Donepezil Combined with DL-3-n-Butylphthalide Delays Cognitive Decline in Patients with Mild to Moderate Alzheimer’s Disease: A Multicenter,Prospective Cohort Study

Abstract

Background:

Vascular factors and mitochondria dysfunction contribute to the pathogenesis of Alzheimer’s disease (AD). DL-3-n-butylphthalide (NBP) has an effect in protecting mitochondria and improving microcirculation.

Objective:

The aim was to investigate the effect of donepezil combined NBP therapy in patients with mild-moderate AD.

Methods:

It was a prospective cohort study. 92 mild-moderate AD patients were classified into the donepezil alone group (n = 43) or the donepezil combined NBP group (n = 49) for 48 weeks. All patients were evaluated with Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-cog), Clinician’s Interview-Based Impression of Change plus caregiver input (CIBIC-plus), Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL), and Neuropsychiatric Inventory (NPI) every 12 weeks. All patients were monitored for adverse events (AEs). The efficacy was analyzed using multivariate logistic regression analysis.

Results:

The multivariate logistic regression analysis showed that the changes of ADAS-cog score (OR = 2.778, 95% CI: [1.087, 7. 100], p = 0.033) and ADCS-ADL score (OR = 2.733, 95% CI: [1.002, 7.459], p = 0.049) had significant difference between donepezil alone group and donepezil combined NBP group, while the changes of NPI (OR = 1.145, 95% CI: [0.463, 2.829], p = 0.769), MMSE (OR = 1.563, 95% CI: [0.615, 3.971], p = 0.348) and CIBIC-plus (OR = 2.593, 95% CI: [0.696, 9.685], p = 0.156) had no significant difference. The occurrence of AEs was similar in the two groups.

Conclusion:

Over the 48-week treatment period, donepezil combined NBP group had slower cognitive decline and better activities of daily living in patients with mild to moderate AD. These indicated that the multi-target therapeutic effect of NBP may be a new choice for AD treatment.

Keywords

Alzheimer’s disease clinical trial DL–3-n-butylphthalide drug treatment prospective cohort study

INTRODUCTION

Alzheimer’s disease (AD) is the most common cause of dementia. At present, there are no drugs that can cure AD, and current medicines just temporarily improve the symptoms of AD patients. Although the pathogenesis of AD had not been fully understood, recent studies have shown that there are extensive vascular lesions in the brain of AD patients, including asymptomatic infarction and demyelination of the white matter. The blood flow of the cerebral hemispheres (especially the temporal parietal lobe) decreases in the brain of AD patients; risk factors for vascular diseases, such as hypertension, diabetes, hyperlipidemia, and hyperhomocysteine, are closely related to the pathogenesis of AD; and vascular risk factor intervention is currently the main method to prevent AD. These findings suggest that vascular factors play an important role in the occurrence and development of AD [1 –4]. Unlike other tissues and organs in the body, brain tissue consumes a large amount of glucose and oxygen but has no glucose and oxygen reserves. Therefore, brain tissue is particularly sensitive to ischemia and hypoxia [5]. Mitochondria are the main source of energy metabolism in brain tissue, and reduced blood flow in brain tissue will first affect mitochondrial energy metabolism. Therefore, brain microcirculatory disorders and mitochondrial dysfunction may play important roles in the pathogenesis of AD and are hotspots in the current study of the pathogenesis of AD.

