Alternative polyadenylation writer CSTF2 forms a positive loop with FGF2 to promote tubular epithelial-mesenchymal transition and renal fibrosis

Effective therapies for renal fibrosis, the common endpoint for most kidney diseases, are lacking. We previously reported that alternative polyadenylation (APA) drives transition from acute kidney injury to chronic kidney disease, suggesting a potential role for APA in renal fibrogenesis. Here, we found that among canonical APA writers, CSTF2 expression was upregulated in tubular epithelial cells (TEC) of fibrotic kidneys. CSTF2 was also identified as a TGF-β-inducible pro-fibrotic gene. Further analysis revealed that CSTF2 promoted epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) overproduction in TEC by inducing 3’UTR shortening and upregulation of the expression of basic fibroblast growth factor 2 (FGF2). Additionally, 3’UTR shortening stabilised FGF2 mRNA through miRNA evasion. Interestingly, FGF2 enhanced CSTF2 expression, leading to the forming of a CSTF2-FGF2 positive loop in TEC. Furthermore, CSTF2 knockdown alleviated unilateral ureteral obstruction-induced renal fibrosis in vivo. Finally, we developed a CSTF2-targeted antisense oligonucleotide (ASO) and validated its effectiveness in vitro. These results indicate that the expression of the APA writer, CSTF2, is upregulated by TGF-β and CSTF2 facilitates TGF-β-induced FGF2 overexpression, forming a TGF-β-CSTF2-FGF2 pro-fibrotic axis in TEC. CSTF2 is a potentially promising target for renal fibrosis that does not directly disrupt TGF-β.     

Estrogen-induced upregulation and 3′-UTR shortening of CDC6

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.     

PGC-1α inhibits the NLRP3 inflammasome via preserving mitochondrial viability to protect kidney fibrosis

The NLRP3 inflammasome is activated by mitochondrial damage and contributes to kidney fibrosis. However, it is unknown whether PGC-1α, a key mitochondrial biogenesis regulator, modulates NLRP3 inflammasome in kidney injury. Primary renal tubular epithelial cells (RTECs) were isolated from C57BL/6 mice. The NLRP3 inflammasome, mitochondrial dynamics and morphology, oxidative stress, and cell injury markers were examined in RTECs treated by TGF-β1 with or without Ppargc1a plasmid, PGC-1α activator (metformin), and siPGC-1α. In vivo, adenine-fed and unilateral ureteral obstruction (UUO) mice were treated with metformin. In vitro, TGF-β1 treatment to RTECs suppressed the expressions of PGC-1α and mitochondrial dynamic-related genes. The NLRP3 inflammasome was also activated and the expression of fibrotic and cell injury markers was increased. PGC-1α induction with the plasmid and metformin improved mitochondrial dynamics and morphology and attenuated the NLRP3 inflammasome and cell injury. The opposite changes were observed by siPGC-1α. The oxidative stress levels, which are inducers of the NLRP3 inflammasome, were increased and the expression of TNFAIP3, a negative regulator of NLRP3 inflammasome regulated by PGC-1α, was decreased by TGF-β1 and siPGC-1α. However, PGC-1α restoration reversed these alterations. In vivo, adenine-fed and UUO mice models showed suppression of PGC-1α and TNFAIP3 and dysregulated mitochondrial dynamics. Moreover, the activation of oxidative stress and NLRP3 inflammasome, and kidney fibrosis were increased in these mice. However, these changes were significantly reversed by metformin. This study demonstrated that kidney injury was ameliorated by PGC-1α-induced inactivation of the NLRP3 inflammasome via modulation of mitochondrial viability and dynamics.Subject terms: Mechanisms of disease, Experimental models of disease     

Irisin protects against vascular calcification by activating autophagy and inhibiting NLRP3-mediated vascular smooth muscle cell pyroptosis in chronic kidney disease

