Effect and Safety of Naomaili Granules ( (cid:0)(cid:0)(cid:0)(cid:0)(cid:0) ) for the Treatment of Acute Ischemic Stroke: A Randomized, Double-Blind, Placebo-Controlled, Multicenter Trial

Background: It is of vital importance for the treatment and prognosis of Acute Stroke to nd effective Chinese medicine that can be combined with western medicine in the acute stage. The purpose of this study is to investigate the effect and safety of Naomaili Granules ( (cid:0)(cid:0)(cid:0)(cid:0)(cid:0) , NML) for the treatment of acute stroke, hoping to provide a new idea and drug choice for the integrated treatment of Chinese and Western medicine in the acute stage of ischemic stroke, and at the same time to improve the treatment plan in the acute stage of ischemic stroke from the perspective of TCM syndromes. Methods: A total of 187 patients with acute ischemic stroke were randomly divided into the NML group (93 cases) and the placebo group (94 NML mimics), 1 bag (10g/bag), thrice daily for 20 days. Basic medications during the trial: Aspirin enteric-coated tablets, 1 tablet (0.1g/tablet), once a day. After treatment, the modied Rankin scale, the incidence of cardiovascular events and TCM Syndrome effect were the main ecacy indicators. Meanwhile, adverse events (AEs) were evaluated during the whole clinical trial. Results: In the FAS 90 days after the onset, the experimental group was 70.00%, and the control group was 45.24%. There was a statistically signicant difference between the two groups. The incidence of acute cardio-cerebrovascular events was 1 case (1.08%) in the experimental group and 0 in the control group after 20 days of FAS treatment. Conclusion: The combined application of NML in the acute stage of ischemic stroke can effectively improve the prognosis of patients, and improve the independent survival ability of patients, and its safety is reliable, providing a new way of thinking and medication choice for the treatment of acute ischemic stroke with integrated traditional Chinese and western medicine. Time; FIB: Fibrinogen; NIHSS: National Institutes of Health Stroke Scale; BMI: Body Mass Index; AIS: Acute ischemic stroke; QD: Qi Deciency; BSS: Blood Stasis Syndrome; VEGF: Vascular endothelial growth factor.

aspirin is the cornerstone of the prevention and treatment of ischemic stroke recommended by multinational guidelines, some patients may have antiplatelet drug resistance, resulting in ineffective treatment [4][5][6][7] . In addition, due to the narrow time window of intravenous thrombolysis, less than 3% of patients can be treated, and stent restenosis and reperfusion injury, etc are also common. Thus, additional treatment strategies are needed to improve poststroke recovery.
Ischemic stroke is a complicated and multi-factor pathogenesis, so it is di cult to improve the cure rate and reduce the disability rate from one aspect and one level of treatment. Traditional Chinese Medicine (TCM) has been historically used for stroke treatment. On the basis of Western medicine treatment, the integrated of TCM combined with evidence-based Chinese medicine and Western medicine in the treatment of ischemic stroke can overlap and merge in the course of practicing process, and give full play to the advantages of their own treatment, so as to better improve the clinical e cacy, reduce adverse reactions and the incidence of sequelae [9] . According to the recommendation of guideline [10] , the integrated treatment of traditional Chinese and Western medicine should be carried out as soon as possible after thrombolysis. However, currently oral Chinese patent medicine is mainly used in recovery period, and the use of traditional Chinese medicine for the treatment of ischemic stroke only accounts for a very low proportion of total treatment expenditure globally [11,12] . Therefore, it is very important to nd effective Chinese medicine which can be combined with western medicine in the acute stage for the treatment and prognosis of ischemic stroke.
In this research, a randomized, double-blind, placebo-controlled multicenter clinical trial was designed, and the effect and safety of western medicine basic therapy (aspirin) combined with NML were investigated in patients with acute stroke. This study aims to provide a new idea and drug choice for the treatment of acute ischemic stroke with integrated traditional Chinese and Western medicine, and to improve the treatment plan of acute ischemic stroke from the perspective of TCM syndromes.

