PFN4 is required for manchette development and acrosome biogenesis during mouse spermiogenesis.

Profilin 4 (Pfn4) is expressed during spermiogenesis and localizes to the acrosome-acroplaxome-manchette complex. Here, we generated PFN4-deficient mice, with sperm displaying severe impairment in manchette formation. Interestingly, HOOK1 staining suggests that the perinuclear ring is established; however, ARL3 staining is disrupted, suggesting that lack of PFN4 does not interfere with the formation of the perinuclear ring and initial localization of HOOK1, but impedes microtubular organization of the manchette. Furthermore, amorphous head shape and flagellar defects were detected, resulting in reduced sperm motility. Disrupted cis- and trans-Golgi networks and aberrant production of proacrosomal vesicles caused impaired acrosome biogenesis. Proteomic analysis showed that the proteins ARF3, SPECC1L and FKBP1, which are involved in Golgi membrane trafficking and PI3K/AKT pathway, are more abundant in Pfn4-/- testes. Levels of PI3K, AKT and mTOR were elevated, whereas AMPK level was reduced, consistent with inhibition of autophagy. This seems to result in blockage of autophagic flux, which could explain the failure in acrosome formation. In vitro fertilization demonstrated that PFN4-deficient sperm is capable of fertilizing zona-free oocytes, suggesting a potential treatment for PFN4-related human infertility.

Newcastle disease virus induces clathrin-mediated endocytosis to establish infection through the activation of PI3K/AKT signaling pathway by VEGFR2.

The phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/ AKT) signaling pathway constitutes a classical phosphorylation cascade that integrates tyrosine, lipid, and serine acid-threonine phosphorylation, affecting cell function. The pathway is vulnerable to viral infection. Newcastle disease virus (NDV) poses a significant threat to the global poultry industry; however, its mechanism of early viral cell invasion and pathogenesis remain unclear. Previous in vivo and in vitro studies have shown that NDV infection activates PI3K/AKT signaling; however, it remains unclear whether NDV establishes infection through endocytosis regulated by this pathway. This study aimed to examine whether different genotypes of NDV strains could activate the PI3K/AKT signaling pathway within 2 h of in vitro infection. This activation, which relies on PI3K phosphorylation, remains unaffected by the phosphorylation-phosphatase and tensin homolog/phosphatase and tensin homolog (p-PTEN/PTEN) signaling pathway. Moreover, inhibition of PI3K activity impedes NDV replication. Additionally, interfering with the PI3K regulatory subunit p85 has no significant effect on NDV replication. Conversely, the tyrosine kinase activity upstream of PI3K can influence AKT activation and viral replication, particularly through vascular endothelial growth factor receptor 2 (VEGFR2). Additionally, NDV F protein primarily mediates PI3K and AKT phosphorylation to activate the PI3K/AKT signaling pathway. NDV F and VEGFR2 proteins, along with the PI3K p85α subunit, interact and co-localize at the cell membrane. NDV-induced PI3K/AKT signaling pathway activation impacts clathrin-mediated endocytosis, with VEGFR2 playing a pivotal role. In conclusion, this study shows that NDV infection is established early through F protein binding to VEGFR2, activating the PI3K/AKT signaling pathway and inducing clathrin-mediated endocytosis, supporting infection prevention and control measures. IMPORTANCE: Newcastle disease virus (NDV) is a threat to the global poultry industry; however, the mechanisms of NDV infection remain unclear. NDV affects the phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/ AKT) signaling pathway, requiring endocytosis for successful infection. Based on previous studies, we identified a close correlation between NDV infection and replication and the PI3K/AKT signaling pathway activity. This study examined the molecular mechanisms through which NDV activates the PI3K/AKT signaling pathway to regulate endocytosis and facilitate infection. This study showed that early-stage in vitro NDV infection activated the PI3K/AKT signaling pathway, enhancing clathrin-mediated endocytosis, crucial for infection onset. Notably, this process involves the interaction between NDV F protein and the vascular endothelial growth factor receptor 2 tyrosine kinase, leading to the subsequent binding and phosphorylation of the PI3K p85α regulatory subunit. This activation primes PI3K, initiating a cascade that promotes clathrin-mediated endocytosis. Our findings elucidate how NDV capitalizes on the PI3K/AKT signaling pathway to establish infection through endocytosis.

