Effect of electroacupuncture on inhibition of inflammatory response and oxidative stress through activating ApoE and Nrf2 in a mouse model of spinal cord injury

Abstract Introduction Electroacupuncture protects neurons and myelinated axons after spinal cord injury by mitigating the inflammatory response and oxidative stress, but how it exerts these effects is unclear. Methods and results Spinal cord injury was induced in C57BL/6 wild‐type and apolipoprotein E (ApoE) knockout (ApoE –/–) mice, followed by electroacupuncture or ApoE mimetic peptide COG112 treatment. Mice with spinal cord injury suffered loss of myelinated axons and hindlimb motor function through the detections of Basso mouse scale, histology, and transmission electron microscopy; electroacupuncture partially reversed these effects in wild‐type mice but not in ApoE–/– mice. Combining exogenous ApoE administration with electroacupuncture significantly mitigated the effects of spinal cord injury in both mouse strains, and these effects were associated with up‐regulation of anti‐inflammatory cytokines and down‐regulation of pro‐inflammatory cytokines which were detected by quantitative reverse transcription‐polymerase chain reaction. Combination treatment also reduced oxidative stress by up‐regulating ApoE and Nrf2/HO‐1 signaling pathway through the detections of immunofluorescence and western blot analysis. Conclusions These results suggest that electroacupuncture protects neurons and myelinated axons following spinal cord injury through an ApoE‐dependent mechanism.

However, rapid medical treatments can reduce secondary damage, protect neurons that survive primary damage, and improve subsequent functional recovery at the early stage of SCI (Ahuja & Fehlings, 2016).
Some adjunctive therapies are used as an effective treatment of acute SCI clinically, such as neuroprotectant agents (Garcia et al., 2016) and electroacupuncture (EA; Alvarado-Sanchez et al., 2019).
EA is widely used in various acute and chronic diseases, and has shown therapeutic efficacy against central nervous system diseases, especially SCI (Liu et al., 2010;Yan et al., 2011). EA accelerates neural function recovery after SCI by reducing neuronal cell apoptosis (Jin et al., 2019), prompting neural stem cells or precursor cells to proliferate and differentiate into neurons (Ding et al., 2013), and accelerating axon regeneration and remyelination . EA may achieve these outcomes by shifting macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, leading to down-regulation of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β (Zhao et al., 2017). Simultaneous up-regulation of the anti-oxidant enzymes superoxide dismutase and glutathione peroxidase by EA may also play a role in its efficacy (Yu et al., 2010). EA may even inhibit the nuclear factor kappa B (NF-κB) signaling pathway (He et al., 2019), which is consistent with our previous work that EA can reduced inflammation through inhibiting IL-1β and NF-κB expression in C57BL/6 wild-type mice (Dai et al., 2019). Whether these mechanisms allow EA to mitigate the inflammatory response and oxidative stress after SCI has not been well investigated.
Apolipoprotein E (ApoE) is a major apolipoprotein for the regulation of lipid and cholesterol metabolism in central nervous system (CNS; Mahley, 1988), and has been associated with physiopathology of Alzheimer's (Griffin et al., 2019) and atherosclerosis (Rasmiena et al., 2015). ApoE exerts anti-inflammatory, anti-oxidative and antiapoptotic effects in CNS Kitagawa et al., 2002;K. Li et al., 2015) , as well as promotion of axonal regeneration and remyelination after a nerve injury (Vance et al., 2000). Deficiency of ApoE results in exaggerated inflammatory response, reduces anti-oxidants levels, and prompts neuronal apoptosis after SCI in rats (Lomnitski et al., 1997;Yang et al., 2018). Treatment with exogenous ApoE can ameliorate motor deficit and tissue damage as well as modify the inflammatory response (R. Wang et al., 2014). As we found in the previous work, the up-regulation of ApoE by EA can reduce inflammation and oxidative stress reactions (Dai et al., 2019). ApoE is therefore considered to play a neuroprotective function (Cheng et al., 2018).
We hypothesized that EA would exert neuroprotective effects on the CNS after SCI, acting through ApoE and Nrf2 to mitigate inflammation and oxidative stress. We compared functional recovery after SCI in wild-type vs. ApoE-deficient mice, and examined the potential effects of EA alone and in combination with exogenous ApoE mimetic peptide.

