Associations Between Plasma Orexin-A Level and Constipation in Cognitive Impairment

Abstract

Background:

Constipation is a common symptom in dementia, and the cause is controversial. Rare clinical studies focused on plasma orexin-A levels and constipation in dementia.

Objective:

To evaluate the associations between orexin-A and constipation in patients with cognitive impairment.

Methods:

A total of 21 patients with mild cognitive impairment (MCI), 142 with Alzheimer’s disease (AD), and 57 with Lewy body dementia (LBD) were conducted. Besides informant-based history, neurological examinations or neuropsychological assessments, plasma levels of orexin-A, and constipation were assessed. The associations between orexin-A and constipation were evaluated by logistic regression models.

Results:

There were 47/220 (21.36%) cognitive impairment patients having constipation, and the proportion of constipation in LBD (61.40%) was significantly higher than AD (5.63%) and MCI (19.05%). No significant age or sex differences in the prevalence of constipation were found in the MCI, AD, and LBD groups. We found the cognitive impairment patients with constipation had lower levels of plasma orexin-A [1.00 (0.86, 1.28) versus 1.29 (1.01, 1.50) ng/ml, p < 0.001] than those without. And the plasma levels of orexin-A were significantly associated with the occurrence of constipation after adjusting for all variables in all patients with cognitive impairment (OR = 0.151, 95% CI: 0.042–0.537, p = 0.003). And the same finding was more prominent in the LBD group (p = 0.048).

Conclusions:

The decrease of plasma level of orexin-A is closely associated with the occurrence of constipation. Orexin-A has an intestinal protective effect and is involved in the gastrointestinal symptoms of patients with cognitive impairment.

Keywords

Alzheimer’s disease brain-gut axis constipation intestinal epithelial barrier Lewy body dementia orexin-A

INTRODUCTION

The general prevalence of constipation worldwide ranges from 0.7 to 79% [1 –4], with a latest prevalence rate of 10.1% (8.7–11.6) when Rome IV criteria were used [2]. Gender, age, social psychology, diet structure, drugs, alteration of intestinal flora, mucosal immune function changes (low-grade intestinal inflammation, increased intestinal permeability, immune activation), visceral hypersensitivity, and abnormal gastrointestinal motility have an impact on constipation [2, 3]. The alternation of intestinal flora interferes with the metabolism of toxic substances, resulting in impaired intestinal motility and chronic inflammation, which can cause or aggravate constipation [5, 6].

Constipation has been observed in variety of neurodegenerative diseases, such as Lewy body dementia (LBD), Parkinson’s disease (PD), and Alzheimer’s disease (AD) [2, 4]. The pathogeneses of constipation and dementia are unclear, while the brain-gut axis dysfunction and changes in intestinal flora both influenced the two diseases. The gut and CNS play key role in regulating the CNS function via gut microbiome, given that its relationships with regulating neuroendocrine, autonomic nervous system, host immune response, neurobehavioral effects, and detoxification mechanism [7, 8]. In addition to amyloid-β (Aβ) and tau, the postmortem of AD brain also showed bacteria, viruses, fungi, and protist parasites [9]. The mouse models of AD revealed that amyloid produced by the bacterial strain contributes to AD pathology by promoting misfolding of Aβ oligomers and fibrils [9 –11]. Alteration in gut microbial composition enhances the release of amyloids, which may contribute to the pathogenesis of AD [12], especially during aging when both the intestinal epithelial barrier (IEB) and blood-brain barrier (BBB) become more permeable [13]. There were many questions remained regarding the mechanisms by which gut dysfunction leads to the development of α-synucleinopathies. Pathological α-synuclein can be found in CNS and enteric nervous system (ENS), which supports the Braak’s initial theory [14] that “α-synuclein aggregation occurs in the gut and spreads in a prion-like way to the brain”. The increased intestinal permeability and translocation of bacteria and their products are also associated with α-synuclein pathology in LBD and PD [15, 16]. Sampson et al. [17] found short-chain fatty acids and extracellular fibers produced from microbes in the gastrointestinal tract could affect α-synuclein aggregation, indicating the systemic inflammatory processes driven by gastrointestinal dysfunction contribute to α-synuclein.

The neuropeptides orexins A and B, also called hypocretins, derive from prepro-orexin and selectively bind to two types of receptors, OX1 R and OX2 R. Orexin has been observed in neurons and enteroendocrine cells of the gut [18]; thus, it is one of the peptides localized in both the CNS and the ENS [19]. It has been proposed that orexin-A is an intrinsic factor influencing the composition of gut microflora, as well as a chemical mediator in the bidirectional gut-brain axis. The neuroprotective role of orexin-A has been proved in several disease [20 –23]. Tunisi et al. [18] showed the “peripheral” orexin-A released by myenteric neurons acting on OX1 R in enterocytes, could inhibit lipopolysaccharide (LPS)-induced IEB leakage as well as microglia activation when the composition of the microbiome is altered. And the “central” orexin-A also improves LPS-induced intestinal hyperpermeability via the vagal cholinergic pathway, again demonstrating that orexin mediates in bidirectional gut-brain communication [24]. Previous studies have only explored the relationship between intestinal flora and orexin-A or constipation, but there are few studies on orexin-A and constipation.

Due to the overlap in neuropathology and clinical symptoms between LBD and AD [25, 26], as well as the commonality of constipation in AD, LBD, and mild cognitive impairment (MCI) [4], we aimed to explore the association between orexin-A and constipation in patients with AD, LBD, and MCI in current study. These findings may 1) provide new biomarkers for the differential diagnosis of AD, LBD, and MCI to some extent, 2) provide new research targets and clinical evidence for the mechanism of brain-gut axis in dementia, and 3) be beneficial for constipation management in dementia.

MATERIALS AND METHODS

Study participants

The present study included 220 patients with cognitive impairment (21 patients with MCI, 142 patients with AD, and 57 patients with LBD) at the cognitive impairment clinics, Beijing Tiantan Hospital, from January to October 2022.

