Diabetes has become a global epidemic disease now. Its chronic progressive deterioration and the acute and chronic complications affect the quality of the patients' lives seriously. The prevention and treatment of diabetes has become one of the research focuses in recent years. NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome can recognize the metabolic stress signals, and cause caspase-1 activation and interleukin-1β (IL-1β) production, and is closely related to diabetes development. The latest studies have shown that NLRP3 inflammasome will be a new potential target for the treatment of diabetes. This article reviews the activation and regulation of NLRP3 inflammasome, and the effect of NLRP3 inflammasome on glucose metabolism and its targeted therapy in diabetes.
Objective To study the expression of NLRP3 inflammasome and its downstream inflammatory factors in patients with chronic obstructive pulmonary disease (COPD) and healthy controls, and to reveal the effect and significance of NLRP3 inflammasome in the pathogenesis of COPD. Methods Forty patients with acute exacerbation COPD (AECOPD) who were hospitalized from November 2016 to May 2017 were recruited in the AECOPD group, and recruited in the stable COPD group when they entered the stable stage. Forty healthy individuals were recruited in the control group. General information and peripheral blood were collected from each subject. The levels of NLRP3 mRNA and caspase-1 mRNA in peripheral blood mononuclear cells were measured by real-time PCR. The levels of IL-18 and IL-1β were measured by enzyme-linked immunosorbent assay. Results The levels of NLRP3 mRNA, IL-18 and IL-1β in the AECOPD patients were significantly higher than those in the stable COPD group [2.11±0.77, 12.79 (7.10, 43.13) pg/ml, 17.02 (8.36, 52.21) pg/ml vs. 1.60±0.44, 10.66 (6.32, 18.59) pg/ml, 13.34 (7.07, 16.89) pg/ml, all P<0.05] . The levels of NLRP3 mRNA, IL-18 and IL-1β in the AECOPD patients were significantly higher than those in the control group [2.11±0.77, 12.79 (7.10, 43.13) pg/ml, 17.02 (8.36, 52.21) pg/mlvs. 1.00±0.49, 6.29 (4.73, 7.93) pg/ml, 5.93 (4.81, 9.67) pg/ml, all P<0.05]. The levels of NLRP3 mRNA, IL-18 and IL-1β were significantly higher in the stable COPD group than the control group [1.60±0.44, 10.66 (6.32, 18.59) pg/ml, 13.34 (7.07, 16.89) pg/mlvs. (1.00±0.49, 6.29 (4.73, 7.93) pg/ml, 5.93 (4.81, 9.67) pg/ml, all P<0.05]. Correlation analysis showed that the plasma IL-18 level was positive correlated with leukocyte count and neutrophil percentage in the AECOPD group (r=0.372, P<0.05;r=0.386, P<0.05). The expression of NLRP3 mRNA in the AECOPD group and stable COPD group were positively correlated with the CAT score (r=0.387, P<0.05;r=0.399, P<0.05) . Conclusion NLRP3 inflammasome is involved in the inflammatory response in COPD patients.
ObjectiveTo investigate relationship between nod-like-receptor protein 3 (NLRP3) inflammasome and acute pancreatitis induced pancreas and extrapancreatic organs injury.MethodThe related literatures on the relationship between the nod-like-receptor protein 1 inflammasome (NLRP3 inflammasome) and the acute pancreatitis in recent years were reviewed.ResultsThe activation and regulation of NLRP3 inflammatory corpuscle are involved in the injury of various organs in acute pancreatitis. The more the activation of NLRP3 inflammatory corpuscle, the more severe the damage to the body. Through the regulation of the activation mechanism of NLRP3 inflammatory corpuscle, the activation of NLRP3 inflammatory corpuscle can be reduced, and finally the injury of various organs can be reduced.ConclusionThe activation of NLRP3 inflammatory corpuscle is involved in the process of acute pancreatitis, but it still needs to be verified by further clinical studies.
