Silencing ANGPT2 alleviates ulcerative colitis by regulating autophagy-mediated NLRP3 inflammasome inactivation via the mTOR signaling pathway

Ulcerative colitis (UC) is a difficult intestinal disease characterized by inflammation, and its mechanism is complex and diverse. Angiopoietin-like protein 2 (ANGPT2) plays an important regulatory role in inflammatory diseases. However, the role of ANGPT2 in UC has not been reported so far. After exploring the expression level of ANGPT2 in serum of UC patients, the reaction mechanism of ANGPT2 was investigated in dextran sodium sulfate (DSS)-induced UC mice. After ANGPT2 expression was suppressed, the clinical symptoms and pathological changes of UC mice were detected. Colonic infiltration, oxidative stress, and colonic mucosal barrier in UC mice were evaluated utilizing immunohistochemistry, immunofluorescence, and related kits. Finally, western blot was applied for the estimation of mTOR signaling pathway and NLRP3 inflammasome-related proteins. ANGPT2 silencing improved clinical symptoms and pathological changes, alleviated colonic inflammatory infiltration and oxidative stress, and maintained the colonic mucosal barrier in DSS-induced UC mice. The regulatory effect of ANGPT2 on UC disease might occur by regulating the mTOR signaling pathway and thus affecting autophagy-mediated NLRP3 inflammasome inactivation. ANGPT2 silencing alleviated UC by regulating autophagy-mediated NLRP3 inflammasome inactivation via the mTOR signaling pathway.

25-hydroxycholesterol aggravates oxygen-glucose deprivation/reoxygenation-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cardiomyocytes

25-hydroxycholesterol (25-HC) plays a role in the regulation of cell survival and immunity. However, the effect of 25-HC on myocardial ischemia/reperfusion (MI/R) injury remains unknown. Our present study aimed to investigate whether 25-HC aggravated MI/R injury through NLRP3 inflammasome-mediated pyroptosis. The overlapping differentially expressed genes (DEGs) in MI/R were identified from the GSE775, GSE45818, GSE58486, and GSE46395 datasets in Gene Expression Omnibus (GEO) database. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using the database of Annotation, Visualization and Integration Discovery (DAVID). The protein-protein interaction (PPI) network of the overlapping DEGs was established using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These bioinformatics analyses indicated that cholesterol 25-hydroxylase (CH25H) was one of the crucial genes in MI/R injury. The oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was established to simulate MI/R injury. Western blot and RT-qPCR analysis demonstrated that CH25H was significantly upregulated in OGD/R-stimulated H9C2 cardiomyocytes. Moreover, knockdown of CH25H inhibited the OGD/R-induced pyroptosis and nod-like receptor protein 3 (NLRP3) inflammasome activation, as demonstrated by cell counting kit-8 (CCK8), lactate dehydrogenase (LDH), RT-qPCR, and western blotting assays. Conversely, 25-HC, which is synthesized by CH25H, promoted activation of NLRP3 inflammasome in OGD/R-stimulated H9C2 cardiomyocytes. In addition, the NLRP3 inhibitor BAY11-7082 attenuated 25-HC-induced H9C2 cell injury and pyroptosis under OGD/R condition. In conclusion, 25-HC could aggravate OGD/R-induced pyroptosis through promoting activation of NLRP3 inflammasome in H9C2 cells.

