
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12851-4
10.1016/j.heliyon.2024.e36820
e36820
Research Article
LFHP-1c improves cognitive function after TBI in mice by reducing oxidative stress through the PGAM5-NRF2-KEAP1 ternary complex
Shao Wei ab
Wang Jia-jun b
Niu Zi-hui b
Zhang Kang b
Wang Shuai b
Wang Yu-Hao b
Tang Yu-hang b
Wang Cheng-Cheng b
Hou Shi-Qiang b
Zhou Dong-Rui b
Zhang Chao b
Lin Ning lin2007512@vip163.com
b⁎
a Department of Emergency, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou, Zhejiang, 310000, China
b Department of Neurosurgery, The Affiliated Chuzhou Hospital of Anhui Medical University, The First People's Hospital of Chuzhou, Chuzhou, Anhui, 239000, China
⁎ Corresponding author. Department of Neurosurgery, The Affiliated Chuzhou Hospital of Anhui Medical University, The First People's Hospital of Chuzhou, 369 Zuiweng Road, Chuzhou , Anhui, 239000, China. lin2007512@vip163.com
25 8 2024
15 9 2024
25 8 2024
10 17 e3682021 6 2024
22 8 2024
22 8 2024
© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Traumatic brain injury (TBI) is a leading cause of disability and death. Thus, timely and effective secondary brain injury intervention is crucial, with potential to improve the prognosis of TBI. Oxidative stress contributes to post-traumatic secondary cognitive impairment, and the reduction of post-traumatic oxidative stress effectively enhances cognitive function. Phosphoglycerate-mutating enzyme 5 (PGAM5), a member of the phosphoglycerate transporter enzyme family, is upregulated in TBI and induces mitochondrial autophagy. This further exacerbates damage following TBI. The present study focused on the small molecule drug, LFHP-1c, which is a novel inhibitor of PGAM5. The present study used an in vivo mouse model incorporating a controlled cortical impact-induced TBI, to examine the impact of LFHP-1c on oxidative stress and cognitive function. The present study aimed to determine the impact of LFHP-1c on the PGAM5-Kelch-like ECH-associated protein 1 (KEAP1)- nuclear factor erythroid 2-related factor 2 (NRF2) ternary complex within the TBI context. Results of the present study indicated that LFHP-1c suppresses PGAM5 expression and inhibits the development of the PGAM5-KEAP1-NRF2 ternary complex, thereby promoting the release of NRF2 and KEAP1. This in turn promotes the entry of NRF2 into the nucleus following TBI, leading to increased expression of anti-oxidative stress downstream factors, such as heme oxygenase-1, glutathione peroxidase 1 and superoxide dismutase 1. In addition, LFHP-1c also released KEAP1, leading to mitochondrial Rho GTPase 2 degradation and reducing perinuclear aggregation of mitochondria in the cell, which reduced oxidative stress and ultimately improved cognitive function after TBI.

Keywords

Traumatic brain injury
LFHP-1c
PGAM5-NRF2-KEAP1 ternary complex
Oxidative stress
Cognitive function
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pmc1 Introduction

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality globally, imposing a significant burden on patients, their families and society [1]. TBI triggers a complex set of pathophysiological processes that result in severe secondary brain damage, including blood-brain barrier disruption, inflammation, excitotoxicity, necrosis, apoptosis, mitochondrial dysfunction and oxidative stress, within hours or days post-injury [2,3]. This cascade leads to oxidative stress, mitochondrial dysfunction, lipid peroxidation, and DNA and protein oxidation. Given the rapid progression of primary brain injury, effective intervention in the secondary pathology cascade exhibits potential in the treatment of TBI [4]. Therefore, investigating the physiological mechanisms of oxidative stress and cognitive impairment is crucial to alleviate the poor prognosis of patients with TBI.

