- Email: [email protected]
Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice
Journal Pre-proof Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice Yiwei Huang, Keming Yun PII:
S1878-8750(20)30183-2
DOI:
https://doi.org/10.1016/j.wneu.2020.01.165
Reference:
WNEU 14195
To appear in:
World Neurosurgery
Received Date: 26 December 2019 Revised Date:
19 January 2020
Accepted Date: 20 January 2020
Please cite this article as: Huang Y, Yun K, Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice, World Neurosurgery (2020), doi: https:// doi.org/10.1016/j.wneu.2020.01.165. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice Yiwei Huang1, Keming Yun2* 1 Department of Neurology, Zhuji Affiliated Hospital of Shaoxing University, Zhuji, Shaoxing311800, Zhejiang, China; 2 College of Forensic Medicine, Shanxi Medical University, Taiyuan City, 030001, Shanxi Province, China. * Corresponding author: Keming Yun College of Forensic Medicine, Shanxi Medical University, No.56 Xinjiannan Road, Yingze District, Taiyuan, 030001, Shanxi, China. Email: [email protected] Abstract Objective: To investigate the effects and mechanism of lead and high fat diet on cognitive function and central nervous system in mice. Methods: 84 healthy male mice were randomly divided into a control group (n=21) (fed with common diet and free drinking), a lead exposure group (n=21) (fed with common diet and 300mg/L lead acetate solution), a high-fat group (n=21) (fed with high-fat diet and free drinking) and a lead + high-fat group (n=21) (fed with high-fat diet and 300mg/L lead acetate solution). In 10 weeks after lead exposure, the mice of all groups were tested for the cognition, learning and memory abilities, body weight, serum triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL); and also for the contents of lead, interleukin 6 (IL-6), interleukin 17 (IL-17), interferon γ (IFN-γ), advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST) and hydrogen peroxide in the brain tissues. Results: Compared with the control group and the lead-exposed group, the body weights of mice in the high-fat group and the lead + high-fat group increased significantly from the sixth week of the experiment, of which the difference was statistically significant (P<0.05); Compared with the control group and the high-fat group, the lead content in brain tissue of the lead exposure group and the lead + high-fat group increased significantly, of which the difference was statistically significant (P<0.05); Compared with the control group, the escape latent period, TG, LDL, IL-6, IL-17, IFN-γ and AGEs of the remaining three groups increased significantly, but the recognition index, passing platform times, HDL and GSH-ST significantly decreased (P<0.05); the 2nd and 3rd escape latent periods, IL-6, IL-17 and AGEs of lead + high-fat group were obviously higher than the remaining three groups, but the passing platform times were obviously lower than the remaining three groups, of which the difference was statistically significant; The content of hydrogen peroxide in brain tissues had no difference among groups (P>0.05). Conclusion: The lead and high fat diet have resulted in the lipid metabolism disorders and impair the cognitive function and central nervous system by promoting the
secretion of inflammatory factors in glial cells, inducing the inflammatory reaction of brain tissue, inhibiting GSH-ST expression and increasing AGEs content. Keywords: Lead exposure; High fat diet; Cognitive function; Central nervous system; Mechanism
Introduction Lead is a kind of neurotoxic heavy metal. It has been found in studies that lead in the environment can be stored in central nervous system to damage central nervous system and cause cognitive function defects and destroys [1]. Because lead is widely used in production and life, lead exposure has become an increasingly serious environmental and medical problem [2]. Central nervous system is the main target organ of lead injury. Lead exposure can affect the ability of learning and memory, and damage the sensory organs and their innervation. At present, there are many researches on the mechanism of lead nerve injury. For instance, lead can induce oxidative stress to induce cell injury, apoptosis, excitatory toxicity and so on. In addition, high fat diet can result in a significant decrease in the expression level of the hippocampus-related functional proteins and damage hippocampal neurons, thereby affecting the function of the nervous system and causing inattention, decline of learning ability, cognitive function disorder and other symptoms [3, 4]. At present, it is still unclear for the mechanism of the combined effect of lead and high fat diet on the function of central nervous system. There are many studies on the damage of lead and high-fat diet to cerebral cortex, hippocampus, neurons, and central nervous system disorders, but there are few studies on the causes. In this study, the mouse models of lead and high fat diet exposure were established to investigate the changes and mechanisms of cognitive function and central nervous function after the effect of the lead and high fat diet. Furthermore, it provides new basis and target for the prevention of neural injury in the lead-exposed occupational population, especially in the obese population. The innovation of this paper is to take the obese people as the main research object, which has obvious pertinence. Materials and methods Animals 84 healthy adult male adult mice of Grade SPF were selected from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Jing) 2009-0004) and the weight of each mouse was 12-17g. All mice were fed in the independent ventilated animal feeding cage of the laboratory. During the experiment, the temperature of the laboratory was maintained at (23±2)℃, the humidity at (55±5)% and the light time for 12h. All mice were fed with the same common feed and drinking water for one week and then averagely divided into 4 groups by the method of random number table, namely control group, lead exposure group, high-fat group and lead + high-fat group, and each group had 21 mice. There was no significant difference in general data (body weight and monthly age) of the mice in each group. The animal experiments and animal treatment processes involved in this study meet the requirements of animal
experiment standardization and the protocol was approved by the ethics committee of our hospital. Grouping and treatment The control group was fed with ordinary feed and normal drinking water; the lead exposure group with ordinary feed and lead acetate solution (300mg/L); the high-fat group with high fat feed and normal drinking water; the lead + high-fat group with high fat feed and lead acetate solution (300mg/L). For all mice, the diet was not restricted but supplemented in time. The general condition of the mice was observed every day, their diets were recorded respectively and all mice were weighed every 2 weeks. After continuous exposure for 10 weeks, neurological function tests were carried out in each group of mice to test their cognitive, learning and memory abilities. Enzyme linked immunosorbent assay The mice were and then sacrificed to take the serum and brain tissue samples. The contents of serum lipometabolic product and inflammatory factor in the brain tissue were tested by the enzyme linked immunosorbent assay; the corresponding kit was selected to determine the contents of advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST) and