Diagmostic Criteria
Medical diagnostic standards (refer to "Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2010" issued by the Chinese Medical Association Neurology Branch [4] ).
Key points of diagnosis: (1) Acute onset; (2) Focal neurological de cits, a few are general neurological de cits; (3) Symptoms and signs last more than 24 hours; (4) Exclude non-vascular brain lesions; (5) Brain CT or MRI excludes cerebral hemorrhage and other lesions, responsible ischemic lesion.
TCM syndrome diagnostic criteria Reference to Wang, etc., 2011 [19] . Developing Syndrome Differentiation of Qi De ciency (QD) and Blood Stasis Syndrome (BSS) in the Acute Phase of Stroke Disease standard.

Inclusion Criteria
(1) Those who meet the medical diagnostic criteria for acute ischemic stroke (belonging to anterior circulation infarction); (2) The syndromes of QD and blood stasis comply with TCM syndrome differentiation, that is, the scores of QD and BSS are ≥ 10 points; (3) Patients whose disease course is 7-14 days after the onset (including 7 days and 14 days); (4) NIHSS scores between 4 ~ 20 points; (5) Age between 40 ~ 80; (6) Patients with rst or recurrent stroke have fully recovered before onset (mRS points are 0-1); (7) Signed informed consent.

Exclusion Criteria
Patients with any of the following conditions were excluded: (1) History of cerebral hemorrhage in the past six months; (2) With conscious disturbance in clinical manifestations; (3) Treated with blood vessels (such as thrombolysis, arterial thrombectomy, ultra-early thrombus aspiration, and stent forming) within 6 h after the onset; (4) It is con rmed by examination that brain embolism caused by brain tumor, brain trauma, cerebral parasitic disease, metabolic disorder, rheumatic heart disease, coronary heart disease; (5) TIA, cerebral hemorrhage, subarachnoid hemorrhage, and asymptomatic cerebral infarction; (6) Cannot be administered orally; (7) Oral anticoagulants; (8) Combined with other diseases that affect limb function; (9) Complicated with severe hypertension or diabetes and other diseases; (10) With active ulcer and bleeding tendency; (11) With severe heart and lung diseases and chronic liver and kidney dysfunction; (12) Pregnant or lactating women; (13) Disabled patients; (14) Those who are allergic to the ingredients of this medicine and allergies; (15) Those who are not suitable for inclusion or in uence the participation or completion by the investigator; (16) Participating in other clinical trials within 3 months.

Rejection Criteria
Patients with false acceptance, misdiagnosis, no medication, or no inspection records were rejected.

Interventions
The study period is 20 days, and the follow-up period was 90 days after the onset. During the start-up period, patients were prohibited from taking other CM to activate blood or dissolve stasis. Both NML (batch No. 11-141201, 10 g/bag) and the NML simulation granules (batch No. 11-160301) were manufactured by Nanjing Kefeiping Shenghui Pharmaceutical Co., Ltd. and supplied by Jiangsu Kefeiping Pharmaceutical Co., Ltd., China. As a basic medicine, aspirin enteric-coated tablets (batch No. BJ19533, 0.1 g/tablet) are produced by Bayer HealthCare Manufacturing S.r.l. and supplied by Jiangsu Kefeiping Pharmaceutical Co., Ltd. During the study, thrombolytic drugs, anticoagulants, de brinators, neuroprotective agents, and other antiplatelet drugs are not allowed to use. Drugs that must be taken in combination with diseases, such as antihypertensive drugs, lipid-lowering drugs, hypoglycemic drugs, or antibiotics, can remain unchanged.

Primary Outcome
The experimental results are mainly evaluated by the following three methods: Evaluation criteria of improved Rankin scale (1) The t-test/rank and test were used to compare the changes in the Rankin Scale (mRS) scores before and after treatment; (2) mRS ≤ 2 are relatively independent, and mRS > 2 or more points are obvious disability or poor prognosis. The ratio of relative independence can be compared using Fisher variable probability method/χ 2 test.

Evaluation criteria for the incidence of cardiovascular and cerebrovascular events
The Fisher prediction probability method/χ 2 test was used to compare the incidence of cardio-cerebral vascular events (myocardial infarction, cerebral infarction, TIA, etc.) 20 days after the onset of the two groups of patients.

Analysis of e cacy indicators of TCM syndromes
The ischemic stroke QD and BSS dialectical scale was used for evaluation according to the "General Principles of Clinical Research of New Chinese Medicines". The t test/Wilcoxon rank sum test was used to compare the scores and reduction rates of the syndromes of QD and blood stasis before and after the two groups of tests ((pre-treatment score-post-treatment score) Pre-treatment score × 100%).