Irisin protects the cardiovascular system against vascular diseases. However, its role in chronic kidney disease (CKD) -associated vascular calcification (VC) and the underlying mechanisms remain unclear. In the present study, we investigated the potential link among Irisin, pyroptosis, and VC under CKD conditions. During mouse vascular smooth muscle cell (VSMC) calcification induced by β-glycerophosphate (β-GP), the pyroptosis level was increased, as evidenced by the upregulated expression of pyroptosis-related proteins (cleaved CASP1, GSDMD-N, and IL1B) and pyroptotic cell death (increased numbers of PI-positive cells and LDH release). Reducing the pyroptosis levels by a CASP1 inhibitor remarkably decreased calcium deposition in β-GP-treated VSMCs. Further experiments revealed that the pyroptosis pathway was activated by excessive reactive oxygen species (ROS) production and subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in calcified VSMCs. Importantly, Irisin effectively inhibited β-GP-induced calcium deposition in VSMCs in vitro and in mice aortic rings ex vivo. Overexpression of Nlrp3 attenuated the suppressive effect of Irisin on VSMC calcification. In addition, Irisin could induce autophagy and restore autophagic flux in calcified VSMCs. Adding the autophagy inhibitor 3-methyladenine or chloroquine attenuated the inhibitory effect of Irisin on β-GP-induced ROS production, NLRP3 inflammasome activation, pyroptosis, and calcification in VSMCs. Finally, our in vivo study showed that Irisin treatment promoted autophagy, downregulated ROS level and thereby suppressed pyroptosis and medial calcification in aortic tissues of adenine-induced CKD mice. Together, our findings for the first time demonstrated that Irisin protected against VC via inducing autophagy and inhibiting VSMC pyroptosis in CKD, and Irisin might serve as an effective therapeutic agent for CKD-associated VC.Subject terms: Calcification, Chronic kidney disease     

Upregulation of NLRP3 Inflammasome in the Tears and Ocular Surface of Dry Eye Patients

PurposeTo evaluate the mRNA and protein expressions of NLRP3 inflammasome and its downstream inflammatory factors in human dry eye.MethodsWe recruited 54 patients with Sjögren’s syndrome dry eye (SSDE), 50 patients with non-Sjögren’s syndrome dry eye (NSSDE), and 46 healthy controls. Tear film breakup time (TBUT), Schirmer I test, and fluorescein staining (FL) were performed on all subjects. Tear samples were obtained to analyze the inflammatory cytokine levels of IL-1β and IL-18 via enzyme-linked immunosorbent (ELISA). Conjunctival impression cytology (CIC) specimens were collected to detect the mRNA expression of NLRP3, caspase-1, IL-1β, and IL-18 using quantitative RT-PCR, and the protein expression of NLRP3 and caspase-1 by Western blotting.ResultsNLRP3 mRNA expression showed higher levels in both dry eye groups compared with controls, with a comparably significant elevation in the SSDE group (relative 2.47-fold upregulation, p<0.05). NLRP3 protein expression was also increased in SSDE group (relative1.94-fold upregulation) compared with the controls. mRNA expression of caspase-1 was significantly upregulated in both SSDE (relative 1.44-fold upregulation, p<0.05) and NSSDE (relative 1.32-fold upregulation, p<0.05). Procaspase-1 protein level was increased in SSDE (relative 1.84-fold upregulation) and NSSDE (relative 1.12-fold upregulation) versus controls; and caspase-1 protein expression was also increased in SSDE (relative 1.49-fold upregulation) and NSSDE (relative 1.17-fold upregulation) compared with the controls. The patients with SSDE and NSSDE had higher IL-1β and IL-18 mRNA values and protein expressions than the controls did. The relative mRNA expression of IL-1β upregulated 3.59-fold (p<0.001) in SSDE and 2.13-fold (p<0.01) in NSSDE compared with the controls. IL-1β protein level also showed significant upregulation in SSDE (p=0.01; vs. controls groups). IL-18 mRNA expression levels were significantly upregulated in the SSDE (relative 2.97-fold upregulation, p=0.001) and NSSDE (relative 2.05-fold upregulation, p=0.001) groups compared with the controls; tear IL-18 concentrations were also significantly increased in the SSDE (p<0.001) and NSSDE (p<0.05) groups.ConclusionsIn the current study, we found that mRNA and protein expressions of NLRP3 inflammasome were upregulated in human dry eyes, especially in SSDE; the downstream inflammatory factors caspase-1, IL-1β, and IL-18 were also elevated in dry eye patients. These observations suggest the involvement of NLRP3 inflammasome in the onset and development of the inflammation in dry eye.     

