RTA‐408 protects against propofol‐induced cognitive impairment in neonatal mice via the activation of Nrf2 and the inhibition of NF‐κB p65 nuclear translocation

Abstract Objective To explore the effect of RTA‐408 on the propofol‐induced cognitive impairment of neonatal mice via regulating Nrf2 and NF‐κB p65 nuclear translocation. Methods C57BL/6 neonatal mice were randomized into intralipid, propofol, vehicle + propofol, and RTA‐408 + propofol groups. The learning and memory ability was inspected by Morries water maze (MWM) test. TUNEL staining was performed to examine the apoptosis of neurons in hippocampus. The gene and protein expressions in hippocampus were detected by immunohistochemistry, qRT‐PCR, or Western blotting. The activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) were tested by the corresponding kits. Results Propofol prolonged escape latency of mice, decreased the times of crossing the platform, and shortened the time of staying in the target quadrant, while RTA‐408 treatment improved the above‐mentioned situation. Besides, Nrf2 protein in hippocampus of mice induced by propofol was decreased with the increased NF‐κB p65 nuclear translocation, which was reversed by RTA‐408. Meanwhile, RTA‐408 decreased the apoptosis of neurons accompanying with the down‐regulation of Caspase‐3 and the up‐regulations of neuronal‐specific nuclear protein (NeuN), microtubule‐associated protein 2 (Map2), Ca2 +/Calmodulin‐dependent Protein Kinase II (CaMKII), and parvalbumin (PV) immunostaining in hippocampus. Besides, propofol‐induced high levels of proinflammatory cytokines and antioxidase activities in hippocampus were reduced by RTA‐408. Conclusion RTA‐408 improved propofol‐induced cognitive impairment in neonatal mice via enhancing survival of neurons, reducing the apoptosis of hippocampal neurons, mitigating the inflammation and oxidative stress, which may be correlated with the activation of Nrf2 and the inhibition of NF‐κB p65 nuclear translocation.


| INTRODUC TI ON
A series of animal studies have demonstrated that many anesthetics, like ketamine, propofol, etomidate, and isoflurane, could cause damage to the brain neurons during the early postnatal period, further leading to cognitive and neurological abnormalities (Kletecka et al., 2019). Since propofol (2, 6-diisopropylphenol) has rapid onset and short duration of action, it has been widely used in surgical procedure and intensive care units (ICU), as well as the pediatric ICU (Yan et al., 2019), which, however, could trigger the apoptosis or inhibit the neuronal growth in vitro (Wang et al., 2018). Especially, the long-term repeated injection of large-dose propofol has influence on the learning and memory capacity (Xu et al., 2016), which could be used as the regulator of Nrf2 and nuclear factor-κB (NF-κB) pathways (Jiang et al., 2018).
Nrf2 is a basic leucine zipper transcription factor, belonging to the Cap' n' Collar (CNC) transcription factor family, which was functioned as a key transcription factor and a major regulator of cell oxidative stress, inducing the expression of antioxidant enzymes, detoxification enzymes, and downstream protein molecules (Ikram et al., 2019). Currently, the researches of Nrf2 in the central nervous system have become a hot spot (Liu et al., 2019;Shah et al., 2019). NF-κB is a family of dimeric transcription factors made up of five family members, p50 (NF-κB1), RelA (p65), p52 (NF-κB2), c-Rel, and RelB (Oda-Kawashima et al., 2020). Zhong et al. (2014) revealed down-regulation of p65 was involved in the potential mechanisms of propofol-induced neurotoxicity. RTA-408 (Omaveloxolone), a nuclear factor E2 related 2 (Nrf2) activator, is a synthetic triterpeniod with anticancer and anti-inflammatory activities (Probst et al., 2015). Recently, Yang et al. (2019) demonstrated RTA-408 attenuated IL-1β-stimulated p65 nuclear translocation in a rat brain astrocyte (RBA-1) line.
Therefore, we hypothesis that RTA-408 may play a role in the propofol-induced cognitive dysfunction in neonatal mice via affecting Nrf2 and NF-κB p65. In our experiment, C57BL/6 mice aged 7 days were pretreated with RTA-408, and treated with propofol for 6 hr (Mandyam et al., 2017), to explore the treatment of RTA-408 on propofol-induced cognitive dysfunction of neonatal mice.

| Ethical statement
All experiments conducted on animals in the study are in accordance with the management and using principles of local experimental animals, meet the requirements of animal ethics, and follow the Guidelines for the Management and Use of Experimental Animals issued by the National Institutes of Health of the United States (Mason & Matthews, 2012).

