Nod-like Receptor Protein 3 Protein Research Articles

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

Mechanism of intestinal injury induced by acute diquat poisoning in rats

To investigate the effects of diquat (DQ) on the expression of intestinal pyroptosis-related proteins and tight junction proteins in rats,and to analyze the role of pyroptosis in the intestinal injury of rats with acute DQ poisoning. A total of 36 Wistar male rats were randomly divided into control group, and 3 hours, 12 hours, 36 hours and 3 days exposure groups, with 6 rats in each group. Each exposure group was given 1/2 median lethal dose (LD50) of 115.5 mg/kg DQ by one-time gavage. The control group was given the same amount of normal saline by gavage. The control group was anesthetized at 3 hours after DQ gavage to take jejunal tissues; each exposure group was anesthetized at 3 hours, 12 hours, 36 hours, and 3 days after DQ gavage to take jejunal tissues, respectively. The general conditions of the rats were recorded. The pathological changes of jejunum tissue were observed by hematoxylin-eosin (HE) staining. The expression of intestinal pyroptosis-related proteins [NOD-like receptor protein 3 (NLRP3), cysteine aspartate-specific protease 1 (caspase-1), Gasdemin D (GSDMD)] in the intestinal tissues was observed by immunohistochemical staining. Western blotting was used to detect the expression of intestinal pyroptosis-related proteins and intestinal tight junction proteins (Occludin and Claudin-1). Light microscopy showed that pathological changes occurred in jejunum tissue at the early stage of exposure (3 hours), and the injury was the most serious in the 12 hours exposure group, with a large number of inflammatory cells infiltrating in the tissue, and the damage was significantly reduced after 3 days exposure. Immunohistochemical results showed that NLRP3, caspase-1 and GSDMD were expressed in the jejunal mucosa of the control group and the exposure groups, and the positive cells in the control group were less expressed with light staining. The expression of the above proteins in the exposed group was increased significantly and the staining was deep. Western blotting results showed that compared with the control group, the expression of NLRP3 protein in jejunum tissues of all groups was increased, with the most significant increase in the 36 hours group (NLRP3/β-actin: 1.47±0.06 vs. 0.43±0.14, P < 0.01). Compared with the control group, the expression of GSDMD protein in the 3 hours, 12 hours and 36 hours exposure groups increased, and the expression of GSDMD protein in the 3 hours and 12 hours exposure groups increased significantly (GSDMD/β-actin: 1.04±0.40, 1.25±0.15 vs. 0.65±0.25, both P < 0.05). The expression of caspase-1 protein was increased in 36 hours exposure group compared with the control group (caspase-1/β-actin: 1.44±0.34 vs. 0.98±0.19, P > 0.05). Compared with the control group, the expression of Occludin and Claudin-1 proteins in each exposure group decreased, and the expression of Occludin proteins was significantly decreased in the 3 hours, 12 hours, and 36 hours exposure groups decreased significantly (Occludin/β-actin: 0.74±0.17, 0.91±0.20, 0.79±0.23 vs. 1.41±0.08, all P < 0.05). Although the protein expression of Claudin-1 decreased in each exposure group, the difference was not statistically significant. The intestinal injury caused by acute DQ poisoning may be related to the activation of pyroptosis pathway of small intestinal cells and the reduction of the density of intercellular junctions.

Effects of NLRP3 Inflammasome Mediated Pyroptosis on Cardiovascular Diseases and Intervention Mechanism of Chinese Medicine.

Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome signaling pathway is an important mechanism underlying myocardial pyroptosis and plays an important role in inflammatory damage to myocardial tissue in patients with cardiovascular diseases (CVDs), such as diabetic cardiomyopathy, ischemia/reperfusion injury, myocardial infarction, heart failure and hypertension. Noncoding RNAs (ncRNAs) are important regulatory factors. Many Chinese medicine (CM) compounds, including their effective components, can regulate pyroptosis and exert myocardium-protecting effects. The mechanisms underlying this protection include inhibition of inflammasome protein expression, Toll-like receptor 4-NF-κB signal pathway activation, oxidative stress, endoplasmic reticulum stress (ERS), and mixed lineage kinase 3 expression and the regulation of silent information regulator 1. The NLRP3 protein is an important regulatory target for CVD prevention and treatment with CM. Exploring the effects of the interventions mediated by CM and the related mechanisms provides new ideas and perspectives for CVD prevention and treatment.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation

Background and aimsThe pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. MethodsFor in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. ResultsPDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αβ (PDGFRαβ) rather than PDGFRαα or PDGFRββ in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. ConclusionsThe results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

Role and mechanism of MiR-542-3p in regulating TLR4 in nonylphenol-induced neuronal cell pyroptosis

