Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and advanced fibrosis, is a leading cause of chronic liver disease worldwide, progressing to cirrhosis and ultimately hepatocellular carcinoma (HCC). Excessive accumulation of fatty acids in the liver triggers multiple forms of hepatocyte death and exacerbates NAFLD progression, with pyroptosis and apoptosis considered key events. Recent studies show that cysteine aspartic acid specific protease-3 (caspase-3) is a central regulator of both pyroptosis and apoptosis in NAFLD. Activated caspase-3 not only directly induces apoptosis but also cleaves the N-terminal domain of gasdermin E (GSDME), disrupts cell membranes, releases inflammatory factors, and thereby mediates pyroptosis. Inhibiting caspase-3 expression in NAFLD can alleviate hepatocyte injury (such as ballooning degeneration), dampen pro-inflammatory signaling, and reduce apoptosis. Caspase-3 acts as a key node coordinating pyroptosis and apoptosis and may serve as a novel therapeutic target for the prevention and treatment of NAFLD.
Non-alcoholic Fatty Liver Disease/metabolism* ; Humans ; Pyroptosis/physiology* ; Apoptosis/physiology* ; Caspase 3/physiology* ; Animals ; Gasdermins

OBJECTIVES: To explore the mechanism by which histone deacetylase 1 (HDAC1) regulates steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells. METHODS: MLY-O4 cells were treated with 400 nmol/L trichostatin A (TSA) or 1 mmol/L dexamethasone for 24 h or transfected with a HDAC1-overexpressing vector prior to TSA or dexamethasone treatment. The changes in the expressions of HDAC1, SP1, cleaved caspase-3 and Bax, SP1 acetylation level, cell proliferation, and cell apoptosis were examined. The interaction between HDAC1 and SP1 was determined with immunoprecipitation assay and Western blotting. RESULTS: Treatment with dexamethasone significantly increased cell apoptosis, enhanced the expressions of cleaved caspase-3 and Bax, reduced HDAC1 expression, and suppressed proliferation of MLO-Y4 cells. Both TSA and dexamethasone obviously increased SP1 acetylation level and the expression of SP1 in MLO-Y4 cells. HDAC1 overexpression in the cells significantly attenuated the effect of TSA and dexamethasone, promoted cell proliferation, lowered the expressions of SP1, cleaved caspase-3 and Bax, and inhibited dexamethasone-induced cell apoptosis. Immunoprecipitation assay and Western blotting demonstrated the interaction between HDAC1 and SP1 in the cells. CONCLUSIONS HDAC1 inhibits dexamethasone-induced apoptosis and promotes proliferation of cultured mouse osteocytes by suppressing SP1 expression via promoting its deacetylation.
Animals ; Apoptosis/drug effects* ; Mice ; Histone Deacetylase 1/genetics* ; Osteocytes/drug effects* ; Sp1 Transcription Factor/metabolism* ; Acetylation ; Dexamethasone/pharmacology* ; Cell Proliferation/drug effects* ; Caspase 3/metabolism* ; Cell Line ; Hydroxamic Acids/pharmacology* ; bcl-2-Associated X Protein/metabolism*

OBJECTIVES: To investigate the effect of amentoflavone (AF) for alleviating lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and inhibiting NLRP3/ASC/Caspase-1 axis-mediated pyroptosis. METHODS: Female BALB/c mice were randomly divided into control group, LPS group, and AF treatment groups at low, moderate and high doses (n=12). ALI models were established by tracheal LPS instillation, and in AF treatment groups, AF was administered by gavage 30 min before LPS instillation. Six hours after LPS instillation, the mice were euthanized for examining lung tissue histopathological changes, protein levels in BALF, and MPO levels in the lung tissue. In the in vitro experiment, RAW264.7 cells were pretreated with AF, AC (a pyroptosis inhibitor), or their combination for 2 h before stimulation with LPS and ATP. The changes in cell proliferation and viability were detected using CCK-8 assay, and IL-1β, IL-6, IL-18, and TNF-α levels were determined with ELISA. Immunohistochemistry, immunofluorescence assay, and immunoblotting were used to detect the protein levels of NLRP3, ASC, cleaved caspase-1, and GSDMD N in rat lung tissues and the treated cells. RESULTS: In mice with LPS exposure, AF treatment significantly improved lung pathologies and edema, reduced protein levels in BALF and pulmonary MPO level, inhibited the high expression of NLRP3/ASC/Aspase-1 axis, reduced the expression of GSDMD N, and lowered the release of IL-1β, IL-6, IL-18, and TNF‑α. In RAW264.7 cells with LPS and ATP stimulation, AF pretreatment effectively reduced cell death, inhibited activation of the NLRP3/ASC/Aspase-1 axis, and reduced GSDMD N expression and the inflammatory factors. The pyroptosis inhibitor showed a similar effect to AF, and their combination produced more pronounced effects in RAW264.7 cells. CONCLUSIONS Amentoflavone can alleviate ALI in mice possibly by inhibiting NLRP3/ASC/Caspase-1 axis-mediated cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Acute Lung Injury/pathology* ; Mice ; Mice, Inbred BALB C ; Female ; Lipopolysaccharides ; Biflavonoids/pharmacology* ; RAW 264.7 Cells ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Lung

