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Purpose: Myocardial ischemia/reperfusion injury (MIRI) leads to myocardial tissue necrosis, which will increase the size of myocardial infarction. The study examined the protective effect and mechanism of the Guanxin Danshen formula (GXDSF) on MIRI in rats. Methods: MIRI model was performed in rats; rat H9C2 cardiomyocytes were hypoxia-reoxygenated to establish a cell injury model. Results: The GXDSF significantly reduced myocardial ischemia area, reduced myocardial structural injury, decreased the levels of interleukin (IL-1ß, IL-6) in serum, decreased the activity of myocardial enzymes, increased the activity of superoxide dismutase (SOD), and reduced glutathione in rats with MIRI. The GXDSF can reduce the expression of nucleotide- binding oligomerization domain, leucine-rich repeat and pyrin domain containing nod-like receptor family protein 3 (NLRP3), IL-1ß, caspase-1, and gasdermin D (GSDMD) in myocardial tissue cells. Salvianolic acid B and notoginsenoside R1 protected H9C2 cardiomyocytes from hypoxia and reoxygenation injury and reduced the levels of tumor necrosis factor α (TNF-α) and IL-6 in the cell supernatant, decreasing the NLRP3, IL-18, IL-1ß, caspase-1, and GSDMD expression in H9C2 cardiomyocytes. GXDSF can reduce the myocardial infarction area and alleviate the damage to myocardial structure in rats with MIRI, which may be related to the regulation of the NLRP3. Conclusion: GXDSF reduces MIRI in rat myocardial infarction injury, improves structural damage in myocardial ischemia injury, and reduces myocardial tissue inflammation and oxidative stress by lowering inflammatory factors and controlling focal cell death signaling pathways.
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Animals , Rats , Myocardial Reperfusion , Reperfusion Injury , Ginsenosides/administration & dosage , NLR Family, Pyrin Domain-Containing 3 ProteinABSTRACT
Purpose: To observe the effect of puerarin on renal ischemia-reperfusion (I/R) injury in rats, and to explore its mechanism based on NLRP3/Caspase-1/GSDMD pathway. Methods: Twenty-one Sprague-Dawley rats were divided into three groups: sham-operated group (sham), model group (RIRI), and puerarin treatment group (RIRI + Pue). The model of acute renal I/R injury was established by cutting the right kidney and clamping the left renal pedicle for 45 min. Results: Renal function parameters were statistically significant in group comparisons. The renal tissue structure of rats in sham group was basically normal. Pathological changes were observed in the RIRI group. The renal pathological damage score and apoptosis rate in the RIRI group were higher than those in the sham group, and significantly lower in the RIRI + Pue group than in the RIRI group. Indicators of oxidative stress-superoxide dismutase, malondialdehyde, and glutathione peroxidase-were statistically significant in group comparisons. Compared with the sham group, the relative expressions of NLRP3, Caspase-1 and GSDMD proteins in the RIRI group were increased. Compared with the RIRI group, the RIRI + Pue group had significant reductions. Conclusions: Puerarin can inhibit the activation of NLRP3/Caspase-1/GSDMD pathway, inhibit inflammatory response and pyroptosis, and enhance the antioxidant capacity of kidney, thereby protecting renal I/R injury in rats.
Subject(s)
Animals , Rats , Reperfusion Injury , Pyroptosis , Inflammation , Kidney/injuriesABSTRACT
In recent years, with the development of metabolic reprogramming research, people have changed their understanding of the biological effects of immune cells. Under the stimulation of inflammatory response, immune cells re-regulate their metabolism and bioenergetics, provide energy and substrates for cell survival, and initiate immune effect functions. Nod-like receptor protein 3 (NLRP3) inflammasome, as an important component of the innate immune system, has been shown to sense metabolites such as uric acid and cholesterol crystals, and can be inhibited by metabolites such as ketones. It is also regulated by mitochondrial reactive oxygen species and glycolytic components (such as hexokinase). Recent studies have shown that a variety of metabolic pathways converge as effective regulators of NLRP3 inflammasome. The paper reviews the metabolic regulatory pathways and specificity of NLRP3 inflammasome activation, and its role in renal diseases.
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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.