Single-cell RNA sequencing reveals vascularization-associated cell subpopulations in dental pulp: PDGFRß+ DPSCs with activated PI3K/AKT pathway.

BACKGROUND: This study aims to address challenges in dental pulp regeneration therapy. The heterogeneity of DPSCs poses challenges, especially in stem cell transplantation for clinical use, particularly when sourced from donors of different ages and conditions. METHODS: Pseudotime analysis was employed to analyze single-cell sequencing data, and immunohistochemical studies were conducted to investigate the expression of fibronectin 1 (FN1). We performed in vitro sorting of PDGFRß+ DPSCs using flow cytometry. A series of functional assays, including cell proliferation, scratch, and tube formation assays, were performed to experimentally validate the vasculogenic capabilities of the identified PDGFRß+ DPSC subset. Furthermore, gene-edited mouse models were utilized to demonstrate the importance of PDGFRß+ DPSCs. Transcriptomic sequencing was conducted to compare the differences between PDGFRß+ DPSCs and P1-DPSCs. RESULTS: Single-cell sequencing analysis unveiled a distinct subset, PDGFRß+ DPSCs, characterized by significantly elevated FN1 expression during dental pulp development. Subsequent cell experiments demonstrated that this subset possesses remarkable abilities to promote HUVEC proliferation, migration, and tube formation. Gene-edited mouse models confirmed the vital role of PDGFRß+ DPSCs in dental pulp development. Transcriptomic sequencing and in vitro experiments demonstrated that the PDGFR/PI3K/AKT signaling pathway is a crucial factor mediating the proliferation rate and pro-angiogenic properties of PDGFRß+ DPSCs. CONCLUSION: We defined a new subset, PDGFRß+ DPSCs, characterized by strong proliferative activity and pro-angiogenic capabilities, demonstrating significant clinical translational potential.

Deep learning-based pathway-centric approach to characterize recurrent hepatocellular carcinoma after liver transplantation.

BACKGROUND: Liver transplantation (LT) is offered as a cure for Hepatocellular carcinoma (HCC), however 15-20% develop recurrence post-transplant which tends to be aggressive. In this study, we examined the transcriptome profiles of patients with recurrent HCC to identify differentially expressed genes (DEGs), the involved pathways, biological functions, and potential gene signatures of recurrent HCC post-transplant using deep machine learning (ML) methodology. MATERIALS AND METHODS: We analyzed the transcriptomic profiles of primary and recurrent tumor samples from 7 pairs of patients who underwent LT. Following differential gene expression analysis, we performed pathway enrichment, gene ontology (GO) analyses and protein-protein interactions (PPIs) with top 10 hub gene networks. We also predicted the landscape of infiltrating immune cells using Cibersortx. We next develop pathway and GO term-based deep learning models leveraging primary tissue gene expression data from The Cancer Genome Atlas (TCGA) to identify gene signatures in recurrent HCC. RESULTS: The PI3K/Akt signaling pathway and cytokine-mediated signaling pathway were particularly activated in HCC recurrence. The recurrent tumors exhibited upregulation of an immune-escape related gene, CD274, in the top 10 hub gene analysis. Significantly higher infiltration of monocytes and lower M1 macrophages were found in recurrent HCC tumors. Our deep learning approach identified a 20-gene signature in recurrent HCC. Amongst the 20 genes, through multiple analysis, IL6 was found to be significantly associated with HCC recurrence. CONCLUSION: Our deep learning approach identified PI3K/Akt signaling as potentially regulating cytokine-mediated functions and the expression of immune escape genes, leading to alterations in the pattern of immune cell infiltration. In conclusion, IL6 was identified to play an important role in HCC recurrence.

RAF1 gene fusions are recurrent driver events in infantile fibrosarcoma-like mesenchymal tumors.

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.

Cervical cancer cell-derived Tie1 expression via PI3K/AKT signaling pathway promotes tumor progression.

