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关键词： Diabetes   Glibenclamide   Vitamin B1   Calcitriol   Nephropathy  

摘要： Glibenclamide is one of the most prescribed insulin secretagogues in diabetes due to its low cost, but its efficacy on suppressing diabetic complications is limited. Here, we examine whether addition of either vitamin B1 or calcitriol to glibenclamide could produce more suppression of diabetic nephropathy. Type 2 diabetes was induced by high fructose (10 % in drinking water), high salt (3 % in diet), and high fat diet (25 % in diet) for 3 weeks, followed by single dose of STZ (40 mg/kg, i.p.). Diabetic rats were treated with either glibenclamide (0.6 mg/kg), vitamin B1 (70 mg/kg), glibenclamide/vitamin B1, calcitriol (0.1 mu g/kg), or glibenclamide/calcitriol. Addition of either vitamin B1 or calcitriol to glibenclamide therapy enabled more suppression of diabetic nephropathy development as evidenced by more preserved creatinine clearance and less renal damage scores. Combination therapy resulted in mild enhancement in the effect of glibenclamide on glucose tolerance without affecting the area under the curve. Combination therapy was associated with more suppression of inflammatory cascades as evidenced by reducing the expression of high mobility group box-1 (HMGB1), toll-like receptor-4 (TLR4), nuclear factor-kappa B (NF-kappa B), and tumor necrosis factor-alpha (TNF-alpha). In addition, combination therapy enhanced the antioxidant mechanisms as evidenced by increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and glutathione content and reducing malondialdehyde and nitric oxide levels. Furthermore, combination therapy provided more suppression of fibrotic pathways as appear from reducing collagen deposition and the expression of alpha- smooth muscle actin (alpha-SMA). In conclusion, addition of vitamin B1 or calcitriol to glibenclamide therapy can enhance the therapeutic efficiency of glibenclamide in suppressing diabetic nephropathy progression to the same extend, the protective effect is mediated through modulating HMGB1/TLR4/ NF-kappa B/TN

关键词： 猪细小病毒   NS1蛋白   多克隆抗体   原核表达  

摘要： 本研究旨在制备PPV-NS1的多克隆抗体，并用猪细小病毒（PPV）和真核质粒pcDNA3.1(+)-NS1-3×Flag进行双重验证。首先将PPV的非结构蛋白编码基因NS1克隆至原核表达载体pETDuet，构建原核质粒pET-PPV-NS1。接着，将原核质粒pET-PPV-NS1转化至感受态*** BL21细胞中，经由1 mmol/L IPTG诱导以后，通过SDS-PAGE和Western-blot对NS1表达进行检测，结果表明，NS1基因在感受态细胞中得到了良好的表达。其次，将纯化后获得的NS1蛋白采用多点注射的方式对大白兔进行免疫，制备获得针对重组NS1蛋白的多克隆抗体。然后，将真核质粒pcDNA3.1(+)-NS1-3×Flag转染至HEK 293T细胞；同时PPV感染ST细胞后收取蛋白样品，用上述制备的NS1多克隆抗体进行验证。结果显示，制备的NS1多克隆抗体具有良好的反应性和特异性。

关键词： Interverbral disc degeneration   IL-32   FAT4   TNF-alpha   YAP  

摘要： Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases;however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-alpha (TNF-alpha). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-alpha-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL

关键词： New therapeutic target   Nicardipine   Liver inflammatory diseases   Chitosan nanoparticles   NF kappa B/NLRP3/IL-1 beta signaling   Liver biodistribution  

摘要： Liver inflammatory diseases are marked by serious complications. Notably, nicardipine (NCD) has demonstrated anti-inflammatory properties, but its benefits in liver inflammation have not been studied yet. However, the therapeutic efficacy of NCD is limited by its short half-life and low bioavailability. Therefore, we aimed to evaluate the potential of NCD-loaded chitosan nanoparticles (ChNPs) to improve its pharmacokinetic profile and hepatic accumulation. Four formulations of NCD-ChNPs were synthesized and characterized. The optimal formulation (NP2) exhibited a mean particle diameter of 172.6 + 1.94 nm, a surface charge of +25.66 + 0.93 mV, and an encapsulation efficiency of 88.86 + 1.17 %. NP2 showed good physical stability as a lyophilized powder over three months. It displayed pH-sensitive release characteristics, releasing 77.15 + 5.09 % of NCD at pH 6 (mimicking the inflammatory microenvironment) and 52.15 + 3.65 % at pH 7.4, indicating targeted release in inflamed liver tissues. Pharmacokinetic and biodistribution studies revealed that NCD-ChNPs significantly prolonged NCD circulation time and enhanced its concentration in liver tissues compared to plain NCD. Additionally, the study investigated the protective effects of NCD-ChNPs in thioacetamide-induced liver injury in rats by modulating the NF kappa B/NLRP3/IL-1(3 signaling axis. NCD-ChNPs effectively inhibited NF kappa B activation, reduced NLRP3 inflammasome activation, and subsequent release of IL-1(3, which correlated with improved hepatic function and reduced inflammation and oxidative stress. These findings highlight the potential of NCD-ChNPs as a promising nanomedicine strategy for the treatment of liver inflammatory diseases, warranting further investigation into their clinical applications, particularly in hypertensive patients with liver inflammatory conditions.