ObjectiveTo observe the effects of four prostaglandin E2 (PGE2) receptors (EP1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment.MethodsThe hRMEC were divided into normal group and high glucose group, and they were cultured in Dulbecco modified Eagle medium containing 5.5 and 30.0 mmol/L glucose, respectively. Flow cytometry was used to observe the apoptosis rate of the high glucose group and the normal group; enzyme chain immunosorbent assay (ELISA) was used to detect the level of PGE2 in the culture supernatant of hRMEC cells. Western blot was used to detect the protein expression of cyclooxyganese (COX2) and EP1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP1-4R agonist group, PGE2+EP1-4R inhibitor group, and dimethylsulfoxide group. According to the group, each group was given the corresponding agonist or inhibitor to continue the culture for 24 h. QRT-PCR was used to detect the expression of nucleotide-binding oligomerization structure-like receptor protein (NLRP3) and pro-interleukin (IL)-1β mRNA in each group of cells. ELISA was used to detect the content of IL-1β and lactic dehydrogenase (LDH) in the cell culture supernatant. Western blot was used to detect the expression of cleaved Caspase-1 in each group of cells. At the same time, hRMEC in a high glucose environment was given IL-1β stimulation for 24 h, and the activity of LDH in the supernatant of the cell culture medium was detected.ResultsThe apoptotic rate, COX2 protein expression, and PGE2 protein content in hRMEC in the high glucose group were significantly higher than those in the normal group, and they were time-dependent. Compared with the normal group, the expression levels of EP1R, EP2R, EP4R protein and mRNA in hRMEC in the high glucose group were higher than those in the normal group (P＜0.05). Compared with the control group, PGE2 group (t=4.627, P＜0.01), EP1-4R agonist group (t=3.889, 3.583, 2.445, 3.216; P＜0.05) hRMEC NLRP3 mRNA expression level was significantly increased; the expression level of pro-IL-1β mRNA increased, however the difference was not statistically significant (PGE2 group: t=1.807, P＞0.05; EP1-4R agonist group: t=1.807, 1.477, 0.302, 1.926, P＞0.05). Compared with the PGE2 group, the expression of NLRP3 mRNA in hRMEC in the PGE2+EP2R inhibitor group was significantly reduced (t=2.812, P＜0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP3R inhibitor group was significantly increased (t=4.113, P＜0.01). The protein content of IL-1β in the cell culture supernatant of the PGE2 group, EP1R agonist group and EP2R agonist group was significantly higher than that of the control group (t=5.155, 4.136, 4.817; P＜0.01). Compared with PGE2 group, the protein content of IL-1β in the cell culture supernatant of the PGE2+EP2R inhibitor group and the PGE2+EP4R inhibitor group were significantly lower than that of the PGE2 group (t=1.964, 4.765; P＜0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2 group and EP2R agonist group was significantly higher than that in the control group (t=5.332, 4.889; P＜0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2+EP2R inhibitor group was significantly lower than that of the PGE2 group (t=6.699, P＜0.01). The LDH activity in the cell culture supernatant of the PGE2 group and the EP2R agonist group was significantly higher than that of the control group (t=4.908, 4.225; P＜0.05). The activity of LDH in the cell culture supernatant of the PGE2+EP2R inhibitor group was significantly lower than that of the PGE2 group (t=5.301, P＜0.01). Compared with the control group, the LDH activity in the culture supernatant of hRMEC cells in the high glucose environment was significantly increased (t=3.499, P＜0.05).ConclusionsThe four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP2R mainly mediates hRMEC damage under high glucose environment.
Objective To observe the effect of NLRP3 inflammasome inhibitor MCC950 intervention on airway Muc5ac level in asthmatic mice, and to explore the role and mechanism of NLRP3 inflammasome in asthmatic airway mucus hypersecretion. Methods A total of 50 SPF grade BALB/c female mice aged 6 - 8 weeks were randomly divided into normal control group (NS group), asthma model group (AS group), dexamethasone group (Dex group), MCC950 high-dose intervention group (MH group) and MCC950 low-dose intervention group (ML group), with 10 mice in each group. Furthermore, the bronchoalveolar lavage fluid (BALF) of mice in each group was counted by total cell count, associated with white blood cell different count. In addition, the concentrations of interleukin (IL)-18 and IL-1β in BALF were tested by enzyme-linked immunosorbent assay; The lung tissues were prepared into paraffin-embedded sections, which were then subject to hematoxylin-eosin (HE) staining, Alcian blue-periodic acid Schiff base staining and Masson staining to observe the pathological changes of lung tissues. Immunohistochemistry was used to detect the protein expression levels of Muc5ac, NLRP3 and caspase-1 in lung tissues. Real-time quantitative polymerase chain reaction was performed to detect the relative mRNA expressions of Muc5ac, NLRP3 and Caspase-1 in lung tissues. Results Compared with NS group, AS group showed significant increase in total cell count of BALF, the percentage of eosinophils, the infiltration score of inflammatory cells around the airway, the positive relative staining area of airway mucus and the deposition area of airway collagen fibers in mice (P<0.05), upregulated protein expression levels of Muc5ac, NLRP3 and Caspase-1 in lung tissues (P<0.05), elevated relative mRNA expressions of Muc5ac, NLRP3 and Caspase-1 in lung tissues (P<0.05), and raised concentrations of IL-18 and IL-1β in BALF (P<0.05). While compared with AS group, the above indicators were reduced in MH group and ML group (P<0.05). Moreover, in relative to Dex group, these indicators were increased in MH group ML group (P<0.05). In addition, no statistically significant difference was observed in the aforementioned indications between MH group and ML group.Conclusions MCC950 intervention can inhibit airway inflammation and airway mucus secretion in asthmatic mice. Its mechanism is speculated to be related to the suppression of NLRP3, Caspase-1, IL-18 and IL-1β expressions, downregulation of Muc5ac expression, and inhibition in airway mucus hypersecretion.
