Protein Expression Of NLRP3 Research Articles

Huang-Pu-Tong-Qiao Formula Alleviates Hippocampal Neuron Damage by Inhibiting NLRP3 Inflammasome-mediated Pyroptosis in Alzheimer's Disease.

Huang-Pu-Tong-Qiao (HPTQ), a Traditional Chinese Medicine formula, has achieved remarkable efficacy in clinically treating Alzheimer's disease (AD). Pyroptosis refers to the inflammatory necrosis of cells, which contributes to AD pathological progression. However, it is unclear whether the therapeutic effect of HPTQ on AD is related to reducing pyroptosis. In this study, the network pharmacology analysis was used to predict the molecular mechanism of HPTQ in treating AD and validated our hypothesis through mice and cell experiments. APP/PS1 transgenic mice and Aβ25-35-injured HT22 cells were used as AD models in vivo and in vitro. The pharmacological effects and mechanisms of HPTQ on AD were evaluated by Morris water maze, Y-maze, transmission electron microscope, immunofluorescence, Hoechst/PI staining, western blot, and ELISA. Network pharmacology reveals the correlation between the therapeutic effect of HPTQ on AD and the NOD-like receptor signaling pathway. In APP/PS1 mice, HPTQ reduced the escape latency and maintained cell membrane integrity. In HT22 cells, 15% HPTQ-medicated serum and 10 µM MCC950 increased cell viability and decreased PI positive rate compared with the Model group. In addition, HPTQ treatment in AD animal and cell models reduced the protein expressions of NLRP3, ASC, cleaved caspase-1, GSDMD, GSDMD-N, IL-1β, and IL-18. The experimental results of MCC950 specifically inhibiting the NLRP3 expression suggested that HPTQ might reduce neuronal pyroptosis by reducing NLRP3 inflammasome. Network pharmacology and experimental validation suggested that HPTQ alleviated NLRP3 inflammasome-mediated neuronal pyroptosis in AD, which could provide valuable candidate drugs for AD clinical treatment.

Preventive effect of imperatorin against doxorubicin-induced cardiotoxicity through suppression of NLRP3 inflammasome activation.

Cardiotoxicity is one of the major obstacles to anthracycline chemotherapy. Anthracycline cardiotoxicity is closely associated with inflammation. Imperatorin (IMP), a furocoumarin ingredient extracted from Angelica dahurica, might have potential activity in preventing anthracycline cardiotoxicity due to its anti-cancer, anti-inflammatory, anti-oxidant, cardioprotective properties. This study aims to reveal the effect of IMP on doxorubicin (DOX)-induced cardiotoxicity and its underlying mechanism. We established a rat model of DOX-induced cardiotoxicity by intraperitoneal injection with DOX (1.25mg/kg twice weekly for 6weeks), and found that both IMP (25mg/kg and 12.5mg/kg) and dexrazoxane 12.5mg/kg relieved DOX-induced reductions in heart weight, change in cardiac histopathology, and elevated serum levels of LDH, AST and CK-MB. Moreover, DOX upregulated mRNA levels of NLRP3, CASP1, GSDMD, ASC, IL-1β and IL-18, elevated protein expressions of NLRP3, ASC, GSDMD-FL, GSDMD-N, pro‑caspase‑1, caspase‑1 p20, pro‑IL‑1β and IL‑1β in heart tissues, as well as increased serum levels of pro-inflammatory cytokines including IL-1β and IL-18, however both of IMP and dexrazoxane suppressed these alterations. In addition, we carried out neonatal rat cardiomyocytes experiments to confirm the results of the in vivo study. Consistently, pretreatment with IMP 25µg/mL relieved DOX (1μg/mL)-induced cardiomyocytes injury, including decreased cell viability and reduced supernatant LDH. IMP inhibited DOX-induced activation of NLRP3 inflammasome in cardiomyocytes. In conclusion, IMP had a protective effect against DOX-induced cardiotoxicity via repressing the activation of NLRP3 inflammasome. These findings suggest that IMP may be a promising alternative or adjunctive drug for the prevention of anthracycline cardiotoxicity.

Quercetin suppresses pyroptosis in mouse fibroblasts by inhibiting the NLRP3/caspase-1/GSDMD pathway

