Identification of SSBP1 as a ferroptosis-related biomarker of glioblastoma based on a novel mitochondria-related gene risk model and in vitro experiments.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor that leads to lethality. Several studies have demonstrated that mitochondria play an important role in GBM and that mitochondria-related genes (MRGs) are potential therapeutic targets. However, the role of MRGs in GBM remains unclear. METHODS: Differential expression and univariate Cox regression analyses were combined to screen for prognostic differentially-expressed (DE)-MRGs in GBM. Based on LASSO Cox analysis, 12 DE-MRGs were selected to construct a risk score model. Survival, time dependent ROC, and stratified analyses were performed to evaluate the performance of this risk model. Mutation and functional enrichment analyses were performed to determine the potential mechanism of the risk score. Immune cell infiltration analysis was used to determine the association between the risk score and immune cell infiltration levels. CCK-8 and transwell assays were performed to evaluate cell proliferation and migration, respectively. Mitochondrial reactive oxygen species (ROS) levels and morphology were measured using a confocal laser scanning microscope. Genes and proteins expression levels were investigated by quantitative PCR and western blotting, respectively. RESULTS: We identified 21 prognostic DE-MRGs, of which 12 DE-MRGs were selected to construct a prognostic risk score model for GBM. This model presented excellent performance in predicting the prognosis of patients with GBM and acted as an independent predictive factor. Functional enrichment analysis revealed that the risk score was enriched in the inflammatory response, extracellular matrix, and pro-cancer-related and immune related pathways. Additionally, the risk score was significantly associated with gene mutations and immune cell infiltration in GBM. Single-stranded DNA-binding protein 1 (SSBP1) was considerably upregulated in GBM and associated with poor prognosis. Furthermore, SSBP1 knockdown inhibited GBM cell progression and migration. Mechanistically, SSBP1 knockdown resulted in mitochondrial dysfunction and increased ROS levels, which, in turn, increased temozolomide (TMZ) sensitivity in GBM cells by enhancing ferroptosis. CONCLUSION: Our 12 DE-MRGs-based prognostic model can predict the GBM patients prognosis and 12 MRGs are potential targets for the treatment of GBM. SSBP1 was significantly upregulated in GBM and protected U87 cells from TMZ-induced ferroptosis, which could serve as a prognostic and therapeutic target/biomarker for GBM.

Tofacitinib enhances IGF1 via inhibiting STAT6 transcriptionally activated-miR-425-5p to ameliorate inflammation in RA-FLS.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, which has been reported closely associated with the dysfunction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. This study aims to explore the potential therapeutic effect of Tofacitinib, a putative JAK/STAT inhibitor, in RA. Tofacitinib suppressed proliferation and accelerated apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) as confirmed by CCK-8, EdU and Western blot assays. Tofacitinib significantly inhibited expression of pro-inflammatory factors including tumor necrosis factor-α (TNF-α), vascular endothelial growth factor A, matrix metalloproteinase 1, matrix metalloproteinase 3, interleukin-6 and interferon gamma in RA-FLS cells. mechanistically, tofacitinib decreased signal transducer and activator of transcription 6 (STAT6), which transcriptionally activates miR-425-5p, and thus increased insulin like growth factor 1 (IGF1) expression, a target of miR-425-5p in RA-FLS. Overexpression of STAT6 restored the expression of pro-inflammatory factors and proliferation inhibited by Tofacitinib in RA-FLS. Overall, Tofacitinib exerted inhibitory effect on proliferation and inflammation of RA-FLS through modulating STAT6/miR-425-5p/IGF1 signal axis. These findings shed light on the novel strategies for improving RA.

Duvira Antarctic polysaccharide inhibited H1N1 influenza virus-induced apoptosis through ROS mediated ERK and STAT-3 signaling pathway.

