Mechanisms of Acupuncture in the Regulation of Oxidative Stress in Treating Ischemic Stroke

4.1. Acupuncture Activates the Inherent Antioxidant Enzyme System

EA therapy can significantly decrease the content of lipid peroxidation end-product malondialdehye (MDA) and 4-hydroxynonenal (4-HNE) in cerebral I/R injury rats [60–62], possibly via increasing the activities of antioxidative enzymes, namely GSH-Px and SOD [63–66]. In addition, the ginger pharma acupuncture or laser acupuncture can significantly reduce MDA production in the cerebral cortex and hippocampus while increasing catalase, GSH-Px, and SOD simultaneously [67, 68]. In multi-infarct rats, Liu et al. [69, 70] conducted two studies to explore whether acupuncture could attenuate memory impairment and ischemia-induced neuronal damage. The data suggested that acupuncture increased the expression of CuZn-SOD and a sensitive marker for oxidative injury within the hippocampus, namely redox effector factor (Ref-1), consequently producing an antioxidant effect. Consistent with the results and using the same animal model, Zhang et al. [71] found that acupuncture exerted an antioxidant effect to improve the cognitive function by upregulating the expressions of total SOD, CuZn-SOD, and Mn-SOD, as well as simultaneously elevating the ratio of reduced to oxidized glutathione (GSH/GSSG) in mitochondria. Therefore, the studies confirmed that acupuncture displayed its antioxidant properties in ischemic stroke via activating the GSH redox system and SOD-mediated dismutation. The abovementioned results are summarized in Table 1.

4.2. Acupuncture Inhibits the Excessive Generation of ROS/RNS

After ischemic stroke, NOX can produce a substantial amount of O₂⁻, which represents a major and initial ROS source. NOX consists of 3 cytosolic subunits (p47phox, p67phox, and p40phox), 2 membrane subunits (p22phox and gp91phox), and the small GTPase protein [34, 72]. In the central nervous system, there are 3 isoforms of the NOX family, namely, NOX1, NOX2, and NOX4 [73]. Of note, NOX4 deletion closely correlates with reduced oxidative stress, BBB disruption, and infarct volume after MCAO [18]. A recent search designed to assess the neuroprotective effects of preischemic EA against cerebral I/R injury was conducted by Jung et al. [28]. The results revealed that EA preconditioning ameliorated neural function loss, diminished BBB permeability, and profoundly reduced NOX-derived O₂⁻ generation through the downregulation of the expression of NOX4 but not NOX2. Furthermore, Shi et al. [74] investigated whether NOX was involved in the neuroprotection of acupuncture in a rat BCCAO model. Acupuncture dramatically decreased the O₂⁻generation and NOX expression, as well as improved the cognitive function. In addition, a NOX antagonist mimicked the effects of acupuncture, whereas gp91phox deletion was able to reverse the antioxidant effect of acupuncture. Similarly, Guo et al. [75] also found that the decreased levels of MDA and ROS by EA pretreatment in diabetic mice with cerebral ischemia were attributable to the inhibition of NOX. Thus, acupuncture has an antioxidant effect in cerebral ischemic injury through inhibiting NOX-mediated oxidative damage, and a complete function of NOX enzyme is required in the process.

Apart from NOX-induced ROS overproduction, uncoupled NOS is another source of superoxide [46]. Moreover, NOS uncoupling can further lead to a dysregulated NO response, whereby a closely related RNS molecule—peroxynitrite—is synthesized by NO in combination with superoxide anion [19], which also exacerbates oxidative/nitrative stress injury during cerebral ischemia [43]. Inducible NOS (iNOS) expression can be upregulated by ischemia and delayed reperfusion after a stroke episode, which plays a pathological role in oxidative/nitrative stress [76]. In I/R injury rats, EA or MA effectively alleviated oxidative/nitrative stress-induced mitochondrial dysfunction and provided neuroprotection against infarct expansion by downregulating iNOS expression and peroxynitrite production, which ameliorated cognitive impairment and improved neurological function [77–79].