ObjectiveTo observe the effects of RasGRP4 gene deletion on the structure and function of the retina in diabetic mice, and to explore the mechanism of RasGRP4 in diabetic retinopathy (DR) by transcriptome sequencing in conjunction with bioinformatics analysis. MethodsA total of 12 male C57BL/6J mice were divided into normal group, diabetic group (DM group), with 6 mice in each group. Six male RasGRP4 knockout mice were uesd as RasGRP4 knockout diabetic group (DM-KO group). Mice in the DM group and DM-KO group were fed with high-fat diet combined with intraperitoneal injection of streptozotocin to establish diabetic model and body weight and blood glucose were monitored regularly. Three months after modeling, optical coherence tomography (OCT) was used to detect the retinal thickness. Electroretinography (ERG) was used to detect the function of the retina in mice under dark-adapted conditions. Total RNA was extracted from the retinas of the DM group and DM-KO group for transcriptome sequencing and bioinformatics analysis. The relative expression of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in retina were detected by real-time quantitative polymerase chain reaction (qRT-PCR). One-way analysis of variance was used to compare groups. ResultsThere was no statistically significant difference in blood glucose and body weight between mice in the DM group and DM-KO group (t=0.12、2.02、0.22、0.10、0.59、0.41、1.35、0.31、1.12、1.58、1.47、1.20、1.24、0.39、0.66、0.14, P＞0.05). Compared with the normal group, the retinal thickness and ganglion cell layer thickness were significantly decreased in the DM group, while the retinal thickness and ganglion cell layer thickness were significantly increased in the DM-KO group compared with the DM group, with statistical significance (F=30.43、7.81, P＜0.0001、0.01). Compared with the normal group, the a-wave and b-wave amplitudes were significantly decreased in the DM group, while the a-wave and b-wave amplitudes were significantly increased in the DM-KO group compared with the DM group, with statistical significance (F=16.46、35.58, P＜0.001、0.0001). Compared with the DM group, 184 differential genes (DEG) were screened in the DM-KO group, among which 39 up-regulated and 145 down-regulated genes were detected, respectively. The results of the MCODE plug-in analysis showed that Col1a2, Fbln1, Fbn1, Col6a3, Fmod, Ogn, Tgfb, Mfap4, Vcan, Nid2, and Col18a1 were core genes in the DEG. Cytohubba plug-in analysis showed that Col1a2, Mrc1, Cd47, Fbn1, Cybb, Cd163, Fbln1, Fmod, Adgre1, and Col6a3 were the core genes in DEG. The results of the GO functional enrichment analysis showed that DEG was mainly involved in hemoglobin complexes, MHC class II protein complex, apical plasma membrane, inflammasome complex, immunological synapse, response to bacterium, inflammatory response, immune system processe, response to hypoxia, and cell adhesion were significantly enriched. qRT-PCR results showed that compared with the normal group, the relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in the retina of mice in DM group were significantly increased, while the relative expression levels of IL-8, TGF-β, IFN-γ, NLRP3, Caspase-1, and IL-1β mRNA in the retina of mice in DM-KO group were significantly decreased compared with the DM group, with statistical significance (F=12.43、15.41、70.09、29.04、11.79、41.28, P＜0.01). Conclusion RasGRP4 deficiency plays a therapeutic role in the development of DR through inhibition of inflammatory factor secretion and NLRP 3 inflammasome pathway activation.
Objective To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment. MethodsExperimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups. ResultsIn vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups (F=43.478, 36.643, 24.464; P＜0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group (F=32.598, P＜0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference (F=47.267, P＜0.01). The mRNA (F=51.563, 32.192, 44.473; P＜0.01) and protein levels (F=63.372, 54.463, 48.412; P＜0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. ConclusionMet can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.