Akt mRNA Research Articles

Sulindac selectively induces autophagic apoptosis of GABAergic neurons and alters motor behaviour in zebrafish

Nonsteroidal anti-inflammatory drugs compose one of the most widely used classes of medications, but the risks for early development remain controversial, especially in the nervous system. Here, we utilized zebrafish larvae to assess the potentially toxic effects of nonsteroidal anti-inflammatory drugs and found that sulindac can selectively induce apoptosis of GABAergic neurons in the brains of zebrafish larvae brains. Zebrafish larvae exhibit hyperactive behaviour after sulindac exposure. We also found that akt1 is selectively expressed in GABAergic neurons and that SC97 (an Akt1 activator) and exogenous akt1 mRNA can reverse the apoptosis caused by sulindac. Further studies showed that sulindac binds to retinoid X receptor alpha (RXRα) and induces autophagy in GABAergic neurons, leading to activation of the mitochondrial apoptotic pathway. Finally, we verified that sulindac can lead to hyperactivity and selectively induce GABAergic neuron apoptosis in mice. These findings suggest that excessive use of sulindac may lead to early neurodevelopmental toxicity and increase the risk of hyperactivity, which could be associated with damage to GABAergic neurons.

The Effect of KATP Openers and Blockers on AKT and mTOR mRNA Levels in the Hippocampus and Cortex of Rats with Penicillin-Induced Epilepsy

The Effect of KATP Openers and Blockers on AKT and mTOR mRNA Levels in the Hippocampus and Cortex of Rats with Penicillin-Induced Epilepsy

Sodium Butyrate Induces Mitophagy and Apoptosis of Bovine Skeletal Muscle Satellite Cells through the Mammalian Target of Rapamycin Signaling Pathway.

Sodium butyrate (NaB) is one of the short-chain fatty acids and is notably produced in large amounts from dietary fiber in the gut. Recent evidence suggests that NaB induces cell proliferation and apoptosis. Skeletal muscle is rich in plenty of mitochondrial. However, it is unclear how NaB acts on host muscle cells and whether it is involved in mitochondria-related functions in myocytes. The present study aimed to investigate the role of NaB treatment on the proliferation, apoptosis, and mitophagy of bovine skeletal muscle satellite cells (BSCs). The results showed that NaB inhibited proliferation, promoted apoptosis of BSCs, and promoted mitophagy in a time- and dose-dependent manner in BSCs. In addition, 1 mM NaB increased the mitochondrial ROS level, decreased the mitochondrial membrane potential (MMP), increased the number of autophagic vesicles in mitochondria, and increased the mitochondrial DNA (mtDNA) and ATP level. The effects of the mTOR pathway on BSCs were investigated. The results showed that 1 mM NaB inhibited the mRNA and protein expression of mTOR and genes AKT1, FOXO1, and EIF4EBP1 in the mTOR signaling pathway. In contrast, the addition of PP242, an inhibitor of the mTOR signaling pathway also inhibited mRNA and protein expression levels of mTOR, AKT1, FOXO1, and EIF4EBP1 and promoted mitophagy and apoptosis, which were consistent with the effect of NaB treatment. NaB might promote mitophagy and apoptosis in BSCs by inhibiting the mTOR signaling pathway. Our results would expand the knowledge of sodium butyrate on bovine skeletal muscle cell state and mitochondrial function.

Duhuo Jisheng decoction alleviates neuroinflammation and neuropathic pain by suppressing microglial M1 polarization: a network pharmacology research

BackgroundNeuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear.MethodsUsing network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia.ResultsOur screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial M1 polarization to improve neuroinflammation, PPARG may have been involved in the process.ConclusionThe multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP.