Jiedu Tongluo Tiaogan Prescription Protects Pancreatic β Cell by Targeting NLRP3 Inflammasome via TGR5/cAMP Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the intervention effect of Jiedu Tongluo Tiaogan prescription （JTTP） in protecting pancreatic β cells by targeting the bile acid Takeda G protein-coupled receptor 5 （TGR5）/cyclic adenosine monophosphate （cAMP） signaling pathway against NOD-like receptor protein 3 （NLRP3） inflammasome. MethodThirty-two male SPF-grade db/db mice were randomly divided into the model group， low-dose JTTP group （3.6 g·kg-1）， high-dose JTTP group （7.2 g·kg-1）， and metformin group （0.2 g·kg-1）. Eight db/m mice were assigned to the blank control group. The mice were treated with drugs for 8 weeks， and fasting blood glucose （FBG） was measured every 2 weeks. Oral glucose tolerance tests （OGTT） were conducted after the last administration. Enzyme-linked immunosorbent assay （ELISA） was performed to detect fasting insulin （FINS）， and the homeostasis model assessment of β-cell function （HOMA-β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and IL-1β levels were calculated. Hematoxylin-eosin （HE） staining was used to observe pathological changes in mouse pancreatic tissue. Immunofluorescence was performed to detect insulin expression in mouse pancreatic tissue. Western blot and real-time quantitative polymerase chain reaction （Real-time PCR） were used to detect the expression of proteins and mRNAs of key targets in the TGR5/cAMP signaling pathway and NLRP3 inflammasome. ResultCompared with blank group， FBG， OGTT， FINS， IL-6， TNF-α and IL-1β in model group were significantly increased （P<0.01）. Compared with model group， after 6 weeks of drug treatment， FBG level in JTTP group and metformin group decreased significantly （P<0.01）. The results of OGTT experiment showed that compared with model group， the blood glucose levels of mice in each administration group were decreased at all time points （P<0.05， P<0.01）， and the levels of FINS， TNF-α and IL-6 in JTTP dose groups and metformin group were significantly decreased. The level of IL-1β in JTTP high-dose group and metformin group was significantly decreased （P<0.01）. Pancreatic pathology showed that the islets in the model group were irregular in shape， uneven in distribution， and showed signs of atrophy. The prognosis of JTTP was that the cell count increased and the boundary was clearer. Immunofluorescence results showed that the islet cells in the blank group were arranged in an orderly and full shape with appropriate insulin secretion， while the islet cells in model group were distorted in shape， atrophy in structure and less insulin secretion. The insulin content of mice in JTTP and metformin group was significantly increased. Compared with blank group， mRNA expressions of NLRP3， apoptosis-related spot-like protein （ASC） and Caspase-1 in pancreatic tissues of model group were significantly increased （P<0.01）. Compared with model group， JTTP high-dose group and metformin group promoted the up-regulation of TGR5 and cAMP mRNA， and down-regulated the mRNA expressions of NLRP3， ASC and Caspase-1 （P<0.05， P<0.01）. Compared with blank group， the expression of TGR5 protein in model group was significantly decreased （P<0.01）. Compared with model group， TGR5 protein in JTTP high-dose group and metformin group was significantly increased （P<0.01）.

The effect of IL-22 on the expression of NLRP3 and caspase-1 mRNA and secretion of IL-18 and IL-1β in macrophages induced by lipopolysaccharide / 中华急诊医学杂志

Objective:To explore the effect of interleukin (IL)-22 on the expression of nucleotide binding oligomerization domain like receptor protein 3 (NLRP3) and caspase-1 mRNA and secretion of IL-18 and IL-1β in macrophages induced by lipopolysaccharide (LPS), RAW264.7 macrophages were cultured in vitro.Methods:Macrophage RAW264.7 was cultured in vitro, and the cultured cells were divided into three groups (control group, LPS group and LPS+IL-22 group), and the experimental cells in each group were intervened, and cultured for 3, 6 and 24 h respectively, and the cells and supernatants in each group were collected. RT-PCR, Western Blot and ELISA were used to detect NLRP3 and caspase-1 when the inflammatory body of macrophage NLRP3 was activated.Results:The expression levels of NLRP3 and caspase-1 mRNA and the secretion levels of IL-1β and IL-18 were increased in the LPS group, and the differences were statistically significant compared with the control group. After LPS and IL-22 co-stimulated macrophages, the expression levels of NLRP3 and caspase-1 mRNA, and the secretion levels of IL-1β and IL-18 were increased to different degrees, which were significantly increased compared with the LPS group.Conclusion:IL-22 could provide a new therapeutic idea for sepsis by enhancing the expression of NLRP3 and caspase-1 mRNA and the secretion of IL-18 and IL-1β in macrophages induced by LPS.

The effects of cannabidiol on the expression of NLRP3 inflammasome following multiple cerebral concussions in rats / 神经解剖学杂志

Objective:To investigate the effects of cannabidiol(CBD)on the NOD-like receptor protein 3(NLRP3)inflammasome in the brains of rats with multiple cerebral concussions(MCC).Methods:Rats were subjec-ted to the MCC model and divided into Sham,MCC,vehicle(MCC+TW),CBD-L(10 mg/kg),and CBD-H(40 mg/kg)groups.Immunofluorescence double staining was used to observe changes in NLRP3 and microglial cells in the brain,and Western Blot was performed to detect the expression changes of the NLRP3 inflammasome.Results:A sig-nificant increase in lectin-positive microglial cells of the cortex with enlarged cell bodies and elevated immunofluores-cence intensity of NLRP3 in the activated microglial cells was revealed by immunofluorescence double staining following MCC(P＜0.05).The immunofluorescence intensity of NLRP3 in the activated microglial cells was downregulated by the administration of CBD,with a more pronounced effect observed in the CBD-H group compared to the CBD-L group(P＜0.05).The expression of NLRP3,caspase-1,and apoptosis-associated speck-like protein(ASC)in the cortex,hippocampus,and basal ganglia of rats following MCC was significantly increased,as shown by Western Blot analysis(P＜0.05),and cortical areas are more elevated.The expression of these proteins in different brain regions was reduced by CBD-10 and CBD-40 intervention(P＜0.05).Conclusion:Cannabidiol can reduce the inflammatory response of multiple cerebral concussions rats through NLRP3 inflammasome and protect nerve tissue.

