The ROS/TXNIP/NLRP3 pathway mediates LPS-induced microglial inflammatory response.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction activated by microglia. The potential pathological changes of SAE are complex, and the cellular pathophysiological characteristics remains unclear. This study aims to explore the ROS/TXNIP/NLRP3 pathway mediated lipopolysaccharide (LPS)-induced inflammatory response in microglia. METHODS: BV-2 cells were pre-incubated with 10 µM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 µg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3. RESULTS: LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1ß, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells. CONCLUSION: ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.

Co-Treatment with Phlorotannin and Extracellular Vesicles from Ecklonia cava Inhibits UV-Induced Melanogenesis.

Hyperpigmentation due to ultraviolet (UV)-induced melanogenesis causes various esthetic problems. Phlorotannin (PT) and extracellular vesicles (EVs) derived from various plants suppress melanogenesis pathways. We used UV-exposed keratinocytes and animal skin to determine if co-treatment with PT and EVs from Ecklonia cava (EVE) could inhibit melanogenesis by reducing UV-induced oxidative stress and the expression of the thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor family pyrin domain containing the 3 (NLRP3)/interleukin-18 (IL-18) pathway, which are upstream signals of the microphthalmia-associated transcription factor. UV exposure increased oxidative stress in keratinocytes and animal skin, as evaluated by 8-OHdG expression, and this effect was reduced by co-treatment with PT and EVE. UV also increased binding between NLRP3 and TXNIP, which increased NLRP3 inflammasome activation and IL-18 secretion, and this effect was reduced by co-treatment with PT and EVE in keratinocytes and animal skin. In melanocytes, conditioned media (CM) from UV-exposed keratinocytes increased the expression of melanogenesis-related pathways; however, these effects were reduced with CM from UV-exposed keratinocytes treated with PT and EVE. Similarly, PT and EVE treatment reduced melanogenesis-related signals, melanin content, and increased basement membrane (BM) components in UV-exposed animal skin. Thus, co-treatment with PT and EVE reduced melanogenesis and restored the BM structure by reducing oxidative stress and TXNIP/NLRP3/IL-18 pathway expression.

Inhibition of Drp1-mediated mitochondrial fission improves contrast-induced acute kidney injury by targeting the mROS-TXNIP-NLRP3 inflammasome axis.

Acute kidney injury (AKI) is a critical complication known for their extremely high mortality rate and lack of effective clinical therapy. Disorders in mitochondrial dynamics possess a pivotal role in the occurrence and progression of contrast-induced nephropathy (CIN) by activating NLRP3 inflammasome. The activation of dynamin-related protein-1 (Drp1) can trigger mitochondrial dynamic disorders by regulating excessive mitochondrial fission. However, the precise role of Drp1 during CIN has not been clarified. In vivo experiments revealed that inhibiting Drp1 through Mdivi-1 (one selective inhibitor of Drp1) can significantly decrease the expression of p-Drp1 (Ser616), mitochondrial p-Drp1 (Ser616), mitochondrial Bax, mitochondrial reactive oxygen species (mROS), NLRP3, caspase-1, ASC, TNF-α, IL-1ß, interleukin (IL)-18, IL-6, creatinine (Cr), malondialdehyde (MDA), blood urea nitrogen (BUN), and KIM-1. Moreover, Mdivi-1 reduced kidney pathological injury and downregulated the interaction between NLRP3 and thioredoxin-interacting protein (TXNIP), which was accompanied by decreased interactions between TRX and TXNIP. This resulted in increasing superoxide dismutase (SOD) and CAT activity, TRX expression, up-regulating mitochondrial membrane potential, and augmenting ATP contents and p-Drp1 (Ser616) levels in the cytoplasm. However, it did not bring impact on the expression of p-Drp1 (Ser637) and TXNIP. Activating Drp-1though Acetaldehyde abrogated the effects of Mdivi-1. In addition, the results of in vitro studies employing siRNA-Drp1 and plasmid-Drp1 intervention in HK-2 cells treated with iohexol were consistent with the in vivo experiments. Our findings revealed inhibiting Drp1 phosphorylation at Ser616 could ameliorate iohexol -induced acute kidney injury though alleviating the activation of the TXNIP-NLRP3 inflammasome pathway.

Polygalasaponin F ameliorates middle cerebral artery occlusion-induced focal ischemia / reperfusion injury in rats through inhibiting TXNIP/NLRP3 signaling pathway.

