Molecular Investigation and Preliminary Validation of Candidate Genes Associated with Neurological Damage in Heat Stroke.

Heat stroke (HS) is a severe medical condition characterized by a systemic inflammatory response that may precipitate multi-organ dysfunction, with a particular predilection for inducing profound central nervous system impairments. We aim to employ bioinformatics techniques for the retrieval and analysis of genes associated with heat stroke-induced neurological damage. We performed a comprehensive analysis of the GSE64778 dataset from the Sequence Read Archive, resulting in the identification of 1178 significantly differentially expressed genes (DEGs). We retrieved 2914 genes associated with heat stroke from the GeneCards database and 2377 genes associated with heat stroke from the Comparative Toxicogenomics Database (CTD). The intersection of the top 300 DEGs in the GSE64778 dataset intersected with the search results of GeneCards and CTD, yielding 25 final candidates for DEGs associated with heat stroke. Gene Ontology functional annotation results indicated that the target genes were mainly involved in apoptosis, stress response, and negative regulation of cellular processes and function in processes such as protein dimerization and protein binding. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed a predominant enrichment of candidate target genes within the PI3K-AKT signaling pathway. Subsequent protein-protein interaction network analysis highlighted HSP90aa1 as a central gene, indicating its pivotal role by possessing the highest number of edges among the genes enriched in the PI3K-AKT signaling pathway. Quantitative reverse transcription-polymerase chain reaction analysis performed on blood samples from patients validated the expression of Hsp90aa1 in individuals exhibiting early neurological damage in HS, consistent with the findings from the mRNA bioinformatics analysis. Additionally, the bioinformatics analysis of the upstream microRNAs (miRNAs) regulating HSP90aa1 and the target miRNAs associated with candidate long non-coding RNAs (lncRNAs) identified three lncRNAs, eight miRNAs, and one mRNA in the regulatory network. The DIANA Tools database and algorithms were employed for pathway enrichment and correlation analysis, revealing a significant association between LOC102547734 and MIR-206-3p, with the latter being identified as a target binding site Moreover, the analysis unveiled a correlation between MIR-206-3p and HSP90aa1, implicating the latter as a potential target binding site within the regulatory network.

Pharmacodynamic material basis and pharmacological mechanisms of Cortex Mori against diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: The application of Cortex Mori (CM) in the treatment of diabetes mellitus (DM) has been extensively documented in traditional medicine. In recent years, the chemical composition of CM has been gradually unraveled, and its therapeutic mechanism in treating DM, diabetic nephropathy, diabetic cardiomyopathy, and other related conditions has been highlighted in successive reports. However, there is no systematic study on the treatment of DM based on the chemical composition of CM. AIM OF THE STUDY: This study was conducted to systematically explore the hypoglycemic activity mechanism of CM based on its chemical composition. METHODS: The material basis of Cortex Mori extract (CME) was investigated through qualitative analyses based on liquid chromatography-mass spectrometry (LC-MS). The possible acting mechanism was simulated using network pharmacology and validated in streptozotocin (STZ) + high fat diet (HFD)-induced diabetic rats and glucosamine-induced IR-HepG2 model with the assistance of molecular docking techniques. RESULTS: A total of 39 compounds were identified in CME by the LC-MS-based qualitative analysis. In diabetic rats, it was demonstrated that CME significantly ameliorated insulin resistance, blood lipid levels, and liver injury. The network pharmacology analysis predicted five major targets, including AKT1, PI3K, FoxO1, Gsk-3ß, and PPARγ. Additionally, three key compounds (resveratrol, protocatechuic acid, and kaempferol) were selected based on their predicted contributions. The experimental results revealed that CME, resveratrol, protocatechuic acid, and kaempferol could promote the expression of AKT1, PI3K, and PPARγ, while inhibiting the expression of FoxO1 and Gsk-3ß. The molecular docking results indicated a strong binding affinity between resveratrol/kaempferol and their respective targets. CONCLUSIONS: CME contains a substantial amount of prenylated flavonoids, which may be the focal point of research on the efficacy of CM in the treatment of DM. Besides, CME is effective in controlling blood glucose and insulin resistance, improving lipid levels, and mitigating liver injury in patients with DM. Relevant mechanisms may be associated with the activation of the PI3K/Akt pathway, the inhibition of the expression of FoxO1 and Gsk-3ß, and the enhancement of PPARγ activity. This study represents the first report on the role of CME in the treatment of DM through regulating PPARγ, FoxO1, and Gsk-3ß.