BACKGROUND: Tie1 orphan receptor has become a focus of research, Tie1 can form a polymer with Tie2, regulate the Ang/Tie2 pathway and play a vital role in pathological angiogenesis and tumor progression, the function of Tie1 has remained uncertain in the progression of cervical cancer (CC). Here, we investigated the functional influences of Tie1 overexpress on CC in vitro and in vivo. METHODS: We used Immunohistochemistry (IHC) analysis to detect the relative expression of Tie1 in CC, and we analyzed its connection with the overall survival (OS) and progression free survival (PFS)of CC patients. To prove the role of Tie1 in cell proliferation and metastatic, Tie1 expression in CC cell lines was upregulated by lentivirus. RESULTS: The high expression of Tie1 in tumor cells of cervical cancer tissues is significantly correlated with FIGO stage, differentiated tumors, tumors with diameters, deep stromal invasion. We found that cell progression was promoted in Tie1-overexpress CC cell lines in vivo and in vitro. Tie1 potentially exerts a commanding influence on the expression of markers associated with epithelial-mesenchymal transition (EMT) and the PI3K/AKT signaling pathway. CONCLUSIONS: Our research indicates that Tie1 is highly connected to CC progression as it may play a role in the EMT process through the PI3K/AKT signaling pathway.

PANX2 promotes malignant transformation of colorectal cancer and 5-Fu resistance through PI3K-AKT signaling pathway.

Colorectal cancer (CRC) is the third deadliest cancer in the world, with a high incidence, aggressiveness, poor prognosis, and resistant to drugs. 5-fluorouracil (5-FU) is the most commonly used drug for the chemotherapeutic of CRC, however, CRC is resistant to 5-FU after a period of treatment. Therefore, there is an urgent need to explore the underlying molecular mechanisms of CRC resistance to 5-FU. In the present study, we found that the expression of PANX2 was increased in CRC tissues and metastatic tissues from the TCGA database. The K-M survival curve showed that the high expression of PANX2 was associated with poor cancer prognosis. GDSC database showed that the IC50 of 5-Fu in the PANX2 high expression group was significantly higher, and the results were verified in CRC cells. In vitro cell function and in vivo tumorigenesis experiments showed that PANX2 promoted CRC cell proliferation, clone formation, migration and tumorigenesis in vivo. WB result revealed that PANX2 may lead to resistance to 5-Fu in CRC by affecting the PI3K-AKT signaling pathway. Overall, PANX2 regulates CRC proliferation, clone formation, migration, and 5-Fu resistance by PI3K-AKT signaling pathway.

Identification of memory mechanism in tissue-resident stem cells via ANGPTL4 beyond immune cells upon viral antigen exposure.

Although memory functions of immune cells characterized by increased resistance to subsequent infections after initial pathogen exposure are well-established, it remains unclear whether non-immune cells, especially tissue-resident stem cells, exhibit similar memory mechanisms. The present study revealed that detrimental effects of initial viral antigen exposure (human papillomavirus [HPV]) on diverse stem cell functions were significantly exacerbated upon subsequent secondary exposure both in vitro and in vivo. Importantly, endometrial stem cells exhibited robust memory functions following consecutive HPV antigen exposures, whereas fully differentiated cells such as fibroblasts and vesicular cells did not show corresponding changes in response to the same antigen exposures. Deficiency of angiopoietin-like 4 (ANGPTL4) achieved through small hairpin RNA knockdown in vitro and knockout (KO) mice in vivo highlighted the critical role of ANGPTL4 in governing memory functions associated with various stem cell processes. This regulation occurred through histone H3 methylation alterations and PI3K/Akt signaling pathways in response to successive HPV antigen exposures. Furthermore, memory functions associated with various stem cell functions that were evident in wild-type mice following consecutive exposures to HPV antigen were not observed in ANGPTL4 KO mice. In summary, our findings strongly support the presence of memory mechanism in non-immune cells, particularly tissue-resident stem cells.

PEBP4 deficiency aggravates LPS-induced acute lung injury and alveolar fluid clearance impairment via modulating PI3K/AKT signaling pathway.