BACKGROUND: Polygalasaponin F (PGSF), an oleanane triterpenoid saponin extracted from Polygala japonica, has been demonstrated with neuroprotective effect. However, the therapeutic effects and mechanisms of PGSF on focal ischemia remain unknown; METHODS: In this study, male Sprague Dawley (SD) rats aged 6-8 weeks were initially selected to establish a rat model of middle cerebral artery occlusion (MCAO) to evaluate the therapeutic effect of PGSF intervention and to investigate the impact of PGSF on the thioredoxin-interacting protein/NOD-, LRR-, and pyrin domain-containing protein 3 (TXNIP/NLRP3) inflammatory pathway. Secondly, brain neuron cells were isolated, and the cells received oxygen-glucose deprivation/reoxygenation (OGD/R) culture to establish the cell injury model in vitro. The mechanism of PGSF on the TXNIP/NLRP3 pathway was further validated; RESULTS: Our results showed that PGSF treatment reduced neurological scores, brain tissue water content and infarct volume and ameliorated the pathological changes in cerebral cortex in MCAO-induced focal ischemia rats. The TNF-α, IL-1ß and IL-6 levels decreased in MCAO-induced focal ischemia rats after PGSF treatment. Moreover, PGSF down-regulated the protein expressions of TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18 in MCAO-induced focal ischemia rats. Meanwhile, PGSF treatment inhibited apoptosis, and reduced the levels of ROS, inflammatory cytokine and TXNIP/NLRP3 pathway-related proteins (TXNIP, NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18) in OGD/R-induced neuronal injury cells. Finally, PGSF treatment also disrupted the interaction between NLRP3 and TXNIP in vitro; CONCLUSIONS: Our study demonstrated the therapeutic effects of PGSF on MCAO-induced focal ischemia rats. Moreover, the neuroprotective mechanism of PGSF on focal ischemia was associated with the inhibition of TXNIP/NLRP3 signaling pathway.

Ezetimibe ameliorates cisplatin-induced nephrotoxicity: A novel therapeutic approach via modulating AMPK/Nrf2/TXNIP signaling.

Cisplatin (Cis) is among the most powerful antineoplastic medications, nevertheless, its serious side effects; particularly nephrotoxicity designates a major concern. Previous studies reported that ezetimibe (Eze), a well-known antihyperlipidemic drug, exerts additional trivial pharmacological effects. In this work, we displayed Eze as an intriguing protective candidate in a cisplatin-induced nephrotoxicity rat model through AMPK activation. Eze (10 mg/kg, p.o.) was administered for two weeks and Cis (10 mg/kg, i.p.) was administered on the 10th day to induce nephrotoxicity in male Wistar rats. Treatment with Eze greatly augmented the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and the antioxidant regulator; nuclear factor erythroid 2-related factor 2 (Nrf2), thus, mitigating oxidative injury through induction of the antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR). As well, Eze relieved inflammation by reducing protein expression of thioredoxin-interacting protein (TXNIP) and nucleotide-binding domain-like receptor protein 3 (NLRP3), which led to a decrease in the release of caspase-1, in addition to, the inflammatory markers IL-18 and IL-1 ß. Besides, Eze ameliorated apoptosis in the renal cells through inhibiting the phosphorylated Apoptosis signal-regulating kinase-1(p-ASK1), caspase-3 and reducing Bax/Bcl2ratio. Correspondingly, histopathological examination corroborated the previous biochemical findings. Collectively, Eze exerts significant renal protection against Cis-induced nephrotoxicity via antioxidant, anti-inflammatory and anti-apoptotic pathways that are probably mediated, at least partly, via activating AMPK/Nrf2/HO-1 pathway and conquering both TXNIP/NLRP3 inflammasome and TXNIP/ASK1 signaling pathways. To confirm the protective effect of Eze via AMPK-activation, an AMPK-inhibitor, dorsomorphin (Dors), when co-administered with Eze abolished its protective effect.

Protective effects of compound M01 on retinal ganglion cells in experimental anterior ischemic optic neuropathy by inhibiting TXNIP/NLRP3 inflammasome pathway.

Apoptotic death of retinal ganglion cells (RGCs) is a common pathologic feature in different types of optic neuropathy, including ischemic optic neuropathy and glaucoma, ultimately leading to irreversible visual function loss. Potent and effective protection against RGC death is determinative in developing a successful treatment for these optic neuropathies. This study evaluated the neuroprotective effect of a HECT domain-E3 ubiquitin ligase inhibitor, M01, on retinal ganglion cells after ischemic injury. Experimental anterior ischemic optic neuropathy (AION) was induced by photothrombotic occlusion of microvessels supplying optic nerve in rats. M01 was administered (100 mg/Kg and 200 mg/Kg) subcutaneously for three consecutive days after AION induction. Administration of M01 (100 mg/Kg) significantly increased RGC survival and preserved visual function after AION induction. The number of TUNEL-positive cells and ED1-positive cells was significantly decreased, and optic disc edema was reduced considerably after ischemic infarction with M01 treatment. Moreover, M01 effectively ameliorated optic nerve demyelination and enhanced M2 microglial polarization after AION induction. M01 enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2); subsequently, downregulated Thioredoxin interacting protein (TXNIP) expression, inhibited NLR family pyrin domain containing 3 (NLRP3) activation, and further decreased inflammatory factors, interleukin (IL)-1ß and IL-6 in the retina after ischemic injury. These findings suggested that M01 has therapeutic potential by modulating Nrf2 and TXNIP/NLRP3 inflammasome pathways in the retina and optic nerve ischemic damage-related diseases.