In addition to inhibiting the ROS-producing enzymes, it has been shown that the antioxidant properties of EA in reducing the production of ROS might be associated with the improvement of respiratory chain function in the mitochondria. Through promoting the activity of respiratory-related enzymes including cytochrome C oxidase, NADH dehydrogenase, and succinic dehydrogenase, EA remarkably improved the mitochondrial respiratory function with an ischemic state, which suggested that it could reduce the generation of ROS [80]. The abovementioned results are summarized in Table 2.

4.3. Acupuncture Protects Proteins and Lipids from Oxidative Damage

In the pathological progression of I/R, plenty of oxygen free radicals impair the stable equilibrium of the intracellular thiol-redox environment, leading to oxidative modification of protein thiols and dysfunction of their biological enzymatic activities such as catalytic and regulatory abilities [81]. The thiol-redox system is composed of thioredoxin, thioredoxin reductase, and NADPH, which exerts an antioxidant effect by reverting protein thiols [82]. A study indicated that EA stimulation could increase thioredoxin significantly in cerebral I/R brain tissues and reduce the oxidative stress-induced protein disulphides [83]. Of note, thioredoxin has a dual action that can keep GSH-Px fully active and increase the generation of Mn-SOD [84], which also enhances the antioxidant effect. It is desirable to explore whether crosstalk exists among the antioxidant effects mediated by acupuncture. The abovementioned results are summarized in Table 3.

4.4. Acupuncture Regulates the Signaling Pathways Involved in Redox Modulation

From the above studies, plenty of researchers have already explored the antioxidant effect of acupuncture in ischemic stroke. However, the precise antioxidant mechanisms of acupuncture are still necessary to be illuminated in more detail. Accordingly, it is crucial to figure out the specific molecular signaling pathways regulated by acupuncture in suppressing oxidative stress.

There exist diverse signaling pathways that participate in the pathological process of oxidative stress which have been confirmed. Therefore, the underlying signaling pathways causing ROS generation may have a close relationship with the antioxidant mechanisms of acupuncture therapy. Under cerebral I/R pathological conditions, the accumulation of damaged mitochondria becomes a major source of toxic ROS [85]. The Pink1/Parkin pathway is an important pathway in regulating mitophagy clearance, which can maintain neuronal mitochondrial quality [86]. By activating Pink1/Parkin-mediated mitophagy clearance, EA could decrease dysfunctional mitochondria and ROS production in transient MCAO models [77]. Moreover, EA also downregulated the level of iNOS via regulating the p38 mitogen-activated protein kinase (MAPK)/nuclear factor-κB (NF-κB) signaling pathway, which contributed salutary effects to the attenuation of nitrative stress as well as inflammation after cerebral I/R [79]. A recently published study found that acupuncture reduced the intracellular OH· generation by inhibiting the activation of p53, which is the downstream target gene of NF-κB [27]. Furthermore, enhancing the activity of the parasympathetic nervous system was found to have conducive effects in cerebral ischemia [87]. Muscarinic receptors are involved in the modulation of the redox state, especially M₂ and M₃ receptors [88]. EA was able to upregulate 5 types of muscarinic receptors, as well as activate the muscarinic receptor-mediated parasympathetic network in the brain after ischemic stroke, which eventually reduced the level of MDA [62]. Further studies to explore the specific muscarinic receptor-mediated signaling pathway that facilitates the antioxidant effect of acupuncture are required.

