyol also suppressed the phosphorylation of PI3K, AKT, NF-κB p65, and IκBα induced by IL-1β. Meanwhile, eriodictyol significantly increased the expression of LXRα and ABCA1. Furthermore, eriodictyol disrupted lipid rafts formation through reducing the cholesterol content. And cholesterol replenishment experiment showed that adding water-soluble cholesterol could reverse the anti-inflammatory effect of eriodictyol. In conclusion, the results indicated eriodictyol inhibited IL-1β-induced inflammation in human osteoarthritis chondrocytes through suppressing lipid rafts formation, which subsequently inhibiting PI3K/AKT/NF-κB signaling pathway.

Effects of Dietary Glutamate on the Growth Performance and Antioxidant Capacity of Juvenile Chinese Mitten Crab (Eriocheir sinensis)

In order to explore the effects of glutamate on the growth performance, antioxidant capacity and protein metabolism of juvenile Chinese mitten crab, 0%, 1% and 2% glutamate were supplemented to low protein (30%) and normal protein (35%) diets, respectively. There were 5 parallel tanks in each treatment, and the feeding duration was 8 weeks. The results showed that dietary glutamate did not significantly affect the weight gain of Chinese mitten crab. Diets supplemented with 2% glutamate significantly decreased the crude protein of crabs. The T-AOC of crabs fed the 30% protein diets was significantly lower than crabs fed the 35% protein diets. At 30% protein level, the superoxide dismutase (SOD) activity significantly increased with the increase in glutamate content. Dietary glutamate significantly down-regulated the relative expressions of mTOR, PI3K, S6K1 and 4EBP at 35% protein level. In conclusion, dietary glutamate cannot significantly increase the growth of Chinese mitten crab, but it can improve the antioxidant capacity in Chinese mitten crab under low protein conditions.

Overexpression of mir-489–3p inhibits proliferation and migration of non-small cell lung cancer cells by suppressing the HER2/PI3K/AKT/Snail signaling pathway

BackgroundLung cancer is a highly prevalent malignancy with significant morbidity and mortality rates. MiR-489–3p, a microRNA, has been identified as a regulator of tumor cell proliferation and invasion. Its expression is downregulated in non-small cell lung cancer (NSCLC). Elucidating the molecular mechanisms underlying miR-489–3p′s role in NSCLC pathogenesis is crucial for identifying potential diagnostic and therapeutic targets. MethodsTo investigate the molecular mechanism of miR-489–3p in NSCLC, this study utilized A549, a commonly used NSCLC cell line. MiR-489–3p mimics and inhibitors were transfected into A549 cells. Additionally, co-transfection experiments using wortmannin, an inhibitor of the PI3K/AKT pathway, were performed. Expression of miR-489–3p and related proteins was analyzed by Western blotting and quantitative real-time PCR (qRT-PCR). Cell migration and proliferation were assessed by wound healing and colony formation assays, respectively. ResultsOverexpression of miR-489–3p significantly inhibited the proliferation and migration of A549 cells. This inhibitory effect was further enhanced upon co-transfected with wortmannin. Analysis of human lung specimens showed increased expression of HER2, PI3K, and AKT in lung adenocarcinoma tissues compared to adjacent non-cancerous tissues. ConclusionsThese findings suggest that miR-489–3p overexpression may inhibit NSCLC cell proliferation and migration by suppressing the HER2/PI3K/AKT/Snail signaling pathway. This study elucidates miR-489–3p′s molecular mechanisms in NSCLC and provides experimental basis for identifying early diagnostic markers and novel therapeutic targets.

Polystyrene nanoplastics induce apoptosis, autophagy, and steroidogenesis disruption in granulosa cells to reduce oocyte quality and fertility by inhibiting the PI3K/AKT pathway in female mice