| Anesthesia
C57BL/6 wild-type male mice aged 7 days (PND 7, purchased from Jackson Laboratory) were fed in clean animal rooms with normal circadian rhythm and free eating and drinking at room temperature 22°C. Mice were randomly divided into four groups: intralipid group, propofol group, vehicle + propofol group, and RTA-408 + propofol group. The animals grouping and the experiment process were shown in Figure 1. The mice in the vehicle + propofol or RTA-408 + propofol were pretreated with either vehicle [10% dimethyl sulphoxide (DMSO)/sterile saline] or RTA-408 (Selleck Chemicals) for 2 hr (Shekh-Ahmad et al., 2018). Then, the mice in intralipid group and propofol groups received intraperitoneal (i.p.) injection of intralipid (vehicle control, 50 mg/kg; Cutter Laboratories) and propofol (50 mg/kg; Sigma-Aldrich) followed by twice injections (25 mg/ kg, i.p.) of intralipid and propofol at 2-hr intervals, respectively, 6 hr of exposure in all (Pearn et al., 2018). One month after propofol exposure, half of the mice randomly chosen in each group were killed, and the others were fed for 3 months after propofol exposure for Morris Water Maze (MWM) test. The body weight was recorded every week.

| Sample collection
The mice in each group were weighted and then killed after blood samples collected from their tail vein. The chest cavity was quickly opened to fully expose the heart. A 16G vein puncture needle was used to insert into the aorta through the left ventrical, and the right auricle cut to be quickly infused with 200 ml of ice normal saline. The brain was taken out, quickly transferred into the ice block formed by sterile double steaming water, and hippocampus, was separated, washed with 4°C sterile water and dried to be frozen in liquid nitrogen, and then transferred to keep it in refrigerator at −80°C. Corresponding kits were used to test the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). The hippocampus was perfused rapidly with heparinized normal saline and fixed with 200 ml of 4% paraformaldehyde.

| qRT-PCR
TRIzol reagent was used to extract the total RNA, which was then reversed into cDNA by the Super Script III cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A SYBR Green PCR Master kit with an ABI Prism 7,500 Sequence Detection System (Applied Biosystems, Foster City, CA) was conducted to perform the PCR reaction. Table 1 shows primer sequences used in the study. The 2 −△△Ct method was utilized to calculate the relative expression of target gene with β-actin as the internal reference.

| Western blotting
The hippocampus was dispersed in precooled RIPA lysis buffer, followed by homogenization. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc.) followed by the quantitation with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the instructions. The same sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane. 5% nonfat milk was added for 1 hr followed by the addition of primary antibodies overnight at 4°C. After the membrane was washed once for three times, secondary antibody was added, and the membrane was washed three times after incubation at 37°C for 1.5 hr (each time for 10 min), followed by the detection with ECL (Amersham Pharmacia Biotech). The densitometric analysis was conducted using ImageJ Software.

| TUNEL staining
The hippocampus was dehydrated with gradient ethanol, cleared in xylene, embedded in paraffin, and sectioned into 4 μm slices. Then, these sections were subjected to conventional dewaxing to water.
The TUNEL kits (Boehringer Mannheim) were applied to conduct TUNEL staining according to the instructions. Observed under the optical microscope, the nucleus of apoptotic cells was stained into brown or yellow. The percentage of TUNEL positive cell number was calculated in five arbitrarily selected fields.

Gene
Primer sequences Then, the tissues were washed with PBS (5 min × 3 times), developed with DAB, counterstained with hematoxylin, dehydrated, rendered transparent, and mounted.

| Morris water maze test
The device adopted a diameter of 214 cm with a height of 50 cm and a depth of 30 cm, and four white mark points divided the pool into four quadrants. Mice were put into the pool from different quadrants every time, and the time from entering water to finding platform was calculated as the escape latency. Mice, which cannot find the platform in 60 s, were placed on the platform to 10 s. After

| Statistical analysis
SPSS 22.0 statistical software was used for conducting analysis.
The measurement was expressed in the way of mean ± standard deviation (SD). One-way or two-way ANOVA was used as a tool for comparison among multiple groups followed by Tukey's honestly significant differences (HSD) test or Holm-Sidak method for pair comparison, respectively. p < .05 was taken as statistically significant.

