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煙酸對心理應激適應能力的影響及機制研究
田惠光 于志杰 程義勇 王冬蘭 李樹(shù)田 陳偉強
天津市衛生局公共衛生監督所，天津 300050；天津衛生學(xué)環(huán)境醫學(xué)研究所，天津 300050
1前言
心理應激誘發(fā)的神經(jīng)內分泌系統反應涉及到體內多種激素和神經(jīng)遞質(zhì)、累及多個(gè)系統和器官，因而對機體健康的影響很大。大量研究表明，長(cháng)期的或嚴重的應激反應在多種疾病的發(fā)生、發(fā)展中有重要作用，精神異常、心臟病、糖尿病、癌癥、高血壓等都與心理應激的影響密切相關(guān)。
煙酸也稱(chēng)為維生素PP或維生素B3是尼克酸和煙酰胺（尼克酸酰胺）及具有煙酰胺生物活性的衍生物的統稱(chēng)，是碳水化合物和脂肪代謝的關(guān)鍵酶。煙酸在體內主要以煙酰胺腺嘌呤二核苷酸（NAD）和煙酰胺腺嘌呤二核苷酸磷酸（NADP）的形式發(fā)揮生理功能。研究表明，體內大約有200多種酶需要NAD和NADP的輔助在氧化還原反應中傳遞電子。早期研究發(fā)現煙酸缺乏糙皮病可同時(shí)伴隨神經(jīng)病癥狀如抑郁、無(wú)情感、頭痛疲勞及喪失記憶，表明煙酸缺乏與心理行為改變密切相關(guān)；近年國外有研究發(fā)現應激反應與煙酰胺代謝過(guò)程相關(guān)，煙酰胺影響神經(jīng)遞質(zhì)作用并增強神經(jīng)系統的抗氧化作用，還有報道煙酰胺具有阻止腦細胞凋亡、壞死的功能。深入了解不同心理應激狀態(tài)下的煙酸營(yíng)養水平，了解補充不同劑量煙酸對應激適應性的影響，將為制定現代社會(huì )我國居民煙酸供給量標準提供科學(xué)依據，有助于提高我國居民的膳食營(yíng)養和身體健康水平。
為了認識機體煙酸營(yíng)養狀況與心理應激的關(guān)系，我們采用動(dòng)物實(shí)驗、人體干預研究和體外神經(jīng)細胞培養相結合的方法，系統觀(guān)察了心理應激對機體煙酸代謝的影響及補充煙酸對心理應激適應能力的影響和可能的作用機制。
2材料和方法
選用雄性Wistar 大鼠１００只，按體重隨機分為10組即對照組，對喂對照組, 煙酰胺缺乏對照組,低劑量煙酰胺補充對照組，高劑量煙酰胺補充對照組和相應應激組（表1）。動(dòng)物飼料根據Zhang等（1993）和Rawling等（1994）報導的配方修改后配制而成。經(jīng)實(shí)驗飼料喂養一周后，對應激組大鼠進(jìn)行束縛應激：每日６h，共２１d。
Table 1 Classification and treatment of rats
|
Study group |
NAM supplement |
Treatment |
|
(abbreviation) |
(mg/kg of diet) |
(number of rats) |
|
Control (C) |
20 |
Un-restrained (10) |
|
Restrained (10) | ||
|
Pair-fed (PF) |
20 |
Un-restrained (10) |
|
Restrained (10) | ||
|
NAM deficiency (ND) |
Not Available |
Un-restrained (10) |
|
Restrained (10) | ||
|
Low-dose
NAM supplement (LNS) |
40 |
Un-restrained (10) |
|
Restrained (10) | ||
|
High-dose
NAM supplement (HNS) |
500 |
Un-restrained (10) |
|
Restrained (10) |
Note, NAM: nicotinamide.