Mollugin activates GLP-1R to improve cognitive dysfunction in type 2 diabetic mice

AimsThe incidence of diabetic cognitive dysfunction is increasing year by year, and it has gradually become a research hot spot. Studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists can improve cognitive dysfunction in diabetic patients. This study focuses on whether small molecule GLP-1R agonists from traditional Chinese medicine (TCM) can improve the diabetic cognitive dysfunction. Materials and methodsThe small molecules from TCM were screened by cell membrane chromatography (CMC) with GLP-1R-HEK293 cell membrane column. MTT assay, flow cytometry, immunofluorescence cytochemistry and other methods were used to determine the effects of mollugin on the apoptosis rate and reactive oxygen species (ROS) level of high glucose (HG)/hydrogen peroxide (H2O2) induced PC12 cells. Real-Time PCR was used to detect mRNA expression in mouse cerebral cortex. Water maze test was further used to confirm the effect of mollugin on cognitive dysfunction in T2DM mice. Key findingsMollugin bound to GLP-1R, promoted Ca2+ influx, increased insulin secretion and cAMP content in β-TC-6 cells. Mollugin enhanced the cell viability, ameliorated apoptosis, reduced intracellular ROS levels in HG/H2O2-injured PC12 cells. Mollugin reduced the T2DM mice's escape latency, improved neuronal cell damage, decreased the expression of Pik3ca, Akt1 and Mapk1 mRNA in the cerebral cortex tissue. SignificanceThe results suggest that mollugin could improve cognitive dysfunction in T2DM mice through activating GLP-1R/cAMP/PKA signal pathway.

The Effect of MMP-9 Inhibitors on the Biological Behavior of Human Oral Squamous Cell Carcinoma SCC15 Cell Line Through PI3K/Akt Signaling Pathway

Objective: To investigate the effect of MMP-9 inhibitor (Mki67) on the biology of human oral squamous cell carcinoma SCC15 cell line and to explore its mechanism of action through PI3K/Akt signaling pathway. Methods: SCC15 cells were extracted, and the supernatant was discarded. The cells were then rinsed twice with PBS, and 0, 2.5, 5, and 10 μL of Mki67 (50 mg/mL) were added to the culture respectively. The inhibition rate of cell proliferation was detected by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) method, and the cell migration was measured by Transwell chamber test. The cell apoptosis rate was detected by cytometry, and the p-Akt protein content in the cells of each group was determined by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit. Results: The cell proliferation rates of the 2.5 μL, 5 μL, and 10 μL dose groups were all lower than the 0 μL group (P < 0.05) before treatment, and the cell proliferation rates in the 2.5 μL, 5μL, and 10μL dose groups decreased overtime (P < 0.05). After 24 h, with the increase of Mki67 concentration, the number of migration and invasion gradually decreased (P < 0.05), and the number of apoptosis gradually increased (P < 0.05); besides, the relative expression of MMP-9, PI3K, and Akt mRNA decreased gradually (P < 0.05), and the expression level of Akt mRNA was not statistically significant (P > 0.05). Conclusion: MMP-9 inhibitor (Mki67) can inhibit the proliferation and migration of SCC15 cell line and induce apoptosis, and its mechanism of action may be related to the inhibition of PI3K/Akt signaling pathway.

Pharmacokinetic investigation on the mechanism of interaction of anti-breast cancer calycosin with albumin: In vitro

It has been documented that calycosin induces anticancer effects against a wide range of cancer cells, in vitro. However, pharmacokinetic characteristics of calycosin based on its interaction with albumin as a main carrier protein as well as corresponding anticancer mechanisms remain largely unknown. Herein, we inquired into the interaction of calycosin with human serum albumin (HSA) by a wide range collection of spectroscopic and theoretical data. Also, anticancer effects of calycosin on breast cancer cells, MDA-MB 231, were explored by cell viability, lactate dehydrogenase (LDH), caspase-3 activity, and real-time PCR assays. The findings demonstrated that the quenching process resulting from the interaction of calycosin and HSA was largely static in nature and that the calycosin-HSA system spontaneously forms. Cellular and molecular studies revealed that calycosin did not induce a significant cytotoxic effect on the viability of normal human breast epithelial cells, (MCF-10A), even at 200 μM concentration at which it was able to inhibit the proliferation of breast cancer MDA-MB 231 cells up to 50%. Then, it was disclosed that calycosin can disrupt the membrane integrity and induces apoptosis in MDA-MB 231 cells through overexpression of caspase-3 mRNA, down regulation of Bcl-2, and elevation of caspase-3 mRNA and activity mediated by downregulation of mTOR and Akt mRNA. Therefore, it was realized that in breast cancer MDA-MB 231 cells, calycosin is able to suppress cellular proliferation via apoptosis, which is controlled by the Akt/mTOR signaling cascade.

