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A Triptolide Derivative Enhances Antitumor Activity in Glioma Cells in Vitro
No full textBackground: Treating glioma with triptolide (TP) produces unsatisfactory outcomes. Previous studies showed that TP combined with succinic acid could sustain antitumor activity during cancer treatment. However, this activity of TP linked with succinic acid has been less investigated during the treatment of glioma. In this study, triptolide-succinic acid ester was synthesized, and its antitumor activity in glioma cells in vitro was determined. Methods: TP was coupled with succinic anhydride (SA) to obtain triptolide-succinic acid ester (TP-SAE). Cell counting kit-8 (CCK-8), transwell, wound healing assays, and flow cytometry analysis of apoptosis were used to evaluate the antitumor activity of TP-SAE in vitro. Results: Results from the cell counting kit-8 assay revealed that TP-SAE rapidly reduced proliferation of glioma cells compared with TP and TP + SA. Transwell and wound healing assays revealed that TP-SAE significantly decreased the invasion and migration of glioma cells compared with TP and TP + SA. The flow cytometry apoptosis assay indicated that apoptosis in glioma cells treated with TP-SAE was significantly higher than in those treated with TP and TP + SA. Conclusions: Triptolide-succinic acid ester could inhibit the proliferation, invasion, and migration activity of glioma cells and promote apoptosis of glioma cells in vitro
Regulatory Mechanisms of IL-19 Modulated Pathways in Osteoarthritis Progression: A Combined Analysis of Differential Gene Expression and WGCNA
No full textBackground: Osteoarthritis (OA) is a common chronic degenerative disease, and its incidence has risen in recent years. However, the specific pathogenesis of OA remains unclear. Studies have shown that interleukin-19 (IL-19) has an inhibitory effect on inflammation caused by colitis, psoriasis, and myocardial infarction. However, whether IL-19 has an inhibitory effect on OA caused by excessive inflammatory mediators is still unknown. This study aimed to investigate the downstream pathway regulation mechanism of IL-19 affecting OA progression by bioinformatics methods. Methods: Differentially expressed genes (DEGs) were identified using R language. Three OA synovial datasets (GSE55235, GSE55457, GSE55584) were downloaded from the Gene Expression Omnibus (GEO) database, including 20 normal and 26 OA synovial tissue samples. After the screening of DEGs, Gene Set Enrichment Analysis (GSEA) was performed. The effects of IL-19 on chondrocytes were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively, to investigate the gene and protein expression of apoptosis and inflammation factors. And weighted gene co-expression network analysis (WGCNA) was performed, followed by functional analysis. The improved Hulth modeling method was used to construct an OA rats model, and the expression levels of IL-19 and Janus kinase 3 (JAK3) mRNA and protein in synovial tissue were detected using qPCR and Western blot. Results: The immune infiltration of DEGs suggested the primary component of M2 macrophages in immune cells, IL-19 was markedly downregulated in OA, and immunofluorescence indicated the co-localization of IL-19 and M2 macrophages. Furthermore, the results revealed that the inhibition of IL-19 on IL-1β induced chondrocyte apoptosis and the expression of inflammatory cytokines. Following, the WGCNA analysis identified 8 distinct gene modules, among which the MEturquoise module showed a significant correlation with OA and was associated with IL-19 expression. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the PI3K-Akt signaling pathway and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, well-known upstream signal pathways of IL-19 were associated with the MEturquoise module. Western blot and qPCR results showed a significant decrease in IL-19 and JAK3 mRNA and protein expression in OA rats. Conclusions: This study revealed the potential role of IL-19 and its upstream JAK3 regulatory pathways in the development of OA using bioinformatics methods
Screening and Validation of Immune Infiltration-Related Prognostic Biomarkers for Cholangiocarcinoma