Results of a previous study demonstrated that pathophysiological changes that occur in the brain after TBI are due to four main factors, including cytokines, excitotoxic activity, reactive oxygen species (ROS) and cell death [5]. TBI also causes mitochondrial dysfunction, resulting in disturbances in intracellular redox balance. During this process, the levels of oxidative stress are markedly increased, and the production of ROS is also increased, resulting in high levels of oxidative stress both inside and outside the cell. Oxidative stress is directly involved in the occurrence and development of secondary injury following TBI, inducing a series of inflammation, apoptosis and other pathophysiological processes [6]. Results of previous studies suggested that oxidative stress plays a key role in cognitive dysfunction after TBI. Oxidative stress-induced cellular damage and inflammatory responses negatively affect neuronal survival and function [7,8]. In addition, oxidative stress may lead to mitochondrial dysfunction, exacerbating cellular vulnerability through impacting energy metabolism and intracellular environmental homeostasis. Oxidative stress induced by TBI disrupts the delicate balance between ROS generation and the antioxidant system, which, to a large extent, leads to cognitive impairment after TBI. Therefore, research is focused on the reduction of oxidative stress and improvements in cognitive impairment following TBI, as these exhibit potential in the treatment of cognitive impairment following TBI.

LFHP-1c is a small-molecule hybrid compound containing 2-(1-hydroxypentyl)- benzoate (HPBA) and temesartan moieties that acts as a novel inhibitor of phosphoglycerate-mutating enzyme 5 (PGAM5). Previous studies have demonstrated the neuroprotective effects of LFHP-1c in stroke models, particularly in maintaining the integrity of the blood-brain barrier via the PGAM5/nuclear factor erythroid 2-related factor 2 (NRF2) axis [9]. However, research into the potential impact on cognitive function following TBI is lacking. PGAM5 is a protein that is present in the outer and inner membrane contact sites of mitochondria. This protein plays a crucial role in regulating mitochondrial functions, such as dynamics and autophagy. It also influences cell death and organelle homeostasis [10,11]. Results of previous studies suggested that the PGAM5-Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 ternary complex plays a crucial role in inhibiting NRF2 nuclear translocation and regulating antioxidant responses. PGAM5, as a novel regulator of mitochondrial homeostasis, forms a complex with KEAP1 and NRF2, influencing NRF2 activity and downstream antioxidant response initiation. KEAP1 acts as a major negative regulator, binding to NRF2 to facilitate its ubiquitination and degradation. Under normal conditions, NRF2 dissociates from KEAP1 and translocates to the nucleus when cells are under oxidative stress. Subsequently, NRF2 binds to antioxidant response elements (AREs), thereby initiating the transcription of antioxidant response genes. However, through the formation of the PGAM5-KEAP1-NRF2 ternary complex, PGAM5 prevents the dissociation and nuclear translocation of NRF2, thereby limiting NRF2 activity and the initiation of antioxidant responses [[12], [13], [14]].

Results of previous studies demonstrated that the microtubule-dependent retrograde transport of mitochondria depends on the intricate PGAM5-KEAP1-NRF2 ternary complex. In addition, the small mitochondrial Rho GTPases, specifically mitochondrial Rho GTPase 2 (Miro2), plays a pivotal role in regulating mitochondrial transport and dynamics, thereby influencing cellular functions and responses to oxidative stress. Specifically, Miro2 is involved in the regulation of mitochondrial motility, distribution and anchorage at cellular locations. It acts as an adaptor protein, linking mitochondria to the cytoskeleton, particularly microtubules, facilitating their movement along the cellular matrix.

Miro2 has been implicated in the modulation of cellular responses to oxidative stress. It participates in the regulation of ROS generation and scavenging within mitochondria. Through its interactions with ROS-producing enzymes and antioxidant defense systems, Miro2 influences ROS levels and cellular redox homeostasis. In addition, results of a previous study suggested that alterations in Miro2 expression or activity may impact nuclear accumulation of mitochondria under conditions of cellular stress, including oxidative stress. This nuclear accumulation of mitochondria may affect cellular functions and signaling pathways, contributing to oxidative stress-related cellular damage and dysfunction [15]. The intricate molecular mechanism inhibits the coupling of KEAP1 to cullin-3 E3 ubiquitin ligase (Cul3), which initiates the subsequent degradation of Miro2. This sequence of events intricately affects the backward movement of mitochondria, highlighting the interplay within the PGAM5-KEAP1-NRF2 axis [16].

To the best of our knowledge, the present study is the first to demonstrate that LFHP-1c inhibits the activity of the PGAM5 enzyme and impacts the intricate interplay within the PGAM5-KEAP1-NRF2 ternary complex. Inhibition of the PGAM5-KEAP1-NRF2 ternary complex was previously observed in rat neurons following trauma, and this led to a significant induction in NRF2 nuclear translocation. In this study, we used Western blot analysis, immunofluorescence analysis and related behavioral experimental methods to verify whether LFHP-1C can improve cognitive function after TBI in rats by affecting the PGAM5-KEAP1-NRF2 ternary complex.

