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.

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.

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.

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。

电针抑制NLRP3炎性小体活化改善缺血性脑卒中的机制研究

目的观察电针对缺血性脑卒中大鼠神经行为学及脑组织结构的影响,探讨电针通过调控NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)炎性小体途径改善缺血性脑卒中大鼠神经功能的机制。方法36只健康雄性SD大鼠采用随机数字表随机分为假手术组12只及手术组24只,手术组大鼠采用Longa等改良线栓法制备大鼠脑缺血再灌注损伤(MCAO)模型。术后2 h行神经行为学评分,造模成功的大鼠再随机分为模型组及电针组。电针组予电针“曲池”“足三里”连续干预7 d,假手术组及模型组不做干预。干预完成后分别评估各组大鼠神经功能缺损情况,HE染色观察脑组织病理学变化,QPCR及Western blot法检测炎性小体相关蛋白NLRP3、ASC、Caspase-1的表达水平,ELISA检测各组大鼠血清IL-1β和IL-18炎性因子表达。结果与假手术组相比,模型组大鼠神经功能评分均显著提高,差异有高度统计学意义(P<0.01),脑组织缺血皮质区神经元胞体缩小变形,核固缩明显,大鼠脑组织缺血周围区炎性因子相关蛋白NLRP3、ASC、Caspase-1的mRNA及蛋白表达水平升高,差异有高度统计学意义(P<0.01),血清IL-1β和IL-18炎症因子表达增加,差异有高度统计学意义(P<0.01);经电针干预7 d后,电针组神经评分功能较模型组显著下降,差异有统计学意义(P<0.05),所见的病理损伤减少;炎性小体相关蛋白NLRP3、ASC、Caspase-1的表达下降,差异有统计学意义(P<0.05),且电针下调了血清IL-1β和IL-18的表达,差异有统计学意义(P<0.05)。结论电针“曲池”“足三里”可减轻缺血性脑卒中大鼠神经功能缺损症状,减少脑组织病理损伤,下调NLRP3、ASC、Caspase-1表达水平,其机制可能与调控NLRP3炎性小体途径抗细胞焦亡,减轻脑缺血再灌注损伤有关。

急性脑梗死患者外周血炎性小体NLRP3表达及与颅内动脉血栓病理成分、预后的相关性分析

目的 探讨急性脑梗死(ACI)患者外周血炎性小体NOD样受体热蛋白结构域相关蛋白3(NLRP3)表达,并分析其与颅内动脉血栓病理成分、预后的相关性。方法 选取2022年1—12月收治的92例ACI作为观察组,另选取健康志愿者92例作为对照组,检测外周血NLRP3 mRNA、蛋白及血清水平。观察组行机械取栓治疗,采用HE染色法半定量分析血栓取出物,并分为红细胞富集血栓者、纤维蛋白富集血栓者。比较不同组别、不同血栓病理成分、不同预后患者外周血炎性小体NLRP3水平,分析其与血栓病理成分相关性及对预后不良风险的关系。结果 观察组NLRP3 mRNA、蛋白表达及血清水平高于对照组,红细胞富集血栓者高于纤维蛋白富集血栓者,且与红细胞富集血栓呈正相关(P<0.05,P<0.01);预后不良者NLRP3 mRNA、蛋白表达及血清水平高于预后良好者(P<0.01);NLRP3 mRNA、蛋白高表达及血清高水平患者预后不良风险分别是低表达、低水平患者的2.182、2.500、2.182倍(P<0.05)。结论 ACI患者外周血炎性小体NLRP3水平升高,且与颅内动脉红细胞富集血栓相关,可增加预后不良发生风险。

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.

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 electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domaincontaining 3 signal pathway

OBJECTIVES: To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome(CPPS) rats by electroacupuncture(EA) of Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS : A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA(n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate(0.1 m L). Electric acupuncture apparatus was applied to stimulate Guanyuan(CV4), Zhongji(CV3), bilateral Huiyang(BL35) and Sanyinjiao(SP6) acupoints in EA group. The general condition of rats was observed and the prostate index(PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and prostaglandin E2(PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay(ELISA). Moreover, the expression levels of purinergic 2X7 receptor(P2X7R), NOD-like receptor pyrin domain-containing 3(NLRP3), caspase-1 and interleukin-18(IL-18) m RNA were quantified by quantitative real-time polymerase chain reaction. RESULTS: Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1β and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 m RNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. CONCLUSION: The effect of EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund’s adjuvant(CFA). This suggests that EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can produce antiinflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.

