Polycytosine RNA-binding protein 1 regulates osteoblast function via a ferroptosis pathway in type 2 diabetic osteoporosis.

BACKGROUND: Recently, type 2 diabetic osteoporosis (T2DOP) has become a research hotspot for the complications of diabetes, but the specific mechanism of its occurrence and development remains unknown. Ferroptosis caused by iron overload is con-sidered an important cause of T2DOP. Polycytosine RNA-binding protein 1 (PCBP1), an iron ion chaperone, is considered a protector of ferroptosis. AIM: To investigate the existence of ferroptosis and specific role of PCBP1 in the development of type 2 diabetes. METHODS: A cell counting kit-8 assay was used to detect changes in osteoblast viability under high glucose (HG) and/or ferroptosis inhibitors at different concentrations and times. Transmission electron microscopy was used to examine the morphological changes in the mitochondria of osteoblasts under HG, and western blotting was used to detect the expression levels of PCBP1, ferritin, and the ferroptosis-related protein glutathione peroxidase 4 (GPX4). A lentivirus silenced and overexpressed PCBP1. Western blotting was used to detect the expression levels of the osteoblast functional proteins osteoprotegerin (OPG) and osteocalcin (OCN), whereas flow cytometry was used to detect changes in reactive oxygen species (ROS) levels in each group. RESULTS: Under HG, the viability of osteoblasts was considerably decreased, the number of mitochondria undergoing atrophy was considerably increased, PCBP1 and ferritin expression levels were increased, and GPX4 expression was decreased. Western blotting results demonstrated that infection with lentivirus overexpressing PCBP1, increased the expression levels of ferritin, GPX4, OPG, and OCN, compared with the HG group. Flow cytometry results showed a reduction in ROS, and an opposite result was obtained after silencing PCBP1. CONCLUSION: PCBP1 may protect osteoblasts and reduce the harm caused by ferroptosis by promoting ferritin expression under a HG environment. Moreover, PCBP1 may be a potential therapeutic target for T2DOP.

Unique quinoline orientations shape the modified aptamer to sclerostin for enhanced binding affinity and bone anabolic potential.

Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility and bone formation. Sclerostin could negatively regulate bone formation by antagonizing the Wnt signal pathway, whereas it imposes severe cardiac ischemic events in clinic. Our team has screened an aptamer that could promote bone anabolic potential without cardiovascular risk. However, the affinity of the aptamer is lower and needs to be improved. In the study, hydrophobic quinoline molecule with unique orientations (seven subtypes) were incorporated into key sites of a bone anabolic aptamer against sclerostin to form a modified aptamer library. Among all the quinoline modifications, 5-quinoline modification could shape the molecular recognition of modified aptamers to sclerostin to facilitate enhancing its binding to sclerostin toward the highest affinity by interacting with newly participated binding sites in sclerostin. Further, 5-quinoline modification could facilitate the modified aptamer attenuating the suppressed effect of the transfected sclerostin on both Wnt signaling and bone formation marker expression levels in vitro, promoting bone anabolism in OI mice (Col1a2+/G610C). The proposed quinoline-oriented modification strategy could shape the molecular recognition of modified aptamers to proteins to facilitate enhancing its binding affinity and therapeutic potency.

A bimolecular modification strategy for developing long-lasting bone anabolic aptamer.

The molecular weight of nucleic acid aptamers (20 kDa) is lower than the cutoff threshold of the renal filtration (30-50 kDa), resulting in a very short half-life, which dramatically limits their druggability. To address this, we utilized 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-hydroxy-2-oxo-2H-chromen-6-yl)propenamide (HC) and 12-((2,5-dioxopyrrolidin-1-yl)oxy)-12-oxododecanoic acid (DA), two newly designed coupling agents, for synergistic binding to human serum albumin (HSA). Both HC and DA are conjugated to a bone anabolic aptamer (Apc001) against sclerostin to form an Apc001OC conjugate with high binding affinity to HSA. Notably, HC and DA could synergistically facilitate prolonging the half-life of the conjugated Apc001 and promoting its bone anabolic potential. Using the designed blocking peptides, the mechanism studies indicate that the synergistic effect of HC-DA on pharmacokinetics and bone anabolic potential of the conjugated Apc001 is achieved via their synergistic binding to HSA. Moreover, biweekly Apc001OC at 50 mg/kg shows comparable bone anabolic potential to the marketed sclerostin antibody given weekly at 25 mg/kg. This proposed bimolecular modification strategy could help address the druggability challenge for aptamers with a short half-life.

