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Objective:To evaluate the role of NOD-like receptor 3 (NLRP3) inflammasome activation-mediated macrophage polarization in myocardial injury after ischemic stroke in diabetic mice.Methods:Wild-type C57BL/6J mice and NLRP3 -/- mice, aged 4-6 weeks, were fed a high fat diet combined with streptozotocin administration to develop the diabetic model. Twenty-four diabetic wild type C57BL/6J mice and 23 diabetic NLRP3 -/- mice were divided into wild type sham operation group (WT D-SHAM group, n=9) , wild type ischemic stroke group (WT D-MCAO group, n=15) , NLRP3 -/- sham operation group (NLRP3 -/-D-SHAM group, n=9) and NLRP3 -/- ischemic stroke group (NLRP3 -/-D-MCAO group, n=14). The ischemic stroke model was developed by middle cerebral artery occlusion in the animals anesthetized with isoflurane. Echocardiography and electrocardiography were carried out at 3, 7, 14 and 28 days after developing the model. Mice were sacrificed under deep anesthesia, and myocardial tissues were taken at 28 days after surgery for determination of the expression of macrophage marker F4/80 and M2 type macrophage marker CD206 mRNA (by real-time fluorescence quantitative polymerase chain reaction). Results:Compared with WT D-SHAM group, the cardiac output, mass of left ventricle and corrected mass of left ventricle were significantly decreased at 28 days after surgery, and QT interval and QTc interval were prolonged at 14 and 28 days after developing the model in WT D-MCAO group ( P<0.05). Compared with NLRP3 -/-D-SHAM group, the cardiac output, mass of left ventricle and corrected mass of left ventricle were significantly decreased, and QT interval and QTc interval were prolonged at 3 days after surgery in NLRP3 -/-D-MCAO group ( P<0.05). There was no significant difference in CD206 and F4/80 mRNA expression between WT D-SHAM group and WT D-MCAO group and between NLRP3 -/-D-SHAM group and NLRP3 -/-D-MCAO group ( P>0.05). Compared with WT D-MCAO group, the QT interval and QTC interval were significantly shortened at 14 and 28 days after developing the model, and the expression of F4/80 mRNA was down-regulated and the expression of CD206 mRNA was up-regulated at 28 days after developing the model in NLRP3 -/-D-MCAO group ( P<0.05). Conclusions:NLRP3 inflammasome activation-mediated polarization of macrophages to M2 phenotype is involved in myocardial injury after ischemic stroke in diabetic mice.
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ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.
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Objective:To explore any effect of repeated transcranial magnetic stimulation (rTMS) on the recovery of neurological functioning and the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and inflammatory factors after ischemic stroke.Methods:Sixty-four C57BL/6J mice were randomly divided into a normal control group, a model group, a sham stimulation group and an observation group, each of 16. All mice except those of the normal control group received middle cerebral artery occlusion using the suture method to model an ischemic stroke. After the modeling the observation group was given 1Hz rTMS daily for 7 consecutive days, while the sham stimulation group was given sham rTMS. After the intervention, Zea-Longa scores were used for all of the groups, and the size of the cerebral infarct was measured using triphenyltetrazolium chloride staining. The expression of NLRP3 around the cerebral infarction was detected using immunofluorescence, while that in the brain tissue was measured using Western blotting. The expression of interleukin-1β and IL-18 in the brain tissue was detected using enzyme-linked immunosorbent assays.Results:Compared with the normal control group, a significant increase was observed in the other groups′ average neurological function impairment scores. Expression of NLRP3, IL-1β and IL-18 in the model and sham stimulation groups also increased, with large cerebral infarcts in the cortex and hippocampus. Compared with the sham stimulation and model groups, there was a significant decrease in the average neurological dysfunction scores, the area of cerebral infarction in the cortex and hippocampus, as well as the expression of NLRP3, IL-1β and IL-18 in the observation group.Conclusions:Low-frequency rTMS can promote the recovery of damaged nerve function after an ischemic stroke, at least in mice. It can reduce the size of cerebral infarction, and inhibit neuronal pyroptosis, which is closely related to the down-regulation of NLRP3, IL-1β and IL-18 expression.
