Role of NLRP3 inflammasome in diabetes mellitus and exercise intervention / 生理学报

Chronic inflammatory reaction has been established as an important sign of the occurrence and development of diabetes mellitus (DM), accompanied by the production of a large number of inflammatory factors, thus aggravating the disease progression. As an important non-invasive intervention measure to inhibit inflammation, exercise plays a very important role in the amelioration of DM. NOD-like receptor protein 3 (NLRP3) inflammasome, a regulatory factor of inflammatory response, can induce a variety of inflammatory cascades and cell death, which are closely related to glucose uptake and dyslipidemia regulation. The development of DM can be postponed with exercise. Previous studies have reported the effects of NLRP3 inflammasome on DM, but the crucial role of exercise in this process remains unclear. Therefore, this paper reviews the research progress on the improving effects of exercise intervention on the symptoms of DM by mediating NLRP3 inflammasome, providing a novel theoretical foundation for understanding the prevention and treatment of DM through exercise.

Guiqi Yiyuan Ointment protects rat left lung from bystander effect of right lung injury induced by ~(12)C~(6+) beam / 中国中药杂志

This study observed the effects of Guiqi Yiyuan Ointment(GQYY) on the left lung subjecting to bystander effect of right lung injury induced by ~(12)C~(6+) beam in rats and decipher the underlying mechanism from NOD-like receptor protein 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/cysteinyl aspartate specific proteinase-1(caspase-1) pathway. Wistar rats were randomized into 7 groups: blank, model, inhibitor [200 mg·kg~(-1), N-acetylcysteine(NAC)], western drug [140 mg·kg~(-1) amifostine(AMI)], and high-, medium-, and low-dose(4.8, 2.4, and 1.2 g·kg~(-1), respectively) GQYY groups. The model of bystander effect damage was established by 4 Gy ~(12)C~(6+) beam irradiation of the right lung(with the other part shielded by a lead plate). The pathological changes in the lung tissue, the level of reactive oxygen species(ROS) in the lung tissue, and the levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum were observed and measured in each group. Furthermore, the mRNA and protein levels of NLRP3, ASC, caspase-1, and phosphorylated nuclear factor-κB p65(p-NF-κB p65)/nuclear factor-κB p65(NF-κB p65) were determined. Compared with the blank group, the model group showed thickened alveolar wall, narrowed alveolar cavity, and presence of massive red blood cells and inflammatory infiltration in the alveolar wall and alveolar cavity. In addition, the model group showed elevated ROS levels in both left and right lungs, elevated MDA level, lowered SOD level, and up-regulated mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. Compared with the model group, the drug administration in all the groups reduced inflammatory cell infiltration in the lung tissue. The inhibitor group and the western drug group showed enlarged alveolar cavity, thinned interstitium, and reduced inflammation. There was a small amount of alveolar wall rupture in the high-and medium-dose GQYY groups and reduced inflammatory cell infiltration in the low dose GQYY group. Compared with the model group, drug administration lowered level of ROS in the left and right lungs, lowered the MDA level, elevated the SOD level, and down-regulated the mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. GQYY can effectively reduce the damage caused by radiation and bystander effect, which may be associated with the ROS-mediated NLRP3 inflammasome activation.

Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway / 中国中药杂志

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1β, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Research progress in effects of pyroptosis on intestinal inflammatory injury / 中南大学学报(医学版)

Inflammatory injury of the intestine is often accompanied by symptoms such as damage to intestinal mucosa, increased intestinal permeability, and intestinal motility dysfunction. Inflammatory factors spread throughout the body via blood circulation, and can cause multi-organ failure. Pyroptosis is a newly discovered way of programmed cell death, which is mainly characterized by the formation of plasma membrane vesicles, cell swelling until the rupture of the cell membrane, and the release of cell contents, thereby activating a drastic inflammatory response and expanding the inflammatory response cascade. Pyroptosis is widely involved in the occurrence of diseases, and the underlying mechanisms for inflammation are still a hot spot of current research. The caspase-1 mediated canonical inflammasome pathway of pyroptosis and caspase-4/5/8/11-mediated non-canonical inflammasome pathway are closely related to the occurrence and development of intestinal inflammation. Therefore, investigation of the signaling pathways and molecular mechanisms of pyroptosis in intestinal injury in sepsis, inflammatory bowel diseases, infectious enteristic, and intestinal tumor is of great significance for the prevention and treatment of intestinal inflammatory injury.

