艾燃烧生成物抗衰老效应机制及嗅觉通路作用途径研究

【作者】 许焕芳；

【导师】 赵百孝；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2012， 博士

【摘要】 目的：探讨艾燃烧生成物的抗衰老作用、适宜干预条件、作用机制及作用途径。方法：(1)以浓度和时间为两因素,采用2×3析因设计,将70只SAMP8小鼠随机分为6个艾烟干预组：低浓度15min组(A1组),低浓度30min组(A2组),中浓度15min组(B1组),中浓度30min组(B2),高浓度15min组(C1组),高浓度30min组(C2组)和模型组(M组),每组各10只。另设10只正常老化小鼠SAMR1为正常组(Z组)。6个艾烟干预组分别用相应浓度和时间的艾烟熏灸4周,M组和Z组只进行相同的抓取。通过避暗实验、Morris水迷宫实验观察小鼠学习记忆行为学的改变；HE染色观察肺组织的病理改变；ELISA法检测脑和血清单胺类神经递质及细胞因子的含量；比色法检测脑和血清抗氧化酶及胆碱类神经递质的含量；RP-HPLC法检测脑内氨基酸类神经递质的含量；PCR-ELISA法检测肝脏端粒酶活性；Western-Blot法检测海马组织中ERK.p38 MAPK和JNK的活性变化。(2)60只昆明小鼠随机分为空白组、艾熏组、假手术组、模型组和模型艾熏组。模型组和模型艾熏组各15只,其余各组均10只。模型组和模型艾熏组采用硫酸锌滴鼻的方法造嗅觉障碍动物模型,假手术组采用蒸馏水滴鼻。以中浓度20min的艾烟干预条件对艾熏组和模型艾熏组进行为期4周的熏灸,其余各组只进行相同的抓取。以埋藏食物小球实验评估造模小鼠的嗅觉障碍,ELISA法检测脑单胺类神经递质和血清细胞因子的含量。结果：艾燃烧生成物抗衰老效应机制研究的实验结果如下：(1)行为学：M组、艾烟干预6组及Z组小鼠避暗实验的潜伏期及错误次数差异无统计学意义。M组、艾烟干预6组及Z组小鼠Morris水迷宫定位航行实验的潜伏期随训练天数的增加呈波动性下降趋势,但8组间差异无统计学意义；训练天数对潜伏期的影响显著,尚不能看出艾烟干预时间、浓度及两者的交互作用对潜伏期的影响；总游程随训练天数的增加呈波动性下降趋势,训练天数对总游程的影响显著,艾烟干预时间和浓度的交互作用可能对其有影响；Morris水迷宫的空间探索实验各指标在8组间差异无统计学意义。(2)细胞因子：M组小鼠IL-4(脑、血清和脾脏)和IL-10(血清和脾脏)等Th2细胞因子的水平显著低于Z组,而IFN-γ(脑、血清和脾脏)、IL-2(血清)及TNF-α(血清和脾脏)等Thl细胞因子的水平显著高于Z组。艾烟干预各组各项指标或优于M组,或与M组无差异。(3)抗氧化能力：M组血清和脑MDA的含量显著高于Z组,但其血清SOD和GSH-Px活性和脑GSH-Px活性则显著低于z组。艾烟干预各组各项指标或优于M组,或与M组无差异。析因分析显示,时间和浓度两因素对血清SOD、血清和脑GSH-Px的影响均存在交互作用,艾烟干预各组中血清SOD活性以B2组(中浓度30min)为优,而血清和脑GSH-Px活性均以B1组(中浓度15min)为优。(4)胆碱类神经递质：M组脑AChE的活力高于z组,而ChAT的活力低于z组,差异有统计学意义。艾烟干预各组小鼠AChE的活力与M组无显著差异,A2组、B1组和B2组小鼠脑ChAT的活力显著高于M组,其余各组与M组无差异。(5)单胺类神经递质：M组血清和脑内3种单胺类神经递质的含量与Z组相比均显著降低。艾烟干预各组单胺类神经递质的含量或优于M组,或与M组无差异。析因分析显示,时间和浓度两因素对3种单胺类神经递质均存在交互作用。艾烟干预各组血清和脑内3种单胺类神经递质的含量均以B1组为最高。(6)氨基酸类神经递质：M组GABA的含量低于Z组,而Glu和Asp的含量则高于Z组,差异均有统计学意义。艾烟干预各组小鼠3种氨基酸类神经递质的含量或与M组无差异,或优于M组。对GABA的析因分析显示,时间和浓度两因素间不存在交互作用,但两因素对GABA的含量均有影响。艾烟干预各组中,以B2组的结果为优。(7)肺组织病理：各组小鼠肺泡腔及间质均可见到不同程度的红细胞及炎性细胞浸润。(8)端粒酶：M组肝脏端粒酶活性显著高于Z组,艾烟干预各组中B1组、B2组和C1组端粒酶活性均低于M组,差异有统计学意义,其余各组与M组无差异。(9)MAPK:M组小鼠海马ERK.P38.JNK的灰度值显著高于z组,艾烟干预各组小鼠海马P38、ERK.JNK的灰度值均显著低于M组。析因分析显示,在对P38的影响中,艾烟的适宜干预参数为：中浓度；对JNK的影响中,艾烟的适宜干预参数为：低浓度,30min。艾燃烧生成物嗅觉通路作用的研究结果如下：(1)体重：实验前各组体重无显著差异,实验后空白组、艾熏组和假手术组3组体重均高于模型组和模型艾熏组。(2)BFPT实验：造模前,各组小鼠BFPT实验及对照实验成绩无显著差异；造模后,模型组和模型艾熏组BFPT实验及对照实验均显著高于其它3组。实验后,模型艾熏组小鼠BFPT实验及对照实验成绩显著高于其它4组,且4组间无显著差异。模型组的成绩较造模后降低,并与假手术组无差异。模型艾熏组小鼠成绩较造模后亦有所降低,但高于模型组,差异有统计学意义。艾熏组小鼠BFPT成绩与空白组和假手术组无显著差异。(3)脑单胺类神经递质：5组间差异无统计学意义。(4)血清细胞因子：模型组和假手术组血清细胞因子的含量未见差异。模型组、模型艾熏组和假手术组血清IFN-γ和IL-2含量均高于空白组,血清IL-4和IL-10的含量与空白组无差异,但有80%的把握发现模型艾熏组血清IL-10的含量低于空白组。此外,艾熏组和空白组间除有80%把握发现艾熏组血清IL-2的含量高于空白组外,其它细胞因子在两组间未见差异。结论：艾燃烧生成物抗衰老效应机制研究的结论如下：(1)艾烟干预不会加重SAMP8小鼠的衰老进程,艾烟干预可通过提高胆碱类和单胺类神经递质的含量,降低氨基酸类神经递质的神经毒性作用,提高抗氧化损伤能力、调整Thl/Th2细胞因子的偏移状态及降低肝脏端粒酶的过表达等起到抗衰老作用。(2)艾烟干预的适宜参数因指标的不同而有所变化：对提高单胺类神经递质含量而言,艾烟的适宜干预参数为中浓度15min；对提高抗氧化能力而言,艾烟的适宜干预参数为中浓度15min或30min；对调整Th1/Th2细胞因子的偏移而言,艾烟的适宜干预参数为中浓度。(3)艾烟干预对SAMP8小鼠学习记忆行为学的影响不显著。(4)艾烟干预可通过MAPK通路起作用,对降低P38灰度值而言,艾烟的适宜干预参数为中浓度,对降低JNK的灰度值而言,艾烟的适宜干预参数为低浓度30min。艾燃烧生成物嗅觉通路作用的结论如下：(1)10μ15％ZnS04连续4天滴鼻确可造成小鼠嗅觉障碍模型,但此模型可逆,嗅觉障碍于4周内逐渐恢复。(2)硫酸锌滴鼻造模方法能引起小鼠应激反应,促使炎症的发生。(3)本实验尚不能看出艾烟的嗅觉通路作用。更多还原