Diagnoses were confirmed by two experienced doctors and if there was disagreement, the subject was excluded. MCI was diagnosed if the participants had 1) subjective cognitive impairment; 2) objective impairment in one or more cognitive domains; 3) slightly impaired complex instrumental daily abilities, but independent daily living abilities; and 4) no dementia according to the clinical criteria recommended by the Winblad & Peterson [27, 28]. Probable AD was diagnosed based on the criteria of the National Institute on Aging and the Alzheimer Association workgroup, and ¹¹C-PIB PET scans (only for assessing Aβ deposition, n = 112) or cerebrospinal fluid tests (n = 52) for AD neuropathological biomarkers were performed [29].

LBD comprised with dementia with Lewy bodies (DLB) and Parkinson’s disease dementia (PDD), probable DLB was diagnosed according to the criteria of McKeith in 2017 [30], and probable PDD was diagnosed according to the clinical criteria of the Movement Disorder Society in 2007 [31]. A probable DLB diagnosis can be made with two or more core symptoms with or without indicative biomarkers, or only one core symptom with one or more indicative biomarker. International consensus suggests that DLB should be diagnosed when cognitive impairment precedes parkinsonism or begins within a year of parkinsonism and PDD should be diagnosed when parkinsonism precedes cognitive impairment by more than one year.

Exclusion criteria were as follows: diagnosis of any other neurological disease except MCI, AD, or LBD; currently using orexin receptor antagonists; patients with hearing loss, aphasia, or an inability to complete a clinical examination or scale assessment; history of mental disorders and illicit drug abuse; patients with acute or chronic liver and kidney dysfunction, malignant tumors, or other serious underlyingdiseases.

The study was designed and conducted in accordance with the Declaration of Helsinki, received ethical approval from the Beijing Tiantan Research Ethics Committee (KYSQ 2021-068-01), and all participants had completed written informed consent.

Assessments of constipation

Constipation was diagnosed according to the revised Rome IV criteria in 2016 [32], and the standard references were all followed constipation assessment indicators. The typical clinical symptoms including straining, having lumpy or hard stools, having sensation of incomplete evacuation, having sensation of anorectal obstruction/blockage, needing manual maneuvers to facilitate defecation more than one quarter of times or having fewer than three spontaneous bowel movements per week. The criteria were fulfilled for the last 3 months, with two or more defecation difficulties onset at least 6 months prior to diagnosis, as our previous report[3].

Clinical assessments

All participants underwent a standardized diagnostic workup that included a semi-structured medical history interview, collection of an informant-based history [including demographic characteristics, lifestyles, comorbidities, family history of cognitive impairment, course of disease, and body mass index (BMI, kg/m²)], clinical and neurological examinations, neuropsychological assessments, as previously described [33]. Neuropsychological assessments included the Mini-Mental State Examination [34], the Montreal Cognitive Assessment [35], and Clinical Dementia Rating [36] used to evaluate global cognitive function and severity of cognitive impairment. The Activities of Daily Living scale [37] was used to measure the functional status.

Sample collection and measurement

After a 12–14 h overnight fast and between 07:30 and 08:30 am before breakfast, peripheral blood from each participant was collected by venipuncture into 6-mL EDTA-containing test tubes. Immediately (within 2 h) after collection, blood was centrifuged (2,200 rpm, 10 min) and stored at –80°C until measurement. Smoking, alcohol consumption, and vigorous activity were prohibited 24 h before the study.

Plasma orexin-A levels were measured using an enzyme-linked immunosorbent assay kit (EK-003-30; Phoenix Pharmaceuticals, Burlingame, CA, USA). The detection range of this kit is 0–100 ng/mL, with a sensitivity of 0.2 ng/mL, and the intra- and inter-assay variability were <10% and <15%, respectively. Samples were analyzed twice on the same plate and the concentration values obtained from all samples were within the detection linearity range (0.12–2.0 ng/mL) of the kit.

Genomic DNA was extracted from peripheral blood stored at –80°C and the Apolipoprotein E (APOE) gene was amplified using polymerase chain reaction [38].

All measurements were determined without knowledge of the patient status.

Statistical analyses

Descriptive analyses were conducted using frequency for qualitative variables and mean [±standard deviation (SD)] or median [interquartile range (IQR)] for quantitative variables. We classified all participants into two groups according to the presence or absence of constipation (“with constipation” or “without constipation”) or three groups according to their diagnosis (“MCI”, “AD” or “LBD”), and then subgrouped them based on sex (“male” and “female”) or age (“≤70 years old” or “>70 years old”). To assess differences in different groups, Student’s t-test was performed to compare two independent groups for normally distributed data and Mann-Whitney U test was used for nonparametric data; the qualitative variables were assessed using a chi-squared test. Binary logistic regression models [odds ratio (OR) with 95% confidence intervals (95% CIs)] were used to analyze the associations between plasma orexin-A levels and constipation.

For statistical analyses, the IBM Statistical Package for the Social Sciences (SPSS) for Windows (version 25.0; IBM Corporation, Armonk, NY, USA) was used. A p-value < 0.05 was considered statistically significant.

RESULTS

Demographic and clinical characteristics of all participants

We conducted 220 patients with cognitive impairment (21 patients with MCI, 142 patients with AD, and 57 patients with LBD) in this study, and the clinical characteristics in the different diagnostic groups are shown in Supplementary Table 1.

In total, there were 47 (21.36%) patients with constipation. We found that patients with constipation had more hypertension (59.57% versus 33.53%, p = 0.001) than those without, while there was no significant difference in other demographic information or scores of neuropsychological assessments (Table 1).

