Introduction

Traumatic brain injury (TBI) has high incidence and mortality rates globally, and is becoming a major public health problem.[1] The most prevalent type of TBI is mild traumatic brain injury (mTBI), accounting for approximately 95% of TBIs.[2] Although mTBI is considered a benign and self-limiting condition, there is a risk of severe short- and long-term sequelae.[3] Of these, cognitive impairment may be the most significant problem because of its long-term impact on daily function.[4,5]

During mTBI, damage results from rapid acceleration and deceleration of the cranium, where shear strains develop as a consequence of inertial forces.[6] Diffuse axonal injury, which occurs over a widespread area with varying degrees of damage, is a major pathophysiological cause of cognitive dysfunction in mTBI.[7–9] At the same time, cognitive dysfunction may also result from the initiation of the neurometabolic cascade, manifested by the extensive release of many neurotransmitters.[10] Activation of N-methyl-D-aspartate receptors is responsible for further depolarization, which releases calcium into the cells and thus causes intracellular mitochondrial calcium overload and oxidative stress. In turn, these phenomena result in impaired synaptic plasticity and subsequent cognitive impairment.[11–13]

Mitochondrial function is thought to be closely related to the sirtuin-1 (SIRT-1)/peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) signaling pathway. PGC-1α can upregulate mitochondrial numbers and intracellular ATP concentrations in a variety of cells, and can induce the expression of superoxide dismutase, whose downregulation increases oxidative stress resulting in subsequent neurodegeneration.[14–16] SIRT-1 can increase the transcriptional activity of PGC-1α through deacetylation, and has a neuroprotective effect in rats with craniocerebral injury.[17,18] This suggests that cognitive dysfunction in mTBI may be linked to impaired SIRT-1/PGC-1α/mitochondrial signaling pathways.

The remarkable curative effect of electroacupuncture in treating neurological diseases has recently gained attention. Electroacupuncture has been reported to be effective in treating mTBI symptoms, however, the biological mechanism underlying its therapeutic effects remains unclear.[19,20] Considering the close relationship between cognitive function and the SIRT-1/PGC-1α/mitochondrial pathway, together with the observation that electroacupuncture improves cognitive symptoms after mTBI, this study aims to evaluate changes in the SIRT-1/PGC-1α/mitochondrial pathway and explore the potential effects of electroacupuncture on cognitive function recovery following mTBI.

Methods Animals

We included forty 6-week-old male Sprague-Dawley rats (SiPeiFu Biotechnology Co., Ltd., Beijing, China) in this study. Ambient room temperature was 22 ± 1°C. Rats were exposed to a daily fixed light-dark cycle (light period: 8:00 a.m. to 8:00 p.m.). Food and water were available ad libitum.

The Ethics Committee of Beijing Jishuitan Hospital reviewed and approved the animal study (approval number: JLKSZ No. 202103-39).

Establishment of the mTBI animal model

We randomly divided rats into four groups: controlled cortical impactor (CCI, n = 10), sham operation (sham, n = 10), electroacupuncture-treated CCI (CCI+EA, n = 10), and electroacupuncture-treated sham (sham+EA, n = 10) group. Randomization was performed by assigning a random number to each rat and using a random number table. This method was based on the protocol adopted by Osier et al.[21] An electromagnetically controlled impacting device was used in the CCI model (PinPointTM PCI3000 Precision, Waltham, MA, USA). We fixed rats to a stereotaxic apparatus (Zhongshi Technology, Beijing, China). Anesthesia was maintained using inhaled isoflurane (2%, 0.4 mL/min) for the duration of the operation. The shaved and prepared area was disinfected with iodophors and 75% (vol/vol) alcohol. A 3-cm incision was made along the midline of the head, and the scalp was fixed bilaterally. The periosteum was peeled off to expose both frontal lobes, and the bone flap was removed at 3 mm from the midline between the anterior fontanel and the lambdoid suture. When removing bone flaps, special care was taken to avoid damaging the dura mater in the process. The sham group underwent craniotomy with no brain consequences [Figure 1]. Generally, the impactor tip was tilted to an angle between 15 and 20 degrees. The following parameters were adopted: velocity, 3.5 m/s; dwell time, 200 ms; and tip depth, 1.5 mm.