hydrogen peroxide in the brain tissue in strict accordance with the instructions, and the content of lead in brain tissue was tested by inductively coupled plasma mass spectrometry (ICP-MS). Testing for cognitive ability 24h before training, firstly put the mice in a special experimental device to freely adapt to the environment, then placed two objects in the same shape, size and color at the left and right ends on a side of the box, put the mice into the box with their backs against the objects, and recorded the cognitive time that the mice respectively smelled, watched and touched the two objects in 3min. The test was carried out in 24h after the training. Replaced one object in the box by a new one with different shape and size, kept other methods similar to the training period and recorded the cognitive time of the mice for two new and old objects. During the whole experiment, it was necessary to keep quiet indoors. After each experiment, washed the device thoroughly with 75% alcohol for fear that odor might affect the test results of mice. Cognitive index (%) = cognitive time of new object / (cognitive time of new object + cognitive time of old object) x 100%. Morris water maze The mice were tested by Morris water maze for Learning and memory ability. Before the beginning of the formal experiment, the adaptive training was carried out for the mice for 1d and the mice were put into the water maze at the 4-quadrant starting point with their faces towards the pool wall. Then, they could freely find the underwater platform and the activity track in 2min was recorded. The formal experiment totally lasted for 4d. In the first 3d, the fixed navigation experiment was
carried out to measure their learning ability. The methods were the same as those of the training period. The time of finding the platform was recorded, namely escape latent period. If the mice did not find the platform in 2min, they were guided to the platform to stay 5s and the escape latent period was recorded as 120s. On the final 1d, the space exploration experiment was carried out to measure the spatial memory ability of mice. The underwater platform was removed and the experimental mice were put in from the opposite quadrant of the platform with their faces towards the pool wall, and the times that the mice passed through the platform in 1min were observed and recorded. Observation indices Include general conditions and body weights; lead content in brain tissue; serum lipid metabolism index, including triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL); neural function, including cognitive ability and learning / memory ability; content of inflammatory factor in brain tissue, including interleukin 6 (IL-6), interleukin 17 (IL-17) and interferon γ (IFN-γ) in brain tissue; AGEs, GSH-ST and hydrogen peroxide contents in brain tissue, of the mice. The content of lead in the blood of mice was detected by inductively coupled plasma-mass spectrometry (ICP-MS). The mice were anesthetized with isoflurane, 200-500uL blood was taken from eyeball and put into 1.5 mL EP tube containing 20uL heparin sodium, which was stored at - 4°C. Take 200uL anticoagulant blood and put it into Teflon digestive tube, add 2mL concentrated nitric acid, and digest it with microwave digestion instrument. After complete digestion, transfer the liquid into 10mL EP tube, and dilute to 5mL with 1% nitric acid for use. TG content in serum of mice was detected by Kit. The color of quinones produced by GPO-PAP method was directly proportional to the content of triglycerides, the OD value was determined by enzyme scale instrument, and the TG content in samples was calculated. Statistical treatment All data were analyzed by SPSS22.0 data analysis software. SNK test was used to compare the groups with homogeneity of variance while Games-Howell test was adopted to compare the groups with irregular variances. When P<0.05, it was considered that the difference was statistically significant. Results Changes of general conditions and body weights of rice The weight changes of the four groups of mice during the experiment are shown in Figure 1. No poisoning or death occurred in all groups during the whole experiment period. There was no significant difference in food intake and drinking water volume. The mice acted normally with smooth hair. The mass of the mice in each group presented an upward trend during the experiment. There was no significant difference for body weights between the control group and the lead exposure group and between the high-fat group and the lead + high-fat group (P>0.05); form the 6th week, the body
weights of the high-fat group and the lead + high-fat group were significantly higher than those of the control group and the lead exposure group, of which the difference was statistically significant (P<0.05). 50
Lead mass (g)
40 30 20 10 0 Before test 实验前
Week 第2周2
Control 对照组 group
Week 第4周4
Lead exposure 铅暴露组 group
Week 6 第6周
Week 8 Week 10 第8周 第10周
High-fat 高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 1 Comparison of Body Weights during Experiment Lead contents in brain tissue of all groups The comparison results of lead content in brain tissue of four groups of mice are shown in Figure 2. There was no significant difference in lead content in the brain tissue between the control group and the high-fat group (P>0.05), and the lead contents in the brain tissues of the two groups were significantly lower than those of the lead exposure group and the lead + high-fat group, of which the difference was statistically significant (P<0.05). 400
Lead content (pg/g)
350 300 250 200 150 100 50 0
Control
对照组 group
Lead exposure 铅暴露组 group
High-fat
高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 2 Comparison of Lead Content in Brain Tissue Lipometabolic product content in serum Table 1 shows the content comparison of serum lipid metabolites of the four groups of mice. The contents of serum TG, HDL and LDL of the high-fat group and
the lead + high-fat group were significantly higher than those of the control group and the lead exposure group, of which there was significant difference (P<0.05); Compared with the control group, the contents of serum TG, HDL and LDL of lead exposure group did not change significantly, of which the difference was not statistically significant (P>0.05). Table 1 Comparison of Lipometabolic Product Content in Serum (mmol/L) Group TG HDL LDL Control group 0.50±0.13 0.71±0.13 0.49±0.15 (n=21) Lead exposure 0.62±0.21 0.63±0.15 0.58±0.12 group (n=21) High-fat group 0.91±0.16① 0.43±0.07① 1.02±0.18① (n=21) Lead + high-fat 0.98±0.25①② 0.37±0.08①② 1.07±0.20①② group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05 Nerve functions of all groups Figure 3 and Table 2 show the comparison of cognitive indexes and learning and memory abilities of the four groups of mice. The cognitive indices of the lead exposure group, the high-fat group and the lead + high-fat group were significantly lower than those of the control group, but the 3d escape latent period was significantly longer than the control group, and the passing platform times significantly decreased, of which the differences were statistically significant (P<0.05); in addition, the 2d and 3d escape latent periods of the lead + high-fat group were significantly longer than those of the lead exposure group and the high-fat group, but the passing platform times decreased significantly, of which the differences were statistically significant (P <0.05).