MiRNA-766-3p inhibits gastric cancer via targeting COL1A1 and regulating PI3K/AKT signaling pathway.

Objective MiRNA-766-3p has been shown to be associated with a variety of cancers. However, few studies have been done in gastric cancer (GC). This study explores the mechanism of miR-766-3p in GC. Methods The potential targets of microRNA (miRNA) were predicted using Tarbase and Targetscan databases. The results are intersected with differential genes (DEGs) (fold change > 1.5, P < 0.05) in gastric cancer to obtain potential core targets. The hub targets screened by constructing PPI networks (degree > 5, expression > 0.5). Validating the differential expression and expression in immunohistochemistry of these targets through the database. And the binding sites between miRNAs and mRNAs were verified using dual-luciferase Assay. Finally, qRT-PCR and Western Blot experiments were conducted to validate the hub targets and signal pathways. Results The potential hub targets from the PPI network were THBS2, COL1A1, FGG, FGB, and PLAU. Combining database, luciferase Assay and experimental validation, miR-766-3p can sponge COL1A1 and it plays the most important role in gastric cancer progression. In GC, COL1A1 was upregulated and the enrichment analysis revealed that COL1A1 regulates PI3K/AKT signal pathway, and AKT is also highly expressed in gastric cancer. Conclusion The miR-766-3p can inhibit the progression of gastric cancer by targeting COL1A1 and regulating the PI3K/AKT signal pathway. It could be a potential therapy option for the GC.

The rapid change of shear rate gradient is beneficial to platelet activation.

Fluid shear plays a key role in hemostasis and thrombosis, and the purpose of this study was to investigate the effect of shear gradient change rate (SGCR) on platelet reactivity and von Willebrand factor (vWF) activity and its mechanism. In this study, we developed a set of microfluidic chips capable of generating different shear gradients and simulated the shear rate distribution in the flow field by COMSOL Multiphysics software. Molecular markers of platelet activation (P-selectin, activated GPIIb/IIIa, phosphatidylserine exposure, and monocyte-platelet aggregate formation) were analyzed by flow cytometry. Platelet aggregation induced by shear gradient was studied by a microfluidic experimental platform, and plasma vWF ristocetin cofactor (vWF: RCO) activity was investigated by flow cytometry. The expression of p-Akt was studied by Western blotting. The results showed that the faster the SGCR, the higher the expression of platelet p-Akt, and the stronger the platelet reactivity and vWF activity. This indicates that fluid shear stress can activate platelets and vWF in a shear gradient-dependent manner through the PI3K/AKT signal pathway, and the faster the SGCR, the more significant the activation effect.

What is the context? Recent studies have shown that fluid shear stress plays a key role in platelet activation and thrombosis. However, its mechanism and effect have not been fully elucidated.The development of microfluidic chip technology enables people to study platelet function in a precisely controlled flow field environment.Previous studies have shown that the PI3K-AKT signal pathway may be a mechanically sensitive signal transduction pathway.What is new?In this study, we designed a microfluidic model with different narrow geometry, and controlled the injection pump to perfuse fluid at the same flow rate, so that the platelets flowing through the model experienced the flow field environment of different shear gradients.We studied the activities of platelets and von Willebrand factor in different flow fields and explored their signal transduction pathways.What is the impact? Our results suggest that vascular stenosis does increase platelet activity and the risk of thrombosis. However, its ability to activate platelets is not only related to the peak shear rate and shear time, but also closely related to the decreasing rate of shear gradient. Even if the peak shear rate at the stenosis is the same, the faster the shear rate decreases, the higher the reactivity of platelets and von Willebrand factor, which may be mediated by the PI3K-AKT signal pathway. This study not only helps clinicians to judge the risk of thrombosis in patients with atherosclerosis or percutaneous coronary intervention, but also helps us to better understand the mechanism of shear-induced platelet activation.

Melatonin Inhibits Testosterone Synthesis in Rooster Leydig Cells by Targeting CXCL14 through miR-7481-3p.