| RTA-408 strengthened the learning and memory capacity of propofol-induced neonatal mice
No significant difference was observed in body weight (p > .05, that the period of propofol-induced escape latency was significantly prolonged, with the decreased number of platform crossings (both p < .05). By comparison with propofol group, the escape latency of mice in RTA-408 + propofol group was reduced, but the number of platform crossings was increased (both p < .05). Besides, when compared with intralipid group, the time of mice in the target quadrant in propofol group was shortened (p < .05), but it was prolonged in RTA-408 + propofol group, as compared to propofol group.

| Comparison of Nrf2 and NF-κB expression in hippocampus of neonatal mice in each group
The result of Western blotting demonstrated the decreased Nrf2

| RTA-408 increased the propofol-induced loss of pyramidal neurons and interneurons in neonatal mice
As shown in Figure 4e-h, the pyramidal neurons (marked by CaMKII) and interneurons (marked by PV) was decreased in the hippocampal CA1 areas of mice form the propofol group as compared with the intralipid group (both p < .05), which was partly reversed by the RTA-408 treatment (both p < .05).

| RTA-408 restricted the inflammation of hippocampus in propofol-induced neonatal mice
Levels of proinflammatory factors (TNF-α, IL-6, and IL-1β) in the hippocampus of each group were determined by qRT-PCR and Western blotting in Figure 5, which showed increased levels of proinflammatory factors in the hippocampus of mice from propofol group than those from intralipid group (all p < .05), but RTA-408 pretreatment could decreased the inflammation of hippocampus in propofol-induced neonatal mice with decreased levels of TNF-α, IL-6, and IL-1β (all p < .05).

| RTA-408 reduced propofol-induced apoptosis and oxidative stress in hippocampus of neonatal mice
RTA-408 pretreatment significantly reduced propofol-induced apoptosis of hippocampus neurons of mice (p < .05, Figure 6a,b). Besides, expression of Caspase-3 was tested in the CA1 area of hippocampus via immunohistochemistry, which showed Caspase-3 expression was increased significantly in other three groups compared with intralipid group, but when compared to vehicle + propofol group, its expression was decreased significantly in RTA-408 + propofol group (all p < .05, Figure 6c,d). Moreover, the activities of GPx, SOD, and CAT in the hippocampus of mice from propofol group and vehicle + propofol group were lower than those from intralipid group, while RTA-408 can significantly improve propofol-induced oxidative stress in hippocampus (all p < .05, Figure 6e-g).

| D ISCUSS I ON
According to the previous study, the brain from a one-week-old mouse developed similar as the human's brain to some extent (Pearn

