Activation Of PI3K-Akt Signaling Pathway Research Articles

Early-life exposure to PM2.5 leads to ASD-like phenotype in male offspring rats through activation of PI3K-AKT signaling pathway

Previous studies have shown that early-life exposure to fine particulate matter (PM2.5) is associated with an increasing risk of autism spectrum disorder (ASD), however, the specific sensitive period of ASD is unknown. Here, a model of dynamic whole-body concentrated PM2.5 exposure in pre- and early-postnatal male offspring rats (MORs) was established. And we found that early postnatal PM2.5 exposed rats showed more typical ASD behavioral characteristics than maternal pregnancy exposure rats, including poor social interaction, novelty avoidance and anxiety disorder. And more severe oxidative stress and inflammatory responses were observed in early postnatal PM2.5 exposed rats. Moreover, the expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN) was down-regulated and the ratios of p-PI3K/PI3K and p-AKT/AKT were up-regulated in early postnatal PM2.5 exposed rats. This study suggests that early postnatal exposure to PM2.5 is more susceptible to ASD-like phenotype in offspring than maternal pregnancy exposure and the activation of PI3K-AKT signaling pathway may represent underlying mechanisms.

Bioactive triterpenoid compounds of Poria cocos (Schw.) Wolf in the treatment of diabetic ulcers via regulating the PI3K-AKT signaling pathway

Ethnopharmacological relevanceDiabetic ulcers represent a chronic condition characterized by prolonged hyperglycemia and delayed wound healing, accompanied by endocrine disorders, inflammatory responses, and microvascular damage in the epidermal tissue, demanding effective clinical treatment approaches. For thousands of years, ancient Chinese ethnopharmacological studies have documented the use of Poria cocos (Schw.) Wolf in treating diabetic ulcers. Recent research has substantiated the diverse pharmacological effects of Poria cocos (Schw.) Wolf, including its potential to alleviate hyperglycemia and exhibit anti-inflammatory, antioxidant, and immune regulatory properties, which could effectively mitigate diabetic ulcer symptoms. Furthermore, being a natural medicine, Poria cocos (Schw.) Wolf has demonstrated promising therapeutic effects and safety in the management of diabetic ulcers, holding significant clinical value. Despite its potential clinical efficacy and applications in diabetic ulcer treatment, the primary active components and underlying pharmacological mechanisms of Poria cocos (Schw.) Wolf remains unclear. Further investigations are imperative to establish a solid foundation for drug development in this domain. Aim of the study and Materials and methodsIn this study, we aimed to identify the active compounds and potential targets of Poria cocos (Schw.) Wolf using UHPLC-Q-TOF-MS and TCMSP databases. Additionally, we attempt to identify targets related to diabetic ulcers. Following enrichment analysis, a network of protein-protein interactions was constructed to identify hub genes based on the common elements between the two datasets. To gain insights into the binding activities of the hub genes and active ingredients, molecular docking analysis was employed. Furthermore, to further validate the therapeutic effect of Poria cocos (Schw.) Wolf, we exerted in vitro experiments using human umbilical vein vascular endothelial cells and human myeloid leukemia monocytes (THP-1). The active ingredient of Poria cocos (Schw.) Wolf was applied in these experiments. Our investigations included various assays, such as CCK-8, scratch test, immunofluorescence, western blotting, RT-PCR, and flow cytometry, to explore the potential of Poria cocos (Schw.) Wolf triterpenoid extract (PTE) in treating diabetic ulcers. ResultsThe findings here highlighted PTE as the primary active ingredient in Poria cocos (Schw.) Wolf. Utilizing network pharmacology, we identified 74 potential targets associated with diabetic ulcer treatment for Poria cocos (Schw.) Wolf, with five hub genes (JUN, MAPK1, STAT3, AKT1, and CTNNB1). Enrichment analysis revealed the involvement of multiple pathways in the therapeutic process, with the PI3K-AKT signaling pathway showing significant enrichment. Through molecular docking, we discovered that relevant targets within this pathway exhibited strong binding with the active components of Poria cocos (Schw.) Wolf. In vitro experiments unveiled that PTE (10 mg/L) facilitated the migration of human umbilical vein vascular endothelial cells (P < 0.05). PTE also increased the expression of CD31 and VEGF mRNA (P < 0.05) while activating the expressions of p-PI3K and p-AKT (P < 0.05). Moreover, PTE demonstrated its potential by reducing the expression of IL-1β, IL-6, TNF-α, and NF-κB mRNA in THP-1 (P < 0.05) and fostering M2 macrophage polarization. These results signify the potential therapeutic effects of PTE in treating diabetic ulcers, with its beneficial actions mediated through the PI3K-AKT signaling pathway. ConclusionsPTE is the main active ingredient in Poria cocos (Schw.) Wolf that exerts therapeutic effects. Through PI3K-AKT signaling pathway activation and inflammatory response reduction, PTE promotes angiogenesis, thereby healing diabetic ulcers.

