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HMGB1 Regulates the Formation of Calcium Oxalate-Induced Kidney Stones via Modulating the PI3K/AKT Signaling Pathway
No full textBackground: High mobility group box 1 (HMGB1) participates in regulating autophagy and cell apoptosis in the cytoplasm and plays a role in various kidney diseases. However, no study has focused on the role of the HMGB1/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB, also called AKT) signaling pathway in kidney stone formation. The aim of this study was to elucidate the role of HMGB1 via the PI3K/AKT axis in calcium oxalate monohydrate (COM)-induced kidney stone formation, which might lead to novel research targets for the treatment of kidney stones. Methods: The human kidney-2 (HK-2) cell line was cultured and treated with si-HMGB1, si-negative control (NC), or PI3K agonist 740 Y-P (1 μM). In vitro kidney stone models were established using COM and calcium oxalate (CaOx) crystals. HMGB1, AKT and PI3K levels in each group of cells were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot. Flow cytometry was utilized to measure the rate of cellular apoptosis. Results: CaOx crystal adhesion was enhanced on the surface of the HK-2 cells stimulated by COM and CaOx, and HMGB1 expression was upregulated. The proportion of apoptotic cells was elevated in HK-2 cells after stimulation with CaOx and COM. Knockdown of HMGB1 inhibited CaOx crystal-cell adhesion and reduced the apoptosis rate in COM-treated HK-2 cells. Activation of the PI3K/AKT pathway counteracted the impact of HMGB1 knockdown on the adhesion of CaOx crystals to cells. Conclusions: Inhibiting the HMGB1/PI3K/AKT axis could reduce the proportion of apoptosis and improve crystal-cell adherence
Neuropilin 1 Drives Gastric Cancer Growth and Metastasis by Regulating Copper Homeostasis
No full textBackground: Gastric cancer (GC) is considered a significant health problem worldwide. The initial diagnosis of GC often occurs at an advanced disease stage and the prognosis is generally poor. The objective of this study is to investigate the role of neuropilin 1 (NRP1) in driving growth and metastasis of gastric cancer through the regulation of copper homeostasis. Our aim is to develop potential novel therapeutic strategies for GC. Methods: Gene expression profiling and clinical data from gastric cancer patients were obtained from The Cancer Genome Atlas (TCGA). Differential analysis was performed using the limma package in R, and a prognostic model was constructed using least absolute shrinkage and selection operator (LASSO) regression. The diagnostic model was established using logistic regression. Cell culture experiments were conducted to validate the findings, including reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), western blotting, cell counting kit (CCK)-8 assay, colony-forming assay, cell scratch test, transwell migration assay, immunofluorescence staining, and apoptosis analysis. Results: NRP1 was found to be overexpressed in gastric cancer cells (p < 0.01). It was identified as a potential regulator of copper homeostasis, as its overexpression led to increased expression levels of ATPase copper transporting alpha (ATP7A; p < 0.01), a copper transporter. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was involved in mediating the effects of NRP1 on ATP7A expression. Functional assays demonstrated that NRP1 promoted cell proliferation (p < 0.05) and migration (p < 0.01), while inhibiting apoptosis (p < 0.05). Furthermore, treatment with a copper chelator sensitized NRP1-overexpressing cells and reduced their proliferation (p < 0.01). Conclusions: This study reveals that NRP1 plays a crucial role in driving the growth and metastasis of gastric cancer through the regulation of copper homeostasis. Targeting NRP1 and copper homeostasis may provide potential therapeutic strategies for the treatment of gastric cancer
Association between Hereditary Spastic Paraplegia and a Novel SPAST Exon 11 Mutation
No full textBackground: Hereditary spastic paraplegias (HSPs) are a heterogeneous and rare group of neurodegenerative disorders characterized by gradually progressing spasticity and weakness in the lower limbs. Various genes are associated with both the pure and the complex types of hereditary spastic paraplegia (HSP). Methods: In this study, next-generation sequencing was employed to explore the case of a male proband suffering from HSP who has been unable to stand or walk since childhood due to an abnormal gait. A thorough examination was conducted, and the sequence data from both the proband and his parents were analyzed. Adhering to the guidelines set by the American College of Medical Genetics and Genomics (ACMG), these data were processed using the Online Mendelian Inheritance in Man (OMIM) database, as well as other resources such as The Human Phenotype Ontology (HPO), Chinese Human Phenotype Ontology (CHPO), and Orphanet. Results: The cause of HSP in the proband was identified as a novel Exon 11 mutation detected at c.1131A>C (p.Asp444Ala). This mutation was determined to be a pathogenic gene and de novo, with no family history of the disorder. Conclusions: This study aimed to uncover a novel genetic basis for HSP and advocate for Spastin (SPAST) testing among patients with the disorder. This research contributes to the understanding and expansion of the phenotype spectrum of SPAST-related diseases