Oral intake of titanium dioxide nanoparticles affect the course and prognosis of ulcerative colitis in mice: involvement of the ROS-TXNIP-NLRP3 inflammasome pathway.

BACKGROUND: Titanium dioxide (TiO2), no matter in nanoscale or micron sizes, has been widely used in food industry as additives for decades. Given the potential impact of TiO2 on the gastrointestinal epithelial and parenchymal cells, including goblet cells, the public consumers may suffer the risk of diseases caused by its widespread dissemination in food products. We therefore set out to investigate the impact of TiO2 NPs on the course and prognosis of ulcerative colitis by oral gavaging TiO2 NPs at the doses levels of 0, 30, 100, and 300 mg/kg during the induction (7 days, from day 1 to day 7) and recovery (10 days, from day 8 to day 17) phases of colitis in mice. RESULTS: The ulcerative colitis (UC) disease model was established by administrating of 2.5% dextran sulfate sodium (DSS) solution. Our results show that TiO2 NPs significantly enhanced the severity of DSS-induced colitis, decreased the body weight, increased the disease activity index (DAI) and colonic mucosa damage index (CMDI) scores, shortened the colonic length, increased the inflammatory infiltration in the colon. The most significant changes occurred in the low dose (30 mg/kg) group of TiO2 NPs exposure during the development phase of UC and the high dose (300 mg/kg) group of TiO2 NPs during UC self-healing phase. Increased reactive oxygen species (ROS) level and upregulation of anti-oxidant enzymes including total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) and catalase (CAT), demonstrate that the TiO2 NP exposure has triggered oxidative stress in mice. Moreover, the upregulation of caspase-1 mRNA and increased expression of thioredoxin interacting protein (TXNIP) further demonstrate the involvement of the ROS-TXNIP-NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in aggravating the development of UC. CONCLUSION: Oral intake of TiO2 NPs could affect the course of acute colitis in exacerbating the development of UC, prolonging the UC course and inhibiting UC recovery.

Memantine mitigates ROS/TXNIP/NLRP3 signaling and protects against mouse diabetic retinopathy: Histopathologic, ultrastructural and bioinformatic studies.

Diabetic retinopathy (DRET) triggers vision loss in adults, however, little therapeutic options are existing. Memantine is an anti-Alzheimer drug that antagonizes the activity of glutamate at N-methyl-D-aspartate (NMDA) receptors. Glutamate and thioredoxin-interacting protein (TXNIP) are known to be overexpressed in diabetic retinas and can produce activation of NOD-like receptor protein 3 (NLRP3) with subsequent secretion of interlukin-1ß. This study repurposed memantine for its neuroprotective effect in experimental DRET and tested its impact on ROS/TXNIP/NLRP3. In addition, KEGG pathway database and STRING database identified the protein-protein interaction between glutamate receptors and TXNIP/NLRP3. Male Swiss albino mice received alloxan (180 mg/kg) to induce DRET. After 9 weeks, we divided the mice into groups: (a) saline, (ii) DRET, (iii and iv) DRET + oral memantine (5 or 10 mg per kg) for 28 days. Then, mice were euthanized, and eyeballs were removed. Retinal samples were utilized for biochemical, histopathological, and electron microscopy studies. Retinal levels of glutamate, TXNIP, NLRP3 and interlukin-1ß were estimated using ELISA technique as well as retinal malondialdehyde. Histopathological and ultrastructural examination demonstrated that oral memantine attenuated vacuolization and restored normal retinal cell layers. Moreover, memantine reduced TXNIP, NLRP3, interleukin-1ß and MDA concentrations. These results provide evidence demonstrating memantine' efficacy in alleviating DRET via suppressing reactive oxygen species/TXNIP/NLRP3 signaling cascade. Therefore, memantine might serve as a potential therapy for retinopathy after adequate clinical research.

Retinoic acid inhibits the pyroptosis of degenerated nucleus pulposus cells by activating Sirt1-SOD2 signaling.

AIM: Intervertebral disc (IVD) degeneration is a common disease initiated by the degeneration of the nucleus pulposus (NP). The pyroptosis of degenerated NP cells (dNPCs) plays an important role in NP degeneration. The purpose of this study is to identify a feasible solution that can inhibit NP cell pyroptosis to therapy the degeneration of the intervertebral disc. METHODS: Cell viability and proliferation were quantified by Cell Counting Kit-8 assay. The measurement of cellular reactive oxygen species (ROS) was detected by 2,7-Dichlorodi-hydrofluorescein diacetate. The death of cells was analyzed by the Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) method of fluorescence analysis. The pyroptosis of cells was assessed by flow cytometry analyses. The contents of sulfate glycosaminoglycans were detected by a blyscan assay kit. RESULT: In this study, we determined the effects of retinoic acid (RA) on dNPCs and investigated the underlying mechanism of RA-mediated pyroptosis in dNPCs. We also verified the effects of RA on IVD degeneration in vivo. Our results demonstrated that RA significantly increased the proliferation and the protein expression of sox9, aggrecan, and collagen II of dNPCs. Pyroptosis-related proteins and the pyroptosis rate of dNPCs were significantly decreased by RA. We found that Sirt1-SOD2 signaling was activated, while ROS generation and TXNIP/NLRP3 signaling in dNPCs were inhibited after the addition of RA. Furthermore, RA also recovered the structure of NP and increased the contents of sulfated glycosaminoglycans and collagen in vivo. CONCLUSION: Our study demonstrated that RA could inhibit the pyroptosis and increase the extracellular matrix synthesis function of dNPCs and verified that RA has a protective effect on IVD degeneration.