Table 1

Sample characteristics

	All participants	Without constipation	With constipation	t/Z/χ²	p
	(n = 220)	(n = 173)	(n = 47)
Age at interviewed, y	70.37±8.18	69.89±8.34	72.13±7.40	–1.731	0.085
Sex (n, %)				0.062	0.804
Men	83 (37.73%)	66 (38.15%)	17 (36.17%)
Women	137 (62.27%)	107 (61.75%)	30 (63.83%)
Education, y (n, %)				0.710	0.701
0	26 (11.82%)	21 (12.14%)	5 (10.64%)
1–6	73 (33.18%)	55 (31.79%)	18 (38.30%)
≥7	121 (55.00%)	97 (56.07%)	24 (51.06%)
Marital status (n, %)				0.434	0.510
Married	180 (81.82%)	140 (80.92%)	40 (85.11%)
Others	40 (18.18%)	33 (19.08%)	7 (14.89%)
BMI, kg/m²	23.31±3.33	23.30±3.00	23.36±4.36	–0.035	0.972
Course of disease, mo	24.00 (12.00, 36.00)	24.00 (12.00, 36.00)	36.00 (24.00, 48.00)	–1.531	0.126
Hypertension	86 (39.09%)	58 (33.53%)	28 (59.57%)	10.532	0.001
T2DM	31 (14.09%)	23 (13.29%)	8 (17.02%)	0.424	0.515
Heart disease	41 (18.64%)	30 (17.34%)	11 (23.40%)	0.896	0.344
Stroke	27 (12.27%)	18 (10.40%)	9 (19.15%)	2.625	0.105
Smoking (n, %)	44 (20.00%)	32 (18.50%)	12 (25.53%)	1.143	0.285
Alcohol consumption (n, %)	30 (13.64%)	25 (14.45%)	5 (10.64%)	0.456	0.499
APOEɛ4 carriers (n, %)	84 (38.18%)	61 (35.26%)	23 (48.94%)	2.929	0.087
Neuropsychological assessments
MMSE	18.00 (12.25, 21.00)	18.00 (11.50, 21.00)	18.00 (13.00, 21.00)	–0.820	0.412
MoCA	13.00 (7.25, 17.00)	13.00 (7.50, 17.00)	14.00 (7.00, 18.00)	–0.781	0.435
ADL	26.00 (22.00, 35.70)	26.00 (21.50, 34.50)	28.00 (24.00, 41.00)	–1.728	0.084
CDR	2.00 (1.00, 2.00)	2.00 (1.00, 2.00)	1.00 (1.00, 2.00)	–1.594	0.111
Plasma orexin-A level, ng/ml	1.24 (0.96, 1.45)	1.29 (1.01, 1.50)	1.00 (0.86, 1.28)	–4.122	<0.001

We also showed that the proportion of constipation in LBD (61.40%) was significantly higher than AD (5.63%, p < 0.001) and MCI (19.05%, p = 0.001). Males had slightly higher proportions of constipation than females in all patients, while we did not find significant differences between female and male subgroups in Fig. 1. In MCI and AD groups, patients≤70 years old had slightly higher proportions of constipation than those > 70 years old; while in LBD group, patients≤70 years old had lower proportion of constipation than those > 70 years old; however, there were no significant differences between age subgroups in each diagnosticgroup.

Fig. 1

The proportions of constipation in three diagnostic groups. Panel a showed the proportions of constipation in MCI (4/21), AD (8/142), and LBD (35/57) group, the proportion in LBD was significantly higher than MCI and AD. Panel b showed the proportions of constipation according to diagnosis-gender and there was no significant difference between female and male subgroups in each diagnostic group. Similarly, there was no significant difference between age subgroups in each diagnostic group according to diagnosis-age in c. MCI, mid cognitive impairment; AD, Alzheimer’s disease; LBD, Lewy body dementia.

As shown in Table 1, we found that patients with constipation had lower levels of plasma orexin-A [1.00 (0.86, 1.28) versus 1.29 (1.01, 1.50) ng/ml, p < 0.001] than those without. When we did a subgroup analysis of the three diagnostic groups (Table 2), we found that plasma orexin-A was highest in patients with AD than those with MCI and LBD (p < 0.001), with a median level of 1.31 (IQR: 1.01, 1.52) ng/ml. We found that the patients with higher plasma levels of orexin-A were more likely to be diagnosed with AD, but not LBD, after adjusting all confounders in regression models (Supplementary Table 2, LBD versus AD: OR = 0.213, 95% CI: 0.074–0.613, p = 0.004). Additionally, there were no significant differences of plasma orexin-A levels between gender and age in each diagnostic group. When subgrouping according to diagnostic-constipation, plasma levels of orexin-A in MCI, AD and LBD patients with constipation were found to be lower than those in the non-constipation group, but there was no significant difference.

Table 2

The distributions of plasma levels of orexin-A (ng/ml) in three diagnostic groups

Groups		Sample size	Plasma Orexin-A levels (ng/ml)		t/Z	p
			Median (IQR)	Mean (SD)
Diagnostics					17.048	<0.001
	MCI	21	1.12 (0.95, 1.28)	1.13±0.24
	AD	142	1.31 (1.01, 1.52)	1.32±0.39
	LBD	57	1.03 (0.89 1.28)	1.14±0.39
Diagnostic-gender
MCI	Male	2	Median = 1.03	1.03±0.32	–0.599	0.549
	Female	19	1.12 (0.99, 1.30)	1.14±0.24
AD	Male	67	1.31 (0.91, 1.54)	1.28±0.39	–1.020	0.308
	Female	75	1.33 (1.13, 1.52)	1.36±0.39
LBD	Male	14	0.95 (0.80, 1.33)	1.10±0.44	–1.138	0.255
	Female	43	1.07 (0.93, 1.28)	1.15±0.38
Diagnostic-age
MCI	≤70 y	12	1.10 (1.00, 1.23)	1.10±0.21	–0.569	0.570
	>70 y	9	1.13 (0.87, 1.48)	1.17±0.28
AD	≤70 y	76	1.29 (1.01, 1.46)	1.29±0.35	–1.014	0.310
	>70 y	66	1.36 (1.01, 1.62)	1.37±0.43
LBD	≤70 y	25	1.04 (0.86, 1.36)	1.16±0.38	–0.571	0.568
	>70 y	32	1.10 (0.90, 1.27)	1.12±0.41
Diagnostic-constipation
MCI	Without	17	1.13 (0.96, 1.38)	1.15±0.25	–0.806	0.420
	With	4	1.07 (0.89, 1.22)	1.06±0.18
AD	Without	134	1.31 (1.07, 1.54)	1.33±0.39	–1.243	0.214
	With	8	1.26 (0.84, 1.41)	1.15±0.28
LBD	Without	22	1.09 (0.99, 1.42)	1.29±0.53	–1.820	0.069
	With	35	0.98 (0.86, 1.28)	1.04±0.23

IQR, interquartile range; SD, standard deviation; MCI, mid cognitive impairment; AD, Alzheimer’s disease; LBD, Lewy body dementia; y, years.