Acute lung injury (ALI) is a common clinical syndrome, which often results in pulmonary edema and respiratory distress. It has been recently reported that phosphatidylethanolamine binding protein 4 (PEBP4), a basic cytoplasmic protein, has anti-inflammatory and hepatoprotective effects, but its relationship with ALI remains undefined so far. In this study, we generated PEBP4 knockout (KO) mice to investigate the potential function of PEBP4, as well as to evaluate the capacity of alveolar fluid clearance (AFC) and the activity of phosphatidylinositide 3-kinases (PI3K)/serine-theronine protein kinase B (PKB, also known as AKT) signaling pathway in lipopolysaccharide (LPS)-induced ALI mice models. We found that PEBP4 deficiency exacerbated lung pathological damage and edema, and increased the wet/dry weight ratio and total protein concentration of bronchoalveolar lavage fluid (BALF) in LPS-treated mice. Meanwhile, PEBP4 KO promoted an LPS-induced rise in the pulmonary myeloperoxidase (MPO) activity, serum interleuin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels, and pulmonary cyclooxygenase-2 (COX-2) expression. Mechanically, PEBP4 deletion further reduced the protein expression of Na+ transport markers, including epithelial sodium channel (ENaC)-α, ENaC-γ, Na,K-ATPase α1, and Na,K-ATPase ß1, and strengthened the inhibition of PI3K/AKT signaling in LPS-challenged mice. Furthermore, we demonstrated that selective activation of PI3K/AKT with 740YP or SC79 partially reversed all of the above effects caused by PEBP4 KO in LPS-treated mice. Altogether, our results indicated the PEBP4 deletion has a deterioration effect on LPS-induced ALI by impairing the capacity of AFC, which may be achieved through modulating the PI3K/AKT pathway.

TIMP1 promotes thyroid cancer cell progression through macrophage phenotypic polarization via the PI3K/AKT signaling pathway.

Increasing evidence suggests that tissue inhibitor of metalloproteinase 1 (TIMP1) played a pivotal role in immune regulation. Our study focused on examining the expression and function of TIMP1 in humans, particularly in its regulation of tumor-associated macrophages (TAMs) in papillary thyroid carcinoma (PTC). We observed an upregulation of TIMP1 in 16 different types of malignancies, including thyroid cancer. TIMP1 shaped the inflammatory TME in PTC. Inhibiting the expression of TIMP1 has been demonstrated to reduce the malignant biological traits of PTC cells. Furthermore, reducing TIMP1 expression impeded M2 macrophage polarization as well as facilitated M1 macrophage polarization in PTC. ELISA results demonstrated that downregulated TIMP1 expression correlated with decreased levels of IL10 and TGF-ß in cell supernatants. Furthermore, the supernatant from polarized macrophages in the TIMP1-silenced group inhibited the motility of wild-type PTC cells. Therefore, TIMP1 may enhance the progression of PTC by stimulating the PI3K/AKT pathway via the secretion of IL10 and TGF-ß, consequently influencing M2-type polarization in TAMs.

Network Pharmacology Analysis, Molecular Docking Integrated Experimental Verification Reveal the Mechanism of Gynostemma pentaphyllum in the Treatment of Type II Diabetes by Regulating the IRS1/PI3K/Akt Signaling Pathway.

Gynostemma pentaphyllum (Thunb.) Makino (GP), a plant with homology of medicine and food, as a traditional Chinese medicine, possesses promising biological activities in the prevention and treatment of type 2 diabetes mellitus (T2DM). However, the material basis and the mechanism of action of GP in the treatment of T2DM have not been fully elucidated. This study aimed to clarify the active components, potential targets and signaling pathways of GP in treating T2DM. The chemical ingredients of GP were collected by combining UPLC-HRMS analysis and literature research. Network pharmacology revealed that GP had 32 components and 326 potential targets in treating T2DM. The results showed that GP affected T2DM by mediating the insulin resistance signaling pathway, PI3K/Akt signaling pathway and FoxO1 signaling pathway, which had a close relationship with T2DM. Molecular docking results showed that STAT3, PIK3CA, AKT1, EGFR, VEGFA and INSR had high affinity with the active compounds of GP. In vitro, GP extracts obviously increased the glucose uptake and glucose consumption in IR-HepG2 cells. GP extracts increased the levels of PI3K, p-AKT, p-GSK3ß and p-FoxO1 and decreased the expression of p-IRS1, p-GS, PEPCK and G6Pase, which indicated that GP could promote glycogen synthesis and inhibit gluconeogenesis by regulating the IRS1/PI3K/Akt signaling pathway. The results demonstrated that GP could improve insulin resistance by promoting glucose uptake and glycogen synthesis and inhibiting gluconeogenesis through regulating the IRS1/PI3K/Akt signaling pathway, which might be a potential alternative therapy for T2DM.