BackgroundThis study aimed to investigate the spatial learning/memory and motor abilities of rats and the alteration of miR-542-3p and pyroptosis in the midbrain nigrostriatal area in vivo after nonylphenol (NP) gavage and to explore the mechanism of miR-542-3p regulation of Toll-like receptor 4 (TLR4) in NP-induced pyroptosis in BV2 microglia in vitro. MethodsIn vivo: Thirty-six specific-pathogen-free-grade Sprague–Dawley rats were divided into three equal groups: blank control group (treated with pure corn oil), NP group (treated with NP, 80 mg/kg body weight per day for 90 days), and positive control group [treated with lipopolysaccharide (LPS), 2 mg/kg body weight for 7 days]. In vitro: The first part of the experiment was divided into blank group (control, saline), LPS group [1 µg/ml + 1 mM adenosine triphosphate (ATP)], and NP group (40 µmol/L). The second part was divided into mimics NC (negative control) group, miR-542-3p mimics group, mimics NC + NP group, and miR-542-3p mimics + NP group. ResultsIn vivo: Behaviorally, the spatial learning/memory and motor abilities of rats after NP exposure declined, as detected via Y-maze, open field, and rotarod tests. Some microglia in the substantia nigra of the NP-treated rats were activated. The downregulation of miR-542-3p was observed in rat brain tissue after NP exposure. The mRNA/protein expression of pyroptosis-related indicators (TLR4), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin-D (GSDMD), cysteinyl aspartate–specific proteinase-1 (caspase-1), and interleukin-1β (IL-1β) in the substantia nigra of the midbrain increased after NP exposure. In vitro: ASC fluorescence intensity increased in BV2 cells after NP exposure. The mRNA and/or protein expression of pyroptosis-related indicators (TLR4, NLRP3, GSDMD, caspase-1, and IL-1β) in BV2 cells was upregulated after NP exposure. The transfection of miR-542-3p mimics inhibited NP-induced ASC expression in BV2 cells. The overexpression of miR-542-3p, followed by NP exposure, significantly reduced TLR4, NLRP3, ASC, caspase-1, and IL-1β gene and/or protein expression. ConclusionsThis study suggested that NP exposure caused a decline in spatial learning memory and whole-body motor ability in rats. Our study was novel in reporting that the upregulation of miR-542-3p targeting and regulating TLR4 could inhibit NLRP3 inflammatory activation and alleviate NP-induced microglia pyroptosis.

Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury

To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

Fuzheng Huayu recipe alleviates liver fibrosis via inhibiting NLRP3 inflammasome activation in macrophages

Ethnopharmacological relevanceFuzheng Huayu recipe (FZHY) is a commonly used Traditional Chinese Medicine formula for treating liver fibrosis in clinical settings. Despite its widespread use, the specific curative effects and underlying pharmacological mechanisms of FZHY in treating liver fibrosis are not yet fully understood. Aim and studyThis study aims to investigate the antifibrotic mechanism of FZHY treatment by exploring its effects on the activation of NOD-like receptor protein 3 (NLRP3) inflammasome in macrophages. Materials and methodsIn order to investigate the impact of FZHY on the activation and priming of NLRP3 inflammasome in clinical trials and animal experiments using immunohistochemistry and Western blotting. Twenty-four C57BL/6 mice were used to induce liver fibrosis by feeding a diet that contained 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). To study inflammasome function, Lipopolysaccharide (LPS)/adenine triphosphate (ATP) induced NLRP3 inflammasome activation was induced in bone marrow-derived macrophages (BMDMs) isolated from wild mice. The effects of macrophage NLRP3 inflammasome activation on the function of hepatic stellate cells (HSCs) were explored by treating primary HSCs with preconditioned media from BMDMs culture. ResultsFZHY treatment resulted in the downregulation of NLRP3 protein expression and inhibition of its priming and activation in both human fibrotic livers and DDC-induced liver fibrosis. Furthermore, FZHY was observed to block the activation of the NLRP3 inflammasome pathway, which can lead to excessive inflammatory cytokine release in supernatants and cell lysates in response to LPS and ATP. Lastly, treatment with FZHY was able to inhibit the activation of HSCs induced by supernatants from macrophages. ConclusionsFZHY has been shown to potentially prevent NLRP3 inflammasome activation in macrophages which can result in the suppression of HSCs activation. Ultimately, these effects may lead to the improvement of liver fibrosis. The ability of FZHY to act on this novel mechanism represents an important aspect of its therapeutic potential for liver fibrosis.

Lactobacillus plantarum postbiotics trigger AMPK-dependent autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome activation.