NLRP3炎性小体水平检测在非霍奇金淋巴瘤患者免疫治疗相关细胞因子风暴及化疗相关间质性肺炎中的预测价值分析

目的分析NOD样受体家族3(NLRP3)炎性小体水平检测在非霍奇金淋巴瘤(NHL)患者免疫治疗相关细胞因子风暴(CRS)及化疗相关间质性肺炎(IP)中的预测价值。方法回顾性分析2019年3月—2021年4月在北京协和医院血液内科接受嵌合抗原受体T细胞(CAR-T)免疫治疗联合化疗的NHL患者125例临床资料,按照CAR-T免疫治疗期间是否出现CRS分为CRS组(n=99)、非CRS组(n=26),比较2组一般资料、基线外周血NLRP3炎性小体各基因mRNA表达、炎性细胞因子、血常规、血生化指标水平,经双变量Preason相关性分析NLRP3炎性小体表达与炎性细胞因子水平的相关性;根据化疗期间是否出现IP分为IP组(n=28)、非IP组(n=97),比较2组一般资料、基线外周血NLRP3炎性小体各基因mRNA表达及血常规、血生化指标水平;多因素Logistic回归分析影响NHL患者CRS、IP发生的危险因素;ROC曲线分析NLRP3炎性小体表达预测NHL患者CRS、IP发生的价值。结果CRS组外周血NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)的mRNA表达水平均高于非CRS组(t/P=9.546/<0.001、3.456/0.001、4.386/<0.001);CRS组外周血C反应蛋白(CRP)、白介素-6(IL-6)、IL-10、干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、乳酸脱氢酶(LDH)水平均高于非CRS组(P<0.01);双变量Preason相关性显示,CRS患者外周血NLRP3、ASC、Caspase-1的mRNA表达均与CRP、IL-6、IL-10、IFN-γ、TNF-α、LDH水平呈正相关(P<0.01)。IP组外周血NLRP3、ASC、Caspase-1的mRNA表达水平均高于非IP组(t=27.931、4.890、6.319,P均<0.001);IP组血常规、血生化相关指标水平与非IP组相比,差异无统计学意义(P>0.05)。多因素Logistic回归分析显示,NLRP3、ASC、Caspase-1的mRNA表达升高均是影响NHL患者CRS、IP发生的危险因素(P<0.05);绘制ROC结果显示,NLRP3、ASC、Caspase-1及三者联合预测NHL患者CRS发生的AUC分别为0.890、0.715、0.800、0.916,预测IP发生的AUC分别为0.901、0.868、0.880、0.935。结论NHL初诊时NLRP3炎性体信号通路的异常活化与炎性细胞因子水平相关,且NLRP3炎性小体表达水平是影响CRS及IP发生的危险因素,可有效预测CRS及IP发生。

基于自噬途径探讨黄芪甲苷抑制糖尿病肾病系膜细胞NLRP3炎症小体活化通路及机制

目的:观察黄芪甲苷对高糖刺激的小鼠肾小球系膜细胞(SV40)核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体活化通路的抑制作用,并基于线粒体自噬途径探究其作用机制。方法:小鼠SV40细胞分别在含有0~80μmol/L黄芪甲苷的DMEM培养基中体外培养48 h,通过细胞活力测试筛选出适宜的黄芪甲苷浓度进行后续实验。实验分为正常对照组(葡萄糖5.6 mmol/L)、高糖组(葡萄糖30 mmol/L)、黄芪甲苷低剂量组(葡萄糖30 mmol/L+黄芪甲苷10μmol/L)、黄芪甲苷中剂量组(葡萄糖30 mmol/L+黄芪甲苷20μmol/L)、黄芪甲苷高剂量组(葡萄糖30 mmol/L+黄芪甲苷40μmol/L),观察黄芪甲苷对小鼠SV40细胞的影响。结果:CCK-8检测结果提示黄芪甲苷浓度达到80μmol/L时小鼠SV40细胞活力明显下降,故本次研究分别选用10、20、40μmol/L作为低、中、高剂量黄芪甲苷进行后续实验。与正常对照组比较,高糖会降低小鼠SV40细胞活力并促进细胞凋亡,并可增加活性氧(ROS)与炎症因子水平,扩大炎症与氧化应激反应,LC3、Beclin1表达水平明显下调,自噬受到抑制。与高糖组比较,黄芪甲苷中、高剂量组细胞凋亡、炎症因子、ROS水平和NLRP3、Caspase-1表达水平均降低(P<0.05或P<0.01),细胞活力、LC3、Beclin-1表达水平均升高(P<0.05或P<0.01)。透射电镜观察发现高糖会减少自噬体产生,黄芪甲苷组自噬体的数目明显多于高糖组。结论:高糖可导致小鼠SV40细胞处于微炎症状态,扩大氧化应激反应并激活NLRP3炎症小体,黄芪甲苷可以抑制NLRP3表达,并提高线粒体自噬,起到减缓DN疾病进展的作用。