Secondary Outcomes
Barthel index e cacy evaluation criteria (1) The t-test/rank sum test was used to compare the changes in the Barthel index (BI) before and after treatment; (2) ≥ 60 are relatively independent, and < 60 means obvious disability or poor prognosis.
Fisher's exact probability method/χ 2 test was used to compare the relative independence of the two groups.
National Institutes of Health Stroke Scale (NIHSS) evaluation criteria (1) The t-test/Wilcoxon rank sum test was used to compare the changes in NIHSS scores before and after treatment; (2) After the treatment, the NIHSS score ≤ 1 is classi ed as good clinical recovery; otherwise, the clinical recovery is poor, and Fisher exact probability method/χ 2 test is used to compare the good clinical recovery ratio between the two groups.

Safety evaluation
The safety indices observed included: (1) General physical examination items, such as body temperature, heart rate, breathing, blood pressure, symptomatic cerebral hemorrhage (bleeding at the infarct site), etc.;

Randomization and Blinding
Randomized design Strati ed block randomization was used. With the help of SAS v9.3 statistical software, given the number of seeds, the random arrangement of subjects (i.e. random coding table) is generated. The corresponding drug number shall be pasted on the conspicuous position of the external package of the drug by the personnel unrelated to the clinical observation, supervision, statistical analysis, etc. of this clinical trial according to the formed processing code. The divided test kits are sent to each test center together.

Blindness
(1) Blind method design: This experiment uses secondary blinding; (2) Management and preservation of blind bottom: The random coding table is established by the clinical trial data management and statistical unit. The blind bottom is sealed in duplicate and submitted to the sponsor and the lead unit for proper storage. The entire drug coding process is written by the blind editor into a le form, that is, a blind edit record, which is saved as one of the documents of the clinical trial. Emergency blind envelopes are kept by the project leaders of each clinical trial unit. No one may unpack it without permission; (3) Emergency blind-breaking: If serious adverse events occur during the test, emergency blind-breaking can be performed. Emergency blindness must be decided by the main investigator of the research center, and the reason, time, and place of blindness should be recorded in detail and signed. After the blindness is broken, the team leader unit and the clinical monitor shall be noti ed in time. Case data should be kept intact.

Sample Size Estimation
The primary outcome measure was the poor prognosis rate (de ned as mRS > 2). According to the relevant literature, it is assumed that the adverse outcome rate of the treatment group through NML and conventional treatment is 20% lower than that of the control group alone. The type I error is set to 0.05 and the control is 80%. According to the 1:1 parallel control design of the experimental group and the control group, the minimum sample size of each group was 93 cases. In consideration of the reasons such as the drop off of the subjects during the clinical trial (assuming 20% drop off rate), the total sample size of the clinical trial of NML in the treatment of acute stroke was designed as 240 cases, 120 cases in the trial group and 120 cases in the control group, which were undertaken by each sub center.

Statistical Analyses
(1) For quantitative data, the data are described by case number, mean, standard deviation, minimum, median, maximum, upper quartile (Q1), lower quartile (Q3), 95% con dence interval (95% CI). Statistical analysis of the data between the two groups or within the group before and after treatment, using t-test or paired t-test; (2) For qualitative data, use frequency tables, percentages or composition ratios to describe the data. Statistical analysis before and after treatment, using χ 2 test, Fisher exact probability method, Wilcoxon rank sum test or Wilcoxon symbol rank sum test; comparison of two classi cation indicators and rank indicators; (3) All hypothesis tests use a two-sided test, taking α = 0.05. All statistical calculations were performed using SAS 9.3 statistical analysis software.

Baseline Characteristics of Patients
A total of 187 patients were enrolled from 16 clinical centers in this study, 93 patients in medication group, 94 patients in control group. The demographic data (age, gender, weight, height, BMI) and vital signs (systolic pressure, diastolic pressure, resting heart rate, respiration, body temperature) of the two groups of subjects were statistically analyzed. There was no signi cant difference on patients' demographic pro les between the two groups (P > 0.05, Table 1).

