NOD-like Receptor Pyrin Domain Containing 3 Research Articles

Up-regulation of microRNA-135 or silencing of PCSK6 attenuates inflammatory response in preeclampsia by restricting NLRP3 inflammasome

ObjectiveNumerous studies have confirmed the correlation of microRNAs (miRNAs) with human disease, yet few have explored the role of miR-135 in preeclampsia (PE). This study intends to discuss miR-135’s function in inflammatory response in PE by modulating proprotein convertase subtilisin/kexin-6 (PCSK6) and NLR pyrin domain containing 3 (NLRP3).MethodsThe venous blood and placental tissues were collected from PE pregnant women and 25 normal ones. The levels of miR-135, PCSK6 and NLRP3 in placenta tissues of patients were detected. Hypoxia/reoxygenation HTR-8/SVneo and HPT-8 models were established to mimic PE in vitro, and cell proliferation, colony formation, apoptosis rate, invasion, migration and inflammation were detected through gain-of and loss-of-function assays.ResultsMiR-135 was down-regulated, and PCSK6 and NLRP3 were up-regulated in PE patients. Up-regulating miR-135 or silencing PCSK6 strengthened colony formation ability, viability, invasion and migration ability, and weakened apoptosis and inflammation of H/R-treated HTR-8/SVneo and HPT-8 cells. Inhibition of NLRP3 negated the effects of silenced PCSK6 in H/R-treated HTR-8/SVneo and HPT-8 cells.ConclusionsAltogether, we demonstrate that up-regulated miR-135 or reduced PCSK6 attenuates inflammatory response in PE by restricting NLRP3 inflammasome, which provides novel therapy for PE treatment.

NLRP3 Inflammasome: A Potential Target in Isoflurane Pretreatment Alleviates Stroke-Induced Retinal Injury in Diabetes.

Ischemic stroke remains a devastating disease which is the leading cause of death worldwide. Visual impairment after stroke is a common complication which may lead to vision loss, greatly impacting life quality of patients. While ischemic stroke is traditionally characterized by a blockage of blood flow to the brain, this may coincide with reduced blood flow to the eye, resulting in retinal ischemia and leading to visual impairment. Diabetes increases the risk of ischemic stroke and induces diabetic retinopathy; the latter may be more sensitive to the ischemic retinal injury. In diabetic status, the underlying mechanism in stroke-induced retinal injury has not been fully clarified. The NLR pyrin domain containing 3 (NLRP3) inflammasome is an important activator of inflammation, which may play a critical role in catalyzing and forming certain pro-inflammatory cytokines in both cerebral and retinal ischemia. Isoflurane has been demonstrated to inhibit the activation of the NLRP3 inflammasome and show neuroprotective effects. In this study, we established a diabetic mouse model and performed the middle cerebral artery occlusion procedure to induce ischemic stroke. Our results revealed that cerebral ischemia-induced retinal injury in the diabetic model. Isoflurane pretreatment alleviated the cerebral and retinal injury after ischemic stroke. Of note, isoflurane pretreatment inhibited the NLRP3 inflammasome activation in the retina, indicating that isoflurane pretreatment may provide substantial retinal protection in stroke-induced retinal injury in diabetes.

Alendronate Augments Lipid A-Induced IL-1α Release via Activation of ASC but Not Caspase-11.

Nitrogen-containing bisphosphonates (NBPs), such as alendronate (ALN), are anti-bone-resorptive drugs that have inflammatory side effects. We previously reported that ALN augmented lipid A-induced interleukin (IL)-1β production and NOD-like receptor pyrin domain-containing-3 (NLRP3)/apoptosis-associated speck-like protein containing a CARD (ASC)-dependent cell death. The present study aimed to examine whether ALN augments lipid A-induced IL-1α release and necroptosis, which is induced by the activation of receptor-interacting protein kinase (RIPK) 3. Treatment of J774.1 cells with ALN augmented lipid A-induced IL-1α release, which was not inhibited by Ac-IETD-CHO, a caspase-8 inhibitor. ALN also activated mixed lineage kinase domain-like (MLKL), a key mediator of the necroptosis pathway, and upregulated the expression of caspase-11, a lipid A receptor. GSK'872, a RIPK3 inhibitor, suppressed the ALN-upregulated expression of caspase-11 and augmented lipid A-induced caspase-8 activation. Moreover, ALN induced the release of NLRP3 and ASC into culture supernatants. GSK'872, but not Ac-IETD-CHO, reduced the ALN-induced release of NLRP3, but not ASC, into culture supernatants, and reduced ALN-induced cell death, but not ALN-induced LDH release. Antibodies against NLRP3 and ASC upregulated caspase-11 expression in the cytosol by inhibiting ALN-induced cell death. However, pretreating cells with an antibody against ASC, but not NLRP3, before ALN addition also inhibited lipid A-induced IL-1α release. Pretreating cells with an antibody against caspase-11 before the addition of ALN or lipid A did not downregulate lipid A-induced production of IL-1α. Taken together, our findings suggest that ALN augments lipid A-induced IL-1α release via activation of ASC, but not caspase-11.