Chrysin ameliorates synovitis and fibrosis of osteoarthritic fibroblast-like synoviocytes in rats through PERK/TXNIP/NLRP3 signaling.

Objective: Synovitis and fibrosis are common pathological features of knee osteoarthritis (KOA). The interaction of synovitis and fibrosis can promote KOA progression. Chrysin (CHR), a natural flavonoid, may treat inflammation and prevent fibrosis. However, the effect and mechanism of CHR in KOA synovitis and fibrosis remains unclear. Methods: The KOA model was established in male SD rats by anterior cruciate ligament transection (ACLT), and histological analysis was used to evaluate synovitis and fibrosis. IL-6, IL-1ß and TNF-α mRNA expression in synovial tissue was measured by qRT‒PCR. Immunohistochemistry (IHC) was performed to detect GRP78, ATF-6 and TXNIP expression in vivo. Synovial fibroblasts (SFs) were treated with TGF-ß1 to stimulate the inflammatory response and fibrosis. CCK-8 assays were used to detect the viability of CHR-treated SFs. The IL-1ß level was detected by immunofluorescence analysis. Coimmunoprecipitation (Co-IP) and double immunofluorescence colocalization were used to detect the physiological interaction between TXNIP and NLRP3. The expression of fibrosis-related mediators and PERK/TXNIP/NLRP3 signaling molecules was detected by western blotting and qRT-PCR. Results: Four weeks after CHR treatment, pathological sections and associated scores showed that CHR improved synovitis and fibrosis in the ACLT model. In vitro, CHR attenuated the TGF-ß1-induced inflammatory response and fibrosis in SFs. Moreover, CHR suppressed the expression of synovial fibrosis markers and PERK/TXNIP/NLRP3 signaling molecules in the synovial tissue of rats with ACLT and cultured SFs. More importantly, we found that CHR inhibited TXNIP-NLRP3 interactions in TGF-ß-induced SFs. Conclusion: Our findings indicate that CHR can ameliorate synovitis and fibrosis in KOA. The underlying mechanism may be related to the PERK/TXNIP/NLRP3 signaling pathway.

Caveolin-1 ameliorates acetaminophen-aggravated inflammatory damage and lipid deposition in non-alcoholic fatty liver disease via the ROS/TXNIP/NLRP3 pathway.

The overuse of acetaminophen (APAP) may cause more severe hepatotoxicity in patients with non-alcoholic fatty liver disease (NAFLD). Caveolin-1 (CAV1), is an essential regulator of metabolic function, which can alleviate liver damage by scavenging reactive oxygen species (ROS). Evidence suggests that the NOD-like receptor family pyrin domain-containing 3 (NLRP3) -mediated pyroptosis is involved in the development of NAFLD. Moreover, thioredoxin-interactive protein (TXNIP) activation is a key event linking ROS to NLRP3 inflammasome. However, whether CAV1 alleviates APAP-aggravated hepatotoxicity in NAFLD via the ROS/TXNIP/NLRP3 pathway remains unclear. An in vivo fatty liver model was established by feeding mice a high-fat diet for 56 days. Additionally, using in vitro approach, AML-12 cells were incubated with free fatty acids for 48 h and APAP was added during the last 24 h. We found that the overuse of APAP in NAFLD not only induced oxidative stress, but also increased TXNIP expression, NLRP3-mediated pyroptosis, and lipid deposition. In addition to inhibiting ROS generation and lipid deposition, overexpression of CAV1 reduced the elevated levels of TXNIP expression and NLRP3-mediated pyroptosis. However, the effect of CAV1 on TXNIP expression, NLRP3-mediated pyroptosis, and lipid deposition was reversed by CAV1 small interfering RNA (siRNA) intervention. Finally, N-acetyl cysteine (NAC) treatment reduced CAV1 siRNA-mediated changes in TXNIP expression and NLRP3-mediated pyroptosis levels. These results demonstrate that the inhibitory effect of CAV1 on NLRP3-mediated pyroptosis may be mediated through the ROS/TXNIP axis. Moreover, the current study provides novel mechanistic insights into the protective effects of CAV1 on APAP-aggravated hepatotoxicity in NAFLD.

Heme oxygenase-1 ameliorates endotoxin-induced acute lung injury by modulating macrophage polarization via inhibiting TXNIP/NLRP3 inflammasome activation.