Hepatitis B virus X protein promotes podocyte pyroptosis in hepatitis B virus-associated glomerulonephritis by down-regulating microRNA -223 targeting NLRP3 inflammasome / 中华肝脏病杂志

Objective: To investigate the potential function and related mechanism of microRNA-223 (miRNA-223) in the podocyte pyroptosis of hepatitis B virus (HBV)-associated glomerulonephritis induced by HBV X protein (HBx). Methods: HBx-overexpressing lentivirus was transfected into human renal podocytes to mimic the pathogenesis of HBV-GN. Real-time fluorescence quantitative PCR and Western blotting experiments were used to detect the mRNA and protein expression of pyroptosis-related proteins [nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1], and inflammatory factors (interleukin-1β and interleukin-18), respectively.TUNEL staining and flow cytometry were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression of podocytes biomarkers desmin and nephrin; Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to measure caspase-1 activity. The dual luciferase reporter gene assay was used to verify the downstream target of miRNA-223. Podocytes were divided into the following nine groups: control group (no special treatment), empty plasmid group (transfected with empty plasmid), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+miRNA-223 mimic group (transfected with HBx overexpression lentivirus and miRNA-223 mimic), HBx overexpression+miRNA-223 inhibitor group (transfected with HBx overexpression lentivirus and miRNA-223 inhibitor), HBx overexpression+miRNA-223 mimic+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 mimic+ NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 mimic and NLRP3 siRNA), HBx overexpression+miRNA-223 inhibitor+NLRP3 group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 overexpression plasmid), HBx overexpression+miRNA-223 inhibitor+NLRP3 siRNA group (transfected with HBx overexpression lentivirus, miRNA-223 inhibitor and NLRP3 siRNA). Results: miRNA-223 was down-regulated in HBx overexpression group compared with the control group (P < 0.05). TUNEL and immunofluorescence staining showed that NLRP3 knockdown attenuated podocyte injury and pyroptosis induced by HBx overexpression (P < 0.05). Dual luciferase reporter gene assay demonstrated that NLRP3 was one of the downstream targets of miRNA-223. Rescue experiments revealed that NLRP3 overexpression weakened the protective effect of miRNA-223 in podocyte injury (P < 0.05). The addition of miRNA-223 mimic and NLRP3 siRNA decreased the expression of NLRP3 inflammasome and cytokines, and reduced the number of pyroptosis cells induced by HBx overexpression (all P < 0.05); The addition of miRNA-223 inhibitor and NLRP3 overexpression plasmid significantly increased the expression of NLRP3 inflammasome and cytokines, caspase-1 activity, and the number of pyroptosis cells (all P < 0.05). Conclusion: HBx may promote podocyte pyroptosis of HBV-GN via downregulating miRNA-223 targeting NLRP3 inflammasome, suggesting that miRNA-223 is expected to be a potential target for the treatment of HBV-GN.

The Chinese herbal prescription JieZe-1 inhibits caspase-1-dependent pyroptosis induced by herpes simplex virus-2 infection in vitro / 中西医结合学报

OBJECTIVE@#JieZe-1 (JZ-1), a Chinese herbal prescription, has an obvious effect on genital herpes, which is mainly caused by herpes simplex virus type 2 (HSV-2). Our study aimed to address whether HSV-2 induces pyroptosis of VK2/E6E7 cells and to investigate the anti-HSV-2 activity of JZ-1 and the effect of JZ-1 on caspase-1-dependent pyroptosis.@*METHODS@#HSV-2-infected VK2/E6E7 cells and culture supernate were harvested at different time points after the infection. Cells were co-treated with HSV-2 and penciclovir (0.078125 mg/mL) or caspase-1 inhibitor VX-765 (24 h pretreatment with 100 μmol/L) or JZ-1 (0.078125-50 mg/mL). Cell counting kit-8 assay and viral load analysis were used to evaluate the antiviral activity of JZ-1. Inflammasome activation and pyroptosis of VK2/E6E7 cells were analyzed using microscopy, Hoechst 33342/propidium iodide staining, lactate dehydrogenase release assay, gene and protein expression, co-immunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay.@*RESULTS@#HSV-2 induced pyroptosis of VK2/E6E7 cells, with the most significant increase observed 24 h after the infection. JZ-1 effectively inhibited HSV-2 (the 50% inhibitory concentration = 1.709 mg/mL), with the 6.25 mg/mL dose showing the highest efficacy (95.76%). JZ-1 (6.25 mg/mL) suppressed pyroptosis of VK2/E6E7 cells. It downregulated the inflammasome activation and pyroptosis via inhibiting the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (P < 0.001) and interferon-γ-inducible protein 16 (P < 0.001), and their interactions with apoptosis-associated speck-like protein containing a caspase recruitment domain, and reducing cleaved caspase-1 p20 (P < 0.01), gasdermin D-N (P < 0.01), interleukin (IL)-1β (P < 0.001), and IL-18 levels (P < 0.001).@*CONCLUSION@#JZ-1 exerts an excellent anti-HSV-2 effect in VK2/E6E7 cells, and it inhibits caspase-1-dependent pyroptosis induced by HSV-2 infection. These data enrich our understanding of the pathologic basis of HSV-2 infection and provide experimental evidence for the anti-HSV-2 activity of JZ-1. Please cite this article as: Liu T, Shao QQ, Wang WJ, Liu TL, Jin XM, Xu LJ, Huang GY, Chen Z. The Chinese herbal prescription JieZe-1 inhibits caspase-1-dependent pyroptosis induced by herpes simplex virus-2 infection in vitro. J Integr Med. 2023; 21(3): 277-288.