淫羊藿苷通过抑制Nod样受体家族蛋白3炎症小体相关蛋白表达抑制BV2小胶质细胞炎症反应

目的研究淫羊藿苷(ICA)靶向Nod样受体家族蛋白3(NLRP3)炎症小体抑制BV2小胶质细胞炎症反应的作用机制。方法用细菌脂多糖(LPS)+干扰素γ(IFN-γ)诱导小鼠BV2小胶质细胞,构建拟神经炎症反应的小胶质细胞模型。BV2细胞用DMEM高糖培养基培养,分为细胞对照组、ICA 10μmol·L^(-1)组、模型组及模型+ICA 5,10和20μmol·L^(-1)组。细胞对照和模型组加入DMEM高糖培养基培,ICA组加入ICA 10μmol·L^(-1),模型+ICA组分别加入相应浓度的ICA,孵育24 h;随后模型和模型+ICA组加入LPS 100μg·L^(-1)和IFN-γ20μg·L^(-1),细胞对照和ICA组用等体积培养基代替,孵育18 h;最后模型+ICA组加入相应浓度的ICA,其他各组加入等体积培养基,继续孵育24 h。用Luminex法检测细胞上清中白细胞介素3(IL-3)、IL-5、IL^(-1)7和单核/巨噬细胞趋化因子11(CCL11)含量;用细胞免疫荧光染色法检测CD16/CD32(M1型小胶质细胞标志物)阳性和D206(M2型小胶质细胞标志物)阳性细胞百分比;Western印迹法检测细胞NLRP3、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶1和胱天蛋白酶原1蛋白表达。结果①Luminex法检测结果显示,与细胞对照组相比,细胞+ICA组细胞上清中IL-3,IL-5,IL^(-1)7和CCL11水平无明显变化,模型组均显著升高(P<0.01)。与模型组相比,模型+ICA 20μmol·L^(-1)组上述细胞因子水平均降低(P<0.05,P<0.01)。②细胞免疫荧光染色结果显示,与细胞对照组相比,ICA组CD16/CD32和CD206阳性细胞百分比无明显变化;模型组CD16/CD32阳性细胞百分比明显增加(P<0.01),CD206阳性细胞百分比显著减少(P<0.01)。与模型组相比,模型+ICA 10和20μmol·L^(-1)组CD16/CD32阳性细胞百分比明显减少(P<0.01),CD206阳性细胞百分比显著增加(P<0.01)。③Western印迹法结果显示,与细胞对照组相比,ICA组NLRP3、ASC、胱天蛋白酶1和胱天蛋白酶原1蛋白表达均无明显变化;模型组NLRP3、ASC和胱天蛋白酶1蛋白表达增多(P<0.05,P<0.01),胱天蛋白酶原1蛋白表达明显减少(P<0.05)。与模型组相比,模型+ICA 5,10和20μmol·L^(-1)组NLRP3、ASC和胱天蛋白酶1蛋白表达减少(P<0.05,P<0.01),胱天蛋白酶原1表达无明显变化。结论ICA可减轻LPS和IFN-γ联合诱导的BV2细胞拟神经炎症反应,其机制可能与抑制NLRP3炎症小体相关蛋白表达有关。