Sodium Houttuyfonate Ameliorates β-amyloid1-42-Induced Memory Impairment and Neuroinflammation through Inhibiting the NLRP3/GSDMD Pathway in Alzheimer's Disease.

Objective Our research is designed to explore the function of sodium houttuyfonate (SH) on Alzheimer's disease (AD) and its potential molecular mechanisms. Methods In our study, the Morris water maze (MWM) test was used to assess the role of SH on spatial learning and memory deficiency in amyloid-β peptide (Aβ)1-42-induced AD mice. We explored the functions of SH on proinflammatory cytokines, neuron apoptosis, and damage in vivo and in vitro by using an enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, western blot, and Nissl staining. Moreover, the effect of SH on oxidative stress in vivo and in vitro was also detected. To explore the underlying molecular mechanisms of SH on AD, the expressions of proteins and mRNA involved in the NOD-like receptor pyrin domain containing-3/gasdermin D (NLRP3/GSDMD) pathway were determined using western blot, immunofluorescence staining, and qRT-PCR. Results Our data demonstrated that SH ameliorated spatial learning and memory deficiency in Aβ1-42-induced AD mice. Moreover, SH significantly improved hippocampal neuron damage and inhibited oxidative stress, neuroinflammation, and neuron apoptosis in Aβ1-42-induced AD mice and PC12 cells. The results also revealed that SH protected Aβ1-42-induced AD through inhibiting the NLRP3/GSDMD pathway. Conclusion The present study demonstrated that SH could ameliorate Aβ1-42-induced memory impairment neuroinflammation and pyroptosis through inhibiting the NLRP3/GSDMD pathway in AD, suggesting that SH may be a potential candidate for AD treatment.

Protective Effect of Joa-Gui Em through the Improvement of the NLRP3 and TLR4/NF-κb Signaling by Ischemia/Reperfusion-Induced Acute Renal Failure Rats.

Joa-gui em (左歸飮, JGE) is known to be effective for treating kidney-yin deficient syndrome. However, there is a lack of objective pharmacological research on improving kidney function. This study was designed to evaluate whether JGE improves renal function and related mechanisms in rats with acute renal injury induced by ischemia/reperfusion (I/R). The acute renal failure (ARF) group was subjected to reperfusion after inserting a clip into the renal artery for 45 min. The ARF + JGE (100 or 200 mg/kg/day) groups were orally administered for four days after their I/R surgery, respectively. JGE treatment suppressed the increase in kidney size in the ARF animal model and alleviated the polyuria symptoms. In addition, to confirm the effect of improving the kidney function of JGE, lactate dehydrogenase levels, blood urea nitrogen/creatinine ratio, and creatinine clearance were measured. As a result, it decreased in the ARF group but significantly improved in the JGE group. Also, as a result of examining the morphological aspects of renal tissue, it was shown that JGE improved renal fibrosis caused by ARF. Meanwhile, it was confirmed that JGE reduced inflammation through the nucleotide-binding oligomerization domain-like receptor pyrin domain containing-3 (NLRP3) and toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathways, which are the major causes of acute ischemic kidney injury, thereby improving renal function disorder. The JGE has a protective effect by improving the NLRP3 and TLR4/NF-κB signaling pathway in rats with acute renal dysfunction induced by I/R injury.