To investigate whether quercetin inhibits pyroptosis of mouse fibroblast NIH-3T3 cells by regulating the NLRP3/caspase-1/GSDMD signaling pathway. NIH-3T3 cells were treated with quercetin or MCC950 (a specific inhibitor of NLRP3) before stimulation with lipopolysaccharide (LPS) and ATP to induce cell pyroptosis. The optimal quercetin concentration and duration were screened using the CCK-8assay after testing various concentrations and times. Morphological changes of the treated cells was observed, and the levels of IL-18 and IL-1β in the cell culture supernatant were detected with ELISA; the protein expressions of NLRP3, cleaved caspase-1, and GSDMD-N and the mRNA levels of NLRP3, caspase-1 and GSDMD were detected using Western blotting and qRT-PCR. The changes in cell pyroptosis were examined with TUNEL staining and LDH release assay. The CCK-8 assay indicated that 24-hour treatment with 20 μmol/L quercetin yielded the most favorable results. LPS and ATP stimulation of NIH-3T3 cells induced obvious swelling, cell membrane rupture and leakage of cell contents, significantly increased IL-18 and IL-1β levels, and enhanced protein expressions of NLRP3, cleaved caspase-1 and GSDMD-N and mRNA levels of NLRP3, caspase-1 and GSDMD. LPS and ATP stimulation also caused a significant increment of TUNEL-positive cell counts and LDH release in NIH-3T3 cells. Treatment with quercetin or MCC950 significantly reduced cell pyroptosis induced by LPS and ATP, lowered the concentrations of IL-18 and IL-1β, decreased the expression levels of NLRP3, caspase-1/cleaved caspase-1, GSDMD/GSDMD-N, and reduced the number of TUNEL-positive cells and LDH release. Quercetin suppresses pyroptosis of mouse fibroblasts stimulated with LPS and ATP and reduces secretion of inflammatory cytokines by inhibiting the NLRP3/caspase-1/GSDMD pathway.

Acylated Anthocyanins From Black Carrots and Their Related Phenolic Acids Diminish Priming and Activation of the NLRP3 Inflammasome in THP-1 Monocytes.

Excessive activation of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to chronic inflammation. Thus, targeting NLRP3 inflammasome activation by anthocyanins may prevent inflammatory diseases. Therefore, the present study determines the influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on the NLRP3 inflammasome. THP-1 monocytes are pretreated with a BCE, cyanidin-3-glucoside (C3G), or hydroxycinnamic acids. NLRP3 inflammasome assembly is initiated by priming THP-1 monocytes with lipopolysaccharide and/or activating the NLRP3 inflammasome with nigericin. Flow cytometry is used to assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, as well as ASC and NLRP3 protein expression. Caspase-1 activity is measured using a bioluminescent assay, and cytokine concentrations are determined by enzyme-linked immunosorbent assays (ELISA). C3G and phenolic acids diminish ASC and NLRP3 protein expression. In addition, C3G and phenolic acids attenuate ASC speck formation. Furthermore, the BCE and C3G decline caspase-1 activity. Consistently, IL-1β and IL-18 secretion are reduced upon NLRP3 inflammasome activation. The present study shows that a BCE with high amounts of acylated anthocyanins and their related phenolic acids diminish priming and activation of the NLRP3 inflammasome in THP-1 monocytes.

FuKe QianJin capsule alleviates endometritis via inhibiting inflammation and pyroptosis through modulating TLR4/ NF-κB /NLRP3 pathway

Ethnopharmacological relevanceFuke Qianjin Capsule (FKC), a traditional Chinese medicine commonly employed for treating endometritis, lacks reported treatment mechanisms. Aim of the studyThe aim of the present study was to explore the role and mechanism of FKC in lipopolysaccharide (LPS)-induced endometritis. Materials and methodsThe main active ingredients of FKC were identified via high-performance liquid chromatography (HPLC) in conjunction with standard substances. Prior to endometritis induction, Sprague Dawley female rats received FKC for 7 days. The endometritis model was established through an intrauterine injection of 1 mg/kg LPS. Concurrently, an LPS-induced RAW264.7 cell inflammation model was utilized, in which the cells were treated with serum containing Fuke Qianjin Capsule. Pathological alterations in the endometrium were assessed via H&E staining and transmission electron microscopy (TEM). The contents of MPO in uterine tissues, and NO release in cells, along with the secretion of IL-18, IL-1β, IL-6, and TNF-α in both tissues and cells, were determined via assay kits. The mRNA levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β in uterine tissues and cells were analyzed via qPCR. The protein levels of TLR4, p65, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β in these samples were evaluated through Western blot analysis. Immunofluorescence was used to assess the protein levels of p-P65 and NLRP3 in uterine tissues and cells. ResultsFive primary active components of FKC were identified. Treatment with FKC in vivo mitigated endometrial pathological damage and significantly decreased the levels of MPO, IL-18, IL-1β, IL-6, and TNF-α, as well as the levels of Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA in tissue samples. Treatment with FKC inhibited the expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β, as well as reduced NLRP3 protein fluorescence intensity, and inhibited P65 phosphorylation. In vitro findings demonstrated that FKC-containing serum reduced IL-18, IL-1β, IL-6, and TNF-α levels, as well as reduced Nlrp3, Caspase-1, Gsdmd, and Il-1β mRNA levels. In addition, FKC-containing serum inhibited the protein expression of TLR4, p-P65, NLRP3, Caspase-1, GSDMD, and IL-1β. FKC-containing serum also reduced NLRP3 protein fluorescence intensity and suppressed P65 phosphorylation. ConclusionFKC reverses the LPS induced NLRP3 inflammasome activation, and mitigates inflammation and pyroptosis through the modulation of the TLR4/NF-κB/NLRP3 pathway, thereby alleviating endometritis.

