Diabetic cardiomyopathy:Importance of direct evidence to support the roles of NOD-like receptor protein 3 inflammasome and pyroptosis

Recently,the roles of pyroptosis,a form of cell death induced by activated NODlike receptor protein 3(NLRP3)inflammasome,in the pathogenesis of diabetic cardiomyopathy(DCM)have been extensively investigated.However,most studies have focused mainly on whether diabetes increases the NLRP3 inflammasome and associated pyroptosis in the heart of type 1 or type 2 diabetic rodent models,and whether various medications and natural products prevent the development of DCM,associated with decreased levels of cardiac NLRP3 inflammasome and pyroptosis.The direct link of NLRP3 inflammasome and associated pyroptosis to the pathogenesis of DCM remains unclear based on the limited evidence derived from the available studies,with the approaches of NLRP3 gene silencing or pharmaceutical application of NLRP3 specific inhibitors.We thus emphasize the requirement for more systematic studies that are designed to provide direct evidence to support the link,given that several studies have provided both direct and indirect evidence under specific conditions.This editorial emphasizes that the current investigation should be circumspect in its conclusion,i.e.,not overemphasizing its role in the pathogenesis of DCM with the fact of only significantly increased expression or activation of NLRP3 inflammasome and pyroptosis in the heart of diabetic rodent models.Only clear-cut evidence-based causative roles of NLRP3 inflammasome and pyroptosis in the pathogenesis of DCM can help to develop effective and safe medications for the clinical management of DCM,targeting these biomarkers.

Dietary saturated fatty acid and polyunsaturated fatty acid oppositely affect hepatic NOD-like receptor protein 3 inflammasome through regulating nuclear factor-kappa B activation

AIM: To investigate the effect of different dietary fatty acids on hepatic inflammasome activation.METHODS: Wild-type C57BL/6 mice were fed either a high-fat diet or polyunsaturated fatty acid (PUFA)-enriched diet. Primary hepatocytes were treated with either saturated fatty acids (SFAs) or PUFAs as well as combined with lipopolysaccharide (LPS). The expression of NOD-like receptor protein 3 (NLRP3) inflammasome, peroxisome proliferator-activated receptor-γ and nuclear factor-kappa B (NF-κB) was determined by real-time PCR and Western blot. The activity of Caspase-1 and interleukine-1β production were measured.RESULTS: High-fat diet-induced hepatic steatosis was sufficient to induce and activate hepatic NLRP3 inflammasome. SFA palmitic acid (PA) directly activated NLRP3 inflammasome and increased sensitization to LPS-induced inflammasome activation in hepatocytes. In contrast, PUFA docosahexaenoic acid (DHA) had the potential to inhibit NLRP3 inflammasome expression in hepatocytes and partly abolished LPS-induced NLRP3 inflammasome activation. Furthermore, a high-fat diet increased but PUFA-enriched diet decreased sensitization to LPS-induced hepatic NLRP3 inflammasome activation in vivo. Moreover, PA increased but DHA decreased phosphorylated NF-κB p65 protein expression in hepatocytes.CONCLUSION: Hepatic NLRP3 inflammasome activation played an important role in the development of non-alcoholic fatty liver disease. Dietary SFAs and PUFAs oppositely regulated the activity of NLRP3 inflammasome through direct activation or inhibition of NF-κB.

Nod-like receptors in the development of intestinal inflammation and cancer

Introduction Inflammatory bowel diseases(IBD),such as Crohn’s disease(CD)and ulcerative colitis(UC),are a group of chronic inflammatory disorders of the gastrointestinal tract[1-2].The symptoms of IBD include abdominal pain,diarrhea,and bloody stool.IBD affects a patient’s quality of life severely,due in part to its frequent recurrence.Colorectal cancer(CRC)is a malignancy in the colon or rectum with symptoms including bloody stool,changes in

Yemazhui(Herba Eupatorii Lindleyani)ameliorates lipopolysaccharide-induced acute lung injury via modulation of the toll-like receptor 4/nuclear factor kappa-B/nod-like receptor family pyrin domain-containing 3 protein signaling pathway and intestinal flor