Figure 1:

Establishment of mTBI animal model and electroacupuncture treatment. mTBI: Mild traumatic brain injury.

Electroacupuncture treatment

Before electroacupuncture, rats were fixed on a wooden board in prone position. Two 0.25 mm stainless steel electroacupuncture needles were placed into the Baihui (GV20) and Yintang acupoints (EX-HN3) at a depth of 5 mm. The output terminals of the electroacupuncture instrument were linked to needles placed at GV20 and EX-HN3. The frequency of sparse and dense waves was 4/20 Hz, with a 2-mA current applied for 10 minutes once a day over a period of 7 consecutive days.

Data sources and collation

We obtained the GSE59645 microarray dataset from the GEO Database hosted by NCBI (https://www.ncbi.nlm.nih.gov/geo/). The GSE59645 gene chip data detection platform is GPL14746. The experimental design involved liquid percussion to induce traumatic brain injury or pseudo-injury in rats, extraction of the hippocampus within 24 hours, and extraction of total RNA for gene expression analysis, with the goal of elucidating and comparing differential gene expression profiles in the hippocampus of rats across four groups: naive, sham-control, TBI, and TBI plus neuroprotective drug treatment. To explore changes in gene expression in the hippocampus region after TBI, we selected sham and TBI groups for subsequent analysis. We used the limma software package from R (version 4.2.3, R Foundation for Statistical Computing, Vienna, Austria; http://www.R-project.org/) to screen differential gene expression between the two groups. We set |log2 fold change (FC)|>2 and P <0.05 as threshold for statistical significance, and used the R package ggrepel and enhanced volcano to plot volcanoes. We performed gene ontology (GO) enrichment analysis using the R packages clusterProfiler and enrichplot, and annotated differentially expressed genes in the biological process (BP), cell component (CC), and molecular function (MF) domains.

Morris water maze (MWM) test

We used the Morris water maze (MWM) test to assess rat cognitive function, including spatial learning and memory. The device comprises a large round container (120 cm in diameter and 30 cm in height) filled with an opaque liquid consisting of water and white milk (22 ± 1°C). The water maze consists of four quadrants of equal area. A white plastic platform of 10 cm diameter was placed 1 cm below the water surface at the same position during all hidden platform tests. First, we pretrained rats four times a day for 5 days to find the hidden platforms. During training, rats were placed at the four water entry points while facing the pool. We recorded the time taken to find the hidden platform and stand on it, which we term the “latency period”. After finding the platform, rats were allowed to rest on it for 10 seconds. If they could not find the underwater platform within 60 seconds, they would be gently pulled out of the water and placed on the platform for 10 seconds. Each rat entered the pool from the four water entry points for training once. The interval between the two training sessions was 30 seconds. On the sixth day, the test was formally conducted. A computer recorded the time it took for the rats to find the platform and the total swimming distance, which was used as an index to evaluate their cognitive function. The platform was removed 24 hours after the last training. We recorded the time it took for rats to reach the original platform position, and the number of times they crossed it. The objective was to measure their ability to retain spatial memory by finding the position of the underwater hidden platform. The following indicators were used for evaluation and measurement: total distance, obtained by tracking the movement trajectory of rats in the pool from the starting point to the platform via computer software and video tracking systems; average speed, calculated as total distance divided by time spent completing the task.

Tissue sampling

We collected tissue samples from all rats 1 day after the final MWM test. We performed biochemical and molecular tests on hippocampal tissues surrounding the brain lesion. Tissues were excised under anesthesia after intracardiac perfusion with an ice-cold saline solution. They were separated on ice and rapidly frozen in liquid nitrogen. Tissue samples were then processed using strategies tailored to the specific detection protocols adopted in this study. We focused on the hippocampus, one of the brain areas most closely associated with cognitive function.