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Objective:To evaluate the role of silent information regulator-1 (SIRT1)/nucleotide-binding domain (NOD)-like receptor protein-3 (NLRP3) signaling pathway in sevoflurane postconditioning-induced attenuation of oxygen-glucose deprivation and restoration (OGD/R) injury in mouse hippocampal neuronal cell line (HT22) cells.Methods:The HT22 cells were seeded in a culture plate (96-well plate, 100 μl/well; 6-well plate, 2 ml/well) at the density of 5×10 4 cells/ml or in a culture dish (6 cm in diameter) and then divided into 4 groups ( n=24 each) using a random number table method: control group (Control group), OGD/R group, sevoflurane postconditioning group (SPC group), and SIRT1 small interfering RNA group (si-SIRT 1 group). In Control group, cells were cultured at 37 ℃ in normal culture atmosphere. In OGD/R group, the culture medium was replaced with glucose-free serum-free culture medium, and cells were exposed to 95% N 2+ 5% CO 2 for 4 h in an incubator at 37 ℃, and then the glucose-free serum-free culture medium was replaced with the primary culture medium, and cells were cultured for 24 h at 37 ℃ in normal culture atmosphere. In SPC group, the glucose-free serum-free culture medium was replaced with the primary cell culture medium after 4-h oxygen and glucose deprivation, the cells were put into the hypoxia incubator chamber which was filled with 2% sevoflurane immediately after start of reoxygenation, then the chamber was placed in an incubator and the cells were cultured for 1 h at 37 ℃ in normal culture atmosphere, and finally the cells were removed from the chamber and cultured for 23 h at 37 ℃ in normal culture atmosphere. In si-SIRT1 group, SIRT1 small interfering RNA 150 pmol was added at 24 h before surgery, cells were then incubated, and the other procedures were the same as those previously described in group SPC. The cell survival rate was determined using MTT assay. TUNEL assay was used to detect cell apoptosis, and the apoptosis rate was calculated. The expression of SIRT1, NLRP3, IL-1β and IL-18 mRNA was determined using polymerase chain reaction. The expression of SIRT1, NLRP3, interleukin-1beta (IL-1β) and IL-18 was detected using Western blot. Results:Compared with Control group, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of SIRT1 protein and mRNA was down-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was up-regulated in OGD/R group ( P<0.05). Compared with OGD/R group, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of SIRT1 protein and mRNA was up-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was down-regulated in SPC group ( P<0.05). Compared with SPC group, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of SIRT1 protein and mRNA was down-regulated, and the expression of NLRP3, IL-1β and IL-18 protein and mRNA was up-regulated in si-SIRT1 group ( P<0.05). Conclusions:Activation of SIRT1-NLRP3 signaling pathway is involved in sevoflurane postconditioning-induced attenuation of OGD/R injury in HT22 cells.
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Objective:To evaluate the role of bilateral superior cervical sympathetic ganglia (SCG) in myocardial ischemia-reperfusion (I/R) injury in mice and the relationship with NOD-like receptor protein 3 (NLRP3) inflammasomes.Methods:Thirty-two healthy SPF male C57BL mice, aged 8-10 weeks, weighing 25-30 g, were divided into 4 groups ( n=8 each) by the random number table method: sham operation group (NS group), myocardial I/R group (NIR group), bilateral SCG excision group (SCGx group) and bilateral SCG excision + myocardial I/R group (SCGx+ IR group). The myocardial I/R injury model was prepared by ligating the anterior descending branch of the left coronary artery for 30 min followed by 24 h reperfusion in isoflurane-anesthetized mice. Bilateral superior cervical sympathectomy was performed at 3 days before reperfusion. Blood samples were collected from the inferior vena cava at 24 h of reperfusion for examination of pathological changes (by HE and WGA staining) and for measurement of serum creatine kinase isoenzymes (CK-MB) activity, cardiac troponin I (cTnI) concentration, norepinephrine (NE) concentration and lactic dehydrogenase (LDH) activity (by enzyme-linked immunosorbent assay), superoxide dismutase (SOD) activity (by colorimetric method), myocardial reactive oxygen species (ROS) level (by DHE method), myocardial infarct size(by TTC method), and expression of interleukin-1beta (IL-1β), IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), NLRP3 mRNA (by quantitativepolymerase chain reaction ), and expression of tyrosine hydroxylase (TH), IL-1β, TNF-α, NLRP3, atrial natriuretic peptide (ANP)and brain natriuretic peptide (BNP) (by Western blot). Results:Compared with NS group, the NE concentration was significantly decreased, and TH expression was down-regulated in SCGx group, and the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS level were significantly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in NIR group ( P<0.05). Compared with SCGx group, the serum CK-MB activity, concentrations of cTnI and NE, LDH activity and myocardial ROS levels were significamtly increased, SOD activity was decreased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was up-regulated, and the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was up-regulated in SCGx+ NIR group ( P<0.05). Compared with NIR group, the serum CK-MB activity, cTnI concentration, LDH activity and myocardial ROS level were significantly decreased, SOD activity was increased, the expression of IL-1β, TNF-α, NLRP3, ANP and BNP was down-regulated, the expression of IL-1β, IL-6, TNF-α and NLPR3 mRNA was down-regulated, and myocardial infarct size was decreased in SCGx+ NIR group ( P<0.05). Conclusions:The mechanism by which bilateral SCG excision attenuates myocardial I/R injury is associated with decreased NLRP3 inflammatory inflammasome activation and inhibition of inflammatory responses in mice.
