Authors' units: 1. China Pharmaceutical University, Nanjing, Jiangsu 210009
2. Guangzhou University of Traditional Chinese Medicine and Materia Medica, Guangzhou, Guangdong 510407
3. Guangdong Gaoming Medical and Medicinal Institute of Encephalopathy, Gaoming, Guangdong 528500
cerebral sequelae with concomitant intellectual disturbance and dysfunction of extremities. Its therapeutic effectiveness is definite and reliable.
In recent years, the in vitro culture of nerve cells has become the commonly used extracorporeal model for studying the mechanism of action of neuronal injury and the protection of drugs, (1) for it can eliminate the experimental errors due to multiple factors from the whole-body animal or brain slice model. Compared with the nerve cell model primarily cultured, the neuroma clone has the advantages of being a single cell without any other cells, rapid growth and reproduction, short culture period, more convenient use and being easily controlled, being the ideal extracorporeal model for studying the neuronal injury and drug action. PC 12 cell is the clone formed by pheochromocytoma of adrenal medulla of rat and has the characteristics of typical neuroendocrine cells, it is extensively used as the experimental material for studying nerve cell differentiation, ion channel, and release of receptor and transmitter, and is one of the most important clones in studying the neurotoxicity. (2)
The purpose of the present experiment was to study the effect of Li's Prescription No.5 on the proliferation of PC 12 cell and on the protection of ischemic-like injury of PC 12 cell caused by NaCN and sugar deficit so as to expound the mechanism of action of Li's Prescription No.5 in treating various diseases that affect the functioning of the brain.
Materials and Methods
Materials
Animal Mice of Kunming strain, weighing 22±3g, with a male-female ratio of fifty-fifty, were provided by the Laboratory Animal Department of China Pharmaceutical University.
Drugs and Reagents
Medicinal powder of Li's Prescription No.5 was supplied by Gaoming Medical and Medicinal Institute of Encephalopathy; PC 12 clone was kindly supplied by Zhu Xingzu, a research fellow of Shanghai Institute of Materia Medica; MTT (3-(4,5-dimethyl-2-thiazoly)2,5-diphenyl-2-tetrazolium bromide) was the product of Fluka; complete DMEM (Dulbecco's Modified Eagle's Medium) was the product of Gibco; trypsinase was the product of Sigma; calf serum was the product of Hangzhou Evergreen Bioengineering Materials Research Institute; nimodipine was the product of Tianjin Central Pharmaceutical Factory; lactic dehydrogenase (LDH) kit was the product of Nanjing Jiancheng Bioengineering Research Institute; and other reagents were all analytical pure preparations on sale.
D-Hanks solution (m mol/l): NaCl 137, KCl 5.0, Na2HPO4 0.6, KH2PO4 0.4, NaHCO3 3.0, pH 7.4.
Sugar-free Earle's solution (m mol/l): NaCl 116.4, KCl 5.4, CaCl2 1.8, MgSO4 0.8, NaH2PO4 2.6, NaHCO3 26.2, Mannitol 5.5, pH 7.4.
Instruments
Model DG3022A enzyme linked immunosorbent assay (ELISA) detector was the product of East China Electron Tube Factory; XSZ-D inverted microscope was the product of Chongqing Optical Instruments Factory; model BB-16 CO2 incubator was the product of HERAEUS; XIDP super clean bench was the product of Suzhou Wuxian Laboratory Animals Equipment Factory; and one set of articles for routine cell culture.
Methods
Preparation of Decoction (containing volatile oil ingredient) of Li's Prescription No.5
Volatile oil was extracted from the medicinal powder of Li's Prescription No.5 by distillation and the dregs of the decoction was decocted to 50 mg/ml as usual. Appropriate amount of Tween 80 was added to the volatile oil to accelerate solution and the mixture was added to the decoction with stirring. The pH value was adjusted to 7.4, and after autoclaving the decoction was stored at 4 ℃ for use.
Preparation of Serum Containing Medicine (3)