Melatonin has been proved to be involved in testosterone synthesis, but whether melatonin participates in testosterone synthesis by regulating miRNA in Leydig cells is still unclear. The purpose of this study is to clarify the mechanism of melatonin on Leydig cells testosterone synthesis from the perspective of miRNA. Our results showed that melatonin could significantly inhibit testosterone synthesis in rooster Leydig cells. miR-7481-3p and CXCL14 were selected as the target of melatonin based on RNA-seq and miRNA sequencing. The results of dual-luciferase reporter assays showed that miR-7481-3p targeted the 3'-UTR of CXCL14. The overexpression of miR-7481-3p significantly inhibited the expression of CXCL14 and restored the inhibitory role of melatonin testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. Similarly, interference with CXCL14 could reverse the inhibitory effect of melatonin on the level of testosterone synthesis and the expression of StAR, CYP11A1, and 3ß-HSD in rooster Leydig cells. The RNA-seq results showed that melatonin could activate the PI3K/AKT signal pathway. Interference with CXCL14 significantly inhibited the phosphorylation level of PI3K and AKT, and the inhibited PI3K/AKT signal pathway could reverse the inhibitory effect of CXCL14 on testosterone synthesis and the expression of StAR, CYP11A1 and 3ß-HSD in rooster Leydig cells. Our results indicated that melatonin inhibits testosterone synthesis by targeting miR-7481-3p/CXCL14 and inhibiting the PI3K/AKT pathway.

Utilizing network pharmacology and experimental validation to explore the potential molecular mechanisms of raw Pinellia ternate in treating esophageal cancer.

Background: Esophageal cancer (EC) is a highly lethal malignancy with a grim prognosis and high mortality rates, primarily treated through surgery and radiotherapy. Herbal remedies are emerging as complementary approaches in cancer therapy. Here, we explore the potential therapeutic benefits of Chinese medicine raw Pinellia ternata (RP) in EC using web-based pharmacological methods and cellular experiments. Methods: The chemical components of RP were obtained by data mining via searches of the systematic pharmacology database, analysis platform, and literature on traditional Chinese medicine (TCM). The properties of the main components of RP were calculated using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). The potential targets of the components were mined and collected through multiple databases, and the relevant potential targets of efficacy were imported into Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database to obtain protein interactions. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment analysis of the potential targets were performed through Metascape. A target-pathway network was established using Cytoscape, and topological analysis was performed on the network so as to obtain the relevant targets and pathways of RP in the treatment of EC. The inhibitory effect of RP on human EC cells was verified by cell experiments. Results: Thirteen bioactive components of RP were screened, 87 related targets were obtained by construction, and 68 co-targets were obtained after taking intersection with EC related genes. The results of the protein-protein interaction (PPI) network analysis of the targets showed that the pharmacodynamic targets of hemicellulose might be closely related to the signaling pathways such as PI3K-Akt, FOS/JUN, and HIF-1. Meanwhile, GO and KEGG enrichment analysis showed that PI3K-Akt was also significantly enriched. The in vitro cellular experiments further indicated that raw hemicrania could inhibit EC through the PI3K-Akt signaling pathway. Conclusions: The pharmacodynamic mechanism of RP in the treatment of esophageal carcinoma was preliminarily revealed, which provided ideas and the basis for further experimental study of RP in the treatment of esophageal carcinoma.

ABCF1/CXCL12/CXCR4 Enhances Glioblastoma Cell Proliferation, Migration, and Invasion by Activating the PI3K/AKT Signal Pathway.

Glioblastoma (GBM) is the most prevalent and fatal form of brain tumor, which is associated with a poor prognosis. ATP-binding cassette subfamily F member 1 (ABCF1) is an E2 ubiquitin-conjugating enzyme, which is implicated in regulating immune responses and tumorigenesis. Aberrant E3 ubiquitylation has been evidenced in GBM. However, the role of ABCF1 in GBM needs to be further explored. The expression of ABCF1, CXC chemokine ligand 12 (CXCL12), and CXC chemokine receptor 4 (CXCR4) in GBM tissues was examined by the GEPIA tool, real-time PCR and Western blotting. HMC3, U251MG, and LN-229 cells were cultured and transfected with shRNA targeting ABCF1 and ABCF1 plasmids. The proliferative, migrative, and invasive ability of cells was detected. Western blotting was used to detect the levels of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (AKT). We observed that GBM tissues had higher ABCF1, CXCL12, and CXCR4 expression levels. The expression levels of CXCL12 and CXCR4 were enhanced by ABCF1 overexpression, which were significantly reversed by silence of ABCF1 in GBM cells. Silencing ABCF1 or CXCR4 inhibition weakened the capacity of GBM cell growth, migration, and invasion, while ectopic ABCF1 expression or CXCL12 treatment enhanced the cellular function of GBM cells. Furthermore, p-PI3K and p-AKT protein levels were downregulated by ABCF1 knockdown or CXCR4 blockade, which were prompted by ABCF1 overexpression or CXCL12 supplement. The ABCF1-CXCL12-CXCR4 axis was identified as a key player in GBM cell survival and metastasis by activating the PI3K/AKT signaling pathway in GBM cells.