ES-PredHSP: Improved Prediction of Heat Shock Proteins Using Machine Learning by Enhanced Sampling Technique
No full textBackground: Heat shock proteins (HSPs) are essential for the growth of various cells. The development of automated and precise machine-learning tools for the quick prediction of HSPs is significant because conventional methods are expensive, and there is abundant protein sequence information accessible in the post-genomic era, which can be easily used to develop machine-learning based tools. Methods: The proposed method utilized the already available dataset from the PredHSP tool. The Composition of k Spaced Amino Acid Pair feature was calculated using iFeature, and an enhanced sampling technique was proposed for the balanced dataset. Six machine learning models were developed to predict HSPs, and their robustness was assessed using a ten-fold cross-validation technique. The best model was finalized among six machine learning models and evaluated by the following metrics: accuracy, precision, recall, f1 score, and area under the curve (AUC). The command line utility for the use was available as a GitHub repository. Results: Six machine learning models were assessed with ten-fold cross-validation, and support vector machine (SVM) outperformed with a higher overall accuracy (87%) compared to existing methods, which can predict all HSP types in one run. For usage, the model was deployed as a command line utility in GitHub. Conclusions: Machine learning is a powerful method to predict the HSPs by identifying hidden patterns inside the sequence. HSPs are important chaperons in research, and their quick prediction will function in many important aspects of biological research. This model helps to predict the heat shock protein in eukaryotes
Cantharidin Inhibits Osteosarcoma Cell Proliferation and Metastasis through the FOXO3A/METTL14/Vimentin Pathway
No full textBackground: Osteosarcoma (OS) is the most prevalent primary malignant bone tumor among adolescents, yet the efficacy of current medications is limited by a lack of understanding of the molecular mechanisms underpinning OS growth and metastasis. In previous studies, cantharidin (CTD), an effective component found in several clinical traditional Chinese medicine formulations, has demonstrated promising pharmacological efficacy against various malignancies. However, the precise biological functions and regulatory mechanisms of CTD in OS, particularly its interaction with the Forkhead box O3A (FOXO3A)/Methyltransferase Like 14 (METTL14)/Type III Intermediate Filament Protein (Vimentin) pathway, remain incompletely understood, warranting further investigation. Methods: Eighteen female BALB/c nude mice (four-week-old; 15 ± 2 g) were utilized in this study and divided into three groups (n = 6/group): Normal control, Control, and CTD groups. Mice in the Normal control group received saline injections, while those in the other groups were injected with U-2 OS cells at the proximal end of the left tibia to establish an osteosarcoma model. Following successful model establishment, mice in the Control group received a daily intraperitoneal injection of saline (1 mg/kg), while those in the CTD group received an intraperitoneal injection of CTD (2.5 mg/kg). The inhibitory effect of CTD on tumor growth was evaluated after 28 days of continuous treatment. Additionally, the effects of CTD on OS cells were assessed using a Cell Counting Kit-8 (CCK-8), colony formation, and Transwell assays. Western blotting and quantitative real-time PCR (qRT-PCR) were employed to quantify FOXO3A/METTL14 expression levels. Results: Lower levels of FOXO3A expression were observed in OS tissues (p < 0.01), with these expression levels significantly correlating with OS patient prognosis (p < 0.01). Compared to controls, CTD significantly suppressed osteosarcoma cell growth and metastasis as well as tumor growth (p < 0.001) by activating the FOXO3A/METTL14 pathway, consequently downregulating Vimentin expression (p < 0.001). Conclusion: Our findings indicate that cantharidin restricts osteosarcoma cell proliferation and metastasis through modulation of the FOXO3A/METTL14/Vimentin pathway