关键词： type 1 diabetes   hybrid insulin peptides   islet-infiltrating CD4(+) T cells   neoepitopes  

摘要： Type 1 diabetes (T1D) is an autoimmune disease that develops when T cells destroy the insulin-producing beta cells that reside in the pancreatic islets. Immune cells, including T cells, infiltrate the islets and gradually destroy the beta cells. Human islet-infiltrating CD4(+)T cells recognize peptide epitopes derived from proinsulin, particularly C-peptide. Hybrid insulin peptides (HIPs) are neoepitopes formed by the fusion of two peptides derived from beta cell granule proteins and are known to be the targets of pathogenic CD4(+)T cells in the non-obese diabetic (NOD) mouse and human islet-infiltrating CD4(+)T cells. Proinsulin is widely recognized as a central antigen in T1D, but its role in forming HIPs is unclear. We developed a method to functionally screen TCRs derived from human islet- infiltrating CD4(+)T cells and applied this to the identification of new proinsulin-derived HIPs. We generated a library of 4,488 candidate HIPs formed by fusion of proinsulin fragments and predicted to bind to HLA-DQ8. This library was screened against 109 islet-infiltrating CD4(+)T cell receptors (TCRs) isolated from four organ donors who had T1D. We identified 13 unique HIPs recognized by nine different TCRs from two organ donors. HIP-specific T cell avatars responded specifically to a peptide extract from human islets. These new HIPs predominantly stimulated CD4(+)T cell proliferation in peripheral blood mononuclear cells from individuals with T1D in contrast to HLA-matched controls. This is the first unbiased functional, islet-infiltrating T cell based, screen to identify proinsulin-derived HIPs. It has revealed many new HIPs and a central role of proinsulin C-peptide in their formation.

关键词： G-CSF   IL-1 beta   BMSC senescence   Osteomyelitis   Bone loss   JNK  

摘要： Staphylococcus aureus (S. aureus)-induced bone loss is a significant challenge in the treatment of osteomyelitis. Our previous study was the first to confirm that granulocyte colony-stimulating factor (G-CSF) mediates S. aureus-induced bone loss. However, the underlying mechanism remains unknown. The objective of this study was to elucidate this. We found G-CSF mediated BMSC senescence and increased IL-1(3 concentration of serum and bone marrow in mice after S. aureus infection. Furthermore, we demonstrated that G-CSF promoted the expression of IL1b in murine bone marrow-derived neutrophils. Notably, we identified that IL-1(3 mediated BMSC (bone marrow mesenchymal stromal cell) senescence in mice after S. aureus infection. Importantly, IL-1(3 neutralizing antibody effectively alleviated BMSC senescence and bone loss caused by S. aureus infection in mice. In terms of molecular mechanism, we found IL-1(3 induced BMSC senescence by JNK/P53 and JNK/BCL2 pathways. Collectively, G-CSF promotes IL-1(3 production which induces BMSC senescence via JNK/P53 and JNK/BCL2 pathways, leading to S. aureus-induced bone loss. This study identified novel targets for preventing and treating S. aureus-induced bone loss in osteomyelitis.

关键词： Dual-mode aptasensor   G-quadruplex   Rolling circle amplification   Interferon gamma  

摘要： Background: Aptasensors have been extensively utilized in target detection due to their advantages of high sensitivity and fast response. However, the reliability of the detection results of the single-mode aptasensor cannot be verified in time. Developing efficient detection methods with cross-validation capability is beneficial to achieving highly reliable detection. This study aims to design a colorimetric and fluorescent dual-mode aptasensor by skillfully engineering G-quadruplex assembly and rolling circle amplification for highly reliable IFN-gamma detection. Results: The complexes of anti -IFN-gamma aptamers and complement sequences (cDNA) were modified on the magnetic beads. In the presence of IFN-gamma, the preferential combination of aptamers with IFN-gamma resulted in the release of cDNAs. The cDNAs were collected by magnetic separation and then used as primers to trigger rolling circle amplification reaction to generate enriched G-quadruplexes. The G-quadruplex could be utilized to combine with hemin to catalyze the oxidation of 3,3 ',5,5 '-tetramethylbenzidine for colormetric mode or to couple with the fluorogenic dye Thioflavin T for fluorescent mode. The developed dual-mode aptasensor displayed a linear range of 1-10000 pM with a detection limit of 0.406 pM for the colormetric mode and a range of 0.1-10000 pM with detection limit of 0.037 pM for the fluorescent mode. Further, the designed aptasensor was applied to IFN-gamma detection in serum samples and achieved satisfactory recoveries. Significance: This innovative dual-mode detection strategy skillfully leverages the effective target-binding ability of aptamer, dual-function of the G-quadruplex and the signal amplifying ability of rolling circle amplification. This approach not only provides a reliable testing tool for the detection of IFN-gamma, but also promotes the development of multimode sensing platforms.