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Objective:To evaluate the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in sepsis-associated encephalopathy (SAE) and the relationship with pyroptosis in microglia of mice.Methods:Twenty-four SPF healthy male C57BL/6J mice, aged 6-8 weeks, weighing 18-22 g, were divided into 3 groups ( n=6 each) using a random number table method: sham operation group (Sham group), SAE group and SAE plus an NLRP3 inhibitor MCC950 group (SAE+ MCC950 group). The mouse model of SAE was prepared by cecal ligation and puncture after anesthesia. MCC950 20 mg/kg was intraperitoneally injected at 1 h after developing the model in SAE+ MCC950 group, and the equal volume of normal saline was given instead in the other groups. Open field tests were conducted at 1 day after developing the model to record the number of rearing and time spent in the central area. Novel object recognition tests were conducted at 2-3 days after developing the model to record the recognition index. After the behavioral experiment on 3 day after developing the model, mice were sacrificed and hippocampal tissues were collected for determination of the expression of NLRP3 (by Western blot), count of cells co-expressing NLRP3 and microglia-specific ionized calcium-binding adaptor molecule 1 (Iba-1) (by immunofluorescence), activity of caspase-1, and contents of interleukin-1beta(IL-1β) and IL-18 (by enzyme-linked immunosorbent assay). Results:Compared with Sham group, the number of rearing was significantly reduced, the time spent in the central area was shortened, the recognition index was decreased, the expression of NLRP3 was up-regulated, the count of NLRP3 + -Iba-1 + cells was increased, and the activity of caspase-1 and contents of IL-1β and IL-18 were increased in SAE and SAE+ MCC950 groups ( P<0.05). Compared with SAE group, the number of rearing was significantly increased, the time spent in the central area was prolonged, the recognition index was increased, the expression of NLRP3 was down-regulated, the count of NLRP3 + -Iba-1 + cells was decreased, and the activity of caspase-1 and contents of IL-1β and IL-18 were decreased in SAE+ MCC950 group ( P<0.05). Conclusions:NLRP3 is involved in the development of SAE, which may be related to the mediation in microglial pyroptosis in mice.
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Objective:To evaluate the relationship between cannabinoid receptor 1 (CB1R) and the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) during the reduction of cerebral ischemia-reperfusion (I/R) injury by electroacupuncture (EA) preconditioning in rats.Methods:Forty SPF healthy male Sprague-Dawley rats, aged 7-9 weeks, weighing 250-280 g, were divided into 5 groups ( n=8 each) according to the random number table method: sham operation group (Sham group), cerebral I/R group (I/R group), EA preconditioning group (EA group), CB1R antagonist AM251+ EA preconditioning group (AM251+ EA group), and CB1R agonist WIN 55, 212-2 group (WIN group). Cerebral I/R was induced by middle cerebral artery occlusion (MCAO) in anesthetized animals. In EA group, EA preconditioning was performed, and the acupoint Baihui (GV20) was stimulated for 30 min with disperse-dense waves, the intensity of 1 mA and frequency of 2/15 Hz once a day for 5 consecutive days, and the model of cerebral I/R injury was developed at 24 h after the last EA. In AM251+ EA group, CB1R antagonist AM251 1 mg/kg was intraperitoneally injected at 30 min before each stimulation, and the remaining operations were the same as those previously described in EA group. CB1R agonist WIN 55, 212-2 1.5 mg/kg was intraperitoneally injected for 5 consecutive days, and the model of cerebral I/R injury was prepared at 24 h after the last injection in WIN group. Neurological behavior was assessed and scored at 3 days of reperfusion. Then the rats were sacrificed, and brains were removed, and the infarct volume was measured by TTC staining, and the tissues in the ischemic penumbra were extracted for determination of the expression of NLRP3, caspase-1 and interleukin-1bata (IL-1β) by Western blot. Results:Compared with Sham group, the percentage of cerebral infarct volume was significantly increased, the neurobehavioral score was decreased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in I/R group ( P<0.05). Compared with I/R group, the percentage of cerebral infarct volume was significantly decreased, the neurobehavioral score was increased, and the expression of NLRP3, caspase-1 and IL-1β was down-regulated in EA and WIN groups ( P<0.05). Compared with EA group, the percentage of cerebral infarct volume was significantly increased, the neurobehavioral score was decreased, and the expression of NLRP3, caspase-1 and IL-1β was up-regulated in AM251+ EA group ( P<0.05). Conclusions:EA preconditioning may inhibit the activation of NLRP3 inflammasomes by activating CB1R, thus alleviating cerebral I/R injury in rats.