BACKGROUND: The H1N1 influenza virus causes acute respiratory tract infection, and its clinical symptoms are very similar to those of ordinary influenza. The disease develops rapidly. If the flu is not treated, complications such as pneumonia, respiratory failure, and multiple organ damage can occur, resulting in a high fatality rate. Influenza virus mutates rapidly. At present, there is no specific drug for H1N1, so it is an urgent need for clinical care to find new drugs to treat H1N1. MATERIALS AND METHODS: The polysaccharide derived from Durvillaea Antarctica green algae has a certain antiviral effect. In this study, the results of CCK-8, apoptosis cycle detection, JC-1 and Western blotting proved that Duvira Antarctic polysaccharide (DAPP) has the ability to inhibit H1N1 infection. RESULTS: CCK-8 test showed that the DAPP with concentration at 32 µg/mL had no toxicity to MDCK cells. In addition, DAPP reduced cell apoptosis by inhibiting the ERK signaling pathway. Meanwhile, DAPP could increase the expression of STAT3 and significantly inhibited proinflammatory cytokines. CONCLUSIONS: In summary, these results suggested that DAPP may be potential with the ability to resist the H1N1 influenza virus.

C-Phycocyanin Suppresses Cell Proliferation and Promotes Apoptosis by Regulating the AMPK Pathway in NCL-H292 Non-Small Cell Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) results in high mortality and has gained increasing attention. C-Phycocyanin (C-PC) has been identified as a potential therapeutic inhibitor for NSCLC, but its underlying mechanism remains obscure. The gene expression of the long noncoding RNA neighbour of BRCAI RNA 2 (NBR2) in NSCLC cells was evaluated by quantitative reverse transcription-PCR. The cell capacity for proliferation and migration was examined by EdU and wound-healing assays. Furthermore, the viability and apoptosis of cells was measured with CCK-8 and annexin V/PI, respectively. Next, the protein level of activation of adenosine monophosphate- activated protein kinase and the rapamycin kinase (mTOR) signalling pathway-associated molecules was evaluated by western blotting. H292 cells were pre-treated with C-PC or transfected with plasmids encoding NBR2 or the shNBR2 plasmid, to over-express or knock down NBR2 expression, respectively. NBR2 expression was robustly down-regulated in NSCLC cell lines compared with a normal cell line (BEAS-2B). NBR2 over-expression inhibited migration and promoted apoptosis of H292 cells. Treatment of H292 cells with C-PC enhanced NBR2 levels in a dose- and time-dependent manner. Downregulation of NBR2 in H292 cells inhibited the activity of C-PC on cell proliferation, viability and clone formation. Further mechanistic investigation showed that the down-regulation of NBR2 abolished the modulatory effects of C-PC on the AMPK/mTOR signalling pathway. In conclusion, C-PC inhibits H292 cell growth by enhancing the NBR2/AMPK signalling pathway.

(pGlu-Gln)-CCK-8[mPEG]: a novel, long-acting, mini-PEGylated cholecystokinin (CCK) agonist that improves metabolic status in dietary-induced diabetes.

BACKGROUND: Cholecystokinin (CCK) is a gastrointestinal hormone that has been proposed as a potential therapeutic option for obesity-diabetes. As such, (pGlu-Gln)-CCK-8 is an N-terminally modified CCK-8 analogue with improved biological effectiveness over the native peptide. METHODS: The current study has examined the in vitro stability, biological activity and in vivo therapeutic applicability of a novel second generation mini-PEGylated form of (pGlu-Gln)-CCK-8, (pGlu-Gln)-CCK-8[mPEG]. RESULTS: (pGlu-Gln)-CCK-8[mPEG] was completely resistant to enzymatic degradation and in addition displayed similar insulinotropic (p<0.05 to p<0.001) and satiating effects (p<0.01 to p<0.001) as (pGlu-Gln)-CCK-8. This confirmed the capability of (pGlu-Gln)-CCK-8[mPEG] to bind to and activate the CCK receptor. Sub-chronic twice daily injection of (pGlu-Gln)-CCK-8[mPEG] in high fat fed mice for 35days significantly decreased body weight gain (p<0.05), food intake (p<0.01 to p<0.001) and triacylglycerol deposition in liver (p<0.001) and muscle (p<0.001). Furthermore, (pGlu-Gln)-CCK-8[mPEG] markedly improved intraperitoneal glucose tolerance (p<0.05) and insulin sensitivity (p<0.001). Despite this therapeutic profile, once daily injection of (pGlu-Gln)-CCK-8[mPEG] in high fat fed mice for 33days, at the same dose, was not associated with alterations in food intake and body weight. In addition, metabolic responses to exogenous glucose and insulin injection were similar to saline treated controls. CONCLUSION: These studies emphasise the therapeutic potential of (pGlu-Gln)-CCK-8[mPEG] and similar molecules. GENERAL SIGNIFICANCE: A more detailed analysis of the dose and administration schedule employed for (pGlu-Gln)-CCK-8[mPEG] could provide a novel and effective compound to treat obesity-diabetes.