Surgical procedure
Mice were anesthetized by injecting 1% sodium pentobarbital (80 mg/kg) intraperitoneally. SCI was performed as described (Paterniti et al., 2018). The skin was incised with a scalpel along the midline of the back to expose the paravertebral muscles. The superficial and deep paravertebral muscles parallel to the spine processes were removed.
T12-L2 spine process and the bilateral laminae were dissected out by surgical forceps without disruption to the dura. Aneurysm clip was inserted around the exposed spinal cord for 25 s to generate an extradural compression of 20× g pressure that causes an acute compression injury. After surgery, muscles and skin were sutured in layers. Mice were kept warm on heating pads until they fully recovered from the anesthesia, and were injected intramuscularly with penicillin (2,000,000 U/kg/day per mouse) for three days after surgery to prevent infection.

EA
Acupuncture at zusanli and sanyinjiao can improve axonal growth and spinal cord plasticity and inhibit inflammatory reaction after SCI (Hong et al., 2021;Huang et al., 2015). Starting on the day after surgery, the mice in EA group and EA+COG112 group were treated with EA stimulation 10 min daily for 6 consecutive days, followed by 1 day off, and lasted for 4 weeks. EA treatment was performed at the bilateral acupoints zusanli (ST 36, located at the anterior aspect of the hindlimb, 2 mm directly below the knee joint) and sanyinjiao (SP 6, located posterior to the tibia, 3 mm above the medial malleolus). Stainless steel needles (0.25 mm × 13 mm, Jiangsu Medical Instruments Inc., China) were inserted into the acupoints with a depth of 3-5 mm below the skin. Then, the needle handles were linked to the output terminals of an electronic acupuncture instrument (SDZ-II, Suzhou Medical Products, Suzhou, China), which offered a pattern of parse-dense waves (60 Hz for 1.05 s and 2 Hz for 2.85 s, alternately). The current intensity was ≤ 5 μA in the animals' body, which was strong enough to induce a mild twitch in the hind limbs.

RMKWKKCLRVRLASHLRKLRKRLL-amide) as synthesized by Gill
Biochemicals (Shanghai, China) and purified by high-performance liquid chromatography to a purity of >95%. COG112 was dissolved in lactated ringer's buffer and injected intraperitoneally (1 mg/kg) 3 h after surgery every other day for 4 weeks (F. Q. .

Hind limb locomotor function
The Basso mouse scale (BMS) is a method to score hind limb locomotor function in spinal cord injured mice (Basso et al., 2006). The score ranges from 0 to 9, in which 0 denotes complete paralysis and 9 denotes normal movements. Two investigators blinded to experimental groups scored the hind limb locomotor function of mice according to ankle joint mobility, coordination, paw status, trunk stability, and tail posture.
Each mouse was observed for 5 min in an open field, and the average of both scores was taken as the final score. This test was performed on days 1, 3, 7, 14, and 28 after SCI.

Tissue preparation
At day 28 after injury, the mice were deeply anesthetized and transcar-

Histology
Tissue sections were deparaffinized with xylene and immersed in hematoxylin solution for 3 min, followed by a quick rinse in distilled water. Then the slides were differentiated in HCl/95% alcohol (1:50) solution for 10 s. After washing in distilled water for 5 min, the sections were stained with 0.5% eosin for 10 s, then dehydrated through a gradient ethanol series from 95% to 100% for 3 min, and cleared by xylene for 1 min. The sections were examined under a light microscope (BX 53, Olympus, Tokyo, Japan).
Sections were scored blindly by two investigators based on edema, hemorrhage, tissue cavity, neuronal apoptosis or necrosis, and inflammatory cell infiltration. A score of 0 was given for no or minor pathology; 1, limited pathology; 2, intermediate pathology; 3, prominent pathology; or 4, extensive pathology.