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Objective:To evaluate the relationship between Sestrin2 and mitochondrial DNA (mtDNA)-NOD-like receptor associated protein 3 (NLRP3) inflammasome pathway during endotoxin-induced myocardial injury in mice.Methods:One hundred and eighty-four clean-grade healthy male ICR mice, aged 8-12 weeks, weighing 20-25 g, were used in this study. One hundred and sixty-eight mice were divided into 7 groups ( n=24 each) using the random number table method: normal control group (N group), lipopolysaccaride(LPS) group (L group), mtDNA group, LPS+ mtDNA group (M group), normal control+ negative control adeno-associated virus (AAV-NC)group (NC group), LPS+ mtDNA+ AAV-NC group (MC group), and LPS+ mtDNA+ Sestrin2 overexpression adeno-associated virus (AAV-Sestrin2) group (MSgroup). Another 10 mice were used to detect the transfection effect of AAV-Sestrin2, and the left 6 mice were used for mtDNA extraction. The model of endotoxemia was developed by intraperitoneal injection of LPS 10 mg/kg. mtDNA 5 mg/kg was intraperitoneally injected in mtDNA group, and mtDNA 5 mg/kg was intraperitoneally injected at 30 min after LPS injection in M group.AAV-Sestrin2 150 μl was injected via the tail vein in MS group, and the equal volume of AAV-NC was injected via the tail vein in MC and NC groups. Four weeks after virus injection, LPS 10 mg/kg was intraperitoneally injected and 30 min later mtDNA 5 mg/kg was intraperitoneally injected in MS and MC groups. Blood samples were collected at 24 h after LPS injection for determination of serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) activities (by biochemical assay), concentrations of serum cardiac troponin I (cTnI), interleukin-18 (IL-18) and interleukin-1beta (IL-1β)(by enzyme-linked immunesorbent assay), and expression of mtDNA (by quantitative real-time polymerase chain reaction). The animals were sacrificed after the end of blood sampling and myocardial tissues were obtained for determination of the contents of reactive oxygen species (ROS), total antioxidant capacity (T-AOC), and adenosine triphosphate (ATP) and expression of NOD-like receptor associated protein 3 (NLRP3), active subunit p20 of caspase-1 (caspase-1p20) and apoptosis-associated microprotein (ASC) in myocardial tissues (by Western blot) and for microscopic examination of the pathological changes after HE staining (with a light microscope). Results:Compared with N group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly increased, the expression of mtDNA was up-regulated, the ROS content in myocardial tissues was increased, the T-AOC and ATP contents in myocardial tissues were decreased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was up-regulated( P<0.05), and the pathological changes of myocardial tissues were aggravated in L group and mtDNA group.Compared with L group and mtDNA group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly increased, the expression of mtDNA was up-regulated, the ROS content in myocardial tissues was increased, the T-AOC and ATP contents in myocardial tissues were decreased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was up-regulated( P<0.05), and the pathological changes of myocardial tissues were aggravated in M group. Compared with M group, the levels of CK-MB, LDH, cTnI, IL-1β and IL-18 in serum were significantly decreased, the expression of mtDNA was down-regulated, the ROS content in myocardial tissues was decreased, the T-AOC and ATP contents in myocardial tissues were increased, the expression of NLRP3, caspase-1p20 and ASC in the myocardial tissues was down-regulated( P<0.05), and the pathological changes of myocardial tissues were significantly attenuated in MS group. Conclusions:Sestrin2 can reduce endotoxin-induced myocardial injury in mice by alleviating mitochondrial damage, inhibiting oxidative stress, protecting mtDNA from oxidative damage, and then inhibiting mtDNA-NLRP3 inflammasome pathway.
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Objective:To explore the potential role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/GATA-binding protein 4 (GATA-4)/vascular endothelial growth factor (VEGF) signal pathway in neovascular age-related macular degeneration (nAMD).Methods:We applied the TRANSFAC Public database to search the human and mouse VEGF promoters and upstream transcription factors, analyzed the transcription factors that may influence the transcriptional activity of VEGF. The RAW264.7 cells were divided into control group and lipopolysaccharide (LPS) stimulated group (LPS group). Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the activation of NLRP3 inflammasome, and the mRNA levels of GATA-4 and VEGFA. Thus, we applied the specific small molecular NLRP3 inhibitor MCC950 pretreated RAW264.7 cells (LPS+ MCC950 group), and detected the gene expression of NLRP3, Caspase-1, interleukin 1β( IL-1β), GATA-4 and VEGFA.Results:There were multiple GATA transcription factor binding sites upstream of human and mouse VEGF promoters. Compared with the control group, mRNA expression of NLRP3, Caspase-1, IL-1β, GATA-4 and VEGFA in LPS group were increased (all P<0.05). Compared with LPS group, mRNA expression of NLRP3, Caspase-1, IL-1β, GATA-4 and VEGFA in LPS+ MCC950 group were significantly decreased (all P<0.05). Conclusions:NLRP3/GATA-4/VEGF signal pathway may play a significant role in the pathologic processes of nAMD.
