Long Non-Coding RNA 1810026B05Rik Mediates Cerebral Ischemia/Reperfusion-Induced Neuronal Injury Through NF-κB Pathway Activation

Figures

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  Figure 1. In vivo involvement of lncRNA-1810026B05Rik in I/R-induced brain injury. (A) Representative TTC-stained brain sections showing infarct areas (magenta: viable tissue; white: infarcted regions). Scale bar = 1 cm. (B) Infarct volume in the brain was quantitatively measured following I/R injury (n = 6). (C) Neurological deficits were evaluated using the modified Neurological Severity Score (mNSS) after I/R (n = 6). (D,E) Neuronal apoptosis was analyzed I/R using TUNEL staining. Scale bar: 200 μm (n = 3). (F) Temporal expression profile of lncRNA-1810026B05Rik in mouse brain tissue at 0, 6, 12, and 24 h after MCAO, as determined by qRT-PCR (n = 3). All data are represented as mean ± SD. One way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 versus Sham group. I/R, ischemia/reperfusion; TTC, triphenyl tetrazolium chloride; mNSS, Neurological Severity Score; qRT-PCR, quantitative real-time polymerase chain reaction; MCAO, cerebral artery occlusion.

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  Figure 2. Subcellular distribution and expression analysis of lncRNA-1810026B05Rik under in vitro I/R-induced hypoxic stress. (A) Neuronal viability following OGD/R was assessed using the CCK-8 assay (n = 6). (B,C) Apoptotic cells were visualized using TUNEL staining post-I/R injury, with nuclei counterstained by Hoechst 33342 (blue) (n = 4). Scale bar: 20 μm. (D) RNA FISH images illustrating the subcellular localization of lncRNA-1810026B05Rik in neurons. Nuclei were counterstained with Hoechst 33342 (blue) (n = 3). Scale bars: 50 μm and 20 μm. (E) Subcellular localization fluorescence intensity of 1810026B05Rik in different lesions induced by OGD/R. (F) 1810026B05Rik expression was detected by qRT-PCR at each time point in cultured neurons subjected to 1 h of OGD and 0, 6, 12, or 24 h of reoxygenation (n = 3). (G) Representative RNA FISH images showing the intracellular localization of lncRNA-1810026B05Rik in rat brain, Nuclei (blue) were counterstained with Hoechst 33342 (n = 3). Scale bar: 0.5 cm/100 μm. All data are represented as mean ± SD. One way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 versus Control group. OGD, oxygen-glucose deprivation; OGD/R, oxygen-glucose…

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  Figure 3. Impact of lncRNA-1810026B05Rik on OGD/R-induced neuronal injury. Primary neurons were transfected with siRNA-NC, siRNA targeting 1810026B05Rik, pcDNA3.1-NC, or pcDNA3.1-1810026B05Rik, followed by OGD/R for 4 h and reoxygenation for 24 h. (A) qRT-PCR analysis confirmed the altered expression of 1810026B05Rik post-transfection (n = 3). (B) Cell viability was assessed using the CCK-8 assay (n = 3). (C) LDH release was measured as an indicator of neuronal injury (n = 3). (D) After 24 h of reoxygenation, the intracellular Ca2+ concentration, ROS levels and apoptosis of neurons were determined using Fluo-4 AM, DCFH-DA or TUNEL staining, Nuclei (blue) were counterstained with Hoechst 33342, respectively. Scale bar: 50 μm. (E) The intracellular Ca2+ fluorescence intensity, demonstrated as the green fluorescence ratio, was calculated (n = 3). (F) The ROS fluorescence intensity was analyzed (n = 3). (G) The apoptotic neurons shown as TUNEL-positive cells were calculated (n = 3). All data are represented as mean ± SD. One way ANOVA, * p < 0.05, ** p < 0.01 versus siRNA-NC or pcDNA3.1-NC group. NC: negative control; LDH, lactate dehydrogenase; DCFH-DA, dichloro-dihydro-fluorescein diacetate;…

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  Figure 4. lncRNA-1810026B05Rik promotes NF-κB pathway activation in neurons subjected to OGD/R. (A) The expression of 1810026B05Rik was evaluated by qRT-PCR in untreated neurons and in OGD/R-treated cells transfected with specific siRNAs or expression plasmids (n = 3). (B,D) Nuclear levels of NF-κB subunits p65 and p50 were analyzed by Western blot. Representative immunoblot images are shown (n = 3). (C,E) Relative protein expression was quantified by densitometry, with Histone H3 as a nuclear loading control (n = 3). (F) mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 was determined by qRT-PCR in the indicated groups (n = 3). (G) Protein expressions of TNF-α, IL-1β, and IL-6 were further validated by Western blot. Representative blots are shown. (H,I) Semi-quantitative analysis of cytokine protein levels was performed, with GAPDH as the loading control (n = 3). Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA; * p < 0.05, ** p < 0.01 versus Mock (OGD/R blank control), siRNA-NC, or pcDNA3.1-NC groups. NF-κB, nuclear factor-kappaB; TNF-α, tumor necrosis factor-α; IL-1β, Interleukin 1 beta.