OBJECTIVE:To investigate the impact of Yemazhui(Herba Eupatorii Lindleyani,HEL)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and explore its underlying mechanism in vivo.METHODS:The chemical constituents of HEL were analyzed by ultra-high performance liquid chromatographyquadrupole time-of-flight mass spectrometry method.Then,HEL was found to suppress LPS-induced ALI in vivo.Six-week-old male Sprague-Dawley rats were randomly divided into 6 groups:control,LPS,Dexamethasone(Dex),HEL low dose 6 g/kg(HEL-L),HEL medium dose 18 g/kg(HEL-M)and HEL high dose 54 g/kg(HEL-H)groups.The model rats were intratracheally injected with 3 mg/kg LPS to establish an ALI model.Leukocyte counts,lung wet/dry weight ratio,as well as myeloperoxidase(MPO)activity were determined followed by the detection with hematoxylin and eosin staining,enzyme linked immunosorbent assay,quantitative real time polymerase chain reaction,western blotting,immunohistochemistry,and immunofluorescence.Besides,to explore the effect of HEL on ALI-mediated intestinal flora,we performed 16s rRNA sequencing analysis of intestinal contents.RESULTS:HEL attenuated LPS-induced inflammation in lung tissue and intestinal flora disturbance.Mechanism study indicated that HEL suppressed the lung coefficient and wet/dry weight ratio of LPS-induced ALI in rats,inhibited leukocytes exudation and MPO activity,and improved the pathological injury of lung tissue.In addition,HEL reduced the expression of tumor necrosis factoralpha,interleukin-1beta(IL-1β)and interleukin-6(IL-6)in bronchoalveolar lavage fluid and serum,and inhibited nuclear displacement of nuclear factor kappa-B p65(NF-κBp65).And 18 g/kg HEL also reduced the expression levels of toll-like receptor 4(TLR4),myeloid differentiation factor 88,NF-κBp65,phosphorylated inhibitor kappa B alpha(phospho-IκBα),nod-like receptor family pyrin domain-containing 3 protein(NLRP3),IL-1β,and interleukin-18(IL-18)in lung tissue,and regulated intestinal flora disturbance.CONCLUSIONS:In summary,our findings revealed that HEL has a protective effect on LPS-induced ALI in rats,and its mechanism may be related to inhibiting TLR4/NF-κB/NLRP3 signaling pathway and improving intestinal flora disturbance.

Compatibility with Fructus Ligustri Lucidi Effectively Mitigates Idiosyncratic Liver Injury of Epimedii Folium by Modulating NOD-like Receptor Family Pyrin Domain Containing 3 Inflammasome Activation

Background: Idiosyncratic drug-induced liver injury(IDILI) is a serious side effect of drugs, Epimedii Folium(EF) is unequivocally implicated in idiosyncratic liver injury onset, potentially due to its ability to perturb the NOD-like receptor family pyrin domain containing 3(NLRP3) inflammasome. Fructus Ligustri Lucidi(FLL), a frequently used medicinal combination with EF, has not yet been investigated for its ability to ameliorate EF-associated hepatotoxicity. Aims and Objectives: Study on the mechanism of compatibility of FLL to alleviate liver injury caused by EF. Materials and Methods: Western blot was used to determine the expression of related proteins, ELISA was used to detect the secretion of related inflammatory factors IL-1β, IL-18, IL-6 and TNF-α, liver injury indexes were detected and liver pathological tissue staining was used to evaluate the liver injury. Results: Our results demonstrated that EF exerted a particular augmenting effect on the stimulation of the NLRP3 inflammasome mediated by nigericin or ATP, whereas FLL suppressed the NLRP3 inflammasome stimulation. Furthermore, an equal EF to FLL ratio significantly reduced the stimulatory effects of EF. Moreover, EF has the potential to induce hepatic injury and augment pro-inflammatory cytokine synthesis in rats subjected to LPS. However, when combined with FLL, the detrimental effects of EF were mitigated. Conclusions: FLL possesses the capacity to attenuate EF-associated hepatotoxicity by suppressing EF-triggered NLRP3 inflammasome activation. Thus, FLL holds promise for improving the clinical safety profile of EF, shedding light on the potential of compatibility and detoxification theories in traditional Chinese medicine.

Increased Expression of the NOD-like Receptor Family, Pyrin Domain Containing 3 Inflammasome in Dermatomyositis and Polymyositis is a Potential Contributor to Their Pathogenesis