Immunofluorescence
For immunofluorescence staining, tissue sections were deparaffinized by xylene, rinsed, and blocked with goat serum (Boster Biological

Statistical analysis
All statistical analyses were performed by GraphPad Prism 5.01 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± SD. Inter-group differences on Basso scores were assessed for significance using ANOVA of repeated measurements, while other inter-group differences were assessed using one-way ANOVA and Bonferroni's post hoc multiple comparison test. Differences were considered statistically significant when p < .05.

EA combined with exogenous ApoE promotes recovery of hindlimb function and neuronal morphology after SCI in mice
As shown in Figure 1, BMS scores of WT and ApoE -/mice in sham groups displayed normal physical movements. No statistically F I G U R E 1 Assessment of locomotor function in C57BL/6 WT and ApoE -/mice subjected to spinal cord injury was assessed by Basso mouse scale (BMS; n = 18 mice per group). (a) Locomotor function of the hind limb was assessed in WT mice over time. (b) Locomotor function of the hind limb was assessed in ApoE -/mice over time. (c) Total scores were assessed in WT and ApoE -/mice at 28 days post-injury. All data are mean ± SD. Abbreviations: ApoE, apolipoprotein E; EA, electroacupuncture; SCI, spinal cord injury; WT, wild type. ** p < .01, *** p < .001 vs. WT EA+COG112 group. ### p < .001 vs. ApoE -/-EA+COG112 group significant differences were found in the baseline studies among SCI, EA, COG112, and EA+COG112 groups at one day after surgery (Figure 1a,b). The mice became spastic, paralyzed, and incontinent. The mean BMS in all groups were increased with the duration of time. The locomotion score in WT EA+COG112 group mice was significantly higher compared with that of the other WT mice at 28 days after injury.
The mice could stand, but not harmoniously. The locomotion score in ApoE -/-EA+COG112 group mice was significantly higher compared with that of the other ApoE -/mice at 28 d after injury. The mice could stand occasionally. The locomotion score in ApoE -/-EA group was significantly lower than that of the ApoE -/-COG112 and EA+COG112 groups at 28 days after injury (Figure 1c). In other words, the presence of ApoE markedly strengthened the ability of EA to restore locomotor function, whereas lack of ApoE significantly weakened its ability to do so.
Histochemistry revealed significant damage to the spinal cord at 28 days after injury in SCI animals, including structural disorganization, tissue cavities, neuronal apoptosis or necrosis, and inflammatory infiltration. Similar signs of damage, but less severe, were seen in EA, COG112, and EA+COG112 groups (Figure 2a sheaths. The myelin sheaths were degraded, loosened and separated from axons, and the neuron was swollen in the SCI group. The demyelination in ApoE -/mice was worse than that in the WT mice. After treatment, axons were wrapped by oligodendrocytes and the demyelination was reversed in EA, COG112, and EA+COG112 groups (Figure 3a,b), with EA+COG112 reversing this demyelination to a significant extent in WT mice (Figure 3c).
Thus, EA combined with exogenous ApoE significantly enhanced hindlimb locomotor function, reduced neural tissue loss and inflammatory response, and suppressed myelin degeneration and axonal demyelination in WT and ApoE -/mice model of SCI.

The expressions of ApoE and Nrf2 in WT and ApoE -/mice model of SCI
Histochemistry also revealed the effect of EA on ApoE and Nrf2 in WT and ApoE -/mice model of SCI. EA induced the activation of ApoE and Nrf2, and the WT EA+COG112 group expressed the highest levels of ApoE and Nrf2 (Figures 4 and 5). The ApoE -/mice showed the opposite. These results directly implicate ApoE and Nrf2 as a downstream mediator of EA after SCI.