2 Materials and methods

2.1 Animals and clinical samples

Adult male C57BL/6 mice (age, 8–10 weeks) and age-matched C57BL/6 female mice were obtained from Qinglongshan Animal Breeding Farm, Jiangning, Nanjing, China. All animal experiments were approved by the Animal Management and Use Committee of Chuzhou Affiliated Hospital of Anhui Medical University. All mice were divided into the Sham group, the 12 h, 1 day (d), 2 d, 3 d, 5 d after TBI groups, and the TBI + LFHP-1c group. Each experimental group consisted of three randomly selected male and three randomly selected female mice.

Clinical samples were obtained intraoperatively from patients with TBI who underwent surgery. When collecting human samples, we mainly selected patient samples of 12h, 1 day and 3 days after TBI. However, it was a pity that the operation time of clinical TBI patients was difficult to grasp led to the limited number of human samples we collected, TBI clinical samples at separate time nodes were not statistically significant. All experiments involving clinical samples were approved by the Ethics Committee of the Chuzhou Affiliated Hospital of Anhui Medical University, which confirmed that all experimental protocols were conducted in accordance with the guidelines and regulations of the experimental facility of the Chuzhou Affiliated Hospital of Anhui Medical University.

2.2 Controlled cortical impact (CCI) modeling and drug administration

To induce TBI in mice in vivo, the CCI device was used. Briefly, mice were anesthetized with 2 % isoflurane and securely positioned in a stereotaxic frame. A 2.2-mm-diameter hole was precisely drilled in the right parietal cortex, situated 2 mm posterior to bregma and 5 mm lateral to the sagittal suture. Subsequently, a 5-mm beveled end was utilized to impact the exposed dura at a speed of 5 m/s, penetrating to a depth of 2.2 mm. After surgery, the wounds were disinfected and sutured, and animals were removed from the stereotaxic device. Animals were subsequently placed on a heating pad until they regained consciousness and resumed gross motor function. Mice were returned to the normal feeding device and monitored.

LFHP-1c was dissolved in 1 % DMSO (Sigma-Aldrich, Merck KgaA) and saline. Previous studies have compared doses of 0.1 and 0.5 mg/kg LFHP-1c, and compared the time of administration at 4 and 12 h after modelling [9]. The results demonstrated that the optimal therapeutic window for the treatment of LFHP-1c is 4 and 24 h after modelling, with an optimal dose of 0.5 mg/kg LFHP-1c. Thus, TBI mice in the present study were fixed and subsequently administered 0.5 mg/kg LFHP-1c via tail vein injection, 4 and 24 h after TBI.

2.3 Neurobehavioral analysis

Cognitive function and behavioral evaluation methods were carried out based on previous studies. The modified Neurological Severity Score (mNSS) and Y-maze experiments were used to measure the behavior of all experimental mice. Mice were divided into a Sham group and drug and TBI groups with six mice in each group.

2.4 Western blot analysis

Total proteins were extracted from hippocampal tissues isolated from the injured side in mice at 12 h, and 1, 2, 3 and 5 d after TBI, and in mice in the TBI group. Tissue samples were weighed and RIPA buffer containing 1 % phenylmethylsulfonyl fluoride (PMSF) was added (Beyotime Institute of Biotechnology) to lysed hippocampal tissues on the injured side. Protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Samples were sonicated and incubated on ice for 30 min, followed by centrifugation at 14000 r/min for 10 min at −4 °C in an ultracentrifuge. The supernatant was collected for protein determination and stored at −20 °C in a refrigerator.