We previously found that Lactobacillus plantarum (LP)-derived postbiotics protected animals against Salmonella infection, but the molecular mechanism remains obscure. This study clarified the mechanisms from the perspective of autophagy. Intestinal porcine epithelial cells (IPEC-J2) were pretreated with LP-derived postbiotics (the culture supernatant, LPC; or heat-killed bacteria, LPB), and then challenged with Salmonella enterica Typhimurium (ST). Results showed that LP postbiotics markedly triggered autophagy under ST infection, as indicated by the increased LC3 and Beclin1 and the decreased p62 levels. Meanwhile, LP postbiotics (particularly LPC) exhibited a strong capacity of inhibiting ST adhesion, invasion and replication. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) led to a significant decrease of autophagy and the aggravated infection, indicating the importance of autophagy in LP postbiotics-mediated Salmonella elimination. LP postbiotics (especially LPB) significantly suppressed ST-induced inflammation by modulating inflammatory cytokines (the increased interleukin (IL)-4 and IL-10, and decreased tumor necrosis factor-α (TNF), IL-1β, IL-6 and IL-18). Furthermore, LP postbiotics inhibited NOD-like receptor protein 3 (NLRP3)inflammasome activation, as evidenced by the decreased levels of NLRP3, Caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Deficits in autophagy resulted in an increase of inflammatory response and inflammasome activation. Finally, we found that both LPC and LPB triggered AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy, and this was further confirmed by AMPK RNA interference. The intracellular infection and NLRP3 inflammasome were aggravated after AMPK knockdown. In summary, LP postbiotics trigger AMPK-mediated autophagy to suppress Salmonella intracellular infection and NLRP3 inflammasome in IPEC-J2 cells. Our findings highlight the effectiveness of postbiotics, and provide a new strategy for preventing Salmonella infection.

Inhibitors of the NLRP3 inflammasome pathway as promising therapeutic candidates for inflammatory diseases (Review).

The inflammasome regulates innate immunity by serving as a signaling platform. The Nod‑like receptor protein 3 (NLRP3) inflammasome, equipped with NLRP3, the adaptor protein apoptosis‑associated speck‑like protein (ASC) and pro‑caspase‑1, is by far the most extensively studied and well‑characterized inflammasome. A variety of stimuli can activate the NLRP3 inflammasome. When activated, the NLRP3 protein recruits the adaptor ASC protein and activates pro‑caspase‑1, resulting in inflammatory cytokine maturation and secretion, which is associated with inflammation and pyroptosis. However, the aberrant activation of the NLRP3 inflammasome has been linked to various inflammatory diseases, including atherosclerosis, ischemic stroke, Alzheimer's disease, diabetes mellitus and inflammatory bowel disease. Therefore, the NLRP3 inflammasome has emerged as a promising therapeutic target for inflammatory diseases. In the present review, systematic searches were performed using 'NLRP3 inhibitor(s)' and 'inflammatory disease(s)' as key words. By browsing the literature from 2012 to 2022, 100 articles were retrieved, of which 35 were excluded as they were reviews, editorials, retracted or unavailable online, and 65 articles were included. According to the retrieved literature, the current understanding of NLRP3 inflammasome pathway activation in inflammatory diseases was summarize, and inhibitors of the NLRP3 inflammasome pathway targeting the NLRP3 protein and other inflammasome components or products were highlighted. Additionally, the present review briefly discusses the current novel efforts in clinical research.

Exosomal Lnc NEAT1 from endothelial cells promote bone regeneration by regulating macrophage polarization via DDX3X/NLRP3 axis

BackgroundBone regeneration is a complex procedure that involves an interaction between osteogenesis and inflammation. Macrophages in the microenvironment are instrumental in bone metabolism. Amount evidence have revealed that exosomes transmitting lncRNA is crucial nanocarriers for cellular interactions in various biotic procedures, especially, osteogenesis. However, the underlying mechanisms of the regulatory relationship between the exosomes and macrophages are awaiting clarification. In the present time study, we aimed to explore the roles of human umbilical vein endothelial cells (HUVECs)-derived exosomes carrying nuclear enrichment enriched transcript 1 (NEAT1) in the osteogenesis mediated by M2 polarized macrophages and elucidate the underlying mechanisms.ResultsWe demonstrated HUVECs-derived exosomes expressing NEAT1 significantly enhanced M2 polarization and attenuated LPS-induced inflammation in vitro. Besides, the conditioned medium from macrophages induced by the exosomes indirectly facilitated the migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Mechanically, Exos carrying NEAT1 decreased remarkably both expression of dead-box helicase 3X-linked (DDX3X) and nod-like receptor protein 3 (NLRP3). The level of NLRP3 protein increased significantly after RAW264.7 cells transfected with DDX3X overexpression plasmid. Additionally, the knockdown of NEAT1 in exosomes partially counteracted the aforementioned effect of Exos. The results of air pouch rat model demonstrated that HUVECs-derived exosomes increased anti-inflammatory cytokines (IL-10) and decreased pro-inflammatory cytokines (IL-1β and IL-6) significantly in vivo, contributing to amelioration of LPS-induced inflammation. Afterwards, we further confirmed that the HUVECs-derived exosomes encapsulated in alginate/gelatin methacrylate (GelMA) interpenetrating polymer network (IPN) hydrogels could promote the bone regeneration, facilitate the angiogenesis, increase the infiltration of M2 polarized macrophages as well as decrease NLRP3 expression in the rat calvarial defect model.ConclusionsHUVECs-derived exosomes enable transmitting NEAT1 to alleviate inflammation by inducing M2 polarization of macrophages through DDX3X/NLRP3 regulatory axis, which finally contributes to osteogenesis with the aid of alginate/GelMA IPN hydrogels in vivo. Thus, our study provides insights in bone healing with the aid of HUVECs-derived exosomes-encapsulated composite hydrogels, which exhibited potential towards the use of bone tissue engineering in the foreseeable future.

