Prognostic value of inflammation and immune-related gene NOD2 in clear cell renal cell carcinoma.

Inflammation and immune responses play important roles in cancer development and prognosis. We identified 59 upregulated inflammation- and immune-related genes (IIRGs) in clear cell renal cell carcinoma (ccRCC) from The Cancer Genome Atlas database. Among the upregulated IIRGs, nucleotide binding oligomerization domain 2 (NOD2), PYD and CARD domain (PYCARD) were also confirmed to be upregulated in the Oncomine database and in three independent GEO data sets. Tumor immune infiltration resource database analysis revealed that NOD2 and PYCARD levels were significantly positively correlated with infiltration levels of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells. Multivariate Cox hazards regression analysis indicated that based on clinical variables (age, gender, tumor grade, pathological TNM stage), NOD2, but not PYCARD, was an independent, unfavorable ccRCC prognostic biomarker. Functional enrichment analyses (GSEA) showed that NOD2 was involved in innate immune responses, inflammatory responses, and regulation of cytokine secretion. Meanwhile, mRNA and protein levels of NOD2 were elevated in four ccRCC cell lines (786-O, ACHN, A498 and Caki-1), and its knockdown significantly inhibited IL-8 secretion, thereby inhibiting ccRCC cell proliferation and invasion. Furthermore, results showed that miR-20b-5p targeted NOD2 to alleviate NOD2-mediated IL-8 secretion. In conclusion, NOD2 is a potential prognostic biomarker for ccRCC and the miR-20b-5p/NOD2/IL-8 axis may regulate inflammation- and immune-mediated tumorigenesis in ccRCC.

Identifying functional dysregulation of NOD2 variant Q902K in patients with Yao syndrome.

BACKGROUND AND OBJECTIVES: The study investigated the pathogenesis of Yao syndrome (YAOS), a rare systemic autoinflammatory disease associated with the nucleotide-binding oligomerization domain containing 2 (NOD2) gene variants. METHODS: RNA sequencing analyses were used to detect transcriptomic profile changes. Immunoblot and immunohistochemistry were used to examine the NOD2-mediated inflammatory signaling pathways and ELISA was used to detect cytokines. RESULTS: Transcriptome analysis of YAOS revealed NOD-like receptor signaling pathway enrichment. Compared with HCs, P-RIP2, p-p65, p-p38, p-ERK, and p-JNK notably increased in PBMCs of a patient with YAOS. P-RIP2, p-p65, and p-p38 elevated in small intestinal mucosa tissues. P-p65 and p-p38 in synovial tissues from YAOS were higher than those in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum interleukin (IL)-6 level along with tumor necrosis factor (TNF)-α and IL-6 secreted from PBMCs were markedly higher in patients with YAOS in comparison to healthy controls (HCs). The supernatants of synovial cells from a patient with YAOS showed substantially higher IL-1ß and IL-6 levels than those of RA and OA. Canakinumab therapy of a Q902K heterozygous patient with YAOS resulted in notable clinical improvement. CONCLUSION: Overproduction of pro-inflammatory cytokines and the hyperactivation of NOD2-mediated signaling pathways were found in the NOD2 variant Q902K patient with YAOS. NOD2-RIP2-MAPK pathway might play a pivotal role in the pathogenesis of YAOS. These results provide new perspectives for targeted therapies in YAOS.

A closer look at ligand specificity for cellular activation of NOD2 with synthetic muramyl dipeptide analogues.

To further understand the specificity of muramyl dipeptide (MDP) sensing by NOD2, we evaluated the compatibility of synthetic MDP analogues for cellular uptake and NAGK phosphorylation, the pre-requisite steps of intracellular NOD2 activation. Our results revealed that these two prior steps do not confer ligand stereoselectivity; yet NAGK strictly discriminates against the disaccharide NOD2 agonists for phosphorylation in vitro, despite it being indispensable for the cellular NOD2-stimulating effects of these analogues, implying potential glycosidase cleavage as a novel intermediate step for cellular activation of NOD2.

The expanding clinical spectrum of autoinflammatory diseases with NOD2 variants: a case series and literature review.