Effect of NLRP3 Knockdown on LPS-induced Inflammatory Response in BV2 Microglia / 华中科技大学学报(医学版)

Objective To evaluate the impact and clinical significance of NOD-like receptor pyrin domain-containing protein 3(NLRP3)in the activation of lipopolysaccharide(LPS)-induced BV2 microglia cells.Methods shRNA plasmids were devised for BV2 microglia transfection,and the most effective transfection segment was identified via RT-PCR and Western blot assays.NLRP3 expression in the cell line was detected by Western blotting,while light microscopy was used to observe morpho-logical alterations in BV2 cells transfected with NLRP3-shRNA.Furthermore,enzyme-linked immunosorbent assay(ELISA)was used to quantify levels of inflammatory cytokines IL-18,IL-1β,TNF-α and NO in cell supernatants.Immunofluorescence staining was used to observe the expression and localization of microglial activation markers iNOS,Arg-1,Iba1,and NLRP3.Results NLRP3 was highly expressed in LPS-induced BV2 cells.The Western blot result showed that the mRNA expression level was the lowest and transfection was the least effective in NLRP3-mus-727 group.Microscopic examination revealed a round or short spindle-shaped morphology in BV2 cells transfected with shNLRP3,which was akin to resting state cells in the blank control group.ELISA showed that pro-inflammatory mediators IL-18,IL-1β,TNF-α and NO levels were decreased in BV2 cells trans-fected with shNLRP3(all P＜0.05).Immunofluorescence staining indicated a relative decrease in iNOS and Iba1 expression,with an upregulation of Arg-1 in BV2 cells transfected with shNLRP3.Conclusion NLRP3 is highly expressed in LPS-induced BV2 cells.Inhibition of NLRP3 expression can suppress the inflammatory response of BV2 cells induced by LPS,promoting their polarization towards the M2 phenotype.

Dapagliflozin attenuates endothelial cell pyroptosis and dysfunction induced by oxidized low-density lipoprotein / 中国组织工程研究

BACKGROUND:Dapagliflozin,an inhibitor of sodium-glucose cotransporter 2,can delay the progression of atherosclerosis by regulating glucose metabolism,inhibiting inflammation and improving endothelial cell function. OBJECTIVE:To study the effect of dapagliflozin on cell pyroptosis and endothelial dysfunction induced by oxidized low-density lipoprotein. METHODS:Human umbilical vein endothelial cells were divided into a control group(no intervention),a model group(treated with oxidized low-density lipoprotein for 24 hours),and a dapagliflozin group(treated with oxidized low-density lipoprotein + dapagliflozin for 24 hours).Endothelial cell proliferation activity was measured by cell counting kit-8 assay.The levels of intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 in cell supernatant were detected using ELISA.Nitric oxide level in the cells was detected by nitrate reductase assay.The pyroptosis rate and characteristics of endothelial cells were detected by Hoechst 33342/PI fluorescence co-staining and lactate dehydrogenase release assay.The protein expression levels of NLRP3,caspase-1,GSDMD,interleukin-1β,and interleukin-18 were detected by western blot assay. RESULTS AND CONCLUSION:(1)Oxidized low-density lipoprotein could cause pyroptosis and dysfunction of endothelial cells.(2)Compared with the control group,the level of nitric oxide and cell activity were decreased(P<0.05),while lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly increased in the model group(P<0.05).Compared with the model group,cell activity and nitric oxide levels significantly increased(P<0.05),but lactate dehydrogenase,intercellular adhesion molecule 1,vascular cell adhesion molecule 1,and monocyte chemotactic protein-1 levels were significantly diminished in the dapagliflozin group(P<0.05).(3)Compared with the model group,cell pyroptosis rate and the protein expression of pyroptosis factor NLRP3,caspase-1,GSDMD,interleukin-18 and interleukin-1β significantly reduced in the dapagliflozin group(P<0.05).(4)The results indicate that dapagliflozin inhibits oxidized low-density lipoprotein-induced endothelial pyroptosis and ameliorates endothelial cell dysfunction.