Cognitive index (%)
0.8
0.6
0.4
0.2
0
Control
对照组 group
Lead exposure 铅暴露组 group
High-fat
高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 3 Comparison of Cognitive Index Table 2 Comparison of Learning and Memory Abilities
Group
1d
Escape latent period (s) 2d
3d
Passing platform times (times)
Control group 65.97±11.24 53.68±11.95 33.01±7.88 4.52±1.37 (n=21) Lead exposure ① 100.13±8.92① 83.33±17.41① 70.14±12.96① 2.63±0.76 group (n=21) High-fat group 104.51±14.03 ① ① ① 69.18±11.21 2.25±0.77 86.73±20.89 ① (n=21) Lead + high-fat 105.26±19.58 103.06±22.79① 91.49±15.84① 1.21±0.63①②③ ① ②③ ②③ group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05 Inflammatory factor content in brain tissue Table 3 and Figure 4 show the comparison results of inflammatory factor content in brain tissue of four groups of mice. The contents of IL-6, IL-17 and IFN-γ in the brain tissues of the lead exposure group, the high-fat group and the lead + high-fat group were significantly higher than that of the control group, of which the differences were statistically significant (P <0.05). In addition, the contents of serum IL-6 and IL-17 in the lead + high-fat group were significantly higher than those of the lead exposure group and the high-fat group, of which the differences were statistically significant (P <0.05). Table 3 Comparison of Inflammatory Factor Contents in Brain Tissue Group IL-6 IL-17 IFN-γ Control group 0.35±0.05 0.27±0.03 0.06±0.01 (n=21) Lead exposure 0.40±0.07① 0.31±0.04① 0.08±0.01① group (n=21) High-fat group 0.39±0.05① 0.30±0.05① 0.08±0.02① (n=21) Lead + high-fat ①②③ ①②③ ① 0.45±0.08 0.35±0.05 0.09±0.02 group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05.
IL-6 IL-17 IFN-y
Content
0.4
0.3
0.2
0.1
0.0
group ad exposure igh-fat group d + high-fat Control H Le Lea
Figure 4 Comparison of IL-6, IL-17 and IFN - γ Contents in Brain Tissues of Four Groups of Mice. AGEs, GSH-ST and hydrogen peroxide contents in brain tissue Table 4 shows the comparison results of AGE, GSH-ST, and hydrogen peroxide contents in brain tissues of four groups of mice. The contents of AGEs in the brain tissues of the lead exposure group, the high-fat group and the lead + high-fat group were significantly higher than that of the control group, but the GSH-ST content decreased, of which the differences were statistically significant (P <0.05). In addition, the contents of AGEs in the lead + high-fat group were significantly higher than those of the lead exposure group and the high-fat group, of which the differences were statistically significant (P <0.05). There was no significant difference (P>0.05) for hydrogen peroxide contents in the brain tissues of all groups. Table 4 Comparison of AGEs, GSH-ST and Hydrogen Peroxide Contents in Brain Tissue Hydrogen peroxide Group AGEs（ng/mg） GSH-ST（U/mg ） （mmol/g ） Control group 10.17±1.12 41.03±7.01 3.09±0.23 (n=21) Lead exposure 11.97±1.20① 26.81±6.73① 3.21±0.26 group (n=21) High-fat group 14.68±1.37① 25.96±8.07① 3.10±0.31 (n=21) Lead + high-fat 17.01±1.31①②③ 25.99±5.49① 3.12±0.27 group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05.
Discussion By comparing the lead exposure and high fat diet mice models, this study has found that the lead content in the brain tissues of the lead exposure group and the lead + high-fat group increased significantly and the body weights of the high-fat group and the lead + high-fat group increased as well. In addition, compared with the control group, the escape latent periods were obviously prolonged and the passing platform times decreased obviously in the other three groups, of which the change was the most significant in the lead + high-fat group. It indicated that lead and high fat diet might lead to decrease of the cognitive ability and learning / memory ability of the mice and obvious damage to the function of the nervous system. Body weight is always adopted to reflect the health condition of laboratory animals during the animal experiment [5]. As one of fat ingredients, TG mainly comes from decomposition of the fat in the in-taken food. TG can be widely accumulated in blood vessels, subcutaneous tissues and visceral organs. Appropriate TG has the effects of keeping body temperature and buffering the pressure, but too much TG in the serum may result in dyslipidemia and atherosclerosis [6, 7]. Main physiological function of HDL is to transport the lipoprotein molecules in the blood to cells of all body tissues, effectively remove the cholesterol in the blood vessels and prevent arteriosclerosis [8]. LDL is a lipoprotein responsible for transporting fatty acid molecules in the blood and its cholesterol level is positively correlated with the incidence of various cardiovascular diseases [9]. The results of this study showed that the body weights of the mice fed with high fat diet increased significantly from the sixth week of the experiment, the contents of serum TG and LDL increased significantly, but the content of HDL decreased significantly. Compared with the high-fat group, the above changes were more obvious in the lead + high-fat group, which means that lead and high fat diet exposure could aggravate the disorder of lipid metabolism in mice. Many studies have shown that lead and high fat diet mainly endanger the cortex and hippocampus areas in brain tissue, damage neurons and cause disorder of the central nervous system [10, 11]. Glial cells in the central nervous system can secrete inflammatory