Unless otherwise indicated, the continuous variables conforming to normal distribution were described by mean (±SD), and the non-normal distribution continuous variables were expressed by median (IQR). Comparison between the “without constipation” and “with constipation” groups was performed by t-test or Mann–Whitney U test, respectively. The Chi-square test was used to compare the categorical variables between groups.

SD, standard deviation; IQR, interquartile range; T2DM, type 2 diabetes mellitus; APOE, Apolipoprotein E; MMSE, the Mini-Mental State Examination; MoCA, the Montreal Cognitive Assessment; ADL, the Activity of Daily Living Scale; CDR, the clinical dementia rating; y, years; mo, months

Associations between plasma levels of orexin-A and constipation

In order to explore the associations between plasma levels of orexin-A and constipation, we established different binary regression analysis models in Table 3. We found that plasma levels of orexin-A were significantly associated with the occurrence of constipation in all patients with cognitive impairment (both in Model 1 and Model 2, p < 0.001), even after adjusting for gender, age, hypertension, and diagnostic variables in Model 3 (OR = 0.151, 95% CI: 0.042–0.537, p = 0.003). We also found that the plasma levels of orexin A were significantly associated with constipation in the LBD group, with the OR = 0.147 (95% CI: 0.025–0.867, p = 0.034) in Model 1 and OR = 0.162 (95% CI: 0.026–0.987, p = 0.048) in Model 2. Unfortunately, we did not find a significant association between plasma levels of orexin-A and constipation in the MCI and AD groups.

Table 3

Associations between plasma levels of orexin-A and constipation in variable models

Groups	Parameter	Model 1	Model 2	Model 3
		ORs (95% CI)	ORs (95% CI)	ORs (95% CI)
All participants	Plasma orexin-A level	0.098 (0.030–0.320)^***	0.116 (0.035–0.387)^***	0.151 (0.042–0.537)^**
	Gender (female versus male)		1.165 (0.564–2.408)	0.678 (0.299–1.536)
	Age		1.031 (0.986–1.079)	1.031 (0.982–1.082)
	Hypertension (yes versus no)		2.280 (1.132–4.595)^*	1.437 (0.663–3.119)
	Diagnosis
	MCI (Reference)	–
	AD			0.278 (0.062–1.243)
	LBD			7.960 (2.158–29.363)^**
MCI	Plasma orexin-A level	0.161 (0.001–23.651)	0.017 (0.000–35.067)
	Gender (female versus male)		0.196 (0.006–6.774)
	Age		0.794 (0.481–1.308)
AD	Hypertension (yes versus no)		na
	Plasma orexin-A level	0.236 (0.026–2.127)	0.328 (0.037–2.946)
	Gender (female versus male)		0.265 (0.049–1.418)
	Age		0.970 (0.891–1.055)
LBD	Hypertension (yes versus no)		3.174 (0.698–14.432)
	Plasma orexin-A level	0.147 (0.025–0.867)^*	0.162 (0.026–0.987)^*
	Gender (female versus male)		0.549 (0.126–2.392)
	Age		1.049 (0.969–1.136)
	Hypertension (yes versus no)		1.097 (0.324–3.715)

MCI, mild cognitive impairment; AD, Alzheimer’s disease; LBD, Lewy body dementia; ORs, odd ratios; CI, Confidence Interval. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Model 1: Crude model; Model 2: Adjusted gender, age, history of hypertension; Model 3: Adjusted gender, age, history of hypertension and diagnosis.

DISCUSSION

With regard to the role of central orexin in digestive functions, it had been proved to act centrally to stimulate gastrointestinal secretion or motility [39, 40]. To our knowledge, we firstly explored the association between orexin-A and constipation based on clinical level in the present study, and pointed out that in patients with cognitive impairment, lower plasma levels of orexin-A was associated with the occurrence of constipation. This finding was particularly strong among people with LBD, and it provides a new biomarker for the differential diagnosis of AD, LBD and MCI to some extent.

In this study, we found 21.36% patients with cognitive impairment had constipation, the proportion was higher than the prevalence rate of general population [10.1% (8.7–11.6)] [2], as well as our previous report (17.6%) in old population≥65 years old based on community study [3]. A recent view had emerged that degenerative component increases the occurrence of constipation, and constipation had been observed in a variety of neurodegenerative diseases, especially in DLB and PD [41, 42]. We found that the proportion of constipation in patients with LBD were 61.4%, and significantly higher than AD and MCI groups (both p = 0.001). LBD is a major neurocognitive dementia with α-synuclein deposition/Lewy bodies, constipation is the most frequently dysautonomia symptom and the earlier manifestation of LBD. We previously reported that 71/185 patients with LBD had constipation in their mild-moderate stage, with PDD (50/93) being more pronounced than DLB (21/92) [43]. In the cohort of Honolulu Heart Program, the prevalence of PD increased significantly with decreasing bowel movement frequency, indicating that a history of constipation was associated with PD [44]. Additionally, our multicenter cross-sectional survey revealed [4] that the prevalence rates of constipation were 19.2% (95% CI, 17.3–21.0), 19.1% (95% CI, 16.8–21.5), 14.4% (95% CI, 12.8–15.9), and 13.8% (95% CI, 13.0–14.6) in the all-cause dementia, non-amnestic-MCI, amnestic-MCI and normal cognition populations, all the prevalence rates were significantly lower than LBD. Traditionally, loss of dietary fiber consumption and reduction of physical exercise can lead to constipation, and the prevalence of constipation increases significantly with age [45]. After subgrouping by age, we found the patients≤70 years old had slightly higher proportions of constipation than those > 70 years old in MCI and AD groups; while in LBD group, patients≤70 years old had lower proportion of constipation than those > 70 years old. However, there were no significant differences between age or gender subgroups in each diagnostic group. In addition to LBD, we found similar trends but without significant difference in AD and MCI as in previous studies; In addition, the results in the LBD group in our study were inconsistent with previous studies, possibly because of the small sample size.