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NLRP3/caspase-1通路研究木犀草素对脂多糖诱导的肠上皮细胞焦亡的影响

目的探讨木犀草素对脂多糖(LPS)诱导的肠上皮细胞焦亡及核苷酸结合寡聚化结构域样受体家族吡啉结构域蛋白3(NLRP3)/半胱氨酸蛋白酶1(caspase-1)通路的影响。方法体外培养人肠上皮细胞HIEC-6,对其进行(1.0μg/mL)LPS诱导,并将细胞分为对照组、LPS组、LPS+(25、50、100μmol/L)木犀草素组、木犀草素+NLRP3激活剂尼日利亚菌素钠盐(NSS)组,除对照组外均添加1.0μg/mL的LPS。MTT法检测各组细胞活力;电阻仪检测单层上皮跨膜电阻(TEER);酶联免疫吸附(ELISA)法检测各组细胞上清液乳酸脱氢酶(LDH)、肿瘤坏死因子α(TNF-α)、白介素-6(IL-6)水平;蛋白免疫印迹(WB)法检测各组细胞焦亡相关蛋白(GSDMD、GSDMD-N)及NLRP3、caspase-1、IL-1β的表达。结果与对照组比较,LPS组HIEC-6细胞活力、TEER值显著降低,细胞上清液LDH水平、TNF-α、IL-6、蛋白GSDMD、GSDMD-N、NLRP3、caspase-1、IL-1β表达显著升高(P<0.05);与LPS组比较,LPS+(25、50、100μmol/L)木犀草素组HIEC-6细胞活力、TEER值显著升高,细胞上清液LDH水平、TNF-α、IL-6、GSDMD、GSDMD-N、NLRP3、caspase-1、IL-1β表达显著降低(P<0.05);与LPS+100μmol/L木犀草素组比较,木犀草素+NSS组HIEC细胞活力、TEER值显著降低,细胞上清液LDH水平、TNF-α、IL-6、GSDMD、GSDMD-N、NLRP3、caspase-1、IL-1β表达显著升高(P<0.05)。结论木犀草素能够减轻LPS诱导的人肠上皮细胞HIEC-6焦亡和细胞炎性损伤,可能与NLRP3/caspase-1信号通路的抑制有关。

Activation of the Macrophage-Associated Inflammasome Exacerbates Myocardial Fibrosis Through the 15-HETE-Mediated Pathway in Acute Myocardial Infarction

This investigation elucidates the spatiotemporal dynamics of NOD-like receptor family pyrin domain con-taining 3(NLRP3)inflammasome activation following myocardial infarction(MI),a process that has not been fully characterized.We revealed early activation of the NLRP3 inflammasome in mice with MI and characterized its dynamic temporal expression.Notably,the knockout and inhibition of Nlrp3 expression were found to significantly mitigate infarct size and enhance cardiac function.Furthermore,our analysis of the spatial characteristics of inflammasome activation revealed predominant activation in macro-phages and subsequent activation in fibroblasts on the third day post-MI.To elucidate the nexus between macrophage-associated NLRP3 inflammasome activation and myocardial fibrosis,we employed targeted metabolomics analyses of inflammatory oxylipins,small interfering RNA(siRNA)interference experi-ments,and various molecular assays.These findings revealed that macrophage-associated inflammasome activation facilitates the conversion of fibroblasts into myofibroblasts via the 15-hydroxy-5,8,11,13-eicosatetraenoic acid(15-HETE)-mediated small mother against decapentaplegic(Smad)pathway.Additionally,both mass spectrometry imaging(MSI)and targeted metabolomics analyses confirmed the significant increase in 15-HETE levels in mice with MI and in patients with MI and acute coronary syndrome(ACS).Our comprehensive dataset suggests that NLRP3 inflammasome activation in MI is char-acterized by distinct temporal and spatial patterns.These insights mark a significant advancement toward precise MI prevention and treatment strategies,particularly early myocardial fibrosis intervention.

