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

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.

清解化攻方调控NLRP3/TLR4/NF-κB信号通路对重症急性胰腺炎小鼠模型胰腺组织的保护作用

目的观察清解化攻方对重症急性胰腺炎(SAP)小鼠模型的治疗作用,探索清解化攻方抗炎症反应的作用机制。方法将36只C57BL/6J雄性小鼠随机分成空白组,模型组,清解化攻方低、中、高剂量组,西药组(乌司他丁),每组6只,除空白组小鼠,余各组小鼠采用逆行胰胆管注射5%牛黄胆酸钠建立SAP模型,清解化攻方低、中、高剂量组在造模后分别予以清解化攻方1、2、4 g/kg灌胃,西药组在造模后予以腹腔注射乌司他丁(5×10^(4) U/kg),共干预7 d。采用苏木素-伊红染色观察胰腺组织病理改变;酶联免疫吸附测定法(ELISA)检测小鼠α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α水平;RTqPCR检测胰腺组织NOD样受体蛋白3(NLRP3)、Toll样受体4(TLR4)、核因子-κB(NF-κB)mRNA表达水平;免疫组化检测胰腺组织NLRP3、TLR4、NF-κB的阳性表达率;Western Blot技术检测NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白的表达水平。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果与空白组相比,模型组小鼠胰腺组织结构弥漫性破坏、胰腺小叶间隔局灶性扩张、腺泡萎缩和大量炎症细胞浸润,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显升高(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显上升(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达均明显上调(P值均<0.05)。与模型组相比,清解化攻方各剂量组和西药组可见小鼠胰腺组织结构稍紧密、完整,胰腺腺泡细胞排列有序,伴少量炎症细胞浸润和胰腺小叶出血灶,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显下降(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显降低(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达水平均明显减弱(P值均<0.05)。结论清解化攻方可能通过抑制NLRP3/TLR4/NF-κB信号通路相关蛋白的激活,减少炎症介质的释放,防止炎症级联反应增强,进而对SAP小鼠胰腺组织发挥保护作用。

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.

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.

基于TLR4/MyD88/NF-κB通路介导的NLRP3炎性小体活性探讨振腹推拿改善CUMS模型大鼠海马组织炎性损伤的手法机制研究

目的 观察振腹推拿对抑郁症模型大鼠海马组织Toll样受体4(Toll-like Receptor 4,TLR4)/髓样分化因子88(myeloiddifferentiationfactor88,MyD88)/核因子-κB(nuclear factor kappa-B,NF-κB)通路介导的NLR蛋白3(NLR protein 3,NLRP3)炎性小体活性的影响,探讨振腹推拿改善抑郁症模型大鼠海马区炎性损伤的作用机制。方法 将40只大鼠随机分为4组,采用慢性不可预见性温和应激方法复制抑郁症大鼠模型。采用离子钙接头蛋白-1(ionized calcium binding adaptor molecule-1,Iba1)免疫荧光染色检测各组大鼠海马组织海马回(cornuammonis, CA)的小胶质细胞活化程度,采用酶联免疫吸附法法检测大鼠海马组织中肿瘤坏死因子(tumor necrosis factor, TNF)-α、白细胞介素(interleukin, IL)-1β、IL-6、IL-10的含量,采用蛋白免疫印迹法和实时荧光定量PCR(Real time-PCR,RT-PCR)法检测各组大鼠海马组织中TLR4、MyD88、磷酸化核因子-κB(phospho-nuclear factor kappa-B,p-NF-κB)、NLRP3、凋亡相关斑点样蛋白(Apoptosis-associated speck-like protein containing CARD,ASC)蛋白及其基因表达。结果 (1)与正常组比较,模型组大鼠海马组织CA1、CA2、CA3、CA4区的活化小胶质细胞占比均显著升高(P<0.01)。与模型组大鼠比较,振腹组、氟西汀组大鼠海马组织CA1、CA2、CA4区的活化小胶质细胞占比均显著降低(P<0.01),氟西汀组大鼠海马组织CA3区的活化小胶质细胞占比与模型组相比降低有统计学差异(P<0.05),振腹组大鼠海马组织CA3区的活化小胶质细胞占比与模型组相比仅有降低趋势。(2)与正常组比较,模型组大鼠海马组织TNF-α、IL-1β、IL-6、含半胱氨酸的天冬氨酸蛋白水解酶-1含量显著升高(P<0.01),IL-10含量显著降低(P<0.01)。与模型组大鼠比较,振腹组、氟西汀组大鼠IL-10含量均显著升高(P<0.01),TNF-α、IL-1β、IL-6、Caspase-1含量均显著降低(P<0.01)。(3)与正常组比较,模型组大鼠海马组织TLR4、MyD88、p-NF-κB、NLRP3、ASC的蛋白和mRNA含量均显著升高(P<0.01)。与模型组大鼠比较,氟西汀组大鼠海马组织TLR4、MyD88、p-NF-κB、NLRP3的蛋白含量均显著降低(P<0.01),ASC的蛋白含量下降有统计学意义(P<0.05);氟西汀组大鼠TLR4、MyD88、p-NF-κB、NLRP3、ASC的mRNA含量均显著降低(P<0.01)。与模型组大鼠比较,振腹组大鼠海马组织TLR4、ASC的蛋白含量下降有统计学意义(P<0.05),MyD88、p-NF-κB、NLRP3的蛋白含量显著降低(P<0.01);振腹组大鼠TLR4、MyD88、NF-κB、NLRP3的mRNA含量均显著降低(P<0.01),ASC的mRNA含量均降低有统计学意义(P<0.05)。结论 振腹推拿缓解抑郁模型大鼠抑郁样行为的其机制与抑制海马组织中TLR4/MyD88/NF-κB通路介导的NLRP3炎性小体活性、改善海马组织炎性损伤有关。