DL-3-n-butylphthalide (NBP), a new drug developed in China, is extracted from celery seeds and has a unique effect in protecting mitochondria and improving microcirculation. It is widely used in acute ischemic stroke patients and can significantly improve neurological damage. NBP was approved by the State Food and Drug Administration of China (SFDA) as a therapeutic drug for treatment of ischemic stroke in 2005. Several multicenter phase II and III randomized controlled clinical trials reported that NBP was effective in alleviating neurologic damage after stroke, with a good safety and tolerability. In in vitro and animal experiments, NBP alleviated the neurotoxicity of amyloid-β, improved the learning and memory function of rats through inhibiting the NF-κB pathway [6, 7], and downregulated autophagy to increase cell viability [8]. The potential mechanisms of NBP regarding its neuroprotective effects include anti-oxidant and anti-inflammatory effects and the stimulation of the proliferation, migration, and differentiation of hippocampal neural stem cells [9]. In clinic trial, NBP was shown to effectively improve cognitive and global function in patients with subcortical vascular cognitive impairment without dementia over a 6-month treatment period [10]. There were some studies investigating the effects of NBP on AD in Chinese scientific literature. The results indicated that NBP improved the cognitive function in mild-moderate AD patients [11 –13]. However, all of these studies were small sample size, and did not use the internationally established assessment tools to evaluate the effects of NBP. Furthermore, all of these studies had a short follow-up time. In the present study, we used Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-cog), Clinician’s Interview-Based Impression of Change plus caregiver input (CIBIC-plus), Alzheimer’s Dis-ease Cooperative Study-Activities of Daily Living (ADCS-ADL), and Neuropsychiatric Inventory (NPI) to assess the effects of NBP globally and followed up the patients for 48 weeks. Our results further documented the effects of combination of donepezil and butylphthalide in treating patients with AD.

METHODS

Study design

This was a prospective cohort study. The patients were enrolled from the Department of Neurology or Geriatrics of 5 hospitals in Xi’an, China. The experimental protocol was reviewed by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University (approval number: XJTU1AF2015LSL-066). All subjects signed an informed consent form. The study was registered in Clinical Trials (registration number: NCT02711683) and was funded by the clinical research project of the First Affiliated Hospital of Xi’an Jiaotong University.

Participants and eligibility criteria

All AD patients were from our memory clinic and in-patients and met the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer‘s Disease and Related Disorders Association (NINCDS-ADRDA) (1984) criteria for probable AD [14]. The diagnosis of AD was mainly based on detail clinical history from caregivers, neuropsychological testing, and brain magnetic resonance imaging (MRI). To make sure the diagnosis for each patient was accurate and reliable, we used a standard three stage diagnostic approach [15] and finally made a diagnosis of AD by a neurologist.

All patients met the following inclusion criteria: 1) age of 50–85 years old (including those who were 50 and 85 years old), either sex; 2) met the diagnostic criteria for suspected AD; 3) mild to moderate AD patients, that is, patients with 11 points ≤MMSE total score ≤26 points (or patients with an elementary school education level: 11 points ≤MMSE total score ≤22 points) [16, 17]; 4) total Hachinski ischemic scale (HIS) [18] score ≤4 points; 5) memory loss for at least 12 months, with a tendency of progressive deterioration; 6) brain MRI scan suggesting a significant possibility of AD (medial temporal lobe atrophy (MTA) visual rating scale [19] grade 2 or higher, Fazekas scale [20] ≤2); 7) no obvious physical signs during nervous system examination; 8) stable and reliable caregivers, with the ability to contact the caregivers frequently (at least 4 days a week, and at least 2 hours a day); the caregivers helped the patients participate throughout trial; 9) elementary school or higher education level and the ability to complete the cognitive ability measurement and other tests specified in the protocol; and 10) signed an informed consent form.

Exclusion criteria were 1) previous nervous system diseases (including stroke, optic neuromyelitis, Parkinson’s disease, epilepsy, etc.); 2) mental illness, according to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (DSM-IV-TR) criteria [21], including schizophrenia and other mental illness, bipolar disorder, and severe depression or paralysis; 3) unstable or severe heart, lung, liver, kidney, or hematopoietic diseases; 4) uncorrectable visual and auditory disorders that affected completing neuropsychological tests and scale assessments; and 5) simultaneous use of cholin-esterase inhibitors or memantine.

Interventions

Patients with mild to moderate AD were assigned to the donepezil alone group or donepezil combined NBP group. The donepezil alone group treated with donepezil 5 mg, once daily, while donepezil combined NBP group treated with donepezil 5 mg daily plus NBP 200 mg, three times a day for 48 weeks. Patient who will been dividing to donepezil alone group or donepezil combined NBP group, was determined according to the patient’s condition, family income, compliance, and other factors.

