雌性大鼠性腺轴系的衰老变化及中药何首乌饮延缓衰老机制的研究
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摘要
衰老作为一种正常而复杂的生命过程,涉及机体的不同组织、细胞的一系列特定程序性变化。下丘脑-垂体-性腺轴(H-P-G-A)的发生发育、成熟与衰老是生命活动的重要方面。免疫-神经-内分泌网络在衰老过程中起重要作用,而H-P-G-A是该网络的重要组成部分。
众多研究表明,细胞衰老过程是借助于信号传导途径实现的,许多细胞因子亦参与到这些信号途径中。当这些途径所涉及的关键调节因子发生改变,细胞将延缓衰老或绕过衰老程序继续增殖。本实验采用D-半乳糖致亚急性衰老的雌性大鼠模型,以细胞衰老信号转导途径中涉及的关键调节因子p53、p19ARF、Rb及p16等为介入点,从分子生物学角度探讨何首乌饮对延缓下丘脑-垂体-卵巢轴(H-P-O-A)衰老的作用机制,并且通过检测下丘脑、垂体、卵巢组织IGF-1、IGF-BP、TNF-α、EGF、EGFR的表达及电镜超微结构变化等,从多层次、多环节阐明哺乳动物衰老机制,以及何首乌饮对下丘脑-垂体-卵巢轴的抗衰老作用。
1.目的
应用RT-PCR、Western blotting、原位杂交及免疫组化方法检测衰老模型大鼠下丘脑、垂体、卵巢组织p53、p19ARF、Rb和p16、EGF、EGFR、TNF-α、IGF-Ⅰ、IGFBP3的表达,并且从下丘脑、垂体和卵巢三个层次来观察H-P-G-A衰老的组织学变化,以及何首乌饮对该轴的作用,从而探讨大鼠性腺轴的衰老机制,分析何首乌饮抗衰老的作用。
2.方法
2.1实验动物的选取、分组及模型的建立:选用8周龄清洁级SD雌性大鼠96只随机分为正常组、模型组、预防组。预防组又分为何首乌饮低剂量组、何首乌饮中剂量组、何首乌饮高剂量组、何首乌丸组,每组动物各16只。用6%的D-半乳糖溶液腹腔注射的方法连续用药60d,建立亚急性衰老大鼠模型。预防组在腹腔注射D-半乳糖溶液的同时给药何首乌饮、何首乌丸。
2.2标本的采集及研究方法:连续用药60d后,取各组大鼠的下丘脑、垂体前叶和卵巢新鲜组织,应用RT-PCR法检测其p53、p19ARF、Rb、p16及IGFBP3的基因水平,应用Western blotting方法检测Rb及p16蛋白的表达。将各组大鼠用不同的固定液进行左心室灌注,最后取其下丘脑、垂体前叶、卵巢组织,应用免疫组化法检测p53、EGF、EGFR、TNF-α蛋白的表达,应用原位杂交技术检测IGF-ⅠmRNA的水平,应用透射电镜观察下丘脑、垂体前叶及卵巢组织超微结构的变化。
3.结果
3.1大鼠下丘脑-垂体-卵巢轴p19ARF/p53/p21Cip1和p16INK4a/Rb衰老途径中关键调节因子发生改变,应用何首乌饮可纠正这些因子的过度表达:
3.1.1 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p53基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p53基因表达比模型组降低(P<0.05)。在下丘脑和垂体前叶,低剂量组p53基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组p53基因表达低于何首乌丸组(P<0.05)。免疫组化结果显示,模型组大鼠下丘脑弓状核、垂体前叶、卵巢组织中p53的染色明显强于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p53染色比模型组降低(P<0.05)。在垂体前叶组织中,低剂量组p53染色弱于何首乌丸组(P<0.05);而在卵巢组织中,高剂量组p53染色弱于何首乌丸组(P<0.05)。
3.1.2 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p19ARF基因明显升高(P<0.01),给予不同剂量的何首乌饮预处理后,与模型组相比,p19ARF基因表达明显降低(P<0.01,P<0.05)。在下丘脑,低剂量组p19ARF基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组p19ARF基因表达低于何首乌丸组(P<0.05)。
3.1.3 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中Rb基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,Rb基因表达明显比模型组降低(P<0.01,P<0.05)。在下丘脑和垂体前叶,低剂量组Rb基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组Rb基因表达低于何首乌丸组(P<0.05)。Western结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织的Rb蛋白表达明显高于正常组(P<0.05),不同剂量的何首乌饮各组下丘脑、垂体前叶、卵巢组织的Rb蛋白表达明显低于模型组(P<0.05)。在垂体前叶,低剂量组Rb蛋白表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组Rb蛋白表达低于何首乌丸组(P<0.05)。