Cerebroprotection by dioscin after experimental subarachnoid haemorrhage via inhibiting NLRP3 inflammasome through SIRT1-dependent pathway.

Dioscin has multiple biological activities and is beneficial for cardiovascular and cerebral vascular diseases. Here, we investigated the protective effects of dioscin against subarachnoid haemorrhage and the molecular mechanisms involved. Dioscin was administered after subarachnoid haemorrhage induced in rats. MCC950, a potent selective nod-like receptor pyrin domain-containing 3 (NLRP3) inhibitor, was used to suppress NLRP3 and EX527 (selisistat) was used to inhibit sirtuin 1 (SIRT1). In vivo, dioscin inhibited acute inflammatory response, oxidative damage, neurological impairment and neural cell degeneration after subarachnoid haemorrhage along with dramatically suppressing NLRP3 inflammasome activation. While pretreatment with MCC950 reduced the inflammatory response and improved neurological outcomes it did not lessen ROS production. However, giving dioscin after MCC950 reduced acute brain damage and ROS production. Dioscin increased SIRT1 expression after subarachnoid haemorrhage, whereas EX527 abolished the up-regulation of SIRT1 induced by dioscin and offset the inhibitory effects of dioscin on NLRP3 inflammasome activation. EX527 pretreatment also reversed the neuroprotective effects of dioscin against subarachnoid haemorrhage. Similarly, in vitro, dioscin dose-dependently suppressed inflammatory response, oxidative damage and neuronal degeneration and improved cell viability in neurons and microglia co-culture system. These effects were associated with inhibition of the NLRP3 inflammasome and stimulation of SIRT1 signalling, which could be inhibited by EX527 pretreatment. Dioscin provides protection against subarachnoid haemorrhage via the suppression of NLRP3 inflammasome activation through SIRT1-dependent pathway. Dioscin may be a new candidate to ameliorate early brain injury after subarachnoid haemorrhage.

Thioredoxin interacting protein regulates age-associated neuroinflammation

Immune system hypersensitivity is believed to contribute to mental frailty in the elderly. Solid evidence indicates NOD-like receptor pyrin domain containing-3 (NLRP3)-inflammasome activation intimately connects aging-associated chronic inflammation (inflammaging) to senile cognitive decline. Thioredoxin interacting protein (TXNIP), an inducible protein involved in oxidative stress, is essential for NLRP3 inflammasome activity. This study aims to find whether TXNIP/NLRP3 inflammasome pathway is involved in senile dementia. According to our studies on sex-matched mice, TXNIP was significantly upregulated in aged animals, paralleled by the NLRP3-inflammasome over-activity leading to enhanced caspase-1 cleavage and IL-1β maturation, in both sexes. This was closely associated with depletion of the anti-aging and cognition enhancing protein klotho, in aged males. Txnip knockout reversed age-related NLRP3-hyperactivity and enhanced thioredoxin (TRX) levels. Further, TXNIP inhibition along with verapamil replicated TXNIP/NLRP3-inflammasome downregulation in aged animals, with FOXO-1 and mTOR upregulation. These alterations concurred with substantial improvements in both cognitive and sensorimotor abilities. Together, these findings substantiate the pivotal role of TXNIP to drive inflammaging in parallel with klotho depletion and functional decline, and delineate thioredoxin system as a potential target to decelerate senile dementia.

Role of P2X7R in eosinophilic and non‑eosinophilic chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammation-mediated disease of the nasal mucosa. P2X7R has been reported to be a potential biomarker for inflammation. The aim of the present study was to explore the role of P2X7R in CRSwNP, and the interaction between P2X7R and the NLRP3 inflammasome in the development of CRSwNP. Firstly, the expression profiles of P2X7R in nasal mucosa were investigated using western blotting (WB), polymerase chain reaction (PCR) and immunofluorescence (IF) staining. Next, the effect of inflammatory stimulation with lipopolysaccharides (LPS) combined with 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate triethylammonium salt (BzATP) on primary human nasal epithelial cells (HNECs) was determined. Then, the therapeutic effect of the selective P2X7R antagonist, A740003, on P3X7R, NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome and IL-1β alterations in HNECs was explored using enzyme-linked immunosorbent assay, WB and PCR. It was found that P2X7R was overexpressed in CRSwNP, especially in eosinophilic CRSwNP, the expression of P2X7R, NLRP3 and IL-1β were upregulated in HNECs after induction by LPS combined with BzATP; but the expression of NLRP3 and IL-1β were downregulated after stimulation with A740003. The aforementioned results indicate that P2X7R-mediated NLRP3 inflammasome activation may have a role in the pathogenesis of CRSwNP.

