Family Pyrin Domain-containing Protein Research Articles

Chang-Kang-Fang alleviates diarrhea predominant irritable bowel syndrome (IBS-D) through inhibiting TLR4/NF-κB/NLRP3 pathway

Ethnopharmacological relevanceChang-Kang-Fang (CKF), originated from traditional Chinese medicine (TCM) formulas, has been utilized to treat diarrhea predominant irritable bowel syndrome (IBS-D) based on clinical experience. However, the underlying mechanism of CKF for treating IBS-D remains unclear and need further clarification. Aim of the studyThe objective of this present investigation was to validate the efficacy of CKF on IBS-D model rats and to uncover its potential mechanism for the treatment of IBS-D. Materials and methodsWe first established the IBS-D rat model through neonatal maternal separation (NMS) in combination with restraint stress (RS) and the administration of senna decoction via gavage. To confirm the therapeutic effect of CKF on treating IBS-D, abdominal withdrawal reflex (AWR) scores, the quantity of fecal pellets, and the fecal water content (FWC) were measured to evaluate the influence of CKF on visceral hypersensitivity and the severity of diarrhea symptom after the intragastric administration of CKF for 14 days. Subsequently, enzyme linked immunosorbent assay (ELISA) was applied to assess the effect of CKF on neuropeptides substance P (SP) and 5-hydroxytryptamine (5-HT), as well as inflammatory cytokines in serum and in intestinal tissues. Further, colonic pathological changes, the amount of colonic mast cells, and the expression level of occludin in rat colon tissues, were investigated by hematoxylin-eosin (HE) staining, toluidine blue staining, and immunohistochemistry, respectively. To explore the underlying mechanisms, alterations in colonic RNA transcriptomics for the normal, model, and CKF treatment groups were assessed using RNA sequencing (RNA-Seq). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays were applied to validate the effect of CKF on predicted pathways in vivo and in vitro. In addition, to elucidate the potential active compounds in CKF, 11 representative components found in CKF were selected, and their anti-inflammation potentials were evaluated using LPS-treated RAW264.7 cell models. ResultsCKF treatment significantly reduced the number of fecal pellets, attenuated visceral hypersensitivity, and decreased 5-HT and SP concentrations in serum and colon tissues, along with a reduction in colonic mast cell counts, correlating with improved symptoms in IBS-D rats. Meanwhile, CKF treatment reduced the colonic inflammatory cell infiltration, lowered the levels of IL-6, TNF-α, and IL-1β in serum and colon tissues, and increased the occludin protein expression in colon tissues to improve inflammatory response and colonic barrier function. RNA-Seq, in conjugation with our previous network pharmacology analysis, indicated that CKF might mitigate the symptoms of IBS-D rats by inhibiting the Toll like receptor 4/Nuclear factor kappa-B/NLR family pyrin domain-containing protein 3 (TLR4/NF-κB/NLRP3) pathway, which was confirmed by WB, IF, and qRT-PCR experiments in vivo and in vitro. Furthermore, coptisine, berberine, hyperoside, epicatechin, and gallic acid present in CKF emerged as potential active components for treating IBS-D, as they demonstrated in vitro anti-inflammatory effects. ConclusionOur findings demonstrate that CKF effectively improves the symptoms of IBS-D rats, potentially through the inhibition of the TLR4/NF-κB/NLRP3 pathway. Moreover, this study unveils the potential bioactive components in CKF that could be applied in the treatment of IBS-D.

Thymoquinone, a Novel Multi-Strike Inhibitor of Pro-Tumorigenic Breast Cancer (BC) Markers: CALR, NLRP3 Pathway and sPD-L1 in PBMCs of HR+ and TNBC Patients.

Breast cancer (BC) is not only a mass of malignant cells but also a systemic inflammatory disease. BC pro-tumorigenic inflammation has been shown to promote immune evasion and provoke BC progression. The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is activated when pattern recognition receptors (PRRs) sense danger signals such as calreticulin (CALR) from damaged/dying cells, leading to the secretion of interleukin-1β (IL-1β). CALR is a novel BC biological marker, and its high levels are associated with advanced tumors. NLRP3 expression is strongly correlated with an elevated proliferative index Ki67, BC progression, metastasis, and recurrence in patients with hormone receptor-positive (HR+) and triple-negative BC (TNBC). Tumor-associated macrophages (TAMs) secrete high levels of IL-1β promoting endocrine resistance in HR+ BC. Recently, an immunosuppressive soluble form of programmed death ligand 1 (sPD-L1) has been identified as a novel prognostic biomarker in triple-negative breast cancer (TNBC) patients. Interestingly, IL-1β induces sPD-L1 release. BC Patients with elevated IL-1β and sPD-L1 levels show significantly short progression-free survival. For the first time, this study aims to investigate the inhibitory impact of thymoquinone (TQ) on CALR, the NLRP3 pathway and sPD-L1 in HR+ and TNBC. Blood samples were collected from 45 patients with BC. The effect of differing TQ concentrations for different durations on the expression of CALR, NLRP3 complex components and IL-1β as well as the protein levels of sPD-L1 and IL-1β were investigated in the peripheral blood mononuclear cells (PBMCs) and TAMs of TNBC and HR+ BC patients, respectively. The findings showed that TQ significantly downregulated the expression of CALR, NLRP3 components and IL-1β together with the protein levels of secreted IL-1β and sPD-L1. The current findings demonstrated novel immunomodulatory effects of TQ, highlighting its potential role not only as an excellent adjuvant but also as a possible immunotherapeutic agent in HR+ and TNBC patients.