P2RY6 deletion promotes UVB-induced skin carcinogenesis by activating the PI3K/AKT signal pathway.

Our previous research has demonstrated that P2RY6 functions as an oncogene in DMBA/TPA-induced two-stage chemical skin carcinogenesis in mice. However, considering that human skin cancer is predominantly attributed to UV radiation from sunlight, additional investigations are needed to elucidate the role of P2RY6 in UVB-induced skin carcinogenesis. Surprisingly, we found that P2ry6-deficient mice exhibited marked promotion to UVB-induced skin papilloma formation compared with wild-type mice, suggesting its tumor-suppressive role in UVB-induced skin cancer. Additionally, a P2ry6 gene knockout promoted skin hyperplasia induced by short-term UVB irradiation, while UDP, the ligand of P2RY6, could inhibit the short-term UVB-induced increase of epiderma thickness in mouse skin. Furthermore, UVB irradiation could significantly upregulate P2RY6 expression in human and mouse skin cells. These results indicated that P2RY6 may play a crucial protective role in resisting the UVB-induced formation of skin tumors. At the molecular level, the loss of the P2RY6 gene inhibits the ubiquitination modification and expression of XPC after UVB irradiation in skin keratinocytes, resulting in the accumulation of CPDs (cyclobutane pyrimidine dimers). We have also demonstrated that P2RY6 deletion activates the PI3K/AKT signaling pathway both in vitro and in vivo. The CPD accumulation and acute inflammatory response enhanced by the loss of the P2RY6 gene can be reversed by an AKT inhibitor. These findings suggest that P2RY6 may act as a tumor suppressor in UVB-induced skin cancer by regulating the PI3K/AKT signaling pathway.

TBC1D10B promotes tumor progression in colon cancer via PAK4­mediated promotion of the PI3K/AKT/mTOR pathway.

This study aimed to explore the expression, function, and mechanisms of TBC1D10B in colon cancer, as well as its potential applications in the diagnosis and treatment of the disease.The expression levels of TBC1D10B in colon cancer were assessed by analyzing the TCGA and CCLE databases. Immunohistochemistry analysis was conducted using tumor and adjacent non-tumor tissues from 68 colon cancer patients. Lentiviral infection techniques were employed to silence and overexpress TBC1D10B in colon cancer cells. The effects on cell proliferation, migration, and invasion were evaluated using CCK-8, EDU, wound healing, and Transwell invasion assays. Additionally, GSEA enrichment analysis was used to explore the association of TBC1D10B with biological pathways related to colon cancer. TBC1D10B was significantly upregulated in colon cancer and closely associated with patient prognosis. Silencing of TBC1D10B notably inhibited proliferation, migration, and invasion of colon cancer cells and promoted apoptosis. Conversely, overexpression of TBC1D10B enhanced these cellular functions. GSEA analysis revealed that TBC1D10B is enriched in the AKT/PI3K/mTOR signaling pathway and highly correlated with PAK4. The high expression of TBC1D10B in colon cancer is associated with poor prognosis. It influences cancer progression by regulating the proliferation, migration, and invasion capabilities of colon cancer cells, potentially acting through the AKT/PI3K/mTOR signaling pathway. These findings provide new targets and therapeutic strategies for the treatment of colon cancer.

GPC2 promotes prostate cancer progression via MDK-mediated activation of PI3K/AKT signaling pathway.