每周用電子天平（最大量程：2000g，精確度0.5g）對實(shí)驗大鼠稱(chēng)量體重。于第一、二、五周末，固定將對照組、應激對照組、煙酰胺缺乏對照組、煙酰胺缺乏應激組、低劑量煙酰胺補充對照組和低劑量煙酰胺補充應激組大鼠各７只置不銹鋼代謝籠內，收集２４h尿液。
束縛應激后２４h，各組大鼠進(jìn)行曠場(chǎng)實(shí)驗（open-field test）。曠場(chǎng)實(shí)驗箱為一個(gè)100cm´100cm´50cm的箱體，箱底畫(huà)有２５個(gè)相等方格。曠場(chǎng)實(shí)驗進(jìn)行３min，將大鼠置于實(shí)驗箱中央格內，觀(guān)察并記錄其在中央格內的停留時(shí)間／潛伏期（latency），以穿格數（crossing）作為水平運動(dòng)得分、直立次數（rearing）作為垂直運動(dòng)得分并記錄修飾次數（modification）。另外還記錄了排便數。
皮質(zhì)醇和胰島素含量用北方生物技術(shù)研究所生產(chǎn)的放免試劑盒測定。尿中N1－甲基煙酰胺（NMN）采用熒光法（王喜生等，１９８7）。血漿總抗氧化能力、一氧化氮含量及肝臟一氧化氮合酶用南京建成生物工程研究所生產(chǎn)的試劑盒測定。血糖含量用中生北控生物科技股份有限公司生產(chǎn)的葡萄糖氧化酶法試劑盒測定。海馬組織中精氨酸含量用酸水解法測定。肝組織金屬硫蛋白含量用鎘飽和法測定。
2.2 人體干預研究
公開(kāi)招募受試對象。85人應征。參試條件設定為：男性，年齡：17-35歲，無(wú)肝病或胃部疾病史，實(shí)驗前一周內無(wú)感冒，發(fā)熱或影響參加實(shí)驗的外傷或軀體障礙；實(shí)驗前六個(gè)月內無(wú)嚴重精神刺激（包括：親屬不幸事件，本人或家庭重大變故等）。
經(jīng)排除女性及35歲以上者，68名17－35歲男性同意參加實(shí)驗。在告知實(shí)驗詳細內容后，受試者被隨機分為兩組，干預組35名，對照組33名；干預組給予NAM（20mg/d），對照組給予相同量淀粉（安慰劑），服用十天；共58名受試者完成了全部試驗程序，干預組30名，對照組28名。
采用隨機對照研究方法。實(shí)驗前，確定了參試人員的年齡、受教育程度、身高、體重、血壓、脈搏并進(jìn)行了隨機分組。采用問(wèn)卷調查、癥狀自評量表（SCL-90）評定、復雜視覺(jué)反應和數字記憶廣度測試對實(shí)驗后兩組有關(guān)心理指征進(jìn)行了比較。收集了實(shí)驗前后的血漿和淋巴細胞；實(shí)驗前后及實(shí)驗中期的晨尿。
2.3 體外神經(jīng)細胞培養
無(wú)菌條件下,采用Sprague-Dawley胎鼠（孕18天）進(jìn)行海馬神經(jīng)元原代培養。在海馬細胞培養的第7天添加干預因素（CORT和NAM）。干預因素加入48h后，測定細胞存活率（MTT法）。每組設6個(gè)復孔，實(shí)驗重復兩次。
用實(shí)驗飼料喂養一周后，不同實(shí)驗組大鼠尿NMN排泄量出現顯著(zhù)變化（表３）。對照組和缺乏組大鼠尿NMN排泄量顯著(zhù)降低，而低劑量補充組顯著(zhù)升高。尿NMN排泄量反映機體煙酸營(yíng)養的充盈程度，實(shí)驗飼料喂養一周后，缺乏組大鼠尿NMN排泄量?jì)H為對照組的1/4已處于煙酸營(yíng)養缺乏狀態(tài)但并未表現出明顯的缺乏癥狀。經(jīng)過(guò)隨后三周的束縛應激，缺乏組和對照應激組大鼠尿NMN排泄量無(wú)明顯變化，補充組大鼠尿NMN排泄量繼續升高。與其相應的對照組比較，對照應激組和補充應激組大鼠的尿NMN排泄量都顯著(zhù)降低，表明應激時(shí)增加了煙酰胺的消耗。應激處理后，缺乏應激組大鼠尿NMN排泄量無(wú)明顯變化，可能提示體內煙酸的耗竭。我們觀(guān)察到在實(shí)驗第四周（束縛應激兩周）時(shí)缺乏組大鼠出現煙酸缺乏癥狀，表現為活動(dòng)減少，毛色暗且粗糙，口周和爪部皮膚粗糙發(fā)紅，實(shí)驗后期甚至有血液滲出等明顯的糙皮病癥狀。