Dysregulated Genes and Signaling Pathways in the Formation and Rupture of Intracranial Aneurysm.

Intracranial aneurysm (IA) has the potential to rupture. Despite scientific advances, we are still not in a position to screen patients for IA and identify those at risk of rupture. It is critical to comprehend the molecular basis of disease to facilitate the development of novel diagnostic strategies. We used transcriptomics to identify the dysregulated genes and understand their role in the disease biology. In particular, RNA-Seq was performed in tissue samples of controls, unruptured IA, and ruptured IA. Dysregulated genes (DGs) were identified and analyzed to understand the functional aspects of molecules. Subsequently, candidate genes were validated at both transcript and protein level. There were 314 DGs in patients with unruptured IA when compared to control samples. Out of these, SPARC and OSM were validated as candidate molecules in unruptured IA. PI3K-AKT signaling pathway was found to be an important pathway for the formation of IA. Similarly, 301 DGs were identified in the samples of ruptured IA when compared with unruptured IAs. CTSL was found to be a key candidate molecule which along with Hippo signaling pathway may be involved in the rupture of IA. We conclude that activation of PI3K-AKT signaling pathway by OSM along with up-regulation of SPARC is important for the formation of IA. Further, regulation of Hippo pathway through PI3K-AKT signaling results in the down-regulation of YAP1 gene. This along with up-regulation of CTSL leads to further weakening of aneurysm wall and its subsequent rupture.

Uncovering the active constituents and mechanisms of Rujin Jiedu powder for ameliorating LPS-induced acute lung injury using network pharmacology and experimental investigations.

Background: Acute lung injury (ALI) is a common clinical disease with high mortality. Rujin Jiedu powder (RJJD) has been clinically utilized for the treatment of ALI in China, but the active constituents in RJJD and its protective mechanisms against ALI are still unclear. Methodology: ALI mice were established by intraperitoneal injection of LPS to test the effectiveness of RJJD in treating ALI. Histopathologic analysis was used to assess the extent of lung injury. An MPO (myeloperoxidase) activity assay was used to evaluate neutrophil infiltration. Network pharmacology was used to explore the potential targets of RJJD against ALI. Immunohistochemistry and TUNEL staining were performed to detect apoptotic cells in lung tissues. RAW264.7 and BEAS-2B cells were used to explore the protective mechanisms of RJJD and its components on ALI in vitro. The inflammatory factors (TNF-α, IL-6, IL-1β and IL-18) in serum, BALF and cell supernatant were assayed using ELISA. Western blotting was performed to detect apoptosis-related markers in lung tissues and BEAS-2B cells. Results: RJJD ameliorated pathological injury and neutrophil infiltration in the lungs of ALI mice and decreased the levels of inflammatory factors in serum and BALF. Network pharmacology investigations suggested that RJJD treated ALI via regulating apoptotic signaling pathways, with AKT1 and CASP3 as crucial targets and PI3K-AKT signaling as the main pathway. Meanwhile, baicalein, daidzein, quercetin and luteolin were identified as key constituents in RJJD targeting on the above crucial targets. Experimental investigations showed that RJJD significantly upregulated the expression of p-PI3K, p-Akt and Bcl-2, downregulated the expression of Bax, caspase-3 and caspase-9 in ALI mice, and attenuated lung tissue apoptosis. Four active constituents in RJJD (baicalein, daidzein, quercetin and luteolin) inhibited the secretion of TNF-α and IL-6 in LPS-induced RAW264.7 cells. Among these components, daidzein and luteolin activated the PI3K-AKT pathway and downregulated the expression of apoptosis-related markers induced by LPS in BEAS-2B cells. Conclusion: RJJD alleviates the inflammatory storm and prevents apoptosis in the lungs of ALI mice. The mechanism of RJJD in treating ALI is related to the activation of PI3K-AKT signaling pathway. This study provides a scientific basis for the clinical application of RJJD.

Single-cell transcriptome analysis identifies Versican(+) myofibroblast as a hallmark for thoracic aortic aneurysm marked by activation of PI3K-AKT signaling pathway

BackgroundThoracic aortic aneurysm (TAA) is a silent but dangerous cardiovascular disease. Understanding molecular mechanisms of TAA on single-cell level might provide new strategies for preventing and treating TAA. MethodsSingle-cell RNA sequencing was performed on control and aneurysmal thoracic aorta to find out specific cell clusters and cell types. Western blot and histological staining were used to verify the findings of single-cell transcriptome analysis. Characteristics of Versican (VCAN) overexpressed myofibroblast was evaluated through bioinformatic methods and experimental validation. ResultsA total of 3 control and 8 TAA specimens were used for single-cell transcriptome analysis including 48,128 thoracic aortic cells. Among these cells, we found out a specific cell cluster containing both hallmarks of smooth muscle cell (SMC) and fibroblast. Thus, we defined these cells as myofibroblast. Further single-cell transcriptome analysis identified VCAN as a cellular marker of myofibroblast. Western blot and histological staining revealed that VCAN(+) myofibroblast was significantly increased in TAA specimens compared with control individuals. Differential analysis, functional, pathway enrichment analysis and cell-cell communication analysis demonstrated that VCAN(+) myofibroblast was closely associated with previous reported TAA associated pathological process including SMC proliferation, SMC migration and extracellular matrix (ECM) disruption. Pathway analysis found out significant activation of PI3K-AKT signaling pathway within VCAN(+) myofibroblast, which was further confirmed by experimental validation. ConclusionsSingle-cell RNA sequencing identified VCAN(+) myofibroblast as a typical cellular hallmark of TAA. These cells might participate in the pathogenesis of TAA through activation of PI3K-AKT signaling pathway to link SMC proliferation, SMC migration and ECM disruption.