Protective effect of combination of anakinra and MCC950 against acute lung injury is achieved through suppression of the NF-κB-mediated-MAPK and NLRP3-caspase pathways

BackgroundIt has been uncovered that the interleukin-1 receptor antagonist anakinra and the NLRP3 inflammasome blocker MCC950 can alleviate acute lung injury (ALI). However, the specific mechanism underlying these effects remains unknown. Thus, we sought to investigate the effects of anakinra and MCC950 in ALI as well as the molecular mechanisms. MethodsWe treated C57BL/6 mice with aerosols of anakinra and/or MCC950 along with lipopolysaccharide (LPS), followed by mechanical ventilation (MV) treatment after 1.5 h of inhalation of aforementioned compounds. Lung injury was assessed by determining the level of inflammatory factors in the alveolar lavage fluid and monitoring blood oxygen saturation. We confirmed our findings of regulation of the ALI model through the nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK)/nucleotide binding domain and leucine-rich repeat (NLR) pyrin domain containing 3 (NLRP3)-caspase pathway in further studies with RelA−/− mice. ResultsCombined treatment of anakinra and MCC950 presented the best therapeutic effect on LPS and MV-induced ALI than did treatment with anakinra or MCC950 alone. Combined therapy with anakinra and MCC950 suppressed MAPK and NLRP3-caspase via inhibition of the NF-κB pathway to improve ALI, but the therapeutic pathway was revoked by knockout of NF-κB. ConclusionTaken together, combined treatment of anakinra and MCC950 was effective in alleviating ALI in the mouse model, highlighting a new insight into ALI treatment.

N-acetylserotonin alleviated the expression of interleukin-1β in retinal ischemia–reperfusion rats via the TLR4/NF-κB/NLRP3 pathway

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1β (IL-1β) in the retina of retinal ischemia–reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p–NF–κB), NLRP3, cleaved Caspase-1, and IL-1β in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4+ and cleaved Caspase-1+ with IL-1β+ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4+, p–NF–κB+, NLRP3+, cleaved Caspase-1+, and IL-1β+ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1β in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p–NF–κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1β+ cells was significantly correlated with those of TLR4+ and cleaved Caspase-1+ cells in the RIRI + NAS group (r2 = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1β by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

Three functional variants in the NLRP3 gene are associated with susceptibility and clinical characteristics of systemic lupus erythematosus.

Nod-like receptor pyrin domain containing 3 (NLRP3) gene encodes an intracellular receptor whose dysregulation in systemic lupus erythematosus (SLE) has been reported in multiple studies. Activation of NLRP3 inflammasome leads to the induction of inflammatory response via cleaving and producing of specific cytokines. In the present study, we assessed the possible association between three functional polymorphisms in this gene and SLE risk in the Iranian population. These variants include two gain of function (rs4612666 and rs10754558) and one loss of function (rs6672995) which are correlated with modulation of expression of NLRP3. A case-control study involving 110 SLE patients and 116 control subjects was undertaken to estimate the frequency of rs4612666, rs10754558, and rs6672995 genotypes using real-time PCR high resolution melting method (HRM). Our findings revealed significant associations between GG genotype and G allele of rs10754558 with increased risk of SLE (OR for GG genotype= 2.82; 95%CI [1.45-5.46]/OR for G allele= 1.97; 95%CI [1.36-2.87]). Although, no significant associations were recognized between allele and genotype frequencies of rs4612666 and rs6672995 polymorphisms with SLE risk (P > 0.05). Also, our analysis revealed that the C allele in rs4612666 and G allele in rs10754558 was correlated with the severity of disease activity (P < 0.001). Moreover, these common variants were associated with lower age of onset and some clinical symptoms in the patient group, such as skin manifestation, neurological symptom and, renal involvement (P < 0.05). This study demonstrates a substantial association between NLRP3 polymorphisms with increased risk, clinical symptoms, and the severity of disease activity of SLE.