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Objective: To investigate the changes of expressions of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in the peripheral blood mononuclear cells and downstream factors interleukin- 1(3 (IL-lβ) and interleukin-18 (IL-18) in serum in the children with asthma, and to explore their significances on assessing the condition of the children. Methods: A total of 176 cases of children with asthma were divided into acute exacerbation group ( n=91), chronic persistent group (n=49) and clinical remission group (n=36) according to the clinical manifestation. During the same period, 60 healthy children were selected from the outpatient physical examination center as control group. The pulmonary function of children was checked with lung function instrument. The expression levels of NLRP3, apoptosis-associated speckflike protein containing a CARD (ASC) and cysteinyl aspartate-specific proteinase-1 (Caspase-1) mRNA in peripheral blood mononuclear cells of the subjects in various groups were detected by using real-time quantitative PCR. The serum levels of IL-1 β and IL-18 of the subjects in various groups were detected by using enzyme-linked immunosorbent assay (ELISA). Results: Compared with control group, the forced expiratory volume in I second percentage of predicted value (FEV1 %) and fixed ratio of forced expiratory volume in the first second/forced vital capacity ( FEV1/FVC) of the children in acute exacerbation, chronic persistent and clinical remission groups were decreased ( P
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Objective:To investigate the relationship between NOD-like receptor pyrin domain containing 3(NLRP3)/cysteine aspartate-specific protease(Caspase)-1 signaling pathway and esophageal inflammation by observing the effect of Xuanfu Daizhe Tang on the composition of inflammatory body and the expression of relevant inflammatory factors in rats with reflux esophagitis (RE), so as to explain the mechanism of Xuanfu Daizhe Tang in treating RE. Method:Sixty healthy male Wistar rats were randomly divided into four groups:the normal control group, the model control group, the Xuanfu Daizhe Tang group (9.89 g·kg-1) and the positive control group (omeprazole enteric-coated tablets+mosapride, 2.58 mg·kg-1), with 15 rats in each group. Except for the blank control group, the remaining rats were operated by " 4.2 mm pyloric clip+2/3 gastric fundus ligation" to establish models. Since the 8th day after the operation, the rats were given corresponding drugs twice a day for 14 days. The arterial blood and esophageal tissues were taken out at the 15th day after the intervention. The pathological morphology of esophagus was observed by naked eyes and under light microscopy. The secretion of cytokines Caspase-1 and interleukin(IL)-1β in serum was detected by enzyme linked immunosorbent assay(ELISA). The expressions of NLRP3, Caspase-1 and IL-1β in esophagus were detected by Western blot. Result:Compared with the normal group, the injury of esophageal mucosa in the model group was the most serious. Compared with the normal group, the levels of Caspase-1 and IL-1β in serum and the expression of NLRP3 protein in esophageal tissue of the model group were significantly increased (PPβ in serum of rats, and down-regulate the expressions of NLRP3, Caspase-1 and IL-1β protein in esophageal tissue (P0.05, PConclusion:Xuanfu Daizhe Tang can regulate the expressions of NLRP3 and Caspase-1, and reduce the content of IL-1β, suggesting that it may antagonize esophageal inflammatory response, reduce esophageal inflammatory injury and treat RE by inhibiting the activation of NLRP3/Caspase-1 signaling pathway.
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Objective:To investigate the changes of expressions of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in the peripheral blood mononuclear cells and downstream factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) in serum in the children with asthma, and to explore their significances on assessing the condition of the children.Methods:A total of 176cases of children with asthma were divided into acute exacerbation group (n=91) , chronic persistent group (n=49) and clinical remission group (n=36) according to the clinical manifestation.During the same period, 60healthy children were selected from the outpatient physical examination center as control group.The pulmonary function of children was checked with lung function instrument.The expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cysteinyl aspartate-specific proteinase-1 (Caspase-1) mRNA in peripheral blood mononuclear cells of the subjects in various groups were detected by using real-time quantitative PCR.The serum levels of IL-1βand IL-18of the subjects in various groups were detected by using enzyme-linked immunosorbent assay (ELISA) .Results:Compared with control group, the forced expiratory volume in 1second percentage of predicted value (FEV1％) and fixed ratio of forced expiratory volume in the first second/forced vital capacity (FEV1/FVC) of the children in acute exacerbation, chronic persistent and clinical remission groups were decreased (P＜0.05) ;acute exacerbation group＜chronic persistent group＜clinical remission group, and there were significant differences between various groups (P＜0.05) .The levels of NLRP3, ASC and Caspase-1mRNA in the peripheral blood mononuclear cells and serum levels of IL-1βand IL-18in the children with asthma were higher than those in control group (P＜0.01) .The expression levels of NLRP3, ASC and Caspase-1mRNA in the peripheral blood mononuclear cells of the children in acute exacerbation group were higher than those in chronic persistent and clinical remission groups (P＜0.05) , and the expression levels of NLRP3, ASC and Caspase-1mRNA in chronic persistent group were higher than those in clinical remission group (P＜0.05) .Pearson correlation analysis showed that the expression level of NLRP3mRNA in the peripheral blood mononuclear cells of asthmatic children was positively correlated with the expression levels of ASC, Caspase-1 mRNA and the serum levels of IL-1βand IL-18 (P＜0.05) , while it was negatively correlated with FEV1％and FEV1/FVC;the expression level of ASC mRNA was positively correlated with the expression level of Caspase-1mRNA and the serum levels of IL-1βand IL-18 (P＜0.05) , while it was negatively correlated with FEV1％and FEV1/FVC (P＜0.05) ;the expression level of Caspase-1 mRNA was positively correlated with the expression level of Caspase-1mRNA and the serum levels of IL-1βand IL-18 (P＜0.05) , while it was negatively correlated with FEV1％and FEV1/FVC (P＜0.05) ;the serum level of IL-1βwas negatively correlated with FEV1％and FEV1/FVC (P＜0.05) , and the serum level of IL-18was negatively correlated with FEV1％and FEV1/FVC (P＜0.05) .Conclusion:The expression levels of NLRP3inflammasome and the downstream factor IL-1βand IL-18in peripheral blood of the children with asthma are increased, and they are related to the clinical stage of the children with asthma.NLRP3inflammasome pathway might promote the pathogenesis of asthma in the children.
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Detection of pathogen by pattern recognition receptors leads to activation of inflammasome which plays a crucial role in immune system. The inflammasome regulates the release of cytokines, such as interleukin (IL)-1beta, IL-18 and high-mobility group box 1 (HMGB1). Double-stranded RNA-dependent protein kinase (PKR) is a critical component of an inflammatory complex. Recently, the critical role of PKR was reported in regulation of multiple inflammasomes.