Loureirin B Reduces Insulin Resistance and Chronic Inflammation in a Rat Model of Polycystic Ovary Syndrome by Upregulating GPR120 and Activating the LKB1/AMPK Signaling Pathway.

Polycystic ovary yndrome (PCOS) is a common metabolic disorder in women, which is usually associated with insulin resistance (IR) and chronic inflammation. Loureirin B (LrB) can effectively improve insulin resistance and alleviate chronic inflammation, and in order to investigate the therapeutic effect of LrB on polycystic ovary syndrome with insulin resistance (PCOS-IR), we conducted animal experiments. A PCOS-IR rat model was established by feeding a high-fat diet combined with letrozole (1 mg/kg·d for 21 days). The rats were treated with the GPR120 agonists TUG-891 and LrB for 4 weeks. Biochemical parameters (fasting blood glucose, total cholesterol, triglycerides, high- and low-density lipoprotein), hormone levels (serum insulin, E2, T, LH, and FSH), and inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-18) were analyzed. Histopathological analyses of ovaries were performed using hematoxylin/eosin (H&E) staining. Real-time PCR and western blotting were used to assess GPR120, NLRP3, and caspase-1 expression in ovaries, and immunohistochemistry was used to evaluate LKB1 and AMPK protein expression. LrB reduced body weight, Lee's index, ovarian index, ovarian area, and volume in PCOS-IR rats. It lowered fasting blood glucose, serum insulin, and HOMA-IR. LrB decreased total serum cholesterol, triglyceride, and LDL levels and increased HDL levels. It reduced serum T, LH, and LH/FSH and raised serum E2 and FSH levels. LrB downregulated the mRNA and protein expression levels of NLRP3 and Caspase-1, increased the protein and mRNA expression levels of GPR120 in rat ovaries, and increased LKB1 and AMPK protein expression in ovaries, ameliorating ovarian histopathological changes in PCOS-IR rats. Taken together, LrB upregulated GPR120, LKB1, and AMPK protein expression, downregulated NLRP3 and Caspase-1 protein expression, reduced insulin resistance and chronic inflammation, and ameliorated histopathological changes in ovarian tissues in PCOS rats, suggesting its potential as a treatment for PCOS.

CBR-470-1 protects against cardiomyocyte death in ischaemia/reperfusion injury by activating the Nrf2–GPX4 cascade

Cardiac ischaemia/reperfusion (I/R) impairs mitochondrial function, resulting in excessive oxidative stress and cardiomyocyte ferroptosis and death. Nuclear factor E2-related factor 2 (Nrf2) is a key regulator of redox homeostasis and has cardioprotective effects against various stresses. Here, we tested whether CBR-470-1, a noncovalent Nrf2 activator, can protect against cardiomyocyte death caused by I/R stress. Compared with vehicle treatment, the administration of CBR-470-1 (2 mg/kg) to mice significantly increased Nrf2 protein levels and ameliorated the infarct size, the I/R-induced decrease in cardiac contractile performance, and the I/R-induced increases in cell apoptosis, ROS levels, and inflammation. Consistently, the beneficial effects of CBR-470-1 on cardiomyocytes were verified in a hypoxia/reoxygenation (H/R) model in vitro, but this cardioprotection was dramatically attenuated by the GPX4 inhibitor RSL3. Mechanistically, CBR-470-1 upregulated Nrf2 expression, which increased the expression levels of antioxidant enzymes (NQO1, SOD1, Prdx1, and Gclc) and antiferroptotic proteins (SLC7A11 and GPX4) and downregulated the protein expression of p53 and Nlrp3, leading to the inhibition of ROS production and inflammation and subsequent cardiomyocyte death (apoptosis, ferroptosis and pyroptosis). In summary, CBR-470-1 prevented I/R-mediated cardiac injury possibly through inhibiting cardiomyocyte apoptosis, ferroptosis and pyroptosis via Nrf2-mediated inhibition of p53 and Nlrp3 and activation of the SLC7A11/GPX4 pathway. Our data also highlight that CBR-470-1 may serve as a valuable agent for treating ischaemic heart disease.