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  Figure 5. Interaction between lncRNA-1810026B05Rik and NF-κB inhibitor alpha (IκBα). (A,B) RNA pull-down assays were conducted in OGD/R-treated neurons using biotin-labeled 1810026B05Rik, control RNA, or no RNA. The presence of IκBα and phosphorylated IκBα (p-IκBα) in the pulled-down protein complex was examined via Western blot. Densitometric quantification was normalized to input protein (n = 3). (C,D) RNA immunoprecipitation (RIP) was performed using antibodies against IκBα, p-IκBα, or IgG as a negative control, followed by qRT-PCR to detect enrichment of 1810026B05Rik (n = 3). (E) Protein levels of IκBα and p-IκBα were detected in the indicated groups by Western blot, and the ratio of p-IκBα/IκBα was quantified (n = 3). (F) Dose-dependent knockdown efficiency of 1810026B05Rik was validated by qRT-PCR in neurons treated with increasing concentrations (30 nM, 60 nM, and 120 nM) of si-1810026B05Rik, with 50 nM si-NC as control (n = 3). All data are represented as mean ± SD. One way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 versus lncRNA-1810026B05Rik, siRNA-NC, Total RNA, IκBα or siRNA-1810026B05Rik group. IκBα, NF-kappa-B inhibitor alpha.

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  Figure 6. Effects of siRNA-1810026B05Rik and the NF-κB inhibitor QNZ on OGD/R-induced neuronal injury. (A) Primary neurons were treated with siRNA-NC, siRNA-1810026B05Rik, QNZ, or ethanol solvent (vehicle control) for 24 h, followed by OGD/R. Immunofluorescence staining was performed to assess p65 localization; nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (B) Neuronal cell death was evaluated using an LDH release assay (n = 3). (C) Apoptosis was quantified using a Histone DNA ELISA assay (n = 3). (D) Early apoptotic cells were detected by Annexin V staining and analyzed via flow cytometry (n = 3). All values are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control, siRNA-NC, or Solvent group.

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  Figure 7. LncRNA-1810026B05Rik aggravated CIRI via activation of the NF-κB pathway. (A) Detection of 1810026B05Rik mRNA expression after siRNA-NC and siRNA-1810026B05Rik intracerebroventricular injection of MCAO/R (n = 3). (B) Representative TTC stained (magenta: healthy tissue; white: damaged tissue) brain tissue sections. Scale bar: 1 cm. (C) Quantitative analysis of mouse cerebral infarction volume (n = 6). (D) Neurological scoring using mNSS after MCAO/R (n = 6). lncRNA-1810026B05Rik regulates neuronal survival and inflammation in the ischemic penumbra following MCAO/R. (E) Representative immunohistochemistry images of NeuN staining illustrate neuronal loss in the peri-infarct cortex. Scale bar = 200 μm. (F) Quantification of neuronal loss in the ischemic penumbra following lncRNA-1810026B05Rik knockdown (n = 3). (G) TUNEL staining was performed to visualize apoptotic cells in the peri-infarct region. Scale bar = 200 μm. (H) Quantitative analysis demonstrated that knockdown of lncRNA-1810026B05Rik significantly elevated neuronal apoptosis in the ischemic cortex (n = 3). (I–K) mRNA and protein levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were measured 24 h…

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  Figure 8. Experimental mechanism diagram 1810026B05Rik, originally exists in the cytoplasm, transports into the nucleus with elevated expression upon cerebral ischemia/reperfusion injury. 1810026B05Rik combined with IκBα and resulted in its phosphorylation. The phosphorylated IκBα promoted the neural expression of p65 and p50 in nucleus and activated NF-κB signaling pathway, which further enhanced downstream expression of TNF-α, IL-1β, Bax and casapse-9. The secretion of pro-inflammatory and proapoptotic factors aggravated neural inflammatory reaction and apoptosis, exacerbating the progression of disease.