BackgroundNanoplastics (NPs) are emerging pollutants that pose risks to living organisms. Recent findings have unveiled the reproductive harm caused by polystyrene nanoparticles (PS-NPs) in female animals, yet the intricate mechanism remains incompletely understood. Under this research, we investigated whether sustained exposure to PS-NPs at certain concentrations in vivo can enter oocytes through the zona pellucida or through other routes that affect female reproduction.ResultsWe show that PS-NPs disrupted ovarian functions and decreased oocyte quality, which may be a contributing factor to lower female fertility in mice. RNA sequencing of mouse ovaries illustrated that the PI3K-AKT signaling pathway emerged as the predominant environmental information processing pathway responding to PS-NPs. Western blotting results of ovaries in vivo and cells in vitro showed that PS-NPs deactivated PI3K-AKT signaling pathway by down-regulating the expression of PI3K and reducing AKT phosphorylation at the protein level, PI3K-AKT signaling pathway which was accompanied by the activation of autophagy and apoptosis and the disruption of steroidogenesis in granulosa cells. Since PS-NPs penetrate granulosa cells but not oocytes, we examined whether PS-NPs indirectly affect oocyte quality through granulosa cells using a granulosa cell–oocyte coculture system. Preincubation of granulosa cells with PS-NPs causes granulosa cell dysfunction, resulting in a decrease in the quality of the cocultured oocytes that can be reversed by the addition of 17β-estradiol.ConclusionsThis study provides findings on how PS-NPs impact ovarian function and include transcriptome sequencing analysis of ovarian tissue. The study demonstrates that PS-NPs impair oocyte quality by altering the functioning of ovarian granulosa cells. Therefore, it is necessary to focus on the research on the effects of PS-NPs on female reproduction and the related methods that may mitigate their toxicity.

The potential neuroprotective effects of Spirulina platensis in a valproic acid-induced experimental model of autism in the siblings of albino rats: targeting PIk3/AKT/mTOR signalling pathway

ABSTRACT Introduction: Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with poor social interaction, communication issues, aberrant motor movements, and limited repetitive interests and behaviour. Spirulina platensis (SP) contains several multi-nutrients and has a wide range of neuroprotective properties. Aim: The target of the current experiment is to detect the protective effects of S. platensis on valproic-induced autism in adult female albino rats’ siblings for the first time. Materials and Methods: Twelve Pregnant rats were separated into four main groups; Group I (control); Group II (S. platensis); Group III (autistic group); and Group IV (autistic SP-treated group). Fifteen offspring pups from each group were sacrificed, brain was divided for biochemical analysis as superoxide dismutase and malondialdehyde were evaluated spectrophotometrically while interleukin-6, interleukin-12, Bcl-2-associated X protein, B-cell lymphoma-2, Beclin-1, brain-derived neurotrophic factor were assessed by ELISA, other division of brain were used for gene expression of PI3k, Akt and mTOR pathway, last division of brain were stained using (H&E) and Giemsa stains. Tumour necrosis factor alpha (TNF- α ) and Synaptophysin (SYN) markers were used for immunohistochemical staining. Results: Autistic Group (III) showed an increment in levels of MDA, IL-6, IL12 and BAX while showing a decrement in SOD, Bcl-2 and Beclin-1 as well as increased PI3k, Akt and mTOR gene expression. Autistic Group (III) also exhibited hypocellularity and disorganization of hippocampal and prefrontal cortex cells. The autistic SP-treated group (IV) showed improvement in these biochemical markers and pathological changes. Our findings suggest that Spirulina platensis will be significant in managing autism.

Insulin promotes the bone formation capability of human dental pulp stem cells through attenuating the IIS/PI3K/AKT/mTOR pathway axis

BackgroundInsulin has been known to regulate bone metabolism, yet its specific molecular mechanisms during the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs) remain poorly understood. This study aimed to explore the effects of insulin on the bone formation capability of human DPSCs and to elucidate the underlying mechanisms.MethodsCell proliferation was assessed using a CCK-8 assay. Cell phenotype was analyzed by flow cytometry. Colony-forming unit-fibroblast ability and multilineage differentiation potential were evaluated using Toluidine blue, Oil red O, Alizarin red, and Alcian blue staining. Gene and protein expressions were quantified by real-time quantitative polymerase chain reaction and Western blotting, respectively. Bone metabolism and biochemical markers were analyzed using electrochemical luminescence and chemical colorimetry. Cell adhesion and growth on nano-hydroxyapatite/collagen (nHAC) were observed with a scanning electron microscope. Bone regeneration was assessed using micro-CT, fluorescent labeling, immunohistochemical and hematoxylin and eosin staining.ResultsInsulin enhanced the proliferation of human DPSCs as well as promoted mineralized matrix formation in a concentration-dependent manner. 10− 6 M insulin significantly up-regulated osteogenic differentiation-related genes and proteins markedly increased the secretion of bone metabolism and biochemical markers, and obviously stimulated mineralized matrix formation. However, it also significantly inhibited the expression of genes and proteins of receptors and receptor substrates associated with insulin/insulin-like growth factor-1 signaling (IIS) pathway, obviously reduced the expression of the phosphorylated PI3K and the ratios of the phosphorylated PI3K/total PI3K, and notably increased the expression of the total PI3K, phosphorylated AKT, total AKT and mTOR. The inhibitor LY294002 attenuated the responsiveness of 10− 6 M insulin to IIS/PI3K/AKT/mTOR pathway axis, suppressing the promoting effect of insulin on cell proliferation, osteogenic differentiation and bone formation. Implantation of 10− 6 M insulin treated DPSCs into the backs of severe combined immunodeficient mice and the rabbit jawbone defects resulted in enhanced bone formation.ConclusionsInsulin induces insulin resistance in human DPSCs and effectively promotes their proliferation, osteogenic differentiation and bone formation capability through gradually inducing the down-regulation of IIS/PI3K/AKT/mTOR pathway axis under insulin resistant states.

