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Contents
Abstract
Foreword
References
Part Ⅰ
A Methodological approach to Sero-pharmacoogy of Chinese Drugs and the
Protective Effect of Naolizhibzo on Inschemic/Anoxic injury of PC 12 Cells
Abstract
Materials and Methods
Animals
Reagents and Instruments
Methods
1. Preparation of Extract of Naolizhibao
2. Preparation of Medicated Serum
3.Effect of Different Frequencies of Administration, Different Blood-collecting
Time and Different Concentrations of Medicated Serum on Ischemic Injury
of PC12 Cells
4.Effect of Naolizhibao on Ischemic Injury of PC12 Cells
5.Effect of Naolizhibao on Anoxic Injury of PC12 Cells Caused by Sodium
Dithionite
Results
Effect of Different Concentrations of Medicated Serum on Ischemic Injury
of PC12 Cells
Effect of Medicated Serum Obtained After Different Frequencies of Administration
and Different Blood-collecting Time on Ischemic Injury of PC12 Cells
Effect of Naolizhibao on Ischemic Injury of PC12 Cells
Effect of Naolizhibao on Anoxic Injury of PC12 Cells Caused by Sodium
Dithionite
Discussion
References
Part Ⅱ
Protection of Naolizhibao on Primary Cultured Cerebral Cortical Nerve
Cell Injury of Rats Caused by Excitatory Amino Acids and NO
Abstract
Materials and Methods
Animal
Drugs and Reagents
Instruments
Methods
1. Primary Culture of Cerebral Cortical Nerve Cells of Rat
2.Glu-induced Nerve Cell Injury
3.SNP-induced Cerebral Cortical Nerve Cell Injury of Rat
4.MTT Determination of Cell Survival Rate
5.Determination of Calcium in Brain Nerve Cells of Newborn Rats
6.Determination of Cell Membrane Fluidity
Results
Morphological Observations
Cell Survival Rate
Determination of LDH
Effect of Naolizhibao on Glu-induced NO and NOS Activities of Primary
Cultured Cerebral Cortical Cells of Rats
Effect of Naolizhibao on Glu-induced Calcium Elevation in Nerve Cells
Effect of Naolizhibao on Glu-induced Nerve Cell Membrane Fluidity
Discussion
References
Part Ⅲ
Effect of Naolizhibao on Simulated Aging Reaction of Primary Cultured
Cerebral Cortical Nerve Cells of Rats Induced by D-galactose and by Serum-free
Medium
Abstract
Materials and Methods
Culture of Primary Cerebral Cortical Nerve Cells of Rat
D-galactose-induced Simulated Aging Reaction
Serum-free Culture-induced Simulated Aging Reaction of Primary Cultured
Nerve Cells
MTT Microdetermination of Cell Survival Rate
Results
Effect of Naolizhibao on D-galactose-induced Simulated Aging Reaction
of Nerve Cells
Effect of Naolizhibao on Serum-free Culture-induced Simulated Aging Reaction
of Nerve Cells
Discussion
References
Part Ⅳ
A Preliminary Approach to the Neurotroophism of Naolizhibao
Abstract
Materials and Methods
Cultured of PC12 Cells and Plotting of Growth Curve
Observation of Axonal Growth of PC12 Cells and MTT Determination of Living
Cell Count
Culture of Primary Cerebral Cortical Nerve Cells of Rat
Effect of Naolizhibao on Growth and Development of Cortical Nerve Cells
Results
Effect of Naolizhibao on Growth of PC12 Cells
Effect of Naolizhibao on Axonal Growth of PC12 Cells and on Cell Survival
Rate
Effect of Naolizhibao on Growth and Development of Primary Cultured Cerebral
Cortical Nerve Cells of Rat
Discussion
References
Part Ⅴ
Effect of Naolizhibao on PC12 Cell Apoptosis Induced by Sodium Nitroprusside
Abstract
Materials and Methods
Drugs and Reagents
Instruments
Methods
1.Preparation of the Extract and Medicated Serum of Naolizhibao
2.Effect of Different Concentrations of sodium Nitroprusside on PC12 Cell
Survival Rate
3.Determination of DNA Breaking Rate by Diphenyl-amine
4.Determination of Cell Survival Rate
5.Nuclear Morphological Observation in the Presence of PC12 Cell Apoptosis
6.Extract of DNA and Electrophoresis
7.Quantitative Analysis of Cell Apoptosis Peak using Flowing Cell Apparatus
8.Immunohistochemical Detection of Bc1-2
9.Detection of Membrane Fluidity