Background： Dermatomyositis （DM） and polymyositis （PM） are common inflammatory myopathies whose immunopathogenic mechanisms remain poorly understood. The NOD-like receptor family, pyrin domain containing 3 （NLRP3） inflammasome is a type of cytoplasmic multiprotein inflammasome and is responsible for the activation of inflammatory reactivations. Responding to a wide range of exogenous and endogenous microbial or sterile stimuli, NLRP3 inflammasomes can cleave pro-caspase- 1 into active caspase- 1, which processes the pro-infammatory cytokines pro-interleukin （IL）-1 β and pro-IL-18 into active and secreted IL-1β and I L-18. The NLRP3 inflammasome is implicated in infectious and sterile inflammatory diseases. However, it remains unclear whether it is involved in the pathogenesis of DM/PM, which we aim to address in our research. Methods： In this study, 22 DM/PM patients and 24 controls were recruited. The protein and RNA expression of IL-113, IL-18, NLRP3, and caspase-1 in serum and muscle samples were tested and compared between the two groups. Results： The serum IL-1 β and IL-18 levels were significantly higher in DM/PM patients than those in the controls by enzyme linked immunosorbent assay （EL1SA, DM vs. control, 25.02 ± 8.29 ng/ml vs. 16.49 ± 3.30 ng/ml, P 〈 0.001 ; PM vs. control, 26.49±7.79 ng/ml vs. 16.49 ± 3.30 ng/ml, P 〈 0.001）. Moreover, the real-time quantitative reverse transcription-polymerase chain reaction （qRT-PCR） showed that DM/PM patients exhibited higher RNA expression of IL-lβ, IL-18, and NLRP3 in the muscle （for IL-1 β, DM vs. control, P 0.0012, PM vs. control, P = 0.0021 ; for IL- 18, DM vs. control, P = 0.0045, PM vs. control, P 0.0031 ; for NLRP3, DM vs. control, P = 0.0017, PM vs. control, P 0.0006）. Moreover, the protein expression of NLRP3 and caspase- 1 in muscle samples of DM/PM patients were also significantly elevated compared to that in the muscles of the controls. Conclusions： Our findings demonstrate that the NLRP3 inflammasome is implicated in the pathogenesis of DM/PM. High NLRP3 expression led to elevated levels of IL-l13 and IL-18 and could be one of the factors promoting disease progress.

A Novel Mutation in the Pyrin Domain of the NOD-like Receptor Family Pyrin Domain Containing Protein 3 in Muckle-Wells Syndrome

Background： Cryopyrin-associated periodic syndrome （CAPS） is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin （IL）-1β-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome （MWS）, and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 （NLRP3）, the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 ofNLRP3 gene. Here, we reported a novel mutation occurred in exon 1 ofNLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism. Methods： Genetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic lever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient＇s parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein （CRP）, erythrocyte sedimentation rate （ESR）, serum levels oflL-1β, immunoglobulin E （lgE）, antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components ofNLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance （SPR） assay. Results： X-ray examination showed no abnormality in the patient＇s knees. Laboratory tests indicated an elevation of CRP （233.24 nag/L） and ESR （67 mm/h） when the patient had fever. Serum IL-1β increased to 24.37 pg/ml, and serum lgE was higher than 2500.00 IU/ml. Other blood tests were normal. Bone marrow aspiration smear was normal. A novel point mutation c.92A〉T in exon 1 of NLRP3 gene was identified, which caused a p.D31V mutation in pyrin domain （PYD） of NLRP3. SPR assay showed that this point mutation may strengthen the interaction between the PYD of NLRP3 and the PYD of the apoptosis-associated speck-like protein. The mutation c.92A〉T in exon 1 of the NLRP3 gene was not lbund in the patient＇s parents and 50 healthy individuals. Conclusions： The rnutation c.92A〉T in exon 1 of the NLRP3 gene is a novel mutation associated with MWS. The p.D31V mutation might promote the activation ofNLRP3 inflammasome and induce MWS in this patient.

Effect of Curcumol on NOD-Like Receptor Thermoprotein Domain 3 Inflammasomes in Liver Fibrosis of Mice

Objective:To investigate the effect of curcumol on NOD-like receptor thermoprotein domain 3(NLRP3)inflammasomes,and analyze the mechanism underlying curcumol against liver fibrosis.Methods:Thirty Kunming mice were divided into a control group,a model group and a curcumol group according to a random number table,10 mice in each group.Mice were intraperitoneally injected with 40% carbon tetrachloride(CCl4:peanut oil,2:3 preparation)at 5 m L/kg for 6 weeks,twice a week,for developing a liver fibrosis model.The mice in the control group were given the same amount of peanut oil,twice a week for 6 weeks.The mice in the curcumol group were given curcumol(30 m L/kg)intragastrically,and the mice in the model and control groups were given the same amount of normal saline,once a day for 6 weeks.Changes in liver structure were observed by hematoxylin and eosin(HE)and Masson staining.Liver function,liver fiber indices,and the expression of interleukin(IL)-10 and tumor necrosis factor-α(TNF-α)levels were determined by automatic biochemical analyzer and enzyme linked immunosorbent assay kit.Immunoblotting and reverse transcription-quantitative PCR(RT-qPCR)were performed to detect the expression of NLRP3 inflammasome-related molecules,TGF-β and collagen.Results:HE and Masson staining results showed that the hepatocytes of the model group were arranged irregularly with pseudo-lobular structure and a large amount of collagen deposition.The mice in the curcumol group had a significant decrease in liver function and liver fibers indices compared with the model group(P<0.05);RT-qPCR and Western blot results reveal that,in the curcumol group,the mRNA and protein expression levels of NLRP3,IL-1β,Caspase 1 and gasdermin D decreased significantly compared with the model group(P<0.05);immunohistochemical results showed that in the curcumol group,the protein expression levels of NLRP3 and IL-1β decreased significantly compared with the model group(P<0.05).Conclusion:A potential anti-liver fibrosis mechanism of curcumol may be associated with the inhibition of NLRP3 inflammasomes and decreasing the downstream inflammatory response.