Role for NLRP3 inflammasome-mediated, Caspase1-dependent response in glaucomatous trabecular meshwork cell death and regulation of aqueous humor outflow

PurposeAcute ocular hypertension (AOH) is the defining feature of acute glaucoma. The mechanical stress and excessive production of reactive oxygen species (ROS) during episodes can directly or indirectly damage the trabecular meshwork (TM). Despite its significance, a clear understanding of its pathogenesis and an effective therapeutic target remain lacking in acute glaucoma. In the present study, we explored the potential molecular mechanisms underlying TM cell death following oxidative damage and AOH. The use of NAC/VX-765 as a potential pharmaceutical intervention for reducing intraocular pressure (IOP) was discussed. MethodsThe levels of NLRP3 and caspase-1 were compared between normal and glaucomatous TM samples. An in vitro oxidative damage model and an AOH rat model were used to investigate the potential molecular mechanism behind TM cell death. The ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to counteract TM damage. ResultsElevated levels of NLRP3 and caspase-1 were observed in patients with acute glaucoma. H2O2 exposure decreased the viability of human trabecular meshwork (HTM) cells and increased intracellular ROS levels. Both Gene and protein expressions of NLRP3, caspase-1, GSDMD-N, and IL-1β were notably upregulated in H2O2-induced HTM cells and the rodent AOH model. Both NAC and VX-765 demonstrated protective effects against TM injury by inhibiting pyroptosis. The IOP-lowering effects of NAC and VX-765 persisted for 7 days. ConclusionsOur findings indicate that the classical pyroptosis pathway, NLRP3/caspase-1/IL-1β, plays a key role in acute glaucomatous TM injury. Targeting pyroptosis provides novel therapeutic avenues for treating AOH-induced irreversible TM injury. This provides not only a promising therapeutic target for glaucoma but also introduces a new approach to intervention.

Investigating the active components and mechanistic effects of Forsythia suspensa Leaf against RSV via the PI3K/Akt-NLRP3 pathway

BackgroundPulmonary infections resulting from respiratory syncytial virus (RSV) continue to pose a significant threat to the well-being of infants and the elderly, but there is no safe, effective and specific treatment except symptomatic treatment. Forsythia Suspensa Leaf (FSL) is cold in nature and bitter in taste, and has the efficacy of clearing away heat and toxic materials. Previous research by our research group showed that the active components in FSL have the pharmacological effect of anti-RSV. Based on that, this study aims further to clarify the anti-RSV active components and mechanism of FSL. MethodsFirstly, we established the BALB/c mouse model of RSV infection, assessed the in vivo anti-RSV efficacy, and determined the optimal dosage of FSL and its active components. Evaluation parameters included body weight changes, organ indices, lung tissue pathological sections, lung tissue viral load, and inflammatory factors. Additionally, we used RT-PCR, Western Blot and other molecular biology techniques to determine the expression changes of key factors such as Nrf2 and NLRP3 in PI3K/Akt-NLRP3 pathway, and revealed the anti-RSV mechanism of FSL and its active components. ResultsPharmacodynamic experiments in animals showed that the FSL Low (0.4 g/kg·d), RosA Low (100 mg/kg·d) and Phillyrin Medium (100 mg/kg·d) groups could effectively improve the pathological conditions of mice with RSV pneumonia, such as weight loss, the level of pulmonary inflammatory factors and the increase of viral load. In addition, oral administration of Phillyrin at a dose of 100 mg/kg d to RSV-infected mice can effectively control the trend that the expression of Nrf2 protein decreases and the expression of NLRP3 protein increases in RSV pneumonia mice. ConclusionPhillyrin, the active component in FSL, can not only directly inhibit the replication of RSV, but also effectively control the inflammatory reaction caused by RSV infection and improve lung injury, which is expected to become a potential drug against RSV pneumonia.

Sacubitril/valsartan attenuated myocardial inflammation, fibrosis, apoptosis and promoted autophagy in doxorubicin-induced cardiotoxicity mice via regulating the AMPKα-mTORC1 signaling pathway.

This study aimed to investigate the potential cardioprotective effects of sacubitril/valsartan (Sac/Val) in mice with doxorubicin (DOX)-induced cardiomyopathy, a common manifestation of cancer therapy-related cardiac dysfunction (CTRCD) associated with DOX. A total of thirty-two mice were equally classified into 4 groups: control group, DOX (total 24mg/kg), Sac/Val (80mg/kg), and Sac/Val + DOX (Sac/Val was given from seven days before doxorubicin administration). Neonatal rat ventricular myocytes was exposed to 5µM of DOX for 6h in vitro to mimic the in vivo conditions. A variety of techniques were used to investigate cardiac inflammation, fibrosis, apoptosis, and autophagy, including western blot, real-time quantitative PCR (RT-qPCR), immunohistochemistry, and fluorescence. Mice with DOX-induced cardiotoxicity displayed impaired systolic and diastolic function, characterized by elevated levels of cardiac inflammation, fibrosis, cardiomyocyte hypertrophy, apoptosis, and autophagy inhibition in the heart. Treatment with Sac/Val partially reversed these effects. In comparison to the control group, the protein expression of NLRP3, caspase-1, collagen I, Bax, cleaved caspase-3, and P62 were significantly increased, while the protein expression of Bcl-2 and LC3-II were significantly decreased in the myocardial tissues of the Dox-induced cardiomyopathy group. The administration of Sac/Val demonstrated the potential to partially reverse alterations in protein expression within the myocardium of mice with DOX-induced cardiotoxicity by modulating the AMPKα-mTORC1 signaling pathway and suppressing oxidative stress. Additionally, Sac/Val treatment may mitigate Dox-induced apoptosis and inhibition of autophagy in primary cardiomyocytes. Sac/Val seems to be cardioprotective against DOX-induced cardiotoxicity in the pre-treatment mice model. These findings could be attributed to the anti-inflammatory, antioxidant, anti-apoptotic, and de-autophagy effects of Sac/Val through regulation of the AMPKα-mTORC1 signaling pathway.