Trial end point and evaluation indexes

The primary end point index was the ADAS-Cog/12 score [22]. Secondary end point indexes were the CIBIC-Plus)scale [23], ADCS-ADL [24], and NPI scale [25]. The ADAS-Cog can assess 6 cognitive fields, including memory, language, orientation, logic, social cognition, and attention. The lower the score is, the lighter the cognitive damage. The CIBIC-Plus reflects the improvement of an individual’s clinical symptoms by interviewing the individual and his/her caregiver. The score ranges from 1 to 7 points, where 1 represents maximum improvement, 4 represents no change, and 7 represents maximum deterioration. The clinician’s impression of the severity of the disease based on the interview with the subject at baseline served as the reference for CIBIC-Plus scoring in follow-ups. Clinicians evaluating the CIBIC-Plus were not aware of the scores of other scales. The ADCS-ADL is a clinician assessment standardized questionnaire composed of 23 items that assesses the actual performance of specific actions and behaviors of the individual observed by the caregiver in the past 4 weeks. The score ranges from 0 to 78 points, and the lower the score is, the more severe the disorder. The NPI includes 10 items regarding behavior and 2 items regarding the autonomic nervous system. The total score ranges from 0 to 144, and the higher the score is, the more severe the damage.

To make ensure the reliable of all assessment, a blind method was used. Neuropsychological assessors did not know what treatment each patient received. Each center at least had two neuropsychological assessors, one responsible for the assessment of the MMSE and ADAS-cog, another responsible for the evaluation of ADL, NPI, and CIBIC-plus. The scale assessors and doctors received uniform training in scale testing and disease diagnosis. The reliability of the cognitive tests and diagnoses between assessors was greater than 0.90. All trainees passed a scale consistency test before participating in the trial.

Safety assessments included physical examinations, vital signs, and adverse event (AE) reports. Each patient attended 5 visits, including baseline, 12 weeks, 24 weeks, 36 weeks, and 48 weeks. At each follow-up, the above scales were evaluated, and AEs were recorded.

Statistical analysis

The calculation of sample size is based on the primary outcome which is the change from baseline in the ADAS-cog score. The clinical use of NBP in AD patients is still in the exploratory stage. Previous studies have found that the effective rate of donepezil in the treatment of AD is 39% [26]. In our previous studies, we found that the effective rate of donepezil combined NBP is 75% in the treatment of mild to moderate AD. Sixty-six participants were needed to provide 80% power at a significant level of 5% using analysis of variance. The total sample size required for the study is 74 (37 each group at least) assuming a 10% loss to follow-up.

The primary and secondary outcome measures were analyzed using data from the intention-to-treat (ITT) population and the per protocol (PP) population. In this study, the ITT population consisted of all patients who had a complete baseline assessment as well as at least one post treatment assessment for the primary outcome variables. Missing values for psychometric score were replaced by use of the last observation carried forward (LOCF) method. The PP population included patients who completed the 48-week treatment and evaluation as planned with no major protocol violations.

The changes of psychometric scores (ADAS-cog, NPI, ADCS-ADL, and CIBIC-plus) from baseline were used as an indicator of changes in cognitive function, mental behavior, daily living ability, and overall efficacy. The ADAS-cog, NPI score change from baseline at week 48 < 0 means improvement, ≥0 means worsening. The MMSE, ADCS-ADL score change from baseline at week 48 > 0 means improvement, ≤0 means worsening. The CIBIC-plus score at 48 week ≤4 means improvement, > 4 means worsening.

All data were used to build a database in EpiData and were analyzed using SPSS 18.0 statistical software. Variables related to the study participants that conformed to an approximate normal distribution are expressed as the mean±standard deviation (x±s), variables that had a severely skewed distribution are expressed in quartiles, and categorical variables are expressed as value (%). The χ² test, t test, or rank sum test was used for comparisons between groups according to the different types of data. Multivariate logistic regression was used for the multivariate analysis. In the logistic regression model, scale score improvement (yes or no) as a dependent variable, age, gender, degree of education, uncontrolled hypertension, and diabetes mellitus as independent variables. Hypertension and diabetes mellitus were defined as a self-reported medical diagnosis, anti-hypertensive/anti-diabetic medication use, or were newly diagnosed according to guidelines [27, 28]. Blood pressure (BP) > 140/85 mmHg was defined as uncontrolled hypertension. Diabetes mellitus was simply as diagnoses present or absent. p < 0.05 (double sided) represented statistical significance for all tests.