Results
Establishment of Models
MTT Microdetermination of Cell Survival Rate
Morphological Observations
Gel Electrophoretic Analysis of DNA Breaking
Quantitative Analysis of Cell Apoptosis Peak Using Flowing Cell Apparatus
Effect of Naolizhibao on SNP-induced bc1-2 Protein Expression in PC12
Cells
Analysis of Membrane Fluidity
Discussion
References
Part Ⅵ
Effect of Naolizhibao on Improvement of D-galactose-induced Subacute Senescent
and the Study of Its Mechanism of Action
Abstract
Materials and Methods
Animals
Drugs and Reagents
Instruments
Methods
1.Preparation of Extract of Naolizhibao
2.Manufacture of Model
3.Learning and Memory Test
4.Determination of Biochemical Criteria of Blood, Tissues and Organs of
Mice
5.Histopathological Examination
6.Ultrastructural Observation
Results
Changes in Body Weight and Brain Weight
Effect of Naolizhibao on Learning and Memory of D-galactose-induced Subacute
Senescent Mice
Effect of Naolizhibao on SOD and MDA Level's in the Brain, Liver and Serum
of D-galactose-induced Subacute Senescent Mice
Effect of Naolizhibao on level of NO and NOS Activity in the Brain of
D-galactose-induced Subacute Senescent Mice
Effect of Naolizhibao on Activity of ATP Enzyme in the Brain and Liver
of D-galactose-induced Subacute Senescent Mice
Effect of Naolizhibao on Level of Lipofuscin in Brain Tissue and Level
of Oxyproline Hydroxyproline in Skin of D-galactose-induced Subacute Senescent
Mice
Effect of Naolizhibao on Protein Content of D-galactose-induced Subacute
Senescent Mice
Histopathological Observation
Ultrastructural Observation
Discussion
References
Foreword
With the progress of human society and the rise of people's living standards
year by year, the age structural ratio of human society is becoming favourable
to old people. In the year of 2000, the total population of China will
amount to 13 hundred millions and the people 60 years old and over will
amount to 10%. According to statistics China has entered into senile society
at a speed of 15 months ahead of time. With the aging of structure and
function of nervous system of old people, the number of patients with
senile dementia increases day by day, and it is estimated that there are
at least three or four million patients senile dementia in China. So numerous
dementia patients not only cause the patients themselves and their families
to have utmost pain but also bring about a heavy pressure to the society
and directly lower the population quality of China. Therefore, the study
of brain aging and of the pathogenesis of senile dementia will necessarily
promote the development of geriatrics and provide a firm theoretical basis
for the prophylaxis and treatment of senile diseases.
Senile dementia is chiefly divided into two types: one is multiple infarctional
dementia (or vascular dementia VD) caused by cerebrovascular disease,
VD is due chiefly to multiple cerebral infarction, lacunar infarction,
solitary infarction in associated cerebral zone and cerebral hemorrhage;
the other is senile dementia or Alzheimer's disease (AD), it is a progressive
retrograde nervous disease with unknown cause and characterized by clinical
manifestations and pathological changes, the pathologic features include
loss of neurons, neurofibrillary tangles (NFT), senile plaques (SP) or
neurite plaques (NP) and amyloid angiopathy (AAP). So far studies on senile
dementia have not revealed a clear pathogenesis. The overall attenuation
of the function and structure of cholinergic nervous system such as loss
of cholinergic neurons and the reduced density of M and N receptors is
generally believed to be an important cause of senile dementia. Recent
studies have shown that calcium overload in nerve cells, nitric oxide
(NO), cell apoptosis and oxygen free radicals are closely related to senile
dementia, there exist mutual link and mutual causality between them.