MiR-146a-5p engineered hucMSC-derived extracellular vesicles attenuate Dermatophagoides farinae-induced allergic airway epithelial cell inflammation.

Allergic asthma is prevalent in children, with Dermatophagoides farinae as a common indoor allergen. Current treatments for allergic airway inflammation are limited and carry risks. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) show promise as a cell-free therapeutic approach. However, the use of engineered MSC-EVs for D. farinae-induced allergic airway epithelial cell inflammation remains unexplored. We generated miR-146a-5p-engineered EVs from human umbilical cord mesenchymal stem cells (hucMSCs) and established D. farinae-induced mouse and human bronchial epithelial cell allergic models. Levels of IL-1β, IL-18, IL-4, IL-5, IL-6, IL-10, IL-33, TNF-α and IgE were detected using ELISA. The relative TRAF6 and IRAK1 mRNA expression was quantified using qPCR assay and the NLRP3, NF-κB, IRAK1 and TRAF6 protein expression was determined using Western blotting. The regulatory effect of IRAK1 and TRAF6 by miR-146a-5p was examined using a dual luciferase reporter assay, and the nuclear translocation of NF-κB p65 into 16-HBE cells was evaluated using immunofluorescence assay. Treatment with hucMSC-EVs effectively reduced allergic inflammation, while miR-146a-5p engineered hucMSC-EVs showed greater efficacy. The enhanced efficacy in alleviating allergic airway inflammation was attributed to the downregulation of IRAK1 and TRAF6 expression, facilitated by miR-146a-5p. This downregulation subsequently led to a decrease in NF-κB nuclear translocation, which in turn resulted in reduced activation of the NLRP3 inflammasome and diminished production of inflammatory cytokines, including IL-6, TNF-α, IL-1β and IL-18. Our study underscores the potential of miR-146a-5p engineered hucMSC-EVs as a cell-free therapeutic strategy for D. farinae-induced allergic airway inflammation, offering a promising avenue for boosting anti-inflammatory responses.

WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after traumatic brain injury.

Traumatic brain injury (TBI) is a common complication of acute and severe neurosurgery. Remodeling of N6-methyladenosine (m6A) stabilization may be an attractive treatment option for neurological dysfunction after TBI. In the present study, the authors explored the epigenetic methylation of RNA-mediated NLRP3 inflammasome activation after TBI. Neurological dysfunction, histopathology, and associated molecules were examined in conditional knockout (CKO) WTAP [flox/flox, Camk2a-cre] , WTAP flox/flox , and pAAV-U6-shRNA-YTHDF1-transfected mice. Primary neurons were used in vitro to further explore the molecular mechanisms of action of WTAP/YTHDF1 following neural damage. The authors found that WTAP and m6A levels were upregulated at an early stage after TBI, and conditional deletion of WTAP in neurons did not affect neurological function but promoted functional recovery after TBI. Conditional deletion of WTAP in neurons suppressed neuroinflammation at the TBI early phase: WTAP could directly act on NLRP3 mRNA, regulate NLRP3 mRNA m6A level, and promote NLRP3 expression after neuronal injury. Further investigation found that YTH domain of YTHDF1 could directly bind to NLRP3 mRNA and regulate NLRP3 protein expression. YTHDF1 mutation or silencing improved neuronal injury, inhibited Caspase-1 activation, and decreased IL-1β levels. This effect was mediated via suppression of NLRP3 protein translation, which also reversed the stimulative effect of WTAP overexpression on NLRP3 expression and inflammation. Our results indicate that WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after TBI and that WTAP/m6A/YTHDF1 downregulation therapeutics is a viable and promising approach for preserving neuronal function after TBI, which can provide support for targeted drug development.