Effects of hydrogen gas on NOD-like receptor protein 3 inflammasomes in the cerebral cortex of rats with traumatic brain injury / 中华危重病急救医学

OBJECTIVE@#To investigate the effect of hydrogen gas on NOD-like receptor protein 3 (NLRP3) inflammasomes in the cerebral cortex of rats with traumatic brain injury (TBI).@*METHODS@#120 adult male Sprague-Dawley (SD) rates were randomly divided into 5 groups (n = 24): sham operation group (S group), TBI model group (T group), TBI+NLRP3 inhibitor MCC950 group (T+M group), TBI+hydrogen gas group (T+H group), TBI+hydrogen gas+MCC950 group (T+H+M group). TBI model was established by controlled cortical impact. NLRP3 inhibitor MCC950 (10 mg/kg) was intraperitoneally injected for 14 consecutive days before TBI operation in T+M and T+H+M groups. 2% hydrogen inhalation was given for 1 hour at 1 hour and 3 hours after TBI operation in T+H and T+H+M groups. At 6 hours after TBI operation, the pericontusional cortex tissues were obtained, the content of Evans blue (EB) was detected to evaluate the permeability of the blood-brain barrier. Water content in brain tissue was detected. The cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) and the neuronal apoptosis index was calculated. The expressions of Bcl-2, Bax, NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 p20 were detected by Western blotting. The levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#Compared with the S group, the content of EB in cerebral cortex, water content in brain tissue, apoptosis index and the expressions of Bax, NLRP3, ASC, caspase-1 p20 in T group were significantly increased, the expression of Bcl-2 was down-regulated, the levels of IL-1β and IL-18 were increased [the content of EB (μg/g): 87.57±6.89 vs. 10.54±1.15, water content in brain tissues: (83.79±2.74)% vs. (74.50±1.19)%, apoptotic index: (62.66±5.33)% vs. (4.61±0.96)%, Bax/β-actin: 4.20±0.44 vs. 1, NLRP3/β-actin: 3.55±0.31 vs. 1, ASC/β-actin: 3.10±0.26 vs. 1, caspase-1 p20/β-actin: 3.28±0.24 vs. 1, Bcl-2/β-actin: 0.23±0.03 vs. 1, IL-1β (ng/g): 221.58±19.15 vs. 27.15±3.27, IL-18 (ng/g): 87.26±7.17 vs. 12.10±1.85, all P < 0.05]. Compared with the T group, the T+M, T+H and T+H+M groups had significant reductions in the content of EB and water content in brain tissue, apoptotic index of the cerebral cortex, the expressions of Bax, NLRP3, and caspase-1 p20 in the brain tissue and the levels of IL-1β and IL-18, significant increases in the expression of Bcl-2. However, there was no significant difference in ASC expression. Compared with the T+H group, the content of EB in the cerebral cortex, water content in brain tissue, and apoptotic index, and the expressions of Bax, NLRP3 and caspase-1 p20 were further down-regulated in T+H+M group, the expression of Bcl-2 was further up-regulated, the levels of IL-1β and IL-18 were further decreased [the content of EB (μg/g): 40.49±3.15 vs. 51.96±4.69, water content in brain tissue: (76.58±1.04)% vs. (78.76±1.16)%, apoptotic index: (32.22±3.44)% vs. (38.54±3.89)%, Bax/β-actin: 1.92±0.16 vs. 2.56±0.21, NLRP3/β-actin: 1.94±0.14 vs. 2.37±0.24, caspase-1 p20/β-actin: 1.97±0.17 vs. 2.31±0.19, Bcl-2/β-actin: 0.82±0.07 vs. 0.52±0.04, IL-1β (ng/g): 86.23±7.09 vs. 110.44±10.48, IL-18 (ng/g): 40.18±3.22 vs. 46.23±4.02, all P < 0.05], but there were no statistical significance in all the indicators between T+M group and T+H group.@*CONCLUSIONS@#The mechanism by which hydrogen gas alleviates TBI may be related to inhibiting NLRP3 inflammasomes in the cerebral cortex of rats.