Luteolin ameliorates LPS-induced acute liver injury by inhibiting TXNIP-NLRP3 inflammasome in mice

BackgroundChemical liver injury is one of the main causes of acute liver failure and death. To date, however, treatment strategies for acute liver injury have been limited. Therefore, there is an urgent need to find new therapeutic targets and effective drugs. NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome is a complex of multiple proteins that has been shown to induce cell death under inflammatory and stress pathologic conditions and is thought to provide new targets for the treatment of a variety of diseases. PurposeThe purpose of this study was to investigate whether luteolin has a protective effect on the liver and further elucidate whether it is realized through the thioredoxin interacting protein (TXNIP)-NLRP3 axis. Study designAcute hepatic injury in mice caused by intraperitoneal injection of lipopolysaccharide (LPS) was treated with or without luteolin. MethodsMale C57BL/6 mice and mouse primary hepatocytes were selected. TXNIP protein knockdown was achieved by siRNA, qPCR and Western blot were performed to explore the mechanism of luteolin in alleviating acute liver injury. ResultsThe results indicated that luteolin had a markedly protective effect on acute liver injury induced by LPS in mice by inhibiting the TXNIP-NLRP3 axis. Luteolin inhibits NLRP3 inflammasome activation by suppressing TXNIP, apoptosis associated speck-like protein containing a CARD domain (ASC), caspase-1, interleukin-1β (IL-1β) and IL-18 to reduce liver injury. In addition, luteolin inhibits LPS-induced liver inflammation by inhibiting the production of inflammation-related gene tumor necrosis factor-α (TNF-α), IL-10, and IL-6. What's more, luteolin alleviated LPS-induced hepatocyte injury by inhibiting oxidative stress and regulating MDA, SOD, and GSH levels. However, the protective effect of luteolin on acute LPS-induced liver injury in mice was blocked by si-TXNIP in vitro. ConclusionsThese combined data showed that luteolin may alleviate LPS-induced liver injury through the TXNIP-NLPR3 axis, providing new therapeutic targets and therapeutic drugs for subsequent studies.

Chlorpromazine and promethazine reduces Brain injury through RIP1-RIP3 regulated activation of NLRP3 inflammasome following ischemic stroke

ABSTRACT Objectives: Stroke is an important cause of death and disability. Recent evidence suggests that post-stroke inflammation is an important factor in stroke pathology and a root cause of its lasting consequences. Phenothiazine drugs, like chlorpromazine and promethazine (C + P), induce hypothermia and have been shown to play a major role in neuroprotection. In the present study, we investigated this neuroprotective mechanism by assessing the anti-inflammatory effect of these drugs. Methods: Adult Sprague-Dawley rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion, with or without C + P (8 mg/kg). Infarct volumes, neurological deficits, along with mRNA and protein quantities of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), NLRPyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β) were assessed, as well as the infiltration of neutrophils and macrophages. Results: C + P induced hypothermia that significantly reduced RIP1, RIP3, NLRP3 and IL-1β expression, infarction, and immune cell infiltration, while C + P treatment with temperature control at 37°C induced lesser effect. Conclusion: These findings suggest that the anti-inflammatory effect of C + P may be dependent on drug-induced hypothermia and regulation of the NLRP3 inflammasome via the RIP1/RIP3 complex. Future investigations are needed regarding C + P as potential treatment of ischemic stroke.

Association of nod-like receptor pyrin domain containing 3 (rs10754558) and protein kinase C zeta (rs2503706) gene polymorphisms with the risk of type 2 diabetes mellitus in Indian population