Atractylenolide-III attenuates osteoarthritis by repolarizing macrophages through inactivating TLR4/NF-κB signaling.

BACKGROUND: As a common chronic musculoskeletal condition, osteoarthritis (OA) presently lacks particular treatment strategies. The aim of this study was to examine how AT-III therapies affected macrophage repolarity in order to slow down the advancement of OA. METHODS: RAW264.7 macrophages were polarized to M1 subtypes then administered with different concentrations of AT-III. Immunofluorescence, qRT-PCR and flow cytometry were used to assess the polarization of the macrophages. The mechanism of AT-III repolarize macrophages was evaluated by western blot. Furthermore, the effects of macrophage conditioned media (CM) on the migration, proliferation, and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were investigated using CCK-8 assays, the scratch test, and alcian blue staining. The effects of macrophage CM on chondrocyte proliferation and degeneration were investigated using CCK-8 and qRT-PCR. In vivo micro-CT and histological observations were performed on rats with anterior cruciate ligament transection and partial medial meniscectomy, either with or without AT-III treatment. RESULTS: AT-III repolarized M1 macrophages to M2 phenotype. Mechanistically, AT-III reduced the expression of Toll-like receptor(TLR) 4 induced by lipopolysaccharide in RAW264.7 and lowered nuclear factor-κB (NF-κB) signaling molecules p-p65 and p-IκBα. The TLR4 agonist RS09 reversed the effects of AT-III on macrophage repolarization. AT-III-induced macrophages CM stimulated BMSCs migration, proliferation and chondrogenic differentiation. AT-III-treated macrophage CM promoted chondrocyte proliferation while inhibiting chondrocyte degeneration. In vivo, AT-III treatment alleviated the degree of synovitis, inhibited subchondral bone remodeling and reduced cartilage destruction in the rat OA model. CONCLUSIONS: AT-III attenuates OA by repolarizing macrophages through inactivating TLR4/NF-κB signaling. These data suggest that AT-III may be an effective therapeutic candidate for OA treatment.

Rare HER2 L796P missense mutation promotes the growth and oncogenic signaling in breast cancer cells.

PURPOSE: This research aimed to find potential HER2 mutations that would have an impact on breast cancer and investigate the underlying mechanism. EXPERIMENTAL DESIGN: This study first investigated 238 pairs of breast cancer and para-cancerous tissue samples from patients on the targeted next-generation sequencing (tNGS) platform. CCK-8 and clone formation assay were used to investigate whether the mutation exerts proliferative effects on breast cancer cells. In addition, mass spectrometry-based comparative proteomic and phosphoproteomic analyses of the mutation types and wild types of MCF-7 cell lines were carried out. RESULTS: Among the identified mutations, a new mutation HER2 L796P promoted the proliferation of breast cancer cells and had resistance to lapatinib using CCK-8 cell proliferation assay and clone formation assay. The bioinformatic analysis showed that RAS family proteins and ERK phosphorylated proteins significantly increased in the L796P mutant cells. The Gene Ontology (GO) analysis revealed that L796P mutation affected the function of breast cancer at the level of upstream genes in the MAPK and PI3K-AKT-TOR pathways. CONCLUSIONS AND CLINICAL RELEVANCE: This study demonstrated that a rare mutation HER2 L796P could be a potential therapeutic target for the clinical management of breast cancer.

Ojeok-san enhances platinum sensitivity in ovarian cancer by regulating adipocyte paracrine IGF1 secretion.

BACKGROUND: Platinum is a commonly used drug for ovarian cancer (OvCa) treatment, but drug resistance limits its clinical application. This study intended to delineate the effects of adipocytes on platinum resistance in OvCa. METHODS: OvCa cells were maintained in the adipocyte-conditioned medium. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, separately. Proliferation and apoptosis-related protein expression were assayed by western blot. The IC50 values of cisplatin and carboplatin were determined using CCK-8. IGF1 secretion and expression were assayed via ELISA and western blot, respectively. A xenograft model was established, and pathological changes were detected by H&E staining. Proliferation and apoptosis-associated protein expression was assessed via IHC. RESULTS: Adipocytes promoted the viability and repressed cell apoptosis in OvCa, as well as enhancing platinum resistance, while the addition of IGF-1 R inhibitor reversed the effects of adipocytes on proliferation, apoptosis, and drug resistance of OvCa cells. Treatment with different concentrations of Ojeok-san (OJS) inhibited the adipocyte-induced platinum resistance in OvCa cells by suppressing IGF1. The combined treatment of OJS and cisplatin significantly inhibited tumour growth in vivo with good mouse tolerance. CONCLUSION: In summary, OJS inhibited OvCa proliferation and platinum resistance by suppressing adipocyte paracrine IGF1 secretion.