Curcumin alleviates nuclear factor-κB/NOD-like receptor protein 3 mediated renal injury caused by acute respiratory distress syndrome through reducing mitochondrial oxidative stress / 中华危重病急救医学

OBJECTIVE@#To evaluate the effect of curcumin on renal mitochondrial oxidative stress, nuclear factor-κB/NOD-like receptor protein 3 (NF-κB/NLRP3) inflammatory body signaling pathway and tissue cell injury in rats with acute respiratory distress syndrome (ARDS).@*METHODS@#A total of 24 specific pathogen free (SPF)-grade healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ARDS model group, and low-dose and high-dose curcumin groups, with 6 rats in each group. The ARDS rat model was reproduced by intratracheal administration of lipopolysaccharide (LPS) at 4 mg/kg via aerosol inhalation. The control group was given 2 mL/kg of normal saline. The low-dose and high-dose curcumin groups were administered 100 mg/kg or 200 mg/kg curcumin by gavage 24 hours after model reproduction, once a day. The control group and ARDS model group were given an equivalent amount of normal saline. After 7 days, blood samples were collected from the inferior vena cava, and the levels of neutrophil gelatinase-associated lipocalin (NGAL) in serum were determined by enzyme-linked immunosorbent assay (ELISA). The rats were sacrificed, and kidney tissues were collected. Reactive oxygen species (ROS) levels were determined by ELISA, superoxide dismutase (SOD) activity was detected using the xanthine oxidase method, and malondialdehyde (MDA) levels were determined by colorimetric method. The protein expressions of hypoxia-inducible factor-1α (HIF-1α), caspase-3, NF-κB p65, and Toll-like receptor 4 (TLR4) were detected by Western blotting. The mRNA expressions of HIF-1α, NLRP3, and interleukin-1β (IL-1β) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Renal cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL). The morphological changes in renal tubular epithelial cells and mitochondria were observed under a transmission electron microscope.@*RESULTS@#Compared with the control group, the ARDS model group exhibited kidney oxidative stress and inflammatory response, significantly elevated serum levels of kidney injury biomarker NGAL, activated NF-κB/NLRP3 inflammasome signaling pathway, increased kidney tissue cell apoptosis rate, and renal tubular epithelial cell damage and mitochondrial integrity destruction under transmission electron microscopy, indicating successful induction of kidney injury. Following curcumin intervention, the injury to renal tubular epithelial cells and mitochondria in the rats was significantly mitigated, along with a noticeable reduction in oxidative stress, inhibition of the NF-κB/NLRP3 inflammasome signaling pathway, and a significant decrease in kidney tissue cell apoptosis rate, demonstrating a certain dose-dependency. Compared with the ARDS model group, the high-dose curcumin group exhibited significantly reduced serum NGAL levels and kidney tissue MDA and ROS levels [NGAL (μg/L): 13.8±1.7 vs. 29.6±2.7, MDA (nmol/g): 115±18 vs. 300±47, ROS (kU/L): 75±19 vs. 260±15, all P < 0.05], significantly down-regulated protein expressions of HIF-1α, caspase-3, NF-κB p65, and TLR4 in the kidney tissue [HIF-1α protein (HIF-1α/β-actin): 0.515±0.064 vs. 0.888±0.055, caspase-3 protein (caspase-3/β-actin): 0.549±0.105 vs. 0.958±0.054, NF-κB p65 protein (NF-κB p65/β-actin): 0.428±0.166 vs. 0.900±0.059, TLR4 protein (TLR4/β-actin): 0.683±0.048 vs. 1.093±0.097, all P < 0.05], and significantly down-regulated mRNA expressions of HIF-1α, NLRP3, and IL-1β [HIF-1α mRNA (2-ΔΔCt): 2.90±0.39 vs. 9.49±1.87, NLRP3 mRNA (2-ΔΔCt): 2.07±0.21 vs. 6.13±1.32, IL-1β mRNA (2-ΔΔCt): 1.43±0.24 vs. 3.95±0.51, all P < 0.05], and significantly decreased kidney tissue cell apoptosis rate [(4.36±0.92)% vs. (27.75±8.31)%, P < 0.05], and significantly increased SOD activity (kU/g: 648±34 vs. 430±47, P < 0.05).@*CONCLUSIONS@#Curcumin can alleviate kidney injury in ARDS rats, and its mechanism may be related to the increasing in SOD activity, reduction of oxidative stress, and inhibition of the activation of the NF-κB/NLRP3 inflammasome signaling pathway.

NLRP3 Inflammasome-Mediated Neuroinflammation and Related Mitochondrial Impairment in Parkinson's Disease / 神经科学通报·英文版

Parkinson's disease (PD) is a common neurodegenerative disorder caused by the loss of dopamine neurons in the substantia nigra and the formation of Lewy bodies, which are mainly composed of alpha-synuclein fibrils. Alpha-synuclein plays a vital role in the neuroinflammation mediated by the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in PD. A better understanding of the NLRP3 inflammasome-mediated neuroinflammation and the related mitochondrial impairment during PD progression may facilitate the development of promising therapies for PD. This review focuses on the molecular mechanisms underlying NLRP3 inflammasome activation, comprising priming and protein complex assembly, as well as the role of mitochondrial impairment and its subsequent inflammatory effects on the progression of neurodegeneration in PD. In addition, the therapeutic strategies targeting the NLRP3 inflammasome for PD treatment are discussed, including the inhibitors of NLRP3 inflammatory pathways, mitochondria-focused treatments, microRNAs, and other therapeutic compounds.

