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Introduction

Alzheimer's disease (AD) is the currently most common neurodegenerative disease. According to the official statistics in 2015, 110,561 individuals succumbed to the disease, rendering AD the 6th most common cause of mortality in the United States and the 5th cause of mortality for Americans aged ≥65 years. The number of patients with AD in the United States are expected to increase to 13.8 million by the middle of the century (1,2). Patients with AD have a number of neurofibrillary tangles and amyloid plaques in their brains. Plaques, astrocyte proliferation, neuronal dystrophy, neuronal loss and vascular variations are signs of the disease. AD results in the loss of mental, behavioral and cognitive functions and learning ability, which causes great distress to patients and nursing staff (3). Patients with AD are mainly treated with acetylcholinesterase inhibitors, which enhance cholinergic neurotransmission in the brain by reducing the degradation rate of acetylcholine in the synaptic cleft (4). Donepezil and rivastigmine are commonly used acetylcholinesterase inhibitors that can improve the treatment of patients with AD (5-8). There are a number of studies available on the separate application of these two drugs. For example, it has been reported that rivastigmine significantly improves cognitive function and daily living ability in patients with AD compared with those administered the placebo (9). In addition, previous research has indicated that compared with the placebo, donepezil significantly improves the overall function of patients with AD, and is safer and well-tolerated (10). At present, however, to the best of our knowledge, there is no evidence available on the effects of the combination of rivastigmine and donepezil in the treatment of AD.

The kallikrein-kinin system (KKS) is considered as an important pathophysiological mediator of cerebrovascular dysfunction, neuroinflammation and amyloid β-protein (Aβ) pathology in AD (11). Bradykinin released by KKS is a pro-inflammatory mediator with a series of physiological actions in the periphery (12). In a previous study, the chronic administration of bradykinin B1 and B2 receptor antagonists was shown to improve amyloidosis-related cerebral hypoperfusion and vascular reactivity, thereby relieving the symptoms of patients with AD (13). Another study demonstrated that bradykinin increased the intracellular calcium concentration [(Ca2+)i] in astrocytes, while donepezil reduced this increase. Donepezil inhibited bradykinin-induced inflammatory responses through the nicotinic acetylcholine receptor (nAChR) and PI3K/Akt pathways in astrocytes, so as to treat AD (14). Therefore, rivastigmine hydrogen tartrate and donepezil hydrochloride may suppress the symptoms of AD by inhibiting the bradykinin level.

In the present study, changes in the cognitive functions and mental behavior of patients with AD were observed through rivastigmine hydrogen tartrate and donepezil hydrochloride, so as to compare their efficacy on AD and to compare the effects between the two drugs. In addition, the effects of the drugs on serum bradykinin levels in the patients were examined.

Patients and methods

Inclusion and exclusion criteria

The present study was a non-randomized controlled trial. A total of 126 patients with AD admitted to Luoyang Central Hospital from January, 2018 to December, 2018 were enrolled. Patients treated with the single-agent donepezil were included in the monotherapy group (n=56), and patients receiving donepezil plus rivastigmine were in the combination group (n=70). Patients were divided into different groups according to the treatment they selected.

The inclusion criteria were as follows: Patients who met the diagnostic criteria for AD (15); patients<80 years of age, but >50 years of age; patients who had a primary school education or higher, and were able to complete the treatments and follow-up tests; patients who did not receive antipsychotic drugs or cholinesterase inhibitors 4 weeks prior to enrollment; patients with a Minimum Mental State Examination (MMSE) score (16) between 12-27 points. The exclusion criteria were as follows: Patients with allergy or contraindications to donepezil and rivastigmine; patients with mental illnesses other than AD; patients with poor treatment compliance; patients with heart, liver and kidney insufficiency; patients who had suffered severe head injuries. In the present study, patients and their families were informed and signed an informed consent form. The study was approved by the Medical Ethics Committee of Luoyang Central Hospital.