RESULTS

General information of patients

One hundred twenty-six outpatients with dementia were selected from the Department of Neurology of 5 hospitals in Xi’an, China, from January 2016 to June 2018, among whom 92 patients with mild to moderate AD met the inclusion criteria. Figure 1 shows the screening and grouping strategy in more detail.

Fig. 1

Trial profile.

At baseline, age in the donepezil combined NBP group was younger than that in the donepezil alone group (p = 0.005). The remaining demographic information and clinical characteristics had no significantly difference between the two groups (Table 1).

Table 1

Baseline characteristics of the participants

	Total	Donepezil	Donepezil combined	p
	(n = 92)	(n = 43)	NBP (n = 49)
Age, mean±SD, y	69.36±8.37	71.93±8.36	67.10±7.79	0.005
Male, n (%)	46 (50.0)	23 (53.5)	23 (46.9)	0.531
Education, n (%)
≤6 y	12 (13.0)	5 (11.6)	7 (14.3)	0.706
> 6 y	80 (87.0)	38 (88.4)	42 (85.7)
Duration, y	3 [2, 4]	3 [2, 5]	3 [2, 3]	0.494
Medical history, n (%)
Hypertension	28 (30.4)	13 (30.2)	15 (30.6)	0.901
BP> 140/85	8 (8.7)	5 (11.6)	3 (6.1)	0.350
Diabetes mellitus	13 (14.1)	7 (16.3)	6 (12.2)	0.579
MRI Fazekas score, n (%)
0	75 (81.5)	31 (72.1)	44 (89.8)
1	15 (16.3)	10 (23.3)	5 (10.2)	0.062
2	2 (2.2)	2 (4.7)	0 (0.0)
MRI MTA score
1	5 (5.4)	3 (7.0)	2 (4.1)	0.477
2	81 (88.0)	36 (83.7)	45 (91.8)
3	6 (6.5)	4 (9.3)	2 (4.1)
Psychometric scores, mean±SD
MMSE	18.78±4.06	18.84±4.09	18.74±4.07	0.905
ADAS-cog	21.63±9.03	21.84±9.73	21.44±8.48	0.832
ADCS-ADL	63.97±9.84	61.98±12.37	65.59±6.32	0.076
NPI	10.01±11.48	9.83±12.21	10.17±10.93	0.892
HAMD	2.23±2.65	2.16±2.48	2.29±2.82	0.826

NBP, dl-3-n-butylphthalide; SD, standard deviation; MRI, magnetic resonance imaging; MTA, medial temporal lobe atrophy; MMSE, Mini-Mental State Examination; ADAS-cog, Alzheimer’s Disease Assessment Scale-Cognitive subscale; ADCS-ADL, Alzheimer’s Disease Cooperative Study Activities of Daily Living scale; NPI, Neuropsychiatric Inventory; HAMD, Hamilton depression scale.

Overview of follow-up

At baseline, there were 43 and 49 patients in the donepezil alone groups and donepezil combined NBP group respectively. During the 48-week follow-up period, both groups had patients who failed to complete the experiment, 43 and 49 patients completed the 12-week follow-up, 42 and 47 patients completed the 24-week follow-up, 40 and 45 patients completed the 36-week follow-up, and 37 and 41 patients completed the 48-week follow-up, respectively. The drop-out rates were 13.95% and 16.33% at 48 weeks, respectively.

Changes of the neuropsychological scores

The change of ADAS-cog score from baseline at week 48 was significant in the Donepezil alone groups than that in Donepezil combined NBP group (1.82±5.20 versus –0.38±4.46, p = 0.048) in PP population and (1.56±4.86 versus –0.40±4.08, p = 0.039) in ITT population. However, the change of ADAS-cog score from baseline had no significant difference between the two groups at other follow-up time points. The change of MMSE score from baseline at week 12 was significantly different between the two groups (–0.48±1.97 versus –0.40±1.93, p = 0.037) in ITT population, while there was no difference between the two groups at other follow-up time points. The changes of ADCS-ADL, NPI, and CIBIC-plus scores from baseline at each follow-up time point also had no difference between the two groups (shown in Table 2). Figure 2 shows this in more detail.