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Purpose: Abnormal activation of NOD-like receptor protein 3 (NLRP3) inflammasome can lead to the occurrence and progression of acute pancreatitis. This study investigated the protective effect of MCC950 on pancreatitis mice. Methods: Eighteen mice were randomly divided into control group, severe acute pancreatitis (SAP) group and SAP+MCC950 group. Serum interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. Hematoxylin and eosin (HE) staining was used to evaluate the pathological damage. Western blotting was used to detect the expression of NLRP3 inflammasome and tight junction proteins in the small intestine and pancreas. Results: MCC950 could reduce the levels of IL-6 and IL-1ß in SAP mice. After treatment with MCC950, the expression levels of NLRP3 inflammasome in the pancreas of SAP mice were significantly reduced and the pathological damage to the pancreas and intestine was alleviated. Compared with the control group, the expression of tight junction protein (ZO-1,occludin and claudin-4) in the intestinal mucosa of SAP mice was decreased, and the expression of claudin-4 and occludin were upregulated after MCC950 treatment. Conclusions: MCC950 can inhibit NLRP3 inflammasome activation and significantly reduce the inflammatory response and delay the process of pancreatitis. It has therapeutic potential in the treatment of acute pancreatitis.
Subject(s)
Animals , Mice , Pancreatitis/drug therapy , Tight Junctions , Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Intestine, Small/pathologyABSTRACT
Purpose: Tanshinone IIA is a well-known lipophilic active constituent refined from traditional Chinese medicines, danshen. It has been previously demonstrated to possess various biological properties, including anti-inflammatory, antioxidant, promoting angiogenesis effect and so on. However, the mechanism of tanshinone IIA on myocardial ischemia-reperfusion injury (MI/RI) remains unclear. In this study, we investigated the effect of tanshinone IIA on MI/RI. Methods: MI/RI rat models were set up. Echocardiographic evaluation and hematoxylin and eosin staining were performed to analyze the cardiac function and morphology of MI/RI. Western blot was conducted for the detection of protein expression of pyrin domain containing 3 (NLRP3) and caspase-1 in heart tissues. Flow cytometry and real-time polymerase chain reaction were used for the detection of proinflammatory cytokines and Th17 cells differentiation. Results: We found that tanshinone IIA alleviated the myocardial damage of MI/RI, ameliorated the overall and local inflammatory reaction, and produced a cardioprotective effect by inhibiting of NLRP3 inflammasome activation and Th17/Treg cells differentiation. Conclusions: Our results highlighted the cardio-protective effect of tanshinone IIA on MI/RI and uncovered its underlying mechanism related to the NLRP3 inflammasome inhibition and the modulation of Th17/Treg cells differentiation.
Subject(s)
Animals , Rats , Myocardial Reperfusion Injury/drug therapy , Myocardial Ischemia/drug therapy , Salvia miltiorrhiza/chemistry , Th17 Cells , NLR Family, Pyrin Domain-Containing 3 Protein , Medicine, Chinese TraditionalABSTRACT
Objective:To evaluate the effect of vitamin K 2 on traumatic brain injury (TBI) and the relationship with nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes in rats. Methods:Thirty-six SPF healthy male Sprague-Dawley rats, weighing 280-300 g, were divided into 3 groups ( n=12 each) by a random number table method: sham operation group (group Sham), group TBI and TBI plus vitamin K 2 group (group TBI+ VK 2). The TBI model was developed using modified Feeney′s method.In TBI+ VK 2 group, vitamin K 2 400 mg/kg (dissolved in dimethyl sulfoxide) was intraperitoneally injected at 30 min after developing TBI model.The equal volume of dimethyl sulfoxide was intraperitoneally injected in group Sham and group TBI.The modified neurological severity score (mNSS) was measured and open field tests were performed at 24 h after development of TBI.The rats were sacrificed after the end of behavioral testing, and brains were obtained for measurement of brain water content (by wet-dry weight method), percentage of brain injury volume (by TTC assay), contents of interleukin-1β (IL-1β), IL-18 and caspase-1 in cortex on the injured side (by enzyme-linked immunosorbent assay) and expression of NLRP3, caspase-1 and IL-18 in cortex on the injured side (by Western blot). Results:Compared with group Sham, the mNSS score was significantly increased, the total distance travelled was reduced, the time spent in the central zone was shortened, the brain water content and percentage of brain injury volume were increased, the contents of IL-1β, IL-18 and caspase-1 in cortex on the injured side were increased, and the expression of NLRP3, caspase-1 and IL-18 was up-regulated in group TBI ( P<0.05 or 0.01). Compared with group TBI, the mNSS score was significantly decreased, the total distance travelled was increased, the time spent in the central zone was prolonged, the brain water content and percentage of brain injury volume were decreased, the contents of IL-1β, IL-18 and caspase-1 in cortex on the injured side were decreased, and the expression of NLRP3, caspase-1 and IL-18 was down-regulated in group TBI+ VK 2 ( P<0.05 or 0.01). Conclusions:Vitamin K 2 can reduce TBI, and the mechanism may be related to inhibition of the activation of NLRP3 inflammasomes in rats.