OBJECTIVES: To study the therapeutic mechanism of Tuihuang Mixture against cholestasis. METHODS: Forty-eight Wistar rats were randomized equally into blank group, model group, ursodeoxycholic acid group and Tuihuang Mixture group. Except for those in the blank group, all the rats were given α‑naphthylisothiocyanate (ANIT) to establish rat models of cholestasis, followed by treatments with indicated drugs or distilled water. Serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL of the rats were determined, and hepatic expressions IL-1β, IL-18, FXR, NLRP3, ASC, Caspase-1 and GSDMD were detected using q-PCR, ELISA or Western blotting. Histopathological changes of the liver tissues were observed using HE staining. RESULTS: The rat models of cholestasis had significantly increased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL with increased mRNA and protein expressions of IL-1β and IL-18, decreased protein and mRNA expressions of FXR, and increased protein expressions of NLRP3 and Caspase-1 and mRNA expressions of NLRP3, ASC, Caspase-1 and GSDMD in the liver tissue, showing also irregular arrangement of liver cells, proliferation of bile duct epithelial cells and inflammatory cells infiltration. Treatment of the rat models with Tuihuang Mixture significantly decreased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL, lowered IL-1β and IL-18 and increased FXR protein and mRNA expressions, and reduced NLRP3, ASC, Caspase-1 and GSDMD proteins and NLRP3, ASC and Caspase-1 mRNA expressions in the liver tissue. Tuihuang Mixture also significantly alleviated hepatocyte injury, bile duct epithelial cell proliferation and inflammatory cell infiltration in the liver of the rat models. CONCLUSIONS Tuihuang Mixture can effectively improve cholestasis in rats possibly by inhibiting NLRP3 inflammatosome-mediated pyroptosis via regulating FXR.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats ; Receptors, Cytoplasmic and Nuclear/metabolism* ; Cholestasis/drug therapy* ; Rats, Wistar ; Inflammasomes/metabolism* ; 1-Naphthylisothiocyanate ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Interleukin-18/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

OBJECTIVES: To study the mechanism mediating the protective effect of asiaticoside (AS) against myocardial ischemia-reperfusion injury (MIRI) in rats. METHODS: Fifty SD rats were randomized into sham-operated group, MIRI model group and AS treatment group. AS treatment was administered at low, moderate and high doses by daily gavage for 2 weeks before MIRI modeling (n=10). Serum levels of lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), interleukin-18 (IL-18) and IL-1β, the volume of myocardial infarction and ischemia, and myocardial pathologies of the rats were determined or observed. The protein expression levels of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the myocardial tissues were detected using Western blotting. The changes in the expression levels of these proteins were also detected in H9C2 cells with AS pretreatment prior to hypoxia-reoxygenation (H/R) injury. RESULTS: The rats models of MIRI exhibited significant myocardial infarction and ischemia with increased serum levels of LDH and CK-MB and myocardial expressions of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18. AS pretreatment effectively reduced myocardial infarction volume in the rat models and significantly reduced serum LDH and CK-MB levels and the protein levels in the myocardial tissue in a dose-dependent manner. In the H9C2 cell model of H/R injury, AS pretreatment significantly suppressed the elevation of the protein expressions of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18. Molecular docking studies showed that AS had a strong binding affinity with NLRP3. CONCLUSIONS Asiaticoside can alleviate MIRI in rats possibly by inhibiting NLRP3 inflammasome-mediated pyroptosis.
Animals ; Myocardial Reperfusion Injury/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis/drug effects* ; Rats, Sprague-Dawley ; Rats ; Inflammasomes/metabolism* ; Triterpenes/pharmacology* ; Interleukin-18/metabolism* ; Male ; Interleukin-1beta/metabolism* ; Caspase 1/metabolism*