The volatile oil extracted from the medicinal powder of Li's Prescription No.5 by routine method was mixed with the decoction with stirring. Mice were divided at random into 5 groups: solvent control group, group of 3g of medicine/kg of body weight, group of 6g of medicine/kg of body weight, group of 12g of medicine/kg of body weight and nimodipine group. The medicine was administered by intubation feeding. Mice in the solvent control group were given equal volume of normal saline and adequate amount of Tween 80, those in the three groups receiving Li's Prescription No.5 were given decoction of different concentrations (containing volatile oil), and those in the nimodipine group were given nimodipine in the dosage of 24mg/kg of body weight. They were given twice a day for 6 successive days. One hour after the last intubation feeding, extraction of eyeballs was made to collect blood, the serum was separated and inactivated at 56℃ for 30 minutes, then the serum was sterilized by suction through 6.22 filter membrane and stored at -20℃ for use.
Revival of PC12 Cells (2,4)
The PC12 clone frozen in liquid nitrogen was removed and rapidly placed into water of 37℃ with continuously shaking to melt it as soon as possible. The frozen tube was removed from water, moved to the super clean benth, and sterilized with 75% alcohol. The cell suspension was removed by aseptic manipulation and poured into sterilized centrifuge tubes, then supplemented with adequate amount of DMEM culture fluid containing 10% calf serum and centrifuged (100 rpm) quickly for 5 minutes. The supernatant fluid was removed and the DMEM culture fluid was again added to the sediment for another centrifugation. The PC12 cells were diluted to a suspension of 105 cells/ml with DMEM culture fluid containing 10% calf serum and inoculated into 4cm×6cm glass culture flasks. The cell suspension was incubated in a CO2 incubator at 37℃ under the condition of 5% CO2. The culture fluid was changed once on the next day and the cell suspension was incubated routinely thereafter.
The Effect of Li's Prescription No.5 on Proliferation of PC12 Cells (3,5)
When PC12 cells formed a single-layer in the culture flasks, the culture fluid was discarded and the single-layer cells were gently washed with D-Hanks solution by blowing for twice, then 1 ml of 0.25% trypsinase solution was added for digestion at 37℃ for 2-3 minutes. As soon as the monolayer cells became loose and floated in ball shape, the DMEM culture fluid containing 10% calf serum was added to terminate the digestion. Cells in the clumps were made to disperse completely with pipette and counted under the inverted microscope. The cells were diluted with culture fluid to 5×105 cells per ml and inoculated in a cell culture plate with 96 wells, 200 μl each well. DMEM complete medium was added to the wells of control group. 50 μl of decoction (containing volatile oil) of different concentrations was added to the wells of the groups of Li's Prescription No.5 to make a final concentration of 0.04 mg/ml, 0.16 mg/ml, 0.63 mg/ml and 2.50 mg/ml, respectively. Mouse serum of normal saline group was added to the medicine-free mouse serum control group, 10 μl of mouse serum per well. To the medicine-containing serum groups of Li's Prescription No.5 were added sera of groups of 3g/kg, 6g/kg and 12g/kg, respectively, 10 μl per well. Eight double wells were set up for each group, the culture plate was incubated at 37℃ under condition of 5% CO2 for 24 hours. Four hours before the end of action, MTT of a final concentration of 0.5 mg/ml was added to each well and the plate was continuously incubated for 4 hours for color reaction, then the original culture fluid was aspirated, 200 μl of 100% dimethyl sulfoxide was added to each well, and when the granules in the wells were completely dissolved optical density (OD) value at 540 nm was determined with Model DG 3022A enzyme linked immunosorbent assay detector. The experiment was repeated twice.
The proliferation rate of PC12 cells for the decoction groups of Li's Prescription No.5 was calculated according to the following formula:

OD of medicine-containing－OD of control
Proliferation rate = ×100%
OD of control

The proliferation rate of PC12 cells for the medicine-containing serum groups of Li's Prescription No.5 was calculated according to the following formula:

OD of medicine-containing serum－OD of medicine-free serum control
Proliferation rate= ×100%
OD of medicine-free serum control


The effect of decoction (containing volatile oil) of Li's Prescription No.5 and of medicine-containing sera of Li's Prescription No.5 on the proliferation of PC12 cells was observed at 48 and 72 hours by using the same method.
The Protection of Li's Prescription No.5 on the Ischemic-like Injury of PC12 Cells
1. Passage of PC12 Cells (2,4)
When PC12 cells formed a single-layer in
the culture flask, the culture fluid was discarded and the single-layer cells were gently washed with D-Hanks solution for twice, then 0.25% trypsinase solution was added for digestion at 37℃ for 2-3 minutes. After digestion, DMEM culture fluid containing 10% calf serum was added to terminate digestion, and the cells were made to disperse completely with pipette and counted under the inverted microscope. The cells were diluted with the complete DMEM culture fluid to 5×106 cells per ml and inoculated into a culture plate with 24 wells. The plate was incubated at 37℃ under the condition of 5% CO2. During a period of 3-4 days, the bottom of the wells was covered with cells, which could be used in the experiment.
2. Effect of Li's Prescription No.5 on
Ischemic-like Injury of PC12 Caused by NaCN and Sugar Deficit (6,7)
The original culture fluid in the 24 wells which were covered with single-layer PC12 cells was discarded, the cells were washed twice with sugat-free Earle's solution and 1 ml of sugar-free Earle's solution was added to each well, the mixture was allowed to stand for 30 minutes. Nimodipine in a final concentration of 5×10－6 mol/l was added to the group of nimodipine. Decoction (containing volatile oil) of Li's Prescription No.5 in the final concentrations of 0.04 mg/ml, 0.16 mg/ml, 0.63 mg/ml and 2.50 mg/ml was added to the decoction groups of Li's Prescription No.5. Serum of the nimodipine group and sera of the Li's Prescription No.5 groups of 3g/kg, 6g/kg and 12g/kg were added to the wells of medicine-containing serum groups, respectively, to make a 5% final concentration of serum. The mixtures were allowed to stand for 20 minutes, then the liquid was aspirated and 1ml of serum-free DMEM was added to each well, the culture plate was allowed to stand for 24 hours. MTT in a final concentration of 0.5mg/ml was added, the plate was incubated for 4 further hours, then the culture fluid was aspirated and 200μl of 100% dimethyl sulfoxide was added to each well. As soon as the blue granules in the wells were completely dissolved, the value of optical density (OD) at 540 nm was determined with enzyme linked immunosorbent assay detector. The experiment was repeated twice.

The inhibition rate of decoction (containing volatile oil) of Li's Prescription No.5 for PC12 cell injury due
to NaCN and sugar deficit was calculated according to the following formula:

OD of medicine-containing group－OD of model group
Inhibition rate = ×100%
OD of control group－OD of model group

The inhibition rate of medicine-containing serum of Li's Prescription No.5 for PC12 cell injury due to NaCN and sugar deficit was calculated according to the following formula:

OD of medicine- OD of medicine- NaCN
－ +
containing serum group free serum group
Inhibition rate = ×100%

OD of medicine- OD of medicine- NaCN
－ +
free serum control group free serum group

Twenty-four hours after development of the injury, 0.5 ml of culture fluid was collected for determination of activity of LDH (8)
The inhibition rate of decoction (containing volatile oil ingredients) and of medicine containing serum of Li's Prescription No.5 for LDH release of PC12 cells due to NaCN and sugar deficit was calculated according to the following formulae:

LDH of model group － LDH of medicine-containing group
Inhibition rate= ×100%
LDH of model group － LDH of control group


LDH of medicine- NaCN LDH of medicine-
+ －
free serum group group containing serum group
Inhibition rate= ×100%
LDH of medicine- NaCN LDH of medicine-
+ －
free serum group group free serum control group

Statistical Treatment
±s was used for experimental data, and t test for comparison among groups.

Results
The Effect of Li's Prescription No.5 on the Proliferation of PC12 Cells
Morphological Observations
It was observed under the inverted microscope that PC12 cells in the control group grew rapidly along the wall of the wells 24 hours after inoculation, the majority of them being shuttle-shaped and the minority being triangular form, that at 48 hours the wells were covered with single-layer cells and that 72 hours the cell growth stagnated, with cells suspending in some wells.
PC12 cells in the decoction groups of Li's Prescription No.5 had neither change in cell morphology nor manifestations of injury and grew well after different concentrations of the decoction were added; at 48 hours the cells grew luxuriantly and the wells were covered with monolayer cells; and at 72 hours the cell growth gradually slowed down.
PC12 cells in the medicine-containing serum groups of Li's Prescription No.5 were still in round form and grew poorly 24 after medicine-containing serum was added, at 48 hours the cells grew luxuriantly in shuttle-shaped and triangular forms and the wells were covered with monolayer cells, and at 72 hours the cell growth gradually slowed down.

Results of MTT Automatic Microdetermination

The effects of decoction (containing volatile oil) of Li's Prescription No.5 on the proliferation of PC12 cells for 24,48 and 72 hours are shown in Table 1.