[Exploration of the Perturbation of PKIG in Lung Squamous Cell Carcinoma and the Role in Tumor Microenvironment Based on Bioinformatics Method].

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide, and patients have limited survival benefits from traditional treatments such as surgery, radiotherapy and chemotherapy. As a new treatment for lung cancer, immunotherapy has significantly prolonged the overall survival (OS) of patients. However, only some patients can benefit from it. We need to explore immunotherapy biomarkers more deeply to screen for advantages. METHODS: The original data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database, and the immunological and prognostic genes of lung squamous cell carcinoma (LUSC) were screened using R software and TIMER database. The expression of target genes was studied in TCGA and GEO databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and correlation analysis with tumor immune characteristics were performed by R software and TISIDB database. RESULTS: We screened out a gene related to immunity and prognosis, cAMP dependent protein kinase inhibitor γ (PKIG), which is significantly differentially expressed in LUSC and normal tissues, and has important reference value for the diagnosis and prognosis assessment of LUSC. PKIG differential genes are mainly concentrated in the regulation of humoral immune response and other processes. The expression of PKIG was positively correlated with the infiltration level of regulatory T cells (Tregs) (r=0.340, P<0.001). In addition, the expression level of PKIG was positively correlated with the expression of chemokines/chemokine receptors such as chemokine C-C motif ligand 2 (CCL2) (r=0.503, P<0.001), CXC chemokine ligand 12 (CXCL12) (r=0.386, P<0.001) and CXC-chemokine receptor 4 (CXCR4) (r=0.492, P<0.001), and immunoinhibitors such as programmed cell death protein 1 (PDCD1) (r=0.359, P<0.001), cytotoxic T-lymphocyte associated antigen 4 (CTLA4) (r=0.375, P<0.001) and T cell immunoglobulin and ITIM domains (TIGIT) (r=0.305, P<0.001) in LUSC. CONCLUSIONS: The immunological and prognostic gene PKIG in lung squamous cell carcinoma was screened through bioinformatics analysis. PKIG is highly correlated with LUSC prognosis and immune microenvironment, and is expected to be a potential biomolecular marker for LUSC immunotherapy.

Polystyrene Microplastics Postpone APAP-Induced Liver Injury through Impeding Macrophage Polarization.

Polystyrene microplastics (PS MPs) are micrometer-scale items degraded from plastics and have been detected in various organisms. PS MPs have been identified as causing cognitive, cardiac, intestinal, and hepatic damage. However, their role in liver regeneration under drug-induced liver injury remains unknown. Thus, the current study aims to evaluate the impact of PS MPs on liver repair during APAP hepatotoxicity. PS MPs pretreatment exacerbates mice mortality and hepatocyte apoptosis, suppresses hepatic cell proliferation, and disturbs the inflammatory response in the APAP-induced damage model. Further mechanism exploration uncovers that prior PS MPs administration is sufficient to recruit neutrophils and macrophages, which are necessary for tissue recovery in the acute liver injury model. However, the polarization capacity of macrophages to anti-inflammatory sub-type is significantly delayed in PS MPs plus APAP group compared to the single APAP group, which is the leading cause of tissue repair suppression. Overall, the current study supports a new insight to realize the toxicity of PS MPs in acute liver injury, which should be considered in health risk assessment.

Mitochondrial Ferritin Deficiency Promotes Osteoblastic Ferroptosis Via Mitophagy in Type 2 Diabetic Osteoporosis.

