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The Experimental Study on the Effect of FRC001 (China No. 1 Tian Xian Liquid)
on Nonspecific Immunity of Mice
Robert W. Bradford D. Sc.1
Kexiang Ding 2
1. Bradford Research Institute, CA, USA
Professor of Capital University, Washington DC
2. FRC Free Radical Biology & Medical Research
Center
ABSTRACT
This paper investigated the effect of FRC001 on
nonspecific immunity of mice and graft versus host
reaction (GVHR). The results shows that FRC001 had
some effect on phagocytic function of mononuclear
macrophage, spleen index and thymus index, splenic
lymphocyte transformation, serum hemolysin formation,
host spleen reaction coefficient and stimulate coefficient
and stimulus index i.e. FRC001 can increase and
enhance humoral immunity and cellular immunity of
experimental basis for the use of FRC001 in regulating
immunity and preventing and curing tumor.
Key Word : FRC001 Nonspecific immunity graft versus
host reaction (GVHR)
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