Comparisons of main e cacy indicators between Two Groups
Improved Rankin's relative independence rate In the FAS 90 days after the onset, the experimental group was 70.00%, and the control group was 45.24%. There was a statistically signi cant difference between the two groups (P < 0.05), the test group is better than the control group. Incidence of acute cardio-cerebral vascular events (1) The incidence of acute cardio-cerebrovascular events was 1 case (1.08%) in the experimental group and 0 in the control group after 20 days of FAS treatment; (2) The incidence of myocardial infarction was 0 in the experimental group and the control group; (3) Cerebral infarction incidence: 1 case (1.08%) in the experimental group and 0 cases in the control group; (4) The number of visits to the FAS test group was 0 in the TIA group (Table 3).  (1) The score of QD and BSS after 20 days of medication was 36.08% ± 28.53% in the FAS test group and 25.03% ± 26.56% in the control group. The difference was statistically signi cant (P < 0.05); (2) The score of BSS score after 20 days of medication was 23.23% ± 35.67% in the FAS test group and 16.86% ± 34.78% in the control group. The difference was not statistically signi cant (P > 0.05); (3) The score of QD syndrome after 20 days of medication was 35.93% ± 31.09% in the FAS test group and 20.80% ± 30.12% in the control group. There was a signi cant difference between the two groups (P < 0.05) ( Table 4). 2. Change in NIHSS score: 90 days after the onset, the experimental group was 4.55 points in the FAS group and 3.14 points in the control group. There was a statistically signi cant difference between the two groups in FAS (P < 0.05).
3. The clinical recovery rate of NIHSS is good: the experimental group was 11.83% in the FAS group after 20 days of treatment, and the control group was 1.06%. There was a statistically signi cant difference between the two groups in FAS (P < 0.05).

Medication compliance
There was no signi cant difference in medication compliance between the two groups (P > 0.05).

Safety Evaluation
A total of 19 adverse events occurred in this study, 8 cases in the test group, and 11 cases in the control group. There was no signi cant difference between the two groups (P > 0.05); There were 3 cases of adverse reactions, 3 cases of the test group, and 0 in the control group. There was no signi cant difference between the two groups (P > 0.05). Among them, 3 cases were serious adverse events in the test group, and 0 in the control group. There was no signi cant difference between the two groups (P > 0.05).