【Abstract】 Objective:To explore anti-aging effect, appropriate intervention condition, mechanism and action pathway of Artemisia burning products.Methods:(1) With time and concentration as two factors,2 x 3 factorial experiment design was applied in the experiment.70 SAMP8 mouse were randomized into 6 intervention groups and 1 model group, with 10 mice in each group. The 6 moxa smoke intervention groups included group with low concentration and 15min (Group Al), group with low concentration and 30min (Group A2), group with middle concentration and 15min (Group B1), group with middle concentration and 30min (Group B2), group with high concentration and 15min (Group Cl), and group with high concentration and 30min (Group C2).10 age-matched SAMR1 mouse were used as normal group. Fume the 6 intervention groups with Artemisia burning products (i.e. Moxa smoke) of different concentration and time for 4 weeks, and group M and Z were just done with the same grasping. The change of learning and memory behavior was tested by passive avoidance experiment and Morris water maze test. HE staining was used to observe the pathological change of lung. Contents of monoamine neurotransmitter and cytokines both in brain and serum were tested by ELISA. Antioxidant enzymes and cholinergic transmitter were tested by colorimetry. Amino acid neurotransmitter was tested by RP-HPLC. The activity of telomerase in liver was tested by PCR-ELISA. The activity of ERK, p38 MAPK and JNK in hippocampus was tested by Western-Blot.(2) 60 KM mouse were randomized into 5 groups:blank group, intervention group, sham operation group, model group and model intervention group. There were 15 mice in model group and model intervention group, and 10 in other 3 groups. Olfactory dysfunction animal model was made by intranasal administration of Zinc surfate solution. Sham operation group was administered with distilled water as control. Fume mice in model group and model intervention group with moxa smoke of middle concentration and 20min for 4 weeks. Other groups were just done with the same grasping. Olfactory dysfunction was assessed by buried food pellet test, and the contents of Monoamine neurotransmitters in brain and cytokines in serum were tested by ELISA.Results:Results of research on anti-aging effect mechanism of Artemisia burning products were as follows: (1) Animal behavior:In passive avoidance test, there was no statistically significant difference in latency and error number between group M,6 intervention groups and group Z. In place navigation test of Morris water maze, latency of group M,6 intervention groups and group Z showed a volatile downtrend with the increase of training days, but there was no statistically significant difference between 8 groups. Training day had a significant influence on latency, while the influence of time, concentration and their interaction on latency still can’t be seen. For the total distance, there was a volatile downtrend with the increase of training days. Training day had a significant influence on total distance, and the interaction between time and concentration may influence total diatance.The detection indexes in spatial probe test showed no significant difference between 8 groups.