Lundquist Institute for Biomedical Innovation at Harbor-University of California at Los Angeles (UCLA) Medical Center and Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and Harbor-UCLA Medical Center, Torrance, CA 90502, USA

Corresponding Author: Sheng-Xing Ma, Ph.D., Professor, Lundquist Institute for Biomedical Innovation at Harbor-Harbor-UCLA Medical Center, 1124 W. Carson Street, E6-room 219, Torrance, CA 90502, USA. Tel: (310) 974-9573; E-mail: sxma@lundquist.org.

Running title: Role of acupuncture against COVID-19

	ABSTRACT
	INTRODUCTION
	THE ROLE OF NITRIC OXIDE (NO) IN THE PATHOPHYSIOLOGY OF COVID-19
	ACUPUNCTURE INDUCED NITRIC OXIDE PRODUCTION AND RELEASE
	CONCLUSION
	ACKNOWLEDGMENTS
	REFERENCES

	ABSTRACT

The ongoing outbreak of COVID-19 has quickly become a daunting challenge to global health. In the absence of satisfied therapy, effective treatment interventions are urgently needed. Previous studies have demonstrated that acupuncture is effective at relieving common symptoms of COVID-19 including breathlessness, nausea, insomnia, leukopenia, fatigue, vomiting, and abdominal pain. Experiments have shown that nitric oxide (NO) inhibits the replication cycle of severe acute respiratory syndrome (SARS) coronavirus with similar structures of COVID-19. Increase in level of NO by using NO gas inhalation has been shown to restore lung function by reducing airway resistance and improving virus-induced lung infections in SARS patients. Recent case report showed that a medical acupuncturist with symptoms consistent with severe COVID pneumonia achieved full recovery by self-administered medical acupuncture and cupping therapy at home. Clinical features and pathophysiology demonstrated that NO deficiency and endothelial dysfunction contribute to the development of COVID-19. Several studies from different groups consistently demonstrated that acupuncture increases NO synthase expression and induces an elevation of NO production and release in plasma and the local skin regions in both animals and humans. It is suggested that exogenous NO supplies or interventions that induce increasing levels of NO can play an important role in protective effects against inflammation and acute lung injury. This article reviews the rationale for mechanisms of NO induction induced by acupuncture in the possible treatment of COVID-19 and highlights its potential for contributing to better clinical outcomes and improving future clinical studies of acupuncture on treatment of COVID-19.

	INTRODUCTION

   The ongoing outbreak of COVID-19, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (2019), has quickly become a daunting challenge to global health. The COVID-19 epidemic is spreading all over the world with a 6.9% mortality rate, but there is no targeted treatment satisfied and confirmed by well-designed clinical trials (1, 2). Supportive treatment is currently being employed for the most common symptoms being fever, chills, shortness of breath, myalgia, headache, anosmia, dysgeusia, cough, and sore throat (3, 4). China has reported that, as of February 17, 2020, about 85% of confirmed COVID-19 patients have been treated with Traditional Chinese Medicine (TCM) (5, 6). In response to a call by the Chinese government to employ TCM to help with prevention, treatment, and rehabilitation, the China Association of Acupuncture-Moxibustion developed guidance for acupuncture and moxibustion intervention on COVID-19 (6, 7). This guidance included advice on how to self-administer acupuncture and moxibustion under the direction of a physician in the various stages of disease process and recovery (7).

