An investigation on the changes of serum CCK-8,substance P,and 5-HT in patients with post-stroke insomnia

Abstract

BACKGROUND:

At present, the pathogenesis of post-stroke insomnia (PSI) is still inconclusive.

OBJECTIVE:

To explore the changes and significance of serum cholecystokinin-8 (CCK-8), substance P (SP), and 5-hydroxytryptamine (5-HT) in patients with PSI.

METHODS:

Ninety-one patients with stroke were selected as the research subjects, and according to the score of the Athens Insomnia Scale (AIS), they were divided into the insomnia group and the non-insomnia group. The serum levels of CCK-8, SP, and 5-HT in the two groups were compared to explore their relationships with PSI.

RESULTS:

Among the 91 patients, 56 were in the insomnia group and 35 were in the non-insomnia group, and the incidence of insomnia was 61.5%. There was no significant difference in the serum levels of CCK-8, SP, and 5-HT between the two groups ( $P=$ 0.696, 0.980, and 0.809, respectively). One-way analysis of variance showed that there was no significant correlation between the serum levels of CCK-8, SP, 5-HT, and the AIS score ( $P=$ 0.7393, 0.9581, and 0.5952, respectively).

CONCLUSION:

The incidence of PSI was relatively high, but it could not be proved that CCK-8, SP, and 5-HT were involved in the pathogenesis of PSI. There might exist other neurotransmitters involved in the pathophysiological process of PSI, which should be further explored.

Keywords

Stroke insomnia cholecystokinin-8 serum substance P 5-hydroxytryptamine

1. Introduction

According to a survey, the incidence of post-stroke sleep disorders (PSSD) in China is approximately 77.23% [1], of which insomnia accounts for 56.7% [2]. There is a close interaction between sleep and stroke. In recent years, foreign studies have shown that PSSD is a sign of severe disease [3], which is not conducive to the recovery of neurological function and the re-learning of limb motor functions [4]. At present, the pathogenesis of post-stroke insomnia (PSI) is still inconclusive [5]. Some researchers believe that it may be due to permanent damage to brain tissue after a stroke caused by ischemia and hypoxia, resulting in the release and accumulation of excitatory neurotransmitters and other harmful substances, affecting the network system and leading to insomnia [6, 7]. Neuropeptides are found to be involved in sleep-wake regulation [8]. Cholecystokinin-8 (CCK-8) and Substance P (SP), collectively known as brain-gut peptides, are related to sleep-wake regulation [9]. The 5-hydroxytryptamine (5-HT) is involved in the regulation of sleep, and its abnormal expression can cause sleep disorders [10]. Currently, there is a lack of clinical research on the correlation between PSI and CCK-8, SP, and 5-HT [11].

Therefore, in the present study, the sleep status of 91 patients with stroke admitted to Nan’ao People’s Hospital of Dapeng New District, Shenzhen from April 2021 to March 2022, was analyzed. The relationship between the serum expression of CCK-8, SP, 5-HT, and PSI was explored to provide a reference for the pathogenesis and evaluation of insomnia disorder in patients with stroke.

2. Material and methods

2.1 General information

Ninety-one patients with stroke admitted to Nan’ao People’s Hospital of Dapeng New District, Shenzhen, from April 2021 to March 2022, were selected and divided into the insomnia group and non-insomnia group according to the Athens Insomnia Scale (AIS) score. The present study protocol was approved by the Medical Ethics Committee and all patients were informed and voluntarily participated in the study.

2.2 The inclusion criteria

The inclusion criteria were: (1) patients with stroke (hemorrhagic stroke or ischemic stroke) who met the stroke diagnostic criteria revised by the Fourth National Cerebrovascular Disease Academic Conference and were diagnosed by computed tomography (CT) or magnetic resonance image (MRI), (2) patients with the first onset of disease and with unilateral brain tissue damage confirmed by cranial CT or MRI, (3) patients with neurological deficit symptoms lasting more than 24 h, (4) patients who were conscious and successfully received the relevant scale assessment two weeks after onset, and (5) patients who signed the informed consent form for the present study. According to the inclusion criteria, a total of 91 patients with stroke were included in this study. There were 56 patients in the insomnia group, including 39 males and 17 females. There were 35 patients were in the non-insomnia group, including 24 males and 11 females. The incidence of insomnia was 61.5% (Table 1).