Prostate cancer is a major medical problem for men worldwide. Advanced prostate cancer is currently incurable. Recently, much attention was paid to the role of GPC2 in the field of oncology. Nevertheless, there have been no investigations of GPC2 and its regulatory mechanism in prostate cancer. Here, we revealed a novel action of GPC2 and a tumor promoting mechanism in prostate cancer. GPC2 was upregulated in prostate cancer tissues and cell lines. Higher expression of GPC2 was correlated with higher Gleason score, lymphatic metastasis, and worse overall survival in prostate cancer patients. Decreased expression of GPC2 inhibited cell proliferation, migration, and invasion in prostate cancer, whereas GPC2 overexpression promoted these properties. Mechanistically, GPC2 promoted the activation of PI3K/AKT signaling pathway through MDK. The rescue assay results in prostate cancer cells demonstrated that overexpression of MDK could attenuate GPC2 knockdown induced inactivation of PI3K/AKT signaling and partly reverse GPC2 knockdown induced inhibition of cell proliferation, migration, and invasion. In all, our study identified GPC2 as an oncogene in prostate cancer. GPC2 promoted prostate cancer cell proliferation, migration, and invasion via MDK-mediated activation of PI3K/AKT signaling pathway. GPC2 might be a promising prognosis predictor and potential therapeutic target in prostate cancer.

Fluvoxamine inhibits Th1 and Th17 polarization and function by repressing glycolysis to attenuate autoimmune progression in type 1 diabetes.

BACKGROUND: Fluvoxamine is one of the selective serotonin reuptake inhibitors (SSRIs) that are regarded as the first-line drugs to manage mental disorders. It has been also recognized with the potential to treat inflammatory diseases and viral infection. However, the effect of fluvoxamine on autoimmune diseases, particularly type 1 diabetes (T1D) and the related cellular and molecular mechanisms, are yet to be addressed. METHOD: Herein in this report, we treated NOD mice with fluvoxamine for 2 weeks starting from 10-week of age to dissect the impact of fluvoxamine on the prevention of type 1 diabetes. We compared the differences of immune cells between 12-week-old control and fluvoxamine-treated mice by flow cytometry analysis. To study the mechanism involved, we extensively examined the characteristics of CD4+ T cells with fluvoxamine stimulation using RNA-seq analysis, real-time PCR, Western blot, and seahorse assay. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. RESULT: Fluvoxamine not only delayed T1D onset, but also decreased T1D incidence. Moreover, fluvoxamine-treated NOD mice showed significantly attenuated insulitis coupled with well-preserved ß cell function, and decreased Th1 and Th17 cells in the peripheral blood, pancreatic lymph nodes (PLNs), and spleen. Mechanistic studies revealed that fluvoxamine downregulated glycolytic process by inhibiting phosphatidylinositol 3-kinase (PI3K)-AKT signaling, by which it restrained effector T (Teff) cell differentiation and production of proinflammatory cytokines. CONCLUSION: Collectively, our study supports that fluvoxamine could be a viable therapeutic drug against autoimmunity in T1D setting.

The combination of SHP099 inhibits the malignant biological behavior of L-OHP/5-FU-resistant colorectal cancer cells by regulating energy metabolism reprogramming.

BACKGROUND AND OBJECTIVE: Colorectal cancer (CRC) is one of the most common malignancies in China. At present, there is a problem that the CRC treatment drugs SHP099, L-OHP and 5-FU are insensitive to tumor cells. Combination medication is an important means to solve the insensitivity of medication alone. The purpose of this project was to explore the effect and molecular mechanism of SHP099 combination on the malignant biological behavior of L-OHP/5-FU resistant strains of CRC. METHODS: HT29 and SW480 cells were cultured in media supplemented with L-OHP or 5-FU to establish drug-resistant strains. HT29 and SW480 drug-resistant cells were subcutaneously injected into the ventral nerves of nude mice at a dose of 5 × 106 to establish CRC drug-resistant animal models. CCK-8, Western blot, flow cytometry, Transwell and kit detection were used to detect the regulatory mechanism of energy metabolism reprogramming in drug-resistant CRC cells. RESULTS: Compared with nonresistant strains, L-OHP/5-FU-resistant strains exhibited greater metabolic reprogramming. Functionally, SHP099 can restrain the metabolic reprogramming of L-OHP/5-FU-resistant strains and subsequently restrain the proliferation, colony formation, migration and spheroid formation of L-OHP/5-FU-resistant strains. Downstream mechanistic studies have shown that SHP099 interferes with the metabolic reprogramming of L-OHP/5-FU drug-resistant strains by suppressing the PI3K/AKT pathway, thereby restraining the malignant biological behavior of L-OHP/5-FU drug-resistant strains and alleviating CRC. CONCLUSION: The combination of SHP099 can restrain the malignant biological behavior of L-OHP/5-FU-resistant CRC cells and alleviate the progression of CRC by interfering with the reprogramming of energy metabolism. This study explored the effect of SHP099 combination on dual-resistant CRC cells for the first time, and provided a new therapeutic idea for solving the problem of SHP099 insensitivity to CRC cells.

