Abstract
This paper studied the influence of exercise on the cognitive ability of AD patients and elucidated potential mechanisms. The expression of SNHG14 was validated by qRT-PCR. The cognitive impairment of mice was examined by MWM Test. ELISA tests were applied to discover the influence of SNHG14 on inflammation. Overexpression of SNHG14 was found in AD patients and underexpression of SNHG14 was identified in these AD patients after exercise. In APP/PS1 double transgenic mice, SNHG14 reversed the protective impacts of exercise on escape latency and distance moved. The upregulation of SNHG14 also inhibited the effects of exercise on the percentage of time spent in the target quadrant and times of platform crossing. Besides, overexpression of SNHG14 reversed the repressed expression of IL-6, IL-1β, and TNF-α. In total, exercise could ameliorate cognitive disorder and inflammation activity by reducing the levels of SNHG14.
Significance Statement and Introduction
Significance Statement The relative levels of SNHG14 were increased in the AD patients and decreased in AD patients after exercise. Enhancement of SNHG14 had a suppressive role in the beneficial impacts of exercise on the cognitive dysfunction and mental situation of AD animal models. SNHG14 alleviated the advantageous impacts of physical exercise on inflammation.
Introduction
As a common irreversible neurodegenerative disease, Alzheimer’s Disease (AD) has a high incidence, which is characterized by cognitive disorder, memory impairment. 1,2 Neuroinflammation is another major characteristic that increases the risk of AD by regulating IFITM3 and thereby increasing amyloid-β. 3 The incidence of AD rises sharply with the increase of age. The incidence of AD in the 60-year-old population is 4%, while that in the 85-year-old population is as high as 30%. 4 Nowadays, the tendency of social aging in China is becoming more and more aggravating, and the consequent incidence of AD is increasing. 5 AD not only affects the life quality of patients but also aggravates their family burden and social burden, so we must pay attention to the treatment and nursing of patients with AD. 6,7 Because of the irreversibility of AD and the burdens from this disease, it is of great significance to find a biomarker associated with the progression of AD.
Physical exercise is identified as one popular invention to ameliorate mental condition and cognitive functions. 8 A study shows that high-intensity aerobic exercise can amend cognitive and executive abilities, indicating the vital roles of exercise in cognitive health. 9 Karssemeijer verifies that physical activity may accelerate the psychomotor speed and thereby mitigate functional decline. 10 Moreover, regular exercise might suppress the aggravation of AD by inhibiting tau hyperphosphorylation. 11 However, the underlying mechanism of exercise on AD is unveiled. It is well known that lncRNA is a kind of long endogenous RNA, participates in many biological activities, including physical exercises. LncRNA MALAT1 is a regulator in the progression of exercise defending from high glucose, which might mediate inflammatory cytokines. 12 The study of lncRNA SNHG14 has been widely published in the literature recently. In retinoblastoma, SNHG14 could be a key regulator in the progression of this malignant cancer and an effective therapeutic target in the treatment of retinoblastoma. 13 An increased level of SNHG14 is already observed in Parkinson’s disease and it plays a key role in the dopaminergic neuron damage. 14 Moreover, the research of 2021 proposes that SNHG14 is highly expressed in the serum of AD patients. 15 But the roles of SNHG14 in the mediation of exercise on AD are unclear.
Therefore, this experiment aimed to provide the expression of SNHG14 in AD patients and the alternation of SNHG14 levels before and after exercise in patients with AD. Furthermore, the APP/PS1 double transgenic mice were put into practice to detect the function of exercise on cognitive impairment and inflammation. The mediated impacts of SNHG14 on the influence of exercise on inflammation and perception were also substantiated to explore the mechanism underlying exercise.
Materials and methods
AD Patients
On the premise of obtaining the approval of the ethics department of Shanghai Municipal Hospital of Traditional Chinese Medicine, we selected 90 patients with AD and 88 healthy volunteers. All individuals read carefully and signed the informed consent form and agreed to participate in this exploration. Patients with AD underwent a 3-month cycling training, during which the heart rate met with 70% of maximum. Before and after this three-month exercise, blood samples were obtained from each AD patient. The serum specimen was collected from each individual after centrifugation.
Animal Models
The APP/PS1 double transgenic mice were purchase from Jackson Laboratory and selected as the AD models. All these mice were allowed access to free food and water to get used to the new situation in an animal room meeting SPF standards. The growth temperature was 24°C with a relative humidity of 55% ∼ 65% and daily light or night for 12 h. All the experimental schemes used in this study have been approved by the Committee on Ethics of Experimental Animals. All animals were divided into controls, voluntary exercise (VE) cohort, exercise and lncRNA negative control (NC) group, and exercise and p-SNHG14 group. A total of 10 mice were included in each group. Mice in the control group were housed in the cages with fixed wheels and mice in other groups were housed in the cages with running wheels. The sequences of SNHG14 or its NC were cloned into pcDNA3.1 plasmids (GenePharma, Shanghai, China) and then recombinant plasmids (p-NC or p-SNHG14) were injected into the bilateral hippocampus of APP/PS1 mice. After 4-week of training, half the mice of each group were selected randomly to be euthanized, the rest rice received the following experiments. The graphical timeline of experimental mice was shown in Figure 1A.