Blocking the Hormone Receptors Modulates NLRP3 in LPS-Primed Breast Cancer Cells

NOD-like receptor protein 3 (NLRP3) may contribute to the growth and propagation of breast cancer (BC). The effect of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) on NLRP3 activation in BC remains unknown. Additionally, our knowledge of the effect of blocking these receptors on NLRP3 expression is limited. We used GEPIA, UALCAN, and the Human Protein Atlas for transcriptomic profiling of NLRP3 in BC. Lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP) were used to activate NLRP3 in luminal A MCF-7 and in TNBC MDA-MB-231 and HCC1806 cells. Tamoxifen (Tx), mifepristone (mife), and trastuzumab (Tmab) were used to block ER-α, PR, and HER2, respectively, on inflammasome activation in LPS-primed MCF7 cells. The transcript level of NLRP3 was correlated with ER-ɑ encoding gene ESR1 in luminal A (ER-α+, PR+) and TNBC tumors. NLRP3 protein expression was higher in untreated and LPS/ATP-treated MDA-MB-231 cells than in MCF7 cells. LPS/ATP-mediated NLRP3 activation reduced cell proliferation and recovery of wound healing in both BC cell lines. LPS/ATP treatment prevented spheroid formation in MDA-MB-231 cells but did not affect MCF7. HGF, IL-3, IL-8, M-CSF, MCP-1, and SCGF-b cytokines were secreted in both MDA-MB-231 and MCF7 cells in response to LPS/ATP treatment. Tx (ER-α inhibition) promoted NLRP3 activation and increased migration and sphere formation after LPS treatment of MCF7 cells. Tx-mediated activation of NLRP3 was associated with increased secretion of IL-8 and SCGF-b compared to LPS-only-treated MCF7 cells. In contrast, Tmab (Her2 inhibition) had a limited effect on NLRP3 activation in LPS-treated MCF7 cells. Mife (PR inhibition) opposed NLRP3 activation in LPS-primed MCF7 cells. We have found that Tx increased the expression of NLRP3 in LPS-primed MCF7. These data suggest a link between blocking ER-α and activation of NLRP3, which was associated with increased aggressiveness of the ER-α+ BC cells.

Protective effect of heat shock transcription factor 1 on acute lung injury in septic rats by regulating NOD-like receptor protein 3 inflammasome activation