Exploring the role of iNOS in HFpEF-Related myocardial fibrosis: Involvement of PTEN-PI3K/AKT signaling pathway.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a challenging condition to treat with myocardial fibrosis being a pivotal pathological component. Previous studies have suggested a role for inducible nitric oxide synthase (iNOS) in the progression of this condition, but the precise mechanisms remain unclear. This study aimed to investigate the role of iNOS in HFpEF-related myocardial fibrosis and identify potential therapeutic targets. METHODS: A 'two-hit' mouse model of HFpEF was established, and echocardiography, histopathology and biochemical analyses were performed. In vitro experiments were conducted in mouse cardiac fibroblasts, with iNOS overexpression and application of iNOS or phosphatidylinositol 3 kinase (PI3K) inhibitors. The iNOS-S-nitrosylated phosphatase and TENsin homolog (SNO-PTEN)-phosphorylated-protein kinase B (p-AKT) pathway was investigated, along with the effects on fibrotic markers and cell proliferation and migration. RESULTS: HFpEF mice exhibited significant cardiac dysfunction and fibrosis, with increased expression of iNOS, SNO-PTEN, and p-AKT, indicative of the activation of the iNOS-SNO-PTEN-p-AKT pathway. iNOS overexpression in mouse cardiac fibroblasts led to increased SNO-PTEN, decreased PTEN, activated phosphorylated PI3K (p-PI3K) and p-AKT, and enhanced cell proliferation and migration, as well as increased collagen I and III expression. The use of an iNOS inhibitor (L-NIL) or a PI3K inhibitor (LY294002) partially reversed these changes. CONCLUSION: Our findings suggest that the iNOS-SNO-PTEN-p-AKT pathway may play a crucial role in HFpEF-related myocardial fibrosis, with iNOS and PI3K inhibitors offering potential therapeutic benefits. These insights may pave the way for the development of effective drug therapies for HFpEF.

Downregulation of Setdb2 promotes alternative activation of macrophages via the PI3K/Akt pathway to attenuate NAFLD after sleeve gastrectomy.

Non-alcoholic fatty liver disease (NAFLD) stands as the most prevalent hepatic disorder, with bariatric surgery emerging as the most effective intervention for NAFLD remission. Sleeve gastrectomy (SG) has notably ascended as the predominant procedure due to its comparative simplicity and consistent surgical outcomes. Nonetheless, the underlying mechanisms remain unclear. In this study, we probed the therapeutic potential of SG for NAFLD induced by a high-fat diet (HFD) in mice, with a focus on its impact on liver lipid accumulation, macrophage polarization, and the role of the histone methyltransferase Setdb2. SG prompted significant weight loss, diminished liver size and liver-to-body weight ratio, and enhanced liver function, evidenced by reduced serum levels of triglycerides (TG), total cholesterol (T-CHO), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Histological examination confirmed a reduction in liver lipid accumulation. Additionally, flow cytometry unveiled an increased proportion of M2 macrophages and a decrease in Setdb2 expression was shown in the SG group, suggesting an association between Setdb2 levels and postsurgical macrophage polarization. Furthermore, the conditional knockout of Setdb2 in mice further mitigated HFD-induced steatosis and promoted the M2 macrophage phenotype. Mechanistically, Setdb2 knockout in bone marrow-derived macrophages (BMDMs) favored M2 polarization, with RNA sequencing and western blotting analyses corroborating the upregulation of the PI3K/Akt signaling pathway. The effects of Setdb2 on macrophage activation were nullified by the PI3K inhibitor LY294002, suggesting that Setdb2 facilitates alternative macrophage activation through the PI3K/Akt signaling pathway. These comprehensive findings underscore the potential of SG as a therapeutic intervention for NAFLD by regulating the critical function of Setdb2 in macrophage polarization and activation, thereby offering novel insights into NAFLD pathogenesis and therapeutic targets.