Glutamate (Glu), and important excitatory amino acid with the highest
content in central nervous system, plays and important role in maintaining
the process of transmission of normal neuronal signals. The velease of
large amount of Glu has marked injury action on nerve cells, chronic neuronal
retrograde diseases (such as Alzheimer's disease) and the pathological
changes in the presence of cerebral ischemia are all related to the neurotoxic
action of Glu. NO, a new-type messenger molecule, mediates neurotoxicity
of Glu as well as transmits of Glu with NMDA receptor causes large opening
of passage of calcium and the inward flow of Ca2+ in large quantity further
activates Ca2+ -dependent calcium regulating protein; NOS synthesizes
excessive NO by means of Glu-NMDA-[ Ca2+]-NOS-NO route. Recent studies,
however, revealed that NO can affect the synthesis and release of Glu
by means of regulating cGMP formation, that is, it causes cell injury
by way of NO-cGMP-Glu-[Ca2+ ]: (1). Therefore, there exist mutual promotion
and mutual causality between Glu and NO. NO is closely related to senile
dementia, NOS inhibitor has protective effect on toxicity of human β-amyloid
fragment (2). Kopa et al (3) reported that the expression of iNOS in patients
wit HIV and dementia showed marked increase. NO can cause overload of
Ca2+ in nerve cells and one of the features of brain aging is Ca2+ overload
in nerve cells, which is the "final common pathway" of causing
nerve cell injury and resulting in cell death. Besides, NO itself is a
free radical, it can provoke peroxidation of membranous lipid; the reaction
of NO and O2 produces OH－ and NO2 free radicals which have greater toxicity;
and the reaction of NO and iron-sulfur center of enzyme or hydrosulfide
group of peptide can cause functional loss of enzyme or polypetptide.
Therefore, peroxidation of NO itself may have greater direct cytotoxicity.
The oxygen metabolism in brain tissue is luxuriant, the cell membrane
of neuron contains large amount of polyunsaturated fatty acid, in the
course of senility, the substance and energy metabolism in brain tissue
leads to production of large amount of free radicals; therefore, there
exists great hazardness that the central nervous system is injured by
free radicals. Autopsies of AD patients found increased formation of free
radicals in brain tissue, severe peroxidation of lipids and injured mitochondria
and mitochondrial DNA. In experiments in vitro, it was also found thatβamyloid
induced cultured nerve cells to form hydrogen peroxide, thus causing cell
damage and that anti-oxidant and redical scavenge protected the nerve
cells from toxicity ofβamyloid (4). Free radical theory about senility
holds that with the increase of age, the reduced metabolism of organism,
functional attenuation of radical scavenge system and increased production
of free radicals bring about structural and functional damage of cells
and marked toxic action of free radicals on nerve cells causes nerve cells
to develop lipofuscin sedimentation, increased senile plaques, vacuolar
degeneration and disturbance of energy metabolism with the result that
the neuronal structure is damaged and the number of neurons decreases,
being similar to the pathology of senile dementia. Therefore, great attention
has been paid to free radical theory about senile dementia.