Biochemical and molecular docking-based assessment of Spirulina platensis's bioactive constituents for their potential application as natural anticancer drug

Spirulina platensis and its extracts possess positive benefits for human health indications from malnutrition, antioxidant properties, and therapeutic properties. This study investigates the antioxidant activity of Spirulina platensis extracts and their cytotoxicity against the breast cancer cell line (MCF-7). The antioxidant and cytotoxicity properties of C- phycocyanin (C-PC) were directly compared with the diethyl ether extract of S. platensis (DEE). Results indicated that C-PC exhibited superior antioxidant activity compared to DEE extract. The observed values for C-PC are 1.453 μg/mL (IC50-value), 299.6 μM AA eq/mL, 86.68 μM TE eq/mL, and 179 μg/g for ABTS assay, DPPH, FRAP and total antioxidant activity, respectively. Moreover, C-PC also demonstrated enhanced cytotoxic activity against MCF-7 tumor cells with IC50 values of 100 μg/mL and 630 μg/mL for C-PC and DEE; respectively. C-PC treatment downregulated the expression of PI3K, Akt, mTOR, and Wnt. C-PC treatment to MCF-7 cells significantly induced caspase 3, caspase 8, and caspase 9. Similar effects, with lower tendency, were also observed for DEE extracts of S. platensis. Molecular docking studies suggest that S. platensis' bioactive constituents can induce apoptosis through intrinsic pathways. The current findings provide evidence for the medical importance of Spirulina platensis active constituents as promising natural anticancer drugs.

Network pharmacology and experimental validation to explore the role and potential mechanism of Liuwei Dihuang Decoction in prostate cancer

ObjectiveTo evaluate the anti-tumor effector of Liuwei Dihuang Decoction (LWDHD) in prostate cancer (PCa) and explore the potential mechanism using experimental validation, network pharmacology, bioinformatics analysis, and molecular docking.MethodsCCK test, Clone formation assay and wound-healing assays were used to determine the effect of LWDHD on prostate cancer growth and metastasis. The active ingredients and targets of LWDHD were obtained from the TCMSP database, and the relevant targets were selected by GeneCards, OMIM and DisGeNET databases for PCa. The cross-targets of drugs and disease were imported into the STRING database to construct protein interactions. The network was also visualized using Cytoscape software and core targets are screened using the Network Analyzer plug-in. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were analyzed using R software. TCGA database was used to analyze the correlation of bioinformatics genes. AutoDock vina was used to predict the molecular docking and binding ability of active ingredients to key targets. Through WB and q-PCR experiments, the above gene targets were detected to verify the effect of LWDHD on PCa.ResultsCCK and scratch tests confirmed that LWDHD could inhibit the proliferation, invasion and migration of prostate cancer cells. Clone formation experiments showed that LWDHD inhibited the long-term proliferative capacity of PC3 cells. LWDHD and PCa had a total of 99 common targets, establishing a “drug-ingredient-common target” network. Through GO and KEGG enrichment analysis, PI3K/AKT, MAPK, TP53 pathway, MYC, TNF pathway and other signaling pathways were found. Bioinformatics analysis showed that MYC gene was highly expressed and CCND1 and MAPK1 were low expressed in prostate cancer tissues. In addition, TP53, AKT1, MYC, TNF and CCND1 were positively correlated with MAPK1, among which AKT1 and CCND1 were most closely correlated with MAPK1. Molecular docking results showed that quercetin, kaempferol, β-sitosterol and other main active ingredients of LWDHD treatment for PCa were combined with core proteins MAPK1 and AKT1 well. WB and q-PCR results showed that LWDHD inhibited the expression of PI3K and AKT in PC3 cells.ConclusionThe mechanism of LWDHD therapy for PCa is a multi-target and multi-pathway complex process, which may be related to the biological processes mediated by MAPK1 and AKT1 pathways, such as cell proliferation and inhibition of metastasis, and the regulation of signaling pathways. The PI3K/AKT signaling pathway may be a central pathway of LWDHD to inhibit prostate cancer proliferation.