No full textBackground: Immune infiltration-related genes have reported to play important roles in the prognosis of cholangiocarcinoma (CHOL). This study aimed to screen for prognostic markers of CHOL and construct a prognostic prediction model based on prognostic markers. Methods: Immune cell infiltration was evaluated in CHOL tumor samples using the single sample Gene Set Enrichment Analysis (ssGSEA) algorithm, followed by immune clustering grouping. Based on immune grouping, differentially expressed genes (DEGs) were selected, and the prognostic markers of CHOL were screened from these DEGs through Cox regression analysis and the least absolute shrinkage and selection operator (LASSO) algorithm. A survival prognostic prediction model was constructed and validated based on the prognostic markers of CHOL. Results: The samples were divided into two clusters, and 349 intersection DEGs were identified between the tumor and normal and cluster 2 groups and cluster 1 comparison groups, which were enriched in immune response, inflammatory response, and cytokine-cytokine receptor interaction-related functions and pathways. Based on these genes, six DEGs were screened to construct a prognostic risk prediction model. In the training and validation datasets, there was a significant correlation between the actual prognosis and the different risk groups of the samples divided based on the prediction model. Conclusion: Our study established a prognostic signature associated with immune cell infiltration in patients with CHOL. This prognostic model may be used for diagnosis and prognosis of this disease
LIG1 Promotes the Development of Colorectal Cancer by Regulating UHRF1 to Promote MEG3 Methylation Level
No full textBackground: DNA ligase 1 (LIG1) can regulate ubiquitin like with PHD and ring finger domains 1 (UHRF1) to the replication site, thereby maintaining DNA methylation, a biological behavior associated with the occurrence of colorectal cancer (CRC). On this basis, this study is engineered to explore whether LIG1 could affect the development of CRC by regulating UHRF1 to promote maternally expressed 3 (MEG3) methylation. Methods: The expressions of LIG1, UHRF1, and MEG3 in CRC were analyzed using bioinformatics and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Co-Immunoprecipitation (Co-IP), Western blot, and Chromatin Immunoprecipitation (ChIP) were used to determine the interaction between UHRF1 and MEG3. Methylation of the MEG3 promoter was determined by Quantitative Methylation-Specific PCR (qMSP). The effects of LIG1 and UHRF1 on CRC cell viability, migration, invasion, and epithelial-mesenchymal transformation (EMT) were studied by loss- and gain-of-function and rescue experiments. Results: LIG1 and UHRF1 levels were up-regulated while MEG3 level was down-regulated in CRC. Overexpression of LIG1 or UHRF1 promoted the migration, invasion, and EMT of CRC cells, while shLIG1 or shUHRF1 had the opposite effect. LIG1 regulated UHRF1 to suppress MEG3 expression and promote MEG3 methylation. There existed negative interactions between shUHRF1 and overexpression of LIG1 and between shLIG1 and overexpression of UHRF1 in the regulation of CRC cells. Conclusion: LIG1 promotes CRC development by regulating UHRF1 to increase MEG3 methylation
Exploring the Effect of Cyclophosphamide on Key Genes and Signaling Pathways in Germinal Center B-Cell Lymphoma Based on Bioinformatics
No full textBackground: The diffuse large B-cell lymphoma (DLBCL) is the most prevalent lymphoma worldwide. Cyclophosphamide (CP) is an important anticancer drug used in various cancer types, including DLBCL, but its use in clinical settings is primarily limited by its dose-related cardiotoxicity. This study aimed to explore prognostic biomarkers of DLBCL in germinal center B cell (GCB) subtype, intending to develop personalized treatment regimens, monitor and assess post-treatment outcome, and improve survival rate. Furthermore, this study aimed to explore the underlying mechanism of CP and devise a more appropriate dosage plan for achieving maximum clinical benefits while minimizing toxicity. Method: GSE87371 dataset was obtained from Gene Expression Omnibus (GEO) to screen prognostic key genes for DLBCL using univariate Cox analysis, Least Absolute Shrinkage and Selection Operation (LASSO), Kaplan-Meier survival analysis, multivariate Cox regression analysis and operating characteristic curve (ROC) with area under the curve (AUC). Differentially expressed genes (DEGs) between the high and low risk scores groups were screened and analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to obtain key pathways involved in progression of DLBCL. Furthermore, the GCB DLBCL cell lines OCI-Ly1 and VAL were cultured and treated with different concentrations of CP. Moreover, cell proliferation was determined using Cell Counting Kit (CCK)-8 and colony-forming assay, cell migration activity was assessed using cell scratch and transwell, and