3.1.4 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p16基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p16基因表达比模型组降低(P<0.01,P<0.05)。在下丘脑,低剂量组p16基因表达低于何首乌丸组(P<0.05)。Western结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织的p16蛋白表达明显高于正常组(P<0.01,P<0.05),不同剂量的何首乌饮各组大鼠下丘脑、垂体前叶、卵巢组织的p16蛋白表达明显低于模型组(P<0.01,P<0.05)。
3.2大鼠下丘脑-垂体-卵巢轴衰老相关细胞因子的表达发生改变,应用何首乌饮可纠正这些因子的异常表达:
3.2.1大鼠免疫组化结果显示,衰老模型大鼠EGF在卵巢中的染色明显弱于正常组(P<0.01),EGFR在下丘脑弓状核、垂体前叶、卵巢中的染色亦明显比正常组减弱(P<0.01)。给予不同剂量的何首乌饮预处理后,EGF及EGFR的染色比模型组增强(P<0.01,P<0.05)。
3.2.2免疫组化结果显示,与正常组相比,衰老模型大鼠TNF-α在下丘脑弓状核、垂体前叶、卵巢中的染色明显增强(P<0.01)。给予不同剂量的何首乌饮预处理后,TNF-α染色明显比模型组减弱(P<0.01,P<0.05)。在下丘脑弓状核和垂体前叶,何首乌饮各组TNF-α的表达低于何首乌丸组(P<0.05)。
3.2.3原位杂交结果显示,衰老模型大鼠卵巢组织中IGF-ⅠmRNA表达明显低于正常大鼠(P<0.01),给予不同剂量的何首乌饮及何首乌丸预处理后,IGF-ⅠmRNA表达比模型大鼠升高(P<0.01)。PCR结果显示,与正常组大鼠相比,模型组大鼠下丘脑、垂体前叶、卵巢组织中IGFBP3基因表达明显上调(P<0.01),给予不同剂量的何首乌饮预处理后,IGFBP3基因表达明显比模型组降低(P<0.01,P<0.05)。
3.3衰老大鼠下丘脑弓状核、垂体前叶、卵巢的形态学发生损伤性改变,应用何首乌饮可减缓这种损伤:
3.3.1下丘脑弓状核形态学改变:光镜下观察,下丘脑弓状核包括神经元细胞和神经胶质细胞。在模型组,尚发现有大量反应胶质细胞。透射电镜下观察,下丘脑弓状核有暗细胞和亮细胞两类神经元。模型组可见核膜凹陷增多,核膜溶解;溶酶体增多;线粒体嵴断裂、膜融合或消失;粗面内质网脱颗粒;高尔基体扩张。突触前、后膜模糊不清,突触间隙融合。预防各组可见部分核膜溶解;可见溶酶体;线粒体嵴部分断裂、膜部分模糊不清;粗面内质网轻度脱颗粒;高尔基体轻度扩张。突触前、后膜尚清晰,突触间隙部分融合。
3.3.2垂体前叶形态学改变:光镜下观察,垂体前叶腺细胞包括嗜酸性细胞和嗜碱性细胞和嫌色细胞。模型组可见腺细胞排列紊乱,血管增生、变性。嗜酸性细胞及嗜碱性细胞数量减少。透射电镜下观察,模型组垂体促性腺激素细胞可见核内异染色质增多,核膜部分溶解;粗面内质网扩张明显;高尔基体增多、扩张;线粒体肿胀,嵴断裂、融合或消失;分泌颗粒增多。预防各组可见促性腺激素细胞粗面内质网丰富,轻度扩张;高尔基体轻度扩张;线粒体肿胀不明显,嵴部分断裂;分泌颗粒轻度增多。
3.3.3卵巢形态学改变:光镜下观察,可见各级卵泡及黄体。衰老模型大鼠卵巢中原始卵泡、生长卵泡及黄体均明显少于正常大鼠(P<0.05,P<0.01)。各级卵泡颗粒细胞层次减少,细胞排列紊乱,囊性扩张的卵泡增多。可见颗粒细胞小巢或条索,尚可见炎细胞浸润。预防各组大鼠卵巢中原始卵泡、生长卵泡及黄体多于模型大鼠(P<0.05,P<0.01)。透射电镜下观察,模型组可见细胞内线粒体减少,呈不同程度的肿胀,嵴部分断裂甚至消失;滑面内质网不丰富,轻度扩张;高尔基体肥大。何首乌饮低剂量组可见线粒体扩张显著;滑面内质网轻度扩张;高尔基复合体扩张;脂滴增多,体积较大。何首乌饮中剂量组可见线粒体数目增多,体积增大,部分线粒体肿胀,核膜扩张;胞质内脂滴较多。滑面内质网数目丰富,中度扩张。何首乌饮高剂量组可见细胞结构接近于正常组,线粒体丰富;胞浆中脂滴电子密度高;滑面内质网扩张;有丰富的高尔基复合体。何首乌丸组可见线粒体数目增多,体积增大,部分线粒体肿胀,核膜扩张;胞质内脂滴较多。
4.结论
1衰老模型大鼠H-P-O-A性腺轴p19ARF/p53/p21Cip1和p16INK4a/Rb衰老途径中关键的调节因子p19ARF、p53、Rb、p16基因及蛋白表达上调,预防性应用何首乌饮对此可以起到抑制作用。
2衰老模型大鼠H-P-O-A性腺轴衰老相关细胞因子EGF及其受体EGFR、IGF-Ⅰ及其结合蛋白IGFBP3表达均降低,预防性应用何首乌饮可增加这些细胞因子的表达。
3衰老模型大鼠H-P-O-A性腺轴衰老相关细胞因子TNF-α蛋白表达增高,预防性应用何首乌饮可以使TNF-α水平下降。
4 D-半乳糖可致大鼠下丘脑弓状核、垂体前叶和卵巢的超微结构发生损伤,预防性应用何首乌饮可以减缓这种超微结构的损伤,从而改善H-P-O-A性腺轴的功能,延缓其衰老。
As a normal and complicated life process, senescence was involved in a series of routine changing in various tissues and cells. The development and aging of hypothalamus- pituitary-gland axis (H-P-G-A) was important in life proceeding. Immune-neuroendocrine network played an important role in aging and H-P-G-A was an important component in this network.