Acute lung injury (ALI) remains a global public health issue without specific and effective treatment options available in the clinic. Alveolar macrophage polarization is involved in the initiation, development and progression of ALI; however, the underlying mechanism remains poorly understood. Heme oxygenase-1 (HO-1) acts as an antioxidant in pulmonary inflammation and has been demonstrated to be linked with the severity and prognosis of ALI. In this study, the therapeutic effects of HO-1 were examined, along with the mechanisms involved, mainly focusing on alveolar macrophage polarization. HO-1 depletion induced higher iNOS and CD86 (M1 phenotype) expression but was significantly decreased in Arg-1 and CD206 (M2 phenotype) expression in BALF alveolar macrophages after equivalent LPS stimulation. We also found that HO-1 deletion distinctly accelerated the expression of inflammasome-associated components NLRP3, ASC and caspase-1 in vivo and in vivo and in vitro. Moreover, on the basis of LPS for MH-S cells, levels of TXNIP, NLRP3, ASC and caspase-1 were increased and HO-1 depletion exacerbated these changes, whereas double depletion of HO-1 and TXNIP partially mitigated these elevations. Also, HO-1 knockdown induced more M1 phenotype and less M2 phenotype compared with LPS alone, whereas double silence of HO-1 and TXNIP partially changed the polarization state. Taken together, we demonstrated that HO-1 could modulate macrophage polarization via TXNIP/NLRP3 signaling pathway, which could be a potential therapeutic target for ALI treatment.

Hypouricemic and nephroprotective effects of palmatine from Cortex Phellodendri Amurensis: A uric acid modulator targeting Keap1-Nrf2/NLRP3 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Palmatine (Pal) is a major bioactive alkaloid originated from ancient Chinese herbal medicine Cortex Phellodendri Amurensis (CPA), which has long been applied to treat hyperuricemia (HUA)-related diseases. Pal possesses potent anti-inflammatory and anti-oxidant effects against metabolic diseases. However, its potential beneficial effect against PO (potassium oxonate)/HX (hypoxanthine)-induced HUA remains elusive. AIM OF THE STUDY: This study aimed to investigate the potential pharmacological effect and mechanism of Pal on PO/HX-induced HUA in mice. MATERIAL AND METHODS: A mouse model of HUA was established by co-administration of PO/HX once daily for 7 consecutive days. The HUA mice were orally given three doses (25, 50 and 100 mg/kg) of Pal daily for a week. Febuxostat (Feb, 5 mg/kg) was given as a positive control. At the scheduled termination of the experiment, the whole blood, liver and kidney were collected for subsequent analyses. The concentrations of uric acid (UA), creatinine (CRE) and blood urea nitrogen (BUN), and activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) were evaluated. Histopathological alterations of the kidney were detected by H&E staining. The inflammatory and oxidative stress status was detected by assay kits. Additionally, key proteins involved in the urate transporter, Keap1-Nrf2 and TXNIP/NLRP3 signaling pathways were evaluated by immunohistochemistry and Western blotting. Finally, molecular docking was employed to probe the binding characteristics of Pal and target proteins Keap1, NLRP3, URAT1 and HO-1. RESULTS: Administration of Pal substantially decreased the elevated kidney weight, lowered UA, CRE and BUN levels, and attenuated abnormal histopathological alterations. Meanwhile, treatment with Pal also dramatically lowered hepatic XOD and ADA activities. Besides, Pal treatment effectively mitigated the renal inflammatory and oxidative stress markers. Further mechanistic investigation indicated Pal distinctly downregulated the protein levels of GLUT9 and URAT1, while up-regulated the expression levels of OAT1 and ABCG2. Pal also restored Nrf2 activation, promoted subsequent expression of anti-oxidative enzymes, and downregulated the expressions of TXNIP, NLRP3, apoptosis-associated speck-like (ASC), caspase-1, IL-1ß and IL-18. Molecular docking analysis also indicated Pal firmly bound with Keap1, NLRP3, URAT1 and HO-1. CONCLUSIONS: These findings indicated that Pal exhibited favorable anti-HUA effect via modulating the expressions of transporter-related proteins and suppressing XOD activity. Furthermore, Pal also alleviated HUA-induced kidney injury, which was at least partially related to restoring Keap1-Nrf2 pathway and inhibiting TXNIP/NLRP3 inflammasome. Our investigation was envisaged to provide experimental support for the traditional application of CPA and CPA-containing classical herbal formulas in the management of HUA-related diseases and might provide novel dimension to the clinical application of Pal.