基于NOD样受体3炎性小体通路对利拉鲁肽在氧化低密度脂蛋白诱导内皮细胞损伤的作用机制研究

背景动脉粥样硬化是世界范围内引起心脑血管疾病最主要的原因,炎症是目前研究热点,其中NOD样受体3(NLRP3)是研究最为深入的炎症小体。胰高糖素样肽1(GLP-1)受体激动剂有抗动脉粥样硬化作用,具体机制尚不明确。目的研究利拉鲁肽通过拮抗氧化低密度脂蛋白(ox-LDL)诱导的内皮细胞损伤的作用机制。方法2022-03-25—05-19培养人脐静脉内皮细胞(HUVEC),取HUVEC加空白血清作为对照组,100μg/mL的ox-LDL干预HUVEC 48 h作为模型组,100μg/mL的ox-LDL干预HUVEC 24 h后分别加入100、200、400 nmol/L利拉鲁肽处理24 h作为利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组。CCK-8法计算细胞增殖率。通过扫描电镜观察焦亡细胞形态。检测乳酸脱氢酶(LDH)活力。酶联免疫吸附试验(ELISA)检测白介素(IL)-1β、IL-18表达水平。蛋白质免疫印迹试验(Western blot)检测NLRP3、接头蛋白凋亡相关斑点样蛋白(ASC)、天冬氨酸蛋白水解酶1(Caspase-1)、焦亡执行蛋白(GSDMD)、N端结构域的焦亡执行蛋白(N-GSDMD)表达水平。结果模型组、利拉鲁肽低浓度组和利拉鲁肽中浓度组细胞增殖率低于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞增殖率高于模型组(P<0.05)。细胞扫描电镜结果示模型组细胞焦亡明显,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞焦亡情况明显改善。模型组、利拉鲁肽低浓度组LDH活力高于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组低于模型组(P<0.05)。模型组、利拉鲁肽低浓度组IL-1β表达水平高于对照组,利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-1β表达水平低于模型组(P<0.05);模型组IL-18表达水平高于对照组,利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-18表达水平低于模型组(P<0.05)。模型组NLRP3、ASC、Caspase-1、GSDMD、N-GSDMD表达水平高于对照组,利拉鲁肽低浓度组ASC、Caspase-1表达水平高于对照组,利拉鲁肽中浓度组NLRP3、ASC表达水平低于模型组,利拉鲁肽高浓度组NLRP3、ASC、Caspase-1表达水平低于模型组(P<0.05)。结论利拉鲁肽显著抑制ox-LDL诱导的内皮细胞NLRP3炎性小体活化,并且能够抑制内皮细胞的焦亡,具有抗动脉粥样硬化作用。

庞氏安胎止血汤通过调控NLRP3炎症小体改善热证自然流产的作用机制

目的研究庞氏安胎止血汤通过调控NOD样受体蛋白3(NLRP3)炎症小体改善热证自然流产的作用机制。方法采用分子对接技术预测庞氏安胎止血汤中13种主要活性成分与NLRP3、凋亡相关斑点样蛋白质(ASC)、胱天蛋白酶1前体(pro-caspase-1)的结合活性。将60只孕1 d大鼠随机分为正常组、模型组、地屈孕酮组(0.002 g/kg)和庞氏安胎止血汤低、中、高剂量组(11.025、22.05、44.10 g/kg),每组10只。各组大鼠灌胃蒸馏水/相应药液,每天1次,连续12 d。除正常组外,其余各组大鼠采用温阳中药和米非司酮诱导热证自然流产模型。末次给药24 h后,检测大鼠血清中三碘甲状腺原氨酸(T3)、甲状腺素(T4)、白细胞介素2(IL-2)、IL-4、IL-6、IL-10、γ干扰素(IFN-γ)水平;计算流产率及子宫系数;观察大鼠妊娠子宫病理形态;检测妊娠子宫中NLRP3、ASC、caspase-1蛋白表达水平。结果分子对接结果显示,庞氏安胎止血汤13种主要活性成分与NLRP3、ASC、pro-caspase-1蛋白的结合能均小于-5 kJ/mol。动物实验结果显示,与正常组比较,模型组大鼠子宫系数和血清中IL-4、IL-6、IL-10水平均显著降低(P<0.05),流产率和血清中T3、T4、IL-2、IFN-γ水平以及妊娠子宫中NLRP3、ASC、caspase-1蛋白表达水平均显著升高(P<0.05),妊娠子宫内膜存在流产病变;与模型组比较,庞氏安胎止血汤各剂量组大鼠上述大部分定量指标均显著逆转(P<0.05),妊娠子宫内膜流产病变均有不同程度改善。结论庞氏安胎止血汤可能通过调节NLRP3炎症小体形成,下调IFN-γ、IL-2等促炎因子,上调IL-4、IL-6、IL-10等抑炎因子,从而影响母胎之间的免疫平衡,进而发挥改善热证自然流产的作用。