薯蓣皂苷元对心肌缺血再灌注损伤大鼠细胞凋亡的影响及相关机制的分析

目的:探究薯蓣皂苷元对心肌缺血再灌注损伤(MIRI)大鼠细胞凋亡的影响,并初步分析其对NOD样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)信号通路的作用。方法:采用左冠状动脉前降支结扎法建立MIRI大鼠模型,将造模成功的72只大鼠,按照随机数字表法分成MIRI组、薯蓣皂苷元低剂量组(50 mg/kg)、薯蓣皂苷元中剂量组(75 mg/kg)和薯蓣皂苷元高剂量组(100 mg/kg),每组18只;另取18只大鼠为假手术组。造模完成后,给予相应药物灌胃,每日1次。末次给药后,记录每组大鼠在不同时间段的心脏血液动力学参数;三苯基四氮唑(TTC)法检测大鼠心肌梗死面积;苏木素-伊红染色检测各组心肌组织病理学变化;TUNEL法检测心肌细胞凋亡情况;ELISA法检测大鼠血清中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的含量;免疫蛋白印迹法检测大鼠心肌组织中NLRP3、Caspase-1、凋亡相关斑点样蛋白(ASC)、IL-1β蛋白表达水平。结果:与假手术组相比,MIRI组大鼠心肌梗死面积更大,心肌细胞凋亡率、左心室舒张末期压力(LVEDP)、血清IL-6、IL-1β、TNF-α含量及心肌组织中NLRP3、Caspase-1、ASC、IL-1β蛋白表达水平均显著升高(P均<0.05),左心室收缩压(LVSP)、左心室射血分数(LVEF)、短轴缩短率(FS)及最大收缩/舒张期压力变化速率(±dp/dtmax)均显著降低(P均<0.05);与MIRI组相比,薯蓣皂苷元不同剂量干预组大鼠心肌梗死面积较小,细胞凋亡率、LVEDP、血清IL-6、IL-1β、TNF-α含量及心肌组织中NLRP3、Caspase-1、ASC、IL-1β蛋白表达水平均显著降低(P均<0.05),LVSP、LVEF、FS及±dp/dtmax均显著升高(P均<0.05)。结论:薯蓣皂苷元可改善MIRI大鼠的心功能,减少炎症反应和细胞凋亡,可能与抑制NLRP3/caspase-1信号通路有关。

核苷酸结合寡聚化结构域样受体蛋白3炎性小体和1-磷酸鞘氨醇对早期糖尿病肾病及其进展情况的诊断价值研究

目的探讨核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)炎性小体和1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)对早期糖尿病肾病及其进展情况的诊断价值。方法选择2018年1月-2020年12月在陕西省人民医院就诊的600例糖尿病患者,将患者分为单纯糖尿病组、早期糖尿病肾病组和临床糖尿病肾病组。采用实时荧光定量聚合酶链反应检测患者空腹静脉血单个核细胞NLRP3信使RNA(messenger RNA,mRNA)表达情况,酶联免疫吸附测定双抗体夹心法检测S1P水平。采用Pearson相关性分析探讨血NLRP3 mRNA和S1P水平的相关性。采用受试者操作特征曲线评价血NLRP3 mRNA和S1P水平对早期糖尿病肾病以及临床糖尿病肾病的诊断价值。结果600例糖尿病患者中单纯糖尿病组205例、早期糖尿病肾病组198例和临床糖尿病肾病组197例。3组患者的年龄和性别差异无统计学意义(P>0.05)。临床糖尿病肾病组患者血NLRP3 mRNA和S1P水平高于早期糖尿病肾病组和单纯糖尿病组,早期糖尿病肾病组患者血NLRP3 mRNA和S1P水平高于单纯糖尿病组,差异有统计学意义[3组患者血NLRP3 mRNA水平分别为2.69±0.64 vs.2.05±0.56 vs.1.76±0.51,S1P水平分别为(1.49±0.27)vs.(1.16±0.13)vs.(0.89±0.07)μmol/L,P<0.05]。患者血NLRP3 mRNA与S1P水平存在正相关关系(r=0.455,P<0.001)。患者血NLRP3 mRNA、S1P水平及二者联合检测均可用于诊断糖尿病患者是否发生早期糖尿病肾病(受试者操作特征曲线下面积分别为0.645、0.968、0.971;P<0.001)以及是否进展至临床糖尿病肾病(受试者操作特征曲线下面积分别为0.825、0.918、0.945;P<0.001)。结论血NLRP3 mRNA和S1P水平可用于诊断早期糖尿病肾病并评估其病情进展情况。