Quantitative real-time polymerase chain reaction (qRT-PCR)

We performed qRT-PCR analysis to measure messenger RNA (mRNA) expression of SIRT-1 and PGC-1α in the hippocampus. We extracted total RNA from the hippocampus using TRIzol (Quanshijin, H10318, Beijing, China). Primer synthesis was performed by Shanghai Jierui Bio-Engineering Co., Ltd. (Shanghai, China) and the FastKing First-strand cDNA Synthesis kit was used for reverse transcription (TIANGEN, KR118-02, Beijing, China). The obtained complementary DNA (cDNA) was subjected to polymerase chain reaction (PCR) amplification using the following primers:

SIRT-1

Forward: 5′-GCTGGCCTAATAGACTTGCAA-3′

Reverse: 5′-TCCGTCAGCTCCAGATCCT-3′

PGC-1α

Forward: 5′-TTGATGCACTGACAGATGGA-3′

Reverse: 5′-CTGAGCAGGGACGTCTTTG-3′

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

Forward: 5′-GATGACATCAAGAAGGTGGTGA-3′

Reverse: 5′-ACCCTGTTGCTGTAGCCATATTC-3′

We then performed qRT-PCR using FastKing-RT SuperMix (TIANGEN). We adopted the following parameters for the reaction protocol: 95°C for 3 min; 40 cycles of 95°C for 30 s, 55°C for 20 s, and 72°C for 20 s; 95°C for 15 s; and 60°C for 15 s. Relative quantification results were normalized to GAPDH expression in each sample using the 2–ΔΔCt method.

Western blotting

We assessed protein expression of SIRT-1 and PGC-1α in the hippocampus using western blot. Rats were euthanized 1 day after the MWM experiment, and the hippocampus was immediately removed to extract total protein. We used a bicinchoninic acid protein assay kit (Bi-Yuntian, Shanghai, China) to measure protein concentration. Proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (IPVH00010; Millipore, Darmstadt, Germany). We administered primary antibodies against SIRT-1 (60303-1-Ig; Proteintech, Chicago, IL, United States) and PGC-1α (66369-1-Ig; Proteintech, Chicago, IL, United States) at a 1:1000 dilution level, and the membrane was incubated at 4°C overnight. GAPDH was used as the internal reference (1:5000, 60004-1-Ig; Proteintech, Rosemont, IL, USA). After washing, membranes were incubated at room temperature (25°C) for 2 h with a secondary antibody. After washing with Tris buffered saline with Tween-20, a chemiluminescence analyzer (Tanon, Shanghai, China) was used to visualize and photograph the protein bands. All experiments were conducted in biological triplicate.

ATP assays

We detected ATP using an ATP content colorimetric kit (Elabscience, E-BC-K157-M; Elabscience, Wuhan, China). We accurately weighed 0.1 g of tissue sample, cut it into pieces, placed it in a 2 mL microcentrifuge tube, and added 0.9 mL of boiling, double-distilled water. The contents were mixed evenly and put into a boiling water bath for 10 min. This mixture was then vortexed for 1 min, and centrifuged for 10 min at 10,000 rotation per minute (RPM) (radius = 15 cm). The supernatant was retrieved and tested following manufacturer’s instructions. We mixed the supernatant with the working solution. Once the reaction was completed, we measured absorbance at 636 nm using an M-200 reader (Tecan, Grödig, Austria). Each scan was repeated three times.

Mitochondrial respiratory chain complex I (MRCC I) assays

The MRCC I kit (ELISA LAB, Wuhan, China) was used to test the reaction of MRCC I. Tissue samples weighing 0.1 g were centrifuged at a speed of 2000–3000 RPM, the centrifugal radius was 15 cm, and the supernatant was carefully collected. Samples were mixed with a working solution, and absorbance at 450 nm was recorded within 15 min of adding the stop solution. The experiment was performed in biological triplicate.

Statistical analysis

Quantitative data with normal distribution were presented as mean ± standard error of the mean. We performed statistical analyses and visualization using GraphPad Prism (GraphPad, San Diego, CA, USA). We assessed the statistical significance of differences between groups using a two-sided Student’s t-test. A P <0.05 was considered to be statistically significant.

Results Effect of electroacupuncture on SIRT-1 and PGC-1α mRNA expression

In terms of mRNA production levels, the CCI group showed significant downregulation of SIRT-1 and PGC-1α compared with the sham surgery group. Compared with the CCI group, the CCI+EA group presented upregulation of mRNA expression for SIRT-1 and PGC-1α [Figure 2].

Figure 2:

Electroacupuncture upregulated the expression of SIRT-1 and PGC-1α mRNA. Expression levels of SIRT-1 (A) and PGC-1 (B) mRNA were significantly higher in the CCI+EA group compared with the CCI group, and were significantly lower in the CCI group compared with the sham group. n = 9 for each group. CCI: Controllable cortical impactor; EA: Electroacupuncture; mRNA: Messenger RNA; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator-1 alpha; SIRT-1: Sirtuin-1. *P <0.001, †P <0.01.