KW-2449 and VPA exert therapeutic effects on human neurons and cerebral organoids derived from MECP2-null hESCs

BackgroundRett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MECP2), is one of the most prevalent neurodevelopmental disorders in girls. However, the underlying mechanism of MECP2 remains largely unknown and currently there is no effective treatment available for RTT.MethodsWe generated MECP2-KO human embryonic stem cells (hESCs), and differentiated them into neurons and cerebral organoids to investigate phenotypes of MECP2 loss-of-function, potential therapeutic agents, and the underlying mechanism by transcriptome sequencing.ResultsWe found that MECP2 deletion caused reduced number of hESCs-derived neurons and simplified dendritic morphology. Moreover, MECP2-KO cortical organoids exhibited fewer neural progenitor cells and neurons at day 60. Electrophysiological recordings showed that MECP2 deletion altered synaptic activity in organoids. Transcriptome analysis of organoids identified many genes in the PI3K-AKT pathway downregulated following MECP2 deletion. Treatment with either KW-2449 or VPA, small molecules for the activation of PI3K-AKT signaling pathway, alleviated neuronal deficits and transcriptome changes in MECP2-KO human neuronal models.ConclusionsThese findings suggest that KW-2449 and VPA might be promising drugs for RTT treatment.

Buyang Huanwu decoction promotes angiogenesis of rat brain microvascular endothelial cells after oxygen-glucose deprivation reperfusion injury via activation of PI3K-AKT signaling pathway.

To investigate the effect and mechanism of Buyang Huanwu decoction (BYHWD) on angiogenesis of rat brain microvascular endothelial cells (RBMECs) after oxygen-glucose deprivation reperfusion (OGD/R) injury. RBMECs were pretreated with BYHWD containing serum 24 h before OGD/R injury was induced. Cells were randomly divided into blank control group, model control group, BYHWD group (provided BYHWD containing serum) and LY294002 group [treated with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 for 1 h before provided BYHWD containing serum]. The cell viability, migration and tube formation abilities of RBMECs were detected by CCK-8, scratch wound healing, Transwell migration and tube formation assays, respectively. The protein expression levels of PI3K, p-PI3K, protein kinase B (AKT), p-AKT, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were determined by Western blotting. Compared with model control group, cell viability, migration and tube formation abilities of RBMECs were significantly improved in BYHWD group (all P<0.01), the protein expression levels of p-PI3K, p-AKT, HIF-1α and VEGF were up-regulated (all P<0.05); while above effects were blocked by LY294002. BYHWD can promote angiogenesis of RBMECs after OGD/R injury, which may be related to the increased protein expression of HIF-1α and VEGF through activation of PI3K-AKT signaling pathway.

EID3 Promotes Cancer Stem Cell-Like Phenotypes in Osteosarcoma through the Activation of PI3K-AKT Signaling Pathway.

The aim of this study is to elucidate molecular mechanism by which E1A-like inhibitor of differentiation 3 (EID3) promotes cancer stem cell-like phenotypes in osteosarcoma. Overexpression of EID3 in osteosarcoma cells generated more spherical clones, enhanced the expression of stemness-associated genes, and promoted chemoresistance, invasion, and metastasis. Furthermore, osteosarcoma cells overexpressing EID3 had increased ability to grow in suspension as osteospheres with high expression of Sox2 and stem cell marker CD133. In addition, knockdown of EID3 reduced sphere formation and inhibited osteosarcoma cell migration and invasion. RNA sequencing and bioinformatics analysis revealed that PI3K-Akt signaling pathway and MAPK pathway­related genes were enriched in osteosarcoma cells with high expression of EID3. Taken together, EID3 promotes osteosarcoma, and EID3–PI3K-Akt axis is a potential therapeutic target for osteosarcoma treatment.