   Acupuncture and electroacupuncture (EA) have long been used in Asia for various types of pain relief, and are increasing in popularity in the West (8-11). Previous randomized controlled trial reported that acupuncture is an effective therapeutic approach for chronic obstructive pulmonary disease (COPD) associated breathlessness (12). The recent systematic review and meta-analysis show that acupuncture can relieve breathlessness in subjects with advanced diseases supported by a number of randomized controlled trials (13-15). Previous clinical studies have also demonstrated that acupuncture is effective at relieving common symptoms including nausea, insomnia, leukopenia, fatigue, vomiting, abdominal pain, abdominal distension, and anxiety disorders (16-22). It is believed that some symptoms of COVID-19, breathlessness, insomnia, leucopenia, fatigue, nausea and vomiting, abdominal pain and abdominal distension as well as anxiety, may respond to acupuncture treatment (15). Recently, a case report indicated a patient who is a frontline anesthesiologist and medical acupuncturist with pulmonary symptoms likely consistent with severe COVID pneumonia achieved full recovery with the help of self-administered medical acupuncture and cupping therapy (23). Acupuncture may play a role in the treatment and rehabilitation of the COVID-19, and relieve the symptoms caused by COVID-19. However, there is no well-designed clinical trial to elucidate the effectiveness and safety of acupuncture with high-quality evidence in the treatment of COVID-19. Relevant studies have recently proved that acupuncture improves multiple symptoms involved in COVID-19 and has a case reporting the therapeutic effects, therefore, it is urgent to introduce novel research ideas into the research of the potential mechanisms of acupuncture for treatment of COVID-19 (11). In this review, the evidence and understanding of the role of nitric oxide (NO) in the pathophysiology of new coronavirus pneumonia have been summarized with an emphasis on recent development of endothelial dysfunction and NO deficiency in the development of COVID-19. Acupuncture and related stimuli-induced NO production and release have been aimed to reveal the possible mechanisms of acupuncture against SARS/COVID-19 through the effects of NO on inhibition of coronavirus, improvements of inflammation and a cytokine storm. The increased interest in the NO on COVID-19 development and treatment has led to an open-minded attitude towards understanding this system, which is fundamental important to establish the valid aspects of scientific basis of the mechanisms and therapeutic effects of acupuncture on treatment of COVID-19.

	THE ROLE OF NITRIC OXIDE (NO) IN THE PATHOPHYSIOLOGY OF COVID-19

   NO deficiency and endothelial dysfunction in the development of COVID-19

   It is well-documented that NO is one of the most important messenger molecules, and NO stimulates guanylyl cyclase to generate cGMP, a second messenger directing vasodilatation (24). Intrinsic vasodilator action of NO-cGMP is critical important in microvascular endothelial function (MEF) and serves as a surrogate index of MEF (25-27). Endothelial dysfunction (ED) contributed to various cardiovascular disorders including essential hypertension, coronary artery disease, and thrombus formation (27-29).

   A recent review (Martel et al., 2020) on strategies suggested to increase airway nitric oxide for treatment and possibly prevention of COVID-19 (30). The clinical presentation of COVID-19 begins with acute respiratory distress in the lungs that moves quickly to vascular networks throughout the gut, kidney, heart, brain and skin with associated ED and abnormally rapid life-threatening blood clotting (2, 30, 31). It is believed that COVID-19 is emerging as a thrombotic and vascular disease targeting endothelial cells throughout the body and is particularly evident in patients with cardiometabolic comorbidities associated with ED (30, 32). Since the endothelium releases NO as the vasodilator and antithrombotic factor, nitric oxide whereas in injured vessels, NO is impaired contributing to hypertension and thrombus formation (31). A hallmark of ED with thrombotic events is the suppression of endothelial nitric oxide synthase (eNOS) with concomitant nitric oxide deficiency. It is suggested that restoring nitric oxide, independent of eNOS, may counter endothelitis and contribute to pulmonary vasodilation, antithrombotic, and direct antiviral activity (30, 32). Moreover, NO has been demonstrated to interfere with the interaction between coronavirus viral S-protein and its cognate host receptor, ACE-2 while NO-mediated S-nitrosylation of viral cysteine proteases and host serine protease, TMPRSS2, which are both critical in viral cellular entry, appear to be NO sensitive (33-36).