苦豆碱通过抑制TLR4/NF-κB/NLRP3通路改善香烟烟雾诱导的人支气管上皮细胞损伤

目的探究苦豆碱(Alo)对香烟烟雾诱导的人支气管上皮细胞损伤的作用及其可能的作用机制。方法16HBE人支气管上皮细胞经100 mL/L香烟烟雾提取物(CSE)和(50、100、200)μmol/L Alo共处理后,CCK-8法检测细胞活力,试剂盒检测乳酸脱氢酶(LDH)活性;原位末端转移酶标记技术(TUNEL)、Western blot法检测细胞凋亡,ELISA检测炎性因子水平;2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针和相关试剂盒检测氧化应激水平;Western blot法检测Toll样受体4(TLR4)/核因子κB(NF-κB)/含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)通路相关蛋白表达水平。16HBE细胞经100 mL/L CSE和200μmol/L Alo共处理后,采用上述方法检测过表达TLR4对TLR4/NF-κB/NLRP3通路、细胞LDH活性、凋亡、炎症反应及氧化应激的影响。结果CSE暴露可降低16HBE细胞活力,增加LDH释放和细胞凋亡,增强炎症反应和氧化应激水平,且激活TLR4/NF-κB/NLRP3通路;经Alo处理后,细胞活性升高,LDH释放减少、凋亡降低、炎症减轻、氧化应激水平下降,且TLR4/NF-κB/NLRP3通路失活;TLR4过表达可逆转Alo处理对CSE诱导的16HBE细胞损伤的保护作用。结论Alo可通过抑制TLR4/NF-κB/NLRP3通路减轻CSE诱导的人支气管上皮细胞损伤。

Puerariae Radix protects against ulcerative colitis in mice by inhibiting NLRP3 inflammasome activation

Ulcerative colitis(UC)is a common inflammatory disease of the gastrointestinal tract.Traditional Chinese medicine(TCM)has long been used in Asia as a treatment for UC and Puerariae Radix(PR)is a reliable anti-diarrheal therapy.The aims of this study were to investigate the protective effect of PR using the dextran sulfate sodium salt(DSS)-induced UC model in mice and identify molecular mechanisms of PR action.The chemical constituents of PR via ultra-performance liquid chromatography/tandem mass spectrometry and identified potential PR and UC targets using a network pharmacology(NP)approach were obtained to guide mouse experiments.A total of 180 peaks were identified from PR including 48 flavonoids,46 organic acids,14 amino acids,8 phenols,8 carbohydrates,7 alkaloids,6 coumarins and 43 other constituents.NP results showed that caspase-1 was the most dysregulated of the core genes associated with UC.A PR dose of 0.136 mg/g administered to DSS treated mice reversed weight loss and decreased colon lengths found in UC mice.PR also alleviated intestinal mucosal shedding,inflammatory cell infiltration and mucin loss.PR treatment suppressed upregulation of NOD-like receptor protein 3(NLRP3),cysteinyl aspartate-specific proteases-1(caspase-1),apoptosis-associated speck-like(ASC)and gasdermin D(GSDMD)at both the protein and m RNA expression levels.The addition of a small molecule dual-specificity phosphatase inhibitor NSC 95397 inhibited the positive effects of PR.These results indicated that PR exerts a protective effect on DSS-induced colitis by inhibiting NLRP3 inflammasome activation in mice.