摘要： Type 1 diabetes (T1D) is a chronic autoimmune disease that is caused by a combination of genetic and environmental risk factors. In this study, we sought to determine whether a known genetic risk factor, the rs1990760 single nucleotide polymorphism (SNP) (A946T) in IFIH1, resulted in a gain of function in the MDA5 protein and the effects of this mutation on the regulation of type I IFNs during infection with the diabetogenic virus coxsackievirus B3. We found that in cell lines overexpressing the risk variant IFIH1946T there was an elevated level of basal type I IFN signaling and increased basal IFN-stimulated gene expression. An investigation into the mechanism demonstrated that recombinant MDA5 with the A946T mutation had increased ATPase activity in vitro. We also assessed the effect of this SNP in primary human PBMCs from healthy donors to determine whether this SNP influenced their response to infection with coxsackievirus B3. However, we observed no significant changes in type I IFN expression or downstream induction of IFN-stimulated genes in PBMCs from donors carrying the risk allele IFIH1946T. These findings demonstrate the need for a deeper understanding of how mutations in T1D-associated genes contribute to disease onset in specific cellular contexts. The Journal of Immunology, 2024, 213: 1415-1428.

关键词： Adipose   single-nucleus RNA sequencing   Glial cells   Macrophages  

摘要： Macrophages in obese adipose tissue have been shown to damage nerve fibers, however, the mechanism underlying how macrophages cause glial cell damage remains unknown. This study aimed to characterize the mechanism by which macrophages induce apoptosis in glial cell during obesity formation in mice by single- nucleus RNA sequencing (snRNA-seq). Cells obtained from paraepididymal adipose tissue in obese mice underwent snRNA-seq. Eighteen different clusters were identified, and 12 cell types were annotated, including glial cells, macrophages, and fibroblasts. There was a negative correlation between the number of glial cells and macrophages in mouse adipose tissue during the formation of obesity. The pro-apoptotic factor PHLPP1 was identified in GO Terms. The interaction between adipose tissue glial cells and macrophages was revealed via indepth analysis, and the cell-cell communication mechanism between the TNF-alpha and NF-KB/PHLPP1 axes was perfected. Apoptosis of glial cell by upregulation of TNF-alpha via obesity-derived macrophages and activation of the NF-kappa B/PHLPP1 axis. We further revealed how macrophages induce apoptosis in glial cells during obesity formation, as well as different changes in the two cell populations. This study provides valuable resources and foundations for understanding the mechanistic effects of macrophages and glial cells during obesity formation, as well as diseases and potential interventions.

关键词： Drug-induced liver injury   Emodin   Human leukocyte antigen   Liver injury   Polygonum multiflorum thunb.   T-cell response  

摘要： Ethnopharmacological relevance: HLA-B*35:01 has been identified as a risk allele for Polygonum multiflorum Thunb.-induced liver injury (PMLI). However, the immune mechanism underlying HLA-B*35:01-mediated PMLI remains unknown. Aim of the study: To characterize the immune mechanism of HLA-B*35:01-mediated PMLI. Materials and methods: Components of P. multiflorum (PM) bound to the HLA-B*35:01 molecule was screened by immunoaffinity chromatography. Both wild-type mice and HLA-B*35:01 transgenic (TG) mice were treated with emodin. The levels of transaminases, histological changes and T-cell response were assessed. Splenocytes from emodin-treated mice were isolated and cultured in vitro. Phenotypes and functions of T cells were characterized upon drug restimulation using flow cytometry or ELISA. Emodin-pulsed antigen-presenting cells (APCs) or glutaraldehyde-fixed APCs were co-cultured with splenocytes from emodin-treated transgenic mice to detect their effect on T-cell activation. Results: Emodin, the main component of PM, could non-covalently bind to the HLA-B*35:01-peptide complexes. TG mice were more sensitive to emodin-induced immune hepatic injury, as manifested by elevated aminotransferase levels, infiltration of inflammatory cells, increased percentage of CD8+T cells and release of effector molecules in the liver. However, these effects were not observed in wild-type mice. An increase in percentage of T cells and the levels of interferon-gamma, granzyme B, and perforin was detected in emodin-restimulated splenocytes from TG mice. Anti-HLA-I antibodies inhibited the secretion of these effector molecules induced by emodin. Mechanistically, emodin-pulsed APCs failed to stimulate T cells, while fixed APCs in the presence of emodin could elicit the secretion of T cell effector molecules. Conclusion: The HLA-B*35:01-mediated CD8+ T cell reaction to emodin through the P-I mechanism may contribute to P. multiflorum-induced liver injury.