Grouping and treatment methods

According to the severity of the patients' symptoms, patients in the monotherapy group were administered 1-2 tablets (5 mg/1 tablet) of donepezil hydrochloride tablets (purchased from Chongqing Zein Pharmaceutical Co., Ltd.), once/day. On the basis of the monotherapy group, patients in the combination group were administered rivastigmine hydrogen tartrate capsules (purchased from Novartis) 1.5 mg/time and twice/day. The dosage could be changed to 6 mg/time and once/day according to the patients' tolerance. Patients in both groups were treated for 6 months.

Scoring standards

Before and after treatment, the MMSE (14) was used to score the patients' memory, attention, language competence and other cognitive functions in the monotherapy and combination groups. A total of 27-30 points indicated normal cognitive functions, whereas <27 points indicated cognitive impairment. The lower the score was, the more severe the cognitive impairment was.

Before and after treatment, the Blessed-Roth Dementia Scale (BRDS) (17) was used to evaluate the patients' ability of social/daily living, cognition of common sense and character changes in the monotherapy and combination groups. A score of ≤7 points indicated no dementia, whereas a score of >7 points indicated dementia. The higher the score was, the more severe the dementia was.

Before and after treatment, the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) (18) was used to score the patients' memory, language competence, ability to use, inferential capability, orientation and other abilities in the monotherapy and combination groups. The scoring system was between 0-70 points. The higher the score was, the more severe the injury was.

Before and after treatment, the AD quality of life scale (QOL-AD) (19) was used to evaluate the quality of life of patients in the 2 groups, including physical condition, energy, mood, memory and other 13 items, with a total score of 52. The higher the score, the greater the quality of life.

Detection of serum bradykinin level before and after treatment

Before and after treatment, 5 ml of fasting venous blood was extracted in the morning from patients in the 2 groups, placed in centrifuge tubes and centrifuged (1,500 x g, 4˚C, 10 min) for multiple times to obtain the supernatant, namely serum. The serum was stored in a refrigerator at -80˚C for later use. Before and after treatment, the serum bradykinin level was determined by enzyme-linked immunosorbent assay (ELISA). An ELISA kit was purchased from Abcam. Standard wells, sample wells to be tested and blank control wells were set up on an ELISA plate. The standard well was supplemented with 50 µl of standard substances with various concentrations, while the sample well to be tested was added with 40 µl of sample diluent and then 10 µl of the samples to be tested. The plate was sealed with a microplate sealer and incubated at 37˚C for 1 h. After the sealer was removed and the liquid in the wells was discarded, the plate was washed with washing liquid over 5 min 3 times and dried with absorbent paper towels. Subsequently, the standard well and the sample well to be tested were added with 50 µl of enzyme-labeled reagents. The plate was sealed with a microplate sealer and incubated at 37˚C for 1 h. After the sealer was removed and the liquid in the wells was discarded, the plate was washed with washing liquid over 5 min 3 times and dried with absorbent paper towels. Subsequently, each well was supplemented with 50 µl of substrates H2O2 and TMB in turn, and then developed at 37˚C for 10-15 min in the dark after the substrates were mixed evenly. Finally, each well was supplemented with 50 µl of stop solution to cease the reaction. The optical density values of each well were detected at 450 nm using a multifunctional microplate reader (CLARIOstar, BMG LABTECH).

Statistical analysis

SPSS 18.0 (IBM Corp.) was used for statistical analysis. In the present study, count data, such as place of residence and the number of adverse reactions were expressed in terms of number/percentage [n (%)]. The comparisons between technical data were performed using the Chi-squared test or Fisher's exact test. ADAS-Cog MMSE scores were expressed as the means ± standard deviation (SD). One-way ANOVA followed by Tukey's HSD test was used for comparison of measurement data, expressed by F values. P<0.05 was considered to indicate a statistically significant difference.

Results

Comparison of general information

No significant differences were observed between the combination and monotherapy groups as regards age, exercise habits, place of residence, nationality, educational level, body weight, marital status and food preference (P>0.05). Further details are presented in Table I.

Table I Comparison of general information [n(%)].

