RESUMO
The secondary amyloidosis of humans is caused by the formation of hSAA fibrils in different organs and tissues. Until now hSAA was thought to have low amyloidogenicity in vitro and the majority of SAA aggregation experiments were done using murine protein or hSAA non-pathogenic isoforms. In this work a novel purification method for recombinant hSAA was introduced, enabling to obtain monomeric protein capable of amyloid aggregation under physiological conditions. The stability and amyloid aggregation of hSAA have been examined using a wide range of biophysical methods. It was shown that the unfolding of monomeric protein occurs through the formation of molten globule-like intermediate state. Polymorphism of hSAA amyloids was discovered to depend on the solution pH. At pH 8.5, rapid protein aggregation occurs, which leads to the formation of twisted short fibrils. Even a slight decrease of the pH to 7.8 results in delayed aggregation with the formation of long straight amyloids composed of laterally associated protofilaments. Limited proteolysis experiments have shown that full-length hSAA is involved in the formation of intermolecular interactions in both amyloid polymorphs. The results obtained, and the experimental approach used in this study can serve as a basis for further research on the mechanism of authentic hSAA amyloid formation.
Assuntos
Amiloide , Amiloidose , Dobramento de Proteína , Proteína Amiloide A Sérica , Humanos , Amiloidose/metabolismo , Concentração de Íons de Hidrogênio , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/isolamento & purificação , Amiloide/química , Amiloide/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genéticaRESUMO
BACKGROUND: MG132, a proteasome inhibitor, is widely used to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity by proteasome-mediated degradation of IκB. It has been marketed as a specific, reversible, cell-permeable and low-cost inhibitor. However, adverse effects of the compound have been reported in the literature. We recently discovered and characterised a point mutation in the acute phase protein serum amyloid A (SAA) in chickens, by overexpressing the protein in chicken hepatocellular carcinoma (LMH) cells. This serine to arginine exchange at amino acid position 90 (SAA.R90S) leads to intra- and extracellular accumulation of SAA, which is surprisingly counteracted by MG132 treatment, independent of SAA's intrinsic promoter. METHODS AND RESULTS: To test, whether low proteasomal degradation of SAA.R90S is responsible for the observed intra- and extracellular SAA accumulation, we intended to inhibit the proteasome in SAA wild type (SAA.WT) overexpressing cells with MG132. However, we observed an unexpected drastic decrease in SAA protein expression at the transcript level. NF-κB gene expression was unchanged by MG132 at the measured time point. CONCLUSIONS: The observed results demonstrate that MG132 inhibits SAA expression at the transcript level, independent of its endogenous promoter. Further, the data might indicate that NF-κB is not involved in the observed MG132-induced inhibition of SAA expression. We, consequently, question in this brief report whether MG132 should truly be categorised as a specific ubiquitin proteasome inhibitor and recommend the usage of alternative compounds.
Assuntos
Carcinoma Hepatocelular , Galinhas , Leupeptinas , Neoplasias Hepáticas , NF-kappa B , Regiões Promotoras Genéticas , Proteína Amiloide A Sérica , Animais , Leupeptinas/farmacologia , Galinhas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Systemic amyloid A (AA) amyloidosis, which is considered the second most common form of systemic amyloidosis usually takes place several years prior to the occurrence of chronic inflammation, generally involving the kidney. Activated HSF1, which alleviated unfolded protein response (UPR) or enhanced HSR, is the potential therapeutic target of many diseases. However, the effect of HSF1 on AA amyloidosis remains unclear. This study focused on evaluating effect of HSF1 on AA amyloidosis based on HSF1 knockout mice. As a result, aggravated amyloid deposits and renal dysfunction have been found in HSF1 knockout mice. In progressive AA amyloidosis, HSF1 deficiency enhances serum amyloid A production might to lead to severe AA amyloid deposition in mice, which may be related to deactivated unfolded protein response as well as enhanced inflammation. Thus, HSF1 plays a significant role on UPR related pathway impacting AA amyloid deposition, which can mitigate amyloidogenic proteins from aggregation pathologically and is the possible way for intervening with the pathology of systemic amyloid disorder. In conclusion, HSF1 could not only serve as a new target for AA amyloidosis treatment in the future, but HSF1 knockout mice also can be considered as a valuable novel animal model for renal AA amyloidosis.