Ameliorative Potential of (-) Pseudosemiglabrin in Mice with Pilocarpine-Induced Epilepsy: Antioxidant, Anti-Inflammatory, Anti-Apoptotic, and Neurotransmission Modulation

One prevalent neurological disorder is epilepsy. Modulating GABAergic/glutamatergic neurotransmission, Nrf2/HO-1, PI3K/Akt, and TLR-4/NF-B pathways might be a therapeutic strategy for epilepsy. Eight-week-old BALB/c mice were administered 12.5, 25, or 50 mg/kg (-) pseudosemiglabrin orally one hour before inducing epilepsy with an i.p. injection of 360 mg/kg pilocarpine. (-) Pseudosemiglabrin dose-dependently alleviated pilocarpine-induced epilepsy, as revealed by the complete repression of pilocarpine-induced convulsions and 100% survival rate in mice. Furthermore, (-) pseudosemiglabrin significantly enhanced mice’s locomotor activities, brain GABA, SLC1A2, GABARα1 levels, glutamate decarboxylase activity, and SLC1A2 and GABARα1mRNA expression while decreasing brain glutamate, SLC6A1, GRIN1 levels, GABA transaminase activity, and SLC6A1 and GRIN1 mRNA expression. These potentials can be due to the suppression of the TLR-4/NF-κB and the enhancement of the Nrf2/HO-1 and PI3K/Akt pathways, as demonstrated by the reduction in TLR-4, NF-κB, IL-1β, TNF-α mRNA expression, MDA, NO, caspase-3, Bax levels, and Bax/Bcl-2 ratio, and the enhancement of Nrf2, HO-1, PI3K, Akt mRNA expression, GSH, Bcl-2 levels, and SOD activity. Additionally, (-) pseudosemiglabrin abrogated the pilocarpine-induced histopathological changes. Interestingly, the (-) pseudosemiglabrin intervention showed a comparable effect to the standard medication, diazepam. Therefore, (-) pseudosemiglabrin can be a promising medication for the management of epilepsy.

Association of physical activity with increased PI3K and Akt mRNA levels in adipose tissues of obese and non-obese adults

Phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway regulates glucose and lipid metabolism. We examined the association of PI3K and Akt expression in visceral (VAT) and subcutaneous adipose tissue (SAT) with daily physical activity (PA) in non-diabetic obese and non-obese adults. In this cross-sectional study, we included 105 obese (BMI ≥ 30 kg/m2) and 71 non-obese (BMI < 30 kg/m2) subjects (aged/ ≥ 18 years). PA was measured using a valid and reliable International Physical Activity Questionnaire(IPAQ)-long-form, and the metabolic equivalent of task(MET) was calculated. Real-time PCR was performed to analyze the mRNA relative expression. VAT PI3K expression had a lower level in obese compared to non-obese (P = 0.015), while its expression was higher in active individuals than inactive ones (P = 0.029). SAT PI3K expression was increased in active individuals compared to inactive ones (P = 0.031). There was a rise in VAT Akt expression in the actives compared to the inactive participants (P = 0.037) and in non-obese/active compared to non-obese/inactive individuals (P = 0.026). Obese individuals had a decreased expression level of SAT Akt compared to non-obsesses (P = 0.005). VAT PI3K was directly and significantly associated with PA in obsesses (β = 1.457, P = 0.015). Positive association between PI3K and PA suggests beneficial effects of PA for obese individuals that can be partly described by PI3K/Akt pathway acceleration in adipose tissue.