Table I

Comparison of general information [n(%)].

Groups	Monotherapy group (n=56)	Combination group (n=70)	χ2	P-value
Age			0.202	0.653
<65 years old	23 (41.07)	26 (37.14)
≥65 years old	33 (58.93)	44 (62.86)
Exercise			0.015	0.902
Yes	21 (37.50)	27 (38.57)
No	35 (62.50)	43 (61.43)
Place of residence			0.161	0.689
City	30 (53.57)	40 (57.14)
Countryside	26 (46.43)	30 (42.86)
Nationality			0.358	0.550
Han	50 (89.29)	60 (85.71)
National minorities	6 (10.71)	10 (15.29)
Educational level			0.129	0.720
<Senior high school	40 (71.43)	52 (74.29)
≥Senior high school	16 (28.57)	18 (25.71)
Body weight			0.082	0.775
<55 KG	21 (37.50)	28 (40.00)
≥55 KG	35 (62.50)	42 (60.00)
Marital status			1.994	0.369
Married	47 (83.93)	52 (74.29)
Unmarried	6 (10.71)	10 (14.29)
Widowed	3 (5.36)	8 (11.42)
Food preference			0.113	0.737
Bland	36 (64.29)	47 (67.14)
Spicy	20 (35.71)	23 (32.86)

Comparison of MMSE score before and after treatment

Patients in both groups cooperated to complete the treatment. Following treatment, the MMSE score in the monotherapy group increased from 16.12±1.74 to 22.23±1.99 (P<0.05), and the score in the combination group increased from 15.85±2.19 to 26.23±2.17 (P<0.05). Following treatment, the MMSE score in the combination group was significantly higher than that in the monotherapy group (P<0.05). Further details are presented in Table II.

Table II Comparison of MMSE score before and after treatment (means ± SD).

Table II

Comparison of MMSE score before and after treatment (means ± SD).

Groups	Monotherapy group (n=56)	Combination group (n=70)
Before treatment	16.12±1.74	15.85±2.19
After treatment	22.23±1.99a	26.23±2.17a,b
F value	16.894	27.783
P-value	<0.001	<0.001

[i] ^aP<0.05, compared with the same group before treatment;

[ii] ^bP<0.05, compared with the monotherapy group after treatment.

Comparison of the BRDS score before and after treatment

Following treatment, the BRDS score in the monotherapy group decreased from 14.78±1.67 to 9.23±1.28 (P<0.05), and the score in the combination group decreased from 14.33±1.81 to 7.18±0.95 (P<0.05). Following treatment, the BRDS score in the combination group was significantly lower than that in the monotherapy group (P<0.05). Further details are presented in Table III.

Table III Comparison of BRDS score before and after treatment (means ± SD).

Table III

Comparison of BRDS score before and after treatment (means ± SD).

Groups	Monotherapy group (n=56)	Combination group (n=70)
Before treatment	14.78±1.67	14.33±1.81
After treatment	9.23±1.28a	7.18±0.95a,b
F value	18.768	28.398
P-value	<0.001	<0.001

[i] ^aP<0.05, compared with the same group before treatment;

[ii] ^bP<0.05, compared with the monotherapy group after treatment.

Comparison of the ADAS-Cog score before and after treatment

Following treatment, the ADAS-Cog score in the monotherapy group decreased from 29.67±3.03 to 22.24±3.98 (P<0.05), and the score in the combination group decreased from 30.15±2.89 to 18.24±3.67 (P<0.05). Following treatment, the ADAS-Cog score in the combination group was significantly lower than that in the monotherapy group (P<0.05). Further details are presented in Table IV.

Table IV Comparison of ADAS-Cog score before and after treatment (means ± SD).

Table IV

Comparison of ADAS-Cog score before and after treatment (means ± SD).

Groups	Monotherapy group (n=56)	Combination group (n=70)
Before treatment	29.67±3.03	30.15±2.89
After treatment	22.24±3.98a	18.24±3.67a,b
F value	10.752	19.278
P-value	<0.001	<0.001

[i] ^aP<0.05, compared with the same group before treatment;

[ii] ^bP<0.05, compared with the monotherapy group after treatment.