Table 2

Univariate analysis of psychometric score changes from baseline in PP and ITT population at week 48

	PP			ITT
	Donepezil	Donepezil combined	p	Donepezil	Donepezil combined	p
	(n = 37)	NBP (n = 41)		(n = 43)	NBP (n = 49)
ADAS-cog	1.82±5.20	–0.38±4.46	0.048	1.56±4.86	–0.40±4.08	0.039
MMSE	–1.16±3.07	–0.02±2.34	0.068	–0.95±2.89	0.00±2.17	0.075
ADCS-ADL	–6.87±9.55	–4.73±7.77	0.286	–6.12±9.07	–3.80±7.43	0.181
NPI	1.54±11.05	–2.68±10.33	0.085	1.19±10.28	–2.49±9.48	0.078
CIBIC-plus	4.19±0.57	4.00±0.55	0.139	4.16±0.53	3.98±0.52	0.099
AE (n, %)	2 (5.4)	2 (4.9)	0.916	4 (9.3)	4 (8.2)	0.847

PP, per protocol; ITT, intention-to-treat; NBP, dl-3-n-butylphthalide; ADAS-cog, Alzheimer’s Disease Assessment Scale-Cognitive subscale; MMSE, Mini-Mental State Examination; ADCS-ADL, Alzheimer’s Disease Cooperative Study Activities of Daily Living scale; NPI, Neuropsychiatric Inventory; CIBIC-plus, Clinician’s Interview-Based Impression of Change plus caregiver input; AE, adverse event.

Fig. 2

Comparison of psychometric score changes from baseline in ITT population at different follow-up periods between the two groups. Missing values for psychometric score were replaced by use of the last observation carried forward (LOCF) method. A) Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-cog) change from baseline; B) Mini-Mental State Examination (MMSE) change from baseline; C) Alzheimer’s Disease Cooperative Study Activities of Daily Living scale (ADCS-ADL) change from baseline; D) Neuropsychiatric Inventory (NPI) change from baseline; E) Clinician’s Interview-Based Impression of Change plus caregiver input (CIBIC-plus). NBP, dl-3-n-butylphthalide.

Multivariate logistic regression analysis

In the multivariable logistic regression analysis, scale score improvement (yes or no) as a dependent variable, age, gender, and degree of education as independent variables in model 1. In model 2, scale score improvement (yes or no) as a dependent variable, age, gender, degree of education, uncontrolled hypertension, and diabetes mellitus as independent variables.

The multivariate logistic regression analysis showed that there was a significant difference for change from baseline at week 48 for the ADAS-cog (OR = 2.778, 95% CI: [1.087, 7.100], p = 0.033), ADCS-ADL (OR = 2.733, 95% CI: [1.002, 7.459], p = 0.049) and no significant difference for change from baseline at week 48 for the MMSE (OR = 1.563, 95% CI: [0.615, 3.971], p = 0.348), NPI (OR = 1.145, 95% CI: [0.463, 2.829], p = 0.769) and CIBIC-plus (OR = 2.593, 95% CI: [0.696, 9.685], p = 0.156) between the two groups in ITT population (Table 3).

Table 3

Multivariate logistic regression analysis of psychometric score changes from baseline in ITT population at week 48

Variables	B	S.E.	Wald	OR	95% CI	p
Model 1
ADAS-cog	0.947	0.458	4.271	2.578	1.050–6.328	0.039
MMSE	0.405	0.468	0.749	1.500	0.599–3.754	0.387
ADCS-ADL	0.953	0.497	3.684	2.594	0.980–6.865	0.055
NPI	0.130	0.459	0.081	1.139	0.463–2.802	0.776
CIBIC-plus	1.002	0.657	2.323	2.723	0.751–9.878	0.127
Model 2
ADAS-cog	1.022	0.479	4.552	2.778	1.087–7.100	0.033
MMSE	0.446	0.476	0.880	1.563	0.615–3.971	0.348
ADCS-ADL	1.006	0.512	3.855	2.733	1.002–7.459	0.049
NPI	0.135	0.461	0.086	1.145	0.463–2.829	0.769
CIBIC-plus	0.953	0.671	2.016	2.593	0.696–9.685	0.156

ADAS-cog, Alzheimer’s Disease Assessment Scale-Cognitive subscale; MMSE, Mini-Mental State Examination; ADCS-ADL, Alzheimer’s Disease Cooperative Study Activities of Daily Living scale; NPI, Neuropsychiatric Inventory; CIBIC-plus, Clinician’s Interview-Based Impression of Change plus caregiver input. Model 1 modified confounding factors (age, gender, degree of education). Model 2 modified confounding factors (age, gender, degree of education, uncontrolled hypertension, diabetes mellitus).