To determine whether heat shock transcription factor 1 (HSF1) ameliorates sepsis induced acute lung injury (ALI) by regulating NOD-like receptor protein 3 (NLRP3) inflammasome activity in rat alveolar macrophages (AM). Twenty-four SPF Sprague-Dawley (SD) rats were divided into control group, lipopolysaccharide (LPS) group, overexpression empty vector+LPS group, and overexpression HSF1+LPS group by random number table method, with 6 rats in each group. The rat model of sepsis-induced ALI was reproduced by intraperitoneal injection of LPS (5 mg/kg); the rats in the control group were given the same volume of normal saline. The rats in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were instilled with 100 μL of overexpressed empty vector adenovirus or overexpressed HSF1 adenovirus through the trachea, respectively; the rats in the control group and LPS group were instilled with an equal volume of normal saline at the same time. At 6 hours after the model was reproduced, carotid blood was collected to determine the oxygenation index (PaO2/FiO2); lung tissue was obtained, and hematoxylin-eosin (HE) staining was performed to observe the pathological changes of lung tissue under a light microscope. Lung tissue wet/dry ratio (W/D) was determined. Immunohistochemistry was used to detect the positive expression of macrophage-specific marker antibody CD68. Western blotting was used to detect the protein expressions of HSF1 and NLRP3. Bronchoalveolar lavage fluid (BALF) was collected, and the levels of interleukins (IL-1β, IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). In addition, BALF of normal rats was collected, and primary AM was isolated, cultured and divided into four groups. The AM in the blank control group was cultured normally without any treatment; the LPS group was treated with 1 mg/L LPS for 24 hours to reproduced the LPS stimulation model; AM in the overexpression empty vector+LPS group and the overexpression HSF1+LPS group were transfected with empty vector plasmid or overexpressed HSF1 plasmid, respectively, for 48 hours, and then the AM was treated with 1 mg/L LPS. The cell viability was detected by cell counting kit-8 (CCK-8). The protein expressions of HSF1 and NLRP3 in AM were detected by Western blotting. The levels of IL-1β and IL-18 in AM culture medium were determined by ELISA. Compared with the control group, the rat lung structure in the LPS group was severely injured, the alveolar cavity and pulmonary interstitium were congested and edema, the alveolar walls were significantly thickened and ruptured, accompanied by a large number of inflammatory cells infiltration. The lung W/D ratio increased, the infiltration degree of macrophages increased, PaO2/FiO2 decreased, HSF1 protein expression decreased and NLRP3 protein expression increased in lung tissue and AM, and IL-1β and IL-18 levels in BALF and AM culture medium increased. Compared with the LPS group, the degree of lung injury in the overexpression HSF1+LPS group was significantly improved, the lung W/D ratio was significantly reduced (4.76±0.16 vs. 6.93±0.33, P < 0.05), the macrophage infiltration was reduced, PaO2/FiO2 increased significantly [mmHg (1 mmHg ≈ 0.133 kPa): 397.62±19.46 vs. 280.12±37.42, P < 0.05], HSF1 protein expression was significantly up-regulated in lung tissue and AM (HSF1/GAPDH: 0.90±0.04 vs. 0.61±0.04 in lung tissue, 1.10±0.10 vs. 0.57±0.08 in AM, both P < 0.05), NLRP3 protein expression was significantly down-regulated (NLRP3/GAPDH: 0.75±0.14 vs. 1.05±0.11 in lung tissue, 0.81±0.09 vs. 1.14±0.17 in AM, both P < 0.05), and the contents of IL-1β and IL-18 in BALF and AM medium were significantly decreased [IL-1β (ng/L): 7.82±0.45 vs. 14.09±0.58 in BALF, 11.11±0.46 vs. 16.66±0.96 in AM; IL-18 (ng/L): 50.44±3.30 vs. 66.31±5.67 in BALF, 43.95±0.88 vs. 73.52±1.23 in AM, all P < 0.05]. There was no significant difference in the detection indicators between the overexpression empty vector+LPS group and the LPS group. HSF1 attenuates LPS-induced ALI in rats, and the mechanism may be related to the inhibition of NLRP3 inflammasome activation in AM.

Intervention effect of Jingfang Mixture on urticaria mice based on NF-κB/NLRP3/IL-1β signaling pathway

This study explored the curative effect of Jingfang Mixture on urticaria mice induced by aluminum hydroxide/ovalbumin, and discussed its mechanism. Sixty male Kunming mice were randomly divided into a normal group, a model group, three Jingfang Mixture(low-dose, medium-dose, and high-dose) groups, and a positive drug(cetirizine hydrochloride) group. The urticarial model in mice was induced by the intraperitoneal injection of the mixed solution of ovalbumin and aluminum hydroxide. The degrees of pruritus were observed after the second immunization. Pathological changes were detected by hematoxylin and eosin(HE) staining. Levels of interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum were detected by enzyme linked immunosorbent assay(ELISA). Expressions of NOD-like receptor protein 3(NLRP3) and IL-1β were detected by immunohistochemistry(IHC). Expressions of nuclear factor kappa-B(NF-κB p65), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate-specific proteases 1(caspase-1), and IL-1β proteins were detected by Western blot. The results showed that, except for the normal group, the mice in all groups had different degrees of pruritus. Compared with the model group, the Jingfang Mixture groups and the positive drug group prolonged the scratching latency of mice(P&lt;0.05), and significantly reduced the number of scratching(P&lt;0.05). In addition, the Jingfang Mixture groups and the positive drug group improved the pathological morphology of skin tissue. The expression levels of IL-1β and TNF-α in serum were significantly reduced(P&lt;0.05), and the number of NLRP3 and IL-1β positive cells was decreased(P&lt;0.01). The expressions of p-NF-κB p65, NLRP3, ASC, cleaved caspase-1, and IL-1β protein were significantly down-regulated(P&lt;0.05). The results of the above study indicate that Jingfang Mixture inhibit the inflammatory response in urticaria mice, and the mechanism may be related to the inhibition of activating NF-κB/NLRP3/IL-1β signaling pathway.