Crocus sativus L.produces anti-inflammatory effects and regulates the NLRP3–NF-κB pathway

Objective:This study aimed to evaluate the anti-inflammatory effects of petal and stamen extracts of saffron crocus(Crocus sativus)and explore the underlying mechanism.Methods:Local and systemic inflammation models were used to investigate the anti-inflammatory effects of C.sativus.A xyleneinduced inflammation model or lipopolysaccharide(LPS)-induced inflammation model was used in this study.C.sativus petal and stamen extracts were each administered to the mice in the xylene and LPS models by gavage for 14 d at 0.1 and 0.4 g/kg doses,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to measure the concentrations of tumor necrosis factor(TNF)-αand interleukin(IL)-1βin mouse serum.Hematoxylin and eosin(H&E)staining was used to observe the pathological changes in the ear in the xylene-induced inflammation model and in the spleen in the LPS-induced inflammation model.NOD-like receptor thermal protein domain associated protein 3(NLRP3)protein levels within the nuclear factor-kappa B(NF-κB)pathway were assessed using western blotting.RAW264.7 cells were treated with LPS(5μg/mL)and LPS+C.sativus(0.05,0.1,and 0.2 mg/mL)for 24 h,and a Cell Counting Kit-8 was used to measure cell proliferation.Changes in NLRP3 and NF-κB levels were evaluated by western blotting.Results:Petal and stamen extracts of C.sativus attenuated the anti-inflammatory effects in local or systemic inflammatory models and repaired pathological changes in the ear in the xylene-induced inflammation model and spleen in the LPS-induced inflammation model.These extracts also decreased the concentrations of TNF-αand IL-1βin the mouse serum in the LPS-induced inflammation model.C.sativus downregulated NLRP3 protein level through the NF-κB pathway and downregulated LC-3 and BECLIN1 in vivo and in vitro.Carbonyl Cyanide3-ChloroPhenylhydrazone(CCCP)weakened the effects of C.sativus on the NLRP3–NF-κB pathway.Conclusion:C.sativus has anti-inflammatory effects and regulates the NLRP3-NF-κB pathway.

急性脑出血患者血清sSRA、NLRC4水平与神经功能缺损程度和预后的关系

目的探讨急性脑出血患者血清可溶性清道夫受体A(sSRA)、NLR家族含CARD结构蛋白4(NLRC4)水平与神经功能缺损程度和预后的关系。方法选取2020年1月至2023年10月保山市人民医院诊治的198例急性脑出血患者纳入病例组,根据入院时神经功能缺损程度分为轻度组、中度组及重度组。随访3个月,根据预后情况分为预后良好组和预后不良组。以同期体检的80例健康体检者为对照组。采用酶联免疫吸附试验检测血清sSRA、NLRC4水平。采用Spearman相关分析血清sSRA、NLRC4水平与急性脑出血患者病情程度的相关性。采用Logistic回归分析急性脑出血患者预后的影响因素。采用受试者工作特征曲线分析血清sSRA、NLRC4对急性脑出血患者预后的评估价值。结果病例组血清sSRA、NLRC4水平分别为(6.04±1.22)μg/L、(215.48±32.23)ng/L,高于对照组的(1.13±0.24)μg/L、(55.59±12.36)ng/mL,差异均有统计学意义(P<0.05)。病情程度越重,血清sSRA、NLRC4水平越高。急性脑出血患者血清sSRA、NLRC4水平与病情严重程度呈正相关(r_(s)=0.667、0.712,P<0.001)。预后不良组格拉斯哥昏迷量表(GCS)评分低于预后良好组,NIHSS评分、颅内血肿量、血清sSRA及NLRC4水平高于预后良好组,差异均有统计学意义(P<0.05)。GCS评分、NIHSS评分、颅内血肿量、血清sSRA及NLRC4是急性脑出血患者预后不良的影响因素(均P<0.05)。血清sSRA、NLRC4联合评估急性脑出血预后不良的曲线下面积(AUC)为0.923(0.882~0.965),大于单一指标的AUC[0.857(0.811~0.896)、0.863(0.834~0.890)]。结论急性脑出血患者血清sSRA、NLRC4水平升高,二者与神经功能缺损程度有关,2项联合对急性脑出血患者预后具有较高的评估价值。