The potential mechanism of high proportion of constipation in LBD is “LBD pathology might originate in the bowel” [46], the brain-gut axis dysfunction can affect the occurrence of constipation. A series of pathological reports by Braak [46, 47] showed that PD pathology in the brain starts in the dorsal vagal nucleus (that might regulate bowel function) earlier than in the substantia nigra (that regulates motor function). Gelpi et al. [48] also demonstrated that α-synuclein aggregated in the peripheral nerves, like vagus nerve (86.7%), myenteric plexus (86.7%), and cardiac sympathetic nerve (100%), earlier than in the brain. The same findings were proved by Beach et al. [49] by immunohistochemically staining, they verified that 12/26 LBD (46%) and 32/36 PD (89%) patients performed pSyn-positive in the vagus, 5/30 (17%) LBD and 42/52 (81%) of PD subjects were pSyn-positive in the stomach. And many clinical studies have proven that constipation appears about 10 to 20 years prior to motor symptoms [50], and 3.7 (±9.2) years preceding the onset of memory loss [51]. Meanwhile, recent studies have pointed out that changes in intestinal flora are associated with the occurrence of constipation [7, 52], but the “causal relationship” between them is still worth discussing. Constipation can cause intestinal flora disorder, then disturb the structure and function of IEB, leading to the abnormal aggregation and metabolism of Lewy bodies, as well as aggravating constipation symptom. Moreover, the alteration in gut microbial composition enhances the release of amyloids, which may contribute to the pathogenesis of AD, especially during aging when both the IEB and BBB become more permeable [9 –11].

Orexin-A was initially generated by hypothalamic neurons and participated the neuroprotection mostly through the effect on microglia activation [53]. Many studies have found orexin-A and OX-1 R also presented in peripheral tissues including the gut [54]. The orexin-A-producing neurons in the myenteric plexus can project to OX-1R-expressing enterocytes of the intestinal villi in the IEB [55], possibly indicating their involvement in gut motility, intestinal absorption, and/or secretion of nutrients [56]. Thus, we analyzed plasma levels of orexin-A in different MCI, AD, and LBD groups and found that the plasma levels of orexin-A in patients with AD were significantly higher than those in LBD and MCI. We have previously reported that the plasma levels of orexin-A in MCI-LB and DLB was higher than that in the normal elderly, but there was no statistical difference between the MCI-LB and LBD groups [38]. Our findings indicated that plasma levels of orexin-A have no significant relationship with sex and age.

However, the pathogeneses of constipation and cognitive impairment are unclear, but they both share similar risk factors, such as age, sex, economic status, dietary structure, etc., and several studies have pointed that the neuroendocrine factors, including orexin-A, can affect the occurrence of the two diseases [18 , 57–59]. The present results strongly suggested the decreased plasma levels of orexin-A significantly increased the risk of constipation. Previous literature pointed that orexin-A facilitates distal stomach motility and relaxation of the proximal stomach via the vagus nerve [18 , 57]. Kobashi et al. [60] found orexin-A can induce relaxation of the proximal stomach and phasic contractions in the distal stomach, but the distal gastric contractions fail in animal models with vagal resection. Mouse studies using both mechanical and electrophysiological techniques also confirmed a direct effect of orexin-A on the smooth muscle of the duodenum that may reinforce or compensate for orexinergic signals from neurons of the dorsal motor nucleus of the vagus (reviewed in [24]). Alterations of gut microbiota is also involved in the link between orexin and constipation. The alterations of gut microbiota causing by constipation can increase IEB permeability, then aggravate the leakage of LPS into the systemic circulation. Orexin-A can block LPS translocation to the blood, weaken metabolic endotoxemia and activation of immune cells, as well as the subsequent systemic and central inflammation, which are responsible of BBB dysfunction and microglia activation. Also, orexin-A released by Myenteric neurons can prevent occludin decreasing through interacting with OX-1 R expressed on the basolateral side of enterocytes, thereby preventing intestinal barrier impairment induced by LPS. It follows then that peripheral orexin-A may act as a protective factor of the epithelial barrier, preventing LPS transfer from the gut to the CNS and reducing the occurrence of neuroinflammation. We speculate that the increased permeability of the IEB leads to α-synuclein aggregation within the ENS [61], which further affects the gastric motility, which may be the reason for the high proportion of constipation in LBD.

Although we have verified the effect of orexin-A on constipation in the clinical population for the first time, the universality of the results is limited due to the small sample size of the single-center study and its concentration in the cognitive impairment population. In addition, due to the lack of control group and the lack of records of lifestyle and other information, we cannot determine other causes affecting constipation. We need to further expand the sample size for more detailed and in-depth comparable studies.

The decrease of plasma level of orexin-A is closely related to the occurrence of constipation, and the orexinergic nervous system is involved in the gastrointestinal symptoms of patients with cognitive impairment. These findings suggest that orexin-A acts as a neuroendocrine medium through which the brain-gut axis co-regulates symptoms of cognitive impairment, confirming the role of the brain-gut axis in neurodegeneration. In addition, we also suggest that orexin-A has an intestinal protective effect, focusing on plasma levels of orexin-A can effectively determine the constipation complications. Clinicians should pay attention to the orexinergic nervous system in the treatment and management of cognitive impairment, which will help to improve the quality of life of patients with cognitive impairment.