Objective: To assess the impact conferred by NOD2 variants on the clinical spectrum of patients with systemic autoinflammatory diseases (SAIDs) in Greece. Methods: Consecutive patients (n=167) with confirmed SAIDs who underwent screening by next generation sequencing (NGS) targeting 26 SAID-associated genes, and carried at least one NOD2 gene variant, were retrospectively studied. The demographic, clinical and laboratory parameters were recorded. Results: In total, 24 rare NOD2 variants in 23/167 patients (14%) were detected. Notably, 18 patients had at least one co-existing variant in 13 genes other than NOD2. Nine patients had juvenile- and 14 adult-onset disease. All patients presented with symptoms potentially induced by the NOD2 variants. In particular, the candidate clinical diagnosis was Yao syndrome (YAOS) in 12 patients (7% of the whole SAID cohort). The clinical spectrum of patients with YAOS (mean episode duration 8 days) was fever (n=12/12), articular symptoms (n=8), gastrointestinal symptoms (n=7; abdominal pain/bloating in 7; diarrhea in 4; oral ulcers in 3), serositis (n=7), and rash (n=5), while the inflammatory markers were elevated in all but one patient. Most of these patients showed a poor response to nonsteroidal anti-inflammatory drugs (n=7/9), colchicine (n=6/8) and/or anti-TNF treatment (n=3/4), while a complete response was observed in 6/10 patients receiving steroids and 3/5 on anti-IL1 treatment. Another 8 patients were diagnosed with either FMF (n=6) or PFAPA syndrome (n=2) presenting with prominent diarrhea (n=7), oral ulcers (n=2), periorbital swelling and sicca-like symptoms (n=1), or maculopapular rash (n=1). One patient had a clinically undefined SAID, albeit characterized by oral ulcers and diarrhea. Finally, one patient presented with chronic relapsing urticaria with periorbital edema and inflammatory markers, and another one had a Crohn-like syndrome with good response to anti-IL-1 but refractory to anti-TNF treatment. Conclusion: NOD2 variants were detected in 1 out of 7 SAID patients and seem to have an impact on disease phenotype and treatment response. Further studies should validate combined molecular and clinical data to better understand these distinct nosological entities.

Discovery of 2,3-Diaminoindole Derivatives as a Novel Class of NOD Antagonists.

NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.

Novel role of peptidoglycan recognition protein 2 in activating NOD2-NFκB inflammatory axis in coronary artery disease.

BACKGROUNDS AND AIMS: The role of inflammation in driving atherosclerosis is well-established. It exerts systemic effects beyond the local site of plaque formation. In the context of coronary artery disease (CAD), the proteins that show altered levels in the plasma, are potentially important for understanding the key regulatory mechanism in the pathogenesis of atherosclerosis. A case-control study revealed that plasma soluble Peptidoglycan Recognition Protein 2 (PGLYRP2) primarily produced by the liver, is increased in subjects with CAD. Furthermore, the concentration of PGLYRP2 in the blood correlates with the severity of coronary artery disease. Thus, it raises interest in understanding the exact role of the protein in aortic inflammation and plaque progression. METHODS: We evaluated the plasma concentration of PGLYRP2 in three distinct groups: patients with CAD (N = 68), asymptomatic individuals (N = 34), and healthy volunteers (N = 20). Furthermore, we investigated the correlation between disease severity and PGLYRP2 levels in CAD patients. To identify potential binding partners of PGLYRP2, we employed computational analysis. We verified the PGLYRP2-NOD2 interaction in macrophage cells and elucidated the inflammatory pathways activated by PGLYRP2 within these cells. To assess the impact of PGLYRP2, we examined its effects in the atherosclerotic mice model (ApoE-/-). RESULTS: In this study, we report for the first time that Nucleotide-binding Oligomerization domain 2 (NOD2) which is expressed on the surface of macrophages, is a receptor of PGLYRP2. The N-terminal domain of PGLYRP2 directly binds to NOD2 and activates the NOD2-RIP2-NFκB cascade that promotes the secretion of proinflammatory cytokines like TNFα, IL1ß, and IL-8. In the atherosclerotic mice model (ApoE-/-) we demonstrate that elevated PGLYRP2 level is parallel with increased proinflammatory cytokines in the plasma when fed a High Cholesterol Diet (HCD). Immunohistochemical analysis reveals that PGLYRP2 is co-localized with NOD2 on the macrophages at the site of the lesion. CONCLUSIONS: Taken together, our data demonstrate that NOD2 acts as a receptor of PGLYRP2 on macrophages, which mediates the activation of the NOD2-RIP2-NFκB pathway and promotes inflammation, thus significantly contributing to the development and progression of atherosclerosis.