Proteins were separated via SDS-PAGE and transferred to PVDF membranes (MilliporeSigma). Subsequently, samples were blocked with 5 % skimmed milk (Biofrox) for 2 h, and incubated overnight at 4 °C with primary antibodies against PGAM5 (cat. no. 28445-1-Ap; Proteintech Group, Inc.), NRF2 (cat. no. 16396-1-Ap; Proteintech Group, Inc.), KEAP1 (cat. no. 10503-2-Ap; Proteintech Group, Inc.), Miro2 (cat. no. 11237-1-AP; Proteintech Group, Inc.), HO-1 (cat. no. 10701-1-Ap; Proteintech Group, Inc.), SOD1 (cat. no. 10269-1-AP; Proteintech Group, Inc.) and GPX1 (cat. no. 29329-1-AP; Proteintech Group, Inc.). Following overnight incubation, membranes were washed three times with TBST for 10 min each time, and subsequently incubated with horseradish peroxidase-labelled goat anti-rabbit secondary antibody for 2 h. Washing with TBST was repeated for a further three times for 10 min each time. Protein bands were visualized using an ECL kit (MilliporeSigma) and images were captured using a ChemiDoc MP imaging system (Bio-rad Laboratories, Inc.). Protein bands were analyzed using ImageJ (National Institutes of Health).

2.5 Nucleus protein extraction

Injured lateral hippocampal tissues were isolated from mice in the post-TBI treatment group and immediately added to the homogenate for lysis. Cytosolic proteins were extracted using the Cytosolic Protein and Cytosolic Plasma Protein Extraction Kit (cat. no. P0028; Beyotime Institute of Health). SDS-PAGE Protein Supersampling Buffer (cat. no. LT101S; EpiZyme) was added and samples were visualized. Subsequently, samples were stored in a −20 °C refrigerator.

2.6 Immunofluorescence analysis

Frozen sections of brain tissue were obtained following transcardiac perfusion, reheated at 37 °C for 60 min and permeabilized using PBST for 20 min. Subsequently, sections were blocked with 10 % skimmed milk for 2 h. The sealing solution was removed, and dilutions of the following antibodies were added in a dropwise manner: anti-Pgam5 (1:200; cat. no. 28445-1-AP; Proteintech Group, Inc.), anti-NRF2 (1:200; cat. no. ab62352; Abcam), anti-KEAP1 (1:200; cat. no. D6B12; CST Biological Reagents Co., Ltd.) and anti-Mitofilin (1:200; cat. no. ab137057; Abcam). Samples were incubated overnight in a wet box. Following recovery at room temperature, primary antibodies were discarded and samples were washed three times with PBS for 5 min each time. The corresponding fluorescent secondary antibody (1:500) was added in the dark, and samples were incubated for 2 h at room temperature. After discarding the secondary antibody, cells were washed with PBS three times for 5 min each time, and the nuclei were stained with DAPI prior to three rounds of washing with PBS. Cells were subsequently blocked using an anti-fluorescent bursting agent and incubated in the dark. Images were captured using a fluorescence microscope (cat. no. LSM 900; ZEISS GmbH).

2.7 Y-maze spontaneous alternation experiments

The Y-maze spontaneous alternation test was used to assess the spatial memory capability of mice. This test relies on the inherent tendency of mice to explore novel situations. The Y-maze, which was constructed on-site, featured three indistinguishable arms (50 x 10 × 20 cm), with an angle of 120° between each pair of arms. There were three removable partitions in the center of the maze, and the bottom of the maze was painted white. Mice were placed at the end of any of the arms of the Y-maze and allowed to explore freely for 5 min. Behavioral changes of the animals were recorded for 5 min through a camera system. Mice alternated the rotations between each wall based on their ability to memorize. Between experiments, 75 % alcohol was used to eliminate odors and bright light interference was avoided throughout the experiment. The following metrics were recorded: i) Total number of arm entries [the number of animals entering the maze arm (measured as all four feet of the mouse entering the arm)]; ii) number of spontaneous alternations (all three arms of the Y-maze entered sequentially at one time); iii) maximum number of spontaneous alternations (total number of arm entries −2). The percentage of spontaneous alternation performance (SAP %) was calculated as the number of spontaneous alternations/maximum number of spontaneous alternations x 100. SAP% is an index to assess the memory and cognitive ability of mice in the Y maze. It measures the cognitive ability of mice by calculating the percentage of mice choosing different arm paths in successive arm path choices. Higher SAP% indicates that mice show better spatial memory and cognitive flexibility in the Y maze task.

2.8 Statistical analysis

All statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, Inc.) and SPSS 22.0 software (IBM Corp.). Data are presented as the mean ± standard deviation, and statistical analyses were carried out using one-way ANOVA and Student's t-tests. P < 0.05 was considered to indicate a statistically significant difference.