MSU crystallization promotes fibroblast proliferation and renal fibrosis in diabetic nephropathy via the ROS/SHP2/TGFβ pathway

Monosodium urate (MSU) crystallisation deposited in local tissues and organs induce inflammatory reactions, resulting in diseases such as gout. MSU has been recognized as a common and prevalent pathology in various clinical conditions. In this study, we investigated the role of MSU in the pathogenesis of diabetic kidney disease (DKD). We induced renal injury in diabetic kidney disease mice using streptozotocin (STZ) and assessed renal histopathological damage using Masson's trichrome staining and Collagen III immunofluorescence staining. We measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and uric acid (UA) using ELISA. Protein expression levels of NLRP3, p-NF-κB, SHP2, p-STAT3, and p-ERK1/2 were analyzed by Western blot. To further investigate the role of MSU in diabetic kidney disease, we conducted in vitro experiments. In our in vivo experiments, we found that compared to the Model group, there was a significant increase in interstitial fibrosis in the kidneys of mice after treatment with MSU, accompanied by elevated levels of MDA, SOD, and UA. Furthermore, the protein expression of NLRP3, p-NF-NB, SHP2, p-STAT3, and p-ERK1/2 was upregulated. In our subsequent studies on mouse fibroblasts (L929 cells), we discovered that high glucose, MSU, and TGF-β could promote the expression of P22, GP91, NLRP3, NF-κB, p-NF-κB, p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins. Additionally, we found that SHP2 could counteract the upregulation trend induced by MSU on the expression of p-SHP2, p-EGFR, p-STAT3, and Collagen-III proteins, and inhibitors YQ128, NAC, and Cetuximab exhibited similar effects. Furthermore, immunofluorescence results indicated that SHP2 could inhibit the expression of the fibrosis marker α-SMA in L929 cells. These findings suggest that MSU can promote renal fibroblast SHP2 expression, induce oxidative stress, activate the NLRP3/NF-κB pathway, and enhance diabetic kidney disease fibroblast proliferation through the TGFβ/STAT3/ERK1/2 signaling pathway, leading to renal fibrosis.

Britannilactone 1-O-acetate induced ubiquitination of NLRP3 inflammasome through TRIM31 as a protective mechanism against reflux esophagitis-induced esophageal injury

BackgroundReflux esophagitis (RE) is a disease in which inflammation of the esophageal mucosa owing to the reflux of gastric contents into the esophagus results in cytokine damage. Britannilactone 1-O-acetate (Brt) has anti-inflammatory effects, significantly inhibiting the activation of the NLRP3 inflammasome, leading to a decrease in inflammatory factors including IL-1 β, IL-6, and TNF-α. However, the mechanism underlying its protective effect against RE-induced esophageal injury remains unclear. In the present study, we investigated the protective mechanism of TRIM31 against NLRP3 ubiquitination-induced RE both in vivo and in vitro.MethodsA model of RE was established in vivo in rats by the method of “4.2 mm pyloric clamp + 2/3 fundoplication”. In vitro, the mod was constructed by using HET-1A (esophageal epithelial cells) and exposing the cells to acid, bile salts, and acidic bile salts. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used to screen the concentration of administered drugs, and the viability of HET-1A cells in each group. HE staining was used to assess the degree of pathological damage in esophageal tissues. Toluidine blue staining was used to detect whether the protective function of the esophageal epithelial barrier was damaged and restored. The enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of IL-1 β, IL-6, and TNF-α factors in serum. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3 in esophageal tissues. The molecular docking and Co-immunoprecipitation assay (Co-IP assay) were used to detect the TRIM31 interacts with NLRP3. Western blotting detected the Claudin-4, Claudin-5, The G-protein-coupled receptor calcium-sensitive receptor (CaSR), NLRP3, TRIM31, ASC, C-Caspase1, and Caspase1 protein expression levels.ResultsBrt could alleviate RE inflammatory responses by modulating serum levels of IL-1 β, IL-6, and TNF-α. It also activated the expression of NLRP3, ASC, Caspase 1, and C-Caspase-1 in HET-1A cells. Brt also attenuated TRIM31/NLRP3-induced pathological injury in rats with RE through a molecular mechanism consistent with the in vitro results.ConclusionsBrt promotes the ubiquitination of NLRP3 through TRIM31 and attenuates esophageal epithelial damage induced by RE caused by acidic bile salt exposure. This study provides valuable insights into the mechanism of action of Brt in the treatment of RE and highlights its promising application in the prevention of NLRP3 inflammatory vesicle-associated inflammatory pathological injury.Graphical

Yinqin Qingfei granules alleviate Mycoplasma pneumoniae pneumonia via inhibiting NLRP3 inflammasome-mediated macrophage pyroptosis.