Glyphosate potentiates insulin resistance in skeletal muscle through the modulation of IRS-1/PI3K/Akt mediated mechanisms: An in vivo and in silico analysis

Herbicides have been linked to a higher risk of developing diabetes. Certain herbicides also operate as environmental toxins. Glyphosate is a popular and extremely effective herbicide for weed control in grain crops that inhibits the shikimate pathway. It has been shown to negatively influence endocrine function. Few studies have demonstrated that glyphosate exposure results in hyperglycemic and insulin resistance; but the molecular mechanism underlying the diabetogenic potential of glyphosate on skeletal muscle, a primary organ that includes insulin-mediated glucose disposal, is unknown. In this study, we aimed to evaluate the impact of glyphosate on the detrimental changes in the insulin metabolic signaling in the gastrocnemius muscle. In vivo results showed that glyphosate exposure caused hyperglycemia, dyslipidemia, increased glycosylated hemoglobin (HbA1c), liver function, kidney function profile, and oxidative stress markers in a dose-dependent fashion. Conversely, hemoglobin and antioxidant enzymes were significantly reduced in glyphosate-induced animals indicating its toxicity is linked to induce insulin resistance. The histopathology of the gastrocnemius muscle and RT-PCR analysis of insulin signaling molecules revealed glyphosate-induced alteration in the expression of IR, IRS-1, PI3K, Akt, β-arrestin-2, and GLUT4 mRNA. Lastly, molecular docking and dynamics simulations confirmed that glyphosate showed a high binding affinity with target molecules such as Akt, IRS-1, c-Src, β-arrestin-2, PI3K, and GLUT4. The current work provides experimental proof that glyphosate exposure has a deleterious effect on the IRS-1/PI3K/Akt signaling pathways, which in turn causes the skeletal muscle to become insulin resistant and eventually develop type 2 diabetes mellitus.

Paliurus spina-christi Mill fruit extracts improve glucose uptake and activate the insulin signaling pathways in HepG2 insulin-resistant cells

BackgroundPaliurus spina-christi Mill. (PSC) fruit is frequently used in the treatment of diabetes mellitus in Mediterranean regions. Here, we investigated the effects of various PSC fruit extracts (PSC-FEs) on glucose consumption and some key mediators of insulin signaling pathways in high glucose and high insulin-induced insulin-resistant HepG2 cells.MethodsThe effects of methanolic, chloroform and total extracts on cell proliferation were assessed by the MTT assay. The potential of non-toxic extracts on glucose utilization in insulin-resistant HepG2 cells was checked using a glucose oxidase assay. AKT and AMP-activated protein kinase (AMPK) pathway activation and mRNA expression levels of insulin receptor (INSR), glucose transporter 1 (GLUT1), and glucose transporters 4 (GLUT4) were determined by western blotting and real-time PCR, respectively.ResultsWe found that high concentrations of methanolic and both low and high concentrations of total extracts were able to enhance glucose uptake in an insulin-resistant cell line model. Moreover, AKT and AMPK phosphorylation were significantly increased by the high strength of methanolic extract, while total extract raised AMPK activation at low and high concentrations. Also, GLUT 1, GLUT 4, and INSR were elevated by both methanolic and total extracts.ConclusionsUltimately, our results shed new light on methanolic and total PSC-FEs as sources of potential anti-diabetic medications, restoring glucose consumption and uptake in insulin-resistant HepG2 cells. These could be at least in part due to re-activating AKT and AMPK signaling pathways and also increased expression of INSR, GLUT1, and GLUT4. Overall, active constituents present in methanolic and total extracts of PCS are appropriate anti-diabetic agents and explain the use of these PSC fruits in traditional medicine for the treatment of diabetes.

Network pharmacology reveals that Berberine may function against Alzheimer's disease via the AKT signaling pathway.