Polydatin protects against atherosclerosis by activating autophagy and inhibiting pyroptosis mediated by the NLRP3 inflammasome.

Polydatin is a bioactive ingredient extracted from the roots of the Reynoutria japonica Houtt, and it is a natural precursor of resveratrol. Polydatin is a useful inhibitor of inflammation and acts as a regulator of lipid metabolism. However, the specific mechanisms of action of polydatin in atherosclerosis (AS) remains poorly explained. The aim of this study was to assess the efficacy of polydatin on inflammation induced by the inflammatory cell death and autophagy in AS. Apolipoprotein E knockout (ApoE-/-) mice were fed with a high-fat diet (HFD) for 12 weeks to induce the formation of atherosclerotic lesions. The ApoE-/- mice were then randomly divided into the following six groups: (1) model group, (2) simvastatin group, (3) MCC950 group, (4) low dose polydatin group (Polydatin-L), (5) medium dose polydatin group (Polydatin-M), (6) and high dose polydatin group (Polydatin-H). The C57BL/6J mice were treated as controls and administered a standard chow diet. All mice were gavaged once daily for 8 weeks. The distribution of aortic plaques was observed by En Oil-red-O staining and hematoxylin and eosin staining (H&E). Oil-red-O staining was used to observe lipid content in the aortic sinus plaque; Masson trichrome staining was used to gauge collagen content in the plaque; and immunohistochemistry was used to evaluate smooth muscle actin (α-SMA) and CD68 macrophages marker expression levels in the plaque, which were used to assess the vulnerability index of the plaque. The lipid levels were measured using an enzymatic assay with an automatic biochemical analyzer. The level of inflammation was detected by enzyme-linked-immunosorbent assay (ELISA). Autophagosomes were detected by transmission electron microscopy (TEM). Pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/caspase-1 and other proteins related to the expression levels of autophagy and pyroptosis were detected by Western blot analysis. Nucleotide oligomerization (NOD)-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome activation leads to pyroptosis, including the cleavage of caspase-1, interleukin (IL)-1β and IL-18 production, and the co-expression of TUNEL/caspase-1-all of these are inhibited by polydatin, whose inhibitory effect is similar to that of MCC950, a specific inhibitor of NLRP3. Further, polydatin decreased the protein expression of NLRP3 and the phosphorylated mammalian target of rapamycin (p-mTOR), and increased the number of autophagosomes as well as the increased the cytoplasmic microtubule-associated protein light chain 3 (LC3)/autophagosome membrane-type LC3 ratio. Moreover, the protein expression levels of p62 decreased, suggesting that polydatin can increase autophagy. Polydatin can inhibit the activation of the NLRP3 inflammasome and cleavage of caspase-1, thereby inhibiting pyroptosis and secretion of inflammatory cytokines, and promoting autophagy through NLRP3/mTOR pathway in AS.

Pharmacological inhibition of the NLRP3 inflammasome as a potential target for multiple sclerosis induced central neuropathic pain.

The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is implicated in the pathogenesis of multiple diseases including neuroinflammation associated with multiple sclerosis (MS). However, the extent to which NLRP3 has a pathobiological role in MS-associated central neuropathic pain (CNP) is unknown. Hence, the present study was designed to address this issue using an optimised relapsing-remitting experimental encephalomyelitis (RR-EAE)-mouse model of MS-associated neuropathic pain. RR-EAE mice with fully developed mechanical allodynia in the bilateral hindpaws (paw withdrawal thresholds (PWTs)≤1g) at day 16 post-immunisation (p.i.) were administered single oral bolus doses of MCC950, a selective and potent small-molecule inhibitor of NLRP3, once daily for 21 consecutive days. Following administration of the first dose of MCC950 at 50 mgkg-1, the mean (±SEM) peak anti-allodynic effect was observed at~1h post-dosing with a duration of action of~2h. Following chronic dosing with MCC950, mechanical allodynia in the bilateral hindpaws was progressively reversed by oral treatment with MCC950 (50 mgkg-1day-1), but not vehicle. Specifically, by day 25 p.i. and continuing until study completion on day 36 p.i., bilateral hindpaw PWTs of RR-EAE mice treated with MCC950 (50 mgkg-1day-1) did not differ significantly (P>0.05) from the corresponding hindpaw PWTs for the sham (control) group. In addition, MCC950 at 50 mgkg-1day-1 attenuated disease relapses in RR-EAE mice indicated by tail limpness as well as hindlimb weakness. Together, our findings suggest that inhibition of NLRP3 inflammasome activation may be a potential therapeutic approach to alleviate MS-associated CNP and disease relapses in patients with RR-MS.