Hsp27 binding to the 3′UTR of bim mRNA prevents neuronal death during oxidative stress–induced injury: a novel cytoprotective mechanism

Neurons face a changeable microenvironment and therefore need mechanisms that allow rapid switch on/off of their cytoprotective and apoptosis-inducing signaling pathways. Cellular mechanisms that control apoptosis activation include the regulation of pro/antiapoptotic mRNAs through their 3′-untranslated region (UTR). This region holds binding elements for RNA-binding proteins, which can control mRNA translation. Here we demonstrate that heat shock protein 27 (Hsp27) prevents oxidative stress–induced cell death in cerebellar granule neurons by specific regulation of the mRNA for the proapoptotic BH3-only protein, Bim. Hsp27 depletion induced by oxidative stress using hydrogen peroxide (H₂O₂) correlated with bim gene activation and subsequent neuronal death, whereas enhanced Hsp27 expression prevented these. This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27. Finally, we determined that enhanced Hsp27 expression in neurons exposed to H₂O₂ or glutamate prevented the translation of a reporter plasmid where bim-3′UTR mRNA sequence was cloned downstream of a luciferase gene. These results suggest that repression of bim mRNA translation through binding to the 3′UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.     

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1β (IL-1β) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1β activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1β secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1β expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1β secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1β. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.Subject terms: Biochemistry, Cell biology     

Activation of the NLRP3 Inflammasome Complex is Not Required for Stress-Induced Death of Pancreatic Islets

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H₂O_2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.     

Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377

High dietary fructose is an important causative factor in the development of metabolic syndrome-associated glomerular podocyte oxidative stress and injury. Here, we identified microRNA-377 (miR-377) as a biomarker of oxidative stress in renal cortex of fructose-fed rats, which correlated with podocyte injury and albuminuria in metabolic syndrome. Fructose feeding increased miR-377 expression, decreased superoxide dismutase (SOD) expression and activity, and caused O₂⁻ and H₂O₂ overproduction in kidney cortex or glomeruli of rats. This reactive oxygen species induction increased p38 MAPK phosphorylation and thioredoxin-interacting protein (TXNIP) expression and activated the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome to produce interleukin-1β in kidney glomeruli of fructose-fed rats. These pathological processes were further evaluated in cultured differentiated podocytes exposed to 5 mM fructose, or transfected with miR-377 mimic/inhibitor and TXNIP siRNA, or co-incubated with p38 MAPK inhibitor, demonstrating that miR-377 overexpression activates the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway to promote oxidative stress and inflammation in fructose-induced podocyte injury. Antioxidants pterostilbene and allopurinol were found to ameliorate fructose-induced hyperuricemia, podocyte injury, and albuminuria in rats. More importantly, pterostilbene and allopurinol inhibited podocyte miR-377 overexpression to increase SOD1 and SOD2 levels and suppress the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway activation in vivo and in vitro, consistent with the reduction of oxidative stress and inflammation. These findings suggest that miR-377 plays an important role in glomerular podocyte oxidative stress, inflammation, and injury driven by high fructose. Inhibition of miR-377 by antioxidants may be a promising therapeutic strategy for the prevention of metabolic syndrome-associated glomerular podocyte injury.     

Depletion of TMEM65 leads to oxidative stress,apoptosis, induction of mitochondrial unfolded protein response,and upregulation of mitochondrial protein import receptor TOMM22

Mutation in the transmembrane protein 65 gene (TMEM65) results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype. However, neither the function of TMEM65 nor the cellular responses to its depletion have been fully elucidated. Hence, we knocked down TMEM65 in human cultured cells and analyzed the resulting cellular responses. Depletion of TMEM65 led to a mild increase in ROS generation and upregulation of the mRNA levels of oxidative stress suppressors, such as NFE2L2 and SESN3, indicating that TMEM65 knockdown induced an oxidative stress response. A mild induction of apoptosis was also observed upon depletion of TMEM65. Depletion of TMEM65 upregulated protein levels of the mitochondrial chaperone HSPD1 and mitochondrial protease LONP1, indicating that mitochondrial unfolded protein response (UPR^mt) was induced in response to TMEM65 depletion. Additionally, we found that the mitochondrial protein import receptor TOMM22 and HSPA9 (mitochondrial Hsp70), were also upregulated in TMEM65-depleted cells. Notably, the depletion of TMEM65 did not lead to upregulation of TOMM22 in an ATF5-dependent manner, although upregulation of LONP1 reportedly occurs in an ATF5-dependent manner. Taken together, our findings suggest that depletion of TMEM65 causes mild oxidative stress and apoptosis, induces UPR^mt, and upregulates protein expression of mitochondrial protein import receptor TOMM22 in an ATF5-independent manner.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.     