Mechanism Study on Chaihuang Qingyi Huoxue Granules Reduce Inflammatory Damage in Severe Acute Pancreatitis Rats by Suppressing NLRP3 Inflammasome Activation / 中药新药与临床药理

Objective To investigate the protective effect and mechanism of Chaihuang Qingyi Huoxue Granules on pancreatic tissue of rats with severe acute pancreatitis,and to observe its regulation on NLRP3 inflammasome activation.Methods Sixty-four SD rats were randomly divided into sham-surgery(SO)group,severe acute pancreatitis model(SAP)group,Chaihuang Qingyi Huoxue Granules(CH)group,and MCC950(NLRP3 inhibitor)group.Each group was further divided into 12-hour and 24-hour subgroups,with rats in each group.The SAP group,CH group,and MCC950 group were retrogradely injected with 3.5%sodium taurocholate(2 mL·kg-1)into the pancreatic ducts to establish SAP model.The MCC950 group was immediately intraperitoneally injected with MCC950(1 mg·mL-1)after model preparation.After awakening from anesthesia,the CH group was administrated by gavage with Chaihuang Qingyi Huoxue Granules solution(0.35 g·mL-1)once every 6 hours.Ascites,abdominal aortic blood,and pancreatic tissue were collected at 12 hours and 24 hours after SAP model construction.The serum amylase and lipase activities were detected using an automated biochemical analyzer.HE staining was used to observe pancreatic injury.Serum levels of IL-18 and IL-1β were detected by ELISA.The expressions of gene and proteins related to the activation of NLRP3 inflammasome were analyzed by IHC,qRT-PCR and Western Blot.Results Compared with the SAP group,the pathological damage of pancreatic tissues in the CH and MCC950 groups was significantly reduced,and the pathological score was significantly reduced(P＜0.05).The levels of serum lipase,amylase,IL-18,and IL-1β were also significantly decreased(P＜0.05).After treatment with Chaihuang Qingyi Huoxue Granules or intraperitoneal injection of NLRP3 inhibitor,the positive expressions of NLRP3,ASC and Caspase-1 in pancreatic tissues,as well as the mRNA levels of NLRP3,ASC and Caspase-1,the protein levels of NLRP3,ASC,Pro-Caspase-1 and Caspase-1 were significantly reduced compared to the SAP group(P＜0.05).Conclusion Chaihuang Qingyi Huoxue Granules can inhibit the activation of NLRP3 inflammasome,reduce the mRNA and protein expressions of NLRP3,ASC and Caspase-1 in pancreatic tissues,and suppress the release of the downstream inflammatory factors IL-18 and IL-1β and alleviate pancreatitis damage in SAP model rats.

Research progress on the role of NLRP3 inflammasome signaling pathway in the occurrence and development of retinal diseases / 国际眼科杂志(Guoji Yanke Zazhi)

The nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome is an inflammatory protein complex, and can participate into the inflammatory response. Upon activation, these inflammasomes can lead to Caspase-1 activation, thereby inducing a cascade of inflammatory factor activation and further cell pyroptosis. Excessive activation of inflammasomes will induce the overexpression of inflammatory factors, persistently triggering immune dysregulation and inflammatory chain reactions, even causing severe damage. The recent studies have confirmed a close association between retinal diseases, such as diabetic retinopathy(DR), retinal ischemia-reperfusion injury(RIRI), and proliferative vitreoretinopathy(PVR)with immune dysregulation and inflammatory responses, which is serving as crucial factors in the progression of retinal diseases. This article reviews the NLRP3 inflammasome signaling pathway and its role in the occurrence and development of retinal diseases, in order to provide new ideas for the pathogenesis and prevention of retinal diseases.

Research progress of NLRP3 inflammasome in the treatment of uveitis / 国际眼科杂志(Guoji Yanke Zazhi)

The NLRP3 inflammasome is a cellular multimeric protein complex that plays a crucial role in inflammation and immune responses. It consists of three main components: Nod-like receptor protein 3(NLRP3), apoptosis-associated speck-like protein containing(ASC)and cysteine protease 1(caspase-1). Uveitis is a broad term encompassing a range of inflammatory diseases that primarily affect the iris, ciliary body, vitreous, retina and choroid. It is considered a major cause of blindness globally. Numerous studies have demonstrated the involvement of NLRP3 inflammasome in the onset and progression of uveitis, indicating its potential as a significant therapeutic target for uveitis in the future. This article provides an overview of the structure, biological functions and activation pathways of the NLRP3 inflammasome, as well as the current research progress on its association with different types of uveitis. Additionally, it discusses the application potential of the NLRP3 inflammasome in the treatment of uveitis.