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Age-related macular degeneration (AMD), the leading cause of central vision loss among people aged 50 years and older, is one of the major eye diseases causing blindness in the world.Clinically, advanced AMD is divided into two types, non-exudative AMD with manifestation of geographic atrophy and exudative AMD with manifestation of choroidal neovascularization.The pathogenesis of AMD is complex, and the para-inflammation is recognized as an important risk factor.Nucleotide-binding oligomerization domain like receptors 3 (NLRP3) inflammasome is a cytoplasmic pattern recognition receptor and is expressed in several kings of cells, including retinal pigment epithelium (RPE) cells, microglial cells, Müller glia cells and retinal vascular endothelial cells.Recent studies have suggested that NLRP3 inflammasome plays an important role in the pathophysiology of both non-exudative and exudative AMD.The role of the NLRP3 inflammasome and its effector cytokines interleukin (IL)-1β and IL-18 in AMD were reviewed in this article to provide guidance on future prevention and therapy of AMD.
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Objective:To investigate the effect of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome on the proliferation and apoptosis of human retinal pigment epithelial cell line ARPE-19 exposed to high glucose and its mechanism.Methods:ARPE-19 cells cultured in vitro were divided into normal control group and high-glucose group, and were cultured in conventional medium and medium containing 30 mmol/L glucose for 48 hours, respectively.The content of reactive oxygen species (ROS) were detected by fluorescent probe, and the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were tested by biochemical assay.The cells of the two groups were cultured with 0, 2, 5, 10, 15 and 20 μmol/L NLRP3 inhibitor CY-09 for 48 hours, respectively.The proliferation rate of ARPE-19 cells under various concentrations of CY-09 treatment was detected by cell counting kit-8, and the appropriate concentration of CY-09 was determined.ARPE-19 cells were divided into normal control group, normal+ CY-09 group, high-glucose group and high glucose+ CY-09 group.The culture medium in the normal+ CY-09 group and high glucose+ CY-09 group was supplemented with 15 μmol/L CY-09.Flow cytometry was used to detect the apoptosis rate of each group, and Western blot was used to detect the relative expression levels of NLRP3, apoptosis-associated point protein (ASC), Caspase-1 precursor (pro-Caspase-1) and active fragments (cleaved-Caspase-1), B lymphocytoma-2 protein (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3 precursor (pro-Caspase-3) and active fragments (cleaved-Caspase-3). Results:The intensity of ROS fluorescence and MDA concentration were 120 020±3 245, (4.92±0.09) nmol/mg in the high-glucose group, which were both significantly higher than 35 426±811 and (1.78±0.03) nmol/mg in the normal control group, and the SOD activity was (35.65±1.22) μmol/(min·mg) in the high-glucose group, which was significantly lower than (74.96±1.41) μmol/(min·mg) in the normal control group, showing statistically significant differences between the two groups ( t=35.760, 46.960, 29.830; all at P<0.05). The proliferation rate of RPE cells in high-glucose group was significantly lower than that in normal control group, and the difference was statistically significant ( t=18.820, P<0.05). With the increase of CY-09 concentration, the proliferation rate of cells in the high-glucose group was gradually increased.The proliferation rates of cells treated with 10, 15 and 20 μmol/L CY-09 were all significantly higher than those treated with 0 μmol/L CY-09, showing statistically significant differences between them (all at P<0.05). The proliferation rates of cells treated with 15 μmol/L and 0 μmol/L CY-09 were not significantly different in the normal control group ( P>0.05). The apoptosis rate of cells in the high-glucose group was (21.68±0.41)%, which was significantly higher than (6.67±1.05)% in the normal control group and (13.96±0.07)% in the high-glucose+ CY-09 group, and the differences were statistically significant (both at P<0.05). The relative expression levels of NLRP3, ASC, cleaved-Caspase-1, cleaved-Caspase-3 and Bax proteins were significantly higher and the relative expression levels of Bcl-2 protein was significantly lower in the high-glucose group compared with the normal control group, and the differences were statistically significant (all at P<0.05). The relative expression levels of NLRP3, ASC, the active fragment of cleaved-Caspase-1, Bax and cleaved-Caspase-3 proteins were decreased and the relative expression levels of Bcl-2 protein were increased in the normal+ CY-09 group and high glucose+ CY-09 group compared with the normal control group and high glucose group, and the differences were statistically significant (all at P<0.05). Conclusions:NLRP3 inflammasome mediates the high glucose induced RPE cells apoptosis through ROS/NLRP3/Caspase-1 signaling pathway.