HSH2D Promotes the Progression and Chemoresistance of CRC by Regulating the YAP1
No full textBackground: Colorectal cancer (CRC) stands out as one of the most widespread and life-threatening malignancies globally. In recent years, significant attention has been directed towards the Yes-associated protein 1 (YAP1), identified as the central effector molecule in the Hippo signaling pathway, highlighting its crucial involvement in CRC development. Nevertheless, the role of hematopoietic SH2 domain-containing (HSH2D) in CRC, and its impact on tumor progression and resistance to chemotherapy by modulating YAP1, remains unclear. Therefore, this study aims to explore the role of HSH2D in CRC and assess its potential therapeutic significance. Methods: The study included the analysis of HSH2D expression levels in CRC tissues and cells by immunohistochemistry and western blot techniques. The influence of HSH2D on CRC cell proliferation and migration was examined using RNA interference and overexpression systems. The regulatory impact of HSH2D on YAP1 expression was assessed through real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and immunoblotting. In vitro cell experiments were conducted to unravel the mechanism by which HSH2D promotes CRC progression through YAP1. Lastly, the study investigated the role of HSH2D in modulating the sensitivity of CRC cells to cisplatin by chemosensitivity experiments. Results: The experimental findings demonstrated a notable elevation in HSH2D expression within both CRC tissues and cells. Overexpression of HSH2D was found to enhance the proliferation and migration capabilities of CRC cells, whereas silencing HSH2D had the opposite effect. Subsequent investigations unveiled that HSH2D upregulated YAP1 expression, and the tumor-promoting influence of HSH2D was, in part, dependent on YAP1. Furthermore, heightened HSH2D expression correlated with increased resistance of CRC cells to cisplatin. Conclusion: This study marks the initial identification of HSH2D upregulation in CRC and its role in advancing the progression and chemoresistance of CRC by modulating YAP1. HSH2D emerges as a promising novel target for CRC treatment, suggesting a potential therapeutic strategy. Future research should delve deeper into the specific interaction mechanisms between HSH2D and YAP1, aiming to effectively target this pathway for enhanced CRC treatment
KOR-Based Effect of Smo/Gli1 on the Development of Osteoarthritis Inflammation via Hedgehog (Hh) and Mechanism Study
No full textBackground: Osteoarthritis (OA) is a chronic progressive joint disease with a complex pathogenesis in which inflammation plays a vital role in its development. This study aims to investigate the function of the Kappa Opioid Receptor (KOR) in the Hedgehog (Hh) signaling pathway, as well as its effects and mechanisms on the development of inflammation in osteoarthritis through the Smoothened (Smo)/Glioma-associated oncogene homolog 1 (Gli1) signaling pathway. Methods: The expression of KOR, Smo, Gli1, and Hh was detected by Western Blotting (WB) and Polymerase Chain Reaction (PCR). Interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were monitored by enzyme-linked immunosorbent assay (ELISA). The expression of apoptotic proteins B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cysteine aspartic acid-specific protease-3 (Caspase-3) was detected by WB, and cell growth was assessed using the Cell Counting Kit-8 (CCK-8) assay. The chondrocyte injury was observed by toluidine blue staining, and apoptosis was detected by flow cytometry. A rat OA model was established using the meniscectomy method, and Hematoxylin and Eosin (HE) staining and Modified Safranin-fast green staining were used to observe the histopathological structure of cartilage in OA rats. The histopathological assessment of the severity of cartilage osteoarthritis lesions was made by Mankin scoring. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) staining was used to identify apoptosis in cartilage tissues. Results: In cellular experiments, the osteoarthritis group showed elevated levels of Smo, Gli1, and Hh, reduced expression of KOR, decreased proliferation of cartilage cells, increased apoptosis, and increased inflammatory response (p < 0.05). The addition of JT09 or cyclopamine amplified cell viability and diminished the expression of pro-apoptotic proteins, which was more effective when dual action was applied to osteoarthritis. In animal experiments, concerning the osteoarthritis group, the addition of JT09 or cyclopamine increased the expression of KOR, and reduced the expression of Smo, Gli1, and Hh. It also increased Bcl-2, declined Bax and Caspase-3, and diminished the inflammatory response, and the effect of both simultaneous actions was significant (p < 0.05). These indicated that JT09 synergistic treatment with cyclopamine declined the inflammatory response in rat osteoarthritis. Conclusions: KOR mediates Hedgehog (Hh) to promote osteoarthritis inflammation progression and exacerbates bone damage through the Smo/Gli1 pathway