factors such as IL-6, IL-17 and IFN-γ [12, 13]. AGEs can damage normal physiological structure of the protein and destroy protein performance by cross-linking with protein. Meanwhile, AGEs can specifically combine with receptors on the cell surface, changing physiological function of cells [14]. GSH-ST can catalyze the binding of glutathione with intermediate products transformed by exogenous chemical poisons in living bodies, and thus play a role in detoxification and protecting DNA from being damaged [15]. This study has found that, compared with the control group, the IL-6, IL-17 and IFN-γ contents in serum of other three groups increased significantly, and the lead + high-fat group were the most obvious; in addition, compared with the control group, the content of AGEs in other three groups increased significantly, but GSH-ST content decreased obviously, of which the high-fat group showed the most obvious change, suggesting that lead and high fat diet would aggravate oxidative stress and inflammatory reaction and damage the central nervous system in mice. The results of this study are basically consistent with those of
Mcdermott et al. The ability of autonomous inquiry of obese mice was decreased, and the retention time of lead exposure obese mice in the central area was more obvious than that of alone obese and lead exposure mice. In conclusion, the combined exposure of lead and high fat diet can cause lipid metabolism disorder and has obvious damage to cognitive ability and central nervous system, of which the mechanism is that lead and high fat diet promote glial cells to secrete inflammatory factors and induce inflammatory reaction in brain tissue to cause the damage, inhibit GSH-ST expression and increase AGEs, which further aggravate the injury of brain tissue. This study provides a basis for the prevention of neurological damage in lead-exposed population. In addition, this study is aimed at obese people, which has a strong pertinence, and can provide suggestions for the daily life habits of obese people. However, the related signaling pathways of Th17, Treg and other inflammatory cells involved in the occurrence and development of neurodegenerative diseases need to be further explored, which will be the specific direction of follow-up studies. References [1] Xing L I, Ning L I, Sun H L, et al. Maternal Lead Exposure Induces Down-regulation of Hippocampal Insulin-degrading Enzyme and Nerve Growth Factor Expression in Mouse Pups[J]. Biomedical and Environmental Sciences, 2017, 30(3):215-219. [2] Mao Weifeng, Yang Dajin, Sui Haixia, et al. Assessment of Lead Exposure Risk in Diets of Chinese Adult Residents [J]. Chinese Journal of Food Hygiene, 2016, 28 (1): 107-110. [3] Owein G L, Julien M, Amandine E, et al. High-fat diet feeding differentially affects the development of inflammation in the central nervous system:[J]. Journal of Neuroinflammation, 2016, 13(1):206. [4] Raider K, Ma D, Harris J L, et al. A high fat diet alters metabolic and bioenergetic function in the brain: A magnetic resonance spectroscopy study[J]. Neurochemistry International, 2016, 97:172. [5] Wu Xiang, Hu Yaohua, Xing Hongyu, et al. Effect of Resveratrol on Expression of Inflammatory Factors Related to Pulmonary Hypertension of Experimental Rabbits [J]. China Journal of Modern Medicine, 2016, 26 (23): 10-15. [6] Khetarpal S A, Rader D J. Triglyceride-Rich Lipoproteins and Coronary Artery Disease Risk[J]. Arteriosclerosis Thrombosis & Vascular Biology, 2015, 35(2):3-9. [7] Nordestgaard B G. Triglyceride-Rich Lipoproteins and Atherosclerotic Cardiovascular Disease: New Insights From Epidemiology, Genetics, and Biology[J]. Circulation Research, 2016, 118(4):547-563. [8] Quispe R, Martin S S, Jones S R. Triglycerides to high-density lipoprotein-cholesterol ratio, glycemic control and cardiovascular risk in obese patients with type 2 diabetes.[J]. Current Opinion in Endocrinology Diabetes & Obesity, 2016, 23(2):1. [9] Leibowitz M, Karpati T, Cohenstavi C J, et al. Association Between Achieved
Low-Density Lipoprotein Levels and Major Adverse Cardiac Events in Patients With Stable Ischemic Heart Disease Taking Statin Treatment.[J]. Jama Intern Med, 2016, 176(8):1105-1113. [10] Kang Beipei, Zhao Fang, Su Peng, et al. Role of XIAP Expression Change in Lead-induced Mouse Hippocampal Neuron Cell Injury [J]. Practical Preventive Medicine, 2015, 22 (5): 513-516. [11] Petrov D, Pedrós I, Artiach G, et al. High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents.[J]. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2015, 1852(9):1687-1699. [12] Tang Tingting, Yu Wenfeng, Guan Zhizhong. Enhanced Expression of Inflammatory Cytokines and Nuclear Factor-κB in Microglia by Overdose Fluoride [J]. Chinese Journal of Endemiology, 2015, 34 (11): 785-789. [13] Wang J B, Li H, Wang L L, et al. Role of IL-1β, IL-6, IL-8 and IFN-γ in pathogenesis of central nervous system neuropsychiatric systemic lupus erythematous[J]. International Journal of Clinical & Experimental Medicine, 2015, 8(9):16658. [14] Song J, Lee W T, Park K A, et al. Receptor for advanced glycation end products (RAGE) and its ligands: focus on spinal cord injury.[J]. International Journal of Molecular Sciences, 2014, 15(8):13172. [15] Roncalli V, Cieslak M C, Passamaneck Y, et al. Glutathione S-Transferase (GST) Gene Diversity in the Crustacean Calanus finmarchicus--Contributors to Cellular Detoxification.[J]. Plos One, 2015, 10(5):e0123322.