To investigate the mechanism underlying the effects of berberine (BBR) in the treatment of Alzheimer's disease (AD). 3 × Tg AD mice were treated with BBR for 3 months, then the open field test (OFT), the novel object recognition test (NOR) and the Morris water maze (MWM) test were performed to assess behavioral performance. Hematoxylin-eosin (HE) staining, Nissl staining were used to examine histopathological changes. The pharmacological and molecular properties of BBR were obtained from the TCMSP database. BBR-associated AD targets were identified using the PharmMapper (PM), the comparative toxicogenomics database (CTD), DisGeNet and the human gene database (GeneCards). Core networks and BBR targets for the treatment of AD were identified using PPI network and functional enrichment analyses. AutoDock software was used to model the interaction between BBR and potential targets. Finally, RT-qPCR, western blotting were used to validate the expression of core targets. Behavioral experiments, HE staining and Nissl staining have shown that BBR can improve memory task performance and neuronal damage in the hippocampus of AD mice. 117 BBR-associated targets for the treatment of AD were identified, and 43 genes were used for downstream functional enrichment analysis in combination with the results of protein-protein interaction (PPI) network analysis. 2,230 biological processes (BP) terms, 67 cell components (CC) terms, 243 molecular function (MF) terms and 118 KEGG terms were identified. ALB, EGFR, CASP3 and five targets in the PI3K-AKT signaling pathway including AKT1, HSP90AA1, SRC, HRAS, IGF1 were selected by PPI network analysis, validated by molecular docking analysis and RT-q PCR as core targets for further analysis. Akt1 mRNA expression levels were significantly decreased in AD mice and significantly increased after BBR treatment (p < 0.05). Besides, AKT and ERK phosphorylation decreased in the model group, and BBR significantly increased their phosphorylation levels. AKT1, HSP90AA1, SRC, HRAS, IGF1 and ALB, EGFR, CASP3 were core targets of BBR in the treatment of AD. BBR may exert a neuroprotective effect by modulating the ERK and AKT signaling pathways.

Hypericin Ameliorates Depression-like Behaviors via Neurotrophin Signaling Pathway Mediating m6A Epitranscriptome Modification.

Hypericin, one of the major antidepressant constituents of St. John's wort, was shown to exert antidepressant effects by affecting cerebral CYP enzymes, serotonin homeostasis, and neuroinflammatory signaling pathways. However, its exact mechanisms are unknown. Previous clinical studies reported that the mRNA modification N6-methyladenosine (m6A) interferes with the neurobiological mechanism in depressed patients, and it was also found that the antidepressant efficacy of tricyclic antidepressants (TCAs) is related to m6A modifications. Therefore, we hypothesize that the antidepressant effect of hypericin may relate to the m6A modification of epitranscriptomic regulation. We constructed a UCMS mouse depression model and found that hypericin ameliorated depressive-like behavior in UCMS mice. Molecular pharmacology experiments showed that hypericin treatment upregulated the expression of m6A-modifying enzymes METTL3 and WTAP in the hippocampi of UCMS mice. Next, we performed MeRIP-seq and RNA-seq to study m6A modifications and changes in mRNA expression on a genome-wide scale. The genome-wide m6A assay and MeRIP-qPCR results revealed that the m6A modifications of Akt3, Ntrk2, Braf, and Kidins220 mRNA were significantly altered in the hippocampi of UCMS mice after stress stimulation and were reversed by hypericin treatment. Transcriptome assays and qPCR results showed that the Camk4 and Arhgdig genes might be related to the antidepressant efficacy of hypericin. Further gene enrichment results showed that the differential genes were mainly involved in neurotrophic factor signaling pathways. In conclusion, our results show that hypericin upregulates m6A methyltransferase METTL3 and WTAP in the hippocampi of UCMS mice and stabilizes m6A modifications to exert antidepressant effects via the neurotrophin signaling pathway. This suggests that METTL3 and WTAP-mediated changes in m6A modifications may be a potential mechanism for the pathogenesis of depression and the efficacy of antidepressants, and that the neurotrophin signaling pathway plays a key role in this process.