Recent studies have shown that there exists an additional mode of death
different from cytonecrosis in addition to cytonecrosis in the process
of death of neurons, that is, under the induction of some physiological
or pathological factors membrane signal system is activated and genes
associated with cell apoptosis or programmed death are further initiated
and controlled with the final result that cells undergo self-destruction
and self-death according to certain programme control, namely cell apoptosis,
the biochemical mechanism of which is related to intracellular increase
of calcium and free radicals. More and more evidence shows that some retrograde
nervous diseases such as senile dementia are due to pathological apoptosis
developed in central nervous system (5). Apoptosis is also related to
the pathological process of central nerve injury in cerebral ischemia
and brain injury. Nerve cell apoptosis is the principal cause of evoking
loss of nerve cells. The relation of NO to apoptosis is the forward field
of present biological study of NO, results of the study show that apoptosis
is the mode of death of NO-dependent cells. It has been demonstrated experimentally
that in primary cultured rat nerve cells SNP could simulate NMDA and cause
apoptosis or death of dose-and time-dependent cells (6). Cell apoptosis
is a complex course of high-precise regulation and involves expression
and regulation of many genes, for example, bc1-2 gene is the suppressor
gene of apoptosis. Bc1-2 is a longevous gene, its product does not change
cell multiplication rate but resists cell death of various forms, prolongs
cell longevity and increases the number of cells. Excessive expression
of bc1-2 gene by artificial means can also prolong the survival of nerve
cells. Carcia et at (7,8) gave microinjection of plasmids which express
bc1-2 gene to in vitro cultured sympathetic neurons which depends on growth
factor and found that apoptosis induced by removal of nerve growth factor
could be prevented. The anti-apoptosis effect of bc1-2 gene may be related
to inhibiting the production of intracellular activated oxygen and intracellular
Ca 2+ (9,10) Bc 1-2 gene was shown to have the effect of promoting nerve
regeneration in mammals in addition to the effect of anti-apoptosis, loss
of the effect of bc1-2 led to lack of axonal gwowth; and the promotion
of axonal growth by bc1-2 was found to be independent on its anti-apoptosis
effect, bc1-2 controlling axonal growth of central nervous system as a
regulator of gene programme (11). It is worth mentioning that recently
Zhang et al (12) advanced the hypothesis that bc1-2 gene may be the associated
gene of the kidney essence in terms of traditional Chinese medicine. This
not only provides a new route for studying material basis of the kidney
in traditional Chinese medicine but also provides the possibility for
using modern medical theories to make a further development of traditional
Chinese medicine. If bc1-2 is the associated gene of the kidney essence,
remedies which interfere with bc1-2 gene will necessarily affect the renal
function in terms of traditional Chinese medicine, many remedies are currently
found to affect bc1-2 gene and these remedies are possibly used as clinical
medicines to regulate the renal function in terms of traditional Chinese
medicine. The senility theory in traditional Chinese medicine is based
on the theory of reduced renal function. In traditional Chinese medicine
deficiency of the kidney is believed to be the principal cause. As stated
in《Orthodox Medical Problems》(written by Yu Tuan): "With excess of
the kidney qi, one can promise longervity; with deficiency of the kidney
qi, longevity cannot be promised." Old people are in the state of
"physiological deficiency of the kidney qi." The kidney essence
in terms of traditional Chinese medicine are the foundation of senility
theory of traditional Chinese medicine. If the hypothesis is tenable,
it will open a new way for the study and development of remedies for treating
senile dementia.
Nervous system plays a leading role in adapting organism to internal and
external environments and, therefore, has an important influence on the
process of senility of organism. Among the various systems of the organism,
the central nervous system is the one with the most stability and the
longest survival time. Because all neurons undergo the process of cell
division, has high specificity and no longer has further division, their
survival time is identical with the lifetime of the entire organism. In
the course of senility, the changes of central nervous system in structure,
function and biochemistry can lead to significant changes in activities
of other systems and organs in the body. There are notable structural
changes in human brain tissue in the course of senility, for example,
the weight of brain reduces by 7%-10% in geratic period (70-80 years of
age), the number of neurons in cerebral cortex of old people (77 years
of age) is, on the average, 20%-25% less than that of young people (9-28
years of age) (14). Substances are the basis of functions, senile dementia
is the dysfunction in learning and memory caused by degenerative death
of neurons due to multiple causes. Some Chinese drugs have been shown
to improve directly the morphology of brain tissue of animals with simulated
dementia, thus improving the brain function. Yang Ying et al (15), using
quantitative synapse determination, found that long-term administration
of ginsenoside significantly increased the number of synapse in the upper
layer of pyramidal cells in zone CA3 of rat hippocampus. Recent studies
showed that senile dementia is related to lack of nerve growth factor
(NGF). NGF can effectively prevent degenerative death of neurons, and
it has been known that NGF, as the nutrient and growth-promoting factor
of neurons, takes effect by means of activating receptor. Some traditional
Chinese medicines with the action of postponing senility such as epimedium,
fleece-flower root, bighead atractylodes rhizome, dodder seed, astragalus
root and poria have been shown to have marked activation on I-NGF. Among
these Chinese drugs the crude extract of cynomorium has the strongest
action. This suggests that the above-mentioned Chinese drugs may possibly
have excitant of NGF receptor (16). The failure of such neuontrophic factors
as NGF to pass blood-brain barrier lead to difficulty in their use. Therefore,
it is of important theoretical and practical significance to seek from
Chinese drugs materia medica which themselves or receptor excitants of
which have trophism on nerve cells.