Many studies indicated that cellular senescence was implemented via some signaling transduction pathways which a lot of cytokines were involved in. When the key regulators in these pathways were changed, cellular senescence would be delayed. In this study, we had investigated the changes of some key regulators including p53, p19ARF, Rb, p16, IGF-Ⅰ, IGF-BP, TNF-α, EGF, EGFR and the changes of the ultrastructure of arcuate nucleus in the hypothalamus, adenohypophysis, ovary after taking traditional Chinese medicine Heshouwuyin (HSWY) by using the sub-acutely aging female model rats induced by D-galactose, so as to find the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) and the mechanism of HSWY in anti-aging.
1. Objective
We deteceted the level of mRNA p53, p19ARF, Rb, p16 and IGFBP3 by reverse transcription-polymerase chain reaction (RT-PCR), and examined the expression of protein Rb and p16 by Western-blotting, and evaluated the expression of p53, TNF-α, EGF and EGFR by immunohistochemistry (IH), and analysised the mRNA level of IGF-Ⅰby hybridization in situ (HIS) in order to find out the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) of the aging model rats and the effect of HSWY in anti-aging, so as to provide theoretical and experiment evidence to anti-aging in the female’sexual gland.
2. Methods
2.1 Choosing of the experimental animal and establishment of the aging model. Eight-week-old female SD rats (n=96) were separated into three groups at random: normal group, aging model group, preventing aging group. The rats in preventing aging group were re-divided into four groups: HSWY low dosage group, HSWY medium dosage group, HSWY high dosage group and Heshouwan(HSWW) group (n=16 in each group). The sub-acutely aging model rats were induced by D-galactose, and the rats in preventing aging group accepted intragastric administration of HSWY and HSWW while accepting D-galactose intraperioneal injection for 60 days.
2.2 Experimental procedures. Hypothalamus, adenohypophysis and ovary tissues of each group were obtained after the rats undergoing experiment for 60 days. We detected the gene mRNA expression of p53, p19ARF, Rb, p16 and IGFBP3 by RT-PCR, and examined the protein expression of Rb and p16 by Western-blotting, evaluated the expression of p53, TNF-α, EGF and EGFR by IH, analysised the level of mRNA IGF-Ⅰby HIS, and observed the ultrastructure by transmission electron microscope (TEM).
3. Results
3.1 Key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were changed in aging model, and the overexpression of these regulators could be retrieved by using HSWY.
3.1.1 The result of PCR had shown that gene expression of p53 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p53 were significantly decreased (P<0.05). In the hypothalamus and adenohypophysis tissues, the gene expression of p53 in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p53 was significantly lower than in HSWW group (P<0.05). The results of IH had shown that immunostaining for p53 in arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for p53 were significantly decreased (P<0.05). In the adenohypophysis tissue, the immunostaining for p53 in HSWY low dosage group was significantly weaker than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the immunostaining for p53 was significantly weaker than in HSWW group (P<0.05).
3.1.2 The result of PCR had shown that gene expression of p19ARF in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p19ARF were significantly decreased (P<0.01, P<0.05). In the hypothalamus tissue, the gene expression of p19ARF in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p19ARF was significantly lower than in HSWW group (P<0.05).
3.1.3 The result of PCR had shown that gene expression of Rb in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of Rb were significantly decreased (P<0.01, P<0.05). In the hypothalamus and the adenohypophysis tissues, the gene expression of Rb in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of Rb was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein Rb in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein Rb were significantly decreased (P<0.05). In the adenohypophysis tissue, the expression of protein Rb in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the expression of protein Rb was significantly lower than in HSWW group (P<0.05).
3.1.4 The result of PCR had shown that gene expression of p16 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p16 were significantly decreased (P<0.01, P<0.05). In the adenohypophysis tissue, the gene expression of p16 in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein p16 in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein p16 were significantly decreased (P<0.05).
3.2 Cytokines related to senescence in H-P-O-A were changed in aging model, and the abnormal expression could be retrieved by using HSWY.