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Objective:To investigate whether hepatitis B virus X protein (HBx) mediates the podocyte injury through reactive oxygen species (ROS) /nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway.Methods:HBx-overexpressing lentivirus was transfected into renal podocytes of mouse to mimic the pathogenesis of hepatitis B virus-associated glomerulonephritis. Podocytes were divided into the following five groups: blank control group (no special treatment), negative control group (transfected with control lentivirus), HBx overexpression group (transfected with HBx overexpression lentivirus), HBx overexpression+NLRP3 siRNA group (transfected with HBx overexpression lentivirus and NLRP3 siRNA), and HBx overexpression+ROS inhibitor group (transfected with HBx overexpression lentivirus and adding ROS inhibitor). The morphological changes of podocytes were observed with electron microscope. The generation of ROS was detected by dichlorodihydrofluorescein diacetate assay (DCFH-DA). Hoechst 33342 staining was used to observe the morphological and quantitative changes of podocyte nuclei. Enzyme-linked immunosorbent assay was used to detect caspase-1 activity, and the levels of lactate dehydrogenase, interleukin (IL)-1β and IL-18. Quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression levels of mRNA and protein of pyroptosis-related protein, such as NLRP3, apoptosis-associated speck-like protein containing card (ASC), caspase-1, IL-1β and IL-18. TUNEL staining and flow cytometer were used to detect the number of pyroptosis cells. Immunofluorescence staining was used to detect the expression levels of desmin and nephrin.Results:After successful infection of podocytes with HBx-overexpressing lentivirus, pyroptosis-related morphological changes in the cells were observed under electron microscope. The level of ROS in the HBx overexpression group was significantly higher compared to the negative control group ( P<0.05). Hoechst 33342 staining revealed condensed nuclei in the HBx overexpression group. TUNEL staining and flow cytometer demonstrated that podocytes underwent increased pyroptosis in the HBx overexpression group. The mRNA and protein expression levels of pyroptosis-related proteins such as NLRP3, ASC, caspase-1, IL-1β and IL-18 were up-regulated upon HBx overexpression (all P<0.05). Caspase-1 enzyme activity, lactate dehydrogenase and desmin expression levels were enhanced after HBx overexpression (all P<0.05). However, NLRP3 knockdown or addition of ROS inhibitors attenuated the pyroptosis and increased expression levels of pyroptosis-related proteins caused by HBx overexpression (all P<0.05). Conclusion:ROS/NLRP3 pathway plays an important role in HBx-induced podocyte pyroptosis.
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Objective:To evaluate the relationship between nuclear receptor subfamily 1, group D, member 1 (Rev-erbα) and NOD-like receptor protein 3 (NLRP3) inflammasome during myocardial ischemia-reperfusion (I/R) in diabetic rats.Methods:SPF-grade healthy male Sprague-Dawley rats, weighing 210-240 g, in which 1% streptozotocin 60 mg/kg was intraperitoneally injected to develop the model of type 1 diabetes mellitus.Eighteen non-diabetic rats were divided into 2 groups by the random number table method: non-diabetic sham operation group (NS group, n=6) and non-diabetic myocardial I/R group (NIR group, n=12). Thirty diabetic rats were divided into 3 groups by the random number table method: diabetic sham operation group (DS group, n=6), diabetic myocardial I/R group (DIR group, n=12), and diabetic myocardial I/R + Rev-erbα inhibitor SR8278 group (DIR+ SR group, n=12). Myocardial I/R model was developed by ligation of left anterior descending coronary artery for 30 min followed by reperfusion for 120 min.In DIR+ SR group, SR8278 2 mg/kg was injected via the femoral vein at 1 h before ischemia.At the end of reperfusion, blood samples from the right carotid artery were collected for determination of serum creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) levels (by enzyme-linked immunosorbent assay). Then the rats were sacrificed, hearts were removed and myocardial tissues were obtained for determination of the percentage of myocardial infarct size (by TTC method) and expression of Rev-erbα, NLRP3 and IL-1β (by Western blot) and for microscopic examination of pathologic changes (by HE staining). Results:Compared with sham-operated rats, the serum concentrations of CK-MB, LDH and cTnI were significantly increased, the expression of Rev-erbα, NLRP3 and IL-1β in myocardial tissues was up-regulated ( P<0.05), and the pathological injury of myocardial tissues was obvious in myocardial I/R rats.Compared with NIR group, the percentage of myocardial infarct size and levels of serum CK-MB, LDH and cTnI were significantly increased, the expression of Rev-erb α, NLRP3 and IL-1β was up-regulated ( P<0.05), and the pathological injury of myocardial tissues was aggravated in DIR group.Compared with DIR group, the percentage of myocardial infarct size and serum CK-MB, LDH and cTnI levels were significantly decreased, the expression of Rev-erbα, NLRP3 and IL-1β was down-regulated ( P<0.05), and the pathological injury of myocardial tissues was reduced in DIR+ SR group. Conclusions:Rev-erbα can promote activation of NLRP3 inflammasome and is involved in the pathophysiological mechanism of myocardial I/R injury in diabetic rats.