Assuntos
Amiloidose , Fatores de Transcrição de Choque Térmico , Rim , Camundongos Knockout , Resposta a Proteínas não Dobradas , Animais , Amiloidose/metabolismo , Amiloidose/genética , Amiloidose/patologia , Fatores de Transcrição de Choque Térmico/metabolismo , Fatores de Transcrição de Choque Térmico/genética , Camundongos , Rim/patologia , Rim/metabolismo , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Modelos Animais de Doenças , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/genética , Nefropatias/etiologia , Camundongos Endogâmicos C57BLRESUMO
Early detection of drug-induced kidney injury is essential for drug development. In this study, multiple low-dose aristolochic acid (AA) and cisplatin (Cis) injections increased renal mRNA levels of inflammation, fibrosis, and renal tubule injury markers. We applied a serum amyloid A3 (Saa3) promoter-driven luciferase reporter (Saa3 promoter-luc mice) to these two tubulointerstitial nephritis models and performed in vivo bioluminescence imaging to monitor early renal pathologies. The bioluminescent signals from renal tissues with AA or CIS injections were stronger than those from normal kidney tissues obtained from normal mice. To verify whether the visualized bioluminescence signal was specifically generated by the injured kidney, we performed in vivo bioluminescence analysis after opening the stomachs of Saa3 promoter-luc mice, and the Saa3-mediated bioluminescent signal was specifically detected in the injured kidney. This study showed that Saa3 promoter activity is a potent non-invasive indicator for the early detection of drug-induced nephrotoxicity.
Assuntos
Ácidos Aristolóquicos , Luciferases , Regiões Promotoras Genéticas , Proteína Amiloide A Sérica , Animais , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Camundongos , Luciferases/metabolismo , Luciferases/genética , Ácidos Aristolóquicos/toxicidade , Genes Reporter , Cisplatino/toxicidade , Cisplatino/efeitos adversos , Medições Luminescentes/métodos , Masculino , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Rim/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Hypercholesterolaemia is a systemic metabolic disease, but the role of organs other than liver in cholesterol metabolism is unappreciated. The phenotypic characterization of the Tsc1Dmp1 mice reveal that genetic depletion of tuberous sclerosis complex 1 (TSC1) in osteocytes/osteoblasts (Dmp1-Cre) triggers progressive increase in serum cholesterol level. The resulting cholesterol metabolic dysregulation is shown to be associated with upregulation and elevation of serum amyloid A3 (SAA3), a lipid metabolism related factor, in the bone and serum respectively. SAA3, elicited from the bone, bound to toll-like receptor 4 (TLR4) on hepatocytes to phosphorylate c-Jun, and caused impeded conversion of cholesterol to bile acids via suppression on cholesterol 7 α-hydroxylase (Cyp7a1) expression. Ablation of Saa3 in Tsc1Dmp1 mice prevented the CYP7A1 reduction in liver and cholesterol elevation in serum. These results expand the understanding of bone function and hepatic regulation of cholesterol metabolism and uncover a potential therapeutic use of pharmacological modulation of SAA3 in hypercholesterolaemia.
Assuntos
Colesterol , Fígado , Osteoblastos , Osteócitos , Proteína Amiloide A Sérica , Animais , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Camundongos , Colesterol/metabolismo , Fígado/metabolismo , Osteócitos/metabolismo , Osteoblastos/metabolismo , Hipercolesterolemia/metabolismo , Modelos Animais de Doenças , Colesterol 7-alfa-Hidroxilase/metabolismo , Colesterol 7-alfa-Hidroxilase/genética , MasculinoRESUMO
T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.
Assuntos
Linfócitos T CD8-Positivos , Hepatócitos , Interleucina-6 , Fator de Transcrição STAT3 , Proteína Amiloide A Sérica , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Hepatócitos/metabolismo , Hepatócitos/imunologia , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Microambiente Tumoral/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Evasão Tumoral , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linhagem Celular TumoralRESUMO
Matrix Metalloproteinases (MMPs) have been demonstrated to be essential in facilitating the migration and metastasis of clear cell renal cell carcinoma (ccRCC). However, the ability of the MMP family to predict clinical outcomes and guide optimal therapeutic strategies for ccRCC patients remains incompletely understood. In this investigation, we initially conducted a thorough examination of the MMP family in pan-cancer. Notably, MMPs exhibited distinctive significance in ccRCC. Following this, we undertook an extensive analysis to evaluate the clinical value of MMPs and potential mechanisms by which MMPs contribute to the progression of ccRCC. A novel stratification method and prognostic model were developed based on MMPs in order to enhance the accuracy of prognosis prediction for ccRCC patients and facilitate personalized treatment. By conducting multi-omics analysis and transcriptional regulation analysis, it was hypothesized that SAA1 plays a crucial role in promoting ccRCC migration through MMPs. Subsequently, in vitro experiments confirmed that SAA1 regulates ccRCC cell migration via the ERK-AP1-MMPs axis. In conclusion, our study has explored the potential value of the MMP family as prognostic markers for ccRCC and as guides for medication regimens. Additionally, we have identified SAA1 as a crucial factor in the migration of ccRCC.
