Antipyretic and anti-inflammatory effects of inosine, an active component of Kangfuxin.

Kangfuxin has been widely recognized for its use in treating ulcerative conditions and mucositis, primarily due to its anti-inflammatory properties, which promote cell proliferation, granulation tissue growth, and angiogenesis. However, the exact mechanisms underlying these effects remain poorly understood. In this study, we employed high-throughput mass spectrometry to identify 11 compounds in Kangfuxin, including uracil, hypoxanthine, xanthine, inosine, glutamic acid, glycine, alanine, valine, isoleucine, leucine, and lysine. Notably, the antipyretic and anti-inflammatory properties of inosine, one of these compounds, have not been well characterized. To address this gap, we induced fever in vivo using lipopolysaccharide (LPS) and conducted various experiments, including the analysis of endogenous mediators, inflammatory factors, quantitative polymerase chain reaction (QPCR), Western blotting, and hematoxylin and eosin (HE) staining. Our findings indicate that inosine significantly reduces LPS-induced fever, inhibits the expression of inflammatory factors, and alleviates the inflammatory response. These results suggest that inosine may serve as a potential therapeutic target for inflammatory diseases.

RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP.

BACKGROUND: Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. METHODS: The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. RESULTS: The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. CONCLUSION: RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

MCM6 is a Poor Prognostic Biomarker and Promotes Progression in Breast Cancer.

BACKGROUND: Breast cancer is the commonest global malignancy and the primary cause of carcinoma death. MCM6 is vital to carcinogenesis, but the pathogenesis of MCM6 remains unclear. METHODS: MCM6 expression in patients with breast cancer was examined through The Cancer Genome Atlas (TCGA) database, immunohistochemistry, Quantitative Real-Time PCR (qRT‒PCR) and Western blotting. The prognostic factors were assessed by the Kaplan‒Meier method and Cox regression. On the basis of the key factors selected by multivariable Cox regression analysis, a nomogram risk prediction model was adopted for clinical risk assessment. The TCGA database was utilized to determine how MCM6 is correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, along with tumor mutation burden (TMB) and methylation. The impact of MCM6 on carcinoma cells was investigated in terms of proliferation, cell cycle as well as migrating and invasive behavior through CCK assays, flow cytometry, wound healing assays, Transwell assays and xenotransplantation experiments. RESULTS: MCM6 expression was upregulated, which is closely associated with the size of the tumor (p = 0.001) and lymph node metastasis (p = 0.012) in patients with breast cancer. Multivariate analysis revealed MCM6 to be an independent risk factor for prognosis in patients with breast carcinoma. The nomograph prediction model included MCM6, age, ER, M and N stage, which displayed good discrimination with a C index of 0.817 and good calibration. Overexpression of MCM6 correlated with chemotherapy sensitivity, immune checkpoint-related genes (ICGs), tumor-infiltrating immune cells, tumor mutation burden (TMB), and methylation. Silencing MCM6 significantly inhibited proliferation, prolonged the G1 phase of the cell cycle, and restrained the proliferation, migration and invasive behavior of cancerous cells and inhibited tumor growth in vivo. CONCLUSIONS: Our research shows that MCM6 is highly expressed in breast cancer and can be used as an independent prognostic factor, which is expected to become a new target for the treatment of breast cancer in the future.

Establishment of a N1-methyladenosine-related risk signature for breast carcinoma by bioinformatics analysis and experimental validation.

OBJECTIVES: Breast carcinoma (BRCA) has resulted in a huge health burden globally. N1-methyladenosine (m1A) RNA methylation has been proven to play key roles in tumorigenesis. Nevertheless, the function of m1A RNA methylation-related genes in BRCA is indistinct. METHODS: The RNA sequencing (RNA-seq), copy-number variation (CNV), single-nucleotide variant (SNV), and clinical data of BRCA were acquired via The Cancer Genome Atlas (TCGA) database. In addition, the GSE20685 dataset, the external validation set, was acquired from the Gene Expression Omnibus (GEO) database. 10 m1A RNA methylation regulators were obtained from the previous literature, and further analyzed through differential expression analysis by rank-sum test, mutation by SNV data, and mutual correlation by Pearson Correlation Analysis. Furthermore, the differentially expressed m1A-related genes were selected through overlapping m1A-related module genes obtained by weighted gene co-expression network analysis (WGCNA), differentially expressed genes (DEGs) in BRCA and DEGs between high- and low- m1A score subgroups. The m1A-related model genes in the risk signature were derived by univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analyses. In addition, a nomogram was built through univariate and multivariate Cox analyses. After that, the immune infiltration between the high- and low-risk groups was investigated through ESTIMATE and CIBERSORT. Finally, the expression trends of model genes in clinical BRCA samples were further confirmed by quantitative real-time PCR (RT‒qPCR). RESULTS: Eighty-five differentially expressed m1A-related genes were obtained. Among them, six genes were selected as prognostic biomarkers to build the risk model. The validation results of the risk model showed that its prediction was reliable. In addition, Cox independent prognosis analysis revealed that age, risk score, and stage were independent prognostic factors for BRCA. Moreover, 13 types of immune cells were different between the high- and low-risk groups and the immune checkpoint molecules TIGIT, IDO1, LAG3, ICOS, PDCD1LG2, PDCD1, CD27, and CD274 were significantly different between the two risk groups. Ultimately, RT-qPCR results confirmed that the model genes MEOX1, COL17A1, FREM1, TNN, and SLIT3 were significantly up-regulated in BRCA tissues versus normal tissues. CONCLUSIONS: An m1A RNA methylation regulator-related prognostic model was constructed, and a nomogram based on the prognostic model was constructed to provide a theoretical reference for individual counseling and clinical preventive intervention in BRCA.

