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

1Worldwide, stroke still remains one of the leading causes of mortality and long-term disability, which causes healthcare systems a substantial financial burden. Globally, one in six people may suffer from a stroke within their lifetime, which causes more than 13.7 million cases each year. Ischemic stroke accounts for 71% of all stroke cases, and the proportion in the USA is estimated to be higher, reaching approximately 85-87%. Ischemic stroke is defined as the permanent infarction of brain tissues due to the abrupt loss of cerebral blood flow resulting in an impairment of normal neurologic function. Following the blockage of a cerebral artery by an embolus or in situ thrombosis and ensuing interruption of oxygen and energy supply, ischemic stroke leads to irreversible neuronal impairment and a cascade of molecular responses. At present, timely revascularization therapies including intravenous thrombolysis and endovascular thrombectomy are the main effective treatments in the early stage of ischemic stroke and are associated with improved neurological outcomes, which are recommended by the current clinical guidelines. However, they also have several limitations including the short therapeutic treatment window, rigorous eligibility criteria, and numerous contraindications.

2Cerebral ischemia and ischemia/reperfusion (I/R) induce a battery of biochemical and cellular reactions with the generation of excessive reactive oxygen species (ROS). Oxidative stress due to ROS overproduction plays an essential role in the fundamental pathologic progression of brain damage in ischemic stroke. Oxidative stress will occur when the inherent antioxidant potential to neutralize ROS is inadequate and fails to keep the endogenous redox balance. When oxidative stress takes place, ROS can lead to cytotoxicity through oxidative damage of lipids, proteins, and nucleic acids with harmful consequences for the structure and function of cerebral tissue. In ischemic stroke, oxidative stress can also cause neuronal apoptosis, activation of inflammatory signaling pathways, and impairment of the blood-brain barrier (BBB), all of which promote neurodegeneration and cell death.

3Preclinical studies have unequivocally confirmed that oxidative stress injury should be a feasible therapeutic target of acute stroke, even though clinical trials yielded unfavourable results. Acupuncture, as the main nonpharmaceutical intervention of traditional Chinese medicine (TCM), has been applied widely in China from time immemorial and has become increasingly popular worldwide, especially in treating cerebrovascular diseases. Meanwhile, a published systematic review and meta-analysis showed that acupuncture could reduce infarct size and improve neurological function in an experimental stroke model. Introduced as a novel nonpharmacological antioxidant approach, acupuncture demonstrated a beneficial effect in modulating the redox status of the central nervous system. Recently, emerging experimental studies have demonstrated that acupuncture exerts the neuroprotective role in acute stroke via its antioxidant potential. Even so, the underlying mechanisms have not been extensively reviewed to date. The review will mainly focus on the evidence to illuminate the possible mechanisms of acupuncture therapy in treating acute cerebral ischemia through regulating oxidative stress.

4We conducted this literature review by searching for mechanistic studies concerning the antioxidant effect of acupuncture therapy in experimental models of ischemic stroke. A literature search was undertaken in PubMed and Chinese National Knowledge Infrastructure (CNKI) on March 9, 2020. The following MeSH items or free words were chosen: “acupuncture,” “electroacupuncture (EA),” “manual acupuncture (MA),” “oxidative stress,” “antioxidative,” “antioxidant,” “free radicals,” “reactive oxygen species,” “reactive nitrogen species,” “redox” in combination with “stroke,” “cerebral ischemia,” and “ischemic cerebrovascular disease.” All the publication records in the last 2 decades (from January 2000 to February 2020) were collected.

5A sum of 28 published articles met our inclusion criteria and were further reviewed. Most of these studies used similar animal ischemic stroke models, namely unilateral temporary middle cerebral artery occlusion (MCAO), while others employed bilateral common carotid artery occlusion (BCCAO) or microemboli injection. In these studies, acupuncture was performed either before or after the surgery. Six studies applied MA and 20 studies applied EA, while the other 2 studies applied other types of acupuncture. These studies reveal 4 major categories classifying the underlying mechanisms of acupuncture in the regulation of oxidative stress, namely, (1) activating the inherent antioxidant enzyme system, (2) inhibiting the excessive generation of ROS/RNS, (3) protecting proteins and lipids from oxidative damage, and (4) regulating the signaling pathways involved in redox modulation. The detailed characters of the included studies are shown in Tables 1, 2, 3, and 4.

