梅建勋
论文题目：中药神经保护作用的离体实验研究－“中药脑脊液药理学”实验方法的提出
作者简介：梅建勋，男，1970年11月出生，1997年09月师从于天津中医学院张伯礼教授，于2000年07月获博士学位。
摘 要
目的 应用“血清药理学”方法对中药进行药理研究特别是药效研究目前倍受关注，但考虑到血脑屏障的存在，本文应用血清和脑脊液分别观察了它们对于血脑屏障以内的细胞-神经元和星形胶质细胞的细胞毒性影响，从中优选中药作用于中枢神经系统的最佳实验方法。
中药复方制剂清脑益智方已被临床实验和在体实验证明是一个治疗血管性痴呆/多发梗塞性痴呆的有效方剂，为探求其疗效机制，本实验采用离体实验手段观察中药对于神经元兴奋毒性损伤的保护作用，寻求中药作用的靶点。
目前，众多学者认为在脑损伤条件下，星形胶质细胞可以活化并表达神经营养因子，进而保护神经元免受损伤，寻找内源性神经营养因子促进剂是一个热点问题。本文前述已证明，中药清脑益智方是通过星形胶质细胞介导而发挥其神经保护作用的，因此本实验试图证明中药是否促进了星形胶质细胞神经营养因子（重点是神经生长因子，NGF）的合成与分泌。
方法 家兔在口服中药复方清脑益智方水煎液后，血清和脑脊液分别被收集，然后按5%、10%、20%浓度加入至体外培养鼠脑皮层星形胶质细胞和神经元培养液中，以台盼蓝染色、乳酸脱氢酶和MTT试验为指标观察正常血清、中药血清、正常脑脊液和中药脑脊液对于上述两种细胞的死亡率和细胞活性的影响。
体外培养鼠脑皮层神经元，将谷氨酸加入细胞培养液中，造成神经元兴奋毒性损伤；家兔在口服中药复方清脑益智方水煎液后，脑脊液被收集，一批按5%、10%、20%浓度加入至损伤神经元培养液中，另一批先按5%、10%、20%浓度加入至体外培养鼠脑皮层星形胶质细胞无血清培养液中，取此种培养液再按5%、10%、20%浓度加入至损伤神经元培养液中，以台盼兰染色、乳酸脱氢酶和MTT试验为指标观察正常脑脊液、中药脑脊液、正常脑脊液胶质细胞培养液和中药脑脊液胶质细胞培养液对于神经元的死亡率和细胞活性的影响。
家兔在口服中药复方清脑益智方水煎液后，脑脊液被收集，按5%、10%、20%浓度加入至体外无血清培养鼠脑皮层星形胶质细胞培养液中，应用酶联免疫吸附技术（ELISA）测定培养液中NGF蛋白含量：应用反转录聚合酶链式反应（RT-PCR）检测星形胶质细胞NGFmRNA表达情况；以3H-TdR掺入技术检测星形胶质细胞的增殖情况。
结果
实验一1.星形胶质细胞：正常血清组和中药血清组与空白对照组比较，细胞死亡率明显升高、细胞孵育液中乳酸脱氢酶含量明显升高、MTT试验OD值明显下降，正常血清组与中药血清组之间比较没有明显差异，说明正常血清和中药血清对星形胶质细胞产生了明显的毒害作用，似乎这种毒性与血清本身有关，与中药的介入无关；而正常脑脊液和中药脑脊液与空白对照组比较，没有明显差异。2.皮层神经元：实验结果同上，正常血清和中药血清都对细胞产生了毒害作用，而正常脑脊液和中药脑脊液未见毒害作用。
实验二：1.模型损伤组与空白对照组比较,神经元死亡率明显升高、培养液中乳酸脱氢酶含量明显升高、MTT试验OD至明显下降，说明模型复制是成功的；2.中药脑脊液组与模型损伤组比较，细胞死亡率、细胞孵育液中乳酸脱氢酶含量、MTT试验OD值无明显变化，而中药脑脊液胶质细胞培养液组与模型损伤组比较，细胞死亡率明显下降、细胞孵育液中乳酸脱氢酶含量明显下降、MTT试验OD值明显升高。
实验三：1。与空白对照组比较，三个不同浓度的干预组均可以增加星形胶质细胞NGF蛋白的表达，尤其以20%和10%浓度明显；2.与空白对照组比较，三个不同浓度的干预组同样可以增加星形胶质细胞NGFmRNA的表达，仍然以20%和10%浓度明显：3.与空白对照组比较，三个不同浓度的干预组在星形胶质细胞的增殖上没有明显变化。
结论
由于血脑屏障的存在，神经胶质细胞和神经元的微观生存环境和屏障以外的其它体细胞有很大差别，本实验证明正常血清和中药血清都对细胞产生了毒害作用，说明“血清药理学”的方法不适合研究中药对上述两种细胞的影响，因为血清本身对于细胞的毒害作用将掩盖中药的药理作用。而参考“血清药理学”的思路，利用脑脊液来进行观察可能比较合理，姑且称这种方法为“脑脊液药理学”。
对于神经元兴奋毒性损伤，中药并非对神经元直接起效，它的神经保护作用是通过星形胶质细胞介导的，即中药神经保护作用的初级作用靶点在星形胶质细胞。
中药清脑益智方可明显促进星形胶质细胞NGF的合成与分泌，对于NGF含量的增加并非以星形胶质细胞的增殖为前提，因此中药清脑益智方中的成分中可能存在某种“内源性神经营养因子”促进剂。
ABSTRACT
Objective The in vitro experiment of pharmacodynamics on Chinese
materia medica usually employs the method of serum pharmacology, however which
might not adapt in the cells behind the blood-brain barrier. To explore a
useful method by which the pharmacological effects of Chinese materia medica on
the CNS could be correctly investigated, the cytotoxity of both serum and
cerebral-spinal fluid (CSF) was observed.