Safety

The occurrence of AEs was similar in the two groups (Table 2). The main adverse reactions are gastrointestinal reactions, such as nausea, vomiting, and anorexia. In general, patients with good tolerance did not affect the continued use of drugs.

DISCUSSION

Currently, AD is still mainly treated with cho-linesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists, which not only improve cognition and global function, but also delay the progression of the disease [29 –33]. All treatments targeting the pathological changes of AD are still in the testing phase [34 –37]. Because the pathogenesis of AD involves a variety of mechanisms, multi-target treatment may be a new direction for AD treatment.

Previous animal experiments have shown that NBP treatment can reduce oxidative stress and soluble Aβ and Aβ oligomers in the brain of APP/PS1 rats, improve synapse plasticity, and reduce learning and memory defects [38]. Some studies also found that in 3xTg-AD mice, NBP could promote the metabolism of APP to non-amyloid formation and reduce the production of Aβ [39], protecting the synapse function of aged APP Tg mice by inhibiting the deposition of Aβ senile plaques and neuroinflammatory reactions [40]. Therefore, NBP shows promising preclinical potential as a multi-target drug for AD.

Although some studies have investigated the effects NBP on AD, however, due to defective design, small sample size, and shorter follow-up period, they did not reach the consistent conclusion. In the present study, we found that the ADAS-cog score decreased significantly at week 48 in donepezil combined NBP group than that in the donepezil alone group. Also ADAS-ADL score in donepezil combined NBP group was significantly superior to donepezil alone group at week 48. This indicated that donepezil combined NBP effectively delayed cognitive decline and improved activities of daily living in patients with mild to moderate AD. Although the effects of NBP was slight, and were not confirmed on MMSE, CIBIC-plus,and NPI score, it should be noted that the effects were compare to donepezil treatment, but not placebo. From an ethical perspective, we designed the study to compare donepezil treatment and NBP combination with donepezil treatment, since many studies have demonstrated that donepezil could delay cognitive decline compared to placebo.

The major adverse reaction in the two groups was side effects in the gastrointestinal tract, which were related to the drug itself. However, the incidence of adverse reactions was low in the two groups, and there was no significant difference between the two groups. These suggested that the donepezil combined NBP therapy was safely tolerated.

In summary, over the 48-week treatment period, donepezil combined NBP effectively delayed cognitive decline and improved activities of daily living in patients with mild to moderate AD. This indicated that the multi-target therapeutic effect of NBP may be a new choice for AD treatment.

Limitations

First, this was a prospective cohort study rather than a randomized controlled trial. It may have selection bias in patient grouping. Because of the strong willingness of treatment, young patients were more inclined to choose combination therapy. But we adjusted age in the multivariate logistic analysis when the influence of age was taken into account. In addition, patients with good economic conditions were more inclined to receive combination therapy. At the same time, families with good economic conditions may pay more attention to the nutritional status of patients and give better care, which may affect the progression of the disease. It was a pity that the participants were unwilling to cooperate with the investigation of economic conditions due to privacy protection, so this factor could not be corrected in the multivariate logistic analysis, which may lead to selection bias. Second, the sample size was small. Third, it was difficult for patients to agree to have CSF drawn before and after NBP treatment, even though some patients agreed to draw CSF for the diagnosis of AD at inclusion stage. In order to clarify the effects of NBP on AD pathophysiology, we launched another clinical trial to assess the effects of NBP on cerebral glucose metabolism using FGD-PET, which is in progress.

Footnotes

ACKNOWLEDGMENTS

The study was supported by the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (No. XJTU1AF-CRF-2015-027; XJTU1AF-CRF-2018-004) and by the First Affiliated Hospital of Xi’an Jiaotong University Foundation (Grant Numbers: 2019QN-22) and by the Nature Science Foundation of China (No. 81771168) and by the Key Research & Development Programs of Shaanxi Province (No. 2017SF-260; 2018ZDXM-SF-052).

Authors’ disclosures available online ().

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