3 Results

3.1 PGAM5 expression is increased in damaged brain tissue following TBI

In the present study, changes in PGAM5 expression were determined in ipsilateral hippocampal tissue post-TBI. Western blot analysis demonstrated a significant elevation in PGAM5 protein levels on the third day post-TBI, compared with the Sham group (Fig. 1A and B). Thus, the third day was selected as the representative time point for the TBI group, with the drug-administered group used as the control. In human brain tissues obtained from patients with TBI, results of the Western blot analysis demonstrated an increase in PGAM5 levels post-TBI, compared with healthy tissues (Fig. 1C and D). Collectively, these results highlighted the potential role of PGAM5 following TBI.Fig. 1 Increased expression of PGAM5 following TBI. (A) Western blot analysis of PGAM5 in the Sham group, and in groups 12 h, 1 d, 2 d, 3 d and 5 d following TBI. (B) PGAM5 expression levels were significantly upregulated in the 3-d post-TBI group compared with the Sham group. (C) Western blot analysis of PGAM5 in Normal and TBI groups. (D) PGAM5 expression levels were higher following TBI compared with the Normal group. *P < 0.05, **P < 0.01, ***P < 0.001. PGAM5, phosphoglycerate-mutating enzyme 5; TBI, traumatic brain injury; d, day.

Fig. 1

3.2 LFHP-1c inhibits PGAM5-mediated increases in NRF2 expression and nuclear entry following TBI

To assess the impact of LFHP-1c on NRF2 post-TBI, Western blot analysis was carried out in the present study. The results demonstrated a notable decrease in PGAM5 expression and a notable increase in NRF2 expression following treatment with LFHP-1c, compared with the TBI group (Fig. 2A and B). In addition, nuclear NRF2 expression levels were elevated compared with the TBI group, suggesting that treatment with LFHP-1c increased NRF2 entry into the nucleus (Fig. 2C and D). Results of the immunofluorescence assay demonstrated that PGAM5 expression was significantly reduced in the neurons of damaged hippocampal tissue following treatment with LFHP-1c, compared with the TBI group. However, nuclear NRF2 expression was significantly increased compared with the TBI group (Fig. 2E and F).Fig. 2 LFHP-1c inhibits PGAM5, and increases NRF2 expression and entry into the nucleus in mice after TBI. (A) Western blot analysis of PGAM5 and NRF2 expression following treatment with LFHP-1c. (B) PGAM5 expression levels were markedly downregulated and NRF2 expression levels were upregulated in the TBI + LFHP-1c group, compared with the TBI group. (C) Results of the Western blot analysis demonstrated that LFHP-1c treatment significantly increased the protein expression of Nuclear-NRF2. (D) Nuclear-NRF2 expression levels were increased in the TBI + LFHP-1c group, compared with the TBI group. (E and F) Fluorescent staining of PGAM5 and NRF2 revealed that LFHP-1c treatment significantly decreased the expression of PGAM5 and increased the entry of NRF2 into the nucleus (scale bar, 20 μm). *P < 0.05, **P < 0.01, ***P < 0.001. PGAM5, phosphoglycerate-mutating enzyme 5; NRF2, nuclear factor erythroid 2-related factor 2; TBI, traumatic brain injury.