cell apoptosis was evaluated using flow cytometry. Additionally, RT-qPCR and Western blot analyses were used to determine the expression levels of key genes and pathways involved in DLBCL. Results: The expressions of leucine zipper motif (APPL) isoform 2 (APPL2), histone cell cycle regulator (HIRA), zinc finger protein 814 (ZNF814), ZYG11A, leucine-tyrosine-arginine motif-containing protein 1 (LYRM1) and valosin-containing protein-interacting protein 1 (VCPIP1) were found to be correlated with the prognosis of DLBCL and were used in constructing prognostic model. It was found that patients in the low-risk group survived longer as compared to those in the high-risk group (p < 0.05). However, the nuclear factor-kappaB (NF-κB) and interleukin (IL)-17 pathways were enriched through functional analysis. Furthermore, CP induced the apoptosis in GCB DLCBL cells (p < 0.05) and inhibited their proliferation (p < 0.05) and migration (p < 0.05). Moreover, CP regulated the expression of 6 key genes. It was observed that CP treatment elevated the expression levels of APPL2, HIRA, ZNF814, and ZYG11A and decreased LYRM1 and VCPIP1, at both mRNA (p < 0.05) and protein (p < 0.05) levels. Additionally, CP decreased the activity of NF-κB (phosphorylation of p65 (p-p65) protein; p < 0.05) and IL-17 (IL-17 mRNA, p < 0.05; IL-17 protein, p < 0.05) pathways. Conclusion: APPL2, HIRA, ZNF814, ZYG11A, LYRM1 and VCPIP1 were identified as key genes involved in the prognosis of DLBCL. Moreover, CP could up-regulate the expressions of APPL2, HIRA, ZNF814, ZYG11A, down-regulate the expressions of LYRM1 and VCPIP1, restrain both NF-κB and IL-17 pathways, suppress cell proliferation, migration while promoting cell apoptosis, thus inhibiting the progression of GCB DLBCL
Anticoccidial Efficacy of Sheep Bile as a Potential Therapy Alternative for Eimeria columbae Infection in Domestic Pigeons: An in Vitro Study
No full textBackground: Eimeria columbae (E. columbae) is a disease that threatens domestic pigeon health and productivity due to resistance and the side effects of treatments. Therefore, there is a pressing need to explore alternative therapies to manage E. columbae infection. This study investigates the possibility of bile application as an alternative treatment to assess its anticoccidial efficacy and determine the concentration with the most substantial effect on oocysts and sporozoites. Methods: We subjected samples of oocysts to several concentrations of bile in a solution containing 2.5% potassium dichromate at 12.5%, 25%, 50%, 75%, and 100% to assay oocyst vitality during the course of 24, 48, 72, and 96 h. In addition, we used 125, 250, 500, 750, and 1000 μg/mL concentrations to determine anti-sporozoite efficacy after 12 and 24 h. Results: The infrared analysis of the bile showed many bioactive components associated with plant secondary metabolites. Furthermore, the phenol and flavonoid totals were estimated. The results showed statistically significant differences at p < 0.05 in the 100% and 75% concentrations whereby sheep bile liquid could inhibit E. columbae oocysts by approximately 94% and 81%, respectively. In addition, bile also demonstrated maximum inhibition of E. columbae sporozoite vitality (92%) at a dose of 1000 g/mL and minimum inhibition of 8% at a concentration of 125 g/mL. The sporulation percentage was highly negatively correlated with the exposure time (R = –0.97, p < 0.0001; R = –0.80, and p < 0.001) at 100% and 75% concentrations, respectively. Thus, inhibiting oocysts was found to prevent infection and enhance the birds health, which contributes to improving the quality of the product, both quantitatively and qualitatively. Conclusions: The overall results of the current study suggest that sheep bile possesses inhibitory properties for oocyst sporulation, meaning that it could be employed in coccidia management
Biological Assessment, GC-MS Analysis, and Molecular Docking Investigation on the Neuropharmacological, Anti-Diarrhoeal, and Cytotoxic Properties of Ficus semicordata Fruits