Screening of Candidate Biomarkers for Primary Membranous Nephropathy Based on Proteomic Analysis of Peripheral Blood Mononuclear Cells
No full textBackground: Primary membranous nephropathy (PMN) is the leading cause of nephrotic syndromes in adults. However, biopsy is a commonly used approach in diagnosing as well as assessing renal disease progression. This study aimed to screen potential PMN biomarkers using proteomic methods on peripheral blood mononuclear cells (PBMCs) and to provide an important reference value for the scientific diagnosis of PMN. Methods: Peripheral blood samples were obtained from both PMN patients and healthy controls. Total RNA was extracted and subsequently reverse-transcribed into cDNA. Meanwhile, by combining proteomics analysis with Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) technique, candidate biomarkers for PMN were screened employing bioinformatics analysis of PBMCs. Furthermore, the expression levels of differentially expressed proteins in both PMN patients and normal controls were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Results: A total of 81 differential expressed proteins (DEPs), of which 29 were up-regulated and 52 were down-regulated, were isolated and screened. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment of DEPs was observed for fatty acid metabolism. Furthermore, four candidate PMN biomarkers were identified, including H2A clustered histone 20 (H2AC20), H2B clustered histone 21 (H2BC21), splicing factor 3a subunit 1 (SF3A1), and serine and arginine rich splicing factor 1 (SRSF1). The mRNA expression levels of these genes were assessed using the qRT-PCR analysis and were found consistent with LC-MS/MS data. Moreover, the mRNA expression levels of H2AC20, H2BC21, SF3A1, and SRSF1 were significantly reduced in PMN patients compared to the healthy controls. Conclusion: In summary, H2AC20, H2BC21, SF3A1, and SRSF1 are the candidate PMN biomarkers using proteomic methods based on PBMCs. More importantly, these findings suggest a potential link between fatty acid metabolic pathways and PMN. Finally, this study provides a scientific reference for the diagnosis of PMN
Chrysin Alleviates Maternal Immune Activation-Induced Autistic-Like Behavior by Altering IL-17A Levels in Fetal and Offspring Brains of C57BL/6J Mice
Get PDFBackground: Immune dysregulation is one of the hypotheses brought up to explain autism spectrum disorder (ASD). Interleukine-17A (IL-17A), a proinflammatory cytokine, has been demonstrated to be a major mediator of immune-related neurodevelopmental impairment of social behavior, including ASD. Chrysin (CHN) is a naturally occurring hydroxylated flavonoid with antioxidant, anti-inflammatory, anti-asthmatic, anticancer, cardioprotective, and neuroprotective activities. The current study investigated the effects of CHN against Polyinosinic:polycytidylic acid (Poly (I:C))-induced autism-like behavior by modulating the fetal serotonin and IL-17A levels in fetal and offspring brains in C57BL/6J mice. Methods: Pregnant C57BL/6J mice (n = 6) were randomly selected. After the confirmation of pregnancy, female mice were divided into two different experimental groups (n = 3 female/group = 4–8 littermates/group). The pups were randomly divided into 5 experimental groups, namely, control (group I), Poly (I:C) (group II), CHN25 (group III) & CHN50 (group IV), and fluoxetine (group V). Group I and II pregnant mice were pre-treated orally with saline for 12 consecutive days (Estrus Day 0.5 (E0.5) to E12.5) and then challenged with saline (group I) and Poly (I:C) [20 mg/kg Body Weight (BW)] (group II) intraperitoneally on the 12th day (E12.5). Group III, IV & V pregnant mice were administered orally with CHN (25 mg/kg & 50 mg/kg BW) and fluoxetine (10 mg/kg, BW), respectively, for 12 consecutive days (E0.5 to E12.5) and then challenged with Poly (I:C) (20 mg/kg BW) intraperitoneally on 12th day (E12.5). In one set of studies, 1 pregnant mouse from each group was sacrificed after 4 h of Poly (I:C) injection to measure the fetal 5-Hydroxytrptophane (5-HT) and IL-17A levels in fetal brains using enzyme-linked immunosorbent assay (ELISA) kits. In the second set of experiments, the remaining pregnant mice were allowed to deliver the pups. Offspring were subjected to different behavior tests, including marble burying