Integrated strategy of network pharmacology, molecular docking, HPLC-DAD and mice model for exploring active ingredients and pharmacological mechanisms of Penthorum chinense Pursh against alcoholic liver injury

Ethnopharmacological relevancePenthorum chinense Pursh (PCP, Saxifragaceae) is an edible plant and frequently-used Chinese herbal medicine, and is commonly used as Miao medicine in China. It showed well effect on alcoholic liver injury (ALI), but studies on its active ingredients and mechanisms against ALI remain at the starting stage. Aim of the studyThis work aims to explore the active ingredients and pharmacological mechanisms of PCP against ALI. Materials and methodsFirst, network pharmacology was applied to decipher the potential active ingredients and pharmacological mechanisms of PCP against ALI by ingredient identification, ADMET evaluation, target identification, network construction and analysis, protein-protein interaction (PPI) analysis, and gene enrichment analysis. Second, molecular docking was used to explore the interaction between key active ingredient and hub protein of PCP against ALI. Then, the ingredient analysis of PCP aqueous extract and semiquantitative analysis of key active ingredient were carried out on HPLC-DAD. Subsequently, mice with ALI were used to investigate the therapeutic effect or verify the predicted mechanisms of PCP or key active ingredient against ALI by analyzing body weight, liver index, ALT and AST activities in serum and liver tissues, oxidation related indices (SOD activity, GSH level and MDA level) in liver tissues, histopathology of liver tissues (oil red O, hematoxylin-eosin and DAB-TUNEL staining), and changes of related proteins (PI3K, Akt, p-Akt, Bax and Bcl-2) in liver tissues with the aid of Western blot. ResultsNetwork pharmacology showed that the active ingredients and related genes of PCP against ALI comprised 10 ingredients and 52 genes. Based on the result of ingredient analysis of PCP aqueous extract, quercitrin was identified as the key active ingredient of PCP against ALI. PPI analysis indicated that AKT1 was the hub gene of PCP against ALI, and molecular docking suggested that there were good interaction between quercetin and Akt1 protein. Gene enrichment analysis showed that the pivotal molecular mechanism of PCP against ALI might be to inhibit hepatocyte apoptosis via activation of PI3K-Akt signaling pathway. PCP and quercitrin showed anti-ALI effect by offsetting weight loss and increase of liver index, and reversing the imbalance of oxidative stress and histopathological changes of liver tissues (abnormal fatty acid metabolism, hepatic cord swelling and inflammatory cell infiltration) in mice with ALI. PCP caused the decrease of DAB-TUNEL-positive cells, upregulated the anti-apoptotic proteins (PI3K, Akt and p-Akt) levels and the ratio of p-Akt/Akt, and downregulated pro-apoptotic protein (Bax) level and the ratio of Bax/Bcl-2 in liver tissues of mice with ALI, indicating that the mechanism of PCP against ALI involved in inhibiting hepatocyte apoptosis via activation of PI3K-Akt signaling pathway. ConclusionPCP and quercitrin showed well anti-ALI effect. The key active ingredient of PCP against ALI was identified as quercitrin. The underlying pharmacological mechanisms of PCP against ALI may be related to PI3K-Akt signaling pathway-mediated inhibition of hepatocyte apoptosis. This work provided new evidence to support the application of PCP in treatment of ALI, and a research basis for the research and development of functional foods or drugs against ALI from PCP.

A Mechanistic Exploratory Study on the Therapeutic Efficacy of Astragaloside IV Against Diabetic Retinopathy Revealed by Network Pharmacology.

Purpose: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus, which nearly happens to all the diabetic sufferers. This study aims to identify the preliminary molecular regulation involved in the therapeutic efficacy of astragaloside IV (AS- IV) for DR. Methods: Diabetic rat models were established and treated with AS-IV. Optical coherence tomography (OCT) and Hematoxylin-eosin (HE) staining was employed to demonstrate the histopathological changes. The main targets of AS-IV were identified by searching from public databases of traditional Chinese medicine (GeneCards, PharmMapper and Swiss Target Prediction). Besides, disease targets of DR were also obtained by integrated data from GEO datasets and predicted from public databases. Protein-protein interaction (PPI) network was constructed by Cytoscape with overlapping genes and 10 core targets were selected, on which Gene Ontology (GO) along with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted. The interaction between AS-IV and these crucial genes were analyzed using molecular docking. RT-qPCR and western blot were used to verify the expression variation of core targets. Results: OCT imaging and HE staining demonstrated that AS-IV administration significantly increased retinal thickness in diabetic rats, obviously alleviating DR induced histopathological changes as well as elevated blood glucose levels. 107 common targets of AS-IV and DR were determined after intersection. PPI network analysis filtered 10 hub genes potentially targeted by AS-IV, including VEGFA, CASP3, HIF1α, STAT3, CTNNB1, SRC, AKT1, EGFR, IL1β and IL6. Enrichment analysis indicated that these genes were mainly enriched in biological processes like T cell activation, epithelial cell proliferation and protein kinase B signaling, and involved in oxidative stress, apoptosis and inflammation-related pathways. The molecular docking prediction suggested that AS-IV exhibited stable binding to these core targets. In addition, mRNA levels of core targets in diabetic rats were differentially expressed before and after AS-IV treatment. Western blot further revealed that AS-IV treatment elevated DR-depressed protein levels of PI3K and AKT. Conclusion: Our study elucidated the effect of AS-IV in attenuating retinopathy induced by diabetes in rats and preliminarily unveiled the therapeutic efficacy of AS-IV in the treatment of DR might be attributed to activation of PI3K-AKT signaling pathway.