Previous studies have suggested that acupuncture therapy can also promote an antioxidant effect by enhancing the function of endogenous enzymatic defenses against oxidative stress. Under normal conditions, the signal transducer and activator of transcription 3 (STAT3) has been confirmed as a transcription factor which can regulate the Mn-SOD gene expression [89]. Through the activation of cannabinoid receptor type 1 (CB1R-) mediated STAT3 phosphorylation at Y705, EA could attenuate the transient ischemic oxidative damage by upregulating the Mn-SOD protein expression and Mn-SOD-positive neuronal cells [90]. Additionally, nuclear factor erythroid-2-related factor 2 (Nrf2), as a master regulator of endogenous antioxidant genes, exerts the critical cytoprotective effect in attenuating oxidative stress [91]. Substantially restored levels of the GSH redox system by EA in cerebral ischemia might be attributed to the activation of the extracellular regulated kinase 1/2 (ERK1/2-) mediated Nrf2 signaling pathway [92]. Glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting biosynthesis of GSH, is regulated by Nrf2 [93]. Several studies investigating neuroprotection against I/R injury of EA suggested that EA activated the Nrf2/GCS signaling pathway and subsequently promoted the expression of GSH remarkably [94–97]. Furthermore, the antioxidant potential of acupuncture may also be associated with the transient receptor potential vanilloid 1 (TRPV-1-) mediated signaling pathway. EA pretreatment could suppress TRPV-1-mediated p38 MAPK activation, which upregulated the expression of SOD and GSH simultaneously [26]. The abovementioned results are summarized in Table 4. A schematic illustration of the signaling pathways regulated by acupuncture in reducing oxidative stress is demonstrated in Figure 1.

5.1. Factors Associated with the Antioxidant Effect of Acupuncture

Different from other pharmacological therapies, acupuncture is one kind of intricate intervention whose therapeutic effects are influenced by a series of factors [98]. Intervention time, type of acupuncture, and acupoint selection or combination may have an impact on the ability of acupuncture to counteract oxidative stress. The results from the preclinical studies indicate that both acupuncture pretreatment and posttreatment are able to alleviate oxidative stress after cerebral ischemia. Of note, a study further evaluated the antioxidant effect against cerebral I/R injury of the EA pretreatment group and posttreatment group in the same experiment, which found that both groups showed preferable results compared with the MCAO control group. It indicates that acupuncture pretreatment might enhance tolerance against ischemic brain insult and provide an excellent antioxidant effect as well as acupuncture posttreatment [94]. In addition, more than half of the researchers employed EA, which is a combination of modern electrical stimulation and acupuncture, while others applied MA. Despite all studies have demonstrated that both MA and EA are beneficial to attenuating oxidative stress, but neither approach has been highlighted as more effective than the other.

Although the acupoint selection varied among the included studies, the following acupoints were applied more frequently by researchers: (1) Baihui (GV20), (2) Dazhui (GV14), and (3) Zusanli (ST36). Thereinto, 70.8% of the studies employed Baihui. A summary of acupoints selected in the reviewed literatures is shown in Table 5. Both of Baihui and Dazhui, located on the Governor vessel, are the meeting points of all yang meridians, which can eliminate interior wind, tonify yang, and promote resuscitation on the view of traditional Chinese medical meridian theory [99]. From the historical perspective, Baihui is the most often-chosen principle acupoint in treating neurological diseases like ischemic stroke and dementia. Nowadays, various basic studies have also confirmed that scalp acupuncture at Baihui plays multiple neuroprotective roles including antiapoptosis, anti-inflammatory, and perfusion augmentation in the pathophysiological process of laboratory models of ischemic stroke, not only regulating oxidative stress [24, 100–102]. It has also been reported that acupuncture at Dazhui could enhance the activity of SOD and GSH-Px [78, 92], improve perfusion in the ischemic zone, and promote the proliferation of neuronal stem cells [101, 103]. Zusanli, as a well-known acupoint good at strengthening the body resistance to eliminate pathogenic factors, could exert multiple neuroprotective roles in the recovery process of ischemic stroke. Acupuncture at Zusanli was found to ameliorate oxidative damage via inhibition of NOX-mediated ROS generation [74], as well as have an antiapoptotic effect and modulate the expression of anti-inflammatory mediators [104, 105]. Moreover, although Baihui plus Dazhui is the most common acupoint combination in this review, which appeared in 29.2% of the studies, there is still no universal standard for the combination of acupoints in the antioxidant studies of acupuncture in treating ischemic stroke. Most of the researchers often determined the acupoint combination according to their own clinical experience. It is necessary to explore the optimum acupoint combination in future studies.