Identification, characterization and the inflammatory regulating effect of NOD1/2 in sturgeon.

As an ancient species with both conservation and commercial value, Sturgeon's inflammatory regulation mechanism is a research point. Nucleotide-binding and oligomerization domain-containing proteins 1 and 2 (NOD1/2) are classical intracellular pattern recognition receptors (PRRs) in immunity of anti-bacterial infection. However, the characterization and function of NOD1/2 in Sturgeon are still unclear. In this study, we analyzed the synteny relationship of NOD1/2 genes between Acipenser ruthenus and representative fishes at the genome-level. Results showed that the ArNOD2 collinear genes pair was present in all representative fishes. The duplicated ArNOD1/2 genes were under purifying selection during evolution as indicated by their Ka/Ks values. To explore the function of NOD1/2, we further investigated their expression patterns and the effects of pathogenic infection, PAMPs treatment, and siRNA interference in Acipenser baerii, the sibling species of A. ruthenus. Results showed that both AbNOD1/2 were expressed at early developmental stages and in different tissues. Pathogenic infection in vivo and PAMPs treatment in vitro demonstrated that AbNOD1/2 could respond to pathogen stimulation. siRNA interference with AbNOD1/2 inhibited expression levels of RIPK2 and inflammatory cytokines compared to the control group after iE-DAP or MDP treatment. This study hinted that the AbNOD1/2 could stimulate the inflammatory cytokines response during evolutionary processes.

Implications of combined NOD2 and other gene mutations in autoinflammatory diseases.

NOD-like receptors (NLRs) are intracellular sensors associated with systemic autoinflammatory diseases (SAIDs). We investigated the largest monocentric cohort of patients with adult-onset SAIDs for coinheritance of low frequency and rare mutations in NOD2 and other autoinflammatory genes. Sixty-three patients underwent molecular testing for SAID gene panels after extensive clinical workups. Whole exome sequencing data from the large Atherosclerosis Risk in Communities (ARIC) study of individuals of European-American ancestry were used as control. Of 63 patients, 44 (69.8%) were found to carry combined gene variants in NOD2 and another gene (Group 1), and 19 (30.2%) were carriers only for NOD2 variants (Group 2). The genetic variant combinations in SAID patients were digenic in 66% (NOD2/MEFV, NOD2/NLRP12, NOD2/NLRP3, and NOD2/TNFRSF1A) and oligogenic in 34% of cases. These variant combinations were either absent or significantly less frequent in the control population. By phenotype-genotype correlation, approximately 40% of patients met diagnostic criteria for a specific SAID, and 60% had mixed diagnoses. There were no statistically significant differences in clinical manifestations between the two patient groups except for chest pain. Due to overlapping phenotypes and mixed genotypes, we have suggested a new term, "Mixed NLR-associated Autoinflammatory Disease ", to describe this disease scenario. Gene variant combinations are significant in patients with SAIDs primarily presenting with mixed clinical phenotypes. Our data support the proposition that immunological disease expression is modified by genetic background and environmental exposure. We provide a preliminary framework in diagnosis, management, and interpretation of the clinical scenario.

Case Report: Methotrexate and hydroxychloroquine in combination for the treatment of NOD2-mutation-associated Blau syndrome.

Mutations in nucleotide binding oligomerization domain containing 2 receptor (NOD2) are associated with Blau syndrome (also known as early-onset sarcoidosis)-a rare autosomal dominant, chronic granulomatous disease that typically presents before 5 years of age. Blau syndrome is characterized by the clinical triad of arthritis, granulomatous dermatitis, and recurrent uveitis. Here, we report a case of NOD2-mutation-associated early-onset sarcoidosis in which a combination of methotrexate and hydroxychloroquine was used to achieve improvement in arthritis, granulomatous dermatitis, and uveitis. A 13-month-old boy presented with a sudden-onset cutaneous eruption affecting the face, trunk, and extremities that initially mimicked papular atopic dermatitis but progressively worsened despite topical steroid therapy. The patient had no other known medical comorbidities or abnormalities except for heterochromia of the right eye. However, prior to presentation to dermatology, the patient began experiencing frequent falls, conjunctival injection, and apparent eye and joint pain. Skin biopsy from the right shoulder demonstrated rounded aggregates of epithelioid histiocytes and multinucleated giant cells without a significant lymphocytic component ("naked granulomas"), consistent with sarcoidal granulomatous dermatitis. Given the concern for Blau syndrome, the patient was sent for evaluation by ophthalmology and was found to have bilateral subconjunctival nodules. Our patient underwent genetic testing and was found to have a mutation in codon 1000 C > T (protein R334W) in the NOD2 gene. The patient responded to oral prednisolone 2 mg/kg/day for 8 weeks, but quickly relapsed, requiring a second 8-week course with taper upon starting methotrexate 7.5 mg subcutaneously weekly with 1 mg folic acid orally daily. After 8 weeks on methotrexate, due to persistent arthritis, conjunctival injection, and pruritus, and in consultation with rheumatology, the patient was started on hydroxychloroquine 75 mg orally daily along with continuation of 7.5 mg methotrexate subcutaneously weekly for 8 weeks, achieving significant reduction in arthritis, pruritus, and uveitis. After 8 weeks of this combination therapy, due to concerns of long-term macular toxicity, hydroxychloroquine was discontinued in favor of continuing methotrexate alone. The patient has remained free of significant side effects and stable with good disease control on 7.5 mg methotrexate weekly injected subcutaneously.