應激處理后，應激組大鼠的血漿皮質(zhì)醇含量與對照組相比明顯升高（圖1）。不同的煙酰胺營(yíng)養狀況與大鼠的血漿皮質(zhì)醇含量也有顯著(zhù)的相關(guān)性。對照組大鼠的血漿皮質(zhì)醇含量最高，低劑量補充組大鼠的含量最低（P <0.001）。
Table 2 Urinary excretion of N1-methylnicotinamide in rats with different nicotinamide nutritures and stress status during the study period (mg/24h, Mean ± SD, n=7)
|
Study group/period |
Week 1 |
Week 2 |
Week 5 | |||
|
|
|
| ||||
|
Un-restrained |
50.8 ± 13.8 |
37.8 ± 13.4 |
45.3 ± 12.2 | |||
|
Restrained |
53.4 ± 15.1 |
31.9 ± 11.9 |
31.5 ± 14.6 | |||
|
|
|
| ||||
|
Un-restrained |
59.2 ± 22.2 |
9.6 ± 4.2 |
9.9 ± 3.6 | |||
|
Restrained |
51.5 ± 15.2 |
8.2 ± 7.4 |
10.3 ± 2.1 | |||
|
NAM supplement1 |
|
|
| |||
|
Un-restrained |
55.1 ± 13.8 |
78.9 ± 46.9 |
135.0 ± 39.6 | |||
|
Restrained |
57.2 ± 24.1 |
84.6 ± 48.3 |
97.7 ± 48.2 | |||
|
P values |
NMN |
NMN ´ nutriture |
NMN ´ stress |
NMN´ nutriture ´ stress | ||
|
|
<0.001 |
<0.001 |
0.519 |
0.013 | ||
1Nicotinamide supplement was 40mg/kg in the diet.
煙酰胺營(yíng)養狀況和應激狀況都對大鼠在曠場(chǎng)實(shí)驗中的行為有顯著(zhù)影響（表3）。對喂組、缺乏組和高劑量補充組大鼠在中央格內的停留時(shí)間明顯長(cháng)于對照組和低劑量補充組。同時(shí)，煙酰胺營(yíng)養狀況和應激狀況對大鼠潛伏期的影響存在著(zhù)交互作用。高劑量補充對照組大鼠潛伏期高于應激組，而在其他各組則結果相反。該現象提示，體內煙酰胺高度蓄積可對正常狀態(tài)下大鼠的行為產(chǎn)生抑制作用。
Figure 1 Relationships between latency time, plasma cortisol and glucose levels. A indicates all rats included, and B indicates only those restrained rats. PF: pair-feed, NAMD: nicotinamide deficiency, NAMS-L: low-dose nicotinamide supplement, NAMS-H: high-dose nicotinamide supplement.