Assuntos
Carcinoma de Células Renais , Movimento Celular , Neoplasias Renais , Metaloproteinases da Matriz , Proteína Amiloide A Sérica , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Humanos , Movimento Celular/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Prognóstico , Linhagem Celular Tumoral , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Feminino , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Transdução de SinaisRESUMO
Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, can be activated by isotonic stimuli, such as infection. It remains unclear, however, whether NFAT5 is required for damage-associated molecular pattern-triggered (DAMP-triggered) inflammation and immunity. Here, we found that several DAMPs increased NFAT5 expression in macrophages. In particular, serum amyloid A (SAA), primarily generated by the liver, substantially upregulated NFAT5 expression and activity through TLR2/4-JNK signalling pathway. Moreover, the SAA-TLR2/4-NFAT5 axis promoted migration and chemotaxis of macrophages in an IL-6- and chemokine ligand 2-dependent (CCL2-dependent) manner in vitro. Intraarticular injection of SAA markedly accelerated macrophage infiltration and arthritis progression in mice. By contrast, genetic ablation of NFAT5 or TLR2/4 rescued the pathology induced by SAA, confirming the SAA-TLR2/4-NFAT5 axis in vivo. Myeloid-specific depletion of NFAT5 also attenuated SAA-accelerated arthritis. Of note, inflammatory arthritis in mice strikingly induced SAA overexpression in the liver. Conversely, forced overexpression of the SAA gene in the liver accelerated joint damage, indicating that the liver contributes to bolstering chronic inflammation at remote sites by secreting SAA. Collectively, this study underscores the importance of the SAA-TLR2/4-NFAT5 axis in innate immunity, suggesting that acute phase reactant SAA mediates mutual interactions between liver and joints and ultimately aggravates chronic arthritis by enhancing macrophage activation.
Assuntos
Artrite , Proteína Amiloide A Sérica , Animais , Camundongos , Artrite/metabolismo , Inflamação/patologia , Fígado/metabolismo , Ativação de Macrófagos , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND & AIMS: Mounting evidence suggests the gastrointestinal microbiome is a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastrointestinal tract and its enteric nervous system. METHODS: To examine the early response, we challenged primary murine myenteric networks with curli, the prototypical bacterial amyloid, and performed shotgun RNA sequencing and multiplex enzyme-linked immunosorbent assay. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. RESULTS: Curli induced a proinflammatory response, with strong up-regulation of Saa3 and the secretion of several cytokines. This proinflammatory state was induced primarily in enteric glia, was accompanied by increased levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant Serum Amyloid A3 (SAA3) was sufficient to recapitulate this specific proinflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Similar to curli, recombinant SAA3 caused a strong up-regulation of Saa3 transcripts, illustrating its self-amplifying potential . Since colonization of curli-producing Salmonella and dextran sulfate sodium-induced colitis triggered a significant increase in Saa3 transcripts as well, we assume SAA3plays a central role in enteric dysfunction. Inhibition of dual leucine zipper kinase, an upstream regulator of the c-Jun N-terminal kinase pathway responsible for SAA3 production, attenuated curli- and recombinant SAA3-induced Saa3 up-regulation, DNA damage, and replication in enteric glia. CONCLUSIONS: Our results position SAA3 as an important mediator of gastrointestinal vulnerability to bacterial-derived amyloids and demonstrate the potential of dual leucine zipper kinase inhibition to dampen enteric pathology.