OBJECTIVES: To explore the mechanism through which moxibustion promotes endometrial repair in rats with in thin endometrium (TE). METHODS: Female SD rats were randomized into control group, 95% anhydrous ethanol-induced TE model group and moxibustion (at "Guan Yuan") group. High-throughput sequencing was used to identify the target genes of TE, and the targeting relationship between miR-223-3p and NLRP3 was verified using a dual luciferase assay. Histopathological of rat uterus was observed with HE staining, and expressions of miR-223-3p and NLRP3 were detected using RT-qPCR; serum levels of IL-1β and IL-18 of the rats were detected using ELISA, and protein expressions of NLRP3, ASC, caspase-1 and GSDMD in the uterus were detected with Western blotting. The pregnancies of the rats after treatment were counted. RESULTS: Enrichment analysis of the differential genes suggested up-regulated inflammatory response in TE, and dual luciferase assay verified targeted inhibition of NLRP3 expression by miR-223-3p. The rat models of TE had significantly decreased endometrial thickness and reduced endometrial glands and blood vessels with enhanced mRNA expression of NLRP3, increased serum levels of IL-1β and IL-18, up-regulated protein expressions of NLRP3, ASC, caspase-1 and GSDMD, lowered pregnancy rates on both the affected and unaffected sides and the overall number of pregnancies. Treatment of the rat models with mo-xibustion obviously increased the endometrial thickness and the density of glands and blood vessels, up-regulated miR-223-3p expression, lowered serum IL-1β and IL-18 levels and the protein expressions of NLRP3, ASC, caspase-1 and GSDMD, and significantly increased the number of pregnancies. CONCLUSIONS Moxibustion at "Guan Yuan" acupoint up-regulates the expression of miR-223-3p, which results in targeted inhibition of NLRP3 to suppress pyroptosis and promote endometrial repair in rat models of TE.
Animals ; Female ; MicroRNAs/genetics* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Endometrium/pathology* ; Rats, Sprague-Dawley ; Rats ; Moxibustion ; Pyroptosis ; Up-Regulation ; Interleukin-1beta/metabolism* ; Interleukin-18 ; Caspase 1/metabolism*

OBJECTIVES: To investigate the therapeutic mechanism of 2,6-dimethoxy-1,4-benzoquinone (DMQ) for alleviating dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. METHODS: Eighteen male C57BL/6J mice were equally randomized into control group, DSS group and DMQ treatment group. In DSS and DMQ groups, the mice were treated with DSS in drinking water to induce UC, and received intraperitoneal injections of sterile PBS or DMQ (20 mg/kg) during modeling. The changes in body weight, disease activity index (DAI), colon length, spleen weight, and colon histological scores of the mice were examined, and the percentages of Th17 and IFN-γ⁺ CD8⁺ T cells in the mesenteric lymph nodes and spleen were analyzed using flow cytometry. The expressions of tight junction proteins (Occludin and ZO-1), proteins associated with inflammasome activation (caspase-1 and p20), IL-1β and TNF-α in the colon tissues were detected using Western blotting or ELISA. In the cell experiment, mouse bone marrow-derived macrophages (BMDMs) primed with lipopolysaccharide (LPS) were treated with DMQ, followed by stmulation with nigericin to activate the classical NLRP3 inflammasome pathway. In cultured human peripheral blood mononuclear cells (PBMCs) treated with either LPS alone or LPS plus nigericin, the effects of DMQ on inflammasome activation, pyroptosis, and cytokine release were evaluated via Western blotting, ELISA, and flow cytometry. RESULTS: In DSS-treated mice, DMQ treatment significantly alleviated DSS-induced body weight loss, colon shortening, spleen enlargement, and colon inflammation. The DMQ-treated mice showed significantly reduced percentages of Th17 cells and IFN-γ⁺ CD8⁺ T cells in the mesenteric lymph nodes and spleen, with increased occludin and ZO-1 expressions and decreased caspase-1 expression in the colon tissue. DMQ obviously inhibited classical NLRP3 inflammasome activation in mouse BMDMs and both the classical and alternative pathways of NLRP3 activation in human PBMCs, causing also suppression of caspase-1-dependent pyroptosis. CONCLUSIONS DMQ ameliorates DSS-induced UC in mice by inhibiting NLRP3 inflammasome activation.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Mice, Inbred C57BL ; Colitis, Ulcerative/metabolism* ; Dextran Sulfate/adverse effects* ; Male ; Inflammasomes/metabolism* ; Mice ; Benzoquinones/therapeutic use* ; Th17 Cells ; Caspase 1/metabolism*