Panax notoginseng saponin Rl attenuates allergic rhinitis through AMPK/DRP1 mediated mitochondrial fission / 中国药理学通报

Aim To investigate whether notoginsenoside Rl (PNS-R1) alleviates allergic rhinitis (AR) through AMP-activated protein kinase (AMPK)/mitochondrial fission critical protein (DRP1) -mediated mitochondrial fission. Methods Different doses of PNSRl were used to treat ovalbumin (OVA) -induced AR model mice,and the inhibitory effect of PNS-R1 on AR was investigated by observing allergic symptoms such as nasal rubbing and sneezing, as well as HE staining of nasal tissues. Serum IgE levels and nasal lavage fluid (NLF) inflammatory cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA) and apoptosis-related proteins were detected by Western blot. In vitro human nasal epithelial cells (HNEpC) were stimulated with IL-13 to observe apoptosis, mitochondrial membrane potential, cellular ROS and mitochondrial ROS production, as well as the expression levels of AMPK/DRP1, expression levels of the TXNIP/NLRP3 inflammasomes and the translocation of DRP1. Results PNS-R1 attenuated allergic symptoms in AR mice, HE staining reduced inflammatory cells and reduced the levels of OVA-specific IgE in serum, and the levels of IL-4, IL-6, and IL-8 in NLF. PNS-R1 attenuated the apoptosis and ROS production of nasal epithelial cells in AR. In vitro PNS-R1 could up-regulate mitochondrial membrane potential after IL-13 stimulation, reduce ROS and mtROS production, the proportion of apoptotic positive cells, and reduce cleaved caspase-3, Bax, and up-regulate Bcl-2 expression, down-regulate DRP1 phosphorylation (Ser 616) and DRP1 translocation at the mitochondrial membrane in an AMPK-dependent manner, reducing TXNIP/NLRP3 expression. Conclusions PNS-R1 can protect mitochondrial integrity by inhibiting the AMPK/DRP1 signaling axis and its subsequent TXNIP/NLRP3 signaling axis,thereby alleviating rhinitis in AR mice.

Role of TXNIP/NLRP3 pathway in renal interstitial fibrosis after renal ischemia-reperfusion injury / 中华急诊医学杂志

Objective:To explore the role of thioredoxin interaction protein (TXNIP)/NOD-like receptor protein 3 (NLRP3) pathway in renal interstitial fibrosis induced by renal ischemia-reperfusion injury (IRI) in mice.Methods:Adult male C57BL/6J mice aged 6 to 8 weeks and TXNIP knockout mice with the same genetic background were selected. The wild type mice were divided into the sham operation (Sham) group and renal IRI group. The TXNIP knockout mice were divided into the sham+TXNIP KO group and IRI+TXNIP KO group, with 12 mice in each group. The model of renal ischemia-reperfusion injury was established by clamping bilateral renal pedicles for 45 min and then restoring perfusion. The sham operation model was only dissociated bilateral renal arteries without other treatment. Blood creatinine, urea nitrogen, kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL), blood transforming growth factor-β (TGF-β) and interleukin 6 (IL-6) were measured on the 1st, 7th and 28th days after reperfusion. The renal cortex was taken on the 1st and 28th days for Masson staining, in which the renal tubule-interstitial injury score was obtained. TGF-β and IL-6 mRNA expression were detected by qPCR, TXNIP, NLRP3, Pro-IL-1β, IL-1β and α-SMA protein expression were detected by Western blot, and MDA and SOD levels were detected by ELISA. Homogeneity test of variance was performed before the statistics of normal distribution measurement data, one-way ANOVA was used for the comparison between multiple groups, and LSD- t test was used for the comparison between the two groups. Results:On the 1st, 7th and 28th days after IRI, compared with the sham group, the Scr, BUN, Kim-1, NGAL, TGF-β and IL-6 were increased continuously in the IRI group ( P<0.05). On the 28th day after IRI, large areas of collagen fibers and inflammatory cell infiltration were observed in the renal interstitium of the IRI group. In the IRI group, the scores of renal tubular injury and renal interstitial fibrosis on the 28th day were significantly higher than those on the 1st day (all P<0.05). On the 1st, 7th and 28th days after IRI, compared with the IRI group, the levels of Scr, BUN, Kim-1, NGAL, TGF-β and IL-6 were significantly decreased in the IRI+TXNIP KO group (all P<0.05). On the 1st and 28th days after IRI, compared to the IRI group, the areas of collagen fibers and inflammatory cell infiltration in the renal interstitium of the IRI+TXNIP KO group were decreased. The renal tubule injury score [Day 1, (192.2 ± 62.4) vs. (103.2 ± 49.1); Day 28, (154.3 ± 93.6) vs. (64.3 ± 24.8), both P<0.05] and interstitial fibrosis score [Day 1, (7.3 ± 3.2) vs. (4.8 ± 1.7); Day 28, (12.8 ± 3.9) vs. (2.3 ± 0.8), both P<0.05] were all decreased. The expression of TGF-β, IL-6 mRNA, TXNIP, NLRP3, Pro-IL-1 β, IL-1 β and α-SMA protein in renal cortex were significantly decreased (both P<0.05). In renal cortex, MDA level was decreased and SOD level was increased (all P<0.05). Conclusions:TXNIP/NLRP3 pathway is involved in the development of renal interstitial inflammation and fibrosis after renal ischemia and reperfusion. Knockout or inhibition of TXNIP can inhibit the progression of acute renal injury to chronic renal disease.