Our previous
clinical study and in vivo experiment has verified that Chinese Herbal compound
QingNaoYiZhi was effective to the vascular dementia/multi-infarct dementia. To
explore its therapeutic mechanism, this in vitro experiment was to observe the
neuroprotection of QingNaoYiZhi from the neuron excitotoxity and find its
effective target.
It is well
known that the activated astrocyte could express kinds of neurotrophic factors,
such as NGF (nerve growth factor), to protect the cortical neuron from the
ischemic damage to the CNS. Therefore, as a new therapeutic approach, finding
an endogenous neurotrophic factor agonist maybe important. The ahead of this
paper manifested that the in vitro neuroprotection of Chinese herbal compound
QingNaoYiZhi was mediated by the astrocyte, so this experiment tried to verify
that this Chinese herbal compound could increase the synthesis and secretion of
NGF in astrocyte.
Methods CSF and serum of the rabbit were taken after administration
of Chinese herbal compound QingNaoYiZhi and added into the medium of cultured
rat cortical neuron and astrocyte in proportion of 5%, 10% and 20%. Typan blue
staining, lactate dehydrogenase (LDH)
mensuration and MTT test were performed to observe the mortality and activity
of the above two kinds of cell.
That the medium of cultured rat cortical neuron was
added by the glutamate resulted in the neuron excitotoxic injury. After the
rabbit was taken orally by Chinese herbal compound (CHC) QingNaoYiZhi, the
cerebral-spinal fluid (CSF) was gained. Some of them were directly added into
the medium of injured neurons in proportion of 5%, 10% and 20%. Others were
first added into the non-serum medium of cultured rat cortical astrocytes and
then their medium was added into the medium of injured neurons in proportion of
5%, 10% and 20%. Typan blue staining, lactate dehydrogenase (LDH) and MTT test were employed to observe the
mortality and activity of neurons.
After the rabbit was taken orally by Chinese herbal
compound QingNaoYiZhi, the cerebral-spinal fluid was gained to add into the
non-serum medium of cultured rat cortical astrocytes in proportion of 5%, 10%
and 20%. The enzyme-linked immunoadsordent assay (ELISA) was used to mensurate the
content of NGF protein in the medium. The reverse transcription polymerase
chain reaction (RT-PCR) was applied to detect the NGF mRNA expression in
astrocyte. Furthermore, the proliferation of astrocyte was determined by 3H-TdR.
Results The serum group and the CHC-serum group
manifested the higher cell death rate, more increased LDH content and more
decreased OD value than the control group on the neuron and astrocyte,
furthermore, there is no difference between those two groups. It meant that
either serum or CHC-serum could product the cytotoxity on the neuron and
astrocyte, which seemed to concern with the serum itself, but not the CHC. In
addition, the CSF group and CHC-CSF group did not show any cytotoxity on the
neuron and astrocyte.
Comparing with those in the
neuron-injury group, the cell death rate, LDH content in the medium and OD
value had no change in the CHC-CSF group, however the cell death rate went
down, LDH content decreased and OD value increased in the CHC-CSF-astrocyte-medium
group.
All of the drug groups in
three kind of concentration could enhance the expression of NGF protein and
mRNA, and the group in 20% and 10% concentration were significantly higher than
in 5% concentration. Comparing with the control group, the drug groups had no
effect on the cell proliferation.
Conclusion Since the serum brought the great cytotoxity to
the cells behind the blood-brain barrier, its toxity will cover the drug effect
if the serum pharmacology is adopted to observe the pharmacodymamics to the
CNS. However, it was deemed that the “Cerebrospinal Fluid Pharmacology of
Chinese Materia Medica” is worth for further study.
For the neuron excitotoxic
injury, the effect of QingNaoYiZhi was not direct. That neuroprotection was
perhaps mediated by the astrocyte, which means the effective target of
QingNaoYiZhi is astrocyte.
Chinese herbal compound could significantly accelerate the
synthesis and secretion of NGF in astrocyte, which did not promote the cell
proliferation. It was suggested that some component of Chinese herbal compound
QingNaoYiZhi might act as an endogenous neurotrophin agonist.