Tranylcypromine upregulates Sestrin 2 expression to ameliorate NLRP3-related noise-induced hearing loss

Noise-induced hearing loss is the primary non-genetic factor contributing to auditory dysfunction.However,there are currently no effective pharmacological interventions for patients with noise-induced hearing loss.Here,we present evidence suggesting that the lysine-specific demethylase 1 inhibitor–tranylcypromine is an otoprotective agent that could be used to treat noise-induced hearing loss,and elucidate its underlying regulatory mechanisms.We established a mouse model of permanent threshold shift hearing loss by exposing the mice to white broadband noise at a sound pressure level of 120 d B for 4 hours.We found that tranylcypromine treatment led to the upregulation of Sestrin2(SESN2)and activation of the autophagy markers light chain 3B and lysosome-associated membrane glycoprotein 1 in the cochleae of mice treated with tranylcypromine.The noise exposure group treated with tranylcypromine showed significantly lower average auditory brainstem response hearing thresholds at click,4,8,and 16 k Hz frequencies compared with the noise exposure group treated with saline.These findings indicate that tranylcypromine treatment resulted in increased SESN2,light chain 3B,and lysosome-associated membrane glycoprotein 1 expression after noise exposure,leading to a reduction in levels of 4-hydroxynonenal and cleaved caspase-3,thereby reducing noise-induced hair cell loss.Additionally,immunoblot analysis demonstrated that treatment with tranylcypromine upregulated SESN2 expression via the autophagy pathway.Tranylcypromine treatment also reduced the production of NOD-like receptor family pyrin domaincontaining 3(NLRP3)production.In conclusion,our results showed that tranylcypromine treatment ameliorated cochlear inflammation by promoting the expression of SESN2,which induced autophagy,thereby restricting NLRP3-related inflammasome signaling,alleviating cochlear hair cell loss,and protecting hearing function.These findings suggest that inhibiting lysine-specific demethylase 1 is a potential therapeutic strategy for preventing hair cell loss and noise-induced hearing loss.

Transcriptome and single-cell profiling of the mechanism of diabetic kidney disease

BACKGROUND The NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome may play an important role in diabetic kidney disease(DKD).However,the exact link remains unclear.AIM To investigate the role of the NLRP3 inflammasome in DKD.METHODS Using datasets from the Gene Expression Omnibus database,30 NLRP3 inflammasome-related genes were identified.Differentially expressed genes were selected using differential expression analysis,whereas intersecting genes were selected based on overlapping differentially expressed genes and NLRP3 inflammasome-related genes.Subsequently,three machine learning algorithms were used to screen genes,and biomarkers were identified by overlapping the genes from the three algorithms.Potential biomarkers were validated by western blotting in a db/db mouse model of diabetes.RESULTS Two biomarkers,sirtuin 2(SIRT2)and caspase 1(CASP1),involved in the Leishmania infection pathway were identified.Both biomarkers were expressed in endothelial cells.Pseudo-temporal analysis based on endothelial cells showed that DKD mostly occurs during the mid-differentiation stage.Western blotting results showed that CASP1 expression was higher in the DKD group than in the control group(P<0.05),and SIRT2 content decreased(P<0.05).CONCLUSION SIRT2 and CASP1 provide a potential theoretical basis for DKD treatment.

P2X7R过表达的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达观察

目的观察嘌呤能受体P2X配体门控离子通道7的配体(P2X7R)过表达白血病细胞诱导分化的巨噬细胞单钠尿酸盐(MSU)晶体诱导痛风炎症反应过程中NOD样受体家族3(NLRP3)蛋白、IL-1β、TNF-α表达情况。方法取人单核细胞白血病细胞系THP-1,并随机分为过表达组、空白组、模型组、对照组;过表达组和空白组分别转染P2X7R过表达质粒、空白载体质粒,转染5 d,将过表达组、空白组、模型组THP-1细胞用100 ng/mL的PMA刺激3 h后分化为巨噬细胞,另将MSU晶体用氢氧化钠溶解配制成浓度为100μg/mL的MSU乳糜状悬液加入培养液中孵育6 h;对照组正常培养。分别采用RT-PCR法和Western blot法测算巨噬细胞P2X7R mRNA、蛋白,ELISA法检测巨噬细胞上清液IL-1β、TNF-α,Western blot法测算巨噬细胞NOD样受体家族3(NLRP3)蛋白。结果与对照组比较,过表达组、空白组、模型组P2X7R mRNA和蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05);与模型组、空白组比较,过表达组P2X7R mRNA、蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05)。结论P2X7R过表达白血病细胞诱导分化的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达增加,IL-1β、TNF-α水平升高可能通过激活NLRP3蛋白来实现。