Effect of electroacupuncture on SIRT-1 and PGC-1α protein expression

We performed western blot analysis to evaluate whether electroacupuncture therapy affects protein expression in the SIRT-1/PGC-1α pathway. There was a significant difference (P <0.001 and P = 0.002, respectively) between the CCI group and the sham group, and similar trends were observed using western blotting and qRT-PCR analysis. Electroacupuncture therapy activated protein expression in the SIRT-1/PGC-1α pathway. Compared with the CCI group, the CCI+EA group presented significant upregulation of SIRT-1 and PGC-1α proteins (P = 0.024 and 0.035, respectively, Figure 3). The results showed that, after electroacupuncture treatment, the expression levels of SIRT-1 and PGC-1α in the CCI+EA group were significantly higher than those in the CCI group, but did not reach the level of the sham operation group.

Figure 3:

Western blotting for SIRT-1 and PGC-1α expression in the hippocampus of the four groups, with corresponding bar graphs. The expression levels of SIRT-1 (A) and PGC-1α (B) were significantly higher in the CCI+EA group compared with the CCI group, while they were significantly lower in the CCI group compared with the sham group. Figure C shows that SIRT-1 and PGC-1α protein immunoblotting levels were significantly higher in the CCI+EA group compared with the CCI group, while they were significantly lower in the CCI group compared with the sham group. n = 9 for each group. CCI: Controlled cortical impactor; EA: Electroacupuncture; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PGC-1α: Proliferator-activated receptor gamma coactivator-1α; SIRT-1: Sirtuin-1. *P <0.001, †P <0.05, ‡P <0.01.

Gene expression data reveal mitochondrial dysfunction in the hippocampal region after TBI

When we selected sham (GSM141403, GSM141404) and TBI groups (GSM1441405, GSM1441406, GSM1414407) from the GSE59645 dataset for screening of differential gene expression, we identified a total of 204 downregulated differentially expressed genes (DEGs) and 97 upregulated DEGs (Figure 4A, |log2FC|>2, P <0.05). Further enrichment analysis showed that the electron transfer chain, respiratory chain complex, and MRCC I were significantly enriched in the BP, CC, and MF domains of GO [Figure 4B]. These results indicate that mitochondrial function in the hippocampus of rats is significantly affected, which should play an essential role in cognitive dysfunction following TBI.

Figure 4:

The figure shows the volcano map of differentially expressed genes between TBI and sham surgery groups. Genes that were significantly up-regulated are labeled in red, and those that were significantly down-regulated are labeled in purple (A). In (B), we conducted gene ontology (GO) enrichment analysis of differentially expressed genes and obtained enrichment results in biological processes (BP), cellular components (CC), and molecular functions (MF). These results provide functional information about differentially expressed genes in various biological processes. ATP: Adenosine triphosphate; NADH: Nicotinamide adenine dinucleotide; Not sig: Not significant; rRNA: Ribosomal RNA.

Effect of electroacupuncture on mitochondrial function

We measured MRCC I and ATP content to analyze mitochondrial function. We measured ATP content using colorimetry. The results indicated that this quantity was significantly reduced in the hippocampal tissues of the CCI group, and increased after electroacupuncture treatment [Figure 5A]. We used a rat MRCC I ELISA kit to measure MRCC I concentration in rat hippocampal tissues. MRCC I concentration in the CCI group was greatly reduced compared with the sham group, indicating that TBI resulted in decreased mitochondrial function. After electroacupuncture treatment, MRCC I content was elevated, suggesting that mitochondrial function had improved [Figure 5B]. Therefore, we speculate that electroacupuncture therapy achieves its therapeutic effect by improving mitochondrial function after mTBI.

Figure 5:

Electroacupuncture treatment for mTBI could achieve its therapeutic effect by improving mitochondrial function. ATP content (A) was detected using colorimetry, and MRCC I content (B) was detected using ELISA. Results show that mitochondrial function decreased after TBI, and that electroacupuncture could increase MRCC I and ATP levels. n = 10 for each group. ATP: Adenosine triphosphate; CCI: Controlled cortical impactor; EA: Electroacupuncture; ELISA: Enzyme linked immunosorbent assay; MRCC I: Mitochondrial respiratory chain complex I; mTBI: Mild traumatic brain injury. Compared with CCI group, *P <0.01, †P <0.05, ‡P <0.005.