   The therapeutic effects of nitric oxide (NO) on SARS-CoV 19

   Previous experiments have shown that NO inhibits the replication cycle of severe acute respiratory syndrome (SARS) coronavirus (37). NO also inhibits the virus by reducing the production of viral RNA and reducing the expression of thioprotein and its binding receptor Angiotensin Converting Enzyme 2 (38). The thioprotein binds to Angiotensin Converting Enzyme 2, which is also the pathogenic target receptor of the 2019 new coronavirus (2). A trial analysis of a coronavirus extracted from SARS clinical patients published in The Lancet in 2003 showed that glycyrrhizin extracted from licorice root produces NO release effect, thereby inhibiting the replication of SARS virus (39). In a small clinical study in Beijing during the SARS prevention and treatment period in 2004, inhaling NO gas in 6 patients reduced airway resistance, increased arterial oxygen partial pressure and oxygen saturation (40). Interestedly, after stopping NO gas inhalation, chest radiograph displayed improvement in the density and area of lung infiltration, and physiological functions continued to be improved. Obviously, NO gas not only reduces airway resistance and improves lung function, but it also has obvious benefits for virus-induced lung infections (40). The 2019-nCoV and SARS-CoV belong to different subtypes of coronaviruses and have similar structures (2). Clinical evidence shows that NO gas inhalation to increase the level of NO which results in restoring lung function by reducing airway resistance and improving virus-induced lung infections. Clinical features, pathology and homology suggest that uncontrolled inflammation and a cytokine storm likely drive COVID-19's unrelenting disease process (2, 30). Interventions that are protective against inflammation, cytokine storm, and acute lung injury can play a critical role for patients and health systems during this pandemic. NO is an antimicrobial and anti-inflammatory molecule with key roles in pulmonary vascular function in the context of viral infections and other pulmonary disease states. Increased evidence supports the rationale for exogenous NO supplements and interventions to increase endogenous NO synthesis/release against the pathogenesis of COVID-19, which is potential for contributing to better clinical outcomes and healthcare capacity.

	ACUPUNCTURE INDUCED NITRIC OXIDE PRODUCTION AND RELEASE

   Acupuncture induces nNOS expression and NO release in acupuncture points (acupoints) and the brain of rats

   Our previous studies demonstrated that neuronal NO synthase (nNOS) expression is increased in the dorsal medulla, the gracile nucleus and nucleus tractus solitarius (NTS), by electroacupuncture (EA) stimulation of ST36 (Zusanli) in rats (41). EA ST36, an acupoint on the leg, induced nNOS-NO generation in the dorsal medulla causes a decrease in arterial blood pressure (42). Withdrawal latencies to mechanical and noxious heat stimuli are reduced in Zucker Diabetic Fatty (ZDF) neuropathic rats and improved by EA ST36 in the rats associated with increased NO release in the gracile nucleus (43). EA ST36-induced alleviation of pain/mechanical hypersensitivities in ZDF neuropathic rats are potentiated by an NO donor and inhibited by a selective inhibitor of neuronal NO synthesis infused into the gracile nucleus. Consistent with the postulated role of the dorsal column pathway in nociceptive regulation, our results show that expressions of transient receptor potential vanilloid type-1 (TRPV1) endowed with nNOS are predominantly increased in the gracile nucleus and in local acupoints following EA ST36 in rats (44, 45). Other study reported that EA stimulation increases the circulating concentration of endothelial NO synthase in rats with hypoxia-induced pulmonary hypertension (46). Consistently, EA stimulation of acupoint ST36 induced antinociception are blocked by both inhibitors of NO synthase and guanylyl cyclase, which suggest that NO-cGMP pathway mediates orofacial antinociception induced by EA ST36 (47). Intraperitoneal administration of specific inhibitors of nNOS, inducible NO synthase and a ATP-sensitive K+ channels blocker reversed the antinociception induced by EA. The results suggest that NO participates of antinociceptive effect of EA through nNOS, iNOS and ATP-sensitive K+ channels activation (48).

   Double immunostaining of transient receptor potential vanilloid type-1 (TRPV1) receptor and nNOS revealed co-localization of TRPV1 and nNOS in both subepidermal nerve fibers and in dermal connective tissue cells (45). A high expression of TRPV1 endowed with nNOS in subepidermal nerve fibers exist in the acupoints and the expression is increased by EA (44, 45). The results suggest that the higher expression of TRPV1 in the subepidermal nerve fibers and its upregulation after EA stimulation may play a key role in mediating the transduction of EA signals to the CNS, and its expression in the subepidermal connective tissue cells may play a role in conducting the local effect of the EA. Other studies have shown that either manual acupuncture or EA at the ST36 acupoint significantly increased components of the TRPV1-related signaling pathway in mice (49), and modified acupuncture-induced reflex excitatory cardiovascular responses in humans (50). The results suggest that the TRPV1 signaling pathway is highly correlated to acupuncture effects and EA-induced expression of TRPV1-nNOS in the ST36 and the NTS/gracile nucleus is involved in the signal transduction of EA stimuli via somatosensory afferents-brain pathways (45, 46, 51).

   Acupuncture induces NO release in humans

   We have demonstrated, using dermal microdialysis in human subjects, that dialysate NO-cGMP releases in the subcutaneous tissue of the forearm skin along the PC acupoints are increased by EA stimulation (52). The results are consistent with the results reported that transcutaneous electrical nerve stimulation (TENS) induces an elevation of NO and cGMP release biocaptured over PC acupoints in humans (53). Recently, the effects of reinforcing method using manual acupuncture (MA) vs. reductive EA on local NO release have been examined using the novel biocapture device over skin regions in humans (54). Results show that NO levels biocaptured over the skin regions are increased following MA by twisting/rotating the needle with gentle amplitude and moderate speed. In contrast, NO levels over the areas of the skin regions are moderately reduced by high-frequency EA (30 Hz), a reduction method. The laser acupuncture (LA) also induces NO release at acupoints with the more level at contralateral side than stimulating site measured by dialysis tube taped to the areas (55). Kimura, et al. reported that acupuncture induces cutaneous vasodilatation in the forearms of humans, which is attenuated by application of NO synthesis inhibitor (56). It is postulated that acupuncture stimulation improves local circulation and allows for a flush of algesic or sensitizing substances, leading to pain relief, thus NO release mediates local effects of acupuncture as therapeutic mechanisms of acupuncture analgesia (57-59).

   These results suggest that MA, EA, LA, and TENS consistently demonstrated an elevation of NO release. It is well-documented that NO improves circulation and microvascular endothelial function, which contribute to various cardiovascular disorders, thrombus formation (27-29), anti-inflammation, and pain relief (57-59).

	CONCLUSION

   The COVID-19 epidemic is spreading all over the world, and the etiological agent of COVID-19 has been confirmed as SARS-CoV-2. The effective treatment interventions are urgently needed since there is no satisfied treatment at the time of this writing. Many studies have been conducted on Chinese herbal remedies with protocols published for various stages of the COVID-19 disease, but acupuncture studies in the treatment of COVID-19 disease are sparse. Acupuncture therapy is relatively easy to practice with minimal risks to the patients but the potential mechanisms of acupuncture for the treatment of COVID-19 are unknown. NO is an antimicrobial and anti-inflammatory molecule with key roles in pulmonary vascular function in the context of viral infections. Previous studies have demonstrated that NO inhibits the replication cycle of SARS coronavirus, and NO gas inhalation to increase the level of NO results in restoring lung function by reducing airway resistance and improving virus-induced lung infections in SARS patients. Recent studies have suggested that NO deficiency and ED contribute to the development of COVID-19. Acupuncture can be effective in treating common symptoms of COVID-19 including breathlessness, nausea, insomnia, leukopenia, fatigue, vomiting, and abdominal pain. Several studies from independent groups consistently demonstrated that MA, EA, LA, and TENS induce local NO-cGMP release in humans. MA and EA also increase NO synthase expression and induces an elevation of NO production in plasma and over local skin regions in both animals and humans. Moreover, it has been suggested that exogenous NO supplies or interventions to elevate NO production may have protective effects against inflammation and acute lung injury for COVID-19 patients and health systems during this pandemic. Whether the therapeutic effects of acupuncture for treatment and prevention of COVID-19 require further investigation by well-designed clinical trials in order to further understand the therapeutic nature of therapy on COVID-19. The possible mechanisms of NO induction induced by acupuncture should contribute to better clinical outcomes and facilitating clinical studies of acupuncture on treatment and prevention of COVID-19.

	ACKNOWLEDGMENTS

   This project was made possible by NIH Grant (AT002478, AT004620, and AT004504) from the National Center for Complementary and Integrative Medicine (to SXM).

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