Assuntos
Sistema Nervoso Entérico , Proteína Amiloide A Sérica , Animais , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Sistema Nervoso Entérico/imunologia , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/genética , Camundongos , Proteínas de Bactérias/metabolismo , Inflamação/imunologia , Inflamação/patologia , Inflamação/metabolismo , Neuroglia/metabolismo , Neuroglia/imunologia , Neuroglia/patologia , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Microbioma Gastrointestinal/imunologia , Camundongos Knockout , Colite/imunologia , Colite/microbiologia , Colite/patologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
BACKGROUND AND AIMS: Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. We reported that deficiency of Serum Amyloid A (SAA) significantly reduces AngII-induced inflammation and AAA in both hyperlipidemic apoE-deficient and obese C57BL/6 mice. The aim of this study is to investigate whether SAA plays a role in the progression of early AAA in obese C57BL/6 mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (60% kcal as fat) throughout the study. After 4 months of diet, the mice were infused with AngII until the end of the study. Mice with at least a 25% increase in the luminal diameter of the abdominal aorta after 4 weeks of AngII infusion were stratified into 2 groups. The first group received a control antisense oligonucleotide (Ctr ASO), and the second group received ASO that suppresses SAA (SAA-ASO) until the end of the study. RESULTS: Plasma SAA levels were significantly reduced by the SAA ASO treatment. While mice that received the control ASO had continued aortic dilation throughout the AngII infusion periods, the mice that received SAA-ASO had a significant reduction in the progression of aortic dilation, which was associated with significant reductions in matrix metalloprotease activities, decreased macrophage infiltration and decreased elastin breaks in the abdominal aortas. CONCLUSIONS: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from the progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.
Assuntos
Angiotensina II , Aneurisma da Aorta Abdominal , Humanos , Masculino , Animais , Camundongos , Angiotensina II/farmacologia , Proteína Amiloide A Sérica/genética , Oligonucleotídeos Antissenso/uso terapêutico , Camundongos Endogâmicos C57BL , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/prevenção & controle , Aorta Abdominal , Obesidade , Modelos Animais de Doenças , Camundongos Knockout , Apolipoproteínas ERESUMO
ABSTRACT: Background: Chronic critical illness (CCI), which was characterized by persistent inflammation, immunosuppression, and catabolism syndrome (PICS), often leads to muscle atrophy. Serum amyloid A (SAA), a protein upregulated in critical illness myopathy, may play a crucial role in these processes. However, the effects of SAA on muscle atrophy in PICS require further investigation. This study aims to develop a mouse model of PICS combined with bone trauma to investigate the mechanisms underlying muscle weakness, with a focus on SAA. Methods: Mice were used to examine the effects of PICS after bone trauma on immune response, muscle atrophy, and bone healing. The mice were divided into two groups: a bone trauma group and a bone trauma with cecal ligation and puncture group. Tibia fracture surgery was performed on all mice, and PICS was induced through cecal ligation and puncture surgery in the PICS group. Various assessments were conducted, including weight change analysis, cytokine analysis, hematological analysis, grip strength analysis, histochemical staining, and immunofluorescence staining for SAA. In vitro experiments using C2C12 cells (myoblasts) were also conducted to investigate the role of SAA in muscle atrophy. The effects of inhibiting receptor for advanced glycation endproducts (RAGE) or JAK2 on SAA-induced muscle atrophy were examined. Bioinformatic analysis was conducted using a dataset from the GEO database to identify differentially expressed genes and construct a coexpression network. Results: Bioinformatic analysis confirmed that SAA was significantly upregulated in muscle tissue of patients with intensive care unit-induced muscle atrophy. The PICS animal models exhibited significant weight loss, spleen enlargement, elevated levels of proinflammatory cytokines, and altered hematological profiles. Evaluation of muscle atrophy in the animal models demonstrated decreased muscle mass, grip strength loss, decreased diameter of muscle fibers, and significantly increased expression of SAA. In vitro experiment demonstrated that SAA decreased myotube formation, reduced myotube diameter, and increased the expression of muscle atrophy-related genes. Furthermore, SAA expression was associated with activation of the FOXO signaling pathway, and inhibition of RAGE or JAK2/STAT3-FOXO signaling partially reversed SAA-induced muscle atrophy. Conclusions: This study successfully develops a mouse model that mimics PICS in CCI patients with bone trauma. Serum amyloid A plays a crucial role in muscle atrophy through the JAK2/STAT3-FOXO signaling pathway, and targeting RAGE or JAK2 may hold therapeutic potential in mitigating SAA-induced muscle atrophy.