Suppression of NLRP3 inflammasome by ivermectin ameliorates bleomycin-induced pulmonary fibrosis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Ivermectin is a US Food and Drug Administration (FDA)-approved antiparasitic agent with antiviral and anti-inflammatory properties. Although recent studies reported the possible anti-inflammatory activity of ivermectin in respiratory injuries, its potential therapeutic effect on pulmonary fibrosis (PF) has not been investigated. This study aimed to explore the ability of ivermectin (0.6 mg/kg) to alleviate bleomycin-induced biochemical derangements and histological changes in an experimental PF rat model. This can provide the means to validate the clinical utility of ivermectin as a treatment option for idiopathic PF. The results showed that ivermectin mitigated the bleomycin-evoked pulmonary injury, as manifested by the reduced infiltration of inflammatory cells, as well as decreased the inflammation and fibrosis scores. Intriguingly, ivermectin decreased collagen fiber deposition and suppressed transforming growth factor-‍β1 (TGF-‍β1) and fibronectin protein expression, highlighting its anti-fibrotic activity. This study revealed for the first time that ivermectin can suppress the nucleotide-binding oligomerization domain (NOD)‍-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, as manifested by the reduced gene expression of NLRP3 and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), with a subsequent decline in the interleukin‍-‍1β (IL‍-‍1β) level. In addition, ivermectin inhibited the expression of intracellular nuclear factor-‍κB (NF‍-‍κB) and hypoxia‑inducible factor‑1α (HIF‍-‍1α) proteins along with lowering the oxidative stress and apoptotic markers. Altogether, this study revealed that ivermectin could ameliorate pulmonary inflammation and fibrosis induced by bleomycin. These beneficial effects were mediated, at least partly, via the downregulation of TGF-‍β1 and fibronectin, as well as the suppression of NLRP3 inflammasome through modulating the expression of HIF‑1α and NF-‍κB.

Ozonated triglyceride protects against septic lethality via preventing the activation of NLRP3 inflammasome / 中南大学学报(医学版)

OBJECTIVES@#Sepsis is a critical dysregulated host response with high mortality and current treatment is difficult to achieve optimal efficacy. Ozone therapy has been revealed to protect infection and inflammation-related diseases due to its role in antibiotic and immunoregulatory effect. Ozonated triglyceride is a key component of ozonated oil that is one of ozone therapy dosage form. However, the potential role of ozonated triglyceride in sepsis remains unclear. This study aims to explore the effect of ozonated triglyceride on septic mouse model and the molecular mechanism.@*METHODS@#Intraperitoneal injection of lipopolysaccharide (LPS), cecal ligation and puncture (CLP) were applied to construct septic mouse model. The mouse serum was obtained for detection of cytokines, and lung tissues were collected for hematoxylin and eosin (HE) staining to evaluate the extent of lung injury in septic mouse with ozonated triglyceride treatment at different time and doses. The survival of septic mice was observed for 96 h and Kaplan-Meier analysis was used to analyze the survival rates. In addition, primary peritoneal macrophages and human acute monocytic-leukemia cell line (THP-1) were treated with inflammasome activators with or without ozonated triglyceride. The level of cytokines was detected by enzyme-linked immunosorbent assay (ELISA). The cleavage of caspase-1 and gasdermin-D (GSDMD) was detected by Western blotting.@*RESULTS@#Ozonated triglyceride at different time and doses reduced the release of inflammasome-related cytokines [interleukin (IL)-1β and IL-18] (all P<0.05) but not pro-inflammatory cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) in septic mice (all P>0.05). Ozonated triglyceride significantly improved the survival rate of septic mice and reduced sepsis-induced lung injury (all P<0.05). Ozonated triglyceride significantly suppressed the canonical and non-canonical activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (all P<0.05) but not affected absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes in vitro (all P>0.05). Ozonated triglyceride reduced the cleavage of caspase-1 and the downstream GSDMD.@*CONCLUSIONS@#Ozonated triglyceride presents a protect effect on sepsis lethality via reducing cytokines release and sepsis-related organ injury. The mechanism is that ozonated triglyceride specifically suppresses the activation of NLRP3 inflammasome. Ozonated triglyceride is a promising candidate for sepsis treatment.