Footnotes

ACKNOWLEDGMENTS

The authors are grateful to all those who participated in this study and wish to acknowledge the valuable assistance obtained from all specialized physicians, as well as the Tianjin Key Medical Discipline (Specialty) Construction Project for their help. We sincerely gratitude Jing Li (Tianjin Huanhu Hospital, Tianjin, China), Xia Yang (Beijing Tiantan Hospital, Capital Medical University), Zhichao Chen (Beijing Friendship Hospital, Capital Medical University), and Moyu Li (Beijing Tiantan Hospital, Capital Medical University) for the data collection and input. The ELISA tests were sponsored by Dr. Sen Liu and his research team at Beijing Pason Pharmaceuticals Inc., including the experimental methods, purchase for diagnostic reagents, and technical support. We would like to express our sincere gratitude to Dr. Sen Liu and his team. All these authors accept responsibility for all aspects of the manuscript and approved the final version of the manuscript.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant number 82171182], Tianjin Science and Technology Plan Project [grant number 22ZYCGSY00840], Tianjin Health Research Project [grant number ZC20121 and TJWJ2023QN060], and Tianjin Key Medical Discipline (Specialty) Construction Project [grant number TJYXZDXK-052B].

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Dr. Ji is an Editorial Board Member of this journal, but was not involved in the peer-review process nor had access to any information regarding its peer-review.

DATA AVAILABILITY

The data supporting the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

The supplementary material is available in the electronic version of this article: .

References

Mugie

, Benninga

, Di Lorenzo

(2011) Epidemiology of constipation in children and adults: A systematic review. Best Pract Res Clin Gastroenterol 25, 3–18.

Barberio

, Judge

, Savarino

, Ford

(2021) Global prevalence of functional constipation according to the Rome criteria: A systematic review and meta-analysis. Lancet Gastroenterol Hepatol 6, 638–648.

, Liu

, Jia

, Wang

, Gan

, Hu

, Zhu

, Song

, Niu

, Ji

(2022) Epidemiology of constipation in elderly people in parts of China: A multicenter study. Front Public Health 10, 823987.

Wang

, Fei

, Hu

, Wang

, Liu

, Zeng

, Zhang

, Lv

, Niu

, Meng

, Cai

, Li

, Gang

, You

, Lv

, Ji

(2021) Prevalence of constipation in elderly and its association with dementia and mild cognitive impairment: A cross-sectional study. Front Neurosci 15, 821654.

Tan

, Dong

, Chen

, Jin

, Yin

, Li

, Shi

, Shao

, Wang

, Chen

, Yang

, Li

(2021) Intestinal microbiota mediates high-fructose and high-fat diets to induce chronic intestinal inflammation. Front Cell Infect Microbiol 11, 654074.

, Li

, Zhou

, Li

, Shao

, Li

(2021) The mechanism of intestinal flora dysregulation mediated by intestinal bacterial biofilm to induce constipation. Bioengineered 12, 6484–6498.

Kesika

, Suganthy

, Sivamaruthi

, Chaiyasut

(2021) Role of gut-brain axis, gut microbial composition, and probiotic intervention in Alzheimer’s disease. Life Sci 264, 118627.

Sharon

, Sampson

, Geschwind

, Mazmanian

(2016) The central nervous system and the gut microbiome. Cell 167, 915–932.

Cattaneo

, Cattane

, Galluzzi

, Provasi

, Lopizzo

, Festari

, Ferrari

, Guerra

, Paghera

, Muscio

, Bianchetti

, Volta

, Turla

, Cotelli

, Gennuso

, Prelle

, Zanetti

, Lussignoli

, Mirabile

, Bellandi

, Gentile

, Belotti

, Villani

, Harach

, Bolmont

, Padovani

, Boccardi

, Frisoni

(2017) Association of brain amyloidosis with pro-inflammatory gut bacterial taxa and peripheral inflammation markers in cognitively impaired elderly. Neurobiol Aging 49, 60–68.

10.

Friedland

, Chapman

(2017) The role of microbial amyloid in neurodegeneration. PLoS Pathog 13, e1006654.

11.

Chen

, Stribinskis

, Rane

, Demuth

, Gozal

, Roberts

, Jagadapillai

, Liu

, Choe

, Shivakumar

, Son

, Jin

, Kerber

, Adame

, Masliah

, Friedland

(2016) Exposure to the functional bacterial amyloid protein curli enhances alpha-synuclein aggregation in aged Fischer 344 rats and Caenorhabditis elegans. Sci Rep 6, 34477.

12.

Chapman

, Robinson

, Pinkner

, Roth

, Heuser

, Hammar

, Normark

, Hultgren

(2002) Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851–855.

13.

Doifode

, Giridharan

, Generoso

, Bhatti

, Collodel

, Schulz

, Forlenza

, Barichello

(2021) The impact of the microbiota-gut-brain axis on Alzheimer’s disease pathophysiology. Pharmacol Res 164, 105314.

14.

Braak

, Rüb

, Gai

, Del Tredici

(2003) Idiopathic Parkinson’s disease: Possible routes by which vulnerable neuronal types may be subject to neuroinvasion by an unknown pathogen. J Neural Transm (Vienna) 110, 517–536.

15.

Kim

, Kwon

, Kam

, Panicker

, Karuppagounder

, Lee

, Kim

, Kook

, Foss

, Shen

, Lee

, Kulkarni

, Pasricha

, Lee

, Pomper

, Dawson

, Ko

(2019) Transneuronal propagation of pathologic α-synuclein from the gut to the brain models Parkinson’s disease. Neuron 103, 627641.e627.

16.

Yang

, Zhang

, Wang

, Zhang

, Liu

, Zhang

, Ren

, Zhou

, Gao

, Zhang

, Feng

(2023) Addition of α-synuclein aggregates to the intestinal environment recapitulates Parkinsonian symptoms in model systems. Acta Pharmacol Sin, doi: 10.1038/s41401-023-01150-2.

17.