Fig. 2

3.3 LFHP-1c inhibits mitochondrial aggregation through increasing KEAP1 protein expression following PGAM5 inhibition

Results of the Western blot analysis indicated that LFHP-1c effectively inhibits PGAM5 and increases KEAP1 protein expression, compared with the TBI group (Fig. 3A). These results suggested that treatment with LFHP-1c may inhibit the formation of the PGAM5-KEAP1-NRF2 ternary complex, leading to increased KEAP1 protein expression. Elevated KEAP1 levels lead to increases in coupling to Cul3, resulting in the degradation of the Miro2 protein. Thus, results of the present study demonstrated that Miro2 protein expression was reduced, compared with the TBI group (Fig. 3B). Results of the immunofluorescence analysis using Mitofilin, a mitochondrial membrane localization protein, demonstrated that mitochondrial aggregation around neurons in damaged hippocampal tissue was significantly reduced following treatment with LFHP-1c (Fig. 3C). Collectively, these results further demonstrated the targeted inhibitory effect of LFHP-1c on PGAM5, which led to an increase in free KEAP1 protein expression (Fig. 3D). The subsequent degradation of the Miro2 protein may further contribute to the reduction in mitochondrial aggregation around the nucleus.Fig. 3 LFHP-1c-mediated inhibition of PGAM5 increases KEAP1 expression in mice following TBI, thereby inhibiting mitochondrial aggregation. (A and B) Western blot analysis of PGAM5, KEAP1 and Miro2 expression following treatment with LFHP-1c. PGAM5 and Miro2 expression levels were significantly reduced, and KEAP1 expression levels were increased in the TBI + LFHP-1c group, compared with the TBI group. (C and D) Fluorescent staining of KEAP1 with Mitofilin revealed that LFHP-1c treatment significantly increased KEAP1 expression and decreased perinuclear aggregation of mitochondria (scale bar, 20 μm). *P < 0.05, **P < 0.01, ***P < 0.001. PGAM5, phosphoglycerate-mutating enzyme 5; KEAP1, Kelch-like ECH-associated protein 1; TBI, traumatic brain injury; Miro2, mitochondrial Rho GTPase 2.

Fig. 3

3.4 Enhanced expression of downstream anti-oxidative stress factors following nuclear entry of NRF2 improves cognitive function in mice after TBI

Results of the present study demonstrated that the LFHP-1c-mediated inhibition of PGAM5 led to a consequential increase in NRF2 expression, which ultimately reduced complex formation and increased nucleation. Subsequent nuclear translocation of NRF2 promoted the transcriptional activation of ARE-regulated genes, leading to increased protein expression of antioxidant stress factors. Results of the present study revealed a substantial increase in HO-1, GPX1 and SOD1 protein expression levels compared with the TBI group (Fig. 4A and B). Functional assessments, including the mNSS and Y-maze spontaneous alternation experiment, demonstrated notable cognitive improvements in mice following treatment with LFHP-1c, compared with the TBI group (Fig. 4C and D). Collectively, results of the presents study revealed that LFHP-1c promoted the post-traumatic nuclear translocation of NRF2, promoting an antioxidant stress response to mitigate secondary damage and improve cognitive function in a TBI model (Fig. 5).Fig. 4 NRF2 increases the expression of anti-oxidative stress-related factors after nuclear entry. (A) Western blot analysis of the expression of anti-oxidative stress factors, HO-1, GPX1 and SOD1. (B) Expression levels of HO-1, GPX1 and SOD1 were significantly upregulated in the TBI + LFHP-1c group, compared with the TBI group. (C and D) Neurological performance was assessed using mNSS score and the T-maze spontaneous alternation test. *P < 0.05, **P < 0.01, ***P < 0.001. NRF2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1; GPX1, glutathione peroxidase 1; SOD1, superoxide dismutase 1; TBI, traumatic brain injury; mNSS, modified Neurological Severity Score.

Fig. 4

Fig. 5 Flowchart demonstrating the mechanism by which LFHP-1C improves cognitive function in mice following TBI, through the regulation of the PGAM5-KEAP1-NRF2 ternary complex. TBI, traumatic brain injury; PGAM5, phosphoglycerate-mutating enzyme 5; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2.

Fig. 5

4 Discussion

TBI, a prevalent craniocerebral disorder worldwide, often manifests with transient or continuous cognitive dysfunction characterized by neuronal loss and consequential brain damage [17]. The cognitive impairment following TBI primarily stems from two major contributors: i) Neuroinflammation, when microglia and astrocytes serve as pivotal agents in instigating neuroinflammation post-TBI. Complement activation mediates this inflammatory response, inducing aberrant microglial activation, thereby precipitating cognitive dysfunction; and ii) mitochondrial dysfunction, where mitochondria, central to energy metabolism, play a critical role in the emergence of cognitive dysfunction subsequent to TBI [18,19]. TBI inflicts mitochondrial damage through the disruption of intracellular calcium (Ca2+) homeostasis, thus promoting the production of ROS [20]. This, in turn, leads to cognitive impairment, creating a cascade effect [21]. Moreover, TBI-induced oxidative stress disrupts the intricate balance between ROS production and the antioxidant system, contributing to impaired cognitive function following TBI [3].