BackgroundType 2 diabetes mellitus (T2DM) is a multifactorial chronic disease, characterized by hyperglycemia due to defect in insulin action, secretion or both. Nod-like receptor pyrin domain containing 3 (NLRP3) and protein kinase C zeta (PRKCZ) genes show a significant role in obesity-induced inflammation and insulin resistance. Polymorphisms of NLRP3 and PRKCZ genes have been reported with contradictory results as genetic risk factor for T2DM in different populations. Therefore, the current study was conducted in North Indian population to investigate the association of single nucleotide polymorphism (SNP) of NLRP3 (rs10754558) and PRKCZ (rs2503706) gene with T2DM. MethodThe study included a total of 270 subjects comprised of 102 healthy controls and 168 T2DM cases and SNPs were genotyped by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) method. Clinical parameters like glycated hemoglobin (HbA1c), fasting blood glucose (FBG), high density lipoprotein cholesterol (HDLC), triglycerides (TG), total cholesterol (CHL) and fasting insulin were determined by enzymatic methods. Result and conclusionA statistically significant association was observed between NLRP3 gene at SNP rs10754558 (C vs. G, OR (95% C.I.) = 1.77 (1.21–2.58), P = 0.002) and T2DM while significant association could not be observed between PRKCZ gene at SNP rs2503706 (C vs. T OR (95% C.I.) = 1.86 (0.94–3.67), P = 0.070) and T2DM. It can be concluded from these results that NLRP3 gene polymorphism at SNP rs10754558 is associated with the development of T2DM.

Low androgen status inhibits erectile function by increasing pyroptosis in rat corpus cavernosum

The mechanism of erectile dysfunction (ED) caused by low androgen status is not fully understood. To investigate whether low androgen status inhibits erectile function of rats by inducing pyroptosis in the corpus cavernosum (CC). Thirty-six eight-weeks-old healthy male Sprague-Dawley rats were equally divided into six groups: sham-operated group (4w sham, 8w sham), castration group (4w cast, 8w cast), and castration + testosterone (T) group (4w cast + T, 8w cast + T). The rats in castration + T groups were injected with testosterone propionate subcutaneously every other day. After 4 and 8weeks, the ratio of maximum intracavernous pressure (ICPmax)/mean arterial pressure (MAP), the level of serum T, the concentration of nitric oxide (NO) and interleukin-1β (IL-1β), the expression of NOD-like receptor pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), Caspase-1 p20, gasdermin D-N (GSDMD-N), transforming growth factor β1 (TGF-β1), collagen-I, and collagen-III, the ratio of smooth muscle/collagen (SM/C), and the proportion of pyroptotic cells in the CC were analyzed. The ratio of ICPmax/MAP (3/5V) and SM/C, the level of NO and serum T was significantly decreased in castration groups when compared to other groups (p<0.01). NLRP3, ASC, Caspase-1, and GSDMD were mainly expressed in the cytoplasm of smooth muscle cells (SMCs) and endothelial cells (ECs) in the CC. The expression of NLRP3, ASC, Caspase-1p20, GSDMD-N, IL-1β, TGF-β1, collagen-I, and collagen-III was significantly increased in castration groups when compared with other groups (p<0.01). The proportion of pyroptotic cells in the CC was increased significantly in castration groups when compared with other groups (p<0.05). Low androgen status inhibits erectile function of rats by promoting CC fibrosis and reducing NO synthesis through pyroptosis of SMCs and ECs in the CC.

Regulatory effect of NLRP3 on airway inflammatory response and pyroptosis in mice with asthma.

To study the regulatory effect of the NOD-like receptor, pyrin domain-containing 3 (NLRP3) on airway inflammatory response and pyroptosis in mice with asthma. The NLRP3 wild-type (WT) C57BL/6J mice were divided into two groups: NLRP3-WT control and NLRP3-WT asthma. The mice with NLRP3 knockout (KO) were divided into two groups: NLRP3-KO control and NLRP3-KO asthma (n=10 each). A model of asthma was prepared by intraperitoneal injection of ovalbumin + aluminium hydroxide for sensitization and ovalbumin inhalation for challenge. Enhanced pause, an index for airway responsiveness, was measured for each group. Hematoxylin and eosin staining was used to observe the histomorphological changes of lungs and determine the inflammation score for each group. Bronchoalveolar lavage fluid was collected from each group to determine the numbers of neutrophils, eosinophils, and lymphocytes and measure the content of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Western blot was used to measure the expression of NLRP3, cleaved caspase-1, and Gasdermin D-N in lung tissue of each group. Compared with the NLRP3-WT control group, the NLRP3-WT asthma group showed morphological changes including airway smooth muscle thickening and inflammatory cell infiltration. Compared with the NLRP3-WT asthma group, the NLRP3-KO asthma group had significant improvements in the above morphological manifestations. Compared with the NLRP3-WT control group, the NLRP3-WT asthma group had significant increases in the enhanced pause, the inflammation score of lung tissue, the numbers of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage fluid, and the levels of IL-1β and IL-18 in bronchoalveolar lavage fluid (P<0.05). The expression of NLRP3, cleaved caspase-1, and Gasdermin D-N in lung tissue also significantly increased in the NLRP3-WT asthma group (P<0.05). The above indices in the NLRP3-KO asthma group were significantly lower than those in the NLRP3-WT asthma group (P<0.05). The overexpression of NLRP3 is associated with the pathogenesis of asthma, which may be related to the molecular mechanisms of the activation of airway inflammatory response and pyroptosis. Citation.