Comparison of independent and combined metabolic effects of chronic treatment with (pGlu-Gln)-CCK-8 and long-acting GLP-1 and GIP mimetics in high fat-fed mice.

AIM: The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) and cholecystokinin (CCK) are gastrointestinal peptides with important physiological effects. However, rapid enzymatic degradation results in short-lived biological actions. METHODS: This study has examined metabolic actions of exendin-4, GIP[mPEG] and a novel CCK-8 analogue, (pGlu-Gln)-CCK-8 as enzymatically stable forms of GLP-1, GIP and CCK, respectively. RESULTS: All peptides significantly (p < 0.01-p < 0.001) stimulated insulin secretion from BRIN BD11 cells, and acute in vivo experiments confirmed prominent antihyperglycaemic and insulinotropic responses to GLP-1 or GIP receptor activation in normal mice. Twice daily injection of (pGlu-Gln)-CCK-8 alone and in combination with exendin-4 or GIP[mPEG] in high fat-fed mice significantly decreased accumulated food intake (p < 0.05-p < 0.01), body weight gain (p < 0.05-p < 0.01) and improved (p < 0.05) insulin sensitivity in high fat-fed mice. However, there was no evidence for superior effects compared to (pGlu-Gln)-CCK-8 alone. Combined treatment of (pGlu-Gln)-CCK-8 and exendin-4 resulted in significantly (p < 0.05) lowered circulating glucose levels and improved (p < 0.05) intraperitoneal glucose tolerance. These effects were superior to either treatment regime alone but not associated with altered insulin concentrations. A single injection of (pGlu-Gln)-CCK-8, or combined with exendin-4, significantly (p < 0.05) lowered blood glucose levels 24 h post injection in untreated high fat-fed mice. CONCLUSION: This study highlights the potential of (pGlu-Gln)-CCK-8 alone and in combination with incretin hormones for the treatment of type 2 diabetes.

[Cigarette Smoke Induces Gefitinib Resistance in NSCLC Cells 
via ROS/Sirt3/SOD2 Pathway].

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC. METHODS: PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8. RESULTS: CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05). CONCLUSIONS: The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.

Beneficial effects of the novel cholecystokinin agonist (pGlu-Gln)-CCK-8 in mouse models of obesity/diabetes.

AIMS/HYPOTHESIS: Cholecystokinin (CCK) is a rapidly degraded gastrointestinal peptide that stimulates satiety and insulin secretion. We aimed to investigate the beneficial weight-lowering and metabolic effects of the novel N-terminally modified CCK analogue, (pGlu-Gln)-CCK-8. METHODS: The biological actions of (pGlu-Gln)-CCK-8 were comprehensively evaluated in pancreatic clonal BRIN BD11 cells and in vivo in high-fat-fed and ob/ob mice. RESULTS: (pGlu-Gln)-CCK-8 was completely resistant to enzymatic degradation and its satiating effects were significantly (p < 0.05 to p < 0.001) more potent than CCK-8. In BRIN-BD11 cells, (pGlu-Gln)-CCK-8 exhibited enhanced (p < 0.01 to p < 0.001) insulinotropic actions compared with CCK-8. When administered acutely to high-fat-fed or ob/ob mice, (pGlu-Gln)-CCK-8 improved glucose homeostasis. Sub-chronic twice daily injections of (pGlu-Gln)-CCK-8 in high-fat-fed mice for 28 days significantly decreased body weight (p < 0.05 to p < 0.001), accumulated food intake (p < 0.05 to p < 0.001), non-fasting glucose (p < 0.05) and triacylglycerol deposition in pancreatic (p < 0.01), adipose (p < 0.05) and liver (p < 0.001) tissue, and improved oral (p < 0.05) and i.p. (p < 0.05) glucose tolerance and insulin sensitivity (p < 0.001). Similar observations were noted in ob/ob mice given twice daily injections of (pGlu-Gln)-CCK-8. In addition, these beneficial effects were not reproduced by simple dietary restriction and were not associated with changes in energy expenditure. There was no evidence for development of tolerance to (pGlu-Gln)-CCK-8, and analysis of histology or blood-borne markers for pancreatic, liver and renal function in mice treated with (pGlu-Gln)-CCK-8 suggested little abnormal pathology. CONCLUSIONS/INTERPRETATION: These studies emphasise the potential of (pGlu-Gln)-CCK-8 for the alleviation of obesity and insulin resistance.