3.2.1 The results of IH had shown that immunostaining for EGF in ovary tissue was significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGF was significantly increased (P<0.01). The results of IH had shown that immunostaining for EGFR in arcuate nucleus, adenohypophysis and ovary tissues were significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGFR were significantly increased (P<0.05).
3.2.2 The results of IH had shown that immunostaining for TNF-αin arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for TNF-αwere significantly decreased (P<0.01, P<0.05). In the arcuate nucleus and adenohypophysis tissues, the immunostaining for TNF-αin HSWY group were significantly weaker than in HSWW group (P<0.05).
3.2.3 The results of HIS had shown that the expression of IGF-ⅠmRNA in ovary tissue was significantly decreased in model rats (P<0.01). After pretreatment of HSWY in preventing aging group, the expression of IGF-ⅠmRNA was significantly increased (P<0.01). The result of PCR had shown that gene expression of IGFBP3 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of IGFBP3 were significantly decreased (P<0.01, P<0.05).
3.3 Morphologic structure of H-P-O-A in aging model was damaged, and the impairment could be restored by using HSWY.
3.3.1 The morphological changes of the arcuate nucleus in the hypothalamus: By light microscope, it could be seen that there were two kinds of cells in arcuate nucleus: neurone and neuroglial cell. In model rats, there were a lot of reactive colloid cells except for the two kinds of cells above-mentioned. By transmission electron microscope(TEM), arcuate nucleus was constituted of dark neurons, pale neurons. In model rats′arcuate nucleus, it was found that the nucleolemma was indented or dissolved, and the number of lysosome was increased, and mitochondriales cristae were broken and even vanished, and rough endoplasmic reticulum losted its granules, and Golgi complex swelled, and pre- and postsynaptic membranes were unclear and synaptic cleft was fusional. In preventing aging rats′arcuate nucleus, part of the nucleolemma was dissolved, and lysosome could be seen. Part of the mitochondriales cristae was broken. Rough endoplasmic reticulum losted its granules lightly. Golgi complex swelled lightly. Pre- and postsynaptic membranes were clear and part of synaptic cleft was fusional.
3.3.2 The morphological changes of the adenohypophysis: By light microscope, it could be seen that the gland cells were including three kinds of cells: acidophil, basophi and chromophobe cells. In model rats, it could be seen that the gland cells were arranged disorderly and the blood vessels were proliferated. The number of acidophil and basophi cells was decreased. By TEM, the gonadotroph of model rats changed obviously. The heterochromatin became more and the nucleolemma was dissolved. GER dilated obviously and Golgi complex increased and swelled. Mitochondriales cristae were broken and even vanished. The secretary granules were increased. In preventing aging rats′gonadotroph, rough endoplasmic reticulum were increased and dilated lightly. Golgi complex swelled lightly and part of the mitochondriales cristae was broken. The secretary granules were increased lightly.
3.3.3 The morphological changes of the ovary: By light microscope, the ovary was characterized by follicles in every grade and corepus luteums. The number of primordial follicles, growing follicles and corepus luteums in model rats was less than that in normal rats. The granulosa cells were arranged disordered and the number of the saccular ectasia follicles was increased. Compared with model rats, the number of primordial follicles, growing follicles and corepus luteums was more in preventing aging rats. By TEM, in the model rats′granulosa cell, it was found that mitochondria were droped and swelled, cristae were broken and even vanished, and smooth endoplasmic reticulum were decreased and dilated, and Golgi complex swelled. In HSWY low dosage rats′granulosa cell, it could be seen that mitochondria were swelled obviously, and smooth endoplasmic reticulum were dilated lightly, and Golgi complex swelled, and lipid droplets were increased. In HSWY medium dosage rats′granulosa cell, it was found that mitochondria were increased and swelled lightly, and many smooth endoplasmic reticulum were dilated lightly, and lipid droplets were increased. The ultrastructure of HSWY high dosage rats′granulosa cell was similar to nomal rats′. And the ultrastructure of HSWW rats′granulosa cell was similar to HSWY medium dosage rats′.
4. Conclusion
4.1 The genes and proteins of key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were overexpressing in aging model, and this could be depressed by using HSWY.
4.2 Cytokines related to senescence—EGF and its receptor EGFR, IGF-Ⅰand its binding protein IGFBP3 were decreased in H-P-O-A in aging model, and the expression of these cytokines could be increased by using HSWY.
4.3 The protein of TNF-α—a cytokine related to senescence was overexpressing in H-P-O-A in aging model, and the level of TNF-αcould be reduced by using HSWY.
4.4 Morphologic structure of H-P-O-A in aging model was damaged by D-galactose. The impairment could be restored by using HSWY, so as to the function of H-P-O-A could be improved and senescence could be delayed.