Table 1 The effect of decoction of Li's Prescription No.5 on the proliferation of PC12 cells
for 24,48 and 72 hours ( ±s) (n=8)
24 hours 48 hours 72 hours
Group Concentration OD540 Proliferation OD540 Proliferation OD540 Proliferation
(mg/ml) rate rate rate
Control 0.75±0.09 1.09±0.07 0.79±0.08
Li's 0.04 0.72±0.12 2.7 1.10±0.05 0.9 0.76±0.07 -3.8
Prescription 0.16 0.85±0.06** 13.3 1.17±0.04* 7.3 0.88±0.05* 11.8
No.5 0.63 0.89±0.09** 18.7 1.25±0.12** 14.7 0.89±0.05** 12.7
2.50 0.97±0.09** 29.3 1.44±0.07*** 32.1 0.93±0.09** 17.71
Note: * p<0.05, ** p<0.01, *** p<0.001, (compared with the control group)

The results of MTT automatic microdetermination showed that decoction of Li's Prescription No.5 promoted the proliferation of PC12 cells in varying degrees after 24, 48 and 72 hours and that the promotion of proliferation had a certain dose-

dependence.
The effects of medicine-containing serum of Li's Prescription No.5 on the proliferation of PC12 cells for 24,48 and 72 hours are presented in Table 2.

Table 2 The effect of medicine-containing serum of Li's Prescription No.5 on the proliferation
of PC12 cells for 24, 48 and 72 hours ( ±s) (n=8)
24 hours 48 hours 72 hours
Group Dosage OD540 Proliferation OD540 Proliferation OD540 Proliferation
(g/kg) rate rate rate
Control 0.75±0.09 1.09±0.07 0.79±0.08
Medicine-free
serum control 0.52±0.16 1.47±0.10 0.87±0.05
Li's Prescription 3 0.70±0.12* 34.6 1.67±0.04*** 13.6 0.93±0.11 7.6
No.5 serum 6 0.73±0.07** 40.4 2.28±0.35*** 55.1 0.99±0.10** 13.8
12 0.81±0.12** 55.8 2.41±0.51*** 63.9 1.05±0.13** 20.7
Note: * p<0.05, ** p<0.01, *** p<0.001, (compared with the medicine-free serum control group)

The results of MTT automatic microdetermination showed that medicine-free serum had a slight inhibition on the growth of PC12 cells at 24 hours but showed a promotion afterwards at 48 and 72 hours and that medicine-containing serum of Li's Prescription No.5 showed a promotion on the proliferation of PC12 cells in all cases.

The Protection of Li's Prescription No.5 on the Ischemic-like Injury of PC12 Cells Caused by NaCN and Sugar Deficit
Morphological Observations
Twenty-four hours after PC12 cells had been incubated in sugar-free Earle's solution containing 20 m mol/l NaCN for 30 minutes, the process structure of the cells was observed to have disappeared under the inverted microscope, with round cell swelling, reduced diopter and cell debris in part of the cells. Cells pretreated with the decoction (containing volatile oil) and medicine-containing serum of Li's Prescription No.5 obviously resisted the morphological change caused by NaCN and sugar deficit, showing decreased disappearance of cell process structure and decreased cell debris.

Results of MTT Automatic Microdetermination and LDH Determination
The results of MTT automatic microdetermination and those of determination of LDH activity are shown in Tables 3 and 4.
Table 3 The effect of Li's Prescription No.5 on the ischemic-like injury of PC12 cells caused by
20 m mol/l NaCN and sugar deficit and on release of LDH by PC12 cells due to NaCN
plus sugar deficit
MTT LDH
Group Concentration OD540 Inhibition rate LDH (u/ml) Inhibition rate
Control 1.99±0.04 272.7±60.7
Model 1.56±0.20*** 694.6±174.2***
Nimodipine 5×10－6 mol/l 1.87±0.05△△ 72.1 395.1±78.8△△ 71.0
Li's Prescription 0.04mg/ml 1.58±0.11 4.7 661.9±75.8 7.7
No.5 0.16mg/ml 1.75 ±0.04 44.2 515.7±114.5 42.4
0.63mg/ml 1.83±0.07△ 62.8 447.0±139.4△ 58.7
2.50 mg/ml 1.96±0.07△△ 93.0 323.6±48.8△△ 87.9
Note: n=6 ***p<0.001 (compared with the control group); △ p<0.05, △△ p<0.01 (campared
with the model group)
Table 4 The effect of medicine-containing serum of Li's Prescription No.5 on the ischemic-like
injury of PC12 cells caused by NaCN and sugar deficit and on release of LDH by PC12
cells due to NaCN plus sugar deficit
MTT LDH
Group Dosage OD540 Inhibition rate LDH (u/ml) Inhibition rate
Control 1.99±0.04 272.7±60.7
Model 1.56±0.20 694.6±174.2
Mouse medicine-
free serum control 1.76±0.11 329.4±80.7
NaCN +mouse
Medicine-free serum 1.01±0.18*** 904.1±143.1***
NaCN+nimodipine
serum 24mg/kg 1.38±0.06△△△ 49.3 598.6±505△△△ 53.2
NaCN+serum of 3g/kg 1.27±0.13△ 34.7 725.3±48.3△ 31.1
Li's Prescription 6g/kg 1.35±0.16△△ 45.3 615.8±32.8△△ 50.2
No.5 12g/kg 1.44±0.08△△△ 57.3 529.9±81.6△△△ 65.1
Note: n=6, ***p<0.001 (compared with mouse medicine-free serum control group), △ p<0.05,
△△ p<0.01, △△△ p<0.001 (compared with NaCN+mouse medicine-free serum group)