衔接蛋白失能同源物2通过抑制NOD样受体热蛋白结构域相关蛋白3对结核性胸膜炎大鼠炎症和氧化应激的影响

目的:探讨衔接蛋白失能同源物2(DAB2)抑制NOD样受体热蛋白结构域相关蛋白3(NLRP3)对结核性胸膜炎大鼠炎症和氧化应激的影响。方法:按照随机数字法将60只SPF级雄性SD大鼠分为四组,每组40只。除正常对照组外,结核性胸膜炎组、DAB2组和pcDNA-NLRP3组进行建模处理,以第2天是否抽出胸腔积液为模型建立成功。正常对照组不注射结核分枝杆菌H37RV悬液,DAB2组建模后第2天静脉注射AAV9-DAB2质粒,每天1次,连续注射7 d,DAB2+pcDNA-NLRP3组在注射AAV9-DAB2质粒500μg/L的同时注射pcDNA-NLRP380μl,正常对照组和结核性胸膜炎组的大鼠尾静脉注射0.9%氯化钠溶液。进行各组呼吸功能指标测定,收集胸腔积液,记录积液量,观察3、5、7 d的胸腔积液粘连性情况,HE染色观察胸膜组织病理学情况,Western blot检测胸膜组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-8(IL-8)、基质金属蛋白酶1(MMP-1)和MMP-9蛋白表达,荧光探针DCFH-DA分析检测活性氧(ROS)的水平。结果:与正常组相比,结核性胸膜炎组的大鼠的胸腔积液和胸膜厚度明显增加,用力肺活量(FVC)、最大呼气流量(PEF)、用力呼气容积(FEV)0.3和FEV0.3/FVC显著降低,TNF-α、IL-8、MMP-1和MMP-9蛋白表达显著升高,基质金属蛋白酶抑制剂(TIMP-1)蛋白表达显著降低,丙二醛(MDA)水平升高,超氧化物歧化酶(SOD)水平降低,ROS累积量明显升高(均P<0.001);与结核性胸膜炎组相比,DAB2组大鼠胸腔积液和胸膜厚度显著降低,FVC、PEF、FEV0.3和FEV0.3/FVC明显升高,DAB2组大鼠胸膜组织中的TNF-α、IL-8、MMP-1和MMP-9蛋白表达明显降低,TIMP-1水平明显升高,MDA水平降低,SOD水平升高,ROS累积量明显降低(均P<0.001);与DAB2组相比,DAB2+pcDNA-NLRP3组大鼠胸腔积液和胸膜厚度明显升高,FVC、PEF、FEV0.3和FEV0.3/FVC明显降低,DAB2+pcDNA-NLRP3组的TNF-α、IL-8、MMP-1和MMP-9蛋白表达明显升高,TIMP-1蛋白表达显著降低,MDA水平明显升高,SOD水平显著降低,ROS累积量显著升高(均P<0.001),各组大鼠在3、5、7 d的胸腔积液粘连性评分比较采用重复测量设计的方差分析,结果显示,不同时间点的胸腔积液粘连性评分比较差异具有统计学意义(均P<0.001);各组胸腔积液粘连性评分比较差异具有统计学意义(均P<0.001);各组胸腔积液粘连性评分变化趋势比较差异有统计学意义(均P<0.001);与模型组相比,DAB2组大鼠的胸膜内和肺间质内血管充血症状减轻,有少量的纤维组织增生,上皮样细胞团以及凝固型坏死也明显减少,淋巴细胞和中性粒细胞浸润也明显减少;与DAB2组相比,DAB2+pcDNA-NLRP3组大鼠的胸膜内和肺间质内血管充血症状加重,出现的纤维组织增生,上皮样细胞团以及凝固型坏死也明显增多,淋巴细胞和中性粒细胞浸润也明显增加。结论:DAB2通过抑制NLRP3的活性促进胸腔积液的吸收,降低胸膜厚度和粘连发生率,降低炎症反应和氧化应激水平,缓解结核性胸膜炎的进展。