Assuntos
Doenças Musculares , Proteína Amiloide A Sérica , Animais , Humanos , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Estado Terminal , Atrofia Muscular/metabolismo , Doença Crônica , Modelos Animais de Doenças , CitocinasRESUMO
At present, although there are tumor markers for hepatocellular carcinoma (HCC), markers with better predictive efficiency are needed. SAA4 gene expression in liver tumor and paracancerous tissues was analyzed using The Cancer Genome Atlas database. The differentially expressed genes (DEGs) were analyzed and visualized by heatmap and volcano plot. Survival analysis was performed based on SAA4 expression. SAA4 expression was compared in patients grouped based on clinicopathological features, and gene set enrichment analysis (GSEA) was conducted. Immunohistochemical staining was used to verify the SAA4 protein staining intensity from The Human Protein Atlas database and our center's samples. The diagnostic value of SAA4 for HCC was evaluated by receiver operating characteristic curves. SAA4 was expressed at low levels in HCC tissues, and low SAA4 expression was associated with a poor prognosis in HCC. In addition, SAA4 expression decreased with HCC progression. There were 188 upregulated DEGs and 1551 downregulated DEGs between the high and low SAA4 expression groups. Complement and coagulation cascades, fatty acid metabolism, and ECM receptor interaction were significantly enriched in the GSEA. SAA4 had good predictive efficacy for HCC and even early HCC and was superior to AFP. In general, low SAA4 expression was associated with advanced HCC stage and a poor prognosis. In addition, SAA4 may be helpful for the diagnosis of early HCC and may become a novel tumor marker with good predictive power for HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Biomarcadores Tumorais/genética , Expressão Gênica , Prognóstico , Proteína Amiloide A Sérica/genéticaRESUMO
Diabetic kidney disease (DKD) is one of the severe complications of diabetes mellitus, yet there is no effective treatment. Exploring the development of DKD is essential to treatment. Podocyte injury and inflammation are closely related to the development of DKD. However, the mechanism of podocyte injury and progression in DKD remains largely unclear. Here, we observed that FTO expression was significantly upregulated in high glucose-induced podocytes and that overexpression of FTO promoted podocyte injury and inflammation. By performing RNA-seq and MeRIP-seq with control podocytes and high glucose-induced podocytes with or without FTO knockdown, we revealed that serum amyloid A2 (SAA2) is a target of FTO-mediated m6A modification. Knockdown of FTO markedly increased SAA2 mRNA m6A modification and decreased SAA2 mRNA expression. Mechanistically, we demonstrated that SAA2 might participate in podocyte injury and inflammation through activation of the NF-κB signaling pathway. Furthermore, by generating podocyte-specific adeno-associated virus 9 (AAV9) to knockdown SAA2 in mice, we discovered that the depletion of SAA2 significantly restored podocyte injury and inflammation. Together, our results suggested that upregulation of SAA2 promoted podocyte injury through m6A-dependent regulation, thus suggesting that SAA2 may be a therapeutic target for diabetic kidney disease.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , Nefropatias Diabéticas , Podócitos , Proteína Amiloide A Sérica , Animais , Camundongos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Nefropatias Diabéticas/genética , Glucose , Inflamação/genética , NF-kappa B , RNA Mensageiro/genética , Transdução de Sinais , Proteína Amiloide A Sérica/genéticaRESUMO
Serum amyloid A (SAA) are major acute-phase response proteins which actively participate in many inflammatory diseases. This study was designed to explore the function of SAA in acute ocular inflammation and the underlying mechanism. We found that SAA3 was upregulated in endotoxin-induced uveitis (EIU) mouse model, and it was primarily expressed in microglia. Recombinant SAA protein augmented intraocular inflammation in EIU, while the inhibition of Saa3 by siRNA effectively alleviated the inflammatory responses and rescued the retina from EIU-induced structural and functional damage. Further study showed that the recombinant SAA protein activated microglia, causing characteristic morphological changes and driving them further to pro-inflammatory status. The downregulation of Saa3 halted the amoeboid change of microglia, reduced the secretion of pro-inflammatory factors, and increased the expression of tissue-reparative genes. SAA3 also regulated the autophagic activity of microglial cells. Finally, we showed that the above effect of SAA on microglial cells was at least partially mediated through the expression and signaling of Toll-like receptor 4 (TLR4). Collectively, our study suggested that microglial cell-expressed SAA could be a potential target in treating acute ocular inflammation.
Assuntos
Microglia , Proteína Amiloide A Sérica , Animais , Camundongos , Proteína Amiloide A Sérica/genética , Inflamação/induzido quimicamente , Retina , Proteínas de Fase Aguda , Endotoxinas/toxicidadeRESUMO
Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.