Electroacupuncture improved cognitive function recovery in mTBI rats

To evaluate rat ability for spatial learning and memory, we used a water maze task. We treated mTBI rats with electroacupuncture stimulation for 7 days, and then trained them for the MWM task. We measured longer latency values in the CCI group compared with the sham surgery group, indicating significant learning and cognitive deficits (P <0.01; Figure 6A). Compared with the CCI group, electroacupuncture significantly increased the number of platform crossings, indicating that electroacupuncture treatment improved spatial learning and memory abilities in rats (P <0.05; Figure 6B). In addition, electroacupuncture shortened the incubation period [Figure 6B], increased the average speed, but had no effect on the total swimming distance [Figure 6C–D].

Figure 6:

Electroacupuncture improved cognitive function recovery in mTBI rats. Rats were trained to remember the location of the hidden platform in the maze during a period of 5 days following mTBI. The platform was removed on day 13. We then examined latency (A), number of platform entries (B), total distance (C), and average velocity (D). The results showed that electroacupuncture treatment decreased latency and increased platform entries and average velocity. CCI: Controlled cortical impactor; EA: Electroacupuncture; mTBI: Mild traumatic brain injury; ns: Not significant. n = 10 for each group. Compared with CCI group, *P <0.05, †P <0.01.

Discussion

In this study, we found that electroacupuncture could enhance mitochondrial function, increase ATP levels, increase protein and mRNA expression of SIRT-1/PGC-1α in brain tissues, and improve cognitive function in mTBI rats.

Patients who develop post-concussion syndrome (PCS) after mTBI present with heterogeneous symptoms, with varying degrees of head and brain injury. Individual patient characteristics may alter the presentation of the injury, indicating that relevant symptoms do not always appear consistently and predictably.[22] Currently, PCS lacks established objective biomarkers. Furthermore, clinical diagnosis of this condition faces several difficulties and suffers from potential bias, especially for inexperienced physicians and primary hospital physicians.[23] Early identification of high-risk patients with PCS after mTBI, assessment of injury, timely intervention and treatment, and improvement of prognosis are therefore key issues in current clinical practice.

Consistent with the known pathological mechanisms underlying most cases of TBI, the pathological changes caused by mTBI include cell death inside the meninges and brain parenchyma, axonal fiber stretching and tearing, and disturbances at white-gray matter junctions. These phenomena are caused by rotational pressures that induce shearing injuries.[24] External forces damage neurons in the brain and neurovascular units responsible for transporting oxygen to the neurons and neuroglia cells. Cerebral blood flow decreases in the injured brain area. Insufficient oxygen supply caused by the decrease in cerebral blood flow triggers a cascade of metabolic reactions that result in secondary cell damage. Normal cellular metabolic regulation is disrupted in the affected brain regions. The metabolic machinery of neurons changes from aerobic metabolism to anaerobic metabolism, resulting in the increased production of reactive oxygen species (ROS).[25] Excess ROS accumulate in neurons to maintain a state of oxidative stress. ROS oxidate cellular proteins, lipids, and DNA, further leading to lipid peroxidation and damage to proteins and DNA.[26,27] Therefore, hypoxia and oxidative stress are two leading causes of cell damage after mTBI.[28]

Current treatments for mTBI include rest after injury or the use of drugs to control symptoms of PCS, such as depression, headache, and sleep disturbance.[29] Recently, the remarkable curative effects of electroacupuncture for the treatment and rehabilitation of nervous system diseases have received substantial attention.[30] According to clinicians and existing literature, acupuncture or electroacupuncture therapy can effectively improve symptoms of PCS after mTBI, such as headache, depression, irritability, and insomnia.[31–34] Baihui (GV20) and Yintang (EX-HN3) are common acupoints for the treatment of nervous system diseases in traditional Chinese medicine.[35,36]

To explore how electroacupuncture can improve cognitive impairment after mTBI, we evaluated relevant metabolic changes in rat neurons before and after mTBI. We determined whether electroacupuncture can counteract oxidative stress and metabolic abnormalities by analyzing mitochondrial function and measuring ATP levels in brain tissue. We found that, compared with the sham group, the mitochondrial function and ATP levels in the hippocampus of mTBI rats were significantly reduced, which is consistent with a previous study.[37] We also found that electroacupuncture improved mitochondrial function and ATP levels in the hippocampus, providing evidence that the cognitive recovery effect of electroacupuncture on mTBI rats may be connected with improved energy metabolism of damaged brain tissue. Because mitochondria play an important role in neuronal function, improving mitochondrial function may represent a new therapeutic direction.