Abbreviations AGEs: advanced glycation end products GSH-ST: glutathione S-transferase HDL: high-density lipoprotein ICP-MS: inductively coupled plasma mass spectrometry IFN-γ: interferon γ IL-17: interleukin 17 IL-6: interleukin 6 LDL: low-density lipoprotein TG: triglyceride
S1878-8750(20)30183-2
DOI:
https://doi.org/10.1016/j.wneu.2020.01.165
Reference:
WNEU 14195
To appear in:
World Neurosurgery
Received Date: 26 December 2019 Revised Date:
19 January 2020
Accepted Date: 20 January 2020
Please cite this article as: Huang Y, Yun K, Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice, World Neurosurgery (2020), doi: https:// doi.org/10.1016/j.wneu.2020.01.165. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Study on Effects and Mechanism of Lead and High Fat Diet on Cognitive Function and Central Nervous System in Mice Yiwei Huang1, Keming Yun2* 1 Department of Neurology, Zhuji Affiliated Hospital of Shaoxing University, Zhuji, Shaoxing311800, Zhejiang, China; 2 College of Forensic Medicine, Shanxi Medical University, Taiyuan City, 030001, Shanxi Province, China. * Corresponding author: Keming Yun College of Forensic Medicine, Shanxi Medical University, No.56 Xinjiannan Road, Yingze District, Taiyuan, 030001, Shanxi, China. Email: [email protected] Abstract Objective: To investigate the effects and mechanism of lead and high fat diet on cognitive function and central nervous system in mice. Methods: 84 healthy male mice were randomly divided into a control group (n=21) (fed with common diet and free drinking), a lead exposure group (n=21) (fed with common diet and 300mg/L lead acetate solution), a high-fat group (n=21) (fed with high-fat diet and free drinking) and a lead + high-fat group (n=21) (fed with high-fat diet and 300mg/L lead acetate solution). In 10 weeks after lead exposure, the mice of all groups were tested for the cognition, learning and memory abilities, body weight, serum triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL); and also for the contents of lead, interleukin 6 (IL-6), interleukin 17 (IL-17), interferon γ (IFN-γ), advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST) and hydrogen peroxide in the brain tissues. Results: Compared with the control group and the lead-exposed group, the body weights of mice in the high-fat group and the lead + high-fat group increased significantly from the sixth week of the experiment, of which the difference was statistically significant (P<0.05); Compared with the control group and the high-fat group, the lead content in brain tissue of the lead exposure group and the lead + high-fat group increased significantly, of which the difference was statistically significant (P<0.05); Compared with the control group, the escape latent period, TG, LDL, IL-6, IL-17, IFN-γ and AGEs of the remaining three groups increased significantly, but the recognition index, passing platform times, HDL and GSH-ST significantly decreased (P<0.05); the 2nd and 3rd escape latent periods, IL-6, IL-17 and AGEs of lead + high-fat group were obviously higher than the remaining three groups, but the passing platform times were obviously lower than the remaining three groups, of which the difference was statistically significant; The content of hydrogen peroxide in brain tissues had no difference among groups (P>0.05). Conclusion: The lead and high fat diet have resulted in the lipid metabolism disorders and impair the cognitive function and central nervous system by promoting the
secretion of inflammatory factors in glial cells, inducing the inflammatory reaction of brain tissue, inhibiting GSH-ST expression and increasing AGEs content. Keywords: Lead exposure; High fat diet; Cognitive function; Central nervous system; Mechanism
Introduction Lead is a kind of neurotoxic heavy metal. It has been found in studies that lead in the environment can be stored in central nervous system to damage central nervous system and cause cognitive function defects and destroys [1]. Because lead is widely used in production and life, lead exposure has become an increasingly serious environmental and medical problem [2]. Central nervous system is the main target organ of lead injury. Lead exposure can affect the ability of learning and memory, and damage the sensory organs and their innervation. At present, there are many researches on the mechanism of lead nerve injury. For instance, lead can induce oxidative stress to induce cell injury, apoptosis, excitatory toxicity and so on. In addition, high fat diet can result in a significant decrease in the expression level of the hippocampus-related functional proteins and damage hippocampal neurons, thereby affecting the function of the nervous system and causing inattention, decline of learning ability, cognitive function disorder and other symptoms [3, 4]. At present, it is still unclear for the mechanism of the combined effect of lead and high fat diet on the function of central nervous system. There are many studies on the damage of lead and high-fat diet to cerebral cortex, hippocampus, neurons, and central nervous system disorders, but there are few studies on the causes. In this study, the mouse models of lead and high fat diet exposure were established to investigate the changes and mechanisms of cognitive function and central nervous function after the effect of the lead and high fat diet. Furthermore, it provides new basis and target for the prevention of neural injury in the lead-exposed occupational population, especially in the obese population. The innovation of this paper is to take the obese people as the main research object, which has obvious pertinence. Materials and methods Animals 84 healthy adult male adult mice of Grade SPF were selected from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Jing) 2009-0004) and the weight of each mouse was 12-17g. All mice were fed in the independent ventilated animal feeding cage of the laboratory. During the experiment, the temperature of the laboratory was maintained at (23±2)℃, the humidity at (55±5)% and the light time for 12h. All mice were fed with the same common feed and drinking water for one week and then averagely divided into 4 groups by the method of random number table, namely control group, lead exposure group, high-fat group and lead + high-fat group, and each group had 21 mice. There was no significant difference in general data (body weight and monthly age) of the mice in each group. The animal experiments and animal treatment processes involved in this study meet the requirements of animal