Network pharmacology-based prediction and validation of the active ingredients and potential mechanisms of the Huangxiong formula for treating ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangxiong Formula (HXF) is composed of four herbs: Rheum palmatum L., Ligusticum striatum DC., Curcuma aromatica Salisb., and Acorus gramineus Aiton. HXF is clinically used for the treatment of ischemic stroke (IS). However, its molecular mechanism remains unclear. AIM OF THE STUDY: A network pharmacology-based strategy combined with experimental study in vivo and in vitro to were used to investigate the bioactive components, potential targets, and molecular mechanisms of HXF in the treatment of IS. MATERIALS AND METHODS: The components of HXF were detected by ultra-performance liquid chromatography (UPLC). The potential active ingredients of HXF were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature, and corresponding targets were discerned through the Swiss TargetPrediction database. IS-related targets were obtained from Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and DisGeNET. The intersection of ingredient and disease targets was screened, and a herbal-compound-target network was constructed. A protein-protein interaction (PPI) network was created, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Based on these analyses, we established a compound-target-pathway (C-T-P) network. A cerebral ischemia-reperfusion (I/R) animal model was established, and the cerebral protective effect of HXF was assessed. The accuracy of the predicted targets was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Hippocampal neuronal injury cell model induced by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of α-Asarone. Furthermore, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to verify whether α-Asarone can bind to PI3K. RESULTS: A total of 44 active ingredients and 795 gene targets were identified through network pharmacology. Network analysis showed that naringenin, eupatin, kaempferol, and α-Asarone were possible drug candidates. SRC, AKT1, TP53, MAPK3, STAT3, HRAS, CTNNB1, EGFR, VEGFA, PIK3R1 could serve as potential drug targets. KEGG analysis implied that the PI3K/AKT signaling pathway might play an important role in treating IS by HXF. Moreover, HXF significantly reduced neurological impairment, cerebral infarct volume, brain index, and brain histopathological damage in I/R rats. The mRNA expression of the top 10 potential targets was verified in the brain tissue. The C-T-P network and UPLC analysis suggested that α-Asarone might be an important component of HXF and can inhibit oxidative stress and apoptosis in HT22 cells by activating the PI3K/AKT signaling pathway. Molecular docking, DARTS, and CETSA assay analysis confirmed that there were direct interactions between α-Asarone and PI3K. CONCLUSION: HXF had a therapeutic effect in IS with multi-component, multi-target, and multi-approach features. α-Asarone, identified as one of the major active components of HXF, could alleviate oxidative stress and apoptosis by targeting PI3K/AKT pathway.

Microrna-206 induces hypoxic necrosis of femoral head by inhibiting VEGF/PI3K/AKT signaling pathway.

The most common form of non-traumatic necrosis of the femoral head is anoxic necrosis of the femoral head, which is a metabolic disease, mainly involving young and middle-aged people. Apoptosis and its related signal regulation pathway play an important role in the occurrence and development of hypoxic necrosis of the femoral head. In order to investigate the possible pathological manifestations of miR-206 and VEGF/PI3K/AKT signal pathway genes and their interactions in hypoxic necrosis of the femoral head, this paper intended to systematically study the expression and regulation mechanism of miR-206 and VEGF/PI3K/AKT signal pathway genes. The interaction between miR-206 and VEGF/PI3K/AKT signaling pathway and its regulation on apoptosis, differentiation and proliferation of human osteoblast cell line hFOB1.19 (SV40 transfer of human osteoblasts) were studied by double luciferase reporter gene analysis, overexpression and inhibition of miR-206, and gene silencing of VEGF/PI3K/AKT signaling pathway. After 24 h and 48 h of intervention with MicroRNA 206 on osteoblasts, it was found that the fluorescence intensity of caspase-3 was higher than that of 0 h group (p < 0.05). This paper has provided an important research basis for the research of femoral head necrosis and the development of new diagnosis and therapeutic drugs for this kind of disease. It also has provided a reference for the further promotion of the chemotherapy drug delivery system.