维生素D_(3)对小鼠支气管哮喘气道炎症和氧化应激反应的作用及其分子机制

目的探究维生素D_(3)(VitD_(3))在小鼠支气管哮喘气道炎症和氧化应激反应中的作用和相关分子机制。方法将28只雌性C57BL/6小鼠随机分为对照组(Ctrl)和模型组。模型组小鼠采用卵清蛋白(OVA)致敏法建立哮喘模型后,将其分为哮喘(Asthma)组、VitD_(3)处理(Asthma+VitD_(3))组和叉头盒O1(FOXO1)抑制剂AS1842856处理(Asthma+AS)组。测定各组小鼠肺阻力(LR)变化。采用ELISA法检测肺泡灌洗液(BALF)中炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-18的含量。Western blot检测肺组织中FOXO1和NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸天冬氨酸酶-1(Caspase-1)和凋亡斑点蛋白(ASC)的表达水平。结果与Ctrl组相比,Asthma组小鼠的LR升高(P<0.01)。与Asthma组相比,Asthma+VitD_(3)组和Asthma+AS组小鼠的LR降低(P<0.05),Asthma+VitD_(3)组与Asthma+AS组小鼠的LR变化差异无统计学意义。与Ctrl组相比,Asthma组、Asthma+VitD_(3)组和Asthma+AS组小鼠BALF中TNF-α、IL-1β与IL-18含量均增加(P<0.01),肺组织中NLRP3、Caspase-1和ASC蛋白表达水平均升高(P<0.01);与Asthma组相比,Asthma+VitD_(3)组和Asthma+AS组小鼠BALF中上述炎症因子含量均减少(P<0.05),肺组织中NLRP3、FOXO1、Caspase-1和ASC蛋白表达均降低(P<0.05);与Asthma+VitD_(3)组相比,Asthma+AS组中除FOXO1蛋白表达水平升高外(P<0.05),上述其他检测指标差异均无统计学意义。结论VitD_(3)可减轻OVA诱导的小鼠哮喘症状,改善气道炎症程度和降低氧化应激水平,且其机制可能与FOXO1/NLRP3轴的下调有关。

3'-Deoxyadenosin alleviates methamphetamine-induced aberrant synaptic plasticity and seeking behavior by inhibiting the NLRP3 inflammasome

Methamphetamine addiction is a brain disorder characterized by persistent drug-seeking behavior, which has been linked with aberrant synaptic plasticity. An increasing body of evidence suggests that aberrant synaptic plasticity is associated with the activation of the NOD-like receptor family pyrin domain containing-3(NLRP3) inflammasome. 3′-Deoxyadenosin, an active component of the Chinese fungus Cordyceps militaris, has strong anti-inflammatory effects. However, whether 3′-deoxyadenosin attenuates methamphetamine-induced aberrant synaptic plasticity via an NLRP3-mediated inflammatory mechanism remains unclear. We first observed that 3′-deoxyadenosin attenuated conditioned place preference scores in methamphetamine-treated mice and decreased the expression of c-fos in hippocampal neurons. Furthermore, we found that 3′-deoxyadenosin reduced the aberrant potentiation of glutamatergic transmission and restored the methamphetamine-induced impairment of synaptic plasticity. We also found that 3′-deoxyadenosin decreased the expression of NLRP3 and neuronal injury. Importantly, a direct NLRP3 deficiency reduced methamphetamine-induced seeking behavior, attenuated the impaired synaptic plasticity, and prevented neuronal damage. Finally, NLRP3 activation reversed the effect of 3′-deoxyadenosin on behavior and synaptic plasticity, suggesting that the anti-neuroinflammatory mechanism of 3′-deoxyadenosin on aberrant synaptic plasticity reduces methamphetamine-induced seeking behavior. Taken together, 3′-deoxyadenosin alleviates methamphetamine-induced aberrant synaptic plasticity and seeking behavior by inhibiting the NLRP3 inflammasome.