METTL14 mediates nerve growth factor-stimulated testosterone synthesis in porcine theca cells†.

Ovarian theca cells produce testosterone, which acts as a vital precursor substance for synthesizing estrogens during follicular development. Nerve growth factor (NGF) has been shown to participate in reproductive physiology, specifically to follicular development and ovulation. There is currently no available data on the impact of NGF on testosterone synthesis in porcine theca cells. Furthermore, m6A modification is the most common internal modification in eukaryotic mRNAs that are closely associated with female gametogenesis, follicle development, ovulation, and other related processes. It is also uncertain whether the three main enzymes associated with m6A, such as Writers, Erasers, and Readers, play a role in this process. The present study, with an in vitro culture model, investigated the effect of NGF on testosterone synthesis in porcine theca cells and the role of Writers-METTL14 in this process. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells. This study will help to further elucidate the mechanisms by which NGF regulates follicular development and provide new therapeutic targets for ovary-related diseases in female animals. Summary Sentence  The present study investigated the effect of NGF on testosterone synthesis in porcine theca cells. It was found that NGF activates the PI3K/AKT signaling pathway through METTL14, which regulates testosterone synthesis in porcine theca cells.

Metabolomics combined with network pharmacology reveals a role for astragaloside IV in inhibiting enterovirus 71 replication via PI3K-AKT signaling.

BACKGROUND: Astragaloside IV (AST-IV), as an effective active ingredient of Astragalus membranaceus (Fisch.) Bunge. It has been found that AST-IV inhibits the replication of dengue virus, hepatitis B virus, adenovirus, and coxsackievirus B3. Enterovirus 71 (EV71) serves as the main pathogen in severe hand-foot-mouth disease (HFMD), but there are no specific drugs available. In this study, we focus on investigating whether AST-IV can inhibit EV71 replication and explore the potential underlying mechanisms. METHODS: The GES-1 or RD cells were infected with EV71, treated with AST-IV, or co-treated with both EV71 and AST-IV. The EV71 structural protein VP1 levels, the viral titers in the supernatant were measured using western blot and 50% tissue culture infective dose (TCID50), respectively. Network pharmacology was used to predict possible pathways and targets for AST-IV to inhibit EV71 replication. Additionally, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used to investigate the potential targeted metabolites of AST-IV. Associations between metabolites and apparent indicators were performed via Spearman's algorithm. RESULTS: This study illustrated that AST-IV effectively inhibited EV71 replication. Network pharmacology suggested that AST-IV inhibits EV71 replication by targeting PI3K-AKT. Metabolomics results showed that AST-IV achieved these effects by elevating the levels of hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-hydroxy-1 H-indole-3- acetamide, oxypurinol, while reducing the levels of PC (14:0/15:0). Furthermore, AST-IV also mitigated EV71-induced oxidative stress by reducing the levels of MDA, ROS, while increasing the activity of T-AOC, CAT, GSH-Px. The inhibition of EV71 replication was also observed when using the ROS inhibitor N-Acetylcysteine (NAC). Additionally, AST-IV exhibited the ability to activate the PI3K-AKT signaling pathway and suppress EV71-induced apoptosis. CONCLUSION: This study suggests that AST-IV may activate the cAMP and the antioxidant stress response by targeting eight key metabolites, including hypoxanthine, 2-ketobutyric acid, adenine, nicotinic acid mononucleotide, prostaglandin H2, 6-Hydroxy-1 H-indole-3-acetamide, oxypurinol and PC (14:0/15:0). This activation can further stimulate the PI3K-AKT signaling to inhibit EV71-induced apoptosis and EV71 replication.