PGAM5, identified as a mitochondrial Ser/Thr phosphatase that is predominantly located in the inner mitochondrial membrane, plays a pivotal role in maintaining organelle homeostasis [22]. However, results of a previous study demonstrated that PGAM5 is also present in the outer mitochondrial membrane, which promotes the activation of inflammasomes in microglia to produce IL-1β mediated neuroinflammation post-TBI, and TBI-induced neuroinflammation may partially depend on the interaction between PGAM5, NLRP3 inflammasomes, and Asc oligomerization [23]. Notably, PGAM5 overexpression promotes physiological and neurological outcomes post-TBI, while knockdown or depletion of PGAM5 yields beneficial effects, ameliorating microglia activation, neuronal damage, tissue injury and neurological deficits ensuing TBI [24].

At present, research focused on the role of PGAM5 is lacking. PGAM5 forms a ternary complex with KEAP1 and NRF2, sequestering NRF2 in the cytoplasm. This, in turn, hinders the nuclear translocation of NRF2, subsequently repressing the transcription of NRF2-associated antioxidant genes [12]. NRF2 is a transcription factor that plays a key role in the body's defense mechanisms. NRF2 promotes the transcription of genes that are mediated by the ARE, and are pivotal in diverse cellular processes, such as redox regulation, proteostasis, detoxification of xenobiotics and primary metabolism, and in neurological diseases [13,25,26]. Results of a previous study revealed that NRF2 activated the autophagy pathway in TBI to provide neuroprotection in vivo and in vitro, and a carotenoid derived from seaweed may alleviate TBI-induced brain damage through activation of the NRF2 autophagy pathway [27]. Moreover, activation of NRF2 may mitigate the production of ROS and enhance the expression of antioxidant genes; thus, exhibiting potential in the prevention of ischemic stroke [28].

Interactions between KEAP1 and PGAM5 serve as mechanisms for sensing superoxide/hydrogen peroxide generated by mitochondria, thereby initiating mitochondrial autophagy [29,30]. Moreover, integrity of the ternary complex of PGAM5, KEAP1 and NRF2 is essential for the microtubule-dependent translocation of mitochondria [15]. Notably, mitochondrial transport factors, Miro1 and Miro2, play a crucial role in linking mitochondrial and microtubule motor proteins, thereby controlling the direction and amplitude of mitochondrial transport [31].

Results of a previous study demonstrated that elevated expression of the mitochondrial transport factor, Miro2, promotes the aggregation of mitochondria toward the nucleus, ultimately resulting in the perinuclear overproduction of ROS [32]. This dynamic redistribution of the mitochondrial network promotes the presence of an intact PGAM5-KEAP1-NRF2 ternary complex [15]. Conversely, the reduction of this complex amplifies the association of KEAP1 with its E3 ligase scaffolding chaperone, Cul3, consequently intensifying the degradation of the mitochondrial transport factor, Miro2 [33,34]. Disruption of this complex, achieved through the depletion of NRF2 or PGAM5, acts as a preventive measure against mitochondrial aggregation. This blockade is crucial as it prevents the degradation of the mitochondrial transporter, Miro2, a critical factor in the process of mitochondrial aggregation [35].

The PGAM5 inhibitor, LFHP-1c, exhibits dual inhibitory effects through suppressing PGAM5 phosphatase activity and disrupting the interaction between PGAM5 and NRF2. This disruption promotes the nuclear translocation of NRF2, ultimately facilitating the downstream production of antioxidant genes and proteins. In addition, LFHP-1c exhibited a protective effect on the blood-brain barrier in a model of cerebral ischaemia [9]. However, the specific association between PGAM5, KEAP1, NRF2 and TBI remain to be fully elucidated. Some studies have hypothesized that LFHP-1c may selectively disrupt the interaction between PGAM5 and NRF2, destabilizing the ternary complex. This disruption may reduce NRF2 ubiquitination, promote nuclear translocation and increase free KEAP1 levels. However, the precise interaction between LFHP-1c and the ternary complex requires further elucidation, through the use of co-crystallisation studies [12].

The present study exhibits limitations. For example, experiments were predominantly conducted in a mouse model; thus, the results may not be applicable to humans, necessitating further clinical validation. In addition, the present study mainly focused on oxidative stress and cognitive function, without exploring other neurobiological processes that may have been impacted by treatment with LFHP-1c. The long-term safety and efficacy of LFHP-1c were not fully assessed; thus, future investigations should focus on the potential side effects and long-term impact of LFHP-1c treatment. Moreover, further investigations are required to determine the specific role of the PGAM5-KEAP1-NRF2 pathway in TBI, and the complex pathological processes that may occur following TBI.