Melatonin inhibits embryonic rat H9c2 cells growth through induction of apoptosis and cell cycle arrest via PI3K‐AKT signaling pathway

Our recent epidemiological study revealed that maternal sleep during the periconceptional period should be involved in the risk of congenital heart disease (CHD) in offspring. Melatonin, a sleep related hormone, has been suggested to play a crucial role in embryonic development based on the emerging evidence. In this study, we set out to assess the effect of melatonin on the embryonic cardiac cell growth and to explore the underlying mechanisms. We observed the effect of different gradient doses of melatonin as 10, 100, or 1,000 μM on cell proliferation in H9c2 embryonic rat cardiac cells. Furthermore, flow cytometry was applied to evaluate the impact on apoptosis and cell cycle. RNA-seq was conducted to screen the changes in expression of mRNA and signaling pathways. Quantitative Real-Time-PCR (qRT-PCR) was then conducted to validate the results. It was observed that melatonin could inhibit H9c2 cell growth, at the doses of 100 and 1,000 μM, but not at 10μM. Moreover, melatonin ranged from 100 to 1,000 μM could instigate cell cycle arrest at G1 phase and simulate apoptosis, in a dose-dependent manner. In addition, melatonin was found to down-regulate the expression of a number of genes, which are related to heart development (SPARC, IFITM3, TNNT2, LOX), and PI3K-Akt signaling pathway activation (FN1, HSP90B1, THBS1, MFGE8, and CLU). Our findings suggested that high level of melatonin could be capable of inhibiting growth through the induction of apoptosis and cell cycle arrest via PI3K-AKT signaling pathway, thereby interfering with embryonic heart development. Considering this study is based on H9c2 embryonic rat cardiac cells, future additional studies using human embryonic cardiac cell are warranted.

Chemerin/ChemR23 regulates cementoblast function and tooth resorption in mice via inflammatory factors.

Periodontitis and orthodontic treatment can lead to inflammatory root resorption (IRR) through an unclear mechanism. Chemerin, a novel chemoattractant protein, is closely associated with inflammation, affects osteoblast and osteoclast differentiation, and may play a role in IRR. We aimed to explore possible roles of the chemerin/ChemR23 interaction in cementoblast function and IRR and reveal a new IRR therapeutic target. Cementoblast function-related gene and protein expression in the immortalized murine cementoblast cell line OCCM-30 after treatment with chemerin and siChemR23 was examined by qRT-PCR and Western blotting. The roles of the MAPK and PI3K-Akt signaling pathways were studied using specific inhibitors. Cementoblast cytokine production under different treatment conditions was measured by enzyme-linked immunosorbent assay and qRT-PCR. Additionally, we modeled IRR in wild-type and chemerin-overexpressing mice and injected transgenic mice with anti-ChemR23 antibody to block ChemR23. We then calculated the root resorption volume and examined periodontal tissue cathepsin K, Runx2, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) expression. Chemerin suppressed cementoblast differentiation and mineralization and exerted a proinflammatory effect on cementoblasts. These effects were partially reversed by siChemR23 and reversed to different extents by p38, Erk1/2 and PI3K-Akt pathway inhibition, suggesting p38, Erk1/2 and PI3K-Akt pathways as signaling pathways downstream of chemerin/ChemR23. In vivo, chemerin overexpression worsened IRR. Moreover, chemerin expression was positively correlated with TNF-α, IL-6, and cathepsin K expression and negatively correlated with Runx2 expression. ChemR23 downregulation reversed these effects. Chemerin/ChemR23 induced TNF-α and IL-6 expression dependent on Erk1/2, p38 MAPK, and PI3K-Akt signaling pathway activation, thereby regulating cementoblast function and affecting IRR.

SNORD126 Promotes Hepatitis C Virus Infection by Upregulating Claudin-1 via Activation of PI3K-AKT Signaling Pathway.

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, some of which promote the infection process. A small nucleolar RNA, C/D box 126 (SNORD126), was previously shown to be associated with hepatocellular carcinoma (HCC). However, the role of SNORD126 in HCV infection, which is one of the primary reasons for HCC development, has not been elucidated. In the present study, using small nucleolar RNA profiling, we observed that SNORD126 was significantly downregulated during HCV infection in both Huh7 and Huh7.5.1 cells. In addition, overexpression of SNORD126 enhanced HCV entry into host cells, whereas SNORD126 knockdown showed the opposite effect, suggesting that SNORD126 promotes HCV infection, especially through viral entry. Further functional analysis revealed that SNORD126 could enhance the expression level of claudin-1 (CLDN1), a key HCV entry factor, by increasing the levels of phosphorylated AKT. Additionally, the function of SNORD126 in HCV infection was associated with ribonucleoprotein (RNP) complexes. In summary, our findings demonstrate that oncogenic SNORD126 levels are decreased during HCV infection probably due to the host defense reaction, and SNORD126 may be important to promote viral entry by increasing CLDN1 expression through activation of the PI3K-AKT pathway, the mechanism of which is partly associated with SNORD126-mediated snoRNA RNP (snoRNP) function. Our work here provides initial evidence that endogenous snoRNA takes part in HCV infection and shows potential as a diagnostic or antiviral agent.