Traditional Chinese medicine is a bright pearl in the world, it has various
kinds of materia medica with wide used and has a history of several thousand
years. It demonstrates clear superiority in preventing and curing gerontal
and chronic diseases. Particularly the more understanding of the toxic
or side effect observed in taking Western medicines spurs many people
on to adopt natural medicines and this provides favourable circumstances
and challenges nerve existed before for bringing the superiority of traditional
Chinese medicine into full play. It is and urgent and heavy task to utilize
modern scientific and technological means and to make traditional Chinese
medicine move towards modernization and internationalization. How to study
the mechanism of action of Chinese drugs and their complex prescription
at cellular, molecular and genic levels is the cause and basis of development
of sero-pharmacological method for Chinese drugs, its occurrence provides
a method of using modern techniques and methods to study traditional Chinese
medicines.
Sero-pharmacological method for Chinese drugs originated in 1987. In Japan,
Iwama et al (17) published a report of using medicated serum of animal
to study Chinese drugs, the method was named "Sero-pharmacology of
Traditional Chinese Medicines." It refers to an experimental method
by which blood is collected from animals after their orally taking medicine
for a given time, serum is separated from the blood and then the medicated
serum is used to make in vitro experiment. Previously crude extract of
Chinese drugs and their complex prescription was directly added to reaction
system (such as cell culture, etc.) for making in vitro experiment. However,
the crude extract of Chinese drugs does not represent the available compositions
which are truly effective in internal environment, and, what is more,
the physicochemical properties of Chinese drugs per se (such as electrolyte,
acie-base scale, etc.) and impurities with exert a certain influence on
cells, thus interfering with experimental results and affecting the truthfulness
and reliability of experimental conclusions. The use of sero-pharmacological
method for studying Chinese drugs can, to some extent, overcome the interference
of experimental results by such factors as physicochemical properties
of the Chinese drugs themselves, it not only reflects the direct effect
of absorbable compositions of drugs but also reflects the indirect results
of metabolites formed in the organism and endogenous substances in the
organism induced by the drugs.
Naolizhibao is a complex prescription consists mainly of tortoise plastron,
wolfberry fruit, earthworm, grassleaved sweetflag rhizome, bitter cardamon,
Chinese angelica root, polygala root, safflower, spiny jujuba seed, curcuma
root, schisandra fruit and fleece-flower root, has the effect of tonifying
the kidney to replenish marrow, promoting qi circulation and activating
the collaterals, promoting generation of blood and strengthening the bone,
and promoting blood circulation by removing blood stasis, and is thus
a prescription of treating both the principal and the secondary aspects
of a disease, its symptoms and cause at the same time and strengthening
the body resistance to eliminate pathogenic factors. Clinically it is
used to treat mental retardation in children, senile dementia, etc. with
satisfactory results. Studies made by Wu Yonjie (18) showed that Naolizhibao
improved microcirculation of human brain, improved energy metabolism of
the brain, promoted development of the brain, raised RNA level in the
brain, promoted protein synthesis in the brain, and enhanced dopaminergic
neurons. It has been used clinically for years, but its mechanism of action
has not yet been studied. It has been known that the bitter cardamon in
the prescription of Naolizhibao contains cardamonol which has the effect
of antagonizing calcium; ferulic acid contained in Chinese angelica root
and fleece-flower root and Siberian solomonseal rhizome can all reduce
the content of lipofuscin, enhance SOD activity in the body and the activity
of Na +－K+－ATP enzyme and has the effect of resisting free radicals; carthamin
and neocarthamin contained in safflower have the effect of antithrombosis
and are used clinically to treat ischemic cerebrovascular disease, cerebral
apoplexy and cerebral arteriosclerosis; fleece-flower root also has the
effect of exciting NGF receptor; and grassleaved sweetflay rhizome can
increase learning capacity and memory and is used clincally to treat dementia.