IMP075 targeting ClpP for colon cancer therapy in vivo and in vitro.

ONC201 is a well-known caseinolytic protease (ClpP)activator with established benefits against multiple tumors, including colorectal cancer (CRC). In this study, we investigated the anticancer effects and associated mechanisms of the ClpP agonist IMP075, derived from ONC201. Acute toxicity and CCK-8 assayswere employed to determine the safety of IMP075. The effectiveness of IMP075 was investigated in HCT116 cells and a mouse xenograft tumor model. Additionally, the properties of IMP075 were evaluated by pharmacokinetic,CYP inhibition, and hERG inhibition assays. Finally, isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF), cellular thermal shift assay (CETSA), molecular dynamics simulations, point mutations, and shRNA experiments were employed to elucidate the potential mechanism of IMP075. Compared with ONC201, IMP075 exhibited similar toxicity and improved antitumor effects in vitro and in vivo. Interestingly, the affinity and agonistic effects of IMP075 on ClpP were superior to ONC201, which allowed IMP075 to disrupt respiratory chain integrity at lower doses in HCT116 cells, leading to mitochondrial dysfunction. Furthermore, molecular dynamics simulations demonstrate that IMP075 forms two pairs of hydrogen bonds with ClpP, maintaining ClpP in an agonistic state. Importantly, the antiproliferative activity of IMP075 significantly decreased following ClpP knockdown. Our findings substantiate that IMP075 exerts excellent antitumor effects against CRC by activating ClpP-mediated impairment of mitochondrial function. Due to its superior properties, IMP075 appears to be have huge prospects for application.

B3GALT4 remodels the tumor microenvironment through GD2-mediated lipid raft formation and the c-met/AKT/mTOR/IRF-1 axis in neuroblastoma.

BACKGROUND: Beta-1,3-galactosyltransferase-4 (B3GALT4) plays a critical regulatory role in tumor biology. However, the role of B3GALT4 in modulating the tumor microenvironment (TME) of neuroblastoma (NB) remains unknown. METHODS: Public datasets and clinical NB samples were collected to evaluate the expression and clinical significance of GD2 and B3GALT4 in NB patients. CCK-8, colony formation, and transwell assays and experiments in tumor-bearing mouse models were conducted to investigate the function of B3GALT4. Flow cytometry, ELISA, immunohistochemistry, immunofluorescence, western blotting, and chemotaxis assays were conducted to ascertain the immunomodulatory mechanism of B3GALT4. The combined therapeutic effect of the lipid raft inhibitor MßCD and anti-GD2 mAb was validated in a murine model of NB. RESULTS: GD2 was overexpressed in NB tissues and high expression of GD2 was associated with poor prognosis in NB patients. B3GALT4 was downregulated in NB tissues, and low expression of B3GALT4 indicated poor prognosis in NB patients. Silencing B3GALT4 significantly enhanced tumor progression both in vitro and in vivo. Meanwhile, the overexpression of B3GALT4 increased the recruitment of CD8+ T lymphocytes via the chemokines CXCL9 and CXCL10. Additionally, B3GALT4 regulated NB-cell GD2 expression and lipid raft formation. Mechanistically, B3GALT4 regulated the expression of CXCL9 and CXCL10 via the c-Met signaling in the lipid rafts and the downstream AKT/mTOR/IRF-1 pathway. The lipid raft inhibitor, MßCD, attenuated B3GALT4 deficiency-induced tumor progression and immune evasion. Last, MßCD combined with anti-GD2 mAb treatment significantly enhanced the antitumor effect and the infiltration of CD8+ T cells. CONCLUSIONS: Upregulation of B3GALT4 promotes the secretion of CXCL9 and CXCL10 to recruit CD8+ T lymphocytes via the GD2-mediated lipid rafts and the c-Met/AKT/mTOR/IRF-1 pathway. Moreover, lipid raft inhibitors may enhance the efficacy of anti-GD2 immunotherapy for NB.