A surface metal ion-modified 3D-printed Ti-6Al-4V implant with direct and immunoregulatory antibacterial and osteogenic activity.

The high concentration of antibacterial metal ions may exhibit unavoidable toxicity to cells and normal tissues. The application of antibacterial metal ions to activate the immune response and induce macrophages to attack and phagocytose bacteria is a new antimicrobial strategy. Herein, 3D-printed Ti-6Al-4V implants modified by copper, and strontium ions combined with natural polymers were designed to treat implant-related infections and osseointegration disorders. The polymer-modified scaffolds rapidly released a large amount of copper and strontium ions. During the release process, copper ions were employed to promote the polarization of M1 macrophages, thus inducing a proinflammatory immune response to inhibit infection and achieve the immune antibacterial activity. Meanwhile, copper and strontium ions promoted the secretion of bone-promoting factors by macrophages, induced osteogenesis and showed immunomodulatory osteogenesis. This study proposed immunomodulatory strategies based on the immunological characteristics of target diseases and provided ideas for the design and synthesis of new immunoregulatory biomaterials.

CMTM6 is highly expressed in lung adenocarcinoma and can be used as a biomarker of a poor diagnosis.

Background: CMTM6 which is chemokine-like factor (CKLF)-like Marvel transmembrane domain containing family member 6 is involved in the occurrence and progression of various tumors. However, the role of CMTM6 is still unclear in lung adenocarcinoma (LUAD). Methods: Immunohistochemical, Western blotting and RT‒PCR methods were used to detect the expression of CMTM6 in LUAD. Cox regression and the Kaplan‒Meier method were performed to assess overall survival. Immunogenic features were evaluated according to immune cell infiltrations, immune checkpoints. The sensitivity to chemotherapy agents was estimated using the pRRophetic package. Results: In LUAD, the expression of CMTM6 was obviously upregulated and was significantly associated with T stage (p = 0.008) and lymph node metastasis (p = 0.018). Multivariate Cox regression analysis demonstrated that CMTM6 was a specialty prognostic risk factor. Based on GSEA enrichment analysis, we found that high expression of CMTM6 is associated with multiple immune signaling pathways. The group with high CMTM6 expression showed a positive association with various types of tumor-infiltrating cells. Moreover, a total of 36 chemotherapeutic drugs were significantly correlated with the expression of CMTM6. Among them, two chemotherapeutic drugs had better therapeutic effects in the high CMTM6 expression group, while 34 chemotherapeutic drugs had therapeutic effects in the low CMTM6 expression group. Conclusion: This study confirmed that CMTM6 is highly expressed in LUAD and is a new independent poor prognostic factor. In addition, the high expression of CMTM6 is closely related to the tumor microenvironment and immunotherapy, providing new ideas for the treatment of posterior LUAD.

ZNF24 regulates the progression of KRAS mutant lung adenocarcinoma by promoting SLC7A5 translation.

Background: Clinical treatment of RAS mutant cancers is challenging because of the complexity of the Ras signaling pathway. SLC7A5 is a newly discovered downstream gene of the Ras signaling pathway, but the regulatory mechanism is unclear. We aimed to explore the molecular mechanism and role in KRAS mutant lung adenocarcinoma progression. Methods: Key gene that regulated SLC7A5 in KRAS mutant lung adenocarcinoma was screened by RNA sequencing and bioinformatics analysis. The effect of this gene on the expression of SLC7A5 was studied by RNAi. The regulatory mechanism between the two genes was investigated by immunofluorescence, CoIP, pulldown and yeast two-hybrid assays. The location of the two genes was determined by inhibiting Ras and the downstream pathways PI3K-AKT and MEK-ERK. By in vivo and in vitro experiments, the effects of the key gene on the biological functions of KRAS mutant lung adenocarcinoma were explored. Results: We found a novel gene, ZNF24, which upregulated SLC7A5 protein expression rather than mRNA expression in KRAS mutant lung adenocarcinoma. Endogenous protein interactions occurred between ZNF24 and SLC7A5. Ras inhibition reduced the expression of ZNF24 and SLC7A5. ZNF24 and SLC7A5 are located downstream of the MEK-ERK and PI3K-AKT pathways. In vivo and in vitro functional experiments confirmed that the ZNF24-SLC7A5 signaling axis promoted the proliferation, invasion and migration of KRAS mutant lung adenocarcinoma. Conclusions: ZNF24 promoted the growth of KRAS mutant lung adenocarcinoma by upregulating SLC7A5 protein expression, which suggested that ZNF24 is a new biomarker of KRAS mutant tumors and could be a new potential therapeutic target for Ras-driven tumors.