ZLN005 Reduces Neuroinflammation and Improves Mitochondrial Function in Mice with Perioperative Neurocognitive Disorders

Abstract

Background: The decrease expression of PGC-1α contributes to perioperative neurocognitive disorders (PND). This study aimed to investigate the effects of the PGC-1α agonist ZLN005 in preventing PND and to explore the potential mechanism.

Methods: C57BL/6 mice were randomly divided into four groups: the control group (Group C), the surgery group (Group S), the surgery and ZLN005 (5 mg/(kg⋅d)) group (Group L), and the surgery and ZLN005 (7.5 mg/(kg⋅d)) group (Group H). Except for Group C, the other three groups received intraperitoneal injections of vehicle or ZLN005 once a day from 3 days before surgery to 3 days after surgery. The open field test, novel object recognition test and fear conditioning test were performed to measure anxiety behaviors, locomotor activity and memory. The levels of IL-6 and IL-1β were measured at 24 hours after surgery. ATP and ROS levels were measured at 3 days post-surgery. PGC-1α, NRF-1, Atp5d, Atp5k and Cox5a were measured at one day or three days post-surgery.

Results: ZLN005 treatment improved the cognitive function of mice in Group L and Group H compared with Group S. The expression of IL-6 and IL-1β in the hippocampus of the S group was increased after surgery, and ZLN005 reduced the expression of IL-6 and IL-1β in the hippocampus of mice one day after surgery. There were parallel decreases in the expression of PGC-1α/NRF-1 and mitochondrial function in the hippocampus of the Group S mice compared with the Group C mice. The expression of PGC-1α/NRF-1 and mitochondrial function were upregulated after ZLN005 treatment.

Conclusion: Neuroinflammation and mitochondrial damage are involved in the occurrence of PND. ZLN005 activates PGC-1α to increase the expression of mitochondrial proteins, improve mitochondrial function, and ultimately ameliorate the cognitive status of mice after surgery.

Keywords: PGC-1α; ZLN005; mitochondrial respiratory chain complex; neuroinflammation; perioperative neurocognitive disorders; respiratory function.

Figure 1

Experimental diagrams. (A) The anxiety level and locomotor activities of the mice were measured in the open field test 6 days post-surgery. The old/new object recognition test was performed 7 days post-surgery, and the training and context tests of the fear conditioning test were performed 8 days and 9 days post-surgery, respectively. (B) On day one- and three-days post-surgery, hippocampal tissues were harvested to detect inflammatory factors, target RNA, and target proteins and to evaluate mitochondrial function in each group. (C) In the groups requiring drug administration, intraperitoneal injection was started from three days before the operation to 3 days after the operation, once a day. Among them, the control group (sham), the S group (surgery and DMSO), the L group (surgery and ZLN005 5 mg/(kg⋅d)) and the H group (surgery and ZLN005 7.5 mg/(kg⋅d)) were treated differently, as shown in the Methods section.

Figure 2

Recognition and memory in mice were evaluated after surgery and ZLN005 treatment. Behaviour tests were performed on postoperative Days 6–9. (A) On Day 7 post-surgery, long-term memory was evaluated by the novel object recognition test (NORT). The recognition index was calculated for each mouse in the four groups at 4 hours after the training. (B) Long-term memory was evaluated by the fear conditioning task. Freezing time was recorded, and the ratio of freezing time to the total testing time was calculated on Day 9 post-surgery. *P < 0.05; **P < 0.01 by one-way analysis of variance (ANOVA); n. s. not significant; error bars denote the standard error of mean (SEM).

Figure 3

Evaluation of neuroinflammation in mice after tibial fracture surgery and drug treatment. Wild-type mice (7–8 months old) were divided into four groups as mentioned before (n=6-8). For the S group, L group and H group, intraperitoneal injections were all performed from 3 days preoperatively to 1 day postoperatively once a day. Hippocampal and cortical tissues were collected 24 hours post-surgery and 30 minutes after drug injection. (A and B) Levels of IL-6 and IL-1β in the hippocampus were determined by ELISA. (C and D) Levels of IL-6 and IL-1β in the cortex were determined by ELISA. *P < 0.05; **P < 0.01 by one-way analysis of variance (ANOVA); n. s. not significant; error bars denote the standard error of mean (SEM).

Figure 4

The expression levels of PGC-1α/NRF-1 and oxidative respiratory chain proteins were measured by Western blotting and qPCR. (A) One day after surgery, the hippocampus was harvested to detect the transcription of PGC-1α and NRF-1 (n=6-7). The grouping is shown before. (B–D) The expression levels of PGC-1α, NRF-1, Atp5k, Atp5d, and Cox5a were measured three days post-surgery in the hippocampus in the four groups (n=4-5). The expression of PGC-1α, NRF-1, Atp5k, Atp5d, and Cox5a was normalized to that of the β-actin internal control. *P < 0.05; **P < 0.01; ***P < 0.001. Mean ± standard error of the mean values is presented for each group.

Figure 5

At 3 days post-surgery, the brain was harvested to detect the expression of PGC-1α and NRF-1 in different groups of mice by immunofluorescence (n=4). The grouping is as shown before. (A and B) Representative images for PGC-1α and NRF-1 in the hippocampus. (C) Quantification of PGC-1α and NRF-1 fluorescence in the hippocampus. *P < 0.05; **P < 0.01. Mean ± standard error of the mean values is presented for each group. Scale bar, 100 μm.

Figure 6

Mitochondrial function was evaluated three days post-surgery in each group. (A and B) On the third day, the fresh hippocampus was harvested to measure ATP (n=9) and ROS (n=6) in the sham (C group), the surgery and DMSO (S group), the surgery and ZLN005 (5 mg/(kg⋅d)) (L group), and the surgery and ZLN005 (7.5 mg/(kg⋅d)) groups (H group). *P < 0.05; **P < 0.01; ***P < 0.001 by one-way analysis of variance (ANOVA); error bars denote the SEM.

Figure 7

The role of ZLN005 in perioperative neurocognitive disorders.