Get PDFBackground: Ficus semicordata Buch. is a well-known ethnomedicinal plant that is used to treat various ailments such as colic pain, urogenital difficulties, gastrointestinal disorders, visceral blockage, leprosy, jaundice, diabetes, and hepatitis. This study aims to investigate the phytochemical contents of the metabolites extracted (methanol) from the fruits of Ficus (F.) semicordata, and determine their neuropharmacological, anti-diarrhoeal, and cytotoxic potencies, using in vivo, in vitro, and in silico methods. Methods: The pharmacological properties of methanol extract of Ficus semicordata fruits (MEFSF) were assessed at different concentrations and its toxicity was determined using the in vitro brine shrimp lethality test. Tail suspension and forced swimming tests were used to investigate the antidepressant activity of MEFSF in mice and elevated plus maze and hole board test models were used to uncover its anxiolytic potentiality. The in vivo anti-diarrhoeal properties of MEFSF were tested on castor oil-induced diarrhoea and gastrointestinal motility models. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted using a mass spectrometer. Based on the GC-MS analysis, 19 phytochemicals were investigated using molecular docking techniques against various target proteins to determine whether they mediate cytotoxic, depressive, anxiolytic, and anti-diarrhoeal effects. Results: MEFSF exhibited moderate toxicity (median lethal dose (LD50): 267.23 μg/mL). In the antidepressant assessment, MEFSF demonstrated a significant (p < 0.0001) dose-dependent decrease in immobility compared to fluoxetine. Similarly, MEFSF exhibited a dose-dependent reduction in anxiolytic-like behaviour in mice, with a 400 mg/kg dose exhibiting vigorous activity. MEFSF also significantly inhibited motility in both anti-diarrhoeal models, with a 400 mg/kg dose exhibiting highly significant (p < 0.0001) suppression. The GC-MS analysis revealed 81 bioactive components. Seven phytochemicals exhibited a strong affinity for various target proteins in the molecular investigation. Notably, beta-D-glucopyranose and 4-O-beta-D-galactopyranosyl manifested a high affinity for hER, K+ channel, SERT3, and M3MAR and exhibited cytotoxic, anxiolytic, antidepressant, and anti-diarrhoeal potential. Conclusion: The findings indicate that MEFSF can potentially contribute to the development of innovative anti-cancer, neuropharmacological, and anti-diarrhoeal treatments. However, additional research is necessary to explore this possibility
SPI1 Regulates Neural Stem Cell Differentiation by Inhibiting FTO-Regulated m6A Modification of IL6R
No full textBackground: The comprehensive role of salmonella pathogenicity island 1 (SPI1) in neural stem cells (NSCs) remains unclear. Therefore, this study aimed to explore the effect of SPI1 on the differentiation of NSCs. Methods: NSCs were administered with lipopolysaccharides (LPS, 100 ng/mL) to construct an NSC model in vitro. Relative mRNA expression levels of SPI1 and fat mass and obesity-associated protein (FTO) were assessed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Protein levels of phosphor (p)-janus kinase 1 (JAK1), p-signal transducer and activator of transcription 3 (STAT3), and interleukin-6 receptor (IL6R) were determined using Western blot analysis. Furthermore, the viability of NSCs was assessed using a cell counting kit-8 assay. NSC identification and neurite outgrowth were observed using immunofluorescence. The N6-methyladenosine (m6A) modification sites of IL6R were determined using the RNA Modification Base Database (RMBase). Additionally, the correlation between FTO and IL6R was confirmed through RNA-binding protein immunoprecipitation-qRT-PCR. Results: NSCs exhibited an increase in mRNA level of SPI1 over time, reaching to peak at day 12 (p < 0.001). Moreover, LPS treatment significantly reduced the expression of SPI1 and neurite growth, and inhibited the JAK1/STAT3 pathway, while enhancing the expression of FTO (p < 0.01). The inhibitory impact of LPS was enhanced by silencing SPI1, while SPI1 overexpression reversed its inhibitory effects on NSCs (p < 0.05). Furthermore, IL6R was predicted to contain m6A modification sites. The increased FTO expression in LPS-treated NSCs inhibited the growth of neurite, the expression levels of the JAK1/STAT3 pathway, and IL6R (p < 0.01). Moreover, silencing SPI1 enhanced the effect of FTO, while overexpression of SPI1 reversed this effect (p < 0.05). Conclusions: SPI1 negatively regulates FTO expression, thereby elevating m6A modification of IL6R, hence promoting NSC differentiation. This finding presents a novel strategy for propelling the clinical application of NSCs
Regulating Mitochondrial Biogenesis via Pachymaran: A Novel Treatment Strategy for Diabetic Cardiomyopathy