另外，缺乏組和高劑量補充組大鼠的穿格次數低于其他組，表明過(guò)低和過(guò)高的煙酰胺營(yíng)養狀況都對大鼠的行為有不良影響。修飾次數以缺乏組最多，對喂組最低，這可能與缺乏組大鼠的糙皮病有關(guān)；對喂組修飾次數過(guò)低可能與其能量攝入不足有關(guān)。
Table 3 Behaviors in the open-field test of rats with different nicotinamide nutritures and stress status (Mean ± SD, n=10)
|
Study group |
Latency |
Crossing |
Rearing |
Modification |
|
|
(s) |
(time) |
(time) |
(time) |
|
|
|
|
| |
|
Un-restrained |
3.7 ± 2.3 |
45.8 ± 21.9 |
16.0 ± 12.6 |
2.6 ± 2.0 |
|
Restrained |
7.3 ± 6.1 |
47.8 ± 18.3 |
17.8 ± 6.6 |
4.8 ± 2.8 |
|
|
|
|
| |
|
Un-restrained |
12.9 ± 13.9 |
46.4 ± 9.6 |
16.0 ± 5.5 |
0.8 ± 1.0 |
|
Restrained |
19.9 ± 19.9 |
37.0 ± 19.4 |
14.0 ± 8.0 |
1.5 ± 1.7 |
|
|
|
|
| |
|
Un-restrained |
8.6 ± 9.9 |
35.9 ± 10.3 |
13.8 ± 5.4 |
5.7 ± 6.5 |
|
Restrained |
19.6 ± 23.3 |
28.1 ± 19.9 |
8.8± 4.8 |
4.2 ± 3.0 |
|
NAM supplement1 |
|
|
|
|
|
Un-restrained |
6.7 ± 5.0 |
44.3 ± 22.0 |
15.5 ± 6.8 |
3.5 ± 2.8 |
|
Restrained |
8.2 ± 5.2 |
50.2 ± 14.9 |
15.7 ± 5.2 |
2.8 ± 2.5 |
|
NAM supplement2 |
|
|
|
|
|
Un-restrained |
15.9 ± 10.7 |
21.3 ± 17.7 |
9.0 ± 7.4 |
2.3 ± 2.3 |
|
Restrained |
7.2 ± 5.1 |
38.8 ± 20.1 |
15.6 ± 6.6 |
2.8 ± 1.9 |
|
P values |
|
|
|
|
|
Nutriture |
0.028 |
0.005 |
0.089 |
0.004 |
|
Stress |
0.150 |
0.650 |
0.830 |
0.667 |
|
Nutriture´ stress |
0.017 |
0.124 |
0.135 |
0.131 |
1Nicotinamide supplement was 40mg/kg in the diet. 2Nicotinamide supplement was 500mg/kg in the diet. Note: Statistic test on the differences in the number of faces was not performed as only five rats had faces in the open-field test; the statistical power could not be reached. When the variances in latency and modification were tested, the naturally logarithmically transformed values were used.
束縛應激可能提高大鼠體內NO水平（P ＝0.055）但對NOS的水平無(wú)影響。增加煙酰胺攝入量可提高大鼠肝臟NOS含量（表5）。
煙酰胺營(yíng)養狀況對大鼠胰島素水平有顯著(zhù)影響（表6）。對喂組大鼠血漿胰島素水平最高，缺乏組最低。束縛應激后對喂應激組大鼠血漿胰島素水平低于其對照組，而其它各組變化趨勢相反。同時(shí)，補充煙酰胺也使補充組大鼠血糖高于其它組。
Table 4 Plasma total anti-oxidative capacity in rats with different nicotinamide nutritures and stress status (unit/ml, Mean ± SD, n=10)
|
Study group |
Un-restrained |
Restrained | |||
|
Control |
7.59 ± 1.48 |
8.88 ± 1.03 | |||
|
Pair-feed |
7.61 ± 1.60 |
6.65 ± 1.58 | |||
|
NAM deficiency |
5.22 ± 1.10 |
6.61 ± 1.79 | |||
|
NAM supplement1 |
8.42 ± 2.05 |
7.89 ± 2.42 | |||
|
NAM supplement2 |
6.61 ± 1.00 |
7.51 ± 1.33 | |||
|
All |
7.09 ± 1.83 |
7.50 ± 1.82 | |||
|
P values |
Nutriture |
Stress |
Nutriture´ stress | ||
|
|
<0.001 |
0.201 |
0.069 | ||
1Nicotinamide supplement was 40mg/kg in the diet. 2Nicotinamide supplement was 500mg/kg in the diet. TAOC: total anti-oxidative capacity.
