Page 40 - 2020年17期

Improvement Effects of Isopimpinelline on PCPA-induced Pineal Gland Injury Model Rats and Its Effects
on Expression of Biological Clock Gene
WANG Danni，YANG Qian，ZHONG Qiming，SONG Meiqing，TONG Liguo，JIA Lili，NIU Yanyan，FENG Mali
（Shanxi Academy of Traditional Chinese Medicine，Taiyuan 030012，China）

ABSTRACT OBJECTIVE：To study the improvement effects of isopimpinelline on p-chlorophenylalanine（PCPA）-induced pineal
injury model rats and its effect on expression of biological clock gene. METHODS：Totally 60 rats were divided into blank control
group（2％ polysorbate solution），model control group（2％ polysorbate solution），positive control group（melatonin，10 mg/kg）
and isopimpinelline high-dose，medium-dose and low-dose groups（3，1.5，0.75 mg/kg）. Except for blank control group，rats in
other groups were given PCPA intraperitoneally（450 mg/kg）to establish pineal injury model. After modeling finished，they were
given relevant medicine intragastrically，once a day，for consecutive 7 d. On the 6th day of administration，the sleep latency and
sleep duration of rats in each group were investigated by pentobarbital sodium coordination sleep test；after last administration，
ELISA assay was used to determine the serum level of melatonin in rats. Fluorescence microscope and electron microscope were
used to observe the pathological tissue and cell ultrastructure changes of the pineal gland. RT-qPCR was used to detect the mRNA
expressions of biological clock gene Clock，Bmal1，Per1，Per2，Per3，Cry1，Cry2 in pineal gland of rats. RESULTS：Compared
with blank control group，model control group had significantly longer sleep latency （P＜0.05）；serum melatonin，mRNA
expressions of Bmal1 and Per1 in pineal gland were significantly decreased（P＜0.05 or P＜0.01）while mRNA expression of Per3
was increased significantly （P＜0.05）. The pineal gland cell arrangement disorder，nuclear pyknosis，vacuolar degeneration
increased and cell number decreased significantly；mitochondria swollen，cristae broken and pyknosis were observed. Compared
with model control group，the sleep latency of isopimpinelline high-dose group was shortened significantly （P＜0.05），sleep
duration time was prolonged significantly （P＜0.05）；the levels of melatonin in serum，mRNA expressions of Clock，Bmal1，
Per1，Cry1 and Cry2 in pineal gland of rats were increased significantly（P＜0.05 or P＜0.01）. In isopimpinelline medium-dose
group，the sleep latency was shortened significantly（P＜0.05）；the levels of melatonin in serum and mRNA expressions of Clock，
Bmal1，Per1，Cry1，Cry2 in pineal gland were increased significantly（P＜0.05 or P＜0.01），while mRNA expression of Per3
was decreased significantly（P＜0.05）. In isopimpinelline low-dose group，the levels of mRNA expressions of Clock，Bmal1，Per2
and Cry2 were increased significantly（P＜0.05），while mRNA expression of Per3 was decreased significantly（P＜0.05）. Cell
arrangement disorder was improved and nuclear pyknosis vacuole degeneration was decreased to some extent in isopimpinelline
groups；mitochondria swelled，cristae fractured，and pyknosis decreased to some extent. CONCLUSIONS：Isopimpinelline can
improve PCPA-induced pineal gland injury in rats；it can up-regulate the expressions of positive regulators Clock，Bmal1 and
negative regulators Per1，Per2，Cry1，Cry2，while down-regulate the expression of negative regulator Per3.
KEYWORDS Isopimpinelline；Pineal gland；Biological clock gene；Rats

松果体（Pineal gland）作为昼夜节律生物钟，主要通 松果体生物钟基因表达尚不明确。鉴于此，本研究通过
过合成、分泌褪黑素进而调节机体昼夜节律、促进睡眠， 腹腔注射对氯苯丙氨酸（PCPA）致大鼠松果体损伤后，
并且其分泌的褪黑素还具有抗氧化、调节免疫、降血压、 观察异虎耳草素对松果体损伤大鼠的睡眠、血清褪黑素
抗肿瘤、延缓衰老等作用 [1-2] 。松果体损伤会导致褪黑素 水平、松果体组织病理学、细胞超微结构和生物钟基因
合成、分泌减少，从而引起机体昼夜节律紊乱。钟振荡器 表达的影响，以期发现含异虎耳草素的中药材或植物药
由一组昼夜节律基因及其编码的蛋白组成，是维持内源 保护松果体的作用及催眠机制，进而为此类药物的临床
[3]
性生物钟运作的核心元件 。目前，针对哺乳动物研究 应用、研发和资源利用提供依据。
较多的生物钟基因分别有Clock、Bmal1、Perl、Per2、Per3、 1 材料
[4]
Cry1、Cry2等，其对昼夜节律生物钟机能有着重要作用 。 1.1 仪器
现代药理学研究表明，蛇床子具有中枢抑制作用， H1型多功能微孔读板机（美国Bio-Tek公司）；BX51
其主要活性成分为蛇床子香豆素类成分，包括异虎耳草 型荧光显微镜（日本Olympus公司）；JEM-1011型透射电
素、蛇床子素、佛手柑内酯等 。本实验室前期研究通过 子显微镜（日本JEOL公司）；7500型实时荧光定量-聚合
[5]
谱-效分析发现，异虎耳草素为蛇床子香豆素类主要活 酶链式反应（PCR）仪、9902 Veriti 型定性 PCR 仪（美国
性成分之一，具有催眠、提高血清褪黑素水平的作用，并 Applied Biosystems 公司）；H1 型全功能酶标仪（美国
可下调原代海马神经元细胞 Bmal1 基因表达，上调 Bio Tek公司）。
Cry1、Per1、Per2 基 因 表 达 ，从 而 调 节 睡 眠 - 觉 醒 周 1.2 药品与试剂
期 [6-8] 。但其催眠活性是否通过改善松果体损伤及调控 异虎耳草素对照品（上海融合医药科技有限公司，

·2082 · China Pharmacy 2020 Vol. 31 No. 17 中国药房 2020年第31卷第17期