(2) Cytokine:Level of Thl type cytokines in group M, like IL-4 (in brain, serum and spleen) and IL-10 (in serum and spleen), was significantly lower than that in the group Z. Level of Thl type cytokines in group M, like IFN-y (in brain, serum and spleen), IL-2 (in serum) and TNF-α(in serum and spleen), was significantly higher than that in group Z. Cytokines in 6 intervention groups were either the same with group M, or better than group M.(3) Antioxidant enzyme:Content of MDA (in brain and serum) in group M was higher than that in group Z, but activity of SOD (in serum) and GSH-Px (in brain and serum) was remarkably lower in group M than that in group Z. Indexes in 6 intervention groups were either the same with group M, or better than group M. According to factorial analysis, there were interaction between time and concentration in the influence to SOD (in serum) and GSH-Px (in brain and serum). Among 6 intervention groups, the activity of SOD (in serum) got the best result in group B2, while the activity of GSH-Px (in brain and serum) got the best result in group B1.(4) Ach:The activity of AChE in group M was higher than that in group Z, while the activity of ChAT in group M was lower than that in group Z. The activity of AChE in 6 intervention groups had no difference with that in group M. The activity of ChAT in group A2, B1 and B2 was higher than that in group M, while other groups had no difference with group M.(5) Monoamine neurotransmitter:Compared with group Z, contents of 3 kind of monoamine neurotransmitter (in brain and serum) in group M decreased. Compared with group M, contents of 3 kind of monoamine neurotransmitter in 6 intervention groups were either the same or much better. According to factorial analysis, time and concentration had an interaction on monoamine neurotransmitter. Among 6 intervention groups, the contents of monoamine neurotransmitter were highest in group B1.(6) Amino acid neurotransmitter:Content of GABA in group M was statistically lower than that in group Z, but contents of Glu and Asp in group M were statistically higher than that in group Z. Contents of 3 kind of amino acid neurotransmitter in 6 intervention groups were either the same with group M or much better. According to factorial analysis on GABA, there were no interaction between time and concentration, while both time and concentration had influence on GABA. Result of group B2 was the best among 6 intervention groups.(7) Pathology of lung:There were RBC and inflammatory cells infiltration in alveolar space and intracellular substance. Overall speaking, lung pathology in 6 intervention groups was better than that in group M.(8) Telomerase:Activity of liver telomerase in group M was higher than that in group Z. Among 6 intervention groups, the activity of telomerase in group B1, B2 and C1 was lower than that in group M, while other groups had no difference with group M.