Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: Aging mouse lenses showed increased lens opacity with increased ROS accumulation and Nlrp3 and its components expression. ( A , B ) Lenses were isolated from young (4 M) and old (21 M) C57BL/6 mice, which were used for the experiment. Isolated lenses from young and old C57BL/6 mice were photographed using a Nikon SMZ745T trinocular stereo microscope connected to a camera and software (Nikon). ( C ) A histogram represents densitometric analysis of lens opacity. 4.79 ± 0.65 (yellow bar); 49.67 ± 0.33 (purple var). ( D ) Aberrant accumulation of ROS levels with aging. ROS levels were measured in intact lenses isolated from mice aged 4 M and 21 M using the H 2 -DCF-DA dye method. ( E ) Enhanced caspase-1 activity in aging lens. Cellular extract prepared from intact lenses isolated from 4 M and 21 M-old C57BL/6 mouse lenses. ( F , G ) Elevated levels of pro-inflammatory cytokines in the aging lenses. Cellular extracts were used to measure the levels of mature IL-1β ( F ) and IL-18 ( G ). ( H ) Upregulation in expression of inflammasome proteins and TXNIP is directly associated with reduced expression of antioxidant genes, Prdx6 and TRX1. Total protein was isolated from young and aged mice lenses and subjected to Western analysis. The analysis showed increased expression of Nlrp3, ASC, Cas-1, IL-1β, IL-18, GSDMD, and TXNIP with downregulation of Prdx6 and TRX1 levels. ( I ) RNA was isolated from young and old C57BL/6 mice lenses, and mRNA expression of Prdx6, Nlrp3, ASC, Cas-1, IL-1β, IL-18, GSDMD, TXNIP, and TRX was analyzed using RT-qPCR as indicated in the Figure and described in . All histograms are presented as mean ± S.D. values derived from four independent experiments. * p < 0.001.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Expressing, Isolation, Microscopy, Software, Activity Assay, Western Blot, Quantitative RT-PCR, Derivative Assay

Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: Age-related increase in intracellular ROS levels was linked to enhanced expression of Nlrp3 inflammasome-associated genes in the human aging lens/LECs. ( A ) Aged human lenses displayed increased accumulation of ROS. Intracellular ROS levels were measured using H 2 -DCF-DA dye methods in young and old human lenses. ( B ) Elevated caspase-1 activity in aging human lenses. Cellular extracts prepared from human aging lens/LECs of different ages were used to measure caspase-1 activity. ( C , D ) Matured form(s) of pro-inflammatory cytokines, IL-1β and IL-18, in the human lenses. Cellular extracts isolated from intact lenses of human subjects were used to measure the IL-1β and IL-18 levels. Enhanced levels of pro-inflammatory cytokines, IL-1β and IL-18, were observed in the aging human lenses/LECs. ( E , F ). Reduced expression of antioxidant genes, Prdx6 and TRX, is directly associated with increased expression of Nlrp3 inflammasome and TXNIP. Total protein and RNA were isolated and subjected to protein and mRNA analyses to assess the expression of Prdx6, Nlrp3, caspase-1, ASC, IL-1β, IL-18, pyroptosis executor GSDMD, TXNIP, and TRX using their specific probes as indicated, respectively. All histograms are presented as mean ± S.D. values derived from four independent experiments. * p < 0.001.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Expressing, Activity Assay, Isolation, Derivative Assay

Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: mLECs overexpressing TXNIP exhibited heightened sensitivity to oxidative stress and showed elevated expression of Nlrp3 inflammasome and its inflammatory genes. mLECs were transfected with pGFP-Vector or pGFP-TXNIP, followed by exposure to H 2 O 2 ( A ) and UVB ( B ) 48 h post-transfection. After 24 h, total RNA was isolated and subjected to RT-qPCR to evaluate the expression profiles of Nlrp3, caspase-1, ASC, IL-1β, IL-18, and GSDMD mRNA as indicated in the figures. Data represent the mean ± S.D. values of three independent experiments. # p < 0.05; * p < 0.001.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: TXNIP knockdown suppressed oxidative stress-induced aberrant expressions of Nlrp3, ASC, Cas-1, IL-1β, IL-18, and GSDMD. Stably infected LV ShControl and/or LV ShTXNIP mLECs were harvested in a 60 mm plate. 24 h later, infectants were exposed to H 2 O 2 ( A ) or UVB ( B ). Total RNA was isolated to assess the expression status of Nlrp3, ASC, Casp-1, IL-1β, IL-18, and GSDMD as indicated in the figure using their specific probes. Data represent the mean ± S.D. values of three independent experiments. * p < 0.001.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Knockdown, Stable Transfection, Infection, Isolation, Expressing

Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: TAT-HA-Prdx6 delivery suppressed oxidative stress-induced aberrantly activated Nlrp3 inflammasome–mediated inflammatory signaling. ( A ) TAT-HA-Prdx6 treatment attenuated H 2 O 2 -induced, accelerated production of caspase-1. Cellular extract prepared from intact lenses exposed to H 2 O 2 in the presence of TAT-HA-Prdx6-inactive mutant or TAT-HA-Prdx6-WT protein, and caspase-1 activity were measured. ( B , C ) TAT-HA-Prdx6 delivery effectively abated H 2 O 2 -induced, increased levels of pro-inflammatory cytokines. C57BL/6 mouse lenses were exposed to H 2 O 2 in the presence of TAT-HA-Prdx6-inactive mutant or TAT-HA-Prdx6-WT protein. After 45 and 90 h, cellular extracts were prepared, and the level of bioactive IL-1β ( B ) and IL-18 ( C ) was measured. ( D ) TAT-HA-Prdx6 suppressed oxidative stress-induced upregulated expression of Nlrp3 inflammasome and TXNIP. C57BL/6 mouse lenses were transduced with TAT-HA-Prdx6-inactive mutant or TAT-HA-Prdx6-WT protein, followed by H 2 O 2 exposure. After 45 h, RNA was isolated and subjected to RT-qPCR analysis to assess the expression of Nlrp3, ASC, Cas-1, IL-1β, IL-18, GSDMD, TXNIP, and TRX levels using their specific probes. All histograms are presented as mean ± S.D. values of six independent experiments. * p < 0.001.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Mutagenesis, Activity Assay, Expressing, Transduction, Isolation, Quantitative RT-PCR

Journal: Antioxidants

Article Title: TXNIP-Deficiency and Prdx6 Delivery Inhibit Aging/Oxidative Stress–Driven TXNIP-Nlrp3 Inflammasome Activation and Mitigate Pyroptosis in Lens Epithelial Cells

doi: 10.3390/antiox15020170

Figure Lengend Snippet: Aging and oxidative stress-driven TXNIP-Nlrp3 inflammasome activation and pyroptotic cell death prevention by Prdx6 delivery or TXNIP silencing. Aging and oxidative stress-driven excessive accumulation of ROS weakens the antioxidant defense system, resulting in elevated expression of TXNIP. Increased TXNIP levels promote aberrant expression and activation of Nlrp3 inflammasome–mediated inflammatory cell death, pyroptosis. Aberrantly activated Nlrp3 recruits the adaptor protein ASCs, which subsequently recruit procaspase-1 to form the Nlrp3-ASC-procaspase1 inflammasome complex, which allows the release of cleaved caspase-1. Activated caspase-1 processes the proinflammatory cytokines pro-IL-1β and pro-IL-18 to their active isomers IL-1β and IL-18, respectively. Also, active caspase-1 allows the cleavage of pro-GSDMD into its active isomer, GSDMD, which forms membrane pores and facilitates the release of bioactive IL-1β and IL-18, leading to pyroptotic cell death. Delivery of Prdx6 or silencing of TXNIP (ShTXNIP) inhibits the Nlrp3 inflammasome activation by enhancing the antioxidant defense and suppressing the ROS accumulation. These interventions represent promising therapeutic strategies for preventing or treating inflammation-driven blinding diseases associated with inflammation.

Article Snippet: The membranes were probed with the following antibodies: Prdx6 and TRX1 (LF-PA0011 and LF-PA0187; 1:2000 dilution, Ab Frontier, Seoul, Republic of Korea), TXNIP (# 14715), caspase-1 (# 24232 and # 3866), ASC (# 13833 and # 67824), IL-1β (# 122452), IL-18 (# 57058 and # 54943), gasdermin D (# 39754S) (1:1000 dilution; all from Cell Signaling Technology, Danvers, MA, USA), Nlrp3 (# PA5-79740; 1:1000 dilution, ThermoFisher Scientific, Waltham, MA, USA), IL-1β, and ASC (sc-52012 and sc-271054; 1:1000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). β-actin (A2066; 1:2000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and tubulin (ab44928; 1:4000 dilution; Abcam Inc., Waltham, MA, USA) were used as internal controls.

Techniques: Activation Assay, Expressing, Membrane