Dihydroisotanshinone I regulates ferroptosis via PI3K/AKT pathway to enhance cisplatin sensitivity in lung adenocarcinoma.

Dihydroisotanshinone I (DT) is a kind of diterpenoid compound extracted from the dried roots of Salvia miltiorrhiza Bunge, and exhibits multiple biological activities including anti-tumor activity. Cisplatin is one of the first-line drugs for the treatment of lung adenocarcinoma (LAUD), but the drug resistance and toxicity limit its efficacy. DT is known to induce apoptosis and ferroptosis, but it is unclear whether DT can inhibit the cisplatin-resistant LAUD cells and reverse the drug resistance in LAUD. Therefore, our study intends to establish the cisplatin-resistant human LAUD cells (A549/DDP), and figure out the influence and related mechanisms of DT reversing cisplatin resistance in A549/DDP cells, so as to provide a theoretical basis for the DT as a new natural candidate for the treatment of LAUD. The establishment of A549/DDP was the continuous stimulation by exposing A549 to gradient concentrations of Cisplatin. The cell viability of A549 and A549/DDP was detected by CCK-8 kit, and the IC50 value was calculated. The morphological changes of A549 and A549/DDP cells were observed by an inverted microscope. The contents of malondialdehyde (MDA) and glutathione (GSH) in A549/DDP cells after drug treatment were detected by related kits. The levels of Fe2+, cytosolic reactive oxygen species (ROS), and lipid reactive oxygen species (lipid ROS) were detected by a fluorescence microplate reader or fluorescence cell imager according to the related fluorescent probe kit instructions. Western blot was used to detect the expressions of PI3K, phospho-PI3K, AKT, phospho-AKT, MDM2, p53, GPX4, and SLC7A11 in A549/DDP after different drug treatments. Our study demonstrated that the inhibitory effect of DT on A549 and A549/DDP cells was time-dependent and concentration-dependent, and DT and DDP had a synergistic effect on inhibiting the proliferation of A549/DDP cells. Furthermore, DT mainly induced ferroptosis in A549/DDP cells and synergized with cisplatin to promote ferroptosis in A549/DDP cells. The result of KEGG pathway analysis, molecular docking and western blot showed that DT could enhance the cisplatin sensitivity of A549/DDP by inhibiting PI3K/MDM2/P53 signaling pathway. Consequently, we concluded that DT promotes ferroptosis in cisplatin-resistant LAUD A549/DDP cells. Additionally, DT reverses cisplatin resistance by promoting ferroptosis via PI3K/MDM2/P53 pathway in A549/DDP cells.

Involvement of &lt;i&gt;Bdnf&lt;/i&gt;, &lt;i&gt;Ntrk2&lt;/i&gt; and &lt;i&gt;Pi3k&lt;/i&gt; in the mechanism of binge eating after psychogenic stressors in ontogenesis