P2X4调控NLRP1/Caspase-1通路在脑出血炎性损伤中的作用机制

目的探讨嘌呤能受体2X4(P2X4)调控含NLR家族pyrin结构域蛋白1(NLRP1)/半胱氨酸天冬氨酸酶1(Caspase-1)通路在脑出血炎性损伤中的作用机制,为寻找脑出血治疗新措施提供实验依据。方法选取100只8~10周C57BL/6雄性小鼠建立脑出血模型,采用随机数字表法分为对照组、模型组、P2X4激动剂组、P2X4拮抗剂组、NLRP1激动剂组,每组各20只。对照组、模型组经小鼠经腹腔给予15μl生理盐水,P2X4激动剂组给予15μl胞苷5′-三磷酸(5′-CTP);P2X4拮抗剂组小鼠经腹腔注射15μl P2X4特异性拮抗剂5-BDBD;NLRP1激动剂组给予15μl胞壁酰二肽(MDP)。在实验第1、3、5、7天进行改良神经功能缺损评分(mNSS),采用ELISA法测量炎症因子[白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)]水平,采用蛋白免疫印迹法(Western blot)测定P2X4、NLRP1、Caspase-1蛋白表达,采用实时荧光定量聚合酶链式反应(qPCR)法测定P2X4、NLRP1、Caspase-1 mRNA表达,并进行组间比较。结果建模后第1、3、5、7天,模型组的mNSS评分高于对照组,P2X4激动剂组、NLRP1激动剂组的mNSS评分高于模型组,而P2X4拮抗剂组的mNSS评分低于模型组,差异有统计学意义(P<0.05)。建模后第1、3、5、7天,模型组的IL-1β、TNF-α水平高于对照组,P2X4激动剂组、NLRP1激动剂组的IL-1β、TNF-α高于模型组,而P2X4拮抗剂组的IL-1β、TNF-α低于模型组,差异有统计学意义(P<0.05)。模型组的P2X4、NLRP1、Caspase-1 mRNA和蛋白表达高于对照组,P2X4激动剂组、NLRP1激动剂组的相应指标均高于模型组,而P2X4拮抗剂组的相应指标低于模型组,差异有统计学意义(P<0.05)。结论P2X4通过调控NLRP1/Caspase-1通路参与脑出血炎性损伤的发生发展,抑制该通路对改善炎症反应有一定积极作用,值得深入研究。

NLRs蛋白家族的研究进展

真核生物具有复杂的免疫识别系统，对病原体的识别主要利用病原相关分子模式（PAMPs）的受体介导的，目前将这些受体称为模式识别受体（Patternrecognition receptor，PRR），

胞内模式识别受体NLR的生物学特性及其在疾病中的作用

Bruce Beutler和Jules Hoffmann因在固有免疫方面的杰出贡献获2011年诺贝尔医学奖。他们发现固有免疫细胞通过TLR（Toll-like receptor）等模式识别受体（Pattern recognition receptor,PRR）识别病原相关分子模式（Pathogen-associated molecular pattern,PAMP）和损伤相关分子模式（Damageassociated molecular pattern,DAMP）而活化,进而产生免疫效应。

甘珀酸抑制eATP-P2X7R-NLRP3信号通路减轻慢性胰腺炎纤维化的实验研究

目的探讨细胞外ATP(eATP)-P2X7R-NLRP3信号通路在慢性胰腺炎(CP)胰腺纤维化中的作用以及ATP抑制剂甘珀酸(CBX)对CP的治疗作用。方法将6周龄雄性C57BL/6小鼠随机均分为5组:正常组,模型组,CBX低、中、高剂量组(分别为25、50、100 mg/kg)。造模结束后,CBX各剂量组小鼠分别腹腔注射相应剂量的药物。末次给药24 h后将小鼠脱颈处死。HE染色评估胰腺组织病理学改变;ATP化学发光法测定小鼠胰腺组织eATP水平;免疫荧光染色检测P2X7R、NLRP3、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)蛋白表达;实时荧光定量逆转录聚合酶链反应(qPCR)检测P2X7R、NLRP3、Caspase-1、缝隙连接蛋白(PAN-1)mRNA的表达。结果HE染色,光镜下可见正常组小鼠胰腺细胞分布紧密,排列规则,无纤维化及炎症细胞浸润。模型组细胞间隙增宽、腺泡萎缩、炎症细胞浸润且有大量胶原纤维生成,组织学评分较正常组显著升高(P<0.01);与模型组相比,CBX中、高剂量组炎症细胞浸润及胶原纤维生成均减少,组织学评分降低。模型组小鼠造模后eATP水平较正常组显著升高(P<0.01),CBX中、高剂量组给药2周后,胰腺组织eATP水平均低于模型组。免疫荧光染色法和qPCR检测均显示,与正常组相比,模型组P2X7R、NLRP3、Caspase-1蛋白和mRNA表达升高(P<0.01);与模型组相比,CBX中、高剂量组表达均下调(P<0.05)。此外,qPCR检测结果还显示,模型组小鼠PAN-1 mRNA表达较正常组显著上调;与模型组相比,CBX中、高剂量组表达均下调(P<0.01)。结论CP病程中eATP水平显著增加并激活P2X7R,促进NLRP3炎性体组装,加重胰腺纤维化,CBX可下调P2X7R、NLRP3、Caspase-1,减轻胰腺炎症损伤及纤维化,从而发挥治疗作用。