F I G U R E 3
Effect of RTA-408 on Nrf2 and NF-κB p65 nuclear translocation in hippocampus from propofol-induced mice. Note: A-B: The expression of Nrf2 and NF-κB p65 nuclear translocation in the hippocampus of mice detected by Western blotting; C: NF-κB p65-positive neurons were detected in CA1 subfield cells using immunohistochemical staining. D: Quantitative analysis of NF-kB p65 nuclear-positive cells. The data are expressed as percentage of total cell number. Data are expressed as means ± SD, and analyzed using One-way ANOVA followed by the Tukey' HSD test, n = 10 mice per group; * p < .05, compared with intralipid group; # p < .05, compared with propofol group and vehicle + propofol group et al., 2018). In addition, propofol has been pointed out to cause the growth cone collapse, axonal transport impairment, loss of synaptic connectivity, and the behavioral deficits of mice at PND 5-7 . Therefore, in this study, we did experiments on PND7 mice, and after being exposed to propofol for 6 hr, the escape latency was prolonged with the decreased number of crossing platform, which was reversed by the treatment of RTA-408, indicating that RTA-408 could strengthen the learning and memory capacity of propofol-induced mice.
In our study, we found the expression of Nrf2 protein in hippocampus was reduced after the propofol injection. Therefore, we speculated that Nrf2 activation might also play a protective role in , and the percent protection of PV (+) cells among the different groups; ml, molecular cell layer; DG, dentate gyrus; sr, stratum radiatum; pcl, pyramidal cell layer; gcl, granule cell layer. Data are expressed as means ± SD, and analyzed using One-way ANOVA followed by the Tukey' HSD test, n = 10 mice per group; * p < .05, compared with intralipid group, # p < .05, compared with propofol group and vehicle + propofol group neurons from a variety of injury (Shi et al., 2018). Debolina Ghosh and his groups also revealed Nrf2 activator treatment additively improve redox glutathione levels and neuron survival in aging and in Alzheimer mouse neurons . RTA-408, as a synthetic triterpenoid compound, is found to be able to potently acti-  (Calkins et al., 2009). We also found propofol induced a significant increase of NF-κB p65 nuclear translocation in the hippocampus of neonatal mice, which was in line with the former researches (Popic et al., 2015). Several researches demonstrated the stimulated nuclear translocation of NF-κB p65 could be suppressed by RTA-408 (Sun et al., 2019;Yang et al., 2019). In our study, the hippocampal CA1 neurons of propofol-induced mice after F I G U R E 5 The levels of proinflammatory factors  in hippocampus of mice in each group measured by qRT-PCR (a) and Western blotting (b-c). Note: Data are expressed as means ± SD and analyzed using one-way ANOVA followed by the Tukey' HSD test, n = 10 mice per group;*p < .05, compared with intralipid group, # p < .05, compared with propofol group and vehicle + propofol group F I G U R E 6 RTA-408 reduced propofol-induced apoptosis of hippocampus neurons of neonatal mice and oxidative stress in the hippocampus. Notes: (a, b) TUNEL staining was used to detect apoptosis in the hippocampal CA1 area of mice; (c,d) Immunohistochemistry was to detect the expression of Caspase-3 in the hippocampal CA1 area of mice; Arrows indicated Caspase-3 positive staining. (e-g) Comparison of oxidative stress indexes (GPx, SOD, and CAT) in hippocampus of mice; Data are expressed as means ± SD and analyzed using One-way ANOVA followed by the Tukey' HSD test, n = 10 mice per group; *p < .05, compared with intralipid group, # p < .05, compared with propofol group and vehicle + propofol group RTA-408 treatment were increased, suggesting the enhance survival of neurons by RTA-408 pretreatment may be related to the activation of Nrf2 and the inhibition of NF-κB p65 nuclear translocation in propofol-induced hippocampal of mice. The proinflammatory cytokines mainly including IL-1, IL-6, IL-8, and TNF are small molecular polypeptides synthesized and secreted by immune and nonimmune cells of the body (Dhanisha et al., 2019;Gebremariam et al., 2019), which were shown to be induced in propofol-induced hippocampus (Milanovic et al., 2016;Yang et al., 2014), being similar as the findings in our study. At present, the anti-inflammatory medicines can inhibit the activation of NF-κB pathway, and antioxidant drugs usually perform cell protection function by activating Nrf2 pathway (Choudhury et al., 2015;Mukherjee et al., 2013). As consistent with previous study, the antioxidase activities of GPx, SOD, and CAT in hippocampus of mice were reduced by propofol, indicating that the oxidative response was serious (Alirezaei et al., 2017). Furthermore, the study of neonatal anesthetics, including propofol, concerning the neurotoxicity mainly focuses on apoptosis . RTA 408 has been reported to play an antiapoptotic role in several studies. The in vitro experiments showed RTA 408 (10 and 100 nM) treatment can reduce H 2 O 2 -induced apoptosis of human retinal pigment epithelial cells , and the mice, underwent unilateral ischemia followed by contralateral nephrectomy, exhibited had the improved renal function and the decreased apoptosis after treated with RTA-408 in vivo (Han et al., 2017).
Meanwhile, caspase-3 had irreplaceable role in cell apoptosis, as the most important end shear enzyme in the process of apoptosis . In our experiment, propofol-induced apoptosis of hippocampal neurons and expression of caspase-3 were inhibited with the treatment of RTA 408 with the decreased loss of NeuN (+) and Map2 (+) principal cells, PV(+) interneurons, and CaMKII (+) pyramidal neurons in the hippocampal CA1 areas of mice, indicating that RTA 408 may play a protective role in propofol-induced cognitive impairment.
To sum up, RTA-408 can be used to improve learning and memory capacity of propofol-induced neonatal mice by increasing the survival of neurons, lowering the inflammatory response and oxidative stress in hippocampus, and reducing the apoptosis of neurons, which may be correlated with the activation of Nrf2 and the inhibition of NF-κB p65 nuclear translocation. However, there exist several limitations: First, we did not measure the actual plasma concentrations of propofol as it is technically difficult to place an intravenous line and obtain blood in PND7 mice; second, the extrahippocampal regions were not examined due to time and funding constraints; finally, further studies in humans will be needed to confirm the ability of RTA-408 in propofol-induced cognitive impairment.

ACK N OWLED G M ENTS
The authors appreciate the reviewers for their useful comments in this study.

CO N FLI C T O F I NTE R E S T
None of the authors have any competing interests.

AUTH O R CO NTR I B UTI O N
Ling Zhang designed and wrote the study. Qian Zhou performed and analyzed the study. Chun-Li Zhou wrote and revised the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets supporting the conclusions of this article are included within the article.