BACKGROUND: The study of the neurochemical mechanisms of food addiction provides experimental modeling of some of its clinical manifestations. AIM: This study aimed to examine the effect of binge eating after maternal deprivation or after rearing in social isolation on the expression of Bdnf, Ntrk2, and Pi3k in the hypothalamus of rats. MATERIALS AND METHODS: Animals aged 2–12 days were weaned from their mother for 10 days at 180 min, and males aged 90–100 days were used in the experiments. Another group of animals was reared in individual cages from day 21 after birth, and males aged 90–100 days were used in the experiments. To induce binge eating, the animals received a high-carbohydrate feed (chocolate spread) for 1 h every day or every third day within 30 days. Fifteen minutes before feeding, the paste was placed 5 cm within visual contact. RESULTS: In groups with intermittent exposure to high-calorie food (the animals received pasta every third day), polymerase chain reaction analysis revealed the expression of the Bdnf, Ntrk2, and Pi3k in the hypothalamus. The expression level of Bdnf was higher in the maternal deprivation group than in the control group. The expression levels of Ntrk2 and Pi3k in rats taking a high-carbohydrate feed were higher in animals reared in isolation than in those reared in the community. CONCLUSIONS: The results present new pathways for the synthesis of peptide drugs associated with the PI3K/AKT/mTOR signaling pathway for the correction of food addiction caused by psychogenic stress in ontogenesis.

Compound Yuye Decoction protects diabetic rats against cardiomyopathy by inhibiting myocardial apoptosis and inflammation via regulating the PI3K/Akt signaling pathway

To explore the therapeutic mechanism of compound Yuye Decoction against diabetic cardiomyopathy (DCM). Drugbank, Gene Cards, OMIM and PharmGKb databases were used to obtain DCM-related targets, and the core targets were identified and functionally annotated by protein-protein interaction network analysis followed by GO and KEGG enrichment analysis. The "Traditional Chinese Medicine-Key Component-Key Target-Key Pathway" network was constructed using Cytoscape 3.9.1, and molecular docking was carried out for the key components and the core targets. In the animal experiment, Wistar rat models of DCM were treated with normal saline or Yuye Decoction by gavage at low (0.29 g/kg) and high (1.15 g/kg) doses for 8 weeks, and the changes in cardiac electrophysiology and histopathology were evaluated. The changes in serum levels of LDH, CK, and CK-MB were examined, and myocardial expressions of PI3K, P-PI3K, Akt, P-AKT, BAX, IL-6, and TNF-α were detected using Western blotting. We identified 61 active compounds in Yuye Decoction with 1057 targets, 3682 DCM-related disease targets, and 551 common targets between them. Enrichment of the core targets suggested that apoptosis, inflammation and the PI3K/Akt pathways were the key signaling pathways for DCM treatment. Molecular docking studies showed that the active components in Yuye Decoction including gold amidohydroxyethyl ester and kaempferol had strong binding activities with AKT1 and PIK3R1. In DCM rat models, treatment with Yuye Decoction significantly alleviated myocardial pathologies, reduced serum levels of LDH, CK and CK-MB, lowered myocardial expressions of BAX, IL-6 and TNF-α, and increased the expressions of P-PI3K and P-AKT. The therapeutic effect of compound Yuye Decoction against DCM is mediated by its multiple active components that act on multiple targets and pathways to inhibit cardiomyocyte apoptosis and inflammatory response by regulating the PI3K/Akt signaling pathway.

Cardioprotective Effects of 'Vasant Kusumakar Rasa,' a Herbo-metallic Formulation, in Type 2 Diabetic Cardiomyopathy in Rats.

Diabetic cardiomyopathy (DCM) is one of the serious complications of type 2 diabetes mellitus. Vasant Kusumakar Rasa (VKR) is a Herbo-metallic formulation reported in Ayurveda, an Indian system of medicine. The present work was designed to study the effect of VKR in cardiomyopathy in type 2 diabetic rats. Diabetes was induced by feeding a high-fat diet (HFD) for 2weeks followed by streptozotocin (STZ) administration (35mg/kg i.p.). VKR was administered orally at dose of28 and 56mg/kg once a day for 16weeks. The results of the study indicated that VKR treatment significantly improved the glycemic and lipid profile, serum insulin, CK-MB, LDH, and cardiac troponin-I when compared to diabetic control animals. VKR treatment in rats significantly improvedthe hemodynamic parameters and cardiac tissue levels of TNF-α, IL-1β, and IL- 6 were also reduced. Antioxidant enzymes such as GSH, SOD, and catalase were improved in all treatment groups. Heart sections stained with H & E and Masson's trichome showed decreased damage to histoarchitecture of the myocardium. Expression of PI3K, Akt, and GLUT4 in the myocardium was upregulated after 16weeks of VKR treatment. The study data suggested the cardioprotective capability of VKR in the management of diabetic cardiomyopathy in rats.