Progress on Regulation of NLRP3 Inflammasome by Chinese Medicine in Treatment of Ulcerative Colitis / 中国结合医学杂志

Ulcerative colitis (UC) is a chronic, non-specific intestinal disease that not only affects the quality of life of patients and their families but also increases the risk of colorectal cancer. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important component of inflammatory response system, and its activation induces an inflammatory cascade response that is involved in the development and progression of UC by releasing inflammatory cytokines, damaging intestinal epithelial cells, and disrupting the intestinal mucosal barrier. Chinese medicine (CM) plays a vital role in the prevention and treatment of UC and is able to regulate NLRP3 inflammasome. Many experimental studies on the regulation of NLRP3 inflammasome mediated by CM have been carried out, demonstrating that CM formulae with main effects of clearing heat, detoxifying toxicity, drying dampness, and activating blood circulation. Flavonoids and phenylpropanoids can effectively regulate NLRP3 inflammasome. Other active components of CM can interfere with the process of NLRP3 inflammasome assembly and activation, leading to a reduction in inflammation and UC symptoms. However, the reports are relatively scattered and lack systematic reviews. This paper reviews the latest findings regarding the NLRP3 inflammasome activation-related pathways associated with UC and the potential of CM in treating UC through modulation of NLRP3 inflammasome. The purpose of this review is to explore the possible pathological mechanisms of UC and suggest new directions for development of therapeutic tools.

Artesunate alleviates hypoxic-ischemic brain damage in neonatal rats by inhibiting NLRP3 inflammasome activation and inflammatory cytokine secretion / 细胞与分子免疫学杂志

Objective To investigate the protective effect of artesunate on hypoxic-ischemic brain damage (HIBD) and its mechanism in neonatal rats. Methods 7-day-old neonatal SD rats were randomly divided into sham operation group, model group, artesunate 5 mg/kg group, artesunate 10 mg/kg group, artesunate 20 mg/kg group and dexamethasone 6 mg/kg group, with 18 rats in each group. HIBD models were established in groups except for the sham operation group. The sham operation group only needed to separate the left common carotid artery without ligation and nitrogen-oxygen mixed gas ventilation. Each group was injected with drug intraperitoneally right after surgery and the rats in the sham operation group and the model group were injected with an equal volume of normal saline (once a day for a total of 5 times). One hour after the last injection, the rats in each group were scored for neurological defects. After the rats were sacrificed, the brain water content was measured and the pathological changes of the brain tissues of rats were observed. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect the neuronal cell apoptosis, and ELISA was applied to detect the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood of each group of rats. Western blot analysis was adopted to detect the protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 in the rats brain tissues of each group. Results Compared with the model group, the neurological deficit score was decreased; the pathological damage of brain tissues was relieved; the brain water content was significantly reduced; the apoptosis number of hippocampal neurons was decreased significantly; the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood were significantly reduced; the protein expression levels of NLRP3, ASC and caspase-1 were significantly lowered in the middle-dose and high-dose artesunate groups and the dexamethasone group. Conclusion Artesunate can improve the neurological function, relieve the brain damage, and alleviate the brain edema in neonatal rats with HIBD. It can protect the HIBD, which may be related to the inhibition of NLRP3 inflammasome activation and reduction of inflammatory cytokine secretion.

Arctigenin mitigates vascular endothelial injury in rats with pregnancy-induced hypertension via autophagy-NLRP3 inflammasome pathway / 中国中药杂志