Sampson

, Debelius

, Thron

, Janssen

, Shastri

, Ilhan

, Challis

, Schretter

, Rocha

, Gradinaru

, Chesselet

, Keshavarzian

, Shannon

, Krajmalnik-Brown

, Wittung-Stafshede

, Knight

, Mazmanian

(2016) Gut microbiota regulate motor deficits andneuroinflammation in a model of Parkinson’s disease. Cell 167, 1469–1480.e1412.

18.

Tunisi

, Forte

, Fernández-Rilo

, Mavaro

, Capasso

, D’Angelo

, Milić

, Cristino

, Di Marzo

, Palomba

(2019) Orexin-A prevents lipopolysaccharide-induced neuroinflammation at the level of the intestinal barrier. Front Endocrinol (Lausanne) 10, 219.

19.

Kirchgessner

(2002) Orexins in the brain-gut axis. Endocr Rev 23, 1–15.

20.

, Kong

, Shao

, Liu

, Zhang

, Kong

, Chen

, Cheng

, Wang

(2021) Orexin-A alleviates astrocytic apoptosis and inflammation via inhibiting OX1R-mediated NF-κB and MAPK signaling pathways in cerebral ischemia/reperfusion injury. Biochim Biophys Acta Mol Basis Dis 1867, 166230.

21.

Liu

, Xue

, Liu

, Diao

, Wang

, Pan

, Chen

(2018) Orexin-A exerts neuroprotective effects via OX1R in Parkinson’s disease. Front Neurosci 12, 835.

22.

, Kim

, Leem

(2022) Protective effects of orexin A in a murine model of cisplatin-induced acute kidney injury. J Clin Med 11, 7196.

23.

Durairaja

, Pandey

, Kahl

, Fendt

(2023) Nasal administration of orexin A partially rescues dizocilpine-induced cognitive impairments in female C57BL/6 J mice. Behav Brain Res 450, 114491.

24.

Okumura

, Nozu

, Ishioh

, Igarashi

, Kumei

, Ohhira

(2020) Brain orexin improves intestinal barrier function via the vagal cholinergic pathway. Neurosci Lett 714, 134592.

25.

Bassil

, Brown

, Pattabhiraman

, Iwasyk

, Maghames

, Meymand

, Cox

, Riddle

, Zhang

, Trojanowski

, Lee

(2020) Amyloid-beta (Aβ) plaques promote seeding and spreading of alpha-synuclein and tau in a mouse model of Lewy body disorders with Aβ pathology. Neuron 105, 260–275.e266.

26.

Foguem

, Manckoundia

(2018) Lewy body disease: Clinical and pathological “overlap syndrome” between synucleinopathies (Parkinson disease) and tauopathies (Alzheimer disease). Curr Neurol Neurosci Rep 18, 24.

27.

Petersen

(2011) Clinical practice. Mild cognitive impairment. N Engl J Med 364, 2227–2234.

28.

Winblad

, Palmer

, Kivipelto

, Jelic

, Fratiglioni

, Wahlund

L-O

, Nordberg

, Bäckman

, Albert

, Almkvist

(2010) Mild cognitive impairment – beyond controversies, towards a consensus: Report of the International Working Group on Mild Cognitive Impairment. J Intern Med 256, 240–246.

29.

McKhann

, Knopman

, Chertkow

, Hyman

, Jack

Jr. , Kawas

, Klunk

, Koroshetz

, Manly

, Mayeux

, Mohs

, Morris

, Rossor

, Scheltens

, Carrillo

, Thies

, Weintraub

, Phelps

(2011) The diagnosis of dementia due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 263–269.

30.

McKeith

, Boeve

, Dickson

, Halliday

, Taylor

, Weintraub

, Aarsland

, Galvin

, Attems

, Ballard

, Bayston

, Beach

, Blanc

, Bohnen

, Bonanni

, Bras

, Brundin

, Burn

, Chen-Plotkin

, Duda

, El-Agnaf

, Feldman

, Ferman

, Ffytche

, Fujishiro

, Galasko

, Goldman

, Gomperts

, Graff-Radford

, Honig

, Iranzo

, Kantarci

, Kaufer

, Kukull

, Lee

VMY

, Leverenz

, Lewis

, Lippa

, Lunde

, Masellis

, Masliah

, McLean

, Mollenhauer

, Montine

, Moreno

, Mori

, Murray

, O’Brien

, Orimo

, Postuma

, Ramaswamy

, Ross

, Salmon

, Singleton

, Taylor

, Thomas

, Tiraboschi

, Toledo

, Trojanowski

, Tsuang

, Walker

, Yamada

, Kosaka

(2017) Diagnosis and management of dementia with Lewy bodies: Fourth consensus report of the DLB Consortium. Neurology 89, 88–100.

31.

Emre

, Aarsland

, Brown

, Burn

, Duyckaerts

, Mizuno

, Broe

, Cummings

, Dickson

, Gauthier

, Goldman

, Goetz

, Korczyn

, Lees

, Levy

, Litvan

, McKeith

, Olanow

, Poewe

, Quinn

, Sampaio

, Tolosa

, Dubois

(2007) Clinical diagnostic criteria for dementia associated with Parkinson’s disease. Mov Disord 22, 1689–1707; quiz 1837.

32.

Lacy

, Mearin

, Chang

, Chey

, Lembo

, Simren

, Spiller

(2016) Bowel disorders. Gastroenterology 150, 1393–1407.e1395.

33.

Liu

, Liu

, Wang

, Ji

(2021) Neuropsychiatric profiles in mild cognitive impairment with Lewy bodies. Aging Ment Health 25, 2011–2017.

34.

Folstein

, Folstein

, McHugh

(1975) “Mini-mental state”: A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12, 189–198.

35.

Nasreddine

, Phillips

, Bédirian

, Charbonneau

, Whitehead

, Collin

, Cummings

, Chertkow

(2005) The Montreal Cognitive Assessment, MoCA: A brief screening tool for mild cognitive impairment. J Am Geriatr Soc 53, 695–699.

36.