EA-mediated improvement in inflammatory response after SCI depends on ApoE
Next, we asked whether the ApoE-dependent recovery induced by EA involves down-regulation of the pro-inflammatory cytokines TNF-α, IL-1β, or IL-6, and up-regulation of the anti-inflammatory cytokines IL-10 and TGF-β1. Indeed, the WT EA+COG112 group expressed the lowest mRNA levels of pro-inflammatory cytokines, but the highest levels of anti-inflammatory cytokines ( Figure 6). The ApoE -/mice showed the opposite.
Exogenous ApoE significantly down-regulated pro-inflammatory cytokine mRNAs and up-regulated anti-inflammatory cytokine mRNAs in WT and ApoE -/animals.

EA-mediated improvement in oxidative stress after SCI depends on ApoE
We examined whether the ApoE-dependent recovery induced by EA involves the Nrf2/ NQO1/HO-1 signaling pathway, which mitigates

DISCUSSION
Our previous work found that EA can reduce inflammation and oxidative stress reactions via up-regulating expression of ApoE, phosphorylated extracellular regulatory protein kinase, and Nrf2/HO-1 and inhibiting IL-1β and NF-κB expression at the early stage after SCI in C57BL/6 wild-type mice (Dai et al., 2019). Our present studies in the knockout (ApoE -/-) mouse model of SCI further suggest that EA can improve locomotor dysfunction, reduce inflammatory response and oxidative stress, and promote remyelination. It can also inhibit inflammatory responses and oxidative stress through activation of the Nrf2/HO-1 pathway. These effects appear to depend on ApoE.
SCI consists of two pathological processes that include immediate primary mechanical injury and subsequent secondary injury, which exacerbates neurological deficits and outcomes (Yip & Malaspina, 2012). Inflammatory responses and oxidative stress are two major types of secondary injury (Heo et al., 2020), during which inflammatory cells such as macrophages, microglia and neutrophils (Kumar et al., 2018) trigger the release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 (Nakamura et al., 2003), leading to cellular necrosis or apoptosis (McPhail et al., 2004) . In addition, neurons of the dorsal horn in the spinal cord release the TGF-β 1, an anti-inflammatory transforming growth factor (Xiyang et al., 2014). The increased levels of IL-6 and IL-1 were recognized as the main cause of the severity of neurological disorders in the Nrf2 knockout mice after SCI (Mao et al., 2012). Inflammatory cells also release excess reactive oxygen and nitrogen species (C. Wang et al., 2019), which cause DNA oxidative damage, protein oxidation and lipid peroxidation (Ahuja et al., 2017), exacerbating necrosis and apoptosis of neurons and glial cells (Alizadeh et al., 2019).
EA, widely used in China, has shown good therapeutic efficacy against SCI and its sequelae (Meng et al., 2015;Zhou et al., 2006).
EA mitigates SCI by reducing edema, inflammatory response, lipid can accelerate the conduction of nerve impulses .
Except for ST36 and SP 6, there are many other acupoints which benefit spinal cord injury. Ding et al. (2013) found Governor Vessel acupoints can also promote neuronal survival and axonal regeneration of injured spinal cord (Xu et al., 2021). Wong et al. (2003) demonstrated that EA at SI3 and BL62 in conjunction with auricular acupoints produced enhanced recovery of bladder function in patients with SCI.
Jia-Ji acupoints may also improve locomotor function by promoting autophagy flux and inhibiting necroptosis (Hongna et al., 2020). In a word, EA plays a key role in the recovery of SCI. The present study confirms these findings and extends them by showing that EA acts through ApoE and the Nrf2/HO-1 pathway. ApoE is an important therapeutic target of EA against SCI and the effect of EA depends on the ApoE.
ApoE has been shown to exert anti-inflammatory, anti-oxidative and anti-apoptotic properties (Laskowitz et al., 1997(Laskowitz et al., , 1998, making it an important modulator of neuronal repair and remodeling in trauma and diseases of the central nervous system (Teng et al., 2017). Loss of ApoE aggravates the inflammatory response and oxidative stress as well as increases neural apoptosis, thus retarding the recovery of locomotor and neurological functions after SCI (Yang et al., 2018). In addition, ApoE has an important role in the remyelination process after experimental demyelination in animals (Boyles et al., 1985). Abnormalities in endogenous ApoE could interfere with the metabolism of myelin lipids.
We showed that the neuroprotective effects of EA were weakened in the absence of ApoE (as a result of gene deletion) and strengthened in its presence (through supplementation with ApoE-like peptide COG112). Our findings are consistent with the reported ability of COG112 to improve neurological and histological outcomes following rat with SCI (R. Wang et al., 2014), which is associated with inhibition of NF-κB-induced inflammation and demyelination in the spinal cord (F. Q. Li et al., 2006;Singh et al., 2011). These data strongly suggest that EA acts via ApoE, which may justify therapies targeting this protein. are recruited to the injury site and reaches peak levels after 7-10 days of injury and persists for several months (Samarghandian et al., 2015).