Ropivacaine inhibits proliferation and invasion and promotes apoptosis and autophagy in bladder cancer cells via inhibiting PI3K/AKT pathway.

Application of a certain concentration of local anesthetics during tumor resection inhibits the progression of tumor. The effects of ropivacaine in bladder cancer (BC) have never been explored. We explored the effects of ropivacaine on the progression of BC in vitro and in vivo. CCK8 assay and EDU staining was conducted to examine cell proliferation. Flow cytometry and transwell assay were performed to evaluate apoptosis and invasion, respectively. Expression of light chain 3 (LC3) was observed through immunofluorescence. Furthermore, the xenograft tumor model of BC was built to detect the effects of ropivacaine in vivo. IHC and TUNEL assay were conducted to detect cell proliferation and apoptosis in vivo. Ropivacaine inhibited the proliferation of T24 and 5639 cells with the 50% inhibitory concentration (IC50) of 20.08 and 31.86 µM, respectively. Ropivacaine suppressed the invasion ability and induces the apoptosis of cells. Besides, ropivacaine triggers obvious autophagy in BC cells. Moreover, ropivacaine blocks the PI3K/AKT signal pathway in BC cells. The impact of ropivacaine on cell viability, motility, and autophagy was reversed by 740 Y-P, the activator of PI3K/AKT signal pathway. The in vivo experiments demonstrated that ropivacaine inhibited the proliferation and mobility of BC. Ropivacaine has anti-carcinoma effects in BC via inactivating PI3K/AKT pathway, providing a new theoretical reference for the use of local anesthetics in the treatment of BC.

Protective effects and mechanism of puerarin targeting PI3K/Akt signal pathway on neurological diseases.

Neurological diseases impose a tremendous and increasing burden on global health, and there is currently no curative agent. Puerarin, a natural isoflavone extracted from the dried root of Pueraria montana var. Lobata (Willd.) Sanjappa and Predeep, is an active ingredient with anti-inflammatory, antioxidant, anti-apoptotic, and autophagy-regulating effects. It has great potential in the treatment of neurological and other diseases. Phosphatidylinositol 3-kinases/protein kinase B (PI3K/Akt) signal pathway is a crucial signal transduction mechanism that regulates biological processes such as cell regeneration, apoptosis, and cognitive memory in the central nervous system, and is closely related to the pathogenesis of nervous system diseases. Accumulating evidence suggests that the excellent neuroprotective effect of puerarin may be related to the regulation of the PI3K/Akt signal pathway. Here, we summarized the main biological functions and neuroprotective effects of puerarin via activating PI3K/Akt signal pathway in neurological diseases. This paper illustrates that puerarin, as a neuroprotective agent, can protect nerve cells and delay the progression of neurological diseases through the PI3K/Akt signal pathway.

Aronia melanocarpa polysaccharide ameliorates liver fibrosis through TGF-ß1-mediated the activation of PI3K/AKT pathway and modulating gut microbiota.

The purpose of this experiment was to investigate the anti-hepatic fibrosis effect of Aronia melanocarpa polysaccharide (AMP) on TAA-induced liver fibrosis mice and its mechanism, as well as the changes in intestinal flora in vivo. This was established with a dose of 200 mg/kg TAA (i.p) once every three days, lasting for eight weeks. Colchicine with 0.4 mg/kg, and AMP (200 and 400 mg/kg) were given by intragastric administration (i.g) after 28 days of intraperitoneal injection of TAA. AMP treatment significantly inhibited the activities of liver injury markers ALT and AST in serum. Histopathological staining demonstrated that AMP significantly reversed TAA-induced hepatocyte necrosis and collagen deposition. In addition, AMP treatment block TGF- ß1/Smads pathway inhibited the production of ECM and alleviates liver fibrosis. Furthermore, AMP treatment enhanced the phosphorylation of PI3K/AKT and decreased the expression of its downstream apoptosis-related proteins in liver, thus effectively alleviating TAA-induced liver fibrosis. In addition, 16S rDNA gene sequencing analysis showed that AMP treatment helped restore the imbalanced ecosystem of gut microbes, increased the proportion of Bacteroidetes and Proteobacteria, and increased species richness. Above findings clearly show that AMP is an effective method for treating liver fibrosis, possibly by improving the gut microbiota.