experiment standardization and the protocol was approved by the ethics committee of our hospital. Grouping and treatment The control group was fed with ordinary feed and normal drinking water; the lead exposure group with ordinary feed and lead acetate solution (300mg/L); the high-fat group with high fat feed and normal drinking water; the lead + high-fat group with high fat feed and lead acetate solution (300mg/L). For all mice, the diet was not restricted but supplemented in time. The general condition of the mice was observed every day, their diets were recorded respectively and all mice were weighed every 2 weeks. After continuous exposure for 10 weeks, neurological function tests were carried out in each group of mice to test their cognitive, learning and memory abilities. Enzyme linked immunosorbent assay The mice were and then sacrificed to take the serum and brain tissue samples. The contents of serum lipometabolic product and inflammatory factor in the brain tissue were tested by the enzyme linked immunosorbent assay; the corresponding kit was selected to determine the contents of advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST) and hydrogen peroxide in the brain tissue in strict accordance with the instructions, and the content of lead in brain tissue was tested by inductively coupled plasma mass spectrometry (ICP-MS). Testing for cognitive ability 24h before training, firstly put the mice in a special experimental device to freely adapt to the environment, then placed two objects in the same shape, size and color at the left and right ends on a side of the box, put the mice into the box with their backs against the objects, and recorded the cognitive time that the mice respectively smelled, watched and touched the two objects in 3min. The test was carried out in 24h after the training. Replaced one object in the box by a new one with different shape and size, kept other methods similar to the training period and recorded the cognitive time of the mice for two new and old objects. During the whole experiment, it was necessary to keep quiet indoors. After each experiment, washed the device thoroughly with 75% alcohol for fear that odor might affect the test results of mice. Cognitive index (%) = cognitive time of new object / (cognitive time of new object + cognitive time of old object) x 100%. Morris water maze The mice were tested by Morris water maze for Learning and memory ability. Before the beginning of the formal experiment, the adaptive training was carried out for the mice for 1d and the mice were put into the water maze at the 4-quadrant starting point with their faces towards the pool wall. Then, they could freely find the underwater platform and the activity track in 2min was recorded. The formal experiment totally lasted for 4d. In the first 3d, the fixed navigation experiment was
carried out to measure their learning ability. The methods were the same as those of the training period. The time of finding the platform was recorded, namely escape latent period. If the mice did not find the platform in 2min, they were guided to the platform to stay 5s and the escape latent period was recorded as 120s. On the final 1d, the space exploration experiment was carried out to measure the spatial memory ability of mice. The underwater platform was removed and the experimental mice were put in from the opposite quadrant of the platform with their faces towards the pool wall, and the times that the mice passed through the platform in 1min were observed and recorded. Observation indices Include general conditions and body weights; lead content in brain tissue; serum lipid metabolism index, including triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL); neural function, including cognitive ability and learning / memory ability; content of inflammatory factor in brain tissue, including interleukin 6 (IL-6), interleukin 17 (IL-17) and interferon γ (IFN-γ) in brain tissue; AGEs, GSH-ST and hydrogen peroxide contents in brain tissue, of the mice. The content of lead in the blood of mice was detected by inductively coupled plasma-mass spectrometry (ICP-MS). The mice were anesthetized with isoflurane, 200-500uL blood was taken from eyeball and put into 1.5 mL EP tube containing 20uL heparin sodium, which was stored at - 4°C. Take 200uL anticoagulant blood and put it into Teflon digestive tube, add 2mL concentrated nitric acid, and digest it with microwave digestion instrument. After complete digestion, transfer the liquid into 10mL EP tube, and dilute to 5mL with 1% nitric acid for use. TG content in serum of mice was detected by Kit. The color of quinones produced by GPO-PAP method was directly proportional to the content of triglycerides, the OD value was determined by enzyme scale instrument, and the TG content in samples was calculated. Statistical treatment All data were analyzed by SPSS22.0 data analysis software. SNK test was used to compare the groups with homogeneity of variance while Games-Howell test was adopted to compare the groups with irregular variances. When P<0.05, it was considered that the difference was statistically significant. Results Changes of general conditions and body weights of rice The weight changes of the four groups of mice during the experiment are shown in Figure 1. No poisoning or death occurred in all groups during the whole experiment period. There was no significant difference in food intake and drinking water volume. The mice acted normally with smooth hair. The mass of the mice in each group presented an upward trend during the experiment. There was no significant difference for body weights between the control group and the lead exposure group and between the high-fat group and the lead + high-fat group (P>0.05); form the 6th week, the body
weights of the high-fat group and the lead + high-fat group were significantly higher than those of the control group and the lead exposure group, of which the difference was statistically significant (P<0.05). 50
Lead mass (g)
40 30 20 10 0 Before test 实验前
Week 第2周2
Control 对照组 group
Week 第4周4
Lead exposure 铅暴露组 group
Week 6 第6周
Week 8 Week 10 第8周 第10周
High-fat 高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 1 Comparison of Body Weights during Experiment Lead contents in brain tissue of all groups The comparison results of lead content in brain tissue of four groups of mice are shown in Figure 2. There was no significant difference in lead content in the brain tissue between the control group and the high-fat group (P>0.05), and the lead contents in the brain tissues of the two groups were significantly lower than those of the lead exposure group and the lead + high-fat group, of which the difference was statistically significant (P<0.05). 400
Lead content (pg/g)
350 300 250 200 150 100 50 0
Control
对照组 group
Lead exposure 铅暴露组 group
High-fat
高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 2 Comparison of Lead Content in Brain Tissue Lipometabolic product content in serum Table 1 shows the content comparison of serum lipid metabolites of the four groups of mice. The contents of serum TG, HDL and LDL of the high-fat group and
the lead + high-fat group were significantly higher than those of the control group and the lead exposure group, of which there was significant difference (P<0.05); Compared with the control group, the contents of serum TG, HDL and LDL of lead exposure group did not change significantly, of which the difference was not statistically significant (P>0.05). Table 1 Comparison of Lipometabolic Product Content in Serum (mmol/L) Group TG HDL LDL Control group 0.50±0.13 0.71±0.13 0.49±0.15 (n=21) Lead exposure 0.62±0.21 0.63±0.15 0.58±0.12 group (n=21) High-fat group 0.91±0.16① 0.43±0.07① 1.02±0.18① (n=21) Lead + high-fat 0.98±0.25①② 0.37±0.08①② 1.07±0.20①② group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05 Nerve functions of all groups Figure 3 and Table 2 show the comparison of cognitive indexes and learning and memory abilities of the four groups of mice. The cognitive indices of the lead exposure group, the high-fat group and the lead + high-fat group were significantly lower than those of the control group, but the 3d escape latent period was significantly longer than the control group, and the passing platform times significantly decreased, of which the differences were statistically significant (P<0.05); in addition, the 2d and 3d escape latent periods of the lead + high-fat group were significantly longer than those of the lead exposure group and the high-fat group, but the passing platform times decreased significantly, of which the differences were statistically significant (P <0.05).