OBJECTIVES: To investigate the regulatory role of the NLRP3 signaling pathway in hepatocyte pyroptosis in nonalcoholic steatohepatitis (NASH) under hypoxia. METHODS: Twenty-four male C57BL/6 mice were randomized equally into hypoxic control (A), hypoxic NASH model (B), hypoxic NASH+NLRP3 inhibitor (C), and hypoxic NASH+caspase-1 inhibitor (D) groups. In groups B-D, the mice were fed a methionine choline-deficient (MCD) diet under hypoxic conditions (to simulate a 5000 m altitude) for 6 weeks; the mice in groups C and D received intraperitoneal injections of the respective inhibitors every other day. RESULTS: Compared with those in group A, the mice in group B showed significantly elevated serum levels of FBG, TC, TG, ALT and AST, increased liver lipid content, inflammatory cell infiltration and collagen fiber deposition, and enhanced hepatic expressions of NLRP3, caspase-1, IL-1β and GSDMD proteins, with obvious swelling, cristae breakage, vacuolization, and outer membrane disruption of the mitochondria, ribosome loss in the cytoplasm, destruction of the nuclear membrane, and pathological changes of the rough endoplasmic reticulum. Treatment with NLRP3 inhibitor and caspase-1 inhibitor both significantly lowered serum levels of TC, TG, ALT and AST (but without significantly affecting FBG) in the mouse models, and reduced liver lipid content, inflammatory cell infiltration, collagen deposition, and expression levels of NLRP3, caspase-1, GSDMD and IL-1β. The treatments also significantly improved pathological changes in the mitochondria, ribosomes and endoplasmic reticulum in liver tissues of the mice. CONCLUSIONS NLRP3 signaling pathway plays a key role in promoting hepatocyte pyroptosis in NASH mice under hypoxic condition, and inhibiting this pathway can effectively reduce liver inflammation, suggesting its potential as a therapeutic target for NASH treatment.
Animals ; Non-alcoholic Fatty Liver Disease/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Pyroptosis ; Mice, Inbred C57BL ; Male ; Hepatocytes/pathology* ; Signal Transduction ; Mice ; Hypoxia/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

OBJECTIVES: To investigate the mechanism underlying the therapeutic effect of electroacupuncture (EA) on post-traumatic stress disorder (PTSD) in rats. METHODS: Forty male SD rats were randomized equally into blank control group, PTSD model group, sham-acupuncture group, paroxetine group, and EA group. In the latter 3 groups, the rat models of PTSD, induced by continuous single-prolonged stress and plantar electrical stimulation, were treated with EA at GV20, GV24, BL18 and BL23 acupoints for 15 min (5 times a week for 3 weeks), sham-acupuncture without electrical stimulation, or gavage with paroxetine suspension on the same schedule. Behavioral changes of the rats were evaluated using open field test (OFT) and elevated plus maze (EPM) test. Hippocampal pathologies and neuronal changes were examined with HE and Nissl staining, and mitochondrial ultrastructure was examined using electron microscopy. The mRNA and protein expression levels of Bcl-2, Bax, and caspase-3 were detected by RT-qPCR and immunofluorescence staining. RESULTS: The rat models of PTSD showed significantly reduced total distance traveled in OFT and distance and time spent in the open arms of the EPM, with decreased hippocampal neurons, obvious neuronal and mitochondrial pathologies, decreased hippocampal expression of Bcl-2, and increased Bax and caspase-3 expressions. Treatments with paroxetine and EA both significantly improved behavioral changes of the rat models, increased the number of Nissl-stained neurons, obviously alleviated pathologies in the hippocampal neurons and mitochondrial ultrastructure, increased hippocampal Bcl-2 expression, and lowered caspase-3 expressions. Paroxetine showed significantly better effect than EA for improving performance of the rats in EPM test, whereas sham-acupuncture did not produce any significant improvement. CONCLUSIONS EA alleviates PTSD in rats possibly by upregulating Bcl-2 and downregulating Bax and caspase-3, thereby ameliorating hippocampal mitochondrial damage.
Animals ; Electroacupuncture ; Stress Disorders, Post-Traumatic/metabolism* ; Hippocampus/pathology* ; Rats, Sprague-Dawley ; Male ; Rats ; Mitochondria/pathology* ; Signal Transduction ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Disease Models, Animal