Prim-O-glucosycimifugin attenuates liver injury in septic mice by inhibiting NLRP3 inflammasome/caspase-1 signaling cascades in macrophages

BackgroundLiver dysfunction and liver failure are serious complications of sepsis, directly leading to septic progression and death. Now, there is no specific therapeutics available for sepsis-related liver dysfunction. Prim-O-glucosylcimifugin (POG), a chromone richest in the roots of Saposhnikovia divaricata (Turcz.) Schischk, is usually used to treat headache, rheumatoid arthritis and tetanus. While, the underlying mechanisms of POG against sepsis-induced liver damage and dysfunction are still not clear. PurposeTo study the anti-sepsis effect of POG, and its pharmacological mechanism to protect liver injury by weakening the function of macrophages in septic livers through inhibiting NOD-like receptor protein 3 (NLRP3) inflammasome pathway. MethodIn vivo experiments, septic mouse model was induced by cecal ligation and puncture (CLP), and then the mortality was detected, liver inflammatory damages and plasma biomarkers of liver injury were evaluated by histopathological staining and biochemical assays, respectively. In vitro experiments, mouse primary peritoneal macrophages were treated with lipopolysaccharide (LPS) and ATP, and then the activated-inflammasomes, macrophage migration and polarization were detected by ASC immunofluorescence staining, transwell and flow cytometry assays, respectively. NLRP3 inflammasome components NLRP3, caspase-1, IL-1β and IL-18 protein expressions were detected using western blot assays, and the contents of IL-1β and IL-18 were measured by ELISA assays. ResultsPOG treatment significantly decreased the mortality, liver inflammatory damages, hepatocyte apoptosis and plasma biomarkers of liver injury in CLP-challenged male WT mice, which were comparable to those in ibuprofen (a putative anti-inflammatory drug)-supplemented septic male WT mice and septic NLRP3 deficient-male mice. POG supplementation significantly suppressed NLRP3 inflammasome activation in septic liver tissues and cultured macrophages, by significantly reducing NLRP3, cleaved-caspase-1, IL-1β and IL-18 levels, the activated-inflammasome ASC specks, and macrophage infiltration and migration, as well as M1-like polarization, but significantly increasing M2-like polarization. These findings were similar to the pharmacological effects of ibuprofen, NLRP3 deficiency, and a special NLRP3 inhibitor, MCC950. ConclusionPOG protected against sepsis by inhibiting NLRP3 inflammasome-mediated macrophage activation in septic liver and attenuating liver inflammatory injury, indicating that it may be a potential anti-sepsis drug candidate.

Pyroptosis of lens epithelial cells in diabetic cataract

Objective: To investigate the possible role of pyroptosis of lens epithelial cells (LECs) in the occurrence of diabetic cataract. Methods: Experimental research. A total of 70 cataract lens anterior capsule and aqueous humor samples were obtained from 70 eyes (70 patients) with cataracts in the operation room of Tianjin Medical University Eye Hospital between March 2020 and November 2020. Patients were divided into the non-diabetic and diabetic groups, with 35 patients (35 eyes) in each group. The expressions of Nod like receptor protein 3 (NLRP3), cysteine-aspartic proteases 1 (caspase-1) and Gasdermin D protein (GSDMD) in the lens anterior capsule were detected by Western blotting, real-time quantitative PCR and immunohistochemical staining. Interleukin (IL)-1β and IL-18 inaqueous humor were detected by enzyme-linked immunosorbent assay (ELISA). The independent sample t-test was used for statistical analysis. Results: The age was (70±9) years in the diabetic group and (71±8) years in the non-diabetic group. There was no significant difference in age and gender distribution between the two groups (both P>0.05). Western blotting analysis showed that the expression levels of NLRP3, caspase-1 and GSDMD protein in the LECs of anterior capsule were 1.11±0.06, 0.95±0.04 and 0.39±0.03 in the diabetic group, significantly higher than those of the non-diabetic group (0.81±0.04, 0.33±0.11 and 0.16±0.04; t=4.38, 5.36, 4.63; all P<0.05). Real-time quantitative PCR showed that the levels of NLRP3, caspase-1 and GSDMD mRNA in the LECs of anterior capsule were 1.98±0.07, 54.36±4.88 and 6.98±1.18 in the diabetic group, significantly higher than those of the non-diabetic group (1.38±0.16, 15.31±1.51 and 2.41±0.95; t=3.49, 7.64, 3.00, all P<0.05). Immunohistochemical staining showed that the average gray values of NLRP3, caspase-1 and GSDMD in the diabetic group were higher than those of the non-diabetic group (all P˂0.01). ELISA showed that the levels of IL-1β and IL-18 in the aqueous humor were (4.178±0.028) fg/L and (20.983±0.018) fg/L in the diabetic group, significantly higher than those of the non-diabetic group [(4.063±0.017) fg/L and (20.509±0.073) fg/L; t=20.63, 37.21; both P˂0.01]. Conclusion: Caspase-1-mediated pyroptosis of LECs and the release of inflammatory mediators induced by pyroptosis may be involved in the occurrence and development of diabetic cataract.