Moreover, to explore the specific mechanism of electroacupuncture intervention in improving abnormal mitochondrial function and energy metabolism in brain cells of mTBI rats, we measured protein and mRNA expression levels of SIRT-1 and PGC-1α. Previous studies have found that SIRT3 plays a significant role in improving apoptosis and mitochondrial fission, and similarly, SIRT1 also plays an important role in oxidative stress and apoptosis.[38] Our results show that electroacupuncture intervention increased protein and mRNA expression of SIRT-1 and PGC-1α in mTBI rats.

SIRT-1 has been shown to regulate mitochondrial function and cellular metabolism. The brain is the most metabolically active organ. A previous study showed that SIRT-1 expression is higher in the brain than in other tissues.[39] SIRT-1 confers a neuroprotective effect to TBI rats by inhibiting the p38/mitogen-activated protein kinase (MAPK) pathway.[18] SIRT-1 can reduce inflammation by regulating the transcription factor nuclear factor-κB (NF-κB), which can promote the transcription of various inflammation-related genes.[40] Overexpression of SIRT-1, or administration of the SIRT-1 agonist resveratrol, protects neurons from potassium withdrawal-induced cell death.[41] The above-mentioned studies reported that SIRT-1 plays an essential role in cellular metabolism, stress response, growth, survival, and apoptosis, which are closely related to the pathologic changes associated with mTBI. Many studies have proven that the SIRT-1 protein has neuroprotective effects in animal models of brain trauma, cerebrovascular diseases, and neurodegenerative diseases.[42–44] Downregulation of SIRT-1 may lead to multiple sclerosis and other neurodegenerative disorders.[45]

SIRT-1 activates PGC-1α by deacetylation. PGC-1α is a transcriptional co-activator, the main regulator of mitochondrial activity.[46] Similar to SIRT-1, PGC-1α would have higher expression in tissues with high energy requirements, such as brain tissue, brown adipose tissue, and heart muscle.[47] These organs and tissues are generally rich in mitochondria. PGC-1α plays an essential role in fighting against oxidative stress and increasing neuronal activity.[48] Its downregulation accelerates the progression of oxidative stress and neurodegeneration.[49] Furthermore, PGC-1α has been reported to be a potent reactive oxygen species (ROS) inhibitor. It can induce the production of ROS-scavenging enzymes, such as superoxide dismutase 2 (SOD2).[50] According to the literature, PGC-1α is essential for inducing the expression of the mitochondria-related ROS-scavenging enzyme SOD2 in nerve cells, which may contribute to neuronal survival.[15] Furthermore, PGC-1α has been proposed as a potential therapeutic target for increasing mitochondrial biogenesis and improving energy metabolism in some neurological diseases, such as Alzheimer’s disease.[51]

Our results are consistent with previous studies, indicating that the SIRT-1/PGC-1α pathway is associated with improved mitochondrial function and increased intracellular ATP concentration. However, this study was only conducted in animal models, so it cannot be directly extended to humans. Furthermore, our study only focused on SIRT-1/PGC-1α and on the relationship between pathways and mitochondrial function, without considering other factors that may affect cognitive function.[52] Future research should incorporate more experimental designs and clinical studies to validate our results, and delve deeper into the potential underlying mechanisms. In summary, we found that electroacupuncture can improve cognitive function in mTBI rats, and our results suggest that the improvement in cognitive function may be related to the SIRT-1/PGC-1α/mitochondrial pathway.

Acknowledgments

The authors thank Editage and Edanz Group (www.edanz.com) for English language editing.

Funding

This research was funded by a scientific research fund from Beijing Jishuitan Hospital (No. ZR-202107).

Conflicts of interest

None.

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