This study aims to investigate the effect and mechanism of arctigenin(ARC) in the treatment of vascular endothelial injury in rats with pregnancy-induced hypertension(PIH). Fifty SD rats pregnant for 12 days were randomly assigned into a control group, a model group, an ARC group, a rapamycin(RAP, autophagy inducer) group, and an ARC+3-methyladenine(3-MA, autophagy inhibitor) group, with 10 rats in each group. The rats in the other groups except the control group were intraperitoneally injected with nitrosyl-L-arginine methyl ester(50 mg·kg~(-1)·d~(-1)) to establish the PIH model on the 13th day of pregnancy. On the 15th day of pregnancy, the rats in ARC, RAP, and ARC+3-MA groups were intraperitoneally injected with ARC(50 mg·kg~(-1)·d~(-1)), RAP(1 mg·kg~(-1)·d~(-1)), and 3-MA(15 mg·kg~(-1)·d~(-1))+ARC(50 mg·kg~(-1)·d~(-1)), respectively. The pregnant rats in the control group and the model group were intraperitoneally injected with the same amount of normal saline. The blood pressure and 24 h urine protein(24 h-UP) of pregnant rats in each group were measured before and after intervention. Cesarean section was performed to terminate pregnancy on day 21, and the body weight and body length of fetal rats were compared among groups. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes of placenta. The expression of endothelin-1(ET-1) and endothelial nitric oxide synthase(eNOS) in placenta was detected by immunohistochemistry. The serum levels of ET-1 and nitric oxide(NO) were determined with corresponding kits. The expression of microtubule-associated protein 1 light chain 3(LC3), Beclin-1, NOD-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein with CARD domain(ASC), caspase-1, interleukin(IL)-1β, and IL-18 was determined by immunofluorescence and Western blot. The level of reactive oxygen species(ROS) in placenta was measured by fluorescence staining. The results showed that on day 12 of pregnancy, the blood pressure and 24 h-UP had no significant differences among groups. On days 15, 19, and 21, the blood pressure and 24 h-UP in the model group were higher than those in the control group(P<0.05). On days 19 and 21, the blood pressure and 24 h-UP in ARC group and RAP group were lower than those in the model group(P<0.05), and they were higher in the ARC+3-MA group than in the ARC group(P<0.05). On day 21, the model group had lower body weight and body length of fetal rats(P<0.05), higher serum level of ET-1, and lower serum level of NO(P<0.05) than the control group. Moreover, the placental tissue showed typical pathological damage, down-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and eNOS(P<0.05), up-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and elevated ROS level. Compared with the model group, ARC and RAP groups showed increased body weight and body length of fetal rats(P<0.05), lowered serum level of ET-1, elevated serum level of NO(P<0.05), reduced pathological damage of placental tissue, up-regulated expression of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and eNOS(P<0.05), down-regulated expression of ET-1, NLRP3, ASC, caspase-1, IL-1β, and IL-18(P<0.05), and lowered ROS level. Compared with ARC group, 3-MA reversed the effects of ARC on the above indicators. In conclusion, ARC can inhibit the activation of NLRP3 inflammasome and mitigate vascular endothelial damage in PIH rats by inducing autophagy of vascular endothelial cells.

"Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy / 中国中药杂志

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Role of NLRP3 inflammasome in diabetic neuropathy and prevention and treatment with traditional Chinese medicine / 中国中药杂志

As one of the most frequent complications of diabetes, diabetic neuropathy often involves peripheral and central nervous systems. Neuroinflammation is the key pathogenic factor of secondary nerve injury in diabetes. NOD-like receptor pyrin domain-containing 3(NLRP3) inflammasome is a group of subcellular multiprotein complexes, including NLRP3, apoptosis associated speck-like protein(ASC), and pro-cysteinyl aspartate specific proteinase 1(pro-caspase-1). NLRP3 inflammasome is an inducer of innate immune responses. Its activation stimulates the inflammatory cascade reaction, promotes the release of inflammatory mediators, triggers cell death and uncontrolled autophagy, activates glial cells, facilitates peripheral immune cell infiltration, and initiates amyoid β(Aβ)-tau cascade reactions. As a result, it contributes to the central nerve, somatic nerve, autonomic nerve, and retinal nerve cell damage secondary to diabetes. Therefore, due to its key role in the neuroinflammation responses of the body, NLRP3 inflammasome may provide new targets for the treatment of diabetic neuropathy. With multi-target and low-toxicity advantages, traditional Chinese medicine plays a vital role in the treatment of diabetic neuropathy. Accumulating evidence has shown that traditional Chinese medicine exerts curative effects on diabetic neuropathy possibly through regulating NLRP3 inflammasome. Although the role of NLRP3 inflammasome in diabetes and related complications has been investigated in the literature, systematical studies on drugs and mechanism analysis for secondary neuropathy are still lacking. In this article, the role of NLRP3 inflammasome in diabetic neuropathy was explored, and the research progress on traditional Chinese medicine in the treatment of diabetic neuropathy through NLRP3 inflammasome was reviewed.

Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and its downstream protein gastermin D (GSDMD) in rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA on the treatment of PDM.@*METHODS@#Forty healthy female SD rats without pregnancy were randomly divided into a control group, a model group, an EA group and an ibuprofen group, 10 rats in each group. PDM model was prepared by injection of estradiol benzoate and oxytocin. Except the control group, the rats in each group were subcutaneously injected with estradiol benzoate for 10 days, and oxytocin was injected on the 11th day. The rats in the EA group were intervened with EA (dense wave, frequency of 50 Hz) at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) at the same time of modeling, once a day, 20 min each time, for 10 consecutive days. The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage (concentration of ibuprofen solution was 1.25 mg/mL) for 10 consecutive days. After modeling, the writhing reaction was observed. After intervention, the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus; ELISA method was used to detect the serum levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α); Western blot method was used to detect the protein expression of NLRP3, apoptosis related spot like protein (ASC), caspase-1, GSDMD, GSDMD-N and inflammatory factors (interleukin [IL]-1β, IL-18) in uterine tissue.@*RESULTS@#In the model group, a large number of vacuolar degeneration and death of endometrial epithelial cells, spiral arterioles congestion in lamina propria and neutrophil infiltration were observed. In the EA group, there was a small amount of vacuolar degeneration and death of endometrial epithelial cells, a small amount of spiral arterioles congestion in the lamina propria, and a small amount of neutrophils infiltration. In the ibuprofen group, there was very small number of degeneration and death of endometrial epithelial cells, and no obvious arterial congestion was found in lamina propria, and neutrophil infiltration was occasionally seen. Compared with the control group, in the model group the number of writhing was increased (P<0.01), the writhing reaction score and serum level of PGF2α and PGF2α/PGE2 value were increased (P<0.01), the level of PGE2 was decreased (P<0.01). Compared with the model group, in the EA group and the ibuprofen group the number of writhing were decreased (P<0.05), the latency of writhing was prolonged (P<0.01), the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased (P<0.05, P<0.01), the levels of PGE2 were increased (P<0.01). Compared with the control group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was increased in the model group (P<0.01). Compared with the model group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group (P<0.01, P<0.05). There was no significant difference between the EA group and the ibuprofen group in the above indexes (P>0.05).@*CONCLUSION@#EA could alleviate pain and uterine tissue injury in rats with PDM. The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues, thereby inhibiting pyroptosis and its inflammatory factors release.