2.3 The exclusion criteria

The exclusion criteria were: (1) patients who had mental and neurological symptoms before the stroke and failed to cooperate with treatment; (2) patients combined with deafness, aphasia, severe cognitive impairment, and who failed to communicate normally; (3) patients with insomnia caused by severe coronary heart disease, respiratory disease, and pain; (4) patients with a history of sleep disorder, depression, and dementia; and (5) patients with recent medication of antidepressants and drugs for anxiety and hypnotic drugs.

2.4 Insomnia assessment criteria

The diagnosis of stroke related insomnia must meet both the diagnostic criteria for stroke and insomnia [12]. The Athens Insomnia Scale (AIS) [13] is a commonly used scale mainly used for self-assessment of sleep quality. It requires participants to evaluate their sleep status over the past month and record items that occur at least three times a week. There were 8 items in the scale, and each item was scored from none to severe according to 0, 1, 2 and 3, respectively, and the scores of each part were accumulated. The total score on this scale ranges from 0 to 24, with higher scores indicating worse sleep quality, a total score of less than 4 indicating no sleep disorder, a total score between 4 and 6 indicating suspected insomnia, and a total score above 6 indicating insomnia.

2.5 Observation indicators

2.5.1 Basic information

Gender, age, stroke type (ischemic or hemorrhagic), and site of the lesion were recorded for all patients after admission. Determine the sleep status of patients based on AIS scores and group them.

2.5.2 Collection and detection of blood samples

A volume of 5 mL of peripheral venous blood was drawn from all patients on a fasting condition the day after admission, and the plasma was extracted by centrifugation at 3,000 r/min. The levels of CCK-8, SP, and 5-HT were detected by enzyme-linked immunosorbent assay using a microplate reader (manufacturer: Shenzhen Mindray Biomedical Electronics Co., Ltd.; model: MR-96A). The above kits were purchased from Shanghai Bohu Biotechnology Co., Ltd., and all operations were carried out in strict accordance with the kit instructions.

2.6 Statistic methods

SPSS Statistics v19.0 software was adopted for statistical analysis. All measurement data satisfied the normal distribution and were expressed as means $\pm$ standard deviations (x $\pm$ s) and tested by the independent sample $t$ -test. The countable data were expressed as percentages (%) and tested by the Chi-squared test. The Pearson method was used for correlation analysis. Univariate analysis was used to explore the related influencing factors for PSI. A value of $P<$ 0.05 was considered statistically significant.

3. Results

3.1 Comparison of the general characteristics between the two groups

Among the 91 patients with stroke, 56 were in the insomnia group and 35 were in the non-insomnia group, and the incidence of insomnia was 61.5%. The results of the Chi-squared test and independent-sample $t$ -test showed that there was no statistically significant difference in terms of gender ratio, age, and the nature of stroke between the two groups. Thus, the data were comparable between the two groups (Table 1).

Table 1
Comparison of the general characteristics between the two groups of patients

	Gender		Age (year)	Nature of stroke
	Male	Female		Ischemic	Hemorrhagic
The insomnia group	39 (69.64%)	17 (30.36%)	67.45 $\pm$ 14.18	33 (58%)	23 (42%)
The non-insomnia group	24 (68.57%)	11 (31.43%)	69.54 $\pm$ 14.68	21 (60%)	14 (40%)
$\chi^{2}/t$	0.000		0.597	0.566
$p$	0.914		0.853	0.111

3.2 Comparison of the serum levels of serum cholecystokinin-8, substance P, and 5-hydroxytryptamine between the two groups

The differences in serum level of CCK-8, SP, and 5-HT were not statistically significant between the two groups ( $P=$ 0.696, 0.980, and 0.809, respectively) (Table 2).