Melatonin Alleviates Radiculopathy Against Apoptosis and NLRP3 Inflammasomes Via the Parkin-Mediated Mitophagy Pathway

Animal experimental study with intervention. To investigate the effect of melatonin on rat radiculopathy model and dorsal root ganglion (DRG) cells, and to elucidate the underlying mechanism. Melatonin has a well-documented efficacy in intervertebral disc degeneration (IVDD) and low back pain. IVDD can also lead to other complications such as disc herniation which will cause radiculopathy. Herniated nucleus pulposus (NP) induced apoptosis and NLR pyrin domain containing 3 (NLRP3) activation in DRG. However, the effect and underlying mechanism of melatonin on radiculopathy and DRG cells are still unclear. Rat radiculopathy model was induced by implanting NP tissue from the tail disc of the same rat into the left L4/5 inter-laminar space near the left DRG. Melatonin was injected intraperitoneally in the treated group to test its function. Apoptosis was determined by Tunnel staining and flow cytometry. NLRP3 inflammasome activation was determined by levels of NLRP3, ASC, GSMDM-N, IL-1β, and Caspase-1. Mitophagy was determined by levels of Parkin, Beclin-1, p62, and LCB-II. Mitophagy was blocked by treatment with Parkin-si or cyclosporine A (CsA). NLRP3 was significantly upregulated in DRG of rat radiculopathy model; moreover, melatonin markedly decreased pain behavior in rat radiculopathy model. Furthermore, melatonin treatment decreases the incidence of apoptosis in DRG cells. Melatonin also promotes mitophagy and inhibits NLRP3 inflammasomes in DRG cells. In addition, mitophagy was blocked by treatment with Parkin-si and CsA. Both Parkin-si and CsA attenuated melatonin's inhibitory effect on apoptosis and the NLRP3 inflammasome, indicating that the beneficial effects of melatonin in DRG cells are mediated through the Parkin-mediated mitophagy. Melatonin alleviates radiculopathy against apoptosis and NLRP3 inflammasomes by promoting Parkin-mediated mitophagy, which may help us provide a potential target for the treatment of radiculopathy.Level of Evidence: N/A.

Possible Protective Role of NLRC4 Inflammasome in Periodontal Diseases: A Preliminary Study

Introduction: Inflammasomes are multiprotein complexes, which regulate proinflammatory cytokines, Interleukin-18 (IL-18), and Interleukin-1β (IL-1β) that are associated with periodontal breakdown. This study investigated the expression of NOD-like receptor pyrin domain-containing-3 (NLRP3) and NOD-like receptor family CARD domain-containing protein 4 (NLRC4) inflammasomes in different periodontal diseases in humans and their potential association with IL-18 release in gingival crevicular fluid (GCF). Materials and Methods: A total of 45 participants (21 males and 24 females) divided into four groups; periodontally healthy (H), gingivitis (G), chronic periodontitis (CP), and aggressive periodontitis (AgP) based on periodontal examination. NLRC4 and NLRP3 expression were detected by immunohistochemistry in gingival tissue samples for all groups. Expression percentage (%) and staining intensity distribution score (SID) were calculated for both NLRC4 and NLRP3. IL-18 was measured in GCF via enzyme linked immunosorbent assay (ELISA). Results: Positive immunoreactivity was seen for NLRC4 and NLRP3 across groups. No differences were found for NLRC4 expression %, but SID scores were slightly higher in G and AgP compared to other groups (P > 0.05). Results showed a significant increase of NRLP3 expression % in group CP compared to group H (P < 0.05) without affecting SID scores (P > 0.05). IL-18 levels were significantly higher in AgP and CP groups compared to H and G groups (P < 0.05). IL-18 significantly and positively correlated with clinical attachment levels across groups. Conclusion: Within the limitations of this preliminary study, we suggest that the NLRC4 platform may have a protective role contrary to the NLRP3 platform influencing IL-18 release and associated periodontal tissue breakdown.