Dissecting the molecular mechanism of cepharanthine against COVID-19, based on a network pharmacology strategy combined with RNA-sequencing analysis, molecular docking, and molecular dynamics simulation.

OBJECTIVES: Recently, it has been reported that cepharanthine (CEP) is highly likely to be an agent against Coronavirus disease 2019 (COVID-19). In the present study, a network pharmacology-based approach combined with RNA-sequencing (RNA-seq), molecular docking, and molecular dynamics (MD) simulation was performed to determine hub targets and potential pharmacological mechanism of CEP against COVID-19. METHODS: Targets of CEP were retrieved from public databases. COVID-19-related targets were acquired from databases and RNA-seq datasets GSE157103 and GSE155249. The potential targets of CEP and COVID-19 were then validated by GSE158050. Hub targets and signaling pathways were acquired through bioinformatics analysis, including protein-protein interaction (PPI) network analysis and enrichment analysis. Subsequently, molecular docking was carried out to predict the combination of CEP with hub targets. Lastly, MD simulation was conducted to further verify the findings. RESULTS: A total of 700 proteins were identified as CEP-COVID-19-related targets. After the validation by GSE158050, 97 validated targets were retained. Enrichment results indicated that CEP acts on COVID-19 through multiple pathways, multiple targets, and overall cooperation. Specifically, PI3K-Akt signaling pathway is the most important pathway. Based on PPI network analysis, 9 central hub genes were obtained (ACE2, STAT1, SRC, PIK3R1, HIF1A, ESR1, ERBB2, CDC42, and BCL2L1). Molecular docking suggested that the combination between CEP and 9 central hub genes is extremely strong. Noteworthy, ACE2, considered the most important gene in CEP against COVID-19, binds to CEP most stably, which was further validated by MD simulation. CONCLUSION: Our study comprehensively illustrated the potential targets and underlying molecular mechanism of CEP against COVID-19, which further provided the theoretical basis for exploring the potential protective mechanism of CEP against COVID-19.

Overexpression of miR-383-3p protects cardiomyocytes against hypoxia/reoxygenation injury via regulating PTEN/PI3K/AKT signal pathway.

MicroRNAs are widely reported as biomarkers and therapeutic targets in cardiovascular diseases. This study is aimed to expound on the regulatory responsibility of miR-383-3p in H/R-induced injury of H9c2 cells. In this study, H9c2 cells were administrated with H/R. MiR-383-3p expression was measured using qRT-PCR. ELISA was used to determine lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) levels. Reactive oxygen species (ROS) were detected with 2,7-Dichlorodihydrofluorescein diacetate probe. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide, flow cytometry, and TUNEL experiments were conducted to measure cell viability and apoptosis. Cleaved caspase-3, caspase-3, Bax, Bcl-2, PTEN, PI3K, p-PI3K, Akt, p-AKT expression levels were examined by Western blot. Cleaved caspase-3 expression was also measured by immunofluorescence staining. Dual-luciferase reporter gene assay was applied to validate the binding sites in miR-383-3p and the 3'UTR of PTEN. We reported that, miR-383-3p expression in H9c2 cells treated with H/R was remarkably decreased. MiR-383-3p overexpression ameliorated oxidative stress and apoptosis and promoted cell viability in H9c2 cells treated with H/R, while miR-383-3p inhibitor showed the reverse effects. PTEN was identified as a target gene of miR-383-3p. Additionally, enhancement of PTEN expression abolished the influences of miR-383-3p on H9c2 cells. MiR-383-3p mimics could significantly decrease PTEN expression in H9c2 cells while increasing p-PI3K expression and p-AKT expression, while the miR-383-3p inhibitors showed the opposed effects. In conclusion, miR-383-3p protected H9c2 cells from H/R-induced injury via regulating PTEN/PI3K/AKT signal pathway.

Lipopolysaccharide downregulates the expression of ZO-1 protein through the Akt pathway.