Multifaceted roles and regulation of nucleotide-binding oligomerization domain containing proteins.

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.

Loss of NOD2 in macrophages improves colitis and tumorigenesis in a lysozyme-dependent manner.

Background: Crohn's disease (CD) is a complex and poorly understood myeloid-mediated disorder. Genetic variants with loss of function in the NOD2 gene confer an increased susceptibility to ileal CD. While Nod2 in myeloid cells may confer protection against T-cell mediated ileopathy, it remains unclear whether it may promote resolution of the inflamed colon. In this study, we evaluated the function of Nod2 in myeloid cells in a model of acute colitis and colitis-associated colon cancer (CAC). Methods: To ablate Nod2 specifically within the myeloid compartment, we generated LysMCre/+;Nod2fl/fl mice. The role of NOD2 was studied in a setting of Dextran Sodium Sulfate (DSS)-induced colitis and in azoxymethane (AOM)/DSS model. Clinical parameters were quantified by colonoscopy, histological, flow cytometry, and qRT-PCR analysis. Results: Upon DSS colitis model, LysMCre/+;Nod2fl/fl mice lost less weight than control littermates and had less severe damage to the colonic epithelium. In the AOM/DSS model, endoscopic monitoring of tumor progression revealed a lowered number of adenomas within the colon of LysMCre/+;Nod2fl/fl mice, associated with less expression of Tgfb. Mechanistically, lysozyme M was required for the improved disease severity in mice with a defect of NOD2 in myeloid cells. Conclusion: Our results indicate that loss of Nod2 signaling in myeloid cells aids in the tissue repair of the inflamed large intestine through lysozyme secretion by myeloid cells. These results may pave the way to design new therapeutics to limit the inflammatory and tumorigenic functions of NOD2.

Receptor Interacting Ser/Thr-Protein Kinase 2 as a New Therapeutic Target.

Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a downstream signaling molecule essential for the activation of several innate immune receptors, including the NOD-like receptors (NOD1 and NOD2). Recognition of pathogen-associated molecular pattern proteins by NOD1/2 leads to their interaction with RIPK2, which induces release of pro-inflammatory cytokines through the activation of NF-κB and MAPK pathways, among others. Thus, RIPK2 has emerged as a key mediator of intracellular signal transduction and represents a new potential therapeutic target for the treatment of various conditions, including inflammatory diseases and cancer. In this Perspective, first, an overview of the mechanisms that underlie RIPK2 function will be presented along with its role in several diseases. Then, the existing inhibitors that target RIPK2 and different therapeutic strategies will be reviewed, followed by a discussion on current challenges and outlook.

Synthesis and validation of click-modified NOD1/2 agonists.

NOD1 and NOD2 sense small bacterial peptidoglycan fragments, often called muropeptides, that access the cytosol. These muropeptides include iE-DAP and MDP, the minimal agonists for NOD1 and NOD2, respectively. Here, we synthesized and validated alkyne-modified muropeptides, iE-DAP-Alk and MDP-Alk, for use in click-chemistry reactions. While it has long been known that many cell types respond to extracellular exposure to muropeptides, it is unclear how these innate immune activators access their cytosolic innate immune receptors, NOD1 and NOD2. The subcellular trafficking and transport mechanisms by which muropeptides access these cytosolic innate immune receptors are a major gap in our understanding of these critical host responses. The click-chemistry-enabled agonists developed here will be particularly powerful to decipher the underlying cell biology and biochemistry of NOD1 and NOD2 innate immune sensing.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Fusobacterium nucleatum-triggered neutrophil extracellular traps facilitate colorectal carcinoma progression.