Oxidized chondroitin sulfate eye drops ameliorate the prognosis of fungal keratitis with anti-inflammatory and antifungal effects.

Fungal keratitis (FK) is a refractory ophthalmic disease that can result in vision impairment and even blindness due to the severe fungal invasiveness and excessive inflammatory response. Therefore, antifungal treatment combined with local immunosuppressive therapy is regarded as the most effective strategy to improve the clinical outcome of FK. Oxidized polysaccharides with aldehyde groups possess obvious inhibitory activity towards microorganisms. Herein, we use chondroitin sulfate (CS), a recognized anti-inflammatory biopolysaccharide, to prepare oxidized chondroitin sulfate (OCS) via sodium periodate (NaIO4) oxidation for the treatment of FK. The chemical structure of OCS was characterized by FTIR, 1H NMR, and XPS, revealing that the O-dihydroxy in the D-glucuronic acid unit of CS was selectively broken by NaIO4, forming active aldehyde groups. The introduction of aldehydes not only retains the anti-inflammatory activity but also confers OCS with antifungal property. In vitro antifungal experiments showed that OCS inhibits the growth, represses the biofilm formation and alters the membrane integrity of A. fumigatus. The toxicity of OCS was evaluated by cytotoxicity tests (CCK-8) and the Draize eye test in vitro and in vivo. qRT-PCR confirmed that OCS had similar anti-inflammatory activity as CS. In mice with A. fumigatus keratitis, OCS versus CS or PBS showed an excellent therapeutic effect, characterized by a lower corneal inflammation score, less fungal load, reduced neutrophil recruitment, and the downregulated expression of pro-inflammatory factors. Our findings demonstrate that OCS improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of fungi, reducing the recruitment of neutrophils and inhibiting the inflammatory response. It provides innovative ideas for the development and application of OCS in medicine and biomaterials fields.

Arsenic trioxide-loaded nanoparticles enhance the chemosensitivity of gemcitabine in pancreatic cancer via the reversal of pancreatic stellate cell desmoplasia by targeting the AP4/galectin-1 pathway.

Pancreatic stellate cells (PSCs) constitute the fibrotic tumor microenvironment composed of the stroma matrix, which blocks the penetration of gemcitabine (GEM) in pancreatic adenocarcinoma (PDAC) and results in chemoresistance. We analyzed the expression of α-SMA, collagen type I, and fibronectin by immunohistochemistry of pancreatic cancer tissues and demonstrated that the abundant interstitial stroma is associated with dismal survival. Two desmoplastic pancreatic tumor models are treated with arsenic trioxide (ATO) and GEM to confirm the sensitizing effect of ATO on GEM. RNA-seq was performed to analyze the potential fibrotic genes regulated by ATO. Western blotting, CCK-8 methods, colony formation, and wound healing and transwell assays were utilized to verify that ATO attenuates the tumor-promoting ability of PSCs by inhibiting its activation and decreasing matrix secretion via the PI3K/AKT/AP4/galectin-1 pathway. Furthermore, we developed targeted ATO-loaded nanoparticles self-assembled by poly (D,L-lactide) and poly(ethylene glycol) (PEG-PDLLA) and modified by the single-chain antibody of FAP-α (scAbFAP-α) (scAb-ATO-NPs) to promote the delivery efficiency of ATO to PSCs and enhance anti-tumor effects with gemcitabine. Herein, we elucidate the mechanism of how ATO inhibits the activation of PSCs and enhances the therapeutic effect of GEM. We propose a novel cocktail therapy including scAb-ATO-NPs and GEM, indicating a new perspective in the treatment of PDAC.

Fibroblast Growth Factor 3 Is Associated with Tongue Squamous Cell Carcinoma: A Controlled Study.