Table 5 Plasma nitric oxide and liver nitric oxide synthase concentrations in rats with different nicotinamide nutritures and stress status (Mean ± SD, n=10)
|
|
NO |
NOS |
|
Study group |
(mmol/L) |
(unit/mg protein) |
|
|
| |
|
Un-restrained |
31.31 ± 10.30 |
0.27 ± 0.10 |
|
Restrained |
40.41± 26.23 |
0.23 ± 0.07 |
|
|
| |
|
Un-restrained |
46.16± 25.23 |
0.23 ± 0.06 |
|
Restrained |
66.70± 45.68 |
0.27 ± 0.11 |
|
|
| |
|
Un-restrained |
43.09± 13.16 |
0.26 ± 0.08 |
|
Restrained |
42.18± 19.67 |
0.31 ± 0.06 |
|
NAM supplement1 |
|
|
|
Un-restrained |
49.78± 31.40 |
0.53 ± 0.18 |
|
Restrained |
53.72± 42.26 |
0.48 ± 0.11 |
|
NAM supplement2 |
|
|
|
Un-restrained |
21.03± 9.67 |
0.59 ± 0.29 |
|
Restrained |
52.86± 37.43 |
0.48 ± 0.15 |
|
P values |
|
|
|
Nutriture |
0.086 |
<0.001 |
|
Stress |
0.055 |
0.475 |
|
Nutriture´ stress |
0.243 |
0.332 |
1Nicotinamide supplement was 40mg/kg in the diet. 2Nicotinamide supplement was 500mg/kg in the diet. Note: When the variances in nitric oxide and nitric oxide synthase concentrations were tested, the naturally logarithmically transformed values were used.
Table 6 Plasma insulin and glucose concentrations in rats with different nicotinamide nutritures and stress status (Mean ± SD, n=10)
|
|
Insulin |
Glucose |
|
Study group |
(mIu/ml) |
(mmol/L) |
|
|
| |
|
Un-restrained |
15.82 ± 2.54 |
8.40 ± 1.95 |
|
Restrained |
16.44± 3.55 |
8.26 ± 1.94 |
|
|
| |
|
Un-restrained |
24.07± 6.32 |
7.97 ± 1.68 |
|
Restrained |
20.16± 6.25 |
7.58 ± 2.82 |
|
|
| |
|
Un-restrained |
9.59± 4.97 |
7.36 ± 1.87 |
|
Restrained |
14.18± 4.86 |
8.15± 1.15 |
|
NAM supplement1 |
|
|
|
Un-restrained |
16.83± 3.60 |
8.64 ± 1.24 |
|
Restrained |
19.84± 2.21 |
9.52 ± 1.38 |
|
NAM supplement2 |
|
|
|
Un-restrained |
18.82± 2.70 |
9.18 ± 1.14 |
|
Restrained |
19.20± 2.72 |
8.94 ± 1.21 |
|
P values |
|
|
|
Nutriture |
<0.001 |
0.031 |
|
Stress |
0.390 |
0.609 |
|
Nutriture´ stress |
0.022 |
0.664 |
1Nicotinamide supplement was 40mg/kg in the diet. 2Nicotinamide supplement was 500mg/kg in the diet.
Figure 2 Liver MT concentrations in rats with different NAM nutritures and stress status (µg/g)
P values were equal to 0.034 for NAM nutritures and 0.043 for stress status, respectively.
3.2補充煙酰胺對人體應激適應性影響的隨機對照研究
經(jīng)計算機隨機分組，干預組和對照組參試人員在年齡、受教育程度、體質(zhì)指數、血壓、脈搏、血漿皮質(zhì)醇、NOS和NO含量等各項指標均無(wú)顯著(zhù)性差異（表7）。
Table 7 Baseline characteristics of the study subjects
|
|
Total
n=68 |
Intervention
n=35 |
Control
n=33 |
|
Age (years) |
21.9 ± 2.8 |
22.2 ± 3.1 |
21.5 ± 2.4 |
|
Education (≥ high school, %) |
78 |
80 |
76 |
|
BMI (kg/m2) |
23.3 ± 2.3 |
23.6 ± 2.5 |
22.9 ± 2.2 |
|
SBP (mmHg) |
103 ± 15 |
105 ± 10 |
101 ± 19 |
|
DBP (mmHg) |
67 ± 9 |
67 ± 11 |
67 ± 8 |
|
HR (min) |
72 ± 8 |
71 ± 8 |
73 ± 9 |
|
Cortisol (ng/ml) |
347.3 ± 87.5 |
360.1 ± 102.6 |
333.7 ± 66.8 |
|
NOS (unit/ml) |
30.8 ± 3.4 |
31.2 ± 3.3 |
30.3 ± 3.5 |
|
NO (µmol/L) |
38.7 ± 36.5 |
35.3 ± 38.7 |
42.7 ± 33.9 |
There is no statistical significance in the parameters between the two study groups.