Effect and Mechanism of Schizandrin A in the Treatment of Liver Cancer Using Network Pharmacology, Molecular Docking, and Target Validation

Background Liver cancer is a common threat to human health. Schizandrin A (SA) has certain therapeutic effect on liver cancer, but its related mechanism of action is not clear. Elucidating this mechanism is of great significance for the development of drugs to target liver cancer. Objective The objective is to study the effect and mechanism of SA on liver cancer. Methods The potential targets of SA in the treatment of liver cancer were screened based on network pharmacology, and the core targets were further screened by protein network interaction. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed based on the core targets to find the key pathways of action. The results obtained for key targets and pathways were verified in vitro by pharmacological experiments with HepG2 cells. Specifically, an Enzyme Linked Immunosorbent assay (ELISA) kit was used to detect the content of key indicators (interleukin-1 (IL-1), Tumor Necrosis Factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), superoxide dismutase [SOD], malondialdehyde [MDA], interferon (IFN), and Glutathione peroxidase (GSH-px)), Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to detect the key messenger Ribonucleic Acid (mRNA) (phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt), vascular endothelial growth factor A (VEGFA), and Transforming Growth Factor Beta-1 (TGF-β1)) after drug intervention, and Western blotting (WB) detected the expression of key target proteins (p-PI3K, PI3K, p-Akt, and Akt). Results The results showed that SA mainly acts on IL-1β, TNF-α, IL-6, IL-10, SOD, MDA, TGF-β1, IFN, and VEGFA, affecting the PI3K-Akt signaling pathway and playing an anti-inflammatory role in the treatment of liver cancer. The experimental results showed that SA significantly reduced the inflammation level of HepG2 cells; improved the oxidative stress state (decreasing SOD level and increasing MDA level of cells); downregulated TGF-β1, VEGFA, PI3K, and Akt mRNA levels; and inhibited the expression of the PI3K-Akt signaling pathway. Conclusions SA has a significant anti-HepG2 effect, which may be achieved by reducing inflammation levels, improving oxidative stress state, and inhibiting the PI3K-Akt pathway.

Shikonin Alleviates Gentamicin-Induced Renal Injury in Rats by Targeting Renal Endocytosis, SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt Cascades.

Gentamicin causes kidney injury due to its accumulation in proximal tubule epithelial cells via the megalin/cubilin/CLC-5 complex. Recently, shikonin has been shown to have potential anti-inflammatory, antioxidant, antimicrobial, and chloride channel-inhibiting effects. The current study investigated the alleviation of gentamicin-induced renal injury by shikonin while preserving its bactericidal effect. Nine-week-old Wistar rats were administered 6.25, 12.5, and 25 mg/kg/day shikonin orally, one hour after the i.p. injection of 100 mg/kg/day gentamicin for seven days. Shikonin significantly and dose-dependently alleviated gentamicin-induced renal injury, as revealed by restoring normal kidney function and histological architecture. Furthermore, shikonin restored renal endocytic function, as indicated by suppressing the elevated renal megalin, cubilin, and CLC-5 and enhancing the reduced NHE3 levels and mRNA expressions induced by gentamicin. These potentials could be attributed to the modulation of the renal SIRT1/Nrf2/HO-1, TLR-4/NF-κB/MAPK, and PI3K/Akt cascades, which enhanced the renal antioxidant system and suppressed renal inflammation and apoptosis, as indicated by enhancements of SIRT1, Nrf2, HO-1, GSH, SOD, TAC, Iκb-α, Bcl-2, PI3K, and Akt levels and mRNA expressions, with reduction of TLR-4, NF-κB, MAPK, IL-1β, TNF-α, MDA, iNOS, NO, cytochrome c, caspase-3, Bax levels, and Bax/Bcl-2 ratio. Therefore, shikonin is a promising therapeutic agent for alleviating gentamicin-induced renal injury.

Study on the action mechanism of Buyang Huanwu Decoction against ischemic stroke based on S1P/S1PR1/PI3K/Akt signaling pathway

Ethnopharmacological relevanceIschemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway. Aim of the studyThe present study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives. Materials and methodsThe BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue. ResultsBHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group.BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously.Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146. ConclusionBHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.