LncUBE2R2-AS1 acts as a microRNA sponge of miR-302b to promote HCC progression via activation EGFR-PI3K-AKT signaling pathway

ABSTRACT Hepatocellular carcinoma (HCC) is a main cause of cancer-related deaths globally. Long non-coding RNAs (lncRNAs) play important roles in diverse cancers. LncRNA-UBE2R2-AS1 has been reported to promote apoptosis in glioma cell. However, the expressions, functions, and mechanisms of action of UBE2R2-AS1 in HCC are still unclear. UBE2R2-AS1 is increased in HCC tissues and cell lines. Increased expression of UBE2R2-AS1 is associated with large tumor size, multiple tumor number, advanced TNM stage, and poor survival of HCC patients. Functional experiments showed that knockdown UBE2R2-AS1 inhibited HCC growth and metastasis through in vitro and in vivo experiments. Regarding the mechanism, UBE2R2-AS1/miR-302b/EGFR established the ceRNA network involved in the modulation of cell progression of HCC cells via activation of PI3K-AKT signaling pathway. Overall, UBE2R2-AS1 may exhibit an oncogenic function in HCC via acting as a sponge for miR-302b to up-regulate EGFR, and may serve as a potential therapeutic target and a prognostic biomarker for HCC patients.

HOTTIP knockdown inhibits cell proliferation and migration via regulating miR-490-3p/HMGB1 axis and PI3K-AKT signaling pathway in ox-LDL-induced VSMCs

AimsAtherosclerosis (AS) is a common cardiovascular disease with complicated pathogenesis. Long non-coding RNAs (lncRNAs) have been reported to be associated with AS progression. We aimed to explore the role and underlying mechanism of HOXA transcript at the distal tip (HOTTIP) in AS. Materials and methodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HOTTIP, miR-490-3p and high mobility group B 1 (HMGB1) in AS patients' sera and oxidized low-density lipoprotein (ox-LDL) induced human aortic vascular smooth muscle cells (HA-VSMCs). Cell Counting Kit-8 (CCK-8) assay and transwell assay were conducted to evaluate the proliferation and migration of HA-VSMCs, respectively. Western blot assay was carried out to determine the levels of proliferating cell nuclear antigen (PCNA), matrix metalloprotein 2 (MMP2), MMP9 and HMGB1. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the targeting association between HOTTIP and miR-490-3p, as well as miR-490-3p and HMGB1. Key findingsHOTTIP and HMGB1 were upregulated and miR-490-3p was downregulated in the sera of AS patients and ox-LDL-stimulated HA-VSMCs. HOTTIP knockdown suppressed ox-LDL induced proliferation and migration in HA-VSMCs. MiR-490-3p was identified as a target of HOTTIP and HOTTIP overexpression abolished the inhibition on cell proliferation and migration mediated by miR-490-3p in ox-LDL-induced HA-VSMCs. Moreover, miR-490-3p inhibition promoted cell proliferation and migration by directly targeting HMGB1 in ox-LDL-induced HA-VSMCs. Besides, HOTTIP knockdown repressed the activation of PI3K-AKT signaling pathway. SignificanceHOTTIP knockdown suppressed cell proliferation and migration by regulating miR-490-3p/HMGB1 axis and PI3K-AKT pathway in ox-LDL-induced HA-VSMCs.

Prediction of the mechanisms of Xiaoai Jiedu Recipe in the treatment of breast cancer: A comprehensive approach study with experimental validation