Mycoplasma pneumoniae pneumonia (MPP) is a prevalent respiratory infectious disease in children. Given the increasing resistance of M. pneumoniae (MP) to macrolide antibiotics, the identification of new therapeutic agents is critical. Yinqin Qingfei granules (YQQFG), a Chinese patent medicine formulated specifically for pediatric MPP, lacks a clear explanation of its mechanism. The primary components of YQQFG were identified using LC-MS/MS. In vitro, RAW264.7 cells infected with MP underwent morphological examination via scanning electron microscopy. Drug-containing serum was prepared, and its intervention concentration was determined using the CCK-8 assay. The active components of YQQFG were molecularly docked with NLRP3 protein using Autodock Vina software. A RAW264.7 cell line overexpressing NLRP3 was created using lentivirus to pinpoint the target of YQQFG. In vivo, MPP model mice were established via nasal instillation of MP. Lung damage was assessed by lung index and H&E staining. Pyroptosis-associated protein levels in cells and lung tissue were measured by western blot, while interleukin (IL)-1β and IL-18 levels in cell supernatants and mouse serum were quantified using ELISA. Immunofluorescence double staining of lung tissue sections was conducted to assess the correlation between NLRP3 protein expression and macrophages. The expression of the community-acquired respiratory distress syndrome toxin (CARDS TX) was evaluated by qPCR. 25 effective components with favorable oral bioavailability were identified in YQQFG. Both in vitro and in vivo studies demonstrated that YQQFG substantially reduced the expression of the NLRP3/Caspase-1/GSDMD pathway, decreasing the release of IL-1β and IL-18, and inhibited MP exotoxin. Molecular docking indicated strong affinity between most YQQFG components and NLRP3 protein. Lentivirus transfection and immunofluorescence double staining confirmed that YQQFG significantly suppressed NLRP3 expression in macrophages, outperforming azithromycin (AZM). The combination of YQQFG and AZM yielded the optimal therapeutic effect for MPP. YQQFG mitigates inflammatory responses by suppressing NLRP3 inflammasome-mediated macrophage pyroptosis, thereby ameliorating MP-induced acute lung injury. YQQFG serves as an effective adjunct and alternative medication for pediatric MPP treatment.

Monocyte NLRP3 inflammasome and interleukin-1β activation modulated by alpha-1 antitrypsin therapy in deficient individuals

IntroductionAltered complement component 3 (C3) activation in patients with alpha-1 antitrypsin (AAT) deficiency (AATD) has been reported. To understand the potential impact on course of inflammation, the aim of this...

Anti-inflammatory Properties of Tongfeng Li'an Granules in an Acute Gouty Arthritis Rat Model.

To examine the anti-inflammatory properties and underlying mechanisms of Tongfeng Li'an Granules (TFLA), a traditional medicine, in acute gouty arthritis using a rat model. We identified 55 major compounds in TFLA via ultrahigh-performance liquid chromatography connected to quadrupole time-of-flight mass spectrometry (UPLC-TQF-MS/MS). Databases were employed for the prediction of potential targets, followed by PPI network construction as well as GO and KEGG analyses. After network-pharmacology-based analysis, a rat gouty arthritis model was used to validate the anti-inflammatory mechanism of TFLA. UPLC-TQF-MS/MS and network pharmacology analyses revealed 55 active ingredients and 160 targets of TFLA associated with gouty arthritis, forming an ingredient-target-disease network. The PPI network identified 20 core targets, including TLR2, TLR4, IL6, NFκB, etc. Functional enrichment analyses highlighted the Toll-like receptor signaling pathway as significantly enriched by multiple targets, validated in in vivo experiments. Animal experiments demonstrated that TFLA improved pathological changes in gouty joint synovium, with decreased ankle joint circumference, serum IL6, IL10, and TNFα levels, as well as reduced protein and mRNA expression of NLRP3, TLR2, and TLR4 in ankle joint synovial tissue observed in the middle- and high-dose TFLA and positive control groups compared to the model group (p < 0.05). This research elucidated the pharmacological mechanisms of TFLA against gouty arthritis, implicating various ingredients, targets, and signaling pathways. Animal experiments confirmed TFLA's efficacy in alleviating inflammation in acute gouty arthritis by modulating Toll-like receptor signaling and NLRP3 expression.

Gasdermin D could be lost in the brain parenchyma infarct core and a pyroptosis-autophagy inhibition effect of Jie-Du-Huo-Xue decoction after stroke.

