The cold-inducible RNA-binding protein-Thioredoxin 1 pathway ameliorates mitochondrial dysfunction and mitochondrial dynamin-related protein 1 level in the hippocampus of aged mice with perioperative neurocognitive dysfunction

Abstract

Background: As a multi-disease model, neuroinflammation, mitochondrial dysfunction, and oxidative stress might be involved in the pathogenic process of perioperative neurocognitive dysfunction (PND). Dynamin-related protein 1 (Drp1) could mediate mitochondrial fission and play important roles in mitochondrial dynamic homeostasis and mitochondria function. The Drp1 may be involved in PND development. The cold-inducible RNA-binding protein (Cirbp) could bind to the 3'-UTR of the thioredoxin 1 (Trx1) mRNA, control oxidative stress, and improve mitochondrial function. In this study, we hypothesized that the Cirbp-Trx1 pathway could ameliorate mitochondrial dysfunction and Drp1 levels in PND mice.

Methods: Differentially expressed genes were screened using the Gene Expression Omnibus (GEO) database GSE95426 and validated using PCR. Eighteen-month-old C57BL/6 mice were subjected to tibial fracture surgery to generate a PND model. Cirbp was upregulated by hippocampal stereotaxic injections of over-Cirbp plasmid according to the manufacturer's instructions for the in vivo DNA transfection reagent. Cirbp expression was measured using western blot (WB) and immunofluorescence (IF). The Morris water maze (MWM) was used to assess cognitive function. After behavioral testing, the hippocampal tissue was extracted to examine changes in mitochondrial Drp1, mitochondrial function, neuroinflammation, and oxidative stress.

Results: Differential gene screening showed that Cirbp expression was significantly downregulated (fold change >1.5, p = 0.003272) in the PND model. In this study, we also found that Cirbp protein levels were downregulated, accompanied by an impairment of cognition, a decrease in superoxide dismutase (SOD) activity, and an increase in malondialdehyde (MDA) content, mitochondrial Drp1 levels, neuroinflammation, and apoptosis. Cirbp overexpression increased Trx1 protein levels and reversed the damage. However, this protective effect was abolished by PX-12 treatment with a Trx1 inhibitor.

Conclusions: The Cirbp-Trx1 pathway may regulate mitochondrial dysfunction and mitochondrial Drp1 expression in the hippocampus of PND mice to ameliorate cognitive dysfunction.

Keywords: Cirbp; Drp1; PND; Trx1; mitochondrial dysfunction.

FIGURE 1

Experimental design and mice were bilaterally injected into the hippocampus with over‐Cirbp. Cirbp expression was downregulated in the hippocampal tissue of PND mice. (A) Experimental flowchart of the experimental design. (B) Diagram of bilateral stereotaxic delivery of over‐Cirbp and immunostaining for Cirbp (green). (C) Volcanic plot of mRNA expression (GSE95426). The abscissa was log₂ (FC value) and the ordinate was ‐log₁₀ (p‐value). Red dots represented upregulated genes, green dots represented downregulated genes, and gray dots represented genes which were the same between the two groups. Screening criteria were as follows: FC >1.5 and p < 0.05. The horizontal line represented the threshold of significant p value. (D) The expression of Cirbp was quantified by qRT‐PCR. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 /group). ***p < 0.001, sham vs. surgery. Down, downregulated genes; FC, fold change; i.p., intraperitoneal injection; No‐diff, no difference; Up, upregulated genes.

FIGURE 2

Cirbp overexpression increased Trx1 protein level. (A,B) Hippocampus micrographs showed immunostaining for Cirbp (green), DAPI (blue), and merged images, and quantified the fluorescence intensity. Scale bar = 100 μm. (C–E) Cirbp and Trx1 protein levels in each group were determined by western blot. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 biological replicates /group). **p < 0.01 and ****p < 0.0001, sham vs. surgery; ^& p < 0.05 and ^&&&& p < 0.0001, surgery vs. surgery+over‐Cirbp; ^# p < 0.05, surgery+over‐Cirbp vs. surgery+over‐vector; ^$$$$ p < 0.0001, surgery+over‐Cirbp vs. surgery+over‐Cirbp+PX‐12; ^@ p < 0.05, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12; n.s, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12. n.s: no statistical difference.