F I G U R E 4 Expression levels of ApoE at 28 d after SCI in spinal cord tissue from WT and ApoE
Persistence inflammatory responses increase the risk of secondary injury progression after SCI (Schwab & Caroni, 1988). Indeed, ApoEdeficiency on its own, significantly up-regulates pro-inflammatory cytokines after SCI (Pandey et al., 2007), and our addition of exogenous COG112 enhanced the effects of EA.
Not only that, our results also identify at least one way in which EA reduces oxidative stress via ApoE: it induces ApoE to activate antioxidative signaling by Nrf2, which has been shown to play a crucial antioxidative role after SCI in animal models Molagoda et al., 2020). Several studies have reported the role of Nrf2 signaling pathways in the mice models of SCI. The Nrf2/HO-1/NQO1 sig-naling axis serves as a robust anti-oxidant pathway in stress-activated and age-related diseases . Z. Li et al. (2019) found that inhibition of triggering receptor expressed on myeloid cells 1 significantly decreased inflammation and oxidative stress by Nrf2/HO-1 expression. The Nrf2/HO-1 signaling pathway was associated with the anti-inflammatory effect of IL-10 (Syapin, 2008). Their association may help explain why ApoE and Nrf2 levels correlate with each other (Mezera et al., 2015) and why they show similar anti-oxidant properties (Pandey et al., 2007), as we observed in the present study. Lack of ApoE reduces the expression of Nrf2 and HO-1 after SCI, resulting in increased oxidative stress (Yang et al., 2018).  Abbreviations: ApoE, apolipoprotein E; DAPI, 4′,6-diamidino-2-phenylindole; EA, electroacupuncture; Nrf2, nuclear factor erythroid 2-related factor; SCI, spinal cord injury; WT, wild type. *** p < .001 vs. WT EA+COG112 group. ### p < .001 vs. ApoE -/-EA+COG112 group

CONCLUSIONS
EA may be an effective treatment to improve motor dysfunction, inflammation, and demyelination after SCI, and it appears to exert these effects by up-regulating anti-inflammatory cytokines, downregulating pro-inflammatory cytokines, and mitigating oxidative stress, all through an ApoE-dependent mechanism.

CONFLICT OF INTEREST
All authors claim that there are no conflicts of interest/competing interests.

AUTHOR CONTRIBUTIONS
Ni Dai and Chenglin Tang performed the whole experiments and molecular studies and drafted the manuscript. Hui Liu performed the molecular biology study. Siqin Huang funded and conceived the study, participated in the study design, and revised the manuscript. All authors read and approved the final version of the manuscript.

DATA AVAILABILITY STATEMENT
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.