Assuntos
Lesão Pulmonar , Sepse , Animais , Camundongos , Lesão Pulmonar/patologia , Proteína Amiloide A Sérica/genética , Sepse/patologia , Pulmão/patologia , Quimiocinas , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Cancer stem cells (CSCs) are believed to be crucial in the initiation, progression, and recurrence of cancer. CSCs are also known to be more resistant to cancer treatments. However, the interaction between CSCs and the immune microenvironment is complex and not fully understood. In current study we used single cell RNA sequence (scRNA-Seq, public dataset) technology to identify the characteristic of CSCs. We found that the lung adenocarcinoma cancer stem population is highly inflammatory and remodels the tumor microenvironment by secreting inflammatory factors, specifically the acute phase protein serum amyloid A (SAA). Next, we developed an ex-vivo autologous patient-derived organoids (PDOs) and peripheral blood mononuclear cells (PBMCs) co-culture model to evaluate the immune biological impact of SAA. We found that SAA not only promotes chemoresistance by inducing cancer stem transformation, but also restricts anti-tumor immunity and promotes tumor fibrosis by driving type 2 immunity, and α-SAA neutralization antibody could restrict treatment resistant and tumor fibrosis. Mechanically, we found that the malignant phenotype induced by SAA is dependent on P2X7 receptor. Our data indicate that cancer stem cells secreted SAA have significant biological impact to promote treatment resistant and tumor fibrosis by driving cancer stemness transformation and type 2 immunity polarization via P2X7 receptor. Notably, α-SAA neutralization antibody shows therapeutic potential by restricting these malignant phenotypes.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Proteína Amiloide A Sérica , Células Th2 , Humanos , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Fibrose , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores Purinérgicos P2X7 , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Microambiente Tumoral , Células Th2/imunologiaRESUMO
BACKGROUND: Ovarian cancer (OC) is a prevalent gynecological malignancy with the highest mortality rate, which generally diagnosed at late stages due to the lack of effective early screening methods and the nonspecific symptoms. Hence, here we aim to identify new metastasis markers and develop a novel detection method with the characteristics of high sensitivity, rapid detection, high specificity, and low cost when compared with other conventional detection technologies. METHODS: Blood from OC patients with or without metastasis were collected and analyzed by 4D Label free LC - MS/MS. Surgically resect samples from OC patients were collected for Single cell RNA sequencing (sc-RNA seq). Short hairpin RNA (shRNA) was used to silence SAA1 expression in SKOV3 and ID8 to verify the relationship between endogenous SAA1 and tumor invasion or metastasis. The functional graphene chips prepared by covalent binding were used for SAA1 detection. RESULTS: In our study, we identified Serum Amyloid A1 (SAA1) as a hematological marker of OC metastasis by comprehensive analysis of proteins in plasma from OC patients with or without metastasis using 4D Label free LC - MS/MS and gene expression patterns from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Further validation using tumor tissues and plasma from human OC and mouse OC model confirmed the correlation between SAA1 and tumor metastasis. Importantly, sc-RNA seq of human OC samples revealed that SAA1 was specifically expressed in tumor cells and upregulated in the metastasis group. The functional role of SAA1 in metastasis was demonstrated through experiments in vitro and in vivo. Based on these findings, we designed and investigated a graphene-based platform for SAA1 detection to predict the risk of metastasis of OC patients. CONCLUSION: Our study suggests that SAA1 is a biomarker of OC metastasis, and we have developed a rapid and highly sensitive platform using graphene chips to detection of plasma SAA1 for the early assessment of metastasis in OC patients.
Assuntos
Grafite , Neoplasias Ovarianas , Animais , Camundongos , Humanos , Feminino , Espectrometria de Massas em Tandem , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismoRESUMO
Deep evolutionary origin of the conserved animal serum amyloid A (SAA) apolipoprotein family leading to yet unknown highly similar SAA-like sequences occurring in certain bacterial genomes is demonstrated in this contribution. Horizontal gene transfer event of corresponding genes between gut bacteria and non-vertebrate animals was discovered in the reconstructed phylogenetic tree obtained with maximum likelihood and neighbor-joining methods, respectively. This detailed phylogeny based on totally 128 complete sequences comprised diverse serum amyloid A isoforms from various animal vertebrate and non-vertebrate phyla and also corresponding genes coding for highly similar proteins from animal gut bacteria. Typical largely conserved sequence motifs and a peculiar structural fold consisting mainly of four α-helices in a bundle within all reconstructed clades of the SAA protein family are discussed with respect to their supposed biological functions in various organisms that contain corresponding genes.