FIGURE 3

Cirbp overexpression ameliorated cognitive impairment in a Trx1‐dependent manner in PND mice. (A) Representative trajectories diagram of mice finding the platform during the MWM training trial. (B) The escape latency during the MWM training trial. n.s: No statistical difference. (C) Representative trajectories diagram of all mice after platform removal in the probe trial at 1 and 3 days postoperatively. (D) The swimming speed in the probe trial at 1 day postoperatively. (E,F) The number of platform crossings and the time spent in the target quadrant in the probe trial at 1 and 3 days postoperatively. Data are expressed as the mean (standard error of the mean [SEM]) (n = 10 /group). *p < 0.05 and **p < 0.01, sham vs. surgery; ^& p < 0.05, surgery vs. surgery+over‐Cirbp; ^$ p < 0.05, surgery+over‐Cirbp vs. surgery+over‐Cirbp+PX‐12; n.s, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12. n.s: No statistical difference.

FIGURE 4

Cirbp overexpression inhibited mitochondrial Drp1 protein level in a Trx1‐dependent manner in PND mice. Drp1 content in cytoplasm and mitochondria was determined by western blot. (A,B) Drp1 level in cytoplasm was determined by western blot. (C,D) Drp1 level in mitochondria was determined by western blot. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 biological replicates/group). *p < 0.05 and **p < 0.01, sham vs. surgery; ^& p < 0.05, surgery vs. surgery+over‐Cirbp; ^$ p < 0.05, surgery+over‐Cirbp vs. surgery+over‐Cirbp+PX‐12; n.s, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12. cyto, cytoplasm; mito, mitochondria; n.s, No statistical difference.

FIGURE 5

Cirbp overexpression inhibited mitochondrial damage in a Trx1‐dependent manner in PND mice. (A) The MDA content was measured in hippocampus. (B) The SOD activity was measured in hippocampus. (C,D) cytc level in cytoplasm was determined by western blot. (E,F) cytc level in mitochondria was determined by western blot. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 biological replicates /group). (G–J) TEM images of mitochondria in the mice hippocampus. (H) Mitochondrial number in the mice hippocampus. (I) Dumbbell‐shaped mitochondrial number in the mice hippocampus. (K) Mitochondrial vacuole area in the mice hippocampus. Scale bar = 1 μm. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 /group). *p < 0.05, **p < 0.01, and ***p < 0.001, sham vs. surgery; ^& p < 0.05 and ^&& p < 0.01, surgery vs. surgery+over‐Cirbp; ^$ p < 0.05 and ^$$ p < 0.01, surgery+over‐Cirbp vs. surgery+over‐Cirbp+PX‐12; n.s, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12. n.s: No statistical difference; cyto: cytoplasm; mito: mitochondria. black arrow: mitochondria; red arrow: dumbbell‐shaped mitochondria.

FIGURE 6

Cirbp overexpression inhibited hippocampus damage in a Trx1‐dependent manner in PND mice. (A,B) Levels of TNF‐α and IL‐6 in hippocampal tissue. Data are expressed as the mean (standard error of the mean [SEM]) (n = 5 /group). (C,D) TUNEL staining demonstrated apoptosis and quantified the number of apoptotic‐positive cells in the DG region of the hippocampus. Scale bars = 100 μm. Data are expressed as the mean (standard error of the mean [SEM]) (n = 3 /group). ^** p < 0.01 and ^*** p < 0.001, sham vs. surgery; ^& p < 0.05 and ^&& p < 0.01, surgery vs. surgery+over‐Cirbp; ^$ p < 0.05 and ^$$ p < 0.01, surgery+over‐Cirbp vs. surgery+over‐Cirbp+PX‐12; n.s, surgery+over‐vector vs. surgery+over‐Cirbp+PX‐12. n.s: No statistical difference.