The results showed that the damaged cells had a reduced ability to uptake MTT and released LDH in large quantities, making the activity of LDH in culture fluid significantly increased and that cells pretreated with decoction of Li's Prescription No.5 and its

medicine-containing serum had an increased ability to absorb MTT and to inhibit the release of LDH in varying degrees and had concentration-and dose-dependence, as shown in Figs 1 and 2.


100

80

60

40
value of LDH
20 value of LDH

0 0.04 0.16 0.63 2.50

Concentration of decoction of Li's Prescription No.5 (mg/ml)

Fig.1 Inhibition of decoction (containing volatile oil) of Li's Prescription No.5 on ischemic-
like injury of PC12 cells (n=6)
100

80

60

40
value of MTT
20 value of LDH


0 3g/kg 6g/kg 12g/kg

Dosage of Li's Prescription No.5

Fig.2 Inhibition of medicine-containing serum of Li's Prescription No.5 on ischemic-like
injury of PC 12 cells (n=6)

Discussion
Cell culture is characterized by short period, easy control of experimental conditions and convenient observation of experimental results and is, therefore, the relatively ideal method for studying pharmacology of Chinese drugs. However, the direct addition of crude preparation of Chinese drugs to the cell culture system for experimental research has many problems in method, such as impurities of Chinese drugs, electrolytes or tannins and pH value, all of which will affect cell growth, thus interfering with experimental results and giving unreliable conclusions. By using sero-pharmacological method, the medicine-containing serum obtained after oral administration and absorption of crude preparation of Chinese drugs is used to make in vitro experiments, this eliminates the interference of various affecting factors, is more close to the real course of pharmacological action produced by the drugs in in vivo environment and improves the confidence of experimental results.
In addition, the failure to absorb from
gastro-intestinal tract the available composition contained in a complex prescription of Chinese drugs (such as some high-molecular compound), or its inactivation following absorption and metabolism in the body, and the production of action of the available composition following its metabolism in the body in the absence of its direct action, or the production of its indirect action by the stimulation of the second messenger can be well reflected by the use of this method. (9)
The results of the present study showed that the PC12 cells grew and reproduced rapidly, thus grew luxuriantly at 48 hours, and grew slowly and stagnantly at 72 hours because of nutritional deficiency. Both the decoction and medicine-containing serum of Li's Prescription No.5 promoted the proliferation of PC12 cells concentration-or dose-dependently after 24,48 and 72 hours, indicating that Li's Prescription No.5 may have the effect of promoting the growth of neurons in the body.
The study showed that the neuroma cells can make full use of anaerobic glycolysis to provide energy, they have a certain tolerance to anoxia, but in the presence of sugar deficit, marked cell injury will develop shortly. Therefore, the injury model of NaCN plus sugar deficit finely reflects the pathological changes of ischemic/anoxic injury. (7)
The results of the present study demonstrated that both the decoction and medicine-containing serum of Li's Prescription No.5 led to vigorous proliferation of nerve cells under normal growth and inhibition and protection of anoxic/ischemic injury of neurons and that this proliferation of nerve cells and the inhibition and protection of anoxic/ischemic injury of neurons had a certain dose-effect relationship, especially when 5% medicine-containing serum was added to the culture fluid of PC12 clone. This demonstrates the clinical therapeutic effectiveness of Li's Prescription No.5 and lays a solid foundation for further study of its mechanism of action.

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