Cognitive index (%)
0.8
0.6
0.4
0.2
0
Control
对照组 group
Lead exposure 铅暴露组 group
High-fat
高脂组 group
Lead + high-fat 铅+高脂组 group
Figure 3 Comparison of Cognitive Index Table 2 Comparison of Learning and Memory Abilities
Group
1d
Escape latent period (s) 2d
3d
Passing platform times (times)
Control group 65.97±11.24 53.68±11.95 33.01±7.88 4.52±1.37 (n=21) Lead exposure ① 100.13±8.92① 83.33±17.41① 70.14±12.96① 2.63±0.76 group (n=21) High-fat group 104.51±14.03 ① ① ① 69.18±11.21 2.25±0.77 86.73±20.89 ① (n=21) Lead + high-fat 105.26±19.58 103.06±22.79① 91.49±15.84① 1.21±0.63①②③ ① ②③ ②③ group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05 Inflammatory factor content in brain tissue Table 3 and Figure 4 show the comparison results of inflammatory factor content in brain tissue of four groups of mice. The contents of IL-6, IL-17 and IFN-γ in the brain tissues of the lead exposure group, the high-fat group and the lead + high-fat group were significantly higher than that of the control group, of which the differences were statistically significant (P <0.05). In addition, the contents of serum IL-6 and IL-17 in the lead + high-fat group were significantly higher than those of the lead exposure group and the high-fat group, of which the differences were statistically significant (P <0.05). Table 3 Comparison of Inflammatory Factor Contents in Brain Tissue Group IL-6 IL-17 IFN-γ Control group 0.35±0.05 0.27±0.03 0.06±0.01 (n=21) Lead exposure 0.40±0.07① 0.31±0.04① 0.08±0.01① group (n=21) High-fat group 0.39±0.05① 0.30±0.05① 0.08±0.02① (n=21) Lead + high-fat ①②③ ①②③ ① 0.45±0.08 0.35±0.05 0.09±0.02 group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05.
IL-6 IL-17 IFN-y
Content
0.4
0.3
0.2
0.1
0.0
group ad exposure igh-fat group d + high-fat Control H Le Lea
Figure 4 Comparison of IL-6, IL-17 and IFN - γ Contents in Brain Tissues of Four Groups of Mice. AGEs, GSH-ST and hydrogen peroxide contents in brain tissue Table 4 shows the comparison results of AGE, GSH-ST, and hydrogen peroxide contents in brain tissues of four groups of mice. The contents of AGEs in the brain tissues of the lead exposure group, the high-fat group and the lead + high-fat group were significantly higher than that of the control group, but the GSH-ST content decreased, of which the differences were statistically significant (P <0.05). In addition, the contents of AGEs in the lead + high-fat group were significantly higher than those of the lead exposure group and the high-fat group, of which the differences were statistically significant (P <0.05). There was no significant difference (P>0.05) for hydrogen peroxide contents in the brain tissues of all groups. Table 4 Comparison of AGEs, GSH-ST and Hydrogen Peroxide Contents in Brain Tissue Hydrogen peroxide Group AGEs（ng/mg） GSH-ST（U/mg ） （mmol/g ） Control group 10.17±1.12 41.03±7.01 3.09±0.23 (n=21) Lead exposure 11.97±1.20① 26.81±6.73① 3.21±0.26 group (n=21) High-fat group 14.68±1.37① 25.96±8.07① 3.10±0.31 (n=21) Lead + high-fat 17.01±1.31①②③ 25.99±5.49① 3.12±0.27 group (n=21) Note: compared with the control group, ① P<0.05; compared with the lead exposure group, ② P<0.05; compared with the high-fat group, ③ P<0.05.
Discussion By comparing the lead exposure and high fat diet mice models, this study has found that the lead content in the brain tissues of the lead exposure group and the lead + high-fat group increased significantly and the body weights of the high-fat group and the lead + high-fat group increased as well. In addition, compared with the control group, the escape latent periods were obviously prolonged and the passing platform times decreased obviously in the other three groups, of which the change was the most significant in the lead + high-fat group. It indicated that lead and high fat diet might lead to decrease of the cognitive ability and learning / memory ability of the mice and obvious damage to the function of the nervous system. Body weight is always adopted to reflect the health condition of laboratory animals during the animal experiment [5]. As one of fat ingredients, TG mainly comes from decomposition of the fat in the in-taken food. TG can be widely accumulated in blood vessels, subcutaneous tissues and visceral organs. Appropriate TG has the effects of keeping body temperature and buffering the pressure, but too much TG in the serum may result in dyslipidemia and atherosclerosis [6, 7]. Main physiological function of HDL is to transport the lipoprotein molecules in the blood to cells of all body tissues, effectively remove the cholesterol in the blood vessels and prevent arteriosclerosis [8]. LDL is a lipoprotein responsible for transporting fatty acid molecules in the blood and its cholesterol level is positively correlated with the incidence of various cardiovascular diseases [9]. The results of this study showed that the body weights of the mice fed with high fat diet increased significantly from the sixth week of the experiment, the contents of serum TG and LDL increased significantly, but the content of HDL decreased significantly. Compared with the high-fat group, the above changes were more obvious in the lead + high-fat group, which means that lead and high fat diet exposure could aggravate the disorder of lipid metabolism in mice. Many studies have shown that lead and high fat diet mainly endanger the cortex and hippocampus areas in brain tissue, damage neurons and cause disorder of the central nervous system [10, 11]. Glial cells in the central nervous system can secrete inflammatory factors such as IL-6, IL-17 and IFN-γ [12, 13]. AGEs can damage normal physiological structure of the protein and destroy protein performance by