Table 2
Comparison of the serum levels of CCK-8, SP, and 5-HT between the two groups of patients

Group	$N$	CCK-8	5-HT	SP
The insomnia group	56	42.50 $\pm$ 34.58	69.45 $\pm$ 53.97	44.57 $\pm$ 46.32
The non-insomnia group	35	39.90 $\pm$ 23.80	69.19 $\pm$ 37.69	42.45 $\pm$ 29.76
$P$		0.696	0.980	0.809

3.3 Correlation between serum levels of serum cholecystokinin-8, substance P, 5-hydroxytryptamine, and the Athens Insomnia Scale (AIS) score

The results of the univariate analysis revealed that there was no obvious correlation between the serum levels of CCK-8, SP, 5-HT, and the AIS score ( $P=$ 0.6941, 0.8070, and 0.9796, respectively) (Table 3).

Table 3
Correlation analysis of the serum levels of CCK-8, SP, 5-HT, and AIS

Group	Statistics	Or (95% CI)	$P$ value
CCK-8	41.50 $\pm$ 30.76	1.00 (0.99, 1.02)	0.6941
5-HT	69.35 $\pm$ 48.13	1.00 (0.99, 1.01)	0.9796
SP	43.76 $\pm$ 40.58	1.00 (0.99, 1.01)	0.8070

4. Discussion

The targeted treatment of patients with PSI is different from that of simple insomnia as they have a longer course of the disease and a complex pathological mechanism. A considerable issue for patients with PSI is refractory insomnia, with the mechanism remaining unclear. Therefore, exploring the pathological mechanism of PSI is a major scientific problem that needs to be solved urgently. Researchers should start from the pathogenesis and take effective intervention measures to deal with the serious problem of PSI. It has been suggested that neurotransmitter disorders may be an important trigger for PSI.

Among 91 patients with stroke included in this study, the incidence of insomnia was 61.5%, which is consistent with other literature reports. The high incidence of insomnia after stroke suggests that we should pay close attention to it in clinical work. The results showed that in the insomnia group, 39 males and 17 females accounted for 69.64% and 30.36% of the total number in the group, respectively. In the non-insomnia group, 24 were male and 11 were female, accounting for 68.57% and 31.43% of the total number in the group, respectively. Further analysis showed that there was no significant difference between different gender groups ( $P>$ 0.05). Therefore, the results of this study suggested that there was no correlation between stroke patients with insomnia and gender differences.

There is abundant expression of SP in the emotion regulation areas in the brain, such as the hypothalamus, amygdala, hippocampus, locus coeruleus, nucleus accumbens, and cerebral cortex, which coordinate with monoamine neurotransmitters and participate in the regulation of circadian biological rhythms [14, 15]. One of the typical brain-gut peptides is CCK-8, which is the peptide with the highest content in the brain so far. It is mainly distributed in the hypothalamus, cerebral cortex, hippocampus, amygdala, and peripheral blood in the central nervous system [16]. Many neuropeptides are involved in sleep-wake regulation [17], such as SP and CCK-8. These peptides are collectively referred to as brain-gut peptides, which play a bi-directional regulating effect between the brain and the gastrointestinal tract and are related to sleep-wake regulation [18]. The roles of SP and CCK-8 were validated in patients with primary insomnia and insomnia rat models and were confirmed to be correlated with insomnia [17, 19, 20, 21]. It is not known whether these neurotransmitters are involved in the pathogenesis of PSI. There are very few studies and literature reports on PSI, and no unified opinion has been formed.