Ethnopharmacological relevanceTraditional Chinese medicine (TCM) holds a great promise for preventing complex chronic diseases through a holistic way. Certain Chinese medicine formulae from TCM are effective for treating and preventing cancer in clinical practice. Xiaoai Jiedu Recipe (XJR) is a Chinese medicine formula that has been used to treat breast cancer (BC). However, its active ingredients and therapeutic mechanisms on tumor are unclear. Therefore, further investigation is necessary. Aim of the studyThis study aims to elucidate the active compounds of XJR and its molecular mechanisms for the treatment of BC. Materials and methodsA comprehensive approach was used to clarify the pharmacodynamic basis of XJR and its pharmacological mechanism, including the acquisition of differentially expressed genes of BC, screening of active ingredients and their targets, construction of complex internetwork between drugs and diseases, and analysis of the key subnetwork. Finally, these results were validated by in vitro experiments and comparison with literature reviews. ResultsBy using bioinformatics, 5211 differentially expressed genes of BC were identified, more than half of them had been reported in previous studies. By using network analysis, 113 potential bioactive compounds in the ten component herbs of XJR and 157 BC-related targets were identified, which were significantly enriched in 85 pathways and 1321 GO terms. The in vitro studies showed that quercetin and ursolic acid, the active components of XJR, could effectively inhibit the proliferation of breast cancer cells, and the combination of the two components could significantly decrease the mitochondrial membrane potential and suppress the activation of PI3K-Akt signaling pathway, thus inducing apoptosis of cancer cells. ConclusionsXJR played an important role in anti-BC through multi-component, multi-target and multi-pathway mechanisms, in which quercetin and ursolic acid may be the key active components. The anticancer effect of multi-component application was better than that of a single component. This study not only deepened our understanding of the role of TCM in the prevention and treatment of diseases, but also provided a reference for the in-depth research, development and application of the ancient medicine.

Phenols fragment of Veronica ciliata Fisch. ameliorate free radical-induced nonalcoholic fatty liver disease by mediating PI3K/Akt signaling pathway

Ethnopharmacological relevanceVeronica ciliata Fisch. is used in numerous of Tibetan medicine prescriptions because of its hepatoprotective effect. Aims of this studyHere, we aimed to investigate the hepatoprotective effect and mechanism of phenolic fraction (PF) of V. ciliata Fisch. on liver injury induced by free radical. Materials and methodsBRL 3A cells were pre-treated with PF and luteolin (Lut) following tert-butyl hydroperoxide (t-BHP) treatment. The cell viability, lactate dehydrogenase (LDH) levels, reactive oxygen species (ROS) generation, apoptosis, cell cycle and autophagy were analyzed. Apoptotic, inflammatory, and autophagy,- related proteins were analyzed using Western blotting. The combination of molecular docking and drug affinity targeting experiments (DARTS) were first utilized to analysis the target protein of Lut. ResultsPF effectively suppressed t-BHP-induced apoptosis caused by mitochondrial dysfunction, which were associated with inhibiting ROS generation. Further investigation indicated that PF significantly suppressed apoptosis, inflammation, and autophagy by regulating the expression of related proteins. The results of molecular docking and drug affinity targeting experiments (DARTS) revealed that PI3K was the target protein of PF and Lut. Further studies have shown that PF relieved liver injury induced by t-BHP via suppressing phosphorylated expression of PI3K. ConclusionOur results indicate that PF effectively protect against hepatotoxicity induced by t-BHP through inhibiting the abnormal activation of PI3K-Akt signaling pathway and highlight the health benefits of PF regarding oxidative stress, proving it to be an important source of bioactive compounds associated with Nonalcoholic fatty liver disease (NAFLD).

Effect and mechanism of adipocyte co-culture on aquaporin-9 expression in HepG2 cells