Berberine interferes with RSV infection of HEp-2 cells by inducing mitophagy autophagy / 中国药理学通报

Aim To explore the effect of berberine (B E) on RSV infected HEp-2 cells and the related mechanism. Methods HEp-2 cells were infected with RSV and treated with BE. Cell viability was assessed using the CCK-8 assay. Protein expression levels of NLRP3, ASC, caspase-1, PINK1, Parkin, Beclinl, p62, LC3 I,LC3 II,and BNIP3 in HEp-2 cells were detected by Western blot. The secretion level of IL-1 p in HEp-2 cells was measured using ELISA. Apoptosis rate and mitochondrial membrane potential of HEp-2 cells were examined by flow cytometry. Mitochondrial ROS (mtROS) in HEp-2 cells was detected through MitoSOX staining. Colocalization of mitochondria and autophagosomes in HEp-2 cells was investigated using immunofluorescence staining. Cyclosporin A was used for validation experiments. Results BE could significantly improve the activity of RSV-infected HEp-2 cells,reduce the apoptosis rate (P < 0. 05), and decrease the activation level of NLRP3 inflammasomes and IL-lp level (P <0. 05); BE improved mitochondrial function by increasing mitochondrial membrane potential and ATP levels,and reduced mtROS. BE significantly promoted the colocalization of mitochondria-autophagosome in RSV infected cells, inducing PINK1/ Parkin and BNIP3 to mediate mitochondrial autophagy; cyclosporine A aggravated RSV infection. Conclusions BE has protective effects on HEp-2 cells infected by RSV. The mechanism may be related to the inhibitory effect of BE on the production of mtROS and the activation of NLRP3 inflammasomes by inducing PINK1/ Parkin and BNIP3-mediated mitochondrial autophagy.

Konjac Mannan Oligosaccharides enhanced intestinal barrier function to improve alcoholic nervous injury / 中国药理学通报

Aim Excessive cerebral inflammation caused by chronic alcohol intake is an important risk factor for central nervous system injury. The purpose of this study was to explore the protective effect of konjac mannan oligosaccharide (KMOS) on central nervous system inflammation in alcohol-fed mice and its mechanism. Methods The chronic alcohol fed model of C57BL/6J mice was established using Gao-binge method. And the different doses of KMOS were gavaged every day for 6 weeks. The neuronal damage and microglia activation were evaluated in cerebral cortex and hippocampus. The damage of colon tissue was assessed and serum LPS concentrations were measured. In vitro, Caco-2 cells were stimulated with LPS to establish intestinal mucosal injury model. Results Chronic alcohol intake can cause brain neuron damage in mice, and different doses of KMOS effectively reduced the activation state of microglia, decreased the expression of inflammatory factors caused by the activation of the NLRP3 inflammasome and alleviated neuronal damage in the brain tissue of alcohol-fed mice. The results of colon tissue analysis showed that the use of KMOS effectively reduced the concentration of endotoxin LPS in serum of alcohol-fed mice, alleviated the pathological injury and inflammatory response of colon tissue, and enhanced the expression of Occludin in intestinal tissue. In vitro experiments also showed that KMOS significantly inhibited the inflammatory reaction of Caco-2 cells exposed to alcohol and increased the expression of Occludin protein. Conclusions KMOS treatment effectively inhibited intestinal inflammation caused by alcohol intake, repaired intestinal barrier to prevent the entry of intestinal LPS into brain tissue, decreased the activation of microglia, and then improved brain neuron damage. KMOS had the potential to prevent alcoholic nerve injury.

Role of NLRP3 Inflammasome in IgA Nephropathy and Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

IgA nephropathy is recognized as the most common primary glomerular disease， with up to 20%-40% of patients developing end-stage kidney disease within 20 years of onset. The deposition of IgA1-containing immune complexes targeting glycosylation defects in the mesangial region and the subsequent inflammation caused by T lymphocyte activation are considered as the main causes of IgA nephropathy， and innate immunity is also involved in the pathogenesis. Nucleotide-binding oligomerization domain （NOD）-like receptor protein 3 （NLRP3） is a newly discovered pattern recognition receptor expressed in renal intrinsic cells such as renal tubular epithelial cells， mesangial cells， and podocytes. Activated by external stimuli， NLRP3 can form NLRP3 inflammasomes with apoptosis-associated speck-like protein containing a caspase recruitment domain （ASC）. The NLRP3 inflammasome can activate cysteine aspartate-specific protease-1 （Caspase-1）， causing the maturation and release of interleukin-18 （IL-18） and interleukin-1β （IL-1β） involved in inflammation. Increasing evidence has suggested that NLRP3 inflammasomes are involved in the pathogenesis and progression of IgA nephropathy and associated with the damage of renal intrinsic cells such as podocytes， mesangial cells， endothelial cells， and renal tubular epithelial cells. Chinese medicine can regulate inflammatory cytokines and their signaling pathways by acting on NLRP3 inflammasomes and related molecules， exerting therapeutic effects on IgA nephropathy. This article introduces the role of NLRP3 inflammasomes in IgA nephropathy and reviews the clinical and experimental research progress of Chinese medicine intervention in IgA nephropathy via NLRP3 inflammasomes， aiming to provide a reference for further research and application of Chinese medicine intervention in the NLRP3 inflammasome as a new therapeutic target.