OBJECTIVE: To elucidate the role and mechanism of microRNA-145-5p (miR-145-5p) in hypoxia-induced pyroptosis of human alveolar epithelial cells. METHODS: In vitro, human alveolar epithelial cell line BEAS-2B was cultured. Cells in the logarithmic growth phase were cultured to 80% confluence and then used for the experiment. (1) BEAS-2B cells were cultured under 1% O₂ hypoxic condition, with a normoxic control group. Western blotting was employed to detect the expressions of pyroptosis marker proteins [NOD-like receptor protein 3 (NLRP3), Gasdermin D N-terminal domain (GSDMD-N), and caspase-1] in cells cultured for 24 hours. Real-time fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-145-5p in cells cultured for 6 hours and 12 hours. (2) Cells were transfected with 30 nmol/L miR-145-5p mimic to overexpress miR-145-5p expression under normoxic condition or 30 nmol/L miR-145-5p inhibitor to suppress miR-145-5p expression under hypoxic condition. Control group and negative control group were respectively set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of pyroptosis marker proteins and nuclear factor-E2-related factor 2 (Nrf2) in cells. Flow cytometry was applied to detect the level of reactive oxygen species (ROS) in cells. The target genes of miR-145-5p were predicted by miR target gene prediction software miRWalk and verified by Western blotting. (3) Under hypoxic condition, cells were transfected with 6.94 ng/μL silent information regulator 5 (Sirt5) overexpression plasmid or pretreated with 12.5 mmol/L N-acetyl-L-cysteine (NAC) as an ROS inhibitor. The empty plasmid group and control group were set up. After 24 hours of cell culture, Western blotting was used to detect the expressions of Sirt5, Nrf2, and pyroptosis marker proteins in cells. Flow cytometry was used to detect the level of ROS in cells. RESULTS: (1) Compared with the normoxic control group, the expression levels of pyroptosis marker proteins in the 24-hour hypoxia group was significantly increased, indicating that hypoxia could induce pyroptosis in BEAS-2B cells. The expression level of miR-145-5p in cells gradually increased with the extension of hypoxia induction time, indicating that hypoxia could cause the increase of miR-145-5p expression level. (2) The expression levels of pyroptosis marker proteins in cells of miR-145-5p mimic group significantly increased under normoxic condition as compared with the control and negative control groups [NLRP3 protein (NLRP3/β-actin): 1.58±0.07 vs. 1.00±0.01, 0.98±0.07, GSDMD-N protein (GSDMD-N/β-actin): 1.71±0.03 vs. 1.01±0.01, 0.85±0.03, caspase-1 protein (caspase-1/β-actin): 2.33±0.04 vs. 1.01±0.01, 1.05±0.04, all P < 0.05], Nrf2 protein expression level was significantly decreased (Nrf2/β-actin: 0.79±0.03 vs. 1.00±0.01, 1.03±0.04, both P < 0.05), ROS level was significantly up-regulated (fluorescence intensity: 1.74±0.03 vs. 1.00±0.01, 0.92±0.03, both P < 0.05). Under hypoxia condition, compared with control group and negative control group, the expression levels of pyroptosis marker proteins in miR-145-5p inhibitor group were significantly decreased [NLRP3 protein (NLRP3/β-actin): 0.21±0.04 vs. 1.70±0.02, 1.63±0.04; GSDMD-N protein (GSDMD-N/β-actin): 1.32±0.02 vs. 2.51±0.02, 2.72±0.03; caspase-1 protein (caspase-1/β-actin): 0.56±0.01 vs. 2.77±0.02, 3.12±0.03; all P < 0.05], Nrf2 protein expression level was significantly increased (Nrf2/β-actin: 1.57±0.04 vs. 1.22±0.01, 1.28±0.04, both P < 0.05), ROS level was significantly down-regulated (fluorescence intensity: 0.64±0.05 vs. 1.87±0.04, 1.70±0.07, both P < 0.05). The results indicated that miR-145-5p could promote cell pyrodeath. The predictive result of miRWalk showed that the 3' untranslated region (3'UTR) of Sirt5 had complementary base binding sites with miR-145-5p. The expression level of Sirt5 protein in cells of miR-145-5p mimic group was significantly lower than that of control group and negative control group under normoxic condition (Sirt5/β-actin: 0.59±0.03 vs. 1.00±0.01, 1.01±0.03, both P < 0.05), which verified that Sirt5 was the target gene of miR-145-5p. (3) The occurrence of pyrodeath could be partially reversed by transfection with Sirt5 overexpression plasmid or adding ROS inhibitor NAC into cells, and Sirt5 overexpression could also up-regulate Nrf2 expression and eliminate intracellular ROS. CONCLUSION In human alveolar epithelial cells, miR-145-5p can down-regulate Nrf2 by targeting Sirt5, thereby increasing ROS expression and inducing pyrodeath.
Humans ; MicroRNAs ; Pyroptosis ; Cell Hypoxia ; Alveolar Epithelial Cells/cytology* ; Cell Line ; NLR Family, Pyrin Domain-Containing 3 Protein ; Caspase 1/metabolism* ; Epithelial Cells/metabolism* ; Gasdermins ; Phosphate-Binding Proteins