6Introduced as a specific term, oxidative stress is defined as a pathologic state in which cells are subjected to excessive reactive oxygen or nitrogen species (ROS/RNS), and they cannot counterbalance the deleterious effects with the antioxidant defense system. Under physiological conditions, a low level of ROS/RNS should be necessary to mediate and modulate cell signaling. ROS/RNS commonly results in detrimental cellular damage and tissue destruction at higher concentrations. Oxidative stress has been involved in the aging and pathological processes of various diseases, including atherosclerosis, hypertension, stroke, and neurodegenerative diseases. Particularly, oxidative stress plays a vital role in the pathogenesis of ischemic stroke and ensuing reperfusion injury, which is ascribed to the susceptibility of the brain to ROS-induced damage. Hence, the specific neuroprotective treatment that can inhibit oxidative stress contributes to preventing neuronal injury associated with ischemic stroke.

7Owing to the existence of unpaired electrons, ROS are short-lived and highly reactive small molecules including superoxide (O2−), hydrogen peroxide (H2O2), and the hydroxyl radical (OH·) that can be produced through many pathways. O2− is the primary and initial ROS product which is formed by the univalent reduction of molecular oxygen. The process is mediated via a variety of oxidative enzymes like nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and xanthine oxidase (XO). Once formed, O2− is able to produce injury directly. However, under normal metabolism, the balance between superoxide production and antioxidant defense system exists, which can eliminate the overproduction of superoxide by dismutation. There are three isoforms of superoxide dismutases (SODs) produced by cells that convert O2− into H2O2: copper-zinc SOD (CuZn-SOD, SOD1) located in the cytoplasm, manganese SOD (Mn-SOD, SOD2) within the mitochondria, and extracellular SOD (EC-SOD, SOD3). H2O2 can be further degraded into water and oxygen via catalase or the glutathione (GSH) redox system, which consists of glutathione reductase (GR), glutathione peroxidase (GSH-Px), and peroxiredoxins (Prdxs). Nonetheless, in combination with reduced transition metals such as ferrous iron (Fe2+), H2O2 may evade enzymatic conversion and form the more highly reactive species, the hydroxyl radical, that can cause cellular dysfunction and damage. In addition to ROS, excessive O2− can also react with nitric oxide (NO) rapidly to generate peroxynitrite (ONOO−), which is a potent oxidizing RNS that in turn can cause oxidative injuries such as deoxyribonucleic acid (DNA) fragmentation and lipid peroxidation. This reaction is much more efficient than the SOD-induced dismutation of superoxide. ONOO− can further increase the production of O2− by inactivating Mn-SOD and uncoupling endothelial nitric oxide synthase (eNOS), and eNOS may function as NOX to produce O2− rather than NO. Because of its ability to produce more superoxide, peroxynitrite might further exacerbate oxidative stress. Once the redox equilibrium between the ROS production and antioxidant enzymes is impaired, it would initiate a synergistic cascade of chain reactions resulting in more secondary ROS accumulation. As a result, high concentrations of ROS can cause significant cellular injury in different forms. A schematic illustration of the mechanisms of ROS/RNS metabolism is demonstrated in Figure 1.

8Following an ischemic stroke, as a result of the sudden interruption or severe reduction of blood flow and oxygen supply to the brain, hypoxia and glucose deprivation lead to lactic acid accumulation due to energy depletion. Increased H+ concentrations provide an acid environment for promoting the prooxidant effect, enhancing the conversion rate of ROS. Simultaneously, an ensuing complex cascade of molecular events such as increased Ca2+ leads to the activation of NOX signaling and mitochondrial dysfunction, which further aggravates oxidative stress. Emerging evidence has shown that elevated oxidative stress is involved in the pathophysiology of ischemic stroke and cerebral ischemia-reperfusion (I/R) injury, and inhibiting oxidative stress may play a protective role in antagonizing stroke-induced complications. In response to ischemic stroke, excessive ROS can cause significant cellular injury as well as vascular effects. In the forms of lipid peroxidation, protein denaturation, and DNA modification as well as inducing detrimental redox-sensitive cell signaling transduction pathways, ROS can disrupt cellular structural integrity and cellular functions, resulting in neuronal tissue destruction and cell apoptosis. High levels of ROS also have a profound vascular effect due to their hazardous effects on cerebral vascular endothelium. Endothelial cell dysfunction induced by overproduction of ROS can result in disruption of the BBB. As a result, increased cerebrovascular permeability can further cause local edema and elevated intracranial pressure, which in turn can hinder the perfusion of the brain tissues.