Luteolin Confers Cerebroprotection after Subarachnoid Hemorrhage by Suppression of NLPR3 Inflammasome Activation through Nrf2-Dependent Pathway.

Luteolin (LUT) possesses multiple biologic functions and has beneficial effects for cardiovascular and cerebral vascular diseases. Here, we investigated the protective effects of LUT against subarachnoid hemorrhage (SAH) and the involvement of underlying molecular mechanisms. In a rat model of SAH, LUT significantly inhibited SAH-induced neuroinflammation as evidenced by reduced microglia activation, decreased neutrophil infiltration, and suppressed proinflammatory cytokine release. In addition, LUT markedly ameliorated SAH-induced oxidative damage and restored the endogenous antioxidant systems. Concomitant with the suppressed oxidative stress and neuroinflammation, LUT significantly improved neurologic function and reduced neuronal cell death after SAH. Mechanistically, LUT treatment significantly enhanced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2), while it downregulated nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation. Inhibition of Nrf2 by ML385 dramatically abrogated LUT-induced Nrf2 activation and NLRP3 suppression and reversed the beneficial effects of LUT against SAH. In neurons and microglia coculture system, LUT also mitigated oxidative stress, inflammatory response, and neuronal degeneration. These beneficial effects were associated with activation of the Nrf2 and inhibitory effects on NLRP3 inflammasome and were reversed by ML385 treatment. Taken together, this present study reveals that LUT confers protection against SAH by inhibiting NLRP3 inflammasome signaling pathway, which may be modulated by Nrf2 activation.

ITRAQ-Based Quantitative Proteomics Indicated Nrf2/OPTN-Mediated Mitophagy Inhibits NLRP3 Inflammasome Activation after Intracerebral Hemorrhage.

Intracerebral hemorrhage- (ICH-) induced secondary brain injury (SBI) is a very complex pathophysiological process. However, the molecular mechanisms and drug targets of SBI are highly intricate and still elusive, yet a clear understanding is crucial for the treatment of SBI. In the current study, we aimed to confirm that nuclear factor-E2-related factor 2 (Nrf2)/Optineurin- (OPTN-) mediated mitophagy alleviated SBI by inhibiting nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation based on the isobaric tag for relative and absolute quantization (iTRAQ) quantification proteomics. Human ICH brain specimens were collected for iTRAQ-based proteomics analysis. Male Nrf2 wild-type (WT) and knockout (KO) mice were employed to establish ICH murine models. The survival rate, hematoma volume, neurofunctional outcomes, blood-brain barrier (BBB) permeability, brain edema, spatial neuronal death, NLRP3 inflammasome, inflammatory response, mitochondrial function, and mitophagy level were evaluated after ICH. The iTRAQ quantification analysis showed that the differentially expressed proteins (DEPs), Nrf2 and NLRP3, were closely associated with the initiation and development of SBI after ICH. The Nrf2 KO mice had a significantly lower survival rate, bigger hematoma volume, worse neurological deficits, and increased BBB disruption, brain edema, and neuronal death when compared with the Nrf2 WT mice after ICH. Furthermore, Nrf2 KO enhanced NLRP3 inflammasome activation and neuroinflammation as evidenced by the NF-κB activation and various proinflammatory cytokine releases following ICH. Moreover, Nrf2 could interact with and modulate the mitophagy receptor OPTN, further mediating mitophagy to remove dysfunctional mitochondria after ICH. Furthermore, OPTN small interfering RNA (siRNA) increased the NLRP3 inflammasome activation by downregulating mitophagy level and enhancing mitochondrial damage in the Nrf2 WT mice after ICH. Together, our data indicated that Nrf2/OPTN inhibited NLRP3 inflammasome activation, possibly via modulating mitophagy, therefore alleviating SBI after ICH.