經(jīng)過(guò)10天的干預后，問(wèn)卷調查顯示干預組反映睡眠時(shí)間增加的比例顯著(zhù)高于對照組（P＝0.046）；SCL-90篩查陽(yáng)性者比例、復雜視覺(jué)反應和數字記憶廣度測試結果及副作用反映率未見(jiàn)差異的顯著(zhù)性（表8,9）。
Table 8 Self-reported reactions to the intervention
|
|
Intervention
n=30 |
Control
n=28 |
Significance
(P value) |
|
Appetite increase (%) |
32 |
18 |
0.205 |
|
Sleep prolonging (%) |
32 |
11 |
0.046 |
|
Sleep quality improvement (%) |
36 |
25 |
0.382 |
|
Mood improvement (%) |
32 |
32 |
0.992 |
|
Nausea (%) |
3 |
7 |
0.494 |
|
Headache (%) |
19 |
11 |
0.357 |
|
Fidget (%) |
16 |
14 |
0.844 |
Table 9 Psychologic assessment after the intervention
|
|
Intervention
n=30 |
Control
n=28 |
Significance
(P value) | ||
|
SCL-90 positivea (%) |
30 |
25 |
0.670 | ||
|
Sight reaction | |||||
|
Time (s) |
3408.1 ± 978.3 |
3676.1 ± 776.9 |
0.255 | ||
|
Error frequency |
10.6 ± 5.7 |
10.4 ± 9.3 |
0.904 | ||
|
Digit memory reactionb |
17.7 ± 3.9 |
16.5 ± 3.5 |
0.207 | ||
Note, a, SCL-90 positive defined as either total score ≥160 or the number of positive items ≥ 43 or both. b, full score is 28.
經(jīng)干預和給予應激刺激后干預組和對照組的血壓和脈搏無(wú)顯著(zhù)性差異；另外未發(fā)現兩組間血漿尿皮質(zhì)醇含量的差異及血漿NOS含量差異的顯著(zhù)性；但干預組血漿NO含量顯著(zhù)高于對照組（表10，圖3）。
Table 10 Biological assessments after the intervention
|
|
Intervention
n=30 |
Control
n=28 |
Significance
(P value) |
|
SBP (mmHg) |
109 ± 11 |
108 ± 12 |
0.725 |
|
DBP (mmHg) |
65 ± 8 |
65 ± 8 |
0.976 |
|
HR (min) |
71 ± 6 |
73 ± 9 |
0.203 |
|
Cortisol (ng/ml) |
291.6 ± 97.1 |
278.5 ± 79.2 |
0.585 |
|
NOS (unit/ml) |
35.6 ± 4.3 |
33.9 ± 5.5 |
0.195 |
Figure 3 Differences in NO concentrations between intervention and control groups after NAM supplementation and psychological stress
由RT-PCR反轉錄擴增得到的GAPDH和iNOS產(chǎn)物，經(jīng)瓊脂糖凝膠電泳后得到的條帶結果未見(jiàn)差異的顯著(zhù)性（圖4）。
Figure 4 Differences in iNOS mRNA expression between intervention and control groups after NAM supplementation and psychological stress
干預組于實(shí)驗中期NMN排出量最高（實(shí)驗前2倍），應激刺激后減少到略高于實(shí)驗前水平。對照組NMN排出量于實(shí)驗后持續降低，應激刺激后僅為實(shí)驗前水平的1/2（圖5）。不同組間NMN排出量及排出量的變化趨勢均有顯著(zhù)性差異。
Figure 5 Urinary excretion of NMN during the study period
Analyzed by the repeated measurement of ANOVA, P = 0.002 for NMN measurements in groups with different nicotinamide nutritures; and P = 0.004 for NMN measurement and nicotinamide nutriture interactions.
3.3 煙酰胺對皮質(zhì)酮致原代培養大鼠海馬神經(jīng)元損傷的保護作用
CORT在10μM濃度即可以造成細胞損傷，存活率顯著(zhù)降低，并隨濃度增加存在劑量－效應關(guān)系（圖6）。
Figure 6 Cell survival rates with different level of corticosterone treatment
* Indicates that cell survival rate is significantly different from the control (P<0.05). P<0.001 for the overall mean.