众多研究表明,细胞衰老过程是借助于信号传导途径实现的,许多细胞因子亦参与到这些信号途径中。当这些途径所涉及的关键调节因子发生改变,细胞将延缓衰老或绕过衰老程序继续增殖。本实验采用D-半乳糖致亚急性衰老的雌性大鼠模型,以细胞衰老信号转导途径中涉及的关键调节因子p53、p19ARF、Rb及p16等为介入点,从分子生物学角度探讨何首乌饮对延缓下丘脑-垂体-卵巢轴(H-P-O-A)衰老的作用机制,并且通过检测下丘脑、垂体、卵巢组织IGF-1、IGF-BP、TNF-α、EGF、EGFR的表达及电镜超微结构变化等,从多层次、多环节阐明哺乳动物衰老机制,以及何首乌饮对下丘脑-垂体-卵巢轴的抗衰老作用。
1.目的
应用RT-PCR、Western blotting、原位杂交及免疫组化方法检测衰老模型大鼠下丘脑、垂体、卵巢组织p53、p19ARF、Rb和p16、EGF、EGFR、TNF-α、IGF-Ⅰ、IGFBP3的表达,并且从下丘脑、垂体和卵巢三个层次来观察H-P-G-A衰老的组织学变化,以及何首乌饮对该轴的作用,从而探讨大鼠性腺轴的衰老机制,分析何首乌饮抗衰老的作用。
2.方法
2.1实验动物的选取、分组及模型的建立:选用8周龄清洁级SD雌性大鼠96只随机分为正常组、模型组、预防组。预防组又分为何首乌饮低剂量组、何首乌饮中剂量组、何首乌饮高剂量组、何首乌丸组,每组动物各16只。用6%的D-半乳糖溶液腹腔注射的方法连续用药60d,建立亚急性衰老大鼠模型。预防组在腹腔注射D-半乳糖溶液的同时给药何首乌饮、何首乌丸。
2.2标本的采集及研究方法:连续用药60d后,取各组大鼠的下丘脑、垂体前叶和卵巢新鲜组织,应用RT-PCR法检测其p53、p19ARF、Rb、p16及IGFBP3的基因水平,应用Western blotting方法检测Rb及p16蛋白的表达。将各组大鼠用不同的固定液进行左心室灌注,最后取其下丘脑、垂体前叶、卵巢组织,应用免疫组化法检测p53、EGF、EGFR、TNF-α蛋白的表达,应用原位杂交技术检测IGF-ⅠmRNA的水平,应用透射电镜观察下丘脑、垂体前叶及卵巢组织超微结构的变化。
3.结果
3.1大鼠下丘脑-垂体-卵巢轴p19ARF/p53/p21Cip1和p16INK4a/Rb衰老途径中关键调节因子发生改变,应用何首乌饮可纠正这些因子的过度表达:
3.1.1 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p53基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p53基因表达比模型组降低(P<0.05)。在下丘脑和垂体前叶,低剂量组p53基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组p53基因表达低于何首乌丸组(P<0.05)。免疫组化结果显示,模型组大鼠下丘脑弓状核、垂体前叶、卵巢组织中p53的染色明显强于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p53染色比模型组降低(P<0.05)。在垂体前叶组织中,低剂量组p53染色弱于何首乌丸组(P<0.05);而在卵巢组织中,高剂量组p53染色弱于何首乌丸组(P<0.05)。
3.1.2 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p19ARF基因明显升高(P<0.01),给予不同剂量的何首乌饮预处理后,与模型组相比,p19ARF基因表达明显降低(P<0.01,P<0.05)。在下丘脑,低剂量组p19ARF基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组p19ARF基因表达低于何首乌丸组(P<0.05)。
3.1.3 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中Rb基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,Rb基因表达明显比模型组降低(P<0.01,P<0.05)。在下丘脑和垂体前叶,低剂量组Rb基因表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组Rb基因表达低于何首乌丸组(P<0.05)。Western结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织的Rb蛋白表达明显高于正常组(P<0.05),不同剂量的何首乌饮各组下丘脑、垂体前叶、卵巢组织的Rb蛋白表达明显低于模型组(P<0.05)。在垂体前叶,低剂量组Rb蛋白表达低于何首乌丸组(P<0.05);而在卵巢,高剂量组Rb蛋白表达低于何首乌丸组(P<0.05)。
3.1.4 PCR结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织中p16基因表达明显高于正常组(P<0.01),给予不同剂量的何首乌饮预处理后,p16基因表达比模型组降低(P<0.01,P<0.05)。在下丘脑,低剂量组p16基因表达低于何首乌丸组(P<0.05)。Western结果显示,模型组大鼠下丘脑、垂体前叶、卵巢组织的p16蛋白表达明显高于正常组(P<0.01,P<0.05),不同剂量的何首乌饮各组大鼠下丘脑、垂体前叶、卵巢组织的p16蛋白表达明显低于模型组(P<0.01,P<0.05)。