Effects of electroacupuncture on ROS-NLRP3 inflammatory pathway and autophagy related proteins in hippocampus of vascular dementia rats

To observe the effect of electroacupuncture (EA) on learning-memory ability, ultrastructural changes of hippocampal CA1 neurons, reactive oxygen species (ROS) level, Nod-like receptor protein 3 (NLRP3) and auto-phagy-related proteins expression in the hippocampus of vascular dementia (VD) rats, so as to reveal its partial mechanisms in treating VD. Male SD rats were randomly divided into sham operation, model, and EA groups (n=10 rats in each group). The VD model was established by permanent ligation of bilateral common carotid arteries. Rats of the EA group were treated with EA at "Baihui" (GV20), "Dazhui" (GV14) and bilateral "Shenshu" (BL23) for 30 min, once a day for 4 weeks. Morris water maze was used to evaluate the learning and memory ability of rats before modeling, 4 weeks after modeling and after intervention. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal CA1 neurons. The level of ROS in hippocampus was detected by DCFH-DA fluorescence probe. The expressions of NLRP3, autophagy-related protein Beclin1 and microtubule-associated protein 1 light chain 3 (LC3) were measured by Western blot. In comparison with the sham operation group, the average escape latency of rats in the model group was prolonged (P<0.01), and the times of crossing the original platform were reduced (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were increased (P<0.01, P<0.05) in the model group. After EA intervention, the average escape latency of rats was significantly shortened (P<0.01), and the times of crossing the original platform were increased (P<0.05), the level of ROS, the expression levels of LC3-Ⅱ/LC3-Ⅰ ratio, Beclin1 and NLRP3 proteins in hippocampus were decreased (P<0.01, P<0.05) in the EA group compared with those of the model group. Outcomes of TEM showed that CA1 neurons in the hippocampus were damaged, chromatin aggregation, mitochondria pyknosis, cristae structure disorder, rough endoplasmic reticulum expanded and degranulated, the number of free ribosomes decreased, and autophagy could be seen in the model group, which were milder in the EA group. EA at GV20, GV14 and BL23 can improve the learning and memory abilities of VD rats, alleviate the ultrastructural damage of neurons in hippocampal CA1 area, and repair the damaged neurons. The mechanism may be related to the reduction of ROS level, LC3-Ⅱ/LC3-Ⅰ ratio, NLRP3 and Beclin1 protein expression, the decrease of neuronal autophagy, inhibition of activation of NLRP3 inflammasome and alleviation of central inflammatory response.

Bone marrow mesenchymal stem cells attenuate pyroptosis lipopolysaccharide-induced renal injury rats

To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC) on pyroptosis of rats with kidney injury. Bone marrow of 4-5 week-old female Sprague-Dawley (SD) rats was isolated in vitro and BMSC was obtained. The third generations of BMSC were used to further experiments. Fifteen 6 week-old SD rats were cluster-randomized divided into control group, kidney injury group and BMSC group (5 rats in each group). Rats in kidney injury group were injected with lipopolysaccharide (LPS) 1 mg/kg via tail vein; the control group was given the same amount of normal saline. BMSC group was injected with 0.5 mL BMSC (including 2×106 BMSC) via tail vein after modeling; the kidney injury group received the same amount of normal saline. On day 3 after these injections, serum creatinine (SCr) was detected by picric acid method, and blood urea nitrogen (BUN) was detected by diacetyl monoxime. The levels of cystatin C (Cys C), interleukins (IL-1β and IL-18) in blood were detected by enzyme-linked immunosorbent assay (ELISA). The rats were then sacrificed and their kidneys were removed for subsequent detection. The mRNA expression levels of NOD-like receptor protein 3 (NLRP3) and cysteinyl aspartate-specific protease-1 (caspase-1) of kidney were detected by quantificational real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting. In vitro, after bone marrow cell suspension was cultured for 24 hours, a large number of round adherent cells and suspended cells appeared in each culture flask. After 4-5 days of culture, a large number of long spindle cells adhered to the wall, and there were still obvious impurity cells. After trypsin digestion and passage to the third generation, the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC. In vivo, compared with the control group, the levels of SCr, BUN, Cys C, IL-1β and IL-18 in kidney injury group were increased [SCr (μmol/L): 85.22±2.29 vs. 21.80±0.59, BUN (mmol/L): 11.50±0.64 vs. 5.86±0.83, Cys C (mg/L): 0.13±0.01 vs. 0.11±0.02, IL-1β (ng/L): 31.49±1.42 vs. 4.74±0.49, IL-18 (ng/L): 29.01±1.95 vs. 1.52±0.03, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly increased [NLRP3 mRNA (2-ΔΔCt): 3.635±0.296 vs. 1.000±0.002, caspase-1 mRNA (2-ΔΔCt): 4.020±0.228 vs. 1.001±0.003; NLRP3 protein (NLRP3/β-actin): 1.560±0.868 vs. 0.902±0.036, caspase-1 protein (caspase-1/β-actin): 1.392±0.097 vs. 0.895±0.046, all P < 0.05]. Compared with kidney injury group, the levels of SCr, BUN, IL-1β and IL-18 in BMSC group were significantly decreased [SCr (μmol/L): 51.64±3.84 vs. 85.22±2.29, BUN (mmol/L): 9.90±0.46 vs. 11.50±0.64, IL-1β (ng/L): 24.20±1.45 vs. 31.49±1.42, IL-18 (ng/L): 12.97±1.25 vs. 29.01±1.95, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly decreased [NLRP3 mRNA (2-ΔΔCt): 1.488±0.136 vs. 3.635±0.296, caspase-1 mRNA (2-ΔΔCt): 1.643±0.143 vs. 4.020±0.228; NLRP3 protein (NLRP3/β-actin): 1.227±0.053 vs. 1.560±0.868, caspase-1 protein (caspase-1/β-actin): 1.159±0.107 vs. 1.392±0.097, all P < 0.05]. In vivo, BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.