較高濃度NAM可使細胞存活率顯著(zhù)下降，而較低濃度則對細胞存活率無(wú)明顯影響（圖7）。
Figure 7 Cell survival rates with different levels of NAM treatment
* Indicates that cell survival rate is significantly different from the control (P<0.05). P<0.001 for the overall mean.
與對照組相比，100μMCORT使細胞存活率顯著(zhù)降低（67±6.8％）。與單純用CORT處理相比，CORT處理前12h用NAM進(jìn)行干預可使細胞存活率顯著(zhù)上升，加入5mM和10mM后細胞存活率分別為94±9.0％和86±5.3％。與前述研究結果一致，加入低劑量NAM未對細胞存活率產(chǎn)生顯著(zhù)影響（5mMNAM：93±15.3％；10mMNAM：90±21.2％）（圖8）。
Figure 8 Cell survival rates with NAM and CORT treatment
* and + indicate that cell survival rate is different from the corresponding control and CORT treated cells, respectively (P<0.05). P<0.001 for the overall mean.
4 總結
本研究采用動(dòng)物實(shí)驗、人體干預研究和細胞實(shí)驗相結合的方法，以煙酰胺為煙酸營(yíng)養補充劑較系統地研究了不同煙酸營(yíng)養水平對應激動(dòng)物曠場(chǎng)行為的影響及可能的作用機制；應激對人體煙酸代謝的影響，補充煙酰胺對人體應激相關(guān)心理、生理和生化指標的影響；預防性給予低濃度煙酰胺對較高濃度皮質(zhì)酮對原代培養海馬神經(jīng)細胞損傷的阻斷作用。
我們采用純化飼料法使雄性Wistar大鼠具有了不同的煙酸營(yíng)養狀態(tài)；21d束縛應激產(chǎn)生動(dòng)物應激模型。實(shí)驗發(fā)現束縛應激可增加大鼠煙酸消耗；適量補充煙酰胺有助于提高大鼠的應激適應性。該作用可能是通過(guò)增加機體NO和血糖水平，增加葡萄糖的利用率和金屬硫蛋白表達，維持機體在束縛應激下的抗氧化能力實(shí)現的。
人體實(shí)驗采用隨機、單盲、安慰劑對照研究方法，煙酰胺（20mg/d）連續補充10天后干預組反映睡眠時(shí)間延長(cháng)的比例顯著(zhù)高于對照組。給予心理應激刺激后，干預組血漿NO含量顯著(zhù)高于對照組。補充煙酰胺使尿中NMN排出量顯著(zhù)增加，應激刺激使其排出量顯著(zhù)減少。提示應激可增加煙酸的消耗，適量補充煙酰胺可在應激狀態(tài)下維持機體良好的煙酸營(yíng)養狀況，有助于提高應激適應能力。另外，復雜視覺(jué)反應和自覺(jué)心理指標反應如食欲增加，睡眠質(zhì)量提高等雖然由于樣本量較小無(wú)差異顯著(zhù)性，但均有改善趨勢。
細胞實(shí)驗研究發(fā)現，低劑量的皮質(zhì)酮即可對原代培養的海馬神經(jīng)元造成損傷，顯著(zhù)降低存活率。較高劑量的煙酰胺也可使神經(jīng)元存活率顯著(zhù)降低。而皮質(zhì)酮加入前用低劑量的煙酰胺進(jìn)行干預可有效防止皮質(zhì)酮對海馬神經(jīng)細胞的損傷作用。
上述結果提示，心理應激可顯著(zhù)增加機體煙酸的消耗；煙酸缺乏和補充過(guò)高劑量的煙酰胺均可對機體的應激適應能力產(chǎn)生不利影響；適量補充煙酰胺不僅可維持機體應激狀態(tài)下的煙酸營(yíng)養水平，而且可提高應激適應能力，有效保護機體免于過(guò)高水平應激激素對機體，特別是神經(jīng)細胞的損傷作用。更深入地了解不同心理應激狀態(tài)下的煙酸營(yíng)養水平，了解補充不同劑量煙酸對應激適應性的影響以及煙酸與其它營(yíng)養素對應激適應能力影響的協(xié)同作用將為制定現代社會(huì )我國居民煙酸供給量標準提供科學(xué)依據，提高我國居民的膳食營(yíng)養水平和身體健康。
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