Abstract 701: Evaluation of an AKT-1 antisense oligonucleotide in combination with Lenvatinib and Everolimus in Renca-luciferase syngeneic orthotopic murine tumor model

Abstract Background: The combination of 18mg Lenvatinib and 5mg Everolimus per day is used in the clinic for advanced renal cancer patients. However, due to the severe side effects of Lenvatinib, the dose is often reduced to 12mg. Archexin, a fully phosphorothioated 20-mer antisense oligonucleotide, inhibits AKT-1 mRNA translation and inhibits tumor growth. Combination of Archexin with reduced-dosed Lenvatinib and Everolimus may reduce adverse reactions while enhancing therapeutic efficacy. Methods: First, Lenvatinib and Everolimus monotherapies were evaluated for antitumor efficacy in an orthotopic Renca-luciferase syngeneic model. 45 mice were randomly divided into 9 groups (5 in each group), vehicle control, Lenvatinib (5, 10, 20, and 30 mg/kg) and Everolimus (0.2, 0.5, 1, 2, and 4 mg/kg) via daily oral administration for 20 days. Next, we investigated the anti-tumor effect of the two- and three-drug combination. 35 mice were randomly divided into 7 groups (5 in each group), including vehicle control, Lenvatinib/Everolimus combinations (5+0.2, 10+0.2 and 10+0.5 mg/kg) and Archexin/Lenvatinib/Everolimus combinations (60+5+0.2, 60+10+0.2 and 60+10+0.5 mg/kg) via tail vein injection (vehicle/Archexin, Q3D*6 times) or oral administration (Lenvatinib and Everolimus, QD*18 days). Body weight was monitored daily. Tumor burden was measured by bioluminescence through whole-body fluorescence images, performed twice a week. Results: The results showed that Everolimus inhibited tumor growth in a dose-dependent manner. Partial response was observed in mice treated with varying doses of Lenvatinib(TGIs: 57.94%,76.65% and 60.22%), and no significant dose-effect correlation was found. The dose of Lenvatinib and Everolimus that partially inhibited the orthotopic tumor growth had also been identified and selected for subsequent study investigating drug combinations. The drugs combination study showed that three drug combinations (Archexin/Lenvatinib/Everolimus) had superior efficacy compared to the Lenvatinib and Everolimus combination. The fluorescence signal intensities (Lg10) of the three-drug combination were significantly lower than that of the two-drug combination group with P value &amp;lt;0.05. No severe adverse effects were observed in the three drug combinations group. Conclusion: Three drugs combination of Archexin, Lenvatinib and Everolimus, demonstrated superior anti-tumor efficacy compared to the two-drug combination currently used in the clinic. This finding suggests the three-drug combination warrant investigation in the clinical trial in the patients with advanced renal cancer. Citation Format: Fengyang Xie, Jia Wei, Lan Deng, Chao Wang, Ben Zhao, Robert J. Lee, Yuxin Angela Men. Evaluation of an AKT-1 antisense oligonucleotide in combination with Lenvatinib and Everolimus in Renca-luciferase syngeneic orthotopic murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 701.

Hesperidin has a protective effect on paclitaxel-induced testicular toxicity through regulating oxidative stress, apoptosis, inflammation and endoplasmic reticulum stress

Paclitaxel (PTX) is widely used to treat a number of malignancies, although it has toxic side effects. Hesperidin (HES) has a wide range of biological and pharmacological properties, including anti-inflammatory and antioxidant abilities. This research aims to investigate the role of HES in PTX-induced testicular toxicity. For 5 days, 2 mg/kg/bw i.p. of PTX was administered to induce testicular toxicity. Rats were administered oral dosages of 100 and 200 mg/kg/bw HES for 10 days after PTX injection. The mechanisms of inflammation, apoptosis, endoplasmic reticulum (ER) stress, and oxidants were investigated using biochemical, genetic, and histological techniques. As a result of PTX administration, decreased antioxidant enzyme (superoxide dismutase, catalase, and glutathione peroxidase) activities and increased malondialdehyde level were regulated, and the severity of oxidative stress was reduced. NF-κB, IL-1β and TNF-α levels, which are among the increased inflammation parameters caused by PTX, decreased with HES administration. Although AKT2 gene expression decreased in PTX administered rats, it was determined that HES administration up-regulated AKT2 mRNA expression. Anti-apoptotic Bcl-2 decreased with PTX administration, and apoptotic Bax and Caspase-3 increased while HES administration reverted these effects towards control level. As a result of toxicity, the increase in ATF6, PERK, IRE1α, GRP78 levels caused prolonged ER stress, and this activity was diminished with HES and tended to regress. While all data were evaluated, Paclitaxel caused damage by increasing inflammation, apoptosis, ER stress and oxidant levels in testicular tissue, and Hesperidin showed a protective effect by correcting the deterioration in these levels.