3.2大鼠下丘脑-垂体-卵巢轴衰老相关细胞因子的表达发生改变,应用何首乌饮可纠正这些因子的异常表达:
3.2.1大鼠免疫组化结果显示,衰老模型大鼠EGF在卵巢中的染色明显弱于正常组(P<0.01),EGFR在下丘脑弓状核、垂体前叶、卵巢中的染色亦明显比正常组减弱(P<0.01)。给予不同剂量的何首乌饮预处理后,EGF及EGFR的染色比模型组增强(P<0.01,P<0.05)。
3.2.2免疫组化结果显示,与正常组相比,衰老模型大鼠TNF-α在下丘脑弓状核、垂体前叶、卵巢中的染色明显增强(P<0.01)。给予不同剂量的何首乌饮预处理后,TNF-α染色明显比模型组减弱(P<0.01,P<0.05)。在下丘脑弓状核和垂体前叶,何首乌饮各组TNF-α的表达低于何首乌丸组(P<0.05)。
3.2.3原位杂交结果显示,衰老模型大鼠卵巢组织中IGF-ⅠmRNA表达明显低于正常大鼠(P<0.01),给予不同剂量的何首乌饮及何首乌丸预处理后,IGF-ⅠmRNA表达比模型大鼠升高(P<0.01)。PCR结果显示,与正常组大鼠相比,模型组大鼠下丘脑、垂体前叶、卵巢组织中IGFBP3基因表达明显上调(P<0.01),给予不同剂量的何首乌饮预处理后,IGFBP3基因表达明显比模型组降低(P<0.01,P<0.05)。
3.3衰老大鼠下丘脑弓状核、垂体前叶、卵巢的形态学发生损伤性改变,应用何首乌饮可减缓这种损伤:
3.3.1下丘脑弓状核形态学改变:光镜下观察,下丘脑弓状核包括神经元细胞和神经胶质细胞。在模型组,尚发现有大量反应胶质细胞。透射电镜下观察,下丘脑弓状核有暗细胞和亮细胞两类神经元。模型组可见核膜凹陷增多,核膜溶解;溶酶体增多;线粒体嵴断裂、膜融合或消失;粗面内质网脱颗粒;高尔基体扩张。突触前、后膜模糊不清,突触间隙融合。预防各组可见部分核膜溶解;可见溶酶体;线粒体嵴部分断裂、膜部分模糊不清;粗面内质网轻度脱颗粒;高尔基体轻度扩张。突触前、后膜尚清晰,突触间隙部分融合。
3.3.2垂体前叶形态学改变:光镜下观察,垂体前叶腺细胞包括嗜酸性细胞和嗜碱性细胞和嫌色细胞。模型组可见腺细胞排列紊乱,血管增生、变性。嗜酸性细胞及嗜碱性细胞数量减少。透射电镜下观察,模型组垂体促性腺激素细胞可见核内异染色质增多,核膜部分溶解;粗面内质网扩张明显;高尔基体增多、扩张;线粒体肿胀,嵴断裂、融合或消失;分泌颗粒增多。预防各组可见促性腺激素细胞粗面内质网丰富,轻度扩张;高尔基体轻度扩张;线粒体肿胀不明显,嵴部分断裂;分泌颗粒轻度增多。
3.3.3卵巢形态学改变:光镜下观察,可见各级卵泡及黄体。衰老模型大鼠卵巢中原始卵泡、生长卵泡及黄体均明显少于正常大鼠(P<0.05,P<0.01)。各级卵泡颗粒细胞层次减少,细胞排列紊乱,囊性扩张的卵泡增多。可见颗粒细胞小巢或条索,尚可见炎细胞浸润。预防各组大鼠卵巢中原始卵泡、生长卵泡及黄体多于模型大鼠(P<0.05,P<0.01)。透射电镜下观察,模型组可见细胞内线粒体减少,呈不同程度的肿胀,嵴部分断裂甚至消失;滑面内质网不丰富,轻度扩张;高尔基体肥大。何首乌饮低剂量组可见线粒体扩张显著;滑面内质网轻度扩张;高尔基复合体扩张;脂滴增多,体积较大。何首乌饮中剂量组可见线粒体数目增多,体积增大,部分线粒体肿胀,核膜扩张;胞质内脂滴较多。滑面内质网数目丰富,中度扩张。何首乌饮高剂量组可见细胞结构接近于正常组,线粒体丰富;胞浆中脂滴电子密度高;滑面内质网扩张;有丰富的高尔基复合体。何首乌丸组可见线粒体数目增多,体积增大,部分线粒体肿胀,核膜扩张;胞质内脂滴较多。
4.结论
1衰老模型大鼠H-P-O-A性腺轴p19ARF/p53/p21Cip1和p16INK4a/Rb衰老途径中关键的调节因子p19ARF、p53、Rb、p16基因及蛋白表达上调,预防性应用何首乌饮对此可以起到抑制作用。
2衰老模型大鼠H-P-O-A性腺轴衰老相关细胞因子EGF及其受体EGFR、IGF-Ⅰ及其结合蛋白IGFBP3表达均降低,预防性应用何首乌饮可增加这些细胞因子的表达。
3衰老模型大鼠H-P-O-A性腺轴衰老相关细胞因子TNF-α蛋白表达增高,预防性应用何首乌饮可以使TNF-α水平下降。
4 D-半乳糖可致大鼠下丘脑弓状核、垂体前叶和卵巢的超微结构发生损伤,预防性应用何首乌饮可以减缓这种超微结构的损伤,从而改善H-P-O-A性腺轴的功能,延缓其衰老。
As a normal and complicated life process, senescence was involved in a series of routine changing in various tissues and cells. The development and aging of hypothalamus- pituitary-gland axis (H-P-G-A) was important in life proceeding. Immune-neuroendocrine network played an important role in aging and H-P-G-A was an important component in this network.