(19)
Therefore, experimental studies on the protective action of Naolizhibao
on the brain, its therapeutic action on senile dementia and its mechanism
of action have been carried out according to the etiological and pathological
theories about senile dementia mentioned above and by means of sero-pharmacological
method for Chinese drugs from the following aspects: a methodological
approach to sero-pharmacology of Chinese drugs and the protective effect
of Naolizhibao on nerve cell injury; studies on the influences of Naolizhibao
on simulated-aging reaction of nerve cells and its in vitro anti-senility
action; and a preliminary approach to the neurotrophism of Naolizhibao
and studies on its anti-apoptosis.
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1 st ed
Part Ⅰ
A Methodological Approach to Sero-pharmacology of Chinese Drugs and the
Protective Effect of Naolizhibao on Ischemic/Anoxic Injury of PC12 Cells
[Abstract] Objective The present study was undertaken to investigate
the protective effect of the extract and medicated serum of Naolizhibao
on ischemic/Anoxic Injury of PC12 cells and to make an approach to some
methodological problems of sero-pharmacology. Methods By using sero-pharmacological
method and PC 12 cell culture in vitro, the effect of Naolizhibao on ischemic/anoxic
injury of PC12 cells caused by NaCN and sodium dithionite plus sugar deprivation
was studied, and an approach was made to different frequencies of administration
in preparationof medicated serum, different blood-collecting time and
different added amount of medicated serum by using model of ischemic injury
of PC12 cells caused by NaCN plus sugar deprivation. Results The results
showed that Naolizibao obviously resisted morphological changes of PC12
cells due to ischemia/anoxia, reduced leakage of LDH and increased cell
survival rate and that medicated serum collected at 0.5 h, 1h, 2h and
3h after a single administration did not have protection on cells, that
collected at 1 h and 2 h after two times of administation at an interval
of 2 h showed marked protection and that collected at different time points
after administration twice daily for three successive days (7 times of
administration ) all had obvious protection on cells. Better results were
obtained with 5% of added amount of serum, drug action was affected with
larger than 10% of added amount of serum.
Key works: Naolizhibao Sero-pharmacological method PC12 cell Sodium dithionite
Sodium cyanide Ischemia Anoxia
Vascular dementia, caused by such pathologic processes as cerebral infarction
and cerebral hemorrhage, occupies an important place in senile dementia.
As shown in literature, about 12%-20% of patients with senile dementia
are those suffering from multiple infarctional dementia, and those with
concurrent Alzheimer's disease account for 16%-20% of the patients. (1,2)
The high dependence on oxygenic metabolism for energy supply and the high
oxygen demand of nerve cells determine the high liability of nerve cells
to ischemic/anoxic injury. Cerebral ischmia/anoxia can result in functional
and structural destruction of brain nerve cells, thus causing the development
of various diseases that affect the functioning of the brain. It is, therefore,
of important significance in the prevention of senile dementia to prevent
cerebral ischemic/anoxic injury. The model of ischemic/anoxic injury of
nerve cells and PC12 cells caused by NaCN plus sugar deprivation and sodium
dithionite plus sugar deprivation is the usual model of screening drugs
with protection on neuronal injury (3,4,5,6) and is suitable for studying
the protection of drugs on ischemic/anoxic injury. Therefore, in the present
study sero-pharmacological method for Chinese drugs and model of ischemic/anoxic
injury of PC12 cells caused by NaCN plus sugar deprivation and sodium
dithionite plus sugar deprivation were used to study the protective effect
of the extract and medicated serum of Naolizhibao on ischemic/injury of
PC12 cells, and an approach was made to the frequencies of administration
of Naolizhibao, time of collecting blood and the amount of serum added
to the reaction system.