Assuntos
Bactérias , Proteína Amiloide A Sérica , Animais , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismo , Filogenia , Bactérias/metabolismo , Genoma Bacteriano , Isoformas de Proteínas/genéticaRESUMO
Trypanosoma species cause animal trypanosomiasis that infects many animals. Trypanosoma evansi is an organism that infects camels. There are many economic problems associated with this disease, including lower milk and meat yields and abortions. The purpose of the current survey was molecular study of the presence of Trypanosoma in dromedary camel blood in the south of Iran, and its effects on the hematologic, and some acute-phase protein changes. Blood samples were aseptically collected from the jugular vein of dromedary camels (n = 100; aged from 1 to 6 years) originating from Fars Province in EDTA-coated vacutainers. Genomic DNA from 100 µL of the whole blood was extracted and amplified using a PCR assay based on ITS1, 5.8S, and ITS2 ribosomal regions. Also, the PCR products obtained were sequenced. Moreover, the changes in hematological parameters and serum acute-phase proteins (serum amyloid A, alpha-1 acid glycoprotein, and haptoglobin) were measured. Among 100 tested blood, nine samples (9%, 95% CI: 4.2-16.4%) were found positive by the PCR assay. The phylogenetic tree and blast analysis showed four different genotypes closely related to the strains (accession numbers: JN896754 and JN896755) previously reported from dromedary camels in Yazd Province, center Iran. Based on hematological analysis, normocytic and normochromic anemia and lymphocytosis were detected in the PCR-positive cases compared with the negative group. Furthermore, alpha-1 acid glycoprotein was significantly increased in the positive cases. There was a substantial and positive relation between the number of lymphocytes, and the levels of alpha-1 acid glycoprotein and serum amyloid A in the blood (p = 0.045, r = 0.223 and p = 0.036, r = 0.234, respectively). A noticeable frequency of T. evansi infection was reported in dromedary camels in south Iran. This is the first report on the genetic diversity of T. evansi in this region. There was a significant association among Trypanosoma infection, lymphocytosis, and alpha-1 acid glycoprotein. Trypanosoma-positive camels had a significant decrease in hematocrit (HCT), hemoglobin (Hb), and red blood cell (RBC) values compared to the non-infected group. Further experimental studies are needed to elucidate the hematological and acute-phase protein alteration during a different phase of Trypanosoma spp. infection.
Assuntos
Linfocitose , Trypanosoma , Tripanossomíase , Animais , Camelus , Irã (Geográfico)/epidemiologia , Filogenia , Proteína Amiloide A Sérica/genética , Trypanosoma/genética , Tripanossomíase/epidemiologia , Tripanossomíase/veterinária , Proteínas de Fase Aguda , Glicoproteínas/genéticaRESUMO
Introduction: Osteopenia has been associated to several inflammatory conditions, including mycobacterial infections. How mycobacteria cause bone loss remains elusive, but direct bone infection may not be required. Methods: Genetically engineered mice and morphometric, transcriptomic, and functional analyses were used. Additionally, inflammatory mediators and bone turnover markers were measured in the serum of healthy controls, individuals with latent tuberculosis and patients with active tuberculosis. Results and discussion: We found that infection with Mycobacterium avium impacts bone turnover by decreasing bone formation and increasing bone resorption, in an IFNγ- and TNFα-dependent manner. IFNγ produced during infection enhanced macrophage TNFα secretion, which in turn increased the production of serum amyloid A (SAA) 3. Saa3 expression was upregulated in the bone of both M. avium- and M. tuberculosis-infected mice and SAA1 and 2 proteins (that share a high homology with murine SAA3 protein) were increased in the serum of patients with active tuberculosis. Furthermore, the increased SAA levels seen in active tuberculosis patients correlated with altered serum bone turnover markers. Additionally, human SAA proteins impaired bone matrix deposition and increased osteoclastogenesis in vitro. Overall, we report a novel crosstalk between the cytokine-SAA network operating in macrophages and bone homeostasis. These findings contribute to a better understanding of the mechanisms of bone loss during infection and open the way to pharmacological intervention. Additionally, our data and disclose SAA proteins as potential biomarkers of bone loss during infection by mycobacteria.