test (MBT), rotarod test, social interaction test (SIT) and sucrose preference test (SPT) at the age of 6, 7 and 12 weeks to investigate the autistic-like behaviors and associated symptoms. Following behavioral studies, the mice were sacrificed to isolate the prefrontal cortex (PFC), hippocampus (HC) and amygdala (AMG) tissues to measure the IL-17A levels through an ELISA kit. Results: The findings of the present study demonstrated that Poly (I:C) administration to pregnant mice resulted in maternal immune activation (MIA), as evidenced by the significant increase in IL-17A (p < 0.05) and decrease in 5-HT levels (p < 0.001) in fetal brains. Pre-treatment of CHN and fluoxetine altered the fetal 5-HT and IL-17A levels significantly (p < 0.001). Offspring of Poly (I:C) injected pregnant mice showed autistic-like behaviors and associated symptoms as evidenced by an increased number of marbles buried in MBT and decreased fall in time in the Rotarod test, sucrose preference in SPT, and social preference in SIT significantly (p < 0.001) which were ameliorated by the chronic pre-treatment of CHN (both the dosages i.e., 25 & 50 mg) and fluoxetine significantly (p < 0.001). Further, results showed the significant elevation of IL-17A levels in PFC (p < 0.001), HC (p < 0.001) and AMG (p < 0.05) of offspring of Poly (I:C) injected pregnant mice, which were attenuated significantly by the chronic pre-treatment of CHN (both the dosage i.e., 25 & 50 mg) and fluoxetine (p < 0.001). Conclusion: Findings of the study demonstrated that chronic pre-treatment of CHN attenuated autistic-like behavior by altering fetal 5-HT and IL-17A in fetal brain, PFC, HC, and AMG of offspring of MIA pregnant C57BL/6J mice. However, further investigation is required to establish the therapeutic applicability of CHN in ASD
Study on the Correlation between MDM2/p53 Interaction and Apoptosis of AEC II in Hyperoxic Acute Lung Injury
No full textBackground: Hyperoxic acute lung injury (HALI) is a pathological condition characterized by lung damage resulting from the inhalation of high concentrations of oxygen. The incidence of this disease has been increasing annually in clinical practice. Therefore, in-depth research into the pathogenesis of HALI is of great significance. This study aims to reveal the underlying regulating mechanism of mouse double minute 2 (MDM2)/p53 on Type II Alveolar Epithelial Cells (AEC II) apoptosis in HALI through in vivo and in vitro experiments. Methods: AEC II cells were isolated, purified, and cultured from healthy male Sprague Dawley (SD) rats. Subsequently, these cells were infected with an adenovirus expressing MDM2/p53 to establish a model of HALI. The proliferation activity of AEC II cells was assessed using the cell counting kit (CCK)-8 assay, and the expressions of MDM2, p53, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, and Caspase-9 mRNA were measured through real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Additionally, cell apoptosis was evaluated through terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry (FCM). Arterial blood gas analysis was performed to measure the oxygenation index (OI) and respiratory index (RI). Early apoptosis was detected by measuring mitochondrial membrane potential (MMP), and expressions of MDM2, p53, Bcl-2, Bax, Caspase-3, and Caspase-9 proteins were examined through Western blot analysis. Finally, lung tissue pathology was assessed using the hematoxylin-eosin (HE) staining method. Results: In H2O2-induced AEC II cells, an increase in apoptosis and a significant decrease in cell viability were observed, accompanied by a notable decrease in MDM2 expression and an evident increase in p53 expression (p < 0.05). Either over-expression of MDM2 or interference with p53 significantly reduced H2O2-induced AEC II apoptosis (p < 0.05), while interfering with MDM2 or over-expressing p53 significantly promoted H2O2-induced AEC II apoptosis (p < 0.05). Over-expression of MDM2 led to a significant reduction in the expressions of p53, Bax, Caspase-3, and Caspase-9, as well as a decrease in the RI and wet-to-dry weight ratio in the HALI model (p < 0.05). It also resulted in an increase in the OI and Bcl-2 expression in the HALI model (p < 0.05), alleviated the pathological state of lung tissue, and restored mitochondrial transmembrane potential in the HALI model (p < 0.05). The p53 molecular inhibitor, Pifithrin-α, significantly promoted the reduction of RI and wet-to-dry weight ratio and increased OI in the HALI model (p < 0.05). Moreover, the Pifithrin-α inhibitor was found to improve lung tissue structure in the HALI model. Conclusion: MDM2 plays a dual role in preventing the apoptosis of AEC II and protecting lung tissue by inhibiting p53 expression