Many studies indicated that cellular senescence was implemented via some signaling transduction pathways which a lot of cytokines were involved in. When the key regulators in these pathways were changed, cellular senescence would be delayed. In this study, we had investigated the changes of some key regulators including p53, p19ARF, Rb, p16, IGF-Ⅰ, IGF-BP, TNF-α, EGF, EGFR and the changes of the ultrastructure of arcuate nucleus in the hypothalamus, adenohypophysis, ovary after taking traditional Chinese medicine Heshouwuyin (HSWY) by using the sub-acutely aging female model rats induced by D-galactose, so as to find the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) and the mechanism of HSWY in anti-aging.
1. Objective
We deteceted the level of mRNA p53, p19ARF, Rb, p16 and IGFBP3 by reverse transcription-polymerase chain reaction (RT-PCR), and examined the expression of protein Rb and p16 by Western-blotting, and evaluated the expression of p53, TNF-α, EGF and EGFR by immunohistochemistry (IH), and analysised the mRNA level of IGF-Ⅰby hybridization in situ (HIS) in order to find out the senescent mechanism of hypothalamus-pituitary-ovary axis (H-P-O-A) of the aging model rats and the effect of HSWY in anti-aging, so as to provide theoretical and experiment evidence to anti-aging in the female’sexual gland.
2. Methods
2.1 Choosing of the experimental animal and establishment of the aging model. Eight-week-old female SD rats (n=96) were separated into three groups at random: normal group, aging model group, preventing aging group. The rats in preventing aging group were re-divided into four groups: HSWY low dosage group, HSWY medium dosage group, HSWY high dosage group and Heshouwan(HSWW) group (n=16 in each group). The sub-acutely aging model rats were induced by D-galactose, and the rats in preventing aging group accepted intragastric administration of HSWY and HSWW while accepting D-galactose intraperioneal injection for 60 days.
2.2 Experimental procedures. Hypothalamus, adenohypophysis and ovary tissues of each group were obtained after the rats undergoing experiment for 60 days. We detected the gene mRNA expression of p53, p19ARF, Rb, p16 and IGFBP3 by RT-PCR, and examined the protein expression of Rb and p16 by Western-blotting, evaluated the expression of p53, TNF-α, EGF and EGFR by IH, analysised the level of mRNA IGF-Ⅰby HIS, and observed the ultrastructure by transmission electron microscope (TEM).
3. Results
3.1 Key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were changed in aging model, and the overexpression of these regulators could be retrieved by using HSWY.
3.1.1 The result of PCR had shown that gene expression of p53 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p53 were significantly decreased (P<0.05). In the hypothalamus and adenohypophysis tissues, the gene expression of p53 in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p53 was significantly lower than in HSWW group (P<0.05). The results of IH had shown that immunostaining for p53 in arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for p53 were significantly decreased (P<0.05). In the adenohypophysis tissue, the immunostaining for p53 in HSWY low dosage group was significantly weaker than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the immunostaining for p53 was significantly weaker than in HSWW group (P<0.05).
3.1.2 The result of PCR had shown that gene expression of p19ARF in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p19ARF were significantly decreased (P<0.01, P<0.05). In the hypothalamus tissue, the gene expression of p19ARF in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of p19ARF was significantly lower than in HSWW group (P<0.05).
3.1.3 The result of PCR had shown that gene expression of Rb in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of Rb were significantly decreased (P<0.01, P<0.05). In the hypothalamus and the adenohypophysis tissues, the gene expression of Rb in HSWY low dosage group were significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the gene expression of Rb was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein Rb in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein Rb were significantly decreased (P<0.05). In the adenohypophysis tissue, the expression of protein Rb in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). However, in the ovary tissue, it was HSWY high dosage group in which the expression of protein Rb was significantly lower than in HSWW group (P<0.05).
3.1.4 The result of PCR had shown that gene expression of p16 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of p16 were significantly decreased (P<0.01, P<0.05). In the adenohypophysis tissue, the gene expression of p16 in HSWY low dosage group was significantly lower than in HSWW group (P<0.05). The result of Western blotting had shown that expression of protein p16 in hypothalamus, adenohypophysis and ovary tissues were increased in aging rats (P<0.05). After pretreatment of HSWY in preventing aging group, the expression of protein p16 were significantly decreased (P<0.05).
3.2 Cytokines related to senescence in H-P-O-A were changed in aging model, and the abnormal expression could be retrieved by using HSWY.
3.2.1 The results of IH had shown that immunostaining for EGF in ovary tissue was significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGF was significantly increased (P<0.01). The results of IH had shown that immunostaining for EGFR in arcuate nucleus, adenohypophysis and ovary tissues were significantly weaker in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for EGFR were significantly increased (P<0.05).