Get PDFBackground: Diabetic cardiomyopathy (DCM) is a common complication among diabetic patients, yet its pathogenesis is not fully understood. Mitochondrial dysfunction and oxidative stress play important roles in the development of DCM. Pachymaran (PPS), a natural compound with various biological properties, has not been extensively investigated in DCM. This study aims to explore the role and mechanism of PPS in DCM. Methods: DCM model mice were initially divided into control, DCM, and PPS treatment groups (50 mg/kg and 100 mg/kg). Subsequently, we evaluated the effects of PPS on DCM via myocardial histopathological analysis, cardiac function assessment, and mitochondrial function detection. In addition, Western blot and real-time quantitative PCR were used to study the regulatory effects of PPS on mitochondrial biogenesis and Sirtuin 3 (SIRT3). Results: PPS treatment in DCM mice exhibited significant myocardial protection, evident from reduced myocardial fibrosis upon histopathological examination (p < 0.05). Furthermore, cardiac function assessment revealed a significant improvement in myocardial contractile function in the PPS treatment group (p < 0.01). Moreover, enhanced activity of mitochondrial respiratory chain complexes and ATP synthesis capacity was observed in the PPS treatment group (p < 0.01), indicating improved mitochondrial function. Further research revealed that PPS significantly increased the expression level of SIRT3 and promoted expression of key regulators of mitochondrial biogenesis (p < 0.01). Conclusion: The results of this study indicate that PPS protects against DCM by activating mitochondrial biogenesis and SIRT3. PPS can inhibit myocardial fibrosis, enhance myocardial contractile function, and improve mitochondrial function. Additionally, PPS can regulate SIRT3 expression and promote mitochondrial biogenesis. Therefore, PPS may be a potential drug for DCM treatment, proposing a new strategy for DCM treatment
Increased FTO Expression Demethylates XBP1 m6A, Thereby Regulating XBP1-C/EBPα and Promoting Hepatocellular Carcinoma Growth
Get PDFBackground: N6-methyladenosine (m6A) modification predominantly occurs in cancer cells mRNA. The X-box binding protein 1 (XBP1) influences hepatocellular carcinoma (HCC) progression, but its m6A regulatory mechanism remains unclear. Furthermore, the dysregulation of CCAAT/enhancer binding proteins alpha (C/EBPα) in liver cancer is influenced by fat mass and obesity-associated protein (FTO) and acts downstream of XBP1. Therefore, this study aims to investigate how FTO catalyzes XBP1 m6A demethylation in HCC regulation. Methods: Initially, HepG2 cells were used to construct FTO overexpression and knockdown cells. The cells were divided into the FTO overexpression group (oe-FTO), overexpression control group (oe-NC), FTO knocked-down group (sh-FTO), and control of FTO knocked-down group (sh-NC) groups. RNA immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR) was used to determine the interaction between FTO and XBP1. Furthermore, quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) analysis were utilized to assess the expression levels of XBP1 and C/EBPα. Additionally, subcutaneous transplanted tumor models were constructed and the tumor size, weight, and occurrence time were monitored. Moreover, Hematoxylin-Eosin (H&E) staining was employed to observe the pathological changes of tumors. m6A immunoprecipitation (MeRIP)-qPCR was used to evaluate the XBP1 m6A modification levels. qRT-PCR and WB analysis were used to determine the expression levels of XBP1 and C/EBPα. Results: We observed that FTO specifically binds to XBP1 mRNA in HCC cells, indicating a potential regulatory role at the RNA level. At the cellular level, compared to the sh-NC and oe-NC groups, the m6A methylation level of XBP1 was significantly increased in the sh-FTO group, while it was decreased in the oe-FTO group (p < 0.05). Furthermore, the mRNA and protein expression levels of FTO, XBP1, and C/EBPα were altered following FTO manipulation. Functional assays demonstrated that FTO overexpression promoted cell proliferation and invasion while inhibiting apoptosis. Conversely, FTO knockdown resulted in decreased cell proliferation and invasion and increased apoptosis. In a mouse xenograft tumor model, we observed rapidly growing tumors in the oe-FTO group, whereas sh-FTO tumors exhibited slower growth. Histological analysis revealed distinct patterns of tumor growth and damage. Collectively, these findings suggest that FTO plays a crucial role in HCC progression through its effects on XBP1 and C/EBPα, providing insights into the potential therapeutic intervention of FTO in hepatocellular carcinoma. Conclusion: FTO overexpression leads to m6A demethylation of XBP1, thereby modulating the expression of XBP1-C/EBPα and suppressing cell apoptosis. This, in turn, facilitates the progression of hepatocellular carcinoma by promoting cell growth