Results
Effect of Different Concentrations of Medicated Serum on Ischemic Injury
of PC12 Cells The results showed that 2.5% medicated serum of Naolizhibao
had protection on ischemic injury of PC12 cells and that 5% medicated
serum of Naolizhibao and medicated serum of nimodipine all had notable
protection on PC12 cells; whereas the addition of 10% mouse serum or over,
whether it was normal serum or medicated serum, all showed marked protection
on PC12 cells, false-positive results developed, and this might be due
to the complex compositions of serum itself. The results are shown in
Table 1-1.
Effect of Medicated Serum Obtained After Different Frequencies of Administration
and at Different Time of Collecting Blood on Ischemic Injury of PC12 Cells
The results showed that medicated serum collected at 0.5 h, 1h, 2h and
3h after a single administration, although it had some protection on cells,
did not show significant statistical difference (p>0.05), that collected
at 1h and 2h after two times of administration showed marked protection,
the difference being significant, and that collected at different time
points after seven times of administration all had obvious protection
on cells, particularly the medicated serum obtained at 0.5h and 1h following
administration (p<0.001). The results are given in Table 1-2.
Effect of Naolizhibao on NaCN-induced Ischemic Injury of PC12 Cells PC12
cells were exposed to Earle's buffer solution containing NaCN without
sugar, 20 minutes later they were observed under inverted microscope and
were found to have had disappearance of cell process structure, cellular
pyknosis, swelling, refractive index, occurrence of cytomembrane breaking
in part of cells, and cytolysis into debris. Results of MTT microcolorimetry
and LDH determination showed that injured cells had obviously reduced
uptake of MTT and that LDH was released in large amount, indicating that
the cells were injured or part of the cells had been dead. Administration
of the extract and medicated serum of Naolizhibao significantly reduced
the disappearance of cell process structure and cell debris and increased
MTT uptake as compared with the drug-free group. The inhibition rates
of extract of Naolizhibao in the concentrations of 0.04mg/ml, 0.16mg/ml,
0.63mg/ml and 2.50mg/ml for NaCN-induced PC12 cell injury were 4.7%, 44.2%,
62.8% and 93.0%, respectively, and those of medicated serum of Naolizhibao
in the doses of 3g/kg, 6g/kg and 12g/kg for PC12 cell injury were 34.7%,
44.2% and 62.8%, respectively. The leakage of LDH after administration
decreased greatly and had better dose-dependence. The inhibition rate
of extract of Naolizhibao in the concentration of 2.50mg/ml and that of
medicated serum of Naolizhibao in the dose of 12g/kg were 87.9% and 65.1%,
respectively. The results are shown in Table 1-3 and 4.
Effect of Naolizhibao on Sodium dithionite-induced PC12 Cell Injury Exposure
of PC12 cells to 2mmol/l sodium dithionite resulted in marked imjury,
decrease or disappearance of cell process structure was visible under
microscope, and the cells became swelling and round. Administration of
extract and medicated serum of Naolizhibao obviously resisted cytomorphological
changes due to ischemia/anoxia, as manifested by decreased disappearance
of cell process structure and decreased cell debris. Treatment of PC12
cells with 2 mmol/l sodium dithionite reduced MTT uptake and the OD values
were significantly lower than those of the control group, as manifested
by damaged cells or cell death in part of the cells. Extract and medicated
serum of Naolizhibao of different administration groups significantly
increased MTT uptake by PC12 cells, made the OD values significantly higher
than those of the model group, increased the number of living cells obviously,
and had better concentration-dependence. However, nimodipine in the concentration
of 5×10－6 mol/l and medicated serum of nimpdipine in the dose of 16 mg/kg
did not show marked difference as compared with the model group. 2mmol/l
sodium dithionite significantly increased LDH activity in culture fluid.
Administration of extract of Naolizhibao and different concentrations
of medicated serum significantly inhibited the release of LDH and showed
obvious dose-dependence. The results are shown in Table 1-5 and 1-6.
Disscussion
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