Ceramide induces pyroptosis through TXNIP/NLRP3/GSDMD pathway in HUVECs.

BACKGROUND: Pyroptosis of endothelial cells is a new cause of endothelial dysfunction in multiple diseases. Ceramide acts as a potential bioactive mediator of inflammation and increases vascular endothelial permeability in many diseases, whether it can aggravate vascular endothelial injury by inducing cell pyroptosis remains unknown. This study was established to explore the effects of C8-ceramide (C8-Cer) on human umbilical vein vascular endothelial cells (HUVECs) and its possible underlying mechanism. METHODS: HUVECs were exposed to various concentrations of C8-Cer for 12 h, 24 h, 48 h. The cell survival rate was measured using the cell counting kit-8 assay. Western blotting and Real-time polymerase chain reaction (RT-PCR) were used to detect the pyroptosis-releated protein and mRNA expressions, respectively. Caspase-1 activity assay was used to detect caspase-1 activity. Hoechst 33342/propidium iodide double staining and flow cytometry were adopted to measure positive staining of cells. Lactate dehydrogenase release assay and enzyme-linked immunosorbent assay were adopted to measure leakage of cellular contents. FITC method was used to detect the permeability of endothelial cells. ROS fluorescence intensity were detected by flow cytometry. RESULTS: The viability of HUVECs decreased gradually with the increase in ceramide concentration and time. Ceramide upregulated the expression of thioredoxin interacting protein (TXNIP), NLRP3, GSDMD, GSDMD-NT, caspase-1 and Casp1 p20 at the protein and mRNA level in a dose-dependent manner. It also enhanced the PI uptake in HUVECs and upregulated caspase-1 activity. Moreover, it promoted the release of lactate dehydrogenase, interleukin-1ß, and interleukin-18. Meanwhile, we found that ceramide led to increased vascular permeability. The inhibitor of NLRP3 inflammasome assembly, MCC950, was able to disrupt the aforementioned positive loop, thus alleviating vascular endothelial cell damage. Interestingly, inhibition of TXNIP either chemically using verapamil or genetically using small interfering RNA (siRNA) can effectively inhibit ceramide-induced pyroptosis and improved cell permeability. In addition, ceramide stimulated reactive oxygen species (ROS) generation. The pretreatment of antioxidant N-acetylcysteine (NAC), ROS scavenger, blocked the expression of pyroptosis markers induced by C8-cer in HUVECs. CONCLUSION: The current study demonstrated that C8-Cer could aggravate vascular endothelial cell damage and increased cell permeability by inducing cell pyroptosis. The results documented that the ROS-dependent TXNIP/NLRP3/GSDMD signalling pathway plays an essential role in the ceramide-induced pyroptosis in HUVECs.

Nicorandil protects against coronary microembolization-induced myocardial injury by suppressing cardiomyocyte pyroptosis via the AMPK/TXNIP/NLRP3 signaling pathway.

BACKGROUND: Coronary microembolization (CME) is a common and intractable complication of coronary revascularization, which leads to perioperative myocardial injury, cardiac dysfunction, and poor prognosis. Nicorandil is widely used for the management of ischemic heart diseases, but the cardioprotective effects of nicorandil beyond anti-angina in CME-induced myocardial injury are worthy of further exploration. Therefore, the present study investigated the effect of nicorandil on CME-induced cardiomyocyte pyroptosis and explored the underlying mechanism. METHODS: A rat model of CME was established via the injection of microspheres into the left ventricle. A cell model of H9c2 cardiomyocytes stimulated by lipopolysaccharide (LPS) and hypoxia mimicked the microenvironment induced by CME. Nicorandil or the adenosine monophosphate-activated protein kinase (AMPK)-specific inhibitor compound C (CC) was administered before CME induction and cell modeling. Cardiac function, histological alterations in the myocardium, myocardial injury biomarkers in serum and cell culture supernatant, cell viability, adenosine triphosphate (ATP) level, superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) activity, mitochondrial membrane potential, and pyroptosis-associated index were assessed after the animal and cell modeling of CME. RESULTS: Nicorandil pretreatment attenuated cardiac dysfunction and myocardial injury following CME. Nicorandil also alleviated oxidative stress and mitochondrial damage. Moreover, nicorandil promoted AMPK activation, reduced the expression of thioredoxin-interacting protein (TXNIP), inhibited the activation of the NOD-like receptor pyrin containing 3 (NLRP3) inflammasome, and mitigated cardiomyocyte pyroptosis. However, co-treatment with CC reversed the cardioprotective effects of nicorandil. CONCLUSION: Nicorandil pretreatment inhibits cardiomyocyte pyroptosis and alleviates CME-induced myocardial injury via the AMPK/TXNIP/NLRP3 signaling pathway.

TXNIP Participated in NLRP3-Mediated Inflammation in a Rat Model of Cervical Spondylotic Myelopathy.