Comparison of the QOL-AD score before and after treatment

Following treatment, the QOL-AD score in the monotherapy group decreased from 26.24±5.89 to 35.24±5.78 (P<0.05), and the score in the combination group decreased from 25.28±6.45 to 43.24±5.35 (P<0.05). Following treatment, the QOL-AD score in the combination group was significantly higher than that in the monotherapy group (P<0.05). Further details are presented in Table V.

Table V Comparison of QOL-AD score before and after treatment (means ± SD).

Table V

Comparison of QOL-AD score before and after treatment (means ± SD).

Groups	Monotherapy group (n=56)	Combination group (n=70)
Before treatment	26.24±5.89	25.28±6.45
After treatment	35.24±5.78a	43.24±5.35a,b
F value	8.852	16.759
P-value	<0.001	<0.001

[i] ^aP<0.05, compared with the same group before treatment;

[ii] ^bP<0.05, compared with the monotherapy group after treatment.

Comparison of adverse reactions

No allergic reactions occurred during the treatment period in the 2 groups, and adverse reactions were treated with symptomatic treatment. No significant differences were observed in the main adverse reactions in appetite loss, headache, vomiting, muscle spasm, insomnia, sleepiness and diarrhea between the 2 groups, and there were no significant differences in the total number of affected patients (with at least 1 adverse event) between the two groups (P>0.05). Further details are presented in Table VI.

Table VI Comparison of adverse reactions [cases (%)].

Table VI

Comparison of adverse reactions [cases (%)].

Groups	Monotherapy group, n=56 [n (%)]	Combination group, n=70 [n (%)]	χ2	P-value
Decreased food appetite	3 (5.36)	5 (7.14)	0.167	0.683
Headache	2 (3.57)	4 (5.71)	0.315	0.575
Vomiting	4 (7.14)	7 (10.00)	0.319	0.572
Muscle spasms	2 (3.57)	5 (7.14)	0.756	0.385
Insomnia	3 (5.36)	6 (8.57)	0.485	0.486
Drowsiness and fatigue	5 (8.93)	4 (5.13)	0.120	0.729
Diarrhea	2 (3.57)	6 (8.57)	1.308	0.253
Total number of affected patients	21 (37.50)	37 (52.86)	2.954	0.086

Comparison of bradykinin level

Following treatment, the bradykinin level in the monotherapy group was 16.48±2.99 µg/l, significantly lower than 22.13±2.18 µg/l before treatment (P<0.05). Following treatment, the level in the combination group was 11.37±2.51 µg/l, significantly lower than 22.55±2.49 µg/l before treatment (P<0.05). Following treatment, the bradykinin level in the combination group was significantly lower than that in the monotherapy group (P<0.05). Further details are presented in Table VII and Fig. 1.

Figure 1 Comparison of bradykinin level. The results of ELISA revealed that following treatment, the levels of bradykinin in the serum of the 2 groups were significantly reduced, and the serum level of bradykinin was significantly lower in the combination group than that in the monotherapy group (P<0.05). *P<0.05, compared with before treatment in the same group; aP<0.05, compared with the monotherapy group after treatment.

Table VII Comparison of bradykinin level (µg/l, means ± SD).

Table VII

Comparison of bradykinin level (µg/l, means ± SD).

Groups	Monotherapy group (n=56)	Combination group (n=70)
Before treatment	22.13±2.18	22.55±2.49
After treatment	16.48±2.99a	11.37±2.51a,b
F value	11.273	25.573
P-value	<0.001	<0.001

[i] ^aP<0.05, compared with the same group before treatment;

[ii] ^bP<0.05, compared with the monotherapy group after treatment.

