Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Mouse hippocampal neurons (HT22 cells) are subjected to OGD/R to mimic CIRI. OGD/R triggers upregulation of CLIC1, which leads to suppression of the Nrf2/HO-1 antioxidant signaling pathway. This results in increased ROS accumulation and disturbance of redox balance, promoting apoptosis by increased Bax and decreased Bcl-2 expression. Additionally, elevated CLIC1 activates the NLRP3 inflammasome and caspase-1 via inhibiting the Nrf2/HO-1 pathway, inducing pyroptosis via upregulation of pyroptosis markers, such as caspase-3, GSDMD, IL-1β, and IL-18. Collectively, these processes contribute to neuronal cell damage after CIRI. ARE, antioxidant response element; CIRI, cerebral ischemia-reperfusion injury; CLIC1, chloride intracellular channel 1; GSDMD, gasdermin D; HO-1, heme oxygenase 1; IL, interleukin; NLRP3, NOD-like receptor family pyrin domain containing 3; Nrf2, nuclear factor erythroid 2–related factor 2; OGD/R, oxygen-glucose deprivation/reoxygenation; ROS, reactive oxygen species.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Expressing

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a) Real-time PCR analysis of CLIC1 gene expression showed significantly higher expression in the OGD/R group compared to the control group, with a marked reduction upon CLIC1 silencing. (b, c) Western blot analysis of CLIC1 protein levels demonstrated increased CLIC1 expression in the OGD/R group and its suppression following CLIC1 silencing. (d-f) Flow cytometry analysis shows that elevated CLIC1 expression in OGD/R-treated HT22 cells led to an increased apoptosis rate (d, e) and higher ROS mean fluorescence intensity (MFI) values (d, f). These effects were reversed by silencing CLIC1. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Control, Western Blot, Flow Cytometry, Fluorescence

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) The activities of SOD (a), CAT (b), and GSH-Px (c) were significantly reduced in OGD/R-treated HT22 cells compared to the control group, with the restoration of these antioxidant enzyme activities upon CLIC1 silencing. (d) The LDH level was elevated in the OGD/R group and decreased following CLIC1 silencing. (e) The CCK-8 OD value was reduced in the OGD/R group and increased with CLIC1 silencing. (f-h) Real-time PCR (f) and WB (g,h) analyses showed that OGD/R treatment decreased anti-apoptotic Bcl-2 expression and increased pro-apoptotic Bax expression, with these effects reversed by CLIC1 silencing. Data are presented as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Control, CCK-8 Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Real-time PCR, WB, and/or ELISA analyses show that the expression levels of inflammation- and pyroptosis-related indicators were all upregulated after the increased expression of CLIC1 in OGD/R-treated HT22 cells, via inhibiting the Nrf2/HO-1 pathway. (a-c) Real-time PCR (a) and WB (b, c) analyses showed upregulation of NLRP3 and caspase-1 expression in OGD/R-treated HT22 cells, with significant downregulation upon CLIC1 silencing. Inhibition of Nrf2/HO-1 signalling or activation of NLRP3 partly reversed the effects of CLIC1 silencing. (d-f) Real-time PCR (d) and WB (e, f) analyses of caspase-3 and GSDMD protein expression showed similar trends with NLRP3 and caspase-1. (g-j) Real-time PCR (g), WB (h,i), and ELISA (j) analyses revealed elevated levels of IL-1β and IL-18 in OGD/R-treated HT22 cells, which were reduced upon CLIC1 silencing. These cytokines were further upregulated with Nrf2/HO-1 inhibition or NLRP3 activation after CLIC1 silencing. Quantitative data were presented as mean ± SEM with three replicate experiments. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Inhibition, Activation Assay, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) Real-time PCR (a) and WB (b, c) analyses showed that Nrf2 and HO-1 expression levels were significantly downregulated in OGD/R-treated HT22 cells compared to the control group. CLIC1 silencing partially restored Nrf2 and HO-1 expression levels, although normal levels were not fully achieved in any group following OGD/R treatment. Data are shown as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: Flow cytometry analysis shows that inhibition of the Nrf2/HO-1 signalling pathway increased both the apoptosis rate (a, b) and ROS mean fluorescence intensity (MFI) value (a, c) in OGD/R-treated HT22 cells. These levels were higher in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Flow Cytometry, Inhibition, Fluorescence

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a-c) Activity of oxidative stress markers of SOD (a), CAT (b), and GSH-Px (c) was reduced following inhibition of the Nrf2/HO-1 signalling pathway. These reductions were more pronounced in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group. (d) CCK-8 OD values were decreased after Nrf2/HO-1 inhibition, with lower values observed in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group than the OGD/R+CLIC1 silencing group. (e) The LDH levels were increased after Nrf2/HO-1 inhibition and were significantly higher in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group compared to the OGD/R+CLIC1 silencing group. (f-h) Real-time PCR (f) and WB (g, h) analyses showed that Bcl-2 expression was downregulated, while Bax expression was upregulated following Nrf2/HO-1 inhibition. These effects were more pronounced in the OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition group than the OGD/R+CLIC1 silencing group. Data were presented as mean ± SEM, with n = 3 per group. * P < 0.05 versus control group; ^ P < 0.05 versus OGD/R group; + P < 0.05 vs OGD/R+CLIC1 silencing group.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Activity Assay, Inhibition, CCK-8 Assay, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: PLOS One

Article Title: CLIC1 down-regulates Nrf2/HO-1 signalling pathway promoting the apoptosis and pyroptosis in OGD/R-treated HT22 cells

doi: 10.1371/journal.pone.0332698

Figure Lengend Snippet: (a) Gene expression heatmap illustrates the relative expression levels of key genes (detected by real-time PCR) across all the six groups [the control, oxygen and glucose deprivation/reoxygenation (OGD/R), OGD/R+ negative control (NC), OGD/R+CLIC1 silencing, OGD/R+CLIC1 silencing+Nrf2/HO-1 inhibition, and OGD/R+CLIC1 silencing+NLRP3 activation]. Each row represents a specific gene, and each column represents a sample. (b) Correlation heatmap showing the Spearman correlation coefficients among the genes detected by real-time PCR. Each cell represents the correlation between two genes, with the colour gradient indicating the strength and direction of the correlation. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The control HT22 cells and OGD/R-induced cells were then infected with either the CLIC1 silencing-negative control virus (WL117218−2, Wanleibio, China) or CLIC1 silencing virus (WL117218−1, Wanleibio, China) to create the NC and CLIC1 silencing groups.

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Control, Negative Control, Inhibition, Activation Assay