cross-linking with protein. Meanwhile, AGEs can specifically combine with receptors on the cell surface, changing physiological function of cells [14]. GSH-ST can catalyze the binding of glutathione with intermediate products transformed by exogenous chemical poisons in living bodies, and thus play a role in detoxification and protecting DNA from being damaged [15]. This study has found that, compared with the control group, the IL-6, IL-17 and IFN-γ contents in serum of other three groups increased significantly, and the lead + high-fat group were the most obvious; in addition, compared with the control group, the content of AGEs in other three groups increased significantly, but GSH-ST content decreased obviously, of which the high-fat group showed the most obvious change, suggesting that lead and high fat diet would aggravate oxidative stress and inflammatory reaction and damage the central nervous system in mice. The results of this study are basically consistent with those of
Mcdermott et al. The ability of autonomous inquiry of obese mice was decreased, and the retention time of lead exposure obese mice in the central area was more obvious than that of alone obese and lead exposure mice. In conclusion, the combined exposure of lead and high fat diet can cause lipid metabolism disorder and has obvious damage to cognitive ability and central nervous system, of which the mechanism is that lead and high fat diet promote glial cells to secrete inflammatory factors and induce inflammatory reaction in brain tissue to cause the damage, inhibit GSH-ST expression and increase AGEs, which further aggravate the injury of brain tissue. This study provides a basis for the prevention of neurological damage in lead-exposed population. In addition, this study is aimed at obese people, which has a strong pertinence, and can provide suggestions for the daily life habits of obese people. However, the related signaling pathways of Th17, Treg and other inflammatory cells involved in the occurrence and development of neurodegenerative diseases need to be further explored, which will be the specific direction of follow-up studies. References [1] Xing L I, Ning L I, Sun H L, et al. Maternal Lead Exposure Induces Down-regulation of Hippocampal Insulin-degrading Enzyme and Nerve Growth Factor Expression in Mouse Pups[J]. Biomedical and Environmental Sciences, 2017, 30(3):215-219. [2] Mao Weifeng, Yang Dajin, Sui Haixia, et al. Assessment of Lead Exposure Risk in Diets of Chinese Adult Residents [J]. Chinese Journal of Food Hygiene, 2016, 28 (1): 107-110. [3] Owein G L, Julien M, Amandine E, et al. High-fat diet feeding differentially affects the development of inflammation in the central nervous system:[J]. Journal of Neuroinflammation, 2016, 13(1):206. [4] Raider K, Ma D, Harris J L, et al. A high fat diet alters metabolic and bioenergetic function in the brain: A magnetic resonance spectroscopy study[J]. Neurochemistry International, 2016, 97:172. [5] Wu Xiang, Hu Yaohua, Xing Hongyu, et al. Effect of Resveratrol on Expression of Inflammatory Factors Related to Pulmonary Hypertension of Experimental Rabbits [J]. China Journal of Modern Medicine, 2016, 26 (23): 10-15. [6] Khetarpal S A, Rader D J. Triglyceride-Rich Lipoproteins and Coronary Artery Disease Risk[J]. Arteriosclerosis Thrombosis & Vascular Biology, 2015, 35(2):3-9. [7] Nordestgaard B G. Triglyceride-Rich Lipoproteins and Atherosclerotic Cardiovascular Disease: New Insights From Epidemiology, Genetics, and Biology[J]. Circulation Research, 2016, 118(4):547-563. [8] Quispe R, Martin S S, Jones S R. Triglycerides to high-density lipoprotein-cholesterol ratio, glycemic control and cardiovascular risk in obese patients with type 2 diabetes.[J]. Current Opinion in Endocrinology Diabetes & Obesity, 2016, 23(2):1. [9] Leibowitz M, Karpati T, Cohenstavi C J, et al. Association Between Achieved
Low-Density Lipoprotein Levels and Major Adverse Cardiac Events in Patients With Stable Ischemic Heart Disease Taking Statin Treatment.[J]. Jama Intern Med, 2016, 176(8):1105-1113. [10] Kang Beipei, Zhao Fang, Su Peng, et al. Role of XIAP Expression Change in Lead-induced Mouse Hippocampal Neuron Cell Injury [J]. Practical Preventive Medicine, 2015, 22 (5): 513-516. [11] Petrov D, Pedrós I, Artiach G, et al. High-fat diet-induced deregulation of hippocampal insulin signaling and mitochondrial homeostasis deficiences contribute to Alzheimer disease pathology in rodents.[J]. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2015, 1852(9):1687-1699. [12] Tang Tingting, Yu Wenfeng, Guan Zhizhong. Enhanced Expression of Inflammatory Cytokines and Nuclear Factor-κB in Microglia by Overdose Fluoride [J]. Chinese Journal of Endemiology, 2015, 34 (11): 785-789. [13] Wang J B, Li H, Wang L L, et al. Role of IL-1β, IL-6, IL-8 and IFN-γ in pathogenesis of central nervous system neuropsychiatric systemic lupus erythematous[J]. International Journal of Clinical & Experimental Medicine, 2015, 8(9):16658. [14] Song J, Lee W T, Park K A, et al. Receptor for advanced glycation end products (RAGE) and its ligands: focus on spinal cord injury.[J]. International Journal of Molecular Sciences, 2014, 15(8):13172. [15] Roncalli V, Cieslak M C, Passamaneck Y, et al. Glutathione S-Transferase (GST) Gene Diversity in the Crustacean Calanus finmarchicus--Contributors to Cellular Detoxification.[J]. Plos One, 2015, 10(5):e0123322.
Abbreviations AGEs: advanced glycation end products GSH-ST: glutathione S-transferase HDL: high-density lipoprotein ICP-MS: inductively coupled plasma mass spectrometry IFN-γ: interferon γ IL-17: interleukin 17 IL-6: interleukin 6 LDL: low-density lipoprotein TG: triglyceride