In the present study, it was found that the differences in the serum levels of CCK-8 and SP were not statistically significant between the PSI group and the non-insomnia group. Univariate analysis revealed that there was no correlation between the serum levels of CCK-8, SP, and the AIS score in the two groups. The above results suggested that there existed differences in the pathogenesis of PSI and primary insomnia, which might be because CCK-8 and SP are closely correlated with gastrointestinal motility. The body would secrete large amounts of CCK-8 and SP to promote gastrointestinal motility when it is inhibited [22]. Both patients with PSI and those without insomnia might affect both CCK-8 and SP due to the presence of gastrointestinal dysfunction, resulting in no significant difference in the comparison of CCK-8 and SP levels between both groups of patients.

As for the lack of a correlation between the levels of CCK-8 and SP and the AIS score as well as no increase or decrease of the above two indicators with the AIS score, it might be correlated with the fact that there existed many neurotransmitters involved in post-stroke sleep regulation together with a very complex regulatory mechanism. Thus, the factors of drug and gastrointestinal function in patients with stroke should be considered during the case enrolment to minimize experimental errors and draw more objective conclusions.

The central neurotransmitter 5-HT has the highest content in the brain and is involved in the regulation of sleep, body temperature, pain, and other physiological functions, and an abnormal expression may result in sleep disorders [23, 24, 25]. In the present study, the difference in the serum level of 5-HT was not statistically significant between the PSI group and the non-insomnia group. The univariate analysis revealed that there was no correlation between the serum level of 5-HT and the AIS score in both groups of patients. Wang et al. [26] found that the serum level of 5-HT in patients with PSSD was negatively correlated with insomnia scores, suggesting that 5-HT expression was related to the occurrence and severity of PSSD. The results of the present study were inconsistent with the findings of Wang et al. [26]. This might be due to the fact that the 5-HT level could be susceptible to a variety of factors and may be associated with post-stroke stress and severity of insomnia. The severity of insomnia was not compared among the two groups, which should be considered in future studies.

5. Conclusion

The innovation of the present study was to analyze the possible neurotransmitter disorders in patients with PSI and to investigate the relevant roles of CCK-8, SP, and 5-HT-related factors in the pathogenesis to provide some reference for the prevention and treatment of PSI. In the present study, it was suggested that the incidence of insomnia in stroke was high, but the involvement of CCK-8, SP, and 5-HT in the occurrence and development of PSI was not confirmed. In a subsequent study, further group comparisons should be conducted based on the severity of insomnia to observe the relationship between CCK-8, SP, and 5-HT and the severity of insomnia to obtain more accurate findings.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the Shenzhen Sanming Project (SZSM201610039).

Ethics statement

This controlled trial was approved by the Ethics Committee of the Shenzhen Dapeng New District Nanao People’s Hospital (2021092101) and registered on the Chinese Clinical Trial Register website (www.chictr.org.cn, ChiCTR2100051820). Written consent was obtained from each patient.

Availability of data and materials

All data generated or analysed during this study is included in this article. Further enquiries can be directed to the corresponding author.

Footnotes

Acknowledgments

We are grateful to everyone who helped with the article.

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An investigation on the changes of serum CCK-8,substance P,and 5-HT in patients with post-stroke insomnia

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Material and methods

2.1 General information

2.2 The inclusion criteria

2.3 The exclusion criteria

2.4 Insomnia assessment criteria

2.5 Observation indicators

2.5.1 Basic information

2.5.2 Collection and detection of blood samples

2.6 Statistic methods

3. Results

3.1 Comparison of the general characteristics between the two groups

Table 1 Comparison of the general characteristics between the two groups of patients

Table 2 Comparison of the serum levels of CCK-8, SP, and 5-HT between the two groups of patients

Table 3 Correlation analysis of the serum levels of CCK-8, SP, 5-HT, and AIS

5. Conclusion

Competing interests

Funding

Ethics statement

Availability of data and materials

Footnotes

Acknowledgments

References

Table 1
Comparison of the general characteristics between the two groups of patients

Table 2
Comparison of the serum levels of CCK-8, SP, and 5-HT between the two groups of patients

Table 3
Correlation analysis of the serum levels of CCK-8, SP, 5-HT, and AIS