BACKGROUND: Neonatal bacterial meningitis is a common neonatal disease with high morbidity, and can cause serious sequelae when left untreated. Escherichia coli is the common pathogen, and its endotoxin, lipopolysaccharide (LPS) can damage the endothelial cells, increasing the permeability of the blood-brain barrier (BBB), leading to intracranial inflammation. However, the specific mechanism of bacterial meningitis induced by LPS damaging BBB remains unclear. In this study, the mouse brain microvascular endothelial (bEND.3) cells were used as a research object to investigate whether LPS damage BBB through the PI3K/Akt pathway. METHODS: The bEND.3 cells were stimulated with different concentrations of LPS for 12 h, and the expression of tight junction proteins (ZO-1, claudin-5, occludin) was detected using western blotting. The cells were challenged with the same concentration of LPS (1ug/ml) across different timepoints (0, 2 h, 4 h, 6 h, 12 h, 24 h). Expression of TJ proteins and signal pathway molecules (PI3K, p-PI3K, Akt, p-Akt) were detected. The distribution of ZO-1 in bEND.3 cells were detected by immunofluorescence staining. RESULTS: A negative correlation is observed between ZO-1 and LPS concentration. Moreover, a reduced expression of ZO-1 was most significant under 1 ug/ml of LPS, and the difference was statistically significant (P < 0.05). Additionally, there is a negative correlation between ZO-1 and LPS stimulation time. Meanwhile, the expression of claudin-5 and occludin did not change significantly with the stimulation of LPS concentration and time. The immunofluorescence assay showed that the amount of ZO-1 on the surface of bEND.3 cells stimulated with LPS was significantly lower than that of the control group. After LPS stimulation, p-Akt protein increased at 2 h and peaked at 4 h. The titer of p-PI3K did not change significantly with time. CONCLUSION: LPS can downregulate the expression of ZO-1; however, its effect on claudin-5 and occludin is minimal. Akt signal pathway may be involved in the regulation of ZO-1 expression induced by LPS in bEND.3 cells.

Bifidobacterium lactis combined with Lactobacillus plantarum inhibit glioma growth in mice through modulating PI3K/AKT pathway and gut microbiota.

Glioma is a common primary aggressive tumor with limited clinical treatment. Recently, growing research suggests that gut microbiota is involved in tumor progression, and several probiotics can inhibit tumor growth. However, evidence for the effect of probiotics on glioma is lacking. Here, we found that Bifidobacterium (B.) lactis combined with Lactobacillus (L.) plantarum reduced tumor volume, prolonged survival time and repaired the intestinal barrier damage in an orthotopic mouse model of glioma. Experiments demonstrated that B. lactis combined with L. plantarum suppressed the PI3K/AKT pathway and down-regulated the expression of Ki-67 and N-cadherin. The glioma-inhibitory effect of probiotic combination is also related to the modulation of gut microbiota composition, which is characterized by an increase in relative abundance of Lactobacillus and a decrease in some potential pathogenic bacteria. Additionally, probiotic combination altered fecal metabolites represented by fatty acyls and organic oxygen compounds. Together, our results prove that B. lactis combined with L. plantarum can inhibit glioma growth by suppressing PI3K/AKT pathway and regulating gut microbiota composition and metabolites in mice, thus suggesting the potential benefits of B. lactis and L. plantarum against glioma.

Orientin Attenuated d-GalN/LPS-Induced Liver Injury through the Inhibition of Oxidative Stress via Nrf2/Keap1 Pathway.

Oxidative stress is involved in the pathogenesis of liver diseases, including liver injury, a serious health problem worldwide. Natural polyphenols have attracted increasing attention as potential agents for the prevention and treatment of liver diseases. Orientin, a flavonoid component with antioxidant capacity, has been regarded as a promising nutraceutical for patients with liver damage. This study aimed to investigate the amelioration effect of orientin on d-galactosamine and lipopolysaccharides (d-GalN/LPS) induced liver injury in mice, with a focus on its underlying mechanisms by using the H2O2-induced oxidative damage model of HepG2 cells. Results indicated that orientin alleviated d-GalN/LPS-induced liver damage by improving the hepatic histological changes and reducing the levels of hepatic and serum alanine aminotransferase and aspartic acid aminotransferase. Additionally, supplementation of orientin improved the antioxidant ability in mice by decreasing the levels of hepatic malondialdehyde, protein carbonyl, myeloperoxidase, nitric oxide, glutathione, glutathione peroxidase, gluathione reductase, and superoxide dismutase. Orientin treatment significantly elevated both the protein and mRNA expressions of nuclear factor erythroid 2-related factor 2, Kelch-like ECH-associated protein-1, heme oxygenase-1, and nicotinamide quinone oxidoreductase 1 in liver and HepG2 cells. The management of orientin also elevated the protein expression of glutathione S-transferase and Maf in HepG2 cells. Taken together, it suggested that orientin played an amelioration effect on liver injury by suppressing oxidative stress, which might be strongly related to the activation of Nrf2/ARE through PI3K/Akt and P38/MAPK signal pathways.

