Acupuncture in the Treatment of Amyotrophic Lateral Sclerosis: A Research Progress in Clinical Trials.

Background: Acupuncture, a complementary and alternative medicine (CAM) modality, shows promise as an integrative therapy for Amyotrophic Lateral Sclerosis (ALS) due to the chronic nature of the disease and its persistent symptoms. Many patients turn to CAM for ALS treatment. Objective: This review assesses acupuncture's efficacy in treating Amyotrophic Lateral Sclerosis. Methods: We searched China National Knowledge Network (CNKI) and PubMed databases for Chinese and English articles, including clinical trials, case studies, cohorts, and randomized controlled trials. The search, performed on March 31, 2023, encompassed literature published up to that date. Keywords used in titles and abstracts were (acupuncture) OR (electro-acupuncture)) AND (Amyotrophic Lateral Sclerosis). Results: Among the 45 articles studied, 34 were included in this research. Acupuncture's benefits primarily lie in neuro-immune system regulation, enhanced quality of life, reduced fatigue, disease progression delay, and fewer relapses. Conclusions: Recent clinical trials highlight the potential of traditional Chinese acupuncture in improving Amyotrophic Lateral Sclerosis symptoms (e.g., fatigue, neural functional deficits) and curtailing relapses. Consequently, acupuncture holds promise as an integrative therapy for ALS patients.

Electro-nape-acupuncture regulates the differentiation of microglia through PD-1/PD-L1 reducing secondary brain injury in acute phase intracerebral hemorrhage rats.

INTRODUCTION: This study aimed to investigate the effect of electro-nape-acupuncture (ENA) on the differentiation of microglia and the secondary brain injury in rats with acute-phase intracerebral hemorrhage (ICH) through the programmed cell death protein-1/ligand-1 (PD-1/PD-L1) pathway. METHODS: A total of 27 male Sprague-Dawley rats were randomly divided into three groups: sham group, ICH group, and ENA group. The autologous blood infusion intracerebral hemorrhage model was used to study the effects of ENA by administering electroacupuncture at GB20 (Fengchi) and Jiaji (EX-B2) acupoints on 24 h after the modeling, once per day for 3 days. The neurological function damage, hematoma lesion, and inflammatory cell infiltration were measured by the beam walking test and hematoxylin-eosin staining. The expression of PD-1, PD-L1, CD86, CD206, and related cytokines around the hematoma was measured by western blot, quantitative reverse transcription polymerase chain reaction, and immunofluorescence. RESULTS: The ICH group had significant neurological deficits (p < .001), hematoma lesions, and inflammatory cell infiltration. The levels of CD86 protein, inflammatory factors tumor necrosis factors (TNF)-α, interleukin (IL)-1ß, and IL-6 were increased (p < .001), while CD206 protein was reduced (p < .01), and the number of CD86+ /CD11b+ cells was also increased (p < .001) compared to the sham group. However, after ENA intervention, there was a significant reduction in neurological function damage (p < .05), infiltration of inflammatory cells, and the expression levels of CD86+ /CD11b+ cells (p < .05), resulting in the increased expression of PD-1 protein and differentiation of M2 phenotype significantly (p < .001). CONCLUSION: The study concludes that ENA could reduce neurological function damage, inhibit the expression of pro-inflammatory cytokines, and improve the infiltration of inflammatory cells to improve secondary brain injury in acute-phase intracerebral hemorrhage rats. These effects could be related to the increased expression of PD-1 around the lesion, promoting the differentiation of microglia from M1 to M2 phenotype.

Electroacupuncture regulates microglial polarization via inhibiting NF-κB/COX2 pathway following traumatic brain injury.

BACKGROUND: Neuroinflammation and oxidative stress are important pathological mechanisms following traumatic brain injury (TBI). The NF-κB/COX2 pathway regulates neuroinflammation and oxidative damage, while microglia also play an important role in neuroinflammation. Since NF-κB is involved in microglial polarization, targeting this pathway and microglial polarization is a critical component of TBI treatment. Currently, electroacupuncture (EA) is widely used to treat various symptoms after TBI, but the mechanisms of EA remain poorly understood. Additionally, the optimal frequency of EA remains unclear, which affects its efficacy. This study focuses on exploring the optimal frequency parameters of EA on TBI and investigating the underlying mechanisms of EA through NF-κB/COX2 pathway and microglial polarization. METHODS: The study was divided into two parts. In Experiment 1, 42 Sprague Dawley (SD) rats were induced and randomly divided into seven groups (n = 6). Except for the sham group, all rats underwent controlled cortical impact (CCI) to establish TBI model. Four EA groups (with different frequencies) and manual acupuncture (without current stimulation) received stimulation on the acupoints of Shuigou (GV26), Fengchi (GB20) and Neiguan (PC6) once a day for 7 days. The neurological function was assessed by modified Neurological Severity Scores (mNSS), and the rats' memory and learning were examined by the Morris water maze (MWM). SOD, MDA, and GSH-Px were detected to evaluate the levels of oxidative stress. The levels of IL-1ß, IL-6, and TNF-α were evaluated by Enzyme Linked Immunosorbent Assay (ELISA). Detection of the above indicators indicated a treatment group that exerted the strongest neuroprotection against TBI, we then conducted Experiment 2 using this screened acupuncture treatment to investigate the mechanism of acupuncture. 48 rats were randomly divided into four groups (n = 12): sham, TBI model, acupuncture and PDTC (NF-κB inhibitor). Evaluations of mNSS, MWM test, SOD, MDA, GSH-Px, IL-1ß, IL-6, TNF-α, and IL-10 were the same as in Experiment 1. Western blot was applied for detecting the expression levels of NF-κB, p-NF-κB, COX2, and Arg-1. TUNEL was used to examine neuronal apoptosis. Brain structure was observed by H&E. Iba-1, COX2, and Arg-1 were investigated by immunofluorescence staining. RESULTS: EA with frequency of 2/100 Hz markedly improved neuronal and cognitive function as compared to the other treatment groups. Moreover, it downregulated the expression of MDA, IL-6, IL-1ß, and TNF-α and upregulated the levels of SOD and GSH-Px. In addition, Both EA with 2/100 Hz and PDTC reduced the levels of p-NF-κB, COX2 and M1 markers (COX2, IL-6, IL-1ß, TNF-α) and increased the levels of M2 markers (Arg-1, IL-10). Moreover, they had similar effects on reducing inflammation, oxidative stress and apoptosis, and improving neuronal and cognitive function. CONCLUSIONS: The collective findings strongly suggest that EA with 2/100 Hz can improve neurologic function by suppressing neuroinflammation, oxidative stress and apoptosis. Additionally, we confirm that EA promotes microglial polarization towards the M2 phenotype through the suppression of NF-κB/COX2 pathway, thus exerting neuroprotective effects after TBI.

Acupuncture for acute migraine attacks in adults: a systematic review and meta-analysis.

Objective To evaluate the effect and safety of acupuncture for acute migraine attacks in adults. DESIGN AND SETTING: We searched PubMed, MEDLINE(OVID), Chinese Biomedical Literature Database, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database and Wanfang database from inception to 15 July 2022. We included randomised controlled trials (RCTs) published in Chinese and English comparing acupuncture alone against sham acupuncture/placebo/no treatment/pharmacological therapy or comparing acupuncture plus pharmacological therapy against the same pharmacological therapy. Results were reported as risk ratios (RRs) for dichotomous outcomes or mean differences (MDs) for continuous outcomes, with 95% CIs. Risk of bias was assessed with the Cochrane tool and the certainty of the evidence (CoE) with GRADE. : Main outcome measures : a) The rate of headache freedom (pain score=0) at 2h after the treatment; b) the rate of headache relief (at least 50% reduction of pain score); c) headache intensity at 2h after the treatment(study data from scales measuring pain intensity, including visual analogue scale, numerical rating scale) d) the improvement of headache intensity at 2h after the treatment; e) the improvement values of migraine-associated symptoms; f) adverse events. RESULTS: We included 21 RCTs from 15 studies with 1926 participants comparing acupuncture against other interventions. Comparing to sham acupuncture or placebo, acupuncture may result in an increase in the rate of headache freedom (RR 6.03, 95% CI 1.62 to 22.41, 180 participants, 2 studies, I2=0%, low CoE) and the improvement of headache intensity (MD 0.51, 95% CI 0.16 to 0.85, 375 participants, 5 studies, I2=13%, moderate CoE) at 2 hours after treatment. It also may result on a higher rate of headache relief (RR 2.29, 95% CI 1.16 to 4.49, 179 participants,3 studies, I2=74%, very low CoE) and greater improvement of migraine-associated symptoms (MD 0.97, 95% CI 0.33 to 1.61, 90 participants, 2 studies, I2=0%, very low CoE) at 2 hours after treatment but the evidence is very uncertain. Meanwhile the analysis indicates acupuncture probably results in little to no difference in adverse events compared with sham acupuncture (RR 1.53, 95% CI 0.82 to 2.87, 884 participants, 10 studies, I2=0%, moderate CoE). In acupuncture plus pharmacological intervention versus the same pharmacological intervention, acupuncture plus pharmacological therapy may result in little to no difference in the rate of headache freedom (RR 1.55, 95% CI 0.99 to 2.42, 94 participants, 2 studies, I2=0%, low CoE), the rate of headache relief (RR 1.20, 95% CI 0.91 to 1.57, 94 participants, 2 studies, I2=0%, low CoE) at 2 hours after treatment and adverse events(RR 1.48, 95% CI 0.25 to 8.92, 94 participants, 2 studies, I2=0%, low CoE). However, it may result in a reduction in headache intensity (MD -1.05, 95% CI -1.49 to -0.62, 129 participants, 2 studies, I2=0%, low CoE) and an increase in the improvement of headache intensity (MD 1.18, 95% CI 0.41 to 1.95, 94 participants, 2 studies, I2=0%, low CoE) at 2 hours after treatment compared with pharmacological therapy only. In comparison to pharmacological intervention, acupuncture may result in little to no difference in the rate of headache freedom (RR 0.95, 95% CI 0.59 to 1.52, 294 participants, 4 studies, I2=22%, low CoE), the rate of headache relief (RR 0.95, 95% CI 0.80 to 1.14, 206 participants, 3 studies. I2=0%, low CoE) at 2 hours and adverse events (RR 0.65, 95% CI 0.35 to 1.22, 294 participants, 4 studies, I2=0%, low CoE) after treatment. The evidence is very uncertain about the effect of acupuncture on the headache intensity (MD -0.07, 95% CI -1.11 to 0.98, 641 participants, 5 studies, I2=98%, very low CoE) and the improvement of headache intensity (MD -0.32, 95% CI -1.07 to 0.42, 95 participants, 2 studies, I2=0%, very low CoE) at 2 hours after treatment compared with pharmacological intervention. CONCLUSION: The body of evidence suggests that acupuncture may be more effective than sham acupuncture in the treatment of migraine. Acupuncture may also be as effective as pharmacological therapy. However, the certainty evidence across outcomes was low to very low and new high-quality studies can provide more clarity. PROSPERO REGISTRATION NUMBER: CRD42014013352.

Acupuncture for the Treatment of Migraine: An Overview of Systematic Reviews.

PURPOSE OF REVIEW: Over the past few years, there have been many systematic reviews (SRs) and meta-analyses (MAs) used to assess the safety and efficacy of acupuncture in the treatment of migraine. Our aim is to assess the methodological and reporting quality of SRs/MAs and make an evaluation about the available evidence of the safety and efficacy of acupuncture in the treatment of migraine. RECENT FINDINGS: Migraine is one of the most common primary headaches, with various symptoms, threatening human health. Acupuncture is a specific treatment of Traditional Chinese Medicine (TCM), and a validated non-pharmaceutical option as well, widely used in the treatment of migraine, and has gained remarkable therapeutic effect. An overview can evaluate research methods and evidence for SRs/MAs in evidence-based medicine, although there is much relevant research, it's still a hard work to synthesis all the evidence or to make robust conclusions, the variation in the methodological and quality evidence in such SRs/MAs play a significant role. For this overview, we searched six electronic databases from inception until 8 September 2022, and without languages restrictions, the results showed that, acupuncture as a more safety and more convenient therapeutic, and it has been shown to be effective in the treatment of migraine, that is worthy of clinical promotion. However, there are also some limitations because of the low-quality evidence of most of the studies. In conclusion, most included SRs/MAs suggested that acupuncture was more effective than the control group in the treatment of migraine. However, the quality evidence of most of the studies still needs to be improved.

Acupuncture for oculomotor nerve palsy:A systematic review and meta-analysis.

BACKGROUND: To evaluate the safety and efficacy of acupuncture in the treatment of oculomotor nerve palsy (ONP). METHODS: The following database will be required from PubMed, Cochrane Library, Medline, Chinese Biomedical Literature Database, China National Knowledge Infrastructure (CNKI), Wanfang data. Randomized controlled trials (RCTs) comparing acupuncture alone versus no treatment/another active therapy/sham acupuncture or comparing acupuncture with another active therapy versus the same active therapy were included. Meta-analysis was conducted according to the 2020 PRISMA guidelines. Data was analyzed using RevMan 5.4 software. Results were reported as risk ratios (RR) for dichotomous outcomes or mean differences (MD) for continuous outcomes, with 95 % confidence intervals (CI). The Cochrane risk of bias tool was used to access the methodological quality of the trails. RESULTS: Eighteen RCTs with 1150 participants comparing acupuncture versus other therapies were included. The results showed a significant differences in the clinical efficiency rate (RR = 1.30, 95 %CI = 1.23-1.37, P < 0.001), scores of diplopia (MD = - 0.78, 95 %CI = - 1.39 to - 0.77, P < 0.001), palpebral fissure size (MD = 1.04, 95 %CI = 0.41-1.68, P = 0.001), the pupil diameter (MD = - 0.56, 95 %CI = - 0.70 to - 0.42, P < 0.001), quality of life (MD = 8.96, 95 %CI = 6.79-11.13, P < 0.001) between the experiment and control groups. However, there were no significant differences in the adverse effects (RR = 0.52, 95 %CI = 0.22-1.22, P = 0.13). The quality of the evidence test by GRADE was low or very low. CONCLUSION: Most included studies suggested that acupuncture was more effective than the control group in the treatment of ONP. However, the quality evidence of most of the studies was low and most of them were performed in China.

[Progress of researches on mechanism of acupuncture for traumatic brain injury].

Acupuncture therapy has been shown to have a positive effect in treating symptoms of patients with traumatic brain injury (TBI) in China. The present paper summarizes findings of recent researches on the underlying mechanisms of acupuncture intervention for improving TBI from 1) inhibiting inflammatory response, 2) regulating neurotransmitters, 3) promoting nerve repair and regeneration, 4) antioxidation, 5) inhibiting intracellular calcium overload, 6) regulating aquaporin content, 7) regulating cellular energy metabolism, 8) improving brain circulation, and 9) inhibiting cell apoptosis.

Acupuncture for patients with atopic dermatitis: A systematic review protocol.

BACKGROUND: The systematic review protocol is aim to evaluate the efficacy and safety of acupuncture in the treatment of atopic dermatitis (AD). METHODS: We will search the database on the Cochrane Library, PubMed, Medline, Excerpta Medica Database, Chinese Biomedical Literature Database, PsychINFO, China National Knowledge Infrastructure, Wanfang data, Chinese Scientific Journal Database, including studies and published systematic review in the reference list and grey. And will use Cochrane Collaboration's tools to evaluate the risk of bias of the included randomized controlled trials. The Review Manager 5.3 will be used to synthesize collected data. RESULTS: This study will assess the safety and effectiveness based on current evidence of acupuncture for AD, especially scoring atopic dermatitis, eczema area, and severity index, patient-oriented eczema measure, and quality of life. CONCLUSION: This study will provide high-quality recently evidence for evaluating the efficacy of acupuncture for patients with AD.PROSPERO registration number: CRD42019135919.

A Pilot Randomized Placebo Controlled Trial of Electroacupuncture for Women with Pure Stress Urinary Incontinence.

BACKGROUND: Acupuncture is a potential conservative therapy for women with stress urinary incontinence (SUI). There is limited evidence to support its effectiveness due to the poor quality of existing studies. METHODS: We performed a pilot randomized, controlled trial to preliminarily assess the efficacy of electroacupuncture (EA) in women with pure SUI. A total of 80 women with pure SUI were randomly assigned to receive EA with deep needling at BL33 and BL35 (n = 40) or sham EA with non-penetrating needling at sham acupoints (n = 40) three sessions per week for 6 weeks. The women were followed for 24 weeks. The primary outcome was the change from baseline in the amount of urine leakage measured by a 1-hour pad test after 6 weeks. The secondary outcomes included the 72-hour incontinence episode frequency (IEF), International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) score, and patient self-evaluation of therapeutic effect. Adverse events (AEs) were monitored throughout the trial. RESULTS: The median decrease from baseline of urine leakage measured by the 1-hour pad test was 2.5 g [interquartile range (IQR): 1.80-14.6 in the EA group, which was greater than the median decrease of 0.05 g (IQR: -2.80-+0.50) in the sham EA group after 6 weeks (p<0.01). The differences between groups in the decrease from baseline of 72-hour IEF became statistically significant at week 30 with a median decrease of 3.25 g (IQR: 1.25-5.69) in the EA group, and a median decrease of 1.00 g (IQR: -0.69-+2.88) in the sham EA group (p = 0.01). The participants in the EA group showed greater decreases in ICIQ-SF score and higher ratings in the help they received from the treatment than those in the sham EA group at weeks 6,18 and 30 (all p<0.05). No obvious AEs were observed in either group. CONCLUSION: EA may effectively and safely relieve urinary incontinence symptoms and improve quality of life in women with pure SUI. EA demonstrated more than a placebo effect. Since this is a pilot study, results should be interpreted with caution. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445573.

Acupuncture for acute migraine attacks in adults: a systematic review protocol.

INTRODUCTION: Acupuncture has been used worldwide for migraine attacks. This systematic review aims to assess if acupuncture is effective and safe in relieving headache, preventing relapse and reducing migraine-associated symptoms for the treatment of acute migraine attacks in adults. METHODS AND ANALYSIS: We will search the following seven databases from inception to February 2015: MEDLINE (OVID), EMBASE, the Cochrane Central Register of Controlled Trials (CENTRAL) and four Chinese databases (Chinese Biomedical Database, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database and Wanfang Database). Any randomised controlled trials in English or Chinese related to acupuncture for acute migraine attacks will be included. Conference abstracts and reference lists of included manuscripts will also be searched. The study inclusion, data extraction and quality assessment will be conducted independently by two reviewers. Meta-analysis will be performed using RevMan V.5.3.5 statistical software. DISSEMINATION: The findings will be disseminated through peer-reviewed publication and/or conference presentations. TRIAL REGISTRATION NUMBER: PROSPERO CRD42014013352.

[Comparison of efficacy on functional constipation treated with electroacupuncture of different acupoint prescriptions: a randomized controlled pilot trial].

OBJECTIVE: To evaluate preliminarily the efficacy on functional constipation treated with electroacupuncture of different acupoint prescriptions. METHODS: One hundred and four patients were randomized into a front-mu and back-shu points group (19 cases), a he-sea points group (34 cases), a he-sea, front-mu and back-shu points group (26 cases) and a western medication control group (25 cases). In the front-mu and back-shu points group, electroacupuncture was applied at bilateral Tianshu (ST 25) and Dachangshu (BL 25). In the he-sea points group, electroacupuncture was applied at bilateral Quchi (LI 11) and Shangjuxu (ST 37). In the he-sea, front-mu and back-shu points group, electroacupuncture was applied at unilateral Tianshu (ST 25), Dachangshu (BL 25), Quchi (LI 11) and Shangjuxu (ST 37). In the three groups above, the treatment was given 5 times a week in the first two weeks and 3 times a week in the next two weeks. In the western medication control group, mosapride citrate tablets were prescribed for oral administration, 1 table (5 mg) each time, 3 times a day, continuously for 4 weeks. The period of research was 9 weeks, including 1 week for baseline evaluation, 4 weeks for treatment and 4 weeks for follow-up. The weekly defecation frequency was taken as primary index, while the defecation difficulty and life quality score were taken as the secondary indices for the efficacy evaluation after treatment and in follow-up. RESULTS: According to the intention-to-treat (ITT) analytic principle, 104 cases were all enrolled in the final analysis. (1) After treatment, the weekly frequency of defecation was all increased significantly in the four groups (P < 0.05, P < 0.01). The efficacy of the three electroacupuncture groups was similar to that of western medication control group (P > 0.05). In follow-up, the increasing effect on the weekly frequency of defecation was maintained in the he-sea points group (P < 0.01), superior to the front-mu and back-shu points group and the western medication control group (P < 0.05, P < 0.01); the weekly frequency of defecation was not improved in the rest three groups (P > 0.05). (2) After treatment, defecation difficulty was relieved in the he-sea points group, the he-sea, front-mu and back-shu points group and the western medication control group (P < 0.05, P < 0.01). In follow-up, the improvements were still significant in the he-sea points group and the he-sea, front-mu and back-shu points group (both P < 0.01). (3) After treatment, the life quality score was significantly improved in the patients of the he-sea points group (P < 0.05). The difference was not significant in the rest three groups as compared with that before treatment (all P > 0.05). CONCLUSION: The weekly frequency of defecation is increased effectively after treatment in the three electroacupuncture groups and the efficacy is similar to mosapride citrate tablets. The bilateral Quchi (LI 11) and Shangjuxu (ST 37) in he-sea acupoints increase significantly the weekly frequency of defecation, relieve defecation difficulty and improve life quality. Acupuncture efficacy is sustained for 4 weeks. This acupoints prescription is the best in the treatment of functional constipation.

Efficacy of electroacupuncture for symptoms of menopausal transition: study protocol for a randomized controlled trial.

BACKGROUND: Previous studies have shown that acupuncture can alleviate postmenopausal symptoms, such as hot flashes, but few studies have assessed symptoms during the menopausal transition (MT) period. Thus, the effect of acupuncture upon MT symptoms is unclear. We designed a large-scale trial aimed at evaluating the efficacy of electroacupuncture for MT symptoms compared with sham electroacupuncture and at observing the safety of electroacupuncture. METHODS/DESIGN: In this multicenter randomized controlled trial, 360 women will be randomized to either an electroacupuncture group or a sham electroacupuncture group. During the 8-week-long treatment, a menopause rating scale, average 24-hour hot flash score, Menopause-Specific Quality of Life Questionnaire score, and level of female hormones will be observed. Follow-ups at the 20th and 32nd week will be made. DISCUSSION: Though there is no completely inert placebo acupuncture and blinding is difficult in acupuncture trials, the placebo effect of EA can still be partially excluded in this study. For the placebo control, we use non-points and a tailor-made sham needle. This needle is different from a retractable needle, which is usually used for sham acupuncture. The needle in this trial is more simply constructed and more acceptable to Chinese people. We expect to evaluate the efficacy of electroacupuncture for MT symptoms and clarify its effect on these symptoms. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01849172 (Date of registration: 05/05/2013).

The efficacy and safety of electroacupuncture for women with pure stress urinary incontinence: study protocol for a multicenter randomized controlled trial.

BACKGROUND: Although available evidence relating to its effectiveness is weak, acupuncture is used as an alternative therapy for stress urinary incontinence. We report a protocol of a randomized controlled trial using electroacupuncture (the passing of a weak current between inserted acupuncture needles) to treat women with pure stress urinary incontinence. METHODS/DESIGN: This is a large-scale multicenter subject-blinded randomized controlled trial. A total of 500 women with pure stress urinary incontinence will be randomly assigned to two groups: a treatment group and a control group. The treatment group will receive electroacupuncture with deep needling at acupuncture points BL33 and BL35. The control group will receive sham electroacupuncture with non-penetrating needling at sham locations for the acupuncture points of BL33 and BL35. Participants will be given three sessions a week for 6 weeks. A 24-week-long follow-up will be conducted. The primary outcome will be the change in amount of urine leakage at the sixth week from a baseline measured by a 1-h pad test. The secondary outcomes include: the 72-h incontinence episode frequency based on a 72-h bladder diary; the score of International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form; the degree of urinary incontinence based on a 72-h bladder diary; self-assessment of the therapeutic effect; weekly consumption of pads; application of other treatments for stress urinary incontinence; and subgroup analysis stratified by incontinence severity. The safety of electroacupuncture will also be assessed. DISCUSSION: This trial will help to identify whether electroacupuncture is effective for stress urinary incontinence, and, if so, whether it is a therapeutic effect rather than a placebo effect. TRIAL REGISTRATION: Clinical Trials.gov NCT01784172.