BACKGROUND: Fusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance. METHODS: Fn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments. RESULTS: Tissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively. CONCLUSIONS: Our findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.

NOD2 silencing promotes cell apoptosis and inhibits drug resistance in chronic lymphocytic leukemia by inhibiting the NF-κB signaling pathway.

Recent years have witnessed increasing studies on the effect of epigenetic silencing of genes in the progression of chronic lymphocytic leukemia (CLL). This study investigates whether the nucleotide binding oligomerization domain containing 2 (NOD2) participates in the cell apoptosis and drug resistance of CLL cells. Cells were treated with adriamycin (ADR), etoposide, aclacinomycin and daunorubicin. After treatment, drug resistance and cell proliferation were examined to detect the inhibitory effect of ADR on cell proliferation; flow cytometry to identify ADR accumulation, the cell cycle distribution and apoptosis after transfection, and rhodamine 123 accumulation and efflux tests to assess P-glycoprotein (P-gp) function. NOD2 silencing or inhibition of the nuclear factor kappa-B (NF-κB) signaling pathway suppressed the multidrug resistance level in CLL, the inhibition rate, and cell proliferation caused by ADR at concentrations of approximately 0.25-1.5 µmol/L. Greater accumulation of ADR was observed in the CLL-AAT cell line than in the CLL-AAT/A02 cell line, but NOD2 silencing or inhibition of the NF-κB signaling pathway further increased the accumulation of ADR drugs in the CLL-AAT cell line and inhibited the drug efflux pump function of P-gp. Additionally, NOD2 silencing or NF-κB signaling pathway inhibition increased the apoptotic rate. The results of this study indicate that NOD2 promotes cell apoptosis and reduces the drug resistance of CLL by inhibiting the NF-κB signaling pathway.

Decoding the IBD paradox: A triadic interplay between REG3, enterococci, and NOD2.

In this issue of Cell Host & Microbe, Jang et al. decode an intriguing paradox: excessive antimicrobial peptide-regenerating family member 3 (REG3) expression in inflammatory bowel disease (IBD) patients depletes protective enterococci. This depletion impairs the NOD2 anti-inflammatory pathway and perpetuates inflammation, thus providing insight into IBD pathogenesis.

Butylphthalide improves brain damage induced by renal ischemia-reperfusion injury rats through Nrf2/HO-1 and NOD2/MAPK/NF-κB pathways.

Renal ischemia-reperfusion (I/R) injury leads to irreversible brain damage with serious consequences. Activation of oxidative stress and release of inflammatory mediators are considered potential pathological mechanisms. Butylphthalide (NBP) has anti-inflammatory and antioxidant effects on I/R injuries. However, it is unclear whether NBP can effectively mitigate renal I/R secondary to brain injury as well as its mechanism, which are the aims of this study. Both renal I/R injury rats and oxygen and glucose deprivation cell models were established and pre-intervened NBP. The Morris water maze assay was used to detect behavior. Hippocampal histopathology and function were examined after renal I/R. Apoptosis and tube-forming capacity of brain microvascular endothelial cells (BMVECs) were tested. Immunohistochemistry and Western blot were used to measure protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme Oxygenase-1 (HO-1) pathway and NOD-like receptor C2 (NOD2)/Mitogen-activated protein kinases (MAPK)/Nuclear factor kappa-B (NF-κB) pathway. NBP treatment attenuated renal I/R-induced brain tissue damage and learning and memory dysfunction. NBP treatment inhibited apoptosis and promoted blood-brain barrier restoration and microangiogenesis. Also, it decreased oxidative stress levels and pro-inflammatory factor expression in renal I/R rats. Furthermore, NBP enhanced BMVECs' viability and tube-forming capacity while inhibiting apoptosis and oxidative stress. Notably, the alleviating effects of NBP were attributed to Nrf2/HO-1 pathway activation and NOD2/MAPK/NF-κB inhibition. This study demonstrates that NBP maintains BBB function by activating the Nrf2/HO-1 pathway and inhibiting the NOD2/MAPK/NF-κB pathway to suppress inflammation and oxidative stress, thereby alleviating renal I/R-induced brain injury.