Celastrol augments sensitivity of NLRP3 to CP-456773 by modulating HSP-90 and inducing autophagy in dextran sodium sulphate-induced colitis in rats.
NLRP3, one of the HSP-90 clients, has been defined as a critical component of IBD. In a rat model of DSS-induced colitis, we investigated the anti-inflammatory potential of the combined therapy with CP-456773 (CP), an NLRP3 inhibitor, and celastrol (CSR), an NF-κB inhibitor. Our results revealed that the CSR/CP combined therapy (CCCT) attenuated colon shortening, DAI and MDI in addition to improvement of the colonic histological picture. Moreover, the CCCT increased the antioxidant defense machinery of the colonic tissue and decreased MPO activity. Furthermore, the inflammation markers such as TNF-α and IL-6 were downregulated. These effects might be attributed to the inhibitory effect of CSR on the priming step of the NLRP3 inflammasome activation by interrupting NF-κB signalling and inhibition of HSP-90 (at the protein and mRNA levels) along with inhibitory effect of CP on the expression of the NLRP3. These latter effects resulted in decreased tissue expression and activity of the caspase-1 and repressing the subsequent release of the active forms of IL-1β and IL-18, hence, the pyroptosis process is restrained. Additionally, the CCCT resulted in inducing autophagy by AMPK/mTOR-dependent mechanisms leading to the accumulation of BECN1 protein and a significant decrease in the levels of p62 SQSTM1. The inhibitory effect on HSP-90 in conjunction with induction of autophagy suggest increased autophagic degradation of NLRP3. This novel approach provides a basis for the clinical application of this combination in IBD treatment and might also be promising for the pharmacological intervention of other NLRP3 inflammasome-dependent inflammatory conditions.
Saber S
，Abd El-Kader EM
，Sharaf H
，El-Shamy R
，El-Saeed B
，Mostafa A
，Ezzat D
，Shata A
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Combining the HSP90 inhibitor TAS-116 with metformin effectively degrades the NLRP3 and attenuates inflammasome activation in rats: A new management paradigm for ulcerative colitis.
Ulcerative colitis (UC) is a prevalent type of inflammatory bowel diseases that may predispose patients to acquire colitis-related cancer if treatment was not effective. Despite the presence of an array of established treatment options, current modalities are not successful for a substanial number of patients. The activation of the NLRP3 inflammasome is critical in the development of inflammatory processes in the colon. Additionally, the regulation of NLRP3 via HSP90 inhibition is a potential target to treat UC. Moreover, during inflammation, autophagy allows the turnover of malfunctioning proteins and therefore stands as a viable strategy for inactivating NLRP3 inflammasomes and halting hyperinflammation. Herein, we evaluated the effect of autophagy induction using metformin in the context of HSP90 inhibition by TAS-116 in the dextran sodium sulfate (DSS)-induced UC in rats. We revealed that TAS-116-induced interruption of the protein complex containing HSP90 and NLRP3 might hamper and delay the start of the inflammatory cascade ensued by the NLRP3 inflammasome oligomerization. In such circumstances, the unprotected NLRP3 is subjected to autophagic degradation in an environment of metformin-promoted autophagic signaling. As a result, such dynamic synergy was efficient in combating colon damage and immune-cell infiltration. This was confirmed by the macroscopic and microscopic investigations. Further, biochemical analysis revealed subdued inflammation cascade and oxidative injury. Therefore, simultaneous administration of TAS-116 and metformin is a new management paradigm aimed at inducing malfunction in the NLRP3 followed by augmenting its autophagic degradation, respectively. However, further studies should be conducted to assess the reliability and consistency of this novel approach.
Shaaban AA
，Abdelhamid AM
，Shaker ME
，Cavalu S
，Maghiar AM
，Alsayegh AA
，Babalghith AO
，El-Ahwany E
，Amin NA
，Mohammed OA
，Eissa H
，Gaafar AGA
，Batiha GE
，Saber S
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Canna x generalis L.H. Bailey rhizome extract ameliorates dextran sulfate sodium-induced colitis via modulating intestinal mucosal dysfunction, oxidative stress, inflammation, and TLR4/ NF-ҡB and NLRP3 inflammasome pathways.
Genus Canna is used in folk medicine as demulcent, diaphoretic, antipyretic, mild laxative and in gastrointestinal upsets therapy. Canna x generalis (CG) L.H. Bailey is traditionally used as anti-inflammatory, analgesic and antipyretic. Besides, CG is used in Ayurvedic medicines' preparations and in the treatment of boils, wounds, and abscess. Nevertheless, its anti-inflammatory effects against ulcerative colitis (UC) are not yet investigated.
This study aimed to investigate the phytoconstituents of CG rhizome ethanol extract (CGE). Additionally, we aimed to comparatively evaluate its therapeutic effects and underlying mechanisms against the reference drug "sulphasalazine (SAS)" in dextran sodium sulfate (DSS)-induced UC in mice.
Metabolic profiling of CG rhizomes was performed via UHPLC/qTOF-HRMS; the total phenolic, flavonoid and steroid contents were determined, and the main phytoconstituents were isolated and identified. Next, DSS-induced (4%) acute UC was established in C57BL/6 mice. DSS-induced mice were administered either CGE (100 and 200 mg/kg) or SAS (200 mg/kg) for 7 days. Body weight, colon length, disease activity index (DAI) and histopathological alterations in colon tissues were examined. Colon levels of oxidative stress (GSH, MDA, SOD and catalase) and pro-inflammatory [Myeloperoxidase (MPO), nitric oxide (NO), IL-1β, IL-12, TNF-α, and INF-γ] markers were colourimetrically determined. Serum levels of lipopolysaccharide (LPS) and relative mRNA expressions of occludin, TLR4 and ASC (Apoptosis-Associated Speck-Like Protein Containing CARD) using RT-PCR were measured. Protein levels of NLRP3 inflammasome and cleaved caspase-1 were determined by Western blot. Furthermore, immunohistochemical examinations of caspase-3, NF-ҡB and claudin-1 were performed.