[Effects of long-chain noncoding RNA Linc00673 overexpression on proliferation and apoptosis of gastric cancer cell line MGC-803].

Objective: To investigate the effects of long-chain noncoding RNA Linc00673 overexpression on proliferation and apoptosis of gastric cancer cells and its mechanisms. Methods: The recombinant lentivirus expressing plasmid pLVX-Linc00673 and the control empty plasmid pLVX-NC were packaged and amplified in 293T cells, and the recombinant lentivirus was transfected into gastric cancer cell line MGC-803 to establish a cell line stably overexpressing Linc00673. The expression of Linc00673 gene was detected by real-time fluorescence quantitative PCR. The growth and proliferation of cells were observed by MTT assay and clone formation assay. Cell cycle and apoptosis were detected by flow cytometry. The expressions of cell cycle related regulatory genes were detected by qPCR. The expressions of key molecules in the PI3K/Akt signaling pathway and tumor proliferation related proteins were detected by Western blot. Results: The expressions of Linc00673 in gastric cancer cell line MGC-803, BGC-823 and AGS were significantly higher than that in normal gastric mucosa cell line GES-1 (P＜0.05). MGC-803 cell line with stable overexpression of LINC00673 was established, and the expression level of LincC00673 was 200 times higher than that of the control empty carrier group. Overexpression of Linc00673 promoted proliferation of MGC-803 cells (P＜0.05) and clone formation (P＜0.05), inhibited cell apoptosis and affected the G1→S phase progression of cell cycle (P＜0.01). Overexpression of Linc00673 could affect the expressions of cell cycle regulatory gene CCNG2, P19 and CDK1 in MGC-803. Western blot showed that Linc00673 overexpression not only promoted the expressions of the key molecule pAkt in PI3K / Akt signaling pathway and its downstream target NF-κ B and Bcl-2 protein, but also up regulated the expressions of tumor related factors ß-catenin and EZH2 proteins. Conclusion: Overexpression of Linc00673 may promote proliferation and inhibit apoptosis of MGC-803 cells through PI3K/Akt signaling pathway.

Andrographolide attenuates Mycoplasma gallisepticum-induced inflammation and apoptosis by the JAK/PI3K/AKT signal pathway in the chicken lungs and primary alveolar type II epithelial cells.

Mycoplasma gallisepticum (MG) is the primary etiologic agent of chronic respiratory disease (CRD) in chickens. Respiratory tract inflammation and apoptosis are the main features of CRD. Andrographolide (Andro), a natural small molecule compound, is known for its excellent anti-pathogenic and anti-inflammatory properties. Hence, this study was to evaluate the anti-inflammation and anti-apoptosis effects of Andro as well as the underlying mechanism in the chicken lungs and primary alveolar type II epithelial cells (AEC II). Results showed Andro had no side effects on AEC II viability at concentrations below 200 µg/ml. Compared with the model group, terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL), western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays (ELISA) results showed Andro treatment significantly reduced apoptosis in the chicken lungs and AEC II, and down-regulated the expression levels of the protein of MG adhesin 1.2 (pMGA1.2), IL-1ß, TNF-α, IL-6, Bax, Caspase 9 and Caspase 3, and up-regulated the expression levels of Bcl-2 and Bcl-xL in the chicken lungs, serum and AEC II (P ≤ 0.05). Moreover, Andro inhibited the MG-induced JAK/PI3K/AKT signal pathway activation in the chicken lungs and AEC II. Inhibiting of the JAK/PI3K/AKT signal pathway significantly alleviated MG-induced inflammation and apoptosis in the AEC II. Andro may exert an anti-inflammatory and anti-apoptotic effect by inhibiting the JAK/PI3K/AKT signal pathway in the chicken lungs and AEC II. In conclusion, Andro could act as a potential agent against MG infection by inhibiting the JAK/PI3K/AKT signal pathway and pMGA1.2 expression in the chickens.