3.2.2 The results of IH had shown that immunostaining for TNF-αin arcuate nucleus, adenohypophysis and ovary tissues were significantly more intense in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the intensity of immunostaining for TNF-αwere significantly decreased (P<0.01, P<0.05). In the arcuate nucleus and adenohypophysis tissues, the immunostaining for TNF-αin HSWY group were significantly weaker than in HSWW group (P<0.05).
3.2.3 The results of HIS had shown that the expression of IGF-ⅠmRNA in ovary tissue was significantly decreased in model rats (P<0.01). After pretreatment of HSWY in preventing aging group, the expression of IGF-ⅠmRNA was significantly increased (P<0.01). The result of PCR had shown that gene expression of IGFBP3 in hypothalamus, adenohypophysis and ovary tissues were up-regulated in model rats than in normal rats (P<0.01). After pretreatment of HSWY in preventing aging group, the gene expression of IGFBP3 were significantly decreased (P<0.01, P<0.05).
3.3 Morphologic structure of H-P-O-A in aging model was damaged, and the impairment could be restored by using HSWY.
3.3.1 The morphological changes of the arcuate nucleus in the hypothalamus: By light microscope, it could be seen that there were two kinds of cells in arcuate nucleus: neurone and neuroglial cell. In model rats, there were a lot of reactive colloid cells except for the two kinds of cells above-mentioned. By transmission electron microscope(TEM), arcuate nucleus was constituted of dark neurons, pale neurons. In model rats′arcuate nucleus, it was found that the nucleolemma was indented or dissolved, and the number of lysosome was increased, and mitochondriales cristae were broken and even vanished, and rough endoplasmic reticulum losted its granules, and Golgi complex swelled, and pre- and postsynaptic membranes were unclear and synaptic cleft was fusional. In preventing aging rats′arcuate nucleus, part of the nucleolemma was dissolved, and lysosome could be seen. Part of the mitochondriales cristae was broken. Rough endoplasmic reticulum losted its granules lightly. Golgi complex swelled lightly. Pre- and postsynaptic membranes were clear and part of synaptic cleft was fusional.
3.3.2 The morphological changes of the adenohypophysis: By light microscope, it could be seen that the gland cells were including three kinds of cells: acidophil, basophi and chromophobe cells. In model rats, it could be seen that the gland cells were arranged disorderly and the blood vessels were proliferated. The number of acidophil and basophi cells was decreased. By TEM, the gonadotroph of model rats changed obviously. The heterochromatin became more and the nucleolemma was dissolved. GER dilated obviously and Golgi complex increased and swelled. Mitochondriales cristae were broken and even vanished. The secretary granules were increased. In preventing aging rats′gonadotroph, rough endoplasmic reticulum were increased and dilated lightly. Golgi complex swelled lightly and part of the mitochondriales cristae was broken. The secretary granules were increased lightly.
3.3.3 The morphological changes of the ovary: By light microscope, the ovary was characterized by follicles in every grade and corepus luteums. The number of primordial follicles, growing follicles and corepus luteums in model rats was less than that in normal rats. The granulosa cells were arranged disordered and the number of the saccular ectasia follicles was increased. Compared with model rats, the number of primordial follicles, growing follicles and corepus luteums was more in preventing aging rats. By TEM, in the model rats′granulosa cell, it was found that mitochondria were droped and swelled, cristae were broken and even vanished, and smooth endoplasmic reticulum were decreased and dilated, and Golgi complex swelled. In HSWY low dosage rats′granulosa cell, it could be seen that mitochondria were swelled obviously, and smooth endoplasmic reticulum were dilated lightly, and Golgi complex swelled, and lipid droplets were increased. In HSWY medium dosage rats′granulosa cell, it was found that mitochondria were increased and swelled lightly, and many smooth endoplasmic reticulum were dilated lightly, and lipid droplets were increased. The ultrastructure of HSWY high dosage rats′granulosa cell was similar to nomal rats′. And the ultrastructure of HSWW rats′granulosa cell was similar to HSWY medium dosage rats′.
4. Conclusion
4.1 The genes and proteins of key regulators—p53, p19ARF, Rb, p16 of signaling transduction pathways: p19ARF/p53/p21Cip1 and p16INK4a/Rb in H-P-O-A were overexpressing in aging model, and this could be depressed by using HSWY.
4.2 Cytokines related to senescence—EGF and its receptor EGFR, IGF-Ⅰand its binding protein IGFBP3 were decreased in H-P-O-A in aging model, and the expression of these cytokines could be increased by using HSWY.
4.3 The protein of TNF-α—a cytokine related to senescence was overexpressing in H-P-O-A in aging model, and the level of TNF-αcould be reduced by using HSWY.
4.4 Morphologic structure of H-P-O-A in aging model was damaged by D-galactose. The impairment could be restored by using HSWY, so as to the function of H-P-O-A could be improved and senescence could be delayed.
引文
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