Major identified constituents of CGE were flavonoids, phenolic acids, phytosterols, beside five isolated phytoconstituents (β-sitosterol, triacontanol fatty alcohol, β-sitosterol-3-O-β-glucoside, rosmarinic acid, 6-O-p-coumaroyl-β-D-fructofuranosyl α-D-glucopyranoside). The percentage of the phenolic, flavonoid and steroid contents in CGE were 20.55, 6.74 and 98.09 μg of gallic acid, quercetin and β-sitosterol equivalents/mg extract, respectively. In DSS-induced mice, CGE treatment ameliorated DAI, body weight loss and colon shortening. CGE attenuated the DSS-induced colonic histopathological alternations, inflammatory cell infiltration and histological scores. CGE elevated GSH, SOD and catalase levels, and suppressed MDA, pro-inflammatory mediators (MPO and NO) as well as cytokines levels in colonic tissues. Moreover, CGE downregulated LPS/TLR4 signaling, caspase-3 and NF-ҡB expressions. CGE treatment inhibited NLRP3 signaling pathway as indicated by the suppression of the protein expression of NLRP3 and cleaved caspase-1, and the ASC mRNA expression in colonic tissues. Additionally, CGE restored tight junction proteins' (occludin and claudin-1) expressions.
Our findings provided evidence for the therapeutic potential of CGE against UC. CGE restored intestinal mucosal barrier's integrity, mitigated oxidative stress, inflammatory cascade, as well as NF-ҡB/TLR4 and NLRP3 pathways activation in colonic tissues. Notably, CGE in a dose of 200 mg/kg was more effective in ameliorating DSS-induced UC as compared to SAS at the same dose.
Mahmoud TN
，El-Maadawy WH
，Kandil ZA
，Khalil H
，El-Fiky NM
，El Alfy TSMA
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Geranylgeranyl diphosphate synthase 1 knockdown suppresses NLRP3 inflammasome activity via promoting autophagy in sepsis-induced acute lung injury.
NOD-like receptor protein 3 (NLRP3) inflammasome activation has emerged as a crucial contributor to sepsis-induced lung injury. Geranylgeranyl diphosphate synthase 1 (GGPPS1) reportedly exerts the pro-inflammatory capability via activation of NLRP3 inflammasome. However, little is known about the role and mechanism of GGPPS1 in sepsis-induced lung injury.
Mice underwent cecal ligation and puncture (CLP) surgery to establish the in vivo model of sepsis. The lung injury of mice was assessed by analyzing the histological changes, the lung wet/dry ratio, PaO2/FiO2 ratio, myeloperoxidase (MPO) activity, total protein content, total cell, and polymorphonuclear leukocyte counts. Mouse alveolar macrophages MH-S were exposed to LPS for developing in vitro model of sepsis. The mRNA and protein expression levels of GGPPS1, beclin-1, and autophagy and inflammasome-related genes were detected using quantitative reverse transcription-polymerase chain reaction and western blot assays. Enzyme-linked immunosorbent assay was conducted to determine the levels of interleukin (IL)-1β and IL-18.
We successfully established sepsis-induced acute lung injury in vivo by CLP surgery. GGPPS1 was upregulated in the lung tissues of CLP-induced septic mice. The activation of autophagy and NLRP3 inflammasome were found in the lung tissues of CLP-induced septic mice. The addition of exogenous GGPP (synthesis products catalyzed by GGPPS1) and autophagic inhibitor 3-MA aggravated sepsis-induced hypoxemia, alveolar inflammatory response, intrapulmonary hemorrhage, and pulmonary edema, as evidenced by increased lung injury score, lung wet/dry weight ratio, MPO activity, total protein content, total cell, and PMNs counts, and decreased PaO2/FiO2 ratio. While NLRP3 inhibitor MCC950 exerted the opposite effects. Additionally, administration of exogenous GGPP could inhibit the activation of autophagy, enhance the activity of NLRP3 inflammasome, and the production of IL-1β and IL-18. Inhibition of autophagy by 3-MA treatment also promoted the activity of NLRP3 inflammasome and the production of IL-1β and IL-18. While MCC950 restrained the activity of NLRP3 inflammasome, but did not affect the activation of autophagy. Notably, the expression of GGPPS1 was unaltered in CLP-induced mice following GGPP, 3-MA, or MCC950 treatment. Moreover, GGPPS1 was upregulated in MH-S cells stimulated with LPS, and GGPPS1 knockdown enhanced the activation of autophagy and inhibited the activity of NLRP3 inflammasome in vitro. Importantly, depletion of GGPPS1 could alleviate LPS-induced inflammatory response by inducing autophagy-dependent NLRP3 inflammasome inhibition.
GGPPS1 knockdown suppressed NLRP3 inflammasome activity via promoting autophagy and then attenuated sepsis-induced acute lung injury, revealing a novel target for treating sepsis-induced lung injury.
Li D
，Li C
，Wang T
，Zhang C
，Zhu Z
，Zhang G
，Fang B
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