5.2. Prospects of Acupuncture as an Antioxidant Therapy for Future Studies

Generally speaking, oxidative stress is a pivotal mechanism closely relevant to early stroke treatment [6]. However, from the clinical standpoint, acupuncture is universally utilized in poststroke rehabilitation at present, which has been adopted by the National Institutes of Health [106]. In fact, acupuncture is likely to have beneficial effects in the acute treatment of cerebrovascular disease as well since ancient times. A famous classical TCM book written by Hong Ge in the Eastern Jin Dynasty (317-420 AD) named Zhouhou Beiji Fang (Handbook of Prescriptions for Emergency), which gave the Nobel Laureate Youyou Tu a novel inspiration of discovering artemisinin in treating malaria [107], strongly recommended acupuncture as a viable first-aid intervention in the early phase of acute stroke. Nonetheless, to date, convincing evidence from clinical trials supporting acupuncture in acute treatment for ischemic stroke may be still insufficient due to the poor methodological quality of current studies [22]. Despite the achievements in animal studies of acupuncture in the regulation of oxidative stress suggest that acupuncture may be helpful in the acute phase, the obstacles hindering successful translation from animal to clinic need to be overcome.

Acupuncture is a novel concept of therapeutically manipulating the redox state via nondrug afferent stimuli [25]. The specific significance of acupuncture as an antioxidant therapy for future clinical research needs to be elaborated. On the one hand, it seems that the antioxidant effect of acupuncture is multifaceted and a common result of different mechanisms, which shows the superiority of acupuncture compared with the single therapeutic target interventions. It may be difficult for us in determining the primary one that the antioxidant effect induced by acupuncture should ascribe to. However, it is not easy to assess the specific oxidative status of one individual patient and determine the most suitable antioxidant agent for that patient either [108], which may make sense in demonstrating the therapeutic efficacy in the current clinical trials of antioxidant interventions for acute ischemic stroke. Therefore, whether acupuncture, as a multiple-target antioxidant therapy in ischemia stroke, can achieve favorable results in clinical studies requires further investigation. On the other hand, limited by the narrow therapeutic treatment window and rigorous contraindications, under clinical conditions, not all patients can always achieve early initiation of revascularization therapy like thrombolysis or thrombectomy [3, 6], even if successful reperfusion after getting these therapeutic modalities may bring the so-called I/R injury, which is likely to be mainly attributable to overproduced ROS [109]. Acupuncture can afford an antioxidant effect but does not have a risk of increasing hemorrhagic transformation and plenty of contraindications. Hence, acupuncture stands a chance of serving as one of the potential complementary approaches for neuroprotection against acute stroke, especially in the early stage.

Several acknowledged limitations exist in our review, which can be explained as follows: (1) All the studies reviewed were conducted using animal models. However, the experimental stroke models cannot be exactly in keeping with the clinical conditions, and the differences between rodent and human brains may influence the extrapolation of the results, such as different antioxidant enzyme concentrations or reactions to acupuncture. More clinical data are needed to determine whether acupuncture is a realistic antioxidant option. (2) Although acupoint specificity remains a crucial matter to the effect of acupuncture, only a few studies set nonacupoint acupuncture or sham acupuncture as a control group in their experiments, which makes it difficult to verify acupoint specificity and the real effect of acupuncture. (3) The number of articles related to our topic and the results from these studies are limited, so the conclusions may be potentially inadequate. Moreover, the quality of some selected studies is not high enough, which may inhibit the reliability of this review.

Even though many advances have been achieved, for further studies, there are still two issues that need to be explored. Firstly, the definite mechanism concerning how the stimulation signal of acupuncture is delivered from peripheral acupoints to the central nervous system so as to exert antioxidant effect remains to be elucidated. Secondly, plenty of variables are present in the current acupuncture-related studies. Researchers need to take into consideration of comparing the antioxidant effects among different types of acupuncture and different acupuncture parameters.