Morris

(1993) The Clinical Dementia Rating (CDR): Current version and scoring rules. Neurology 43, 2412–2414.

37.

Eto

, Tanaka

, Chishima

, Igarashi

, Mizoguchi

, Wada

, Iijima

(1992) [Comprehensive activities of daily living (ADL) index for the elderly]. Nihon Ronen Igakkai Zasshi 29, 841–848.

38.

Gan

, Liu

, Chen

, Yang

, Ma

, Meng

, Wang

, Liu

, Li

, Zhang

, Ji

(2022) Elevated plasma orexin-A levels in prodromal dementia with Lewy bodies. J Alzheimers Dis 88, 1037–1048.

39.

Nozu

, Tuchiya

, Kumei

, Takakusaki

, Ataka

, Fujimiya

, Okumura

(2012) Endogenous orexin-A in the brain mediates 2-deoxy-D-glucose-induced stimulation of gastric motility in freely moving conscious rats. J Gastroenterol 47, 404–411.

40.

Jin

, Jiang

, Luan

, Qu

, Guo

, Gao

, Xu

, Sun

(2020) Exogenous orexin-A microinjected into central nucleus of the amygdala modulates feeding and gastric motility in rats. Front Neurosci 14, 274.

41.

Savica

, Boeve

, Mielke

(2018) When do α-synucleinopathies start? An epidemiological timeline: A review. JAMA Neurol 75, 503–509.

42.

De Pablo-Fernandez

, Tur

, Revesz

, Lees

, Holton

, Warner

(2017) Association of autonomic dysfunction with disease progression and survival in Parkinson disease. JAMA Neurol 74, 970–976.

43.

Gan

, Chen

, Liu

, Shi

, Liu

, Wang

, Liu

, Ji

(2022) The presence and co-incidence of geriatric syndromes in older patients with mild-moderate Lewy body dementia. BMC Neurol 22, 355.

44.

Petrovitch

, Abbott

, Ross

, Nelson

, Masaki

, Tanner

, Launer

, White

(2009) Bowel movement frequency in late-life and substantia nigra neuron density at death. Mov Disord 24, 371–376.

45.

Sakakibara

, Doi

, Fukudo

(2019) Lewy body constipation. J Anus Rectum Colon 3, 10–17.

46.

Braak

, de Vos

, Bohl

, Del Tredici

(2006) Gastric alpha-synuclein immunoreactive inclusions in Meissner’s and Auerbach’s plexuses in cases staged for Parkinson’s disease-related brain pathology. Neurosci Lett 396, 67–72.

47.

Braak

, Sastre

, Del Tredici

(2007) Development of alpha-synuclein immunoreactive astrocytes in the forebrain parallels stages of intraneuronal pathology in sporadic Parkinson’s disease. Acta Neuropathol 114, 231–241.

48.

Gelpi

, Navarro-Otano

, Tolosa

, Gaig

, Compta

, Rey

, Martí

, Hernández

, Valldeoriola

, Reñé

, Ribalta

(2014) Multiple organ involvement by alpha-synuclein pathology in Lewy body disorders. Mov Disord 29, 1010–1018.

49.

Beach

, Adler

, Sue

, Shill

, Driver-Dunckley

, Mehta

, Intorcia

, Glass

, Walker

, Arce

, Nelson

, Serrano

(2021) Vagus nerve and stomach synucleinopathy in Parkinson’s disease, incidental Lewy body disease, and normal elderly subjects: Evidence against the “body-first” hypothesis. J Parkinsons Dis 11, 1833–1843.

50.

Ueki

, Otsuka

(2004) Life style risks of Parkinson’s disease: Association between decreased water intake and constipation. J Neurol 251(Suppl 7), vII18–23.

51.

, Liu

, Wang

, Zhu

, Du

, Ma

, Gan

, Wu

, Wang

, Ji

(2022) Autonomic symptoms are predictive of dementia with Lewy bodies. Parkinsonism Relat Disord 95, 1–4.

52.

, Shih

, Wu

, Hsieh

, Lee

, Lin

, Wang

(2022) Exploring the causal effect of constipation on Parkinson’s disease through mediation analysis of microbial data. Front Cell Infect Microbiol 12, 871710.

53.

Wang

, Wang

, Ji

, Pan

, Xu

, Cheng

, Bai

, Chen

(2018) The orexin/receptor system: Molecular mechanism and therapeutic potential for neurological diseases. Front Mol Neurosci 11, 220.

54.

Voisin

, Rouet-Benzineb

, Reuter

, Laburthe

(2003) Orexins and their receptors: Structural aspects and role in peripheral tissues. Cell Mol Life Sci 60, 72–87.

55.

Kirchgessner

, Liu

(1999) Orexin synthesis and response in the gut. Neuron 24, 941–951.

56.

Dale

, Hoyer

, Jacobson

, Pfleger

KDG

, Johnstone

EKM

(2022) Orexin signaling: A complex, multifaceted process. Front Cell Neurosci 16, 812359.

57.

Mediavilla

(2020) Bidirectional gut-brain communication: A role for orexin-A. Neurochem Int 141, 104882.

58.

Chen

, Zhou

, Wang

, Alam

, Kang

, Ahn

, Liu

, Jia

, Ye

(2021) Gut inflammation triggers C/EBPβ/δ-secretase-dependent gut-to-brain propagation of Aβ and tau fibrils in Alzheimer’s disease. EMBO J 40, e106320.

59.

, de Lecea

(2020) The hypocretin (orexin) system: From a neural circuitry perspective. Neuropharmacology 167, 107993.

60.

Kobashi

, Furudono

, Matsuo

, Yamamoto

(2002) Central orexin facilitates gastric relaxation and contractility in rats. Neurosci Lett 332, 171–174.

61.

Wallen

, Demirkan

, Twa

, Cohen

, Dean

, Standaert

, Sampson

, Payami

(2022) Metagenomics of Parkinson’s disease implicates the gut microbiome in multiple disease mechanisms. Nat Commun 13, 6958.