Objective: To observe the effect of differentiated mature adipocytes on hepatic steatosis and aquaporin-9 (AQP9) expressions in HepG2 cells and further explore its possible mechanism of action. Methods: Human preadipocytes were cultured and differentiated to full maturity. HepG2 cells were co-cultured with non-differentiated adipocytes and differentiated mature adipocytes for 48 h, and then labeled as control group and experimental group. Oil red O staining and intracellular triglyceride content were performed on co-cultured HepG2 cells and simultaneous changes in phosphatidylinositol 3-kinase (PI3K) - serine/threonine kinase (Akt) signaling pathway, and AQP9 mRNA and protein levels were detected. The experimental group was co-cultured with recombinant human insulin-like growth factor-I (IGF-I), with the addition of 100ng/ml PI3K-Akt pathway agonist, labeled as experimental group + IGF-I group. The activation of PI3K-Akt pathway was verified by Western blotting (WB). The expression of AQP9 was detected by RT-q PCR and WB. The recombinant lentivirus LV-AQP9 or empty-loaded virus LV-PWPI was transfected with HepG2 cells by recombinant lentiviral transfection tecnique, and labeled as HepG2-AQP9 and HepG2-PWPI. The transfection efficiency was assessed by confocal laser scanning microscopy and RT-qPCR and WB detected the change of AQP9 expression level after virus transfection. Afterwards, the stable over-expressed HepG2-AQP9 cells and the empty-loaded HepG2-PWPI cells were co-cultured with differentiated mature adipocytes for 48h, and labeled as HepG2-AQP9 co-culture group, and then intracellular triglyceride content were detected with Oil red O staining. Finally, IGF-I was added to the HepG2-AQP9 co-culture group, which was recorded as HepG2-AQP9 co-culture + IGF-I group. Intracellular triglyceride content was detected with Oil red O staining, and WB verified PI3K-Akt signaling pathway activation and changes in AQP9 mRNA and protein levels. A t-test was used to compare the two independent samples. Results: The intracellular lipid droplets and triglyceride content (0.052 ± 0.005) in the experimental group was increased significantly than the control group (0.033 ± 0.003) (t= 5.225,P= 0.006), suggesting that adipocyte co-culture had induced steatosis in HepG2 cells. RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (3.615 ± 0.330) and protein levels (0.072 ± 0.005) in the experimental group were significantly higher than the control group (t= 13.708, 11.225,P= 0.005, < 0.001). WB results showed that the expression level of phosphorylated Akt (p-Akt) protein (0.116±0.003) in the experimental group was significantly lower than the control group (0.202 ± 0.003) (t= 27.136,P< 0.001). The total Akt protein was constant, and the p-Akt/total Akt (0.182 ± 0.017)was significantly lower than the control group (0.327 ± 0.019) (t= 2.431,P= 0.001), suggesting that adipocyte co-culture had inhibited PI3K- Akt signaling pathway in HepG2 cells and up-regulated the expression level of AQP9. WB results indicated that the expression level of p-Akt protein (0.194 ± 0.021) in the experimental group + IGF-I group was significantly higher than the experimental group (0.132 ± 0.003) (t= 5.082,P= 0.007). The total Akt protein was constant, and the p-Akt/total Akt (0.281 ± 0.009) was significantly higher than the control group (0.184 ± 0.132) (t= 10.311,P< 0.001). Simultaneously, RT-qPCR and WB results indicated that the expression levels of AQP9 mRNA (0.327 ± 0.347) and protein levels (0.042 ± 0.004) in the experimental group + IGF-I group were significantly lower than the experimental group (t= 33.573, 5.598,P< 0.001, 0.005), suggesting that adipocyte co-culture had possibility to regulate the expression level of AQP9 through the PI3K-Akt pathway. Confocal laser microscopy analysis showed that the transfection efficiency was more than 90%. RT-q PCR and WB results indicated that the expression levels of AQP9 mRNA and protein levels (0.373 ± 0.221) in HepG2-AQP9 group were significantly higher than HepG2-PWPI group (t=14.953, 28.931,P= 0.002 and 0.000), suggesting that the stable overexpression of AQP9 cell line was successfully constructed. The intracellular lipid droplets and triglyceride content in HepG2-AQP9 co-culture group was significantly increased (t= 5.478, 5.369,P= 0.005) than HepG2-PWPI co-culture group and HepG2-AQP9 co-culture+ IGF-I group, suggesting that the increased expression of AQP9 had promoted HepG2 steatosis in co-cultured adipocytes. WB results showed the expression levels of p-Akt protein (0.168 ± 0.006) and p-Akt/total Akt (0.265±0.009) in HepG2-AQP9 co-culture + IGF-1 group was significantly increased (t= 16.311, 8.769,P< 0.001) than HepG2-AQP9 co-culture group, while the expression levels of AQP9 mRNA (0.327 ± 0.034) and protein (0.375 ± 0.025) was significantly decreased (t= 33.573, 9.146,P< 0.001 and 0.001). Conclusion: Adipocytes co-culture can induce steatosis in HepG2 cells, and may participate in inhibiting PI3K-Akt signaling pathway to upregulate the expression of AQP9 in steatotic HepG2 cells.

Influenza A Virus NS1 Protein Suppresses JNK1-Dependent Autophagosome Formation Mediated by Rab11a Recycling Endosomes.

Autophagy is an essential process for cellular metabolism and homeostasis, but also functions as one of innate immune responses against pathogen infection. However, in contrast to cellular metabolism and homeostasis pathways, less is known about how virus infection leads to autophagosome formation. Here, we showed that influenza A virus NS1 protein inhibits the formation of autophagosomes. The autophagosome formation was induced by infection with NS1 mutant virus lacking the dsRNA-binding activity for inhibition of innate immune responses (R38AK41A) or the activation of PI3K-Akt signaling pathway (Y89F). R38AK41A mutant infection induced phosphorylation of JNK1 and up-regulated the expression of autophagy-related genes which are downstream of JNK1 signaling pathway. We also found that the amount of phosphorylated TSC2, which activates mTOR, increased in wild type-infected cells but not in Y89F mutant-infected cells. These findings suggest that NS1 inhibits the autophagosome formation through both the inhibition of JNK1 and the activation of PI3K-Akt-mTOR pathway. Further, viral ribonucleoprotein (vRNP) complexes were selectively sequestered into autophagosomes, and knockdown of Rab11a, which is responsible for the apical transport of vRNP complexes, impaired not only engulfment of vRNP complexes by autophagosomes but also the formation of autophagosomes in R38AK41A mutant-infected cells. This indicates that Rab11a-positive recycling endosomes function as a donor membrane for the phagophore elongation and an autophagic receptor for the selective engulfment of viral RNP complexes. Based on these results, we propose that NS1 inhibits JNK1-mediated autophagy induction and the sequestration of vRNP complexes into autophagosomes.

Up-Regulation of PI 3-Kinases and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA Hypomethylation Mediated by the Cancer Microenvironment

Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.