Leptin Signaling in the Ovary of Diet-Induced Obese Mice Regulates Activation of NOD-Like Receptor Protein 3 Inflammasome.

Obesity leads to ovarian dysfunction and the establishment of local leptin resistance. The aim of our study was to characterize the levels of NOD-like receptor protein 3 (NLRP3) inflammasome activation in ovaries and liver of mice during obesity progression. Furthermore, we tested the putative role of leptin on NLRP3 regulation in those organs. C57BL/6J female mice were treated with equine chorionic gonadotropin (eCG) or human chorionic gonadotropin (hCG) for estrous cycle synchronization and ovary collection. In diet-induced obesity (DIO) protocol, mice were fed chow diet (CD) or high-fat diet (HFD) for 4 or 16 weeks, whereas in the hyperleptinemic model (LEPT), mice were injected with leptin for 16 days (16 L) or saline (16 C). Finally, the genetic obese leptin-deficient ob/ob (+/? and −/−) mice were fed CD for 4 week. Either ovaries and liver were collected, as well as cumulus cells (CCs) after superovulation from DIO and LEPT. The estrus cycle synchronization protocol showed increased protein levels of NLRP3 and interleukin (IL)-18 in diestrus, with this stage used for further sample collections. In DIO, protein expression of NLRP3 inflammasome components was increased in 4 week HFD, but decreased in 16 week HFD. Moreover, NLRP3 and IL-1β were upregulated in 16 L and downregulated in ob/ob. Transcriptome analysis of CC showed common genes between LEPT and 4 week HFD modulating NLRP3 inflammasome. Liver analysis showed NLRP3 protein upregulation after 16 week HFD in DIO, but also its downregulation in ob/ob−/−. We showed the link between leptin signaling and NLRP3 inflammasome activation in the ovary throughout obesity progression in mice, elucidating the molecular mechanisms underpinning ovarian failure in maternal obesity.

Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1β and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1β and IL-18 in the serum and spleen index(P&lt;0.01), significantly lowered arthritis score and degree of paw edema(P&lt;0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P&lt;0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

NLRP3 Overexpression Associated With Poor Prognosis and Presented as an Effective Therapeutic Target in Osteosarcoma.

Despite the development of diagnostic and treatment strategies, the survival outcome of patients with osteosarcoma remains poor. Nod-like receptor protein 3 (NLRP3) plays a crucial role in the inflammasome pathway, which is related to the progression of various tumors. However, the effect of NLRP3 on osteosarcoma has not yet been well explored. Our study aimed to investigate the role of NLRP3 in the malignant biological behavior of osteosarcoma as well as its therapeutic value. Immunohistochemistry was applied to investigate the NLRP3 expression in osteosarcoma and osteochondroma specimens. Cell Counting Kit-8, colony formation, wound healing, transwell, and flow cytometry assays were used to explore the contribution of NLRP3 to the proliferation, migration, invasion, apoptosis and cell cycle distribution of osteosarcoma cells in vitro. Western blot was performed to evaluate the expression of NLRP3 and the related proteins in osteosarcoma cell lines after the blockade of NLRP3 using CY-09 and lentivirus intervention. Furthermore, tumor formation assay was used to analyze the effect of NLRP3 on the growth of osteosarcoma in vivo. The results showed that the NLRP3 protein was overexpressed in osteosarcoma, which was independently correlated with the poor prognosis of patients. Moreover, NLRP3 suppression by the inhibitor of CY-09 or lentivirus-induced gene knockdown inhibited the cell proliferation, migration, invasion and promoted the cell apoptosis and G1 cell cycle arrest in osteosarcoma via targeting the inflammasome pathway. Our in vivo results confirmed that the inhibition of NLRP3 suppressed the tumor formation of osteosarcoma. In conclusion, NLRP3 may be regarded as an independent prognostic biomarker and a potential therapeutic target for osteosarcoma.