ARRB1/β-arrestin-1 mediates neuroprotection through coordination of BECN1-dependent autophagy in cerebral ischemia

Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation.     

Repression of TXNIP–NLRP3 axis restores intestinal barrier function via inhibition of myeloperoxidase activity and oxidative stress in nonalcoholic steatohepatitis

Dysfunction of the intestinal barrier function occurs in hepatic injury, but the specific mechanisms responsible are largely unknown. Recently, NOD-like receptor 3 (NLRP3) inflammasome functions in impairing endothelial barrier function. In this study, we test the hypothesis that TXNIP–NLRP3 axis repression prevents against intestinal barrier function disruption in nonalcoholic steatohepatitis (NASH). First, lipopolysaccharide (LPS)-induced alterations in expression of ZO-1 and occludin, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) level, and transepithelial electric resistance (TEER) in intestinal epithelial cells (IECs) isolated from C57BL/6 wild-type (WT) and TXNIP^−/− mice were evaluated. The underlying regulatory mechanisms of TXNIP knockout in vivo were investigated with the detection of expressions of TXNIP, NLRP3 and ZO-1, and occludin, the interaction of TXNIP–NLRP3, MPO activity, ROS level, permeability of intestinal mucosa, levels of inflammatory factors in serum, and LPS concentration. We identified that TXNIP knockout promoted ZO-1 and occludin expression, yet reduced MPO activity, ROS level, and cell permeability in IECs, indicating restored the intestinal barrier function. However, LPS upregulated TXNIP and NLRP3 expression, as well as contributed to the interaction between TXNIP and NLRP3 in vitro. Furthermore, TXNIP was significantly upregulated in the intestinal mucosa of NASH mice and its knockout repaired the intestinal barrier disrupt, inhibited expression of inflammatory factors, and reduced LPS concentration as well as hepatic injury in vivo. Taken together, our findings demonstrated that inhibited the activation of the TXNIP–NLRP3 axis reduced MPO activity and oxidative stress and thus restoring the intestinal barrier function in NASH. TXNIP–NLRP3 axis may be a promising therapeutic strategy for the NASH treatment.     

Hydrolyzed camel whey protein alleviated heat stress-induced hepatocyte damage by activated Nrf2/HO-1 signaling pathway and inhibited NF-κB/NLRP3 axis

Liver damage is the most severe complication of heat stress (HS). Hydrolyzed camel whey protein (CWP) possesses bioactive peptides with obviously antioxidant and anti-inflammatory activities. The current study aims to investigate whether CWP that is hydrolyzed by a simulated gastrointestinal digestion process, named S-CWP, protects BRL-3A hepatocytes from HS-induced damage via antioxidant and anti-inflammatory mechanisms. BRL-3A cells were pretreated with S-CWP before being treated at 43 °C for 1 h, and the levels of the cellular oxidative stress, inflammation, apoptosis, biomarkers for liver function, the activities of several antioxidant enzymes, and the cell viability were analyzed. The expression level of pivotal proteins in correlative signaling pathways was evaluated by western blotting. We confirmed that S-CWP alleviated HS-induced hepatocytes oxidative stress by decreased reactive oxygen species (ROS), nitric oxide (NO), 8-Hydroxy-2′-deoxyguanosine (8-OHdG), lipid peroxidation (LPO), protein carbonylation (PCO), and the activities of NADPH oxidase while enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) activities, and GSH content. S-CWP suppressed HS-induced inflammatory response by reducing the phosphorylation of NF-κB p65, the expression of NLRP3, and caspase-1 and finally alleviated caspase-3-mediated apoptosis. S-CWP also alleviated HS-induced hepatocyte injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and restoring Heat Shock Protein 70 (HSP70) expression. Furthermore, S-CWP treatment significantly enhanced the expression of NF-E2-related nuclear factor erythroid-2 (Nrf2) and HO-1. The antioxidant and anti-inflammatory effects of S-CWP were weakened by ML385, a specific Nrf2 inhibitor. Additionally, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, significantly reversed S-CWP-induced reduction in the phosphorylation of NF-κB p65. Thus, our results revealed that S-CWP protected against HS-induced hepatocytes damage via activating the Nrf2/HO-1 signaling pathway and inhibiting NF-κB/NLRP3 axis.