N6-methyladenosine plays a dual role in arsenic carcinogenesis by temporal-specific control of core target AKT1.

High-profile RNA epigenetic modification N6-methyladenosine (m6A), as a double-edged sword for cancer, can either promote or inhibit arsenic-induced skin carcinogenesis. However, the core m6A-target gene determining the duality of m6A and the regulatory mechanism of m6A on the core gene are still poorly understood. Based on m6A microarray detection, integrated multi-omics analysis, and further experiments in vitro and in vivo, we explored the molecular basis for the dual role of m6A in cancer induced by environmental pollutants using models in different stages of arsenic carcinogenesis, including As-treated, As-transformed, and As-tumorigenic cell models. We found that the key proliferative signaling node AKT1 is in the center of the m6A-regulatory network in arsenic carcinogenicity. The m6A level on AKT1 mRNA (3'UTR, CDS, and 5'UTR) dynamically changed in different stages of arsenic carcinogenesis. The m6A writer METTL3-catalyzed upregulation of m6A promotes AKT1 expression by elevating m6A reader YTHDF1-mediated AKT1 mRNA stability in As-treated and As-transformed cells, while the m6A eraser FTO-catalyzed downregulation of m6A promotes AKT1 expression mainly by inhibiting m6A reader YTHDF2-mediated AKT1 mRNA degradation in As-tumorigenic cells. Furthermore, upregulation of m6A inhibits the expression of AKT1 negative regulator PHLPP2 and promotes the expression of AKT1 positive regulator PDK1. These changes in AKT1 regulators result in AKT1 activation by upregulating AKT1 phosphorylation at S473 and T308. Interestingly, the FTO-catalyzed decrease in m6A prevents AKT upregulation in As-treated cells but promotes AKT upregulation in As-tumorigenic cells. Both inhibitors targeting the m6A writer and eraser can inhibit the AKT1-mediated proliferation of As-tumorigenic cells by breaking the balance of m6A regulators. Our results demonstrated that AKT1 is the core hub determining m6A as a double-edged sword. Changed m6A dynamically upregulates the expression and activity of AKT1 in different stages of arsenic carcinogenesis. This study can advance our understanding of the dual role and precise time-specific mechanism of RNA epigenetics involved in the carcinogenesis of hazardous materials.

Semaphorin 4C Plays a Key Role in Colorectal Cancer Cells Development

Objective: Purpose of this work was to discuss effects and mechanisms of Sema 4C in colon cancer development. Results: Sema4C were significantly upregulated in cancer tissues (P &lt;0.001). Following transfection, the expression levels of Sema4C mRNA were significantly downregulated in the si-Sema4C groups compared with those in the si-negative control groups of both HT-29 and SW620 cells (both P &lt;0.001). The apoptotic rate was significantly increased, while the invasive and wound healing rates were significantly suppressed in the si-Sema4C groups of HT-29 and SW620 cells (both P &lt; 0.001). The results of the RT-qPCR and western blotting analyses revealed that PI3K, AKT, MMP-2 and MMP-9 mRNA and protein expression levels, respectively, were significantly downregulated, while caspase-3 mRNA and protein expression levels were significantly upregulated in the si-Sema4C groups of HT-29 and SW620 cells. Conclusion: The findings of the present study demonstrated that the knockdown of Sema4C expression suppressed CRC cell biological activities by regulating the PI3K/AKT signaling pathway. Therefore, Sema4C may act as an oncogene in CRC.