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Objective:To evaluate the role of cathepsin B (CTSB) in mechanical ventilator-induced lung injury (VILI) in rats and the relationship with NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome.Methods:Thirty-six SPF-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-300 g, were divided into 3 groups ( n=12 each) by the random number table method: control group (group C), VILI group (group V) and VILI + CA074-me group (group Me). CA074-me 5 mg/kg was intraperitoneally injected in group Me, while the equal volume of normal saline was given instead in group C and group V. Group C kept spontaneous breathing for 4 h, and the animals were mechanically ventilated (tidal volume 20 ml/kg, respiratory rate 80 breaths/min, fraction of inspired oxygen 21%, PEEP 0 cmH 2O). Blood samples from femoral artery were collected for arterial blood gas analysis before tracheal intubation and after spontaneous breathing or ventilation, and PaO 2 was recorded.Rats were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected and lung tissues were collected for determination of the wet/dry lung weight ratio (W/D ratio), serum interleukin-1beta (IL-1β) and IL-18 concentrations in BALF (by enzyme-linked immunosorbent assay), expression of CTSB, NLRP3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) and caspase-1 mRNA in lung tissues (quantitative real-time polymerase chain reaction), and expression of CTSB, NLRP3, ASC and caspase-1 in lung tissues (by Western blot) and for microscopic examination of the pathological changes (using HE staining). Lung injury was assessed and scored. Results:Compared with group C, PaO 2 was significantly decreased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were increased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was up-regulated in group V and group Me ( P<0.01). Compared with group V, PaO 2 was significantly increased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were decreased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was down-regulated in group Me ( P<0.01). Conclusions:CTSB is involved in VILI in the rats, and the mechanism may be related to activation of NLRP3 inflammasomes.
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ABSTRACT Purpose: To delve into the influence of paeoniflorin (PA) on abating primary biliary cholangitis (PBC)-induced liver fibrosis and its causative role. Methods: Our team allocated the mice to control group, PA group, PBC group and PBC+PA group. We recorded the weight change of mice in each group. We used Masson staining for determining liver fibrosis, immunofluorescence staining for measuring tumor necrosis factor-α (TNF-α) expression, quantitative real-time polymerase chain reaction (qRT-PCR) for assaying related gene expression, as well as Western blot for testing related protein expression. Results: The weight of PBC model mice declined. Twenty-four weeks after modeling, the positive rate of anti-mitochondrial antibody-M2 (AMA-M2) in PBC mice reached 100%. Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyproline (HYP), laminin (LN), procollagen type III (PC III), and malondialdehyde (MDA) contents saliently waxed (p<0.01). Meanwhile, superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activity patently waned (p<0.01). Liver fibrosis levels were flagrantly higher (p<0.01), and TNF-α, NOD-like receptor protein 3 (NLRP3), caspase-1, interleukin-18 (IL-18), and interleukin-1β (IL-1β) protein or gene expression were manifestly up-regulated (p<0.01). PA could restore the weight of PBC mice, strikingly restrain the positive expression of AMA-M2, and down-regulate serum ALP, ALT, AST, HYP, LN, PC III, MDA in PBC mice (p<0.01). PA could also significantly up-regulate SOD and GSH-px levels (p<0.01), down-regulate IL-1β, IL-18, caspase-1, NLRP3, and TNF-α protein or gene expression in PBC mice (p<0.01) and inhibit liver fibrosis levels (p<0.01). Conclusions: PA can reduce PBC-induced liver fibrosis in mice and may function by curbing the formation of NLRP3.
Subject(s)
Animals , Mice , Monoterpenes/pharmacology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Glucosides/pharmacology , Liver Cirrhosis/prevention & control , Liver Cirrhosis/drug therapy , Aspartate Aminotransferases , Liver/pathologyABSTRACT
Objective:To evaluate the role of Nod-like receptor family pyrin domain-containing 3 (NLRP3) in the dorsal root ganglion (DRG) in persistent postoperative pain in rats.Methods:Forty-eight male Sprague-Dawley rats, in which IT catheters were successfully implanted, weighing 200-250 g, aged 2-3 months, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (S group), persistent postoperative pain group (P group), persistent postoperative pain+ NLRP3-siRNA group (P+ siRNA group) and persistent postoperative pain+ NLRP3-scrRNA group (P+ scrRNA group). A persistent postoperative pain model was established by skin/muscle incision and retraction (SMIR) in anesthetized animals.Normal saline 10 μl were intrathecally injected in S group and P group, NLRP3-siRNA 10 μl and NLRP3-scrRNA 10 μl were intrathecally injected in CP+ siRNA group and CP+ scrRNA group, respectively, at 3 days before operation.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation (T 0) and 1, 5, 10, 15 and 20 days after operation (T 1-5). Six rats in each group were randomly selected and sacrificed at T 3, and the L 4-5 DRGs on the operated side were harvested for determination of the expression of NLRP3 and caspase-1 (by Western blot), NLRP3 mRNA expression (by real-time polymerase chain reaction) and content of interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay). Results:Compared with group S, the MWT was significantly decreased at T 2-5, the expression of NLRP3 protein and mRNA in DRGs was up-regulated, and the content of IL-1β was increased in group P ( P<0.05). Compared with group P, the MWT was significantly increased at T 2-5, the expression of NLRP3 protein and mRNA and caspase-1 was down-regulated, and the content IL-1β was decreased in group P+ siRNA ( P<0.05), and no significant change was found in the parameters mentioned above in group P+ scrRNA ( P>0.05). Conclusion:NLRP3 in DRGs is involved in the development of persistent postoperative pain, and the mechanism may be related to the development of NLPR3 inflammasomes which further induces peripheral neuroinflammatory response in rats.