NLRP6诱导胶质瘤细胞生物活性及对TGF-β1/Smad蛋白的作用

目的探讨核苷酸结合寡聚化结构域样受体(NLR)P6诱导胶质瘤细胞生物活性及对转化生长因子(TGF)-β1/Smad蛋白的作用。方法将胶质瘤细胞U251分为正常组(U251细胞)、NC组(U251细胞+NC-空转)和干预组(U251细胞+NLRP6-shRNA),四甲基偶氮唑蓝(MTT)法检测24、48和72 h U251细胞增殖,Transwell小室法、流式细胞法、Western印迹和聚合酶链反应(PCR)法分别检测24 h后U251细胞迁移数目、凋亡率、TGF-β1/Smad蛋白和mRNA表达。结果与正常组比较,NC组及干预组细胞侵袭数目、TGF-β1 mRNA及蛋白表达水平显著降低;细胞凋亡率、Smad4 mRNA及蛋白表达水平显著升高(均P<0.05)。与NC组比较,干预组细胞侵袭数目、TGF-β1 mRNA及蛋白表达水平显著降低;细胞凋亡率、Smad4 mRNA及蛋白水平显著升高(均P<0.05)。结论沉默NLRP6能够降低胶质瘤细胞U251细胞活性,其作用机制可能与抑制TGF-β1水平及激活Smad4水平相关。

Efficacy of Jiangzhi Xiaoban tablet(降脂消斑片)on toll-like receptor 4/nuclear factor-kappa B/nod-like receptor protein 3 signaling pathway in mice with atherosclerosis induced by high-fat diet

OBJECTIVE:To study the effect of Jiangzhi Xiaoban tablet(降脂消斑片,JZXB)on toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/Nod-like receptor protein 3(NLRP3)signaling pathway expression in atherosclerosis(AS)mice by establishing a mouse model of AS,and to explore its mechanism of prevention and treatment of AS.METHODS:Sixty-four male C57BL/6J mice were randomly divided into two groups,12 in the normal control group and 52 in the model group(MOD).Seven weeks later,two mice in each of the above two groups were randomly sacrificed,and the whole aortic tissue of the mice was taken out for hematoxylin-eosin staining.After successful modeling,50 mice in the modeling group were randomly divided into 5 groups:MOD,atorvastatin group(ATO),low-dose group of JZXB(JZXB-L),middle-dose group of JZXB(JZXB-M),and high-dose group of JZXB(JZXB-H),10 mice in each group.The mice in each group were killed after 6 weeks of preventive administration.HE staining was used to observe the pathological changes of aorta in AS mice.The levels of serum triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were detected by automatic biochemical analyzer.The levels of inflammatory factor interleukin-1β(IL-1β)were detected by enzyme linked immunosorbent assay.The expression of TLR4,NF-κB and NLRP3 proteins in aortic tissue was detected by immunohistochemistry.RESULTS:Compared with the MOD,the levels of serum TC,TG and LDL-C in the JZXB-H and ATO were significantly decreased,while the level of HDL-C was significantly increased.The levels of serum TG,LDL-C in the JZXB-M were significantly decreased,and the level of HDL-C was significantly increased.Compared with the MOD,the levels of IL-1βwere significantly decreased,aortic lesions were significantly improved,and the expression of TLR4,NF-κB,and NLRP3 proteins in the aortic tissue was significantly decreased in the JZXB-H,JZXB-M,and ATO.CONCLUSION:JZXB has inhibitory effect on atherosclerosis in mice,and its mechanism may be through regulating the TLR4/NF-κB/NLRP3 signaling pathway and reducing the inflammatory response,so as to play a role in inhibiting atherosclerosis.

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.