Wu-Mei-Wan improves colonic mucosal inflammation and pyroptosis in rats with ulcerative colitis via regulating NLRP3 inflammasome / 中国免疫学杂志

Objective:To study effect and mechanism of Wu-Mei-Wan on improving colonic mucosal inflammatory response and pyroptosis in rats with ulcerative colitis(UC)via regulating NOD-like receptor family pyrin domain containing 3(NLRP3)inflam-masome.Methods:Male SD rats were selected for animal experiments.UC model was established by enema with 2,4,6-trinitroben-zene sulfonic acid.After model was established,different doses of Wu-Mei-Wan were given by gavage,negative control(NC)-lentivi-rus(LV)or NLRP3-LV were injected by tail vein.Disease activity index(DAI)of rats in each group was evaluated,length of colon was measured,pathological changes and histopathological scores,contents of IL-1β,IL-18,GSDMD-N,NLRP3 and Cleaved cas-pase-1 expressions in colon tissues were detected.Results:Typical UC pathological changes were found in colon mucosa of UC group,DAI,histopathological score,IL-1β,IL-18 contents,GSDMD-N,NLRP3 and Cleaved caspase-1 expressions in colon tissues of UC group were higher than control group,and colon length was shorter than control group(P<0.05).UC pathological changes of colon mucosa of rats in different concentrations of Wu-Mei-Wan groups were improved,DAI,histopathological score,IL-1β,IL-18 con-tents,GSDMD-N,NLRP3 and Cleaved caspase-1 expressions in colon tissues were lower than UC group,and colon length was longer than UC group(P<0.05).LV was injected simultaneously with intragastric administration of high-dose Wu-Mei-Wan,pathological changes of UC in colon mucosa of rats in NLRP3-LV+NC+high-dose Wu-Mei-Wan group was aggravated,DAI,histopathological score,IL-1β,IL-18 contents,GSDMD-N,NLRP3 and Cleaved caspase-1 expressions in colon tissues were higher than NC-LV+NC+ high-dose Wu-Mei-Wan group,and colon length was shorter than NC-LV+NC+high-dose Wu-Mei-Wan group(P<0.05).Conclusion:Wu-Mei-Wan improves colonic mucosal inflammation and pyroptosis in UC rats by inhibiting NLRP3 inflammasome.

Mechanism of icariin regulating the NLRP3 inflammasome against cerebral ischemia reperfusion / 中国实验动物学报

Objective To investigate the mechanism of icariin regulating the NLRP3 inflammasome in the treatment of cerebral ischemia-reperfusion injury in rats.Methods A rat model of focal cerebral ischemia-reperfusion was induced using the thread embolism method.At 24 hours post-operation,the rats were randomly allocated into a sham operation group,model group,butylphthalide group(70 mg/kg),ICA-low dose(20 mg/kg),ICA-middle dose(40 mg/kg),and ICA-high dose(80 mg/kg)groups.The corresponding drugs were administered by gavage at 10 mL/kg once a day for 13 consecutive days.One hour after the last administration,neurological function was scored.The cerebral cortex was observed by hematoxylin-eosin(HE)staining.Expression of interleukin(IL)-1β and IL-18 in the cerebral cortex was determined by immunohistochemistry.Expression of NLRP3,ASC,and Caspase-1 in the cerebral cortex was determined by Western Blot.Results In contrast to the sham operation group,there was a notable increase in neural function scores within the model group.The ischemic area around the visible cerebral cortex showed neuron necrosis at various level or glial cell proliferation,and the number of intact neurons was significantly reduced.IL-1β and IL-18 positive cells were significantly increased.Expression of NLRP3,ASC,and Caspase-1 was significantly increased(P＜0.01,P＜0.05).After treatment with icariin,the neural function score was decreased significantly.The degree of neuronal necrosis in the peri-ischemic area was significantly reduced,and the number of intact neurons was significantly increased.IL-1 β and IL-18-positive cells were decreased significantly.Expressions of NLRP3,ASC,and Caspase-1 were significantly decreased(P＜0.01,P＜0.05).Conclusions Treatment of cerebral ischemia-reperfusion injury by icariin may be related to regulation of the NLRP3 inflammasome.