The expression of miR-940 in APP/PS1 mice. one-way analysis of variance was used for this analysis. there were 5 mice in each group. (
RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was exacted from each specimen using TRIzol reagent (Life Technologies, Carlsbad, CA). A reverse Transcription Mix (Tiangen, Beijing, China) was obtained to synthesize cDNA on ice. The qRT-PCR reagents were obtained from Biolab (Beijing, China), which were mixed with cDNA accord to its instruction. The sequences of primers were as follows: SNHG14 forward: 5’-GGGTGTTTACGTAGACCAGAACC-3’, SNHG14 reverse: 5’-CTTCCAAAAGCCTTCTGCCTTAG-3’; GAPDH forward: 5’-GAAGGTGAAGGTCGGAGTC-3’, reverse: 5’-GAAGATGGTGATGGGATTTC-3’. PCR detection system was applied to measure CT value. The reaction conditions were 95° for 3 min; 40 cycles of 95° for 5 sec, 60° for 10 sec, and 72° for 15 sec. SNHG14 expression was calculated by the 2-ddCT method and normalized by GAPDH.
MWM Test
Mice were taken to the Morris water maze test based on the previous method. The mice were respectively put into the water from the start points in the 4 quadrants and received a movement trace within 120 s. The experiment was repeated once a day for 5 days, with the experimental conditions unchanged for each time. In these experiments, the time to reach the platform was recorded. The movement trace and the corresponding movement time were also recorded. After the platform was removed, the mice were respectively put into the water in the 4 quadrants. The time spend in quadrants and the number of times crossing the platform area was calculated for statistical analysis.
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentration of IL-1β, IL-6, and TNF-α was identified by the ELISA kit (Boster Bio, Pleasanton, CA). The absorbance value was measured at 450 nm by the enzyme labeling instrument, and then the content of inflammatory indicators was obtained.
Statistical Analysis
Statistical analysis was performed using SPSS and GraphPad. All data were expressed as mean ± standard deviation (x¯ ± s) or number. One-way analysis of variance was used for the multi-group comparison of experimental data. Student t-test, paired sample t-test, and chi-square test were used for comparison between 2 groups. The test level P < 0.05 meant statistical significance.
Results
Baseline Features of Included Cohort
There were 48 females and 42 males in the AD group, with an average age of 63.64 ± 5.45 years and a BMI of 24.76 ± 2.57 kg/m2 (Table 1). In terms of basic demographic data, the control group and AD group could be compared statistically (P > 0.05). Some clinical parameters were also documented, such as TG, TC, FBG, SBP, and DBP. Statistical results propounded no obvious differences on all clinical parameters between the healthy and control people (P > 0.05). Furthermore, the mental condition and cognitive disorder of patients were judged by the NMSE score, ADAS-cog level, and NPI-Q score. Significantly, the average MMSE score was increased in AD patients after physical exercise (Table 2, P = 0.006), indicating exercise might contribute to cognitive amelioration. Besides, a significant decrease was found in the patients after exercise on the ADAS-cog score (Table 2, P < 0.001) and NPI-Q score (Table 2, P < 0.001).
Clinical Data of the Study Population.
Annotation: AD, Alzheimer’s disease; BMI, body mass index; TG, triglycerides; TC, total cholesterol; FBG, fasting blood glucose; SBP, systolic blood pressure; DBP, diastolic blood pressure; Data are expressed as n or mean ± standard deviation.
Changes of Various Parameters of AD by Exercise.
Annotation: AD, Alzheimer’s disease; MMSE, Minimum Mental State Examination; ADAS-cog, Alzheimer’s Disease Assessment Scale cognition; NPI-Q, Neuropsychiatric Inventory Questionnaire. Data are expressed as n or mean ± standard deviation.
Results of qRT-PCR in AD Patients
The expression of SNHG14 was detected in healthy controls as well as AD patients before and after exercise. Compared with the control group, the relative expression of serum SNHG14 in the AD group increased significantly (Figure 2A, P < 0.001), indicating SNHG14 might be a regulator in the development of AD. Moreover, the levels of SNHG14 were decreased in the AD patients after physical exercise relative to patients before exercise (Figure 2B, P < 0.001), providing that exercise might meliorate the alternation of SNHG14 expression in AD patients.
Student t-test analysis on the results of qRT-PCR. (
SNHG14 Mediated the Function of Exercise on Cognition
The qRT-PCR finding of APP/PS1 mice was shown in Figure 1B, which provided that the expression of SNHG14 was declined in the mice with voluntary exercise (P < 0.001). Furthermore, we accommodated the levels of SNHG14 in the AD model and explored the impacts of SNHG14. Figure 1B also elucidated that the injection of p-SNHG14 could increase the levels of SNHG14 successfully (P < 0.001).