Background: Cervical spondylotic myelopathy (CSM) is a spinal cord disease caused by cervical disc degeneration and related pathological changes. Cervical spondylotic myelopathy may result from inflammation responses and neuronal damage. Thioredoxin-interacting protein (TXNIP)/NOD-like receptor protein 3 (NLRP3) signaling promotes inflammation. However, the effects of TXNIP/NLRP3 on the pathogenesis of CSM have not been reported. Methods: A rat model of chronic cervical cord compression was established to observe changes in the levels of of TNXIP/NeuN and NLRP3/NeuN expression in the damaged anterior horn of the spinal cord following progression of CSM. Rats were injected with TXNIP small interfering RNA (siRNA) and scrambled control to determine the effects of TXNIP inhibition on NLRP3-mediated inflammation in rats with CSM. Behaviors effects and the expression of NLRP3 and pro-caspase-1 in the damaged spinal cord were evaluated. Results: The expression levels of TXNIP and NLRP3 were significantly increased in the damaged anterior horn of the spinal cord following CSM. Injection of TXNIP siRNA significantly improved behavioral measures and decreased apoptosis in the damaged anterior horn of spinal cord. Furthermore, the levels of NLRP3 and pro-caspase-1 in the lesioned area were reduced by the TXNIP siRNA injection. Conclusion: Thioredoxin-interacting protein participated in NLRP3 mediated inflammation in a rat model of CSM, which indicated that TXNIP may be a potential therapeutic target in improving CSM.

Fudosteine attenuates acute lung injury in septic mice by inhibiting pyroptosis via the TXNIP/NLRP3/GSDMD pathway.

There is a dearth of effective pharmacotherapies for sepsis-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS) to which oxidative stress and excessive inflammation are major contributors. We hypothesized that fudosteine, a cysteine derivative, may protect against sepsis-induced ALI/ARDS given its anti-oxidant capacity. This study aimed to investigate the effects and mechanisms of fudosteine in a mouse model of sepsis-induced ALI. Sepsis was induced by cecal ligation and puncture (CLP). The intragastrical administration of fudosteine (25 mg/kg, 50 mg/kg, and 100 mg/kg) dose-dependently decreased proinflammatory cytokine levels in bronchoalveolar lavage fluid (BALF) and serum and reduced BALF/serum albumin and lung wet/dry weight ratios in septic mice. The lung injury score was significantly lowered by fudosteine [e.g., 0.18 ± 0.03 (100 mg/kg) vs. 0.42 ± 0.03 (CLP), P < 0.0001]. Fudosteine also reduced the biomarkers of lung epithelial injury in BALF and markedly improved oxidative stress indicators in lung tissues [e.g., malondialdehyde: 337.70 ± 23.78 (100 mg/kg) vs. 686.40 ± 28.36 (CLP) nmol/mg protein, P < 0.0001]. Lung tissue transcriptomics analyses revealed suppressed inflammatory responses and oxidative stress with fudosteine and the involvement of the inflammasome and pyroptosis pathways. Western blot analyses indicated that fudosteine inhibited the sepsis-induced activation of gasdermin D (GSDMD) and caspase-1 and the upregulation of thioredoxin-interacting protein (TXNIP), nucleotide-binding domain, leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Fudosteine therefore protects against sepsis-induced ALI in mice, and the inhibition of pyroptosis via the TXNIP/NLRP3/GSDMD pathway may be an underlying mechanism.

Paeoniflorin ameliorates diabetic liver injury by targeting the TXNIP-mediated NLRP3 inflammasome in db/db mice.

BACKGROUND: Diabetic liver injury (DLI) is a complication that damages the quality of life in diabetes patients. While paeoniflorin (PF) exhibits anti-inflammatory and antioxidant effects, no data are available on whether PF protects against DLI. Therefore, we evaluated the effects of PF on hepatic steatosis and inflammation in db/db mice, a type 2 diabetes model. METHODS: In this study, we investigated the effects of PF on DLI using diabetic mice model (db/db mice) and high glucose (HG)-induced mouse AML12 cells. The effects of PF on TXNIP-mediated NLRP3 inflammasome in vivo and in vitro were evaluated by Western bloting, RT-PCR, immunohistochemistry (IHC) and immunofluorescence (IF) analysis. Through molecular docking experiments and cellular thermal shift assay (CETSA), we studied the binding ability of PF to thioredoxin-interacting protein (TXNIP). We use TXNIP siRNA to knock down TXNIP in AML12 cells. RESULTS: We found that PF reversed abnormal liver function and liver steatosis in db/db mice, while blocking the release of inflammatory cytokines. These effects are associated with PF inhibition of the TXNIP/NLRP3 signaling pathway. Molecular docking experiments and CETSA also demonstrated that TXNIP is a likely target of PF. In HG-treated AML12 cells, TXNIP knockdown eliminated the beneficial effects of PF. CONCLUSION: Using a combination of animal and in vitro experiments, this study demonstrated for the first time that PF ameliorates DLI through targeting the TXNIP-activated NLRP3 inflammasome. Thus, PF may be a potential therapeutic agent against DLI.