In this study, we have explored the role of LFHP-1C in the context of stress-induced mitochondrial retrograde trafficking. Despite the valuable insights provided by our current findings, it is important to acknowledge a significant limitation of our work: the absence of cellular experiments. The lack of direct cellular validation restricts our ability to fully elucidate the detailed molecular mechanisms through which LFHP-1C exerts its effects. To address this limitation, we propose incorporating cellular models in future research. By conducting experiments at the cellular level, we aim to gain a deeper understanding of the interactions between LFHP-1C and key regulatory proteins involved in mitochondrial trafficking. Cellular assays would enable us to investigate the impact of LFHP-1C on mitochondrial dynamics, including its influence on mitochondrial motility and the functional interplay with proteins such as KEAP1 and Miro2.

The present study focused on the impact of LFHP-1c on the nuclear translocation of NRF2 in the context of TBI. The increased expression of anti-oxidative stress genes controlled by NRF2 led to the increased expression of KEAP1. This, in turn, mitigated the perinuclear aggregation of mitochondria, thereby reducing oxidative stress damage and improving cognitive function following TBI. However, the precise mechanisms by which LFHP-1c acts on the PGAM5-KEAP1-NRF2 ternary complex remain unclear. Results of a previous study revealed that Miro2 is required for retrograde mitochondrial transport [36]. Notably, disruption of the PGAM5-KEAP1-NRF2 complex destabilizes Miro2, initiating retrograde mitochondrial transport. Results of the present study highlighted that LFHP-1c reduced Miro2 expression, consequently mitigating perinuclear aggregation of mitochondria. Further investigations are required to determine whether LFHP-1c also reduces ROS deposition around the nucleus. Notably, while our observations are aligned with the existing body of knowledge, they are based on a limited dataset and do not include direct cellular experiments. This absence of cellular validation is a significant limitation, as it restricts our ability to conclusively determine the mechanisms through which PGAM5 affects KEAP1 and Miro2. The importance of cellular studies in validating these interactions and understanding the detailed molecular pathways cannot be overstated. Future research will prioritize incorporating cellular experiments to provide a more comprehensive understanding of the interactions between PGAM5, KEAP1, and Miro2.

In conclusion, LFHP-1c synergistically enhances intracellular antioxidant stress response through modulating the PGAM5-NRF2-KEAP1 pathway, thereby offering a potential therapeutic mechanism for ameliorating cognitive impairment following TBI. The present study provides a theoretical basis for further elucidating the molecular mechanisms underlying TBI and the development of novel therapeutic strategies.

Funding statement

This present study was supported by 10.13039/501100017668 Anhui Provincial Key Research and Development Plan (Grant No. 2022AH050769 ）, 10.13039/501100018628 Scientific Research Foundation of Education Department of Anhui Province (Grant No. KJ2021ZD0036 , 2022AH050769 ), and Health Scientific Research Foundation of Chuzhou (Grant No. CZWJ2022A001 ).

Data availability statements

Data included in article/suppmaterial/referenced in article.

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Chuzhou Affiliated Hospital of 10.13039/501100002947 Anhui Medical University (Grant No. CZWJ 2022A001 ).

Patient consent for publication

All subjects gave their informed consent for inclusion before they participated in the study.

Preprint statement

We uploaded this manuscript to Research Square as a preprint for peer review at the same time as submission to seek further receive feedback from the community. This is the DOI for this manuscript: https://doi.org/10.21203/rs.3.rs-3921657/v1.

CRediT authorship contribution statement

Wei Shao: Writing – original draft, Data curation, Conceptualization. Jia-jun Wang: Formal analysis. Zi-hui Niu: Project administration, Methodology. Kang Zhang: Supervision. Shuai Wang: Project administration, Methodology. Yu-Hao Wang: Project administration, Methodology. Yu-hang Tang: Formal analysis. Cheng-Cheng Wang: Supervision. Shi-Qiang Hou: Writing – review & editing, Supervision, Formal analysis. Dong-Rui Zhou: Project administration, Methodology. Chao Zhang: Writing – original draft. Ning Lin: Writing – review & editing, Supervision, Resources, Funding acquisition.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

Not applicable.
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