The incidence of type 2 diabetic osteoporosis (T2DOP), which seriously threatens elderly people's health, is rapidly increasing in recent years. However, the specific mechanism of the T2DOP is still unclear. Studies have shown the relationship between iron overload and T2DOP. Mitochondrial ferritin (FtMt) is a protein that stores iron ions and intercepts toxic ferrous ions in cells mitochondria. Ferroptosis, an iron-dependent cell injured way, may be related to the pathogenesis of T2DOP. In this study, we intend to elucidate the effect of FtMt on ferroptosis in osteoblasts and explain the possible mechanism. We first detected the occurrence of ferroptosis in bone tissue and the expression of FtMt after inducing T2DOP rat model. Then we used hFOB1.19 cells to study the influence of high glucose on FtMt, ferroptosis, and osteogenic function of osteoblasts. Then we observed the effect of FtMt on ferroptosis and osteoblast function by lentiviral silencing and overexpression of FtMt. We found ferroptosis in T2DOP rats bone. Overexpression of FtMt reduced osteoblastic ferroptosis under high glucose condition while silent FtMt induced mitophagy through ROS / PINK1/Parkin pathway. Then we found increased ferroptosis in osteoblasts after activating mitophagy by carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP, a mitophagy agonist). Our study demonstrated that FtMt inhibited the occurrence of ferroptosis in osteoblasts by reducing oxidative stress caused by excess ferrous ions, and FtMt deficiency induced mitophagy in the pathogenesis of T2DOP. This study suggested that FtMt might serve as a potential target for T2DOP therapy.

Melatonin Suppresses Ferroptosis Induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis.

Ferroptosis is recently identified, an iron- and reactive oxygen species- (ROS-) dependent form of regulated cell death. This study was designed to determine the existence of ferroptosis in the pathogenesis of type 2 diabetic osteoporosis and confirm that melatonin can inhibit the ferroptosis of osteoblasts through activating Nrf2/HO-1 signaling pathway to improve bone microstructure in vivo and in vitro. We treated MC3T3-E1 cells with different concentrations of melatonin (1, 10, or 100 µM) and exposed them to high glucose (25.5 mM) for 48 h in vitro. Our data showed that high glucose can induce osteoblast cytotoxicity and the accumulation of lipid peroxide, the mitochondria of osteoblast show the same morphology changes as the erastin treatment group, and the expression of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11) is downregulated, but these effects were reversed by ferroptosis inhibitor ferrastatin-1 and iron chelator deferoxamine (DFO). Furthermore, western blot and real-time polymerase chain reaction were used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1); osteogenic capacity was evaluated by alizarin red S staining and the expression of osteoprotegerin, osteocalcin, and alkaline phosphatase; the results showed that the expression levels of these proteins in osteoblasts with 1, 10, or 100 µM melatonins were significantly higher than the high glucose group, but after using Nrf2-SiRNA interference, the therapeutic effect of melatonin was significantly inhibited. We also performed in vivo experiments in a diabetic rat model treated with two concentrations of melatonin (10, 50 mg/kg). Dynamic bone histomorphometry and micro-CT were used to observe the rat bone microstructure, and the expression of GPX4 and Nrf2 was determined by immunohistochemistry. Here, we first report that high glucose induces ferroptosis via increased ROS/lipid peroxidation/glutathione depletion in type 2 diabetic osteoporosis. More importantly, melatonin significantly reduced the level of ferroptosis and improved the osteogenic capacity of MC3T3-E1 through activating the Nrf2/HO-1 pathway in vivo and in vitro.

LC/MS/MS quantitation assay for pharmacokinetics of naringenin and double peaks phenomenon in rats plasma.

A highly sensitive and specific electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for quantitation of naringenin (NAR) and an explanation for the double peaks phenomenon was developed and validated. NAR was extracted from rat plasma and tissues along with the internal standard (IS), hesperidin, with ethyl acetate. The analytes were analyzed in the multiple-reaction-monitoring (MRM) mode as the precursor/product ion pair of m/z 273.4/151.3 for NAR and m/z 611.5/303.3 for the IS. The assay was linear over the concentration range of 5-2500 ng/mL. The lower limit quantification was 5 ng/mL, available for plasma pharmacokinetics of NAR in rats. Accuracy in within- and between-run precisions showed good reproducibility. When NAR was administered orally, only little and predominantly its glucuronidation were into circulation in the plasma. There existed double peaks phenomenon in plasma concentration-time curve leading to the relatively slow elimination of NAR in plasma. The results showed that there was a linear relationship between the AUC of total NAR and dosages. And the double peaks are mainly due to enterohepatic circulation.