电针对功能性消化不良大鼠十二指肠CRHR2、NLRP6表达的影响

目的观察电针对功能性消化不良(functional dyspepsia,FD)大鼠十二指肠促肾上腺皮质激素释放激素受体2(corticotropin-releasing hormone receptor 2,CRHR2)及NOD样受体家族pyrin结构域蛋白6(NOD-like receptor family pyrin domain containing 6,NLRP6)表达水平的影响。方法将40只雄性SD大鼠随机分为空白组、模型组和电针组,每组10只。模型组和电针组均选用多因素干预法复制FD大鼠模型,空白组进行常规饲养。模型复制结束后,电针组大鼠电针“印堂”“内关”“足三里”,每次30 min,每日1次,连续14 d。观察各组大鼠干预前后的一般状态及体质量变化;干预结束后,采用半固体糊灌胃法检测各组大鼠胃排空率及小肠推进率;苏木精—伊红染色法观察大鼠胃窦组织形态变化;蛋白免疫印迹法检测大鼠十二指肠CRHR2、NLRP6蛋白表达水平;阿利新蓝染色法观察大鼠十二指肠形态变化。结果与空白组比较,模型组大鼠精神欠佳,体质量、胃排空率及小肠推进率明显降低(P<0.05);与模型组比较,电针组大鼠活泼好动,体质量、胃排空率及小肠推进率明显增加(P<0.05)。模型组大鼠胃窦黏膜排列疏松,有轻度水肿,存在少量淋巴细胞,十二指肠绒毛上皮细胞间隙增宽,肠绒毛结构破碎,可见散在分布的上皮细胞;电针组大鼠胃窦组织结构完整,固有层排列紧密,无明显炎症反应及病理改变,十二指肠绒毛上皮细胞间隙清晰,排列紧密,组织结构完整。与空白组比较,模型组大鼠十二指肠CRHR2、NLRP6蛋白表达水平明显降低(P<0.05);与模型组比较,电针组CRHR2、NLRP6蛋白表达水平明显升高(P<0.05)。结论电针可改善FD大鼠消化不良症状,提高FD大鼠胃肠动力,降低FD大鼠十二指肠黏膜通透性,减轻大鼠胃肠炎症反应,其机制可能与提升十二指肠CRHR2、NLRP6蛋白表达水平有关。

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

颅脑损伤术后颅脑感染血清NLRP3、SAA及NFκB的临床意义

目的探讨血清寡聚化结构域样受体蛋白3(NLRP3)、淀粉样蛋白A(SAA)及核转录因子(NFκB)在颅脑损伤术后颅脑感染中的诊断价值。方法选择2020年6月至2022年6月江苏省如皋市人民医院接诊的70例颅脑损伤患者为研究对象,根据感染情况将感染者设为感染组(n=19),未感染者设为对照组(n=51),分析血清NLRP3、SAA及NFκB在颅脑损伤术后颅脑感染中的诊断价值。结果感染组患者血清NLRP3、SAA及NFκB水平显著高于对照组,差异有统计学意义(P<0.05);NLRP3、SAA及NFκB水平:轻度感染<中度感染<重度感染,差异有统计学意义(P<0.05);预后不良组患者血清NLRP3、SAA及NFκB水平显著高于预后良好组,差异有统计学意义(P<0.05);ROC结果显示,血清NLRP3预测颅脑损伤术后颅脑感染的AUC为0.634,灵敏度为63.43%,特异度为67.40%,截断值为114.02 pg/mL;血清SAA预测颅脑损伤术后颅脑感染的AUC为0.715,灵敏度73.50%,特异度为69.00%,截断值为30.99 mg/L;血清NFκB预测颅脑损伤术后颅脑感染的AUC为0.914,灵敏度为81.40%,特异度为70.00%,截断值为38.27μg/mL,联合检测较单独检测特异度、准确度更高(P<0.05)。结论血清NLRP3、SAA及NFκB在颅脑损伤术后颅脑感染患者中均异常高表达,且三指标联合检测颅脑损伤术后颅脑感染诊断效能更高,临床应用价值高。

黄芪甲苷经由miR-125a-5p/NLRP1轴减轻椎间盘突出髓核细胞损伤

目的在白介素-1β(IL-1β)诱导退变的人髓核细胞中,探究黄芪甲苷对miR-125a-5p及其靶基因介导的信号通路的作用,揭示黄芪甲苷(astragaloside IV,AS-IV)对髓核细胞增殖、凋亡以及炎症反应的影响。方法实时荧光定量PCR(RT-qPCR)检测miR-125a-5p和核苷酸寡聚化结构域(NOD)样受体蛋白1(nucleotide oligomerization domain(NOD)-like receptor protein 1,NLRP1)在椎间盘突出患者髓核组织中的表达,用IL-1β诱导髓核细胞退变,在20、50和80μg·mL^(-1)黄芪甲苷干预浓度下检测miR-125a-5p和NLRP1的表达,在IL-1β和80μg·mL^(-1)黄芪甲苷处理的髓核细胞中单独或共同转染miR-125a-5p模拟物和NLRP1过表达质粒,然后分别检测细胞增殖、凋亡和炎症因子分泌情况,蛋白质免疫印迹(Western blot)检测细胞中信号通路相关蛋白p56和p38的磷酸化水平。结果椎间盘突出(lumbar disc herniation,LDH)患者的髓核组织中miR-125a-5p表达下调,NLRP1表达上调。黄芪甲苷促进IL-1β处理的髓核细胞中miR-125a-5p表达,减少NLRP1表达以及p56和p38蛋白的磷酸化水平。黄芪甲苷促进髓核细胞增殖,减少凋亡和炎症反应。结论黄芪甲苷通过上调miR-125a-5p表达,抑制NLRP1的表达和NF-κB/MAPK信号通路,减少IL-1β诱导的髓核细胞损伤。