The MWM test results discovered that the escape latency was significantly decreased in the VE group, while overexpression of SNHG14 reversed this beneficial effect (Figure 3A, P < 0.01). The distance moved was also documented, which manifested that the distance in the VE group was reduced, however, the distance in the VE + p-SNHG14 group was reversely increased (Figure 3B, P < 0.01). Additionally, the percentage of time in the objective quadrant was elevated in the mice experimenting exercise, whereas, the upregulation of SNHG14 repressed the enhancement of time (Figure 3C, P < 0.01). The abundance of SNHG14 also reversed the increased number of platform crossing in the VE group caused by exercise (Figure 3D, P < 0.05).
The function of exercise and SNHG14 on cognition in the AD mice. one-way analysis of variance was applied to analyze these results. there were 5 mice in each group. (
SNHG14 Mediated the Function of Exercise on Inflammation
Neuroinflammation is an acknowledged pathological character in the impairment stimulated by AD. 16 In this study, we detected the levels of inflammatory indicators to reveal the influence of physical exercise and SNHG14. Voluntary exercise declined the levels of IL-6, IL-1β, and TNF-α, nonetheless, the injection of p-SNHG14 suppressed the reduced levels of inflammatory cytokines (Figure 4A-C, P < 0.01).
The function of SNHG14 on inflammation was revealed by one-way analysis of variance. exercise promoted the amelioration of inflammation, while SNHG14 inhibited the declined levels of (
Discussion
AD is a common chronic central nervous injury. 17 Such lesions can cause dementia in patients, and most of them occur in elderly patients. 18 Cognitive improvement and antipsychotic drugs are generally used in clinical treatment, but their effects on AD patients are limited. 19 Under this background, some researchers have actively explored non-drug therapies, such as exercise therapy, behavioral therapy, cognitive training, music therapy, and nostalgia therapy, among which exercise intervention is a prominent mean. 20,21 Through scientific and reasonable exercise, AD patients can delay cognitive degradation or improve cognitive function, improve daily living and quality of life, and relieve mental symptoms. 22 Exercise prevention in the treatment of AD has good effects, social benefits, and broad application prospects. However, there are still many problems to be solved in the research of exercise intervention for AD patients.
In the present study, we concluded that the relative expression of SNHG14 was increased in AD patients, providing that SNHG14 might be involved in the progression of AD. An investigation of microarray re-annotation mentions that the expression of SNHG14 is highly expressed in AD patients, which further verifies our conclusion. 15 In another degenerative disease, the levels of SNHG14 are obviously raised in the mice with Parkinson’s disease and its silence can facilitate the restoration of Parkinson’s disease. 14 Previous studies have shown that exercise can improve cognitive function, relieve neuropsychiatric disorders, and improve daily activities. 23 In our publication, we further observed that a three-month training meliorated the impairments of AD on cognition and mental station by the decreases of NMSE score, ADAS-cog level, and NPI-Q score. Moreover, after three months of exercise, the relative levels of SNHG14 in the APP/PS1 AD model were significantly declined, which indicated that exercise could reverse the alternation of SNHG14 expression.
Considering the change of SNHG14 expression in the patients with AD and patients after exercise, the underlying mechanism was got further exploration. More and more evidence shows that voluntary exercise may restore the cognitive dysfunction of the elderly and exercise contributes to the alternation of lncRNAs’ expression. 24 -26 In a publication of heart protection, swimming can mitigate vascular endothelial ischemia-reperfusion injury by regulating FR030200-Col3A1 and FR402720-Rnd1, and the pattern of lncRNA THRIL regulating TNF-α may change after running 27,28 A further study of 2020 indicates that exercise-caused lncRNA MALAT1 protects hippocampal cells and cardiac muscle cells against ischemic diseases 29 In the current investigation, the AD mice were obtained to access the molecular mechanism of SNHG14. In the assay, we found that the expression of SNHG14 in the VE group was decreased, which showed that exercise might reduce the SNHG14 levels of AD mice. More importantly, the overexpression of SNHG14 could inhibit the protective function of exercise on the cognition of AD rice. In a publication of 2020, the beneficial influence of exercise is identified in inflammation 30 SNHG14 is aberrantly expressed in the ApoE-/- mice and participates in the aggression of atherosclerosis. 31 In acute lung injury, the overexpression of SNHG14 promotes the levels of pro-inflammatory proteins. 32 Our experiments also indicated that exercise might repress the inflammation response and upregulation of SNHG14 reversed this trend.
In summary, the relative levels of SNHG14 were increased in the AD patients and decreased in AD patients after exercise. Enhancement of SNHG14 had a suppressive role in the beneficial impacts of exercise on the cognitive dysfunction and mental situation of AD animal models. Additionally, physical exercise might attenuate the inflammation, and increased SNHG14 alleviated this trend.
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Research Ethics and Patient Consent
On the premise of obtaining the approval of the ethics department of Shanghai Municipal Hospital of Traditional Chinese Medicine, we selected 90 patients with AD and 88 healthy volunteers. All individuals carefully read and signed the informed consent form and agreed to participate in the study.