α-mangostin inhibits LPS/ATP-induced NLRP3 inflammasome activation in microglia via the NF- κB pathway / 安徽医科大学学报

Objective @#To investigate the effects and underlying mechanisms of α-mangostin in a spinal cord inj ury model of microglial cell inflammation .@*Methods @#Mouse microglial cell line BV-2 was cultured in vitro , and an in- flammation model was established by co-treatment with lipopolysaccharide and adenosine triphosphate (LPS/ATP) . The CCK-8 assay was used to test the influence of different concentrations (0 , 10 , 20 , 40 , 80 μmol/L) of α-man- gostin on cell proliferation vitality under LPS/ATP stimulation to select an appropriate concentration range of α- mangostin; BV-2 cells were divided into Ctrl group , LPS/ATP group , 40 μmol/L α-mangostin group , and inter- vention groups with different concentrations (10 , 20 , 40 μmol/L) of α-mangostin ( designated as LPS/ATP + 10 μmol/L α-mangostin group , LPS/ATP + 20 μmol/L α-mangostin group , and LPS/ATP + 40 μmol/L α-mangostin group , respectively) . ELISA experiments were conducted to detect the levels of pro-inflammatory cytokines inter- leukin -6/1β/18 (IL-6 , IL-1β, IL-18) and tumor necrosis factor (TNF-α) in the supernatants of each group , and Western blot was used to detect the expression of NLRP3 , ASC , cleaved caspase-1 , IL-1β, and the phosphoryla- tion levels of p65 (p-p65/p65) in the NF- κB pathway , as well as the expression of p65 in the nuclei of BV-2 cells .@*Results @#Compared with the Ctrl group, cell proliferation vitality in the LPS/ATP group was significantly reduced (P < 0. 05) , but low concentrations (10 , 20 , 40 μmol/L) of α-mangostin significantly improved the inhibi- tory effect of LPS/ATP on microglial cell proliferation vitality (P < 0. 05) , while a high concentration (80 μmol/ L) of α-mangostin exacerbated the damage to microglial cells caused by LPS/ATP (P < 0. 05) . C ompared with the Ctrl group , the levels of inflammatory factors IL-6 , IL-1β, IL-18 , TNF-α, and the expression of NLRP3 , ASC , cleaved caspase-1 , IL-1β, and the p-p65/p65 ratio in the 40 μmol/L α-mangostin group , as well as the expression of p65 protein in the nuclei , showed no significant changes ( P > 0 . 05) , whereas these significantly increased in the LPS/ATP group (P < 0. 05) . Compared with the LPS/ATP group , the levels of IL-6 , IL-1β, IL-18 , TNF-α, and the expression of NLRP3 , ASC , cleaved caspase-1 , IL-1β, and the p-p65/p65 ratio in the intervention groups , as well as the expression of p65 protein in the nuclei , decreased in a concentration-dependent manner with increasing α-mangostin concentration , with the most significant reduction ob served in the LPS/ATP + 40 μmol/L α- mangostin group (P < 0. 01) . @*Conclusion @#α-mangostin can inhibit the neuroinflammatory response mediated by NLRP3 inflammasome activation in BV-2 cells through the NF- κB pathway .

Scutellariae Radix-Coptidis Rhizoma Treats Atherosclerosis via NLRP3 Inflammasome-mediated Pyroptosis of Macrophages / 中国实验方剂学杂志