9Overproduction of ROS and consequent oxidative stress have been demonstrated to exert a main role within the pathogenesis of ischemic injury. The highly active metabolism of the brain asks for the constant supply of oxygen and energy. Due to the low storage of oxygen and glucose in the brain, it is especially vulnerable to the sudden interruption of cerebral blood flow. Both hypoxia-ischemia and abrupt reperfusion after ischemia can trigger the plentiful generation of ROS. To date, the effects and underlying mechanisms of acupuncture involved in improving neurological outcomes of ischemic stroke have been reported by many studies. Based on these studies, the therapeutic effect of acupuncture is likely attributable to its antioxidant effect at least partially. The following 4 sections address the means of acupuncture in combating oxidative stress.

10EA therapy can significantly decrease the content of lipid peroxidation end-product malondialdehye (MDA) and 4-hydroxynonenal (4-HNE) in cerebral I/R injury rats, possibly via increasing the activities of antioxidative enzymes, namely GSH-Px and SOD. 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. In multi-infarct rats, Liu et al. 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. 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.

11After 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. In the central nervous system, there are 3 isoforms of the NOX family, namely, NOX1, NOX2, and NOX4. Of note, NOX4 deletion closely correlates with reduced oxidative stress, BBB disruption, and infarct volume after MCAO. A recent search designed to assess the neuroprotective effects of preischemic EA against cerebral I/R injury was conducted by Jung et al.. 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. 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. 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.

12Apart from NOX-induced ROS overproduction, uncoupled NOS is another source of superoxide. 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, which also exacerbates oxidative/nitrative stress injury during cerebral ischemia. 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. 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.

13In 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. The abovementioned results are summarized in Table 2.

14In 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. The thiol-redox system is composed of thioredoxin, thioredoxin reductase, and NADPH, which exerts an antioxidant effect by reverting protein thiols. A study indicated that EA stimulation could increase thioredoxin significantly in cerebral I/R brain tissues and reduce the oxidative stress-induced protein disulphides. Of note, thioredoxin has a dual action that can keep GSH-Px fully active and increase the generation of Mn-SOD, 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.

15From 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.

16There 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. The Pink1/Parkin pathway is an important pathway in regulating mitophagy clearance, which can maintain neuronal mitochondrial quality. By activating Pink1/Parkin-mediated mitophagy clearance, EA could decrease dysfunctional mitochondria and ROS production in transient MCAO models. 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. 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. Furthermore, enhancing the activity of the parasympathetic nervous system was found to have conducive effects in cerebral ischemia. Muscarinic receptors are involved in the modulation of the redox state, especially M2 and M3 receptors. 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. Further studies to explore the specific muscarinic receptor-mediated signaling pathway that facilitates the antioxidant effect of acupuncture are required.

17Previous 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. 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. 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. 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. Glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting biosynthesis of GSH, is regulated by Nrf2. 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. 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. 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.

18As an age-old treatment, acupuncture has been demonstrated beneficial effects for recovery from ischemic stroke, perhaps through reducing oxidative stress. According to all the convincing evidences from the studies we have reviewed, the acupuncture-induced antioxidant effect is mainly produced by enhancing the activity of antioxidative enzymes, as well as suppressing the expression of prooxidant enzymes and inhibiting the oxidative modification of cellular molecules. Modulating the molecular signaling pathways related to the generation or elimination of ROS has been suggested to be closely associated with the antioxidant effect of acupuncture. In addition, acupuncture was also found to improve the mitochondrial respiratory function and mitophagy clearance to decrease ROS production indirectly.

19Different from other pharmacological therapies, acupuncture is one kind of intricate intervention whose therapeutic effects are influenced by a series of factors. 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. 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.

20Although 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. 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. It has also been reported that acupuncture at Dazhui could enhance the activity of SOD and GSH-Px, improve perfusion in the ischemic zone, and promote the proliferation of neuronal stem cells. 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, as well as have an antiapoptotic effect and modulate the expression of anti-inflammatory mediators. 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.

21Generally speaking, oxidative stress is a pivotal mechanism closely relevant to early stroke treatment. However, from the clinical standpoint, acupuncture is universally utilized in poststroke rehabilitation at present, which has been adopted by the National Institutes of Health. 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, 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. 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.

22Acupuncture is a novel concept of therapeutically manipulating the redox state via nondrug afferent stimuli. 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, 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, 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. 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.

23Several 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.

24Even 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.

25In summary, by regulating a battery of molecular signaling pathways involved in redox modulation, acupuncture not only activates the inherent antioxidant enzyme system but also inhibits the excessive generation of ROS. Acupuncture therapy possesses the potential in alleviating oxidative stress caused by cerebral ischemia, which may be linked with the neuroprotective effect of acupuncture.