The Chinese ethnic medicine Jie-Du-Huo-Xue Decoction (JDHXD) is used to alleviate neuroinflammation in cerebral ischemia (CI). Our previous studies have confirmed that JDHXD can inhibit microglial pyroptosis in CI. However, the pharmacological mechanism of JDHXD in alleviating neuroinflammation and pyroptosis needs to be further elucidated. New research points out that there is an interaction between autophagy and inflammasome NLRP3, and autophagy can help clear NLRP3. The NLRP3 is a key initiator of pyroptosis and autophagy. The effect of JDHXD promoting autophagy to clear NLRP3 to inhibit pyroptosis on cerebral ischemia-reperfusion inflammatory injury is currently unknown. We speculate that JDHXD can inhibit pyroptosis in CI by promoting autophagy to clear NLRP3. Chemical characterization of JDHXD was performed using LC-MS. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in SD rats. Neurological deficits, neuron damage, and cerebral infarct volume were evaluated. Western Blot and immunofluorescence were used to detect neuronal pyroptosis and autophagy. 30 possible substance metabolites in JDHXD medicated serum were analyzed by LC-MS (Composite Score > 0.98). Furthermore, JDHXD protects rat neurological function and cerebral infarct size after CI. JDHXD inhibited the expression of pyroptosis and autophagy after CI. Our western blot and immunofluorescence results showed that JDHXD treatment can reduce the expression of autophagy-related factors ULK1, beclin1, and LC3-Ⅱ. The expression of NLRP3 protein was lower in the JDHXD group than in the I/R group. Compared with the I/R group, the expressions of pyroptosis-related factors caspase-1 P 10, GSDMD-NT, IL-18, and IL-1β decreased in the JDHXD group. Furthermore, we observed an unexpected result: immunofluorescence demonstrated that Gasdermin D (GSDMD) was significantly absent in the infarct core, and highly expressed in the peri-infarct and contralateral cerebral hemispheres. This finding challenges the prevailing view that GSDMD is elevated in the ischemic cerebral hemisphere. JDHXD inhibited pyroptosis and autophagy after MCAO/R. JDHXD suppressed pyroptosis and autophagy by inhibiting NLRP3, thereby alleviating CI. In addition, we present a different observation from previous studies that the expression of GSDMD in the infarct core was lower than that in the peri-infarct and contralateral non-ischemic hemispheres on day 3 of CI.

Engagement of specific intracellular pathways in the inflammation-based reprotoxicity effect of small-size silver nanoparticles on spermatogonia and spermatocytes invitro cell models

Male infertility is a serious ongoing problem, whose causes have not yet been clearly identified. However, since human exposure to silver nanoparticles (AgNPs) has recently increased due to their beneficial properties, the present study aimed to determine the impact of small-size AgNPs on mouse spermatogonia (GC-1 spg) and spermatocytes [GC-2 spd(ts)] in vitro models as well as the ability of these nanostructures to induce inflammation. The results showed a significant dose- and time-dependent decrease in the metabolic activity in both cell models, which was correlated with an increase in the intracellular ROS level. Moreover, increased activity of caspase-9 and -3, together with enhanced expression of CASP3 and p(S15)-p53 proteins, was detected. Further studies indicated a decrease in ΔΨm after the AgNP-treatment, which proves induction of apoptosis with engagement of an intrinsic pathway. The PARP1 protein expression, the activity and protein expression of antioxidant enzymes, the GSH level, and the increased level of p-ERK1/2 indicate not only the engagement of DNA damage but also the occurrence of oxidative stress. The small-size AgNPs were able to induce inflammation, proved by increased protein expression of NF-κB, p-IκBα, and NLRP3, which indicate damage to spermatogonia and spermatocyte cells. Moreover, the PGC-1α/PPARγ and NRF2/Keap1 pathways were engaged in the observed effect. The spermatogonial cells were characterized by a stronger inflammation-based response to AgNPs, which may be correlated with the TNFα/TRAF2-based pathway. Summarizing, the obtained results prove that AgNPs impair the function of testis-derived cells by inducing the redox imbalance and inflammation process; therefore, these NPs should be carefully implemented in the human environment.

Hydrogen combined with tetrandrine attenuates silica-induced pulmonary fibrosis via suppressing NF-kappaB/NLRP3 signaling pathway-mediated epithelial mesenchymal transition and inflammation

Silicosis is a progressive disease characterized by interstitial fibrosis resulting from inhalation of silica particles, and currently lacks specific treatment. Hydrogen (H2) has demonstrated antioxidative, anti-inflammatory, and anti-fibrotic properties, yet its efficacy in treating silicosis remains unexplored. In this study, rats exposed to silica were administered interventions of H2 combined with tetrandrine, and euthanized at 14, 28, and 56 days post-intervention. Lung tissues and serum samples were collected for analysis. Histological examination, MDA assay, enzyme-linked immunosorbent assay, hydroxyproline assay, and Western blotting were employed to assess the impact of H2 combined with tetrandrine on pulmonary fibrosis. The results revealed that this combination significantly alleviated inflammation in silicosis-afflicted rats, effectively suppressed levels of MDA, TNF-α, and IL-1β expression, and inhibited epithelial-mesenchymal transition (EMT), thereby ameliorating pulmonary fibrosis. Notably, protein expression level of E-cadherin was increased，however protein expression levels of vimentin and α-SMA were reduced, and TGF-β were reduced, alongside a significant decrease in hydroxyproline content. Furthermore, H2 combined with tetrandrine downregulated protein expression of NF-κB p65, NF-κB p-p65, Caspase-1, ASC, and NLRP3. These findings substantiate the hypothesis that H2 combined with tetrandrine mitigates inflammation associated with silicosis and suppresses the EMT process to ameliorate fibrosis via the NF-κB/NLRP3 signaling pathway. However, the pressure of airway opening was not assessed in this study and dynamic readings of lung physiological function were not obtained, which is a major limitation of this study.