ObjectiveTo investigate the therapeutic effect of Scutellariae Radix-Coptidis Rhizoma （SRCR） on atherosclerosis （AS） in mice and the effect of SRCR on macrophage pyroptosis in plaques via NOD-like receptor thermal protein domain-associated protein 3 （NLRP3） inflammasomes. MethodApoE-/- mice were fed with a high-fat diet for the modeling of AS and randomized into model， atorvastatin （5 mg·kg-1）， and low-， medium-， and high-dose （1.95， 3.9， 7.8 g·kg-1， respectively） SRCR groups. Normal C57BL/6J mice were selected as the control group. After 8 weeks of administration， hematoxylin-eosin staining was used to observe the pathological status of the aortic plaque. The lipid accumulation in aortic plaque was observed by oil red O staining. The serum levels of total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） in mice were measured. Immunofluorescence double staining was employed to detect the co-localized expression of EGF-like module-containing mucin-like hormone receptor-like 1 （EMR1）/NLRP3 and EMR1/gasdermin D （GSDMD）. The serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The protein levels of NLRP3， apoptosis-associated speck-like protein （ASC）， Caspase-1， cleaved Caspase-1， GSDMD， N-terminus of GSDMD （GSDMD-NT）， pro-IL-1β， IL-1β， and IL-18 were determined by Western blot， and the mRNA levels of NLRP3， ASC， Caspase-1， GSDMD， IL-1β， and IL-18 were determined by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the control group， the model group showed obvious plaques， elevated serum levels of TG， TC， LDL-C， IL-1β， and IL-18 （P<0.01）， lowered serum level of HDL-C （P<0.01）， and up-regulated expression of NLRP3 inflammasomes and molecules related to pyroptosis in the aortic plaques （P<0.01）. Compared with the model group， SRCR， especially at the medium and high doses， alleviated the plaque pathology， reduced the lipid content in plaques （P<0.05， P<0.01）， recovered the serum lipid levels （P<0.05）， reduced the macrophage recruitment （P<0.01）， activation of NLRP3 inflammasomes， and pyroptosis in aortic root plaques （P<0.05）， lowered the serum IL-1β and IL-18 levels （P<0.01）， and down-regulated the protein levels of NLRP3， ASC， Caspase-1， cleaved Caspase-1， GSDMD， GSDMD-NT， pro-IL-1β， IL-1β， and IL-18 （P<0.05） and the mRNA levels of NLRP3， ASC， Caspase-1， GSDMD， IL-1β， and IL-18 in the aortic tissue （P<0.05）. ConclusionSRCR exerts a therapeutic effect on high-fat diet-induced AS in mice by inhibiting the activation NLRP3 inflammasomes and reducing the pyroptosis of macrophages in plaques.

Mecanismos y dianas terapéuticas del inflamasoma NLRP3 en la miocardiopatía diabética / NLRP3 inflammasome mechanism and therapeutic targets in diabetic cardiomyopathy

Resumen La cardiomiopatía diabética es una complicación grave de la diabetes causada por estrés oxidativo, inflamación, resistencia a la insulina, fibrosis miocárdica y lipotoxicidad. Se trata de un padecimiento insidioso, complejo y difícil de tratar. El inflamasoma NLRP3 desencadena la maduración y liberación de citoquinas proinflamatorias, participa en procesos fisiopatológicos como la resistencia a la insulina y la fibrosis miocárdica, además de estar estrechamente relacionado con la aparición y progresión de la cardiomiopatía diabética. El desarrollo de inhibidores dirigidos a aspectos específicos de la inflamación sugiere que el inflamasoma NLRP3 puede utilizarse para tratar la cardiomiopatía diabética. Este artículo pretende resumir el mecanismo y las dianas terapéuticas del inflamasoma NLRP3 en la cardiomiopatía diabética, así como aportar nuevas sugerencias para el tratamiento de esta enfermedad.

Abstract Diabetic cardiomyopathy (DCM) is a serious complication of diabetes caused by oxidative stress, inflammation, insulin resistance, myocardial fibrosis, and lipotoxicity; its nature is insidious, complex and difficult to treat. NLRP3 inflammasome triggers the maturation and release of pro-inflammatory cytokines, participates in pathophysiological processes such as insulin resistance and myocardial fibrosis, in addition to being closely related to the development and progression of diabetic cardiomyopathy. The development of inhibitors targeting specific aspects of inflammation suggests that NLRP3 inflammasome can be used to treat diabetic cardiomyopathy. This paper aims to summarize NLRP3 inflammasome mechanism and therapeutic targets in diabetic cardiomyopathy, and to provide new suggestions for the treatment of this disease.

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.

Costunolide covalently targets NACHT domain of NLRP3 to inhibit inflammasome activation and alleviate NLRP3-driven inflammatory diseases

The NLRP3 inflammasome's core and most specific protein, NLRP3, has a variety of functions in inflammation-driven diseases. Costunolide (COS) is the major active ingredient of the traditional Chinese medicinal herb Saussurea lappa and has anti-inflammatory activity, but the principal mechanism and molecular target of COS remain unclear. Here, we show that COS covalently binds to cysteine 598 in NACHT domain of NLRP3, altering the ATPase activity and assembly of NLRP3 inflammasome. We declare COS's great anti-inflammasome efficacy in macrophages and disease models of gouty arthritis and ulcerative colitis via inhibiting NLRP3 inflammasome activation. We also reveal that the α-methylene-γ-butyrolactone motif in sesquiterpene lactone is the certain active group in inhibiting NLRP3 activation. Taken together, NLRP3 is identified as a direct target of COS for its anti-inflammasome activity. COS, especially the α-methylene-γ-butyrolactone motif in COS structure, might be used to design and produce novel NLRP3 inhibitors as a lead compound.