Discussion

There are currently 47 million patients with AD worldwide. The morbidity rate of the disease increases with the aging of the population, which brings great mental and economic burdens to families. It is estimated that 74.7 million individuals will suffer from dementia by the year 2030, and the nursing expenses for these patients will increase to about 2 trillion US dollars (20,21). At present, there is no cure available for AD, although the symptoms can be relieved by drug interventions (4). Therefore, it is of great significance for patients with AD and their families to identify therapeutic regimens with which to attenuate the deterioration of AD symptoms.

Patients with AD exhibit significant choline deficiency (22). Therefore, cholinesterase inhibitors are mainly used to reduce the metabolic rate of acetylcholine and suppress its hydrolysis, thus increasing acetylcholine in the body and improving the choline deficiency, and relieving AD symptoms (23). As a selective and reversible cholinesterase inhibitor widely used in clinical practice, donepezil hydrochloride inhibits the hydrolysis of acetylcholine and increases its concentration in the human body, thereby improving information transfer in the brain (24,25). As a dual cholinesterase inhibitor, rivastigmine hydrogen tartrate inhibits cholinesterase activity and reduces the loss of presynaptic cholinergic function, thus reducing the degradation of acetylcholine in synaptic cleft and increasing cholinergic signal transduction, and then improving cognition and memory (26,27). There are number of previous studies available confirming the efficacy of rivastigmine hydrogen tartrate and donepezil hydrochloride in the treatment of AD, however, their combined use in the treatment of the disease has been rarely studied (28-30). The results of the present study demonstrated that the MMSE, BRDS, ADAS-Cog, and QOL-AD scores of the 2 groups were significantly improved following treatment, and the improvement of the combination group was more significant. No significant differences were found between the 2 groups in the occurrence of adverse reactions. These findings suggest that compared with donepezil hydrochloride alone, rivastigmine hydrogen tartrate combined with donepezil hydrochloride can relieve the symptoms and improve the quality of life of patients with AD more effectively, and the adverse reactions are not increased.

Recently, it has been indicated that bradykinin released by KKS plays an important role in the central nervous system (31). Brain inflammation aggravates the pathology of AD, and bradykinin B1 receptors play a regulatory role in brain inflammation and in amyloid deposition of AD in mice. Therefore, bradykinin B1 receptors may involve microglia/macrophages, thus affecting the progression of AD (32). Bradykinin B2 receptor antagonists provide significant protection for synaptic loss and cognitive impairment induced by Aβ1-40 in mice, by reducing the activation of microglia and pro-inflammatory protein levels, as well as inhibiting MAPK signaling pathway and the activation of transcription factors (33). According to the study by Ashby et al, the bradykinin level was significantly higher in patients with AD, and it may affect cerebral blood flow and vascular permeability (34). This indicates that the reduction of the bradykinin level can relieve the symptoms of patients with AD. In the present study, following treatment, the bradykinin level in the 2 groups significantly decreased and the AD symptoms were relieved, which was consistent with the results of the above-mentioned studies. The decrease in the combination group was stronger compared with that in the monotherapy group, suggesting that the two drugs may relieve AD symptoms by reducing bradykinin level.

In conclusion, compared to treatment with donepezil hydrochloride alone, rivastigmine hydrogen tartrate combined with donepezil hydrochloride can relieve the symptoms and improve the quality of life of patients with AD more effectively, which may be related to the reduction of the bradykinin level. However, in the present study, the efficacy was not compared between the 2 drugs and other drugs. Additionally, the mechanisms of action between rivastigmine hydrogen tartrate, donepezil hydrochloride and bradykinin were not explored. Therefore, the conclusions presented herein are limited. In the future, large-scale randomized controlled trials or prospective cohort studies need to be conducted, and individual differences and potential interference factors will be controlled, in order to further confirm the reliability of the conclusions of the present study.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

XZ conceived the study and wrote the manuscript. RY analyzed and interpreted the patient general data. HW performed ELISA. RZ was responsible for observation indicators analysis. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Luoyang Central Hospital. Patients who participated in this research, signed the informed consent and had complete clinical data. Signed written informed consents were obtained from the patients and/or guardians.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References