10,11-Dehydrocurvularin attenuates inflammation by suppressing NLRP3 inflammasome activation / 中国天然药物

10,11-Dehydrocurvularin (DCV) is a natural-product macrolide that has been shown to exert anti-inflammatory activity. However, the underlying mechanism of its anti-inflammatory activity remains poorly understood. Aberrant activation of the NLRP3 inflammasome is involved in diverse inflammation-related diseases, which should be controlled. The results showed that DCV specifically inhibited the activation of the NLRP3 inflammasome in association with reduced IL-1β secretion and caspase-1 activation, without effect on the NLRC4 and AIM2 inflammasomes. Furthermore, DCV disturbed the interaction between NEK7 and NLRP3, resulting in the inhibition of NLRP3 inflammasome activation. The C=C double bond of DCV was required for the NLRP3 inflammasome inhibition induced by DCV. Importantly, DCV ameliorated inflammation in vivo through inhibiting the NLRP3 inflammasome. Taken together, our study reveals a novel mechanism by which DCV suppresses inflammation, which indicates the potential role of DCV in NLRP3 inflammasome-driven inflammatory disorders.

Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis / 中国中药杂志

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

Effects and clinical significance of NLRP3 inflammasome activated by IL-17A in CRSwNP / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the effects and clinical significance of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activated by interleukin (IL)-17A in chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Patients underwent nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2020 to December 2021 were collected, including 28 CRSwNP (including 19 males and 9 females, aged 19 to 67 years), 22 chronic rhinosinusitis without nasal polyps (CRSsNP) and 22 controls. qRT-PCR was used to detect the expressions of IL-17A, NLRP3, IL-1β and IL-18 in the three groups, and their correlations were analyzed. The positions of IL-17A, NLRP3 and IL-18 in nasal polys were analyzed by immunofluorescence. Western Blotting and ELISA were employed to detect the expression of NLRP3, IL-1β and IL-18 in the human nasal epithelial cells after using IL-17A stimulation or IL-17A receptor inhibitor. Immunofluorescence was used to observe the NLRP3, IL-1β, and IL-18 protein expression after IL-17A stimulating human nasal epithelial cells, and after the use of IL-17A receptor inhibitor and NLRP3 inhibitor MCC950. The correlations between NLRP3, IL-1β, IL-18 and CT scores, nasal endoscopic scores, visual analogue scale (VAS) scores, and sino-nasal outcome test (SNOT) 22 scores of CRSwNP patients were analyzed. SPSS 20.0 software was used for statistical analysis. Results: The expressions of IL-17A, NLRP3, IL-1β and IL-18 in the tissues of CRSwNP patients were significantly higher than those in CRSsNP group(P=0.018,P<0.001,P=0.005, P=0.016) and the control group(all P<0.001). IL-17A was positively correlated with the expression of NLRP3, IL-1β, and IL-18(r ralue was 0.643,0.650,0.629,respectively, all P<0.05). IL-17A, NLRP3, and IL-18 were co-localized in the epithelial propria of polyp tissue. IL-17A stimulated the expressions of NLRP3, IL-1β, and IL-18 in human nasal epithelial cells. After the use of IL-17A receptor inhibitor, the expressions of NLRP3, IL-1β, and IL-18 were significantly down-regulated. After the use of NLRP3 inhibitor MCC950, IL-17A was significantly down-regulated to promote the expression of NLRP3, IL-1β, and IL-18. The expressions of NLRP3, IL-1β and IL-18 were positively correlated with CT, nasal endoscopy, VAS, and SNOT22 scores in patients with CRSwNP. Conclusions: IL-17A promotes the release of IL-1β and IL-18 by activating the NLRP3 inflammasome and aggravates the severity of the disease in CRSwNP.