(9) MAPK:Grey values of hippocampus ERK, P38 and JNK in group M were higher than that in group Z and 6 intervention groups. According to factorial analysis, the appropriate intervention condition for P38 was middle concentration, while for JNK it was low concentration and 30 min.Results of research on olfactory pathway of Artemisia burning products were as follows:(1) Weight:Before the experiment, there was no difference between 8 groups; after the experiment, weight in blank group, intervention group, and sham operation group was higher than that in model group and model intervention group.(2) BFPT:Before preparing model, there were no differences in results of BFPT and control test among 8 groups. After modeling, results of BFPT and control test in model group and model intervention group were higher than that in other groups. After experiment, results of BFPT and control test in model group were higher than that in other 4 groups, and there were no difference between the 4 groups.(3) Monoamine neurotransmitter of brain:There was no difference in brain monoamine neurotransmitter between 5 groups. (4) Cytokine of serum:Contents of IFN-γand IL-2 in model group, model intervention group and sham operation group were higher than that in blank group, and there was 80% certainty on finding content of IL-10 in model intervention group lower than that in blank group. Besides, there was 80% certainty on finding content of IL-2 in intervention group higher than that in blank group, and there were no difference between other cytokines.Conclusion:Conclusions of research on anti-aging effect mechanism of Artemisia burning products are as follows:(1) Intervention by moxa smoke can’t aggravate the senescence procedure of SAMP8 mouse. Instead, intervention by moxa smoke can exert anti-aging effect through the following ways:increasing contents of Ach and monoamine neurotransmitter, reducing neurotoxicity action of amino acid neurotransmitter, enhancing antioxidation, regulating the balance of Thl/Th2 cytokine and decreasing over-expression of liver telomerase, etc.(2) The appropriate intervention condition of moxa smoke varies with detection indexes. To increase content of monoamine neurotransmitter, the appropriate intervention condition is middle concentration and 15min; to enhance antioxidation, it is middle concentration and 15min or30min; to regulate the balance of Th1/Th2 cytokine, it’s middle concentration.(3) The intervention of moxa smoke does no significant influence on learning and memory behavior of SAMP8 mouse.(4) The intervention of moxa smoke can work through MAPK pathway. For P38, the appropriate intervention condition is middle concentration; for JNK, it’s low concentration and 30min.Conclusions of research on olfactory pathway of Artemisia burning products are as follows:(1) The model of olfactory dysfunction can be made by intranasal administration with 10μ15% ZnSO4 for 4 days. However, this model is reversible, and recovery of dysosmia can be seen in 4 weeks.(2) Intranasal administration with Zinc sulfate solution can cause stress on mice, and then results in the occurrence of inflammation.(3) This experiment can’t confirm the role of olfactory pathway in the effect of Artemisia burning products.更多还原