Objective: To explore the effects of fibroblast growth factor 3 (FGF3) on the proliferation, cell cycle, and apoptosis of the tongue squamous cell carcinoma SCC-9 cell line (SCC-9). Methods: We measured the proliferation of SCC-9 cells in a control group, an FGF3 intervention group, and a fibroblast growth factor (FGFR) inhibitor intervention group in cholecystokinin octapeptide (CCK-8) experiments. We studied effects of FGF3 on the cell cycle and apoptosis of tongue cancer cells using flow cytometry. We further explored the IRS1/PI3K/AKT signaling pathway by measuring BCL-2 and Bcl-2 Associated X-protein (BAX) mRNA and protein levels with RT-PCR and western blot, respectively. Results: Results from the CCK-8 experiment showed that survival rates of cells in the control group, FGF3 intervention group, and FGFR inhibitor intervention group were 100.000% ± 4.026%, 136.330% ± 9.779%, and 83.199% ± 4.954%, respectively; survival rates of SCC-9 cells in all three groups were statistically significant (P < 0.05). Compared with that in the control group, the ratio of cells in G0/G1 phase in the FGFR inhibitor intervention group was higher (P < 0.05) and that in G2/M phase was lower, while the FGF3 intervention group showed opposite results (P < 0.05). The apoptosis rate of tongue cancer cells differed significantly between the FGFR inhibitor intervention and the control groups (P < 0.05). The mRNA and protein expression levels of IRS1, PI3K, and BCL-2 were all increased in the FGF3 intervention group (P < 0.05), while BAX mRNA and protein expression levels were decreased (P < 0.05). The mRNA expression levels of protein kinase B (AKT) showed no differences between groups. The p-AKT protein was overexpressed, while the total amount of AKT protein remained stable (P < 0.05). Conclusion: FGF3 contributes to the proliferation of SCC-9 cells by increasing the proportion of cells in G2/M phase. Therefore, appropriately timed inhibition of FGF3 can potentially promote tumor apoptosis through the IRS1/PI3K/AKT signaling pathway. Our results demonstrate the role of FGF3 in the tumor microenvironment in tongue squamous cell carcinoma SCC-9 cells and suggest new therapeutic targets.

Melittin Inhibits Growth of Human Osteosarcoma 143B Cells through Induction of Apoptosis via Suppressing the Wnt/ß-catenin Signaling Pathway.

BACKGROUND AND PURPOSE: Osteosarcoma is the most commonly seen type of primary malignant bone tumors in children and adolescents. Partial patients with osteosarcoma cannot tolerate the side effects of chemotherapy drugs. Hence, it is urgent to find anti-osteosarcoma drugs with low side effects. Melittin is an anti-tumor Traditional Chinese Medicine with low side effects. The purpose of this study was to explore the anti-osteosarcoma effect of melittin and its possible molecular mechanisms. METHODS: The effects of melittin on cell growth were detected by CCK-8, clonal formation, and flow cytometry. The related molecules were also investigated by Real-time PCR and Western blot. A xenograft model in nude mice was established to observe the effects of melittin on tumor growth and the related molecular expression was detected by immunohistochemistry. RESULTS: Melittin can inhibit the proliferation of osteosarcoma 143B cells, reduce colony formation, and induce apoptosis while significantly up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 proteins. Moreover, treatment with melittin significantly reduced the mRNA and protein levels of ß-catenin and Wnt/ß- catenin related genes (LRP5, c-Myc, and Survivin) in osteosarcoma 143B cells in vitro. The xenograft model found that melittin significantly inhibited tumor growth and decreased the protein expression levels of ß-catenin and Wnt/ß- catenin related genes in vivo. CONCLUSION: These findings show that melittin could inhibit the growth of osteosarcoma 143B cells, which may be related to the inhibition of Wnt/ß-catenin signaling pathway activity and induce apoptosis by up-regulating the ratio of Bax/Bcl-2 in osteosarcoma 143B cells. Therefore, melittin is a promising anti-tumor drug for the treatment of osteosarcoma.

Conciliatory Anti-Allergic Decoction Attenuates Pyroptosis in RSV-Infected Asthmatic Mice and Lipopolysaccharide (LPS)-Induced 16HBE Cells by Inhibiting TLR3/NLRP3/NF-κB/IRF3 Signaling Pathway.

Respiratory syncytial virus (RSV) infection can deteriorate asthma by inducing persistent airway inflammation. Increasing evidence elucidated that pyroptosis plays a pivotal role in asthma. Conciliatory anti-allergic decoction (CAD) exhibits an anti-inflammatory effect in ovalbumin (OVA)-induced asthma; however, the effects and mechanisms of CAD in RSV-infected asthmatic mice have not yet been elucidated. The RSV-infected asthmatic mice model and lipopolysaccharide (LPS)-induced 16HBE cell pyroptosis model were established, respectively. Pulmonary function, ELISA, and histopathologic analysis were performed to assess the airway inflammation and remodeling in mice with CAD treatment. Furthermore, ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) was conducted to identify the chemical compounds of high-dose CAD (30 g/kg). Cell viability and apoptosis of 16HBE cells were assessed by CCK-8 and flow cytometry assays, respectively. Finally, the expression levels of apoptosis-, pyroptosis-, and TLR3/NLRP3/NF-κB/IRF3 signaling-related genes were measured with qRT-PCR or western blotting, respectively. Pulmonary function tests showed that CAD significantly ameliorated respiratory dysfunction, airway hyperresponsiveness, inflammation cell recruitment in BALF, pulmonary inflammation, collagen deposition, and cell death in lung tissues. CAD significantly decreased the content of TNF-α, IL-13, IL-4, IL-1ß and IL-5 in the bronchoalveolar lavage fluid (BALF), IL-17, IL-6, and OVA-specific IgE in serum and increased serum IFN-γ in asthma mice. The results of UPLC-Q-TOF/MS showed that high-dose CAD had 88 kinds of chemical components. In vitro, CAD-contained serum significantly suppressed LPS-induced 16HBE cell apoptosis. Additionally, CAD and CAD-contained serum attenuated the up-regulated expressions of Bax, Cleaved caspase-3, NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-18, IL-1ß, TLR3, p-P65, p-IκBα, and IRF3 but increased Bcl-1 and GSDMD levels in the asthma mice and LPS-induced 16HBE cells, respectively. These results illustrated that CAD may have a potential role in improving airway inflammation and pyroptosis through inhibition of the TLR3/NLRP3/NF-κB/IRF3 signaling pathway.

Carboxylesterase 2 induces mitochondrial dysfunction via disrupting lipid homeostasis in oral squamous cell carcinoma.

OBJECTIVE: Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear. METHODS: The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC. RESULTS: CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2high OSCC patients showed better overall survival than CES2low OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2. CONCLUSIONS: We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC.

Cholecystokinin octapeptide induces hypothermia and improves outcomes in a rat model of cardiopulmonary resuscitation.

OBJECTIVES: To investigate the effects of cholecystokinin octapeptide on thermoregulation, postresuscitation myocardial function, neurologic outcome, and duration of survival in a rat model of cardiopulmonary resuscitation. DESIGN: : Prospective, randomized, placebo-controlled experimental study. SETTING: University-affiliated animal research laboratory. SUBJECTS: Ten male Sprague-Dawley rats. INTERVENTIONS: Ventricular fibrillation was induced and untreated for 6 mins. Defibrillation was attempted after 8 mins of cardiopulmonary resuscitation. Animal temperature was adjusted to 37.0 °C with the aid of a heating lamp. At 30 mins after resuscitation, animals were randomized to receive an intravenous injection of either cholecystokinin octapeptide (200 µg/kg in 0.3 mL saline) or vehicle placebo (0.3 mL saline). The ambient temperature settings and that of the distance of the heating lamp from the animal remained the same in both groups throughout the entire experiment. MEASUREMENTS AND MAIN RESULTS: Body temperature, hemodynamic measurements, and postresuscitation myocardial function, including cardiac output, left ventricular ejection fraction, and myocardial performance index, were measured together with neurologic deficit scores and duration of survival. RESULTS: After injection of cholecystokinin octapeptide, blood temperature decreased progressively from 37.0 °C to 34.8 °C 5 hrs after resuscitation and returned to 37.0 °C at 9 hrs after injection. In the control group, blood temperature was sustained at 37.0 °C ± 0.2 °C during the same period of observation. Myocardial and neurologic function and duration of survival were significantly better in the cholecystokinin octapeptide-treated animals when compared to the control group. CONCLUSIONS: : In a rat model of cardiopulmonary resuscitation, cholecystokinin octapeptide induced mild hypothermia, attenuated postresuscitation myocardial dysfunction, and improved neurologic outcome and duration of survival.