Discussion
In recent years, the incidence of cerebrovascular diseases in China has been on the rise. According to statistics, the mortality rate in 2014 was higher than that of cardiovascular, tumor and other diseases.
Acute ischemic stroke (AIS) is a group of clinical syndromes, which is caused by various reasons, such as blood supply disorder of brain tissue, ischemic anoxic necrosis and neurological dysfunction, accounting for 60% − 80% of stroke. According to TCM, the basic pathogenesis is the disorder of Qi and blood, which is committed to the brain and the brain's gods are not used.
In this study, western medicine curative effect evaluation system and TCM syndrome change were simultaneously used to evaluate the e cacy of patients. It re ects the advantages of integrated traditional Chinese and western medicine treatment, and makes up for the de ciency of previous studies that only do western medicine curative effect judgment but lack the effectiveness evaluation of traditional Chinese medicine syndromes [20][21][22] . The research results of Huang Yan et al. [23] showed that wind syndrome, phlegm syndrome, BSS and QD syndrome were the main syndromes of ischemic stroke within 30 days after the onset of ischemic stroke. The combination of two syndromes was dominant in 4-10d and 11-30d, and the combination of BSS and QD syndrome appears most frequently. The second is the combination of phlegm syndrome and QD syndrome, among which QD syndrome is the root cause of ischemic stroke; The research results of Zhang Teng et al. [24] showed that among the patients with clear consciousness in the acute stage of ischemic stroke, those with QD had more severe neurological impairment in the acute stage and convalescence stage, and the long-term prognosis was worse. Therefore, it is bene cial to improve the long-term prognosis of patients to pay attention to the treatment of QD syndrome in acute stage. In this experiment, the syndrome of QD and blood stasis were evaluated, and QD and blood stasis were analyzed respectively. The nal results showed that there was statistical difference in the score reduction rate of QD and BSS and QD syndrome after treatment. At 90 days after onset, there was a statistically signi cant difference in NIHSS score between the two groups, it suggests that NML can signi cantly improve the acute QD syndrome and play an important role in the long-term prognosis.
In ammatory response is one of the important mechanisms in many complex factors of ischemic stroke, which mainly occurs in the central nervous system and its surrounding tissues. Its pathogenesis is closely related to immune cells and their secreted in ammatory mediators. The abnormal cellular energy metabolism, the activation of ion channels and the release of oxygen free radicals caused by ischemiareperfusion can all lead to systemic in ammatory response [25][26][27][28] . The immune in ammatory response induced by ischemia plays a major role, while ischemia itself plays a secondary role [29] . After cerebral ischemia, the production of in ammatory mediators, the destruction of blood-brain barrier, the activation and in ltration of in ammatory cells can all induce and aggravate the in ammatory reactions, which leads to a series of complex pathophysiological processes and brain injury [30][31][32] . NML can inhibit the release of tumor necrosis factor (TNF-α), interleukin (IL-6) and other in ammatory factors, reduce the level of adhesion molecule (ICAM-1), thus reducing leukocyte adhesion ,so it has an obvious antiin ammatory effect [33] .
The primary therapeutic principle for ischemic stroke is to restore blood ow and reperfusion to the ischemic brain tissue as soon as possible to regain blood oxygen supply. However, when cerebrovascular recanalization occurs, ischemia reperfusion injury will follow [34] . Ischemia-reperfusion injury is a complicated pathological process, which is closely related to many factors, It mainly includes energy metabolism disorder, oxidative stress, Ca 2+ overload, excessive synthesis of NO, cell apoptosis, etc [35] .
Some studies have shown that in the process of ischemic stroke, due to the damage of blood-brain barrier, ferritin and free iron ions exudate and accumulate in the endothelial cells and penumbra, causing iron dependent oxidative stress, producing a large amount of superoxide (ROS), which leads to neuronal apoptosis and injury [36][37][38] . In addition, 24 hours after the onset of acute ischemic stroke, MDA content in the serum was signi cantly increased and SOD activity was signi cantly reduced, indicating that oxidative stress injury is one of the important pathogenesis mechanisms in the acute stage, and may be related to the prognosis of patients [39] . NML can signi cantly reduce the area of cerebral infarction, reduce the score of cerebral histopathology, reduce the content of H 2 O 2 and MDA in brain tissue, and improve the ability of anti-superoxide anion free radical and anti-hydroxyl free radical. In addition, the 6 g/kg dose group can signi cantly reduce the neurological damage, decrease the brain water content, increase the GSH content in brain tissue, and increase the GSH-Px and SOD activity in rats with cerebral ischemia-reperfusion [16] . This suggests that its anti-oxidative stress injury may be the main mechanism to improve the neurological function and prognosis of patients.
At present, angiogenesis is one of the main strategies for the treatment of ischemic stroke at functional recovery stage, a series of studies suggest that endogenous VEGF, EPC, etc. are closely related to the recovery after injury of ischemic stroke [40,41] . VEGF and its receptor changes are important factors affecting endogenous angiogenesis after stroke, and are also one of the most important neurotrophic factors affecting neurogenesis after stroke [42] . Recent research reports [15] show that promoting angiogenesis may be a potential target for the treatment of ischemic stroke, and may promote the improvement of symptoms and prognosis of patients. The pharmacological study of NML showed that it could signi cantly promote angiogenesis by increasing the expression of VEGFR1, it suggested that the potential therapeutic mechanism of improving the function and prognosis of patients may be related to its role in promoting angiogenesis, and play a positive role in its e cacy.
According to the results of improved Rankin independence rate, the difference between the two groups was statistically signi cant 90 days after the onset of the disease, 70.00% in the trial group and 45.24% in the control group. It is suggested that NML has signi cant effect on the improvement of limb activity in the long term after acute ischemic stroke. This indicated the effectiveness of the treatment, which may be closely related to the above mechanisms.
Although this study proved that cerebral pulsation has better clinical treatment advantages, in terms of the incidence of acute cardiovascular and cerebrovascular events. Owing to our study strictly limited the combination of drugs in acute phase and prohibited the use of other drugs with similar effects, as a result, there are some di culties in enrolling patients into the group, resulting in a small sample size.
Therefore, there was no signi cant difference between the two groups in the study of incidence of acute cardiovascular and cerebrovascular events. In future studies, the study sample size can be increased and the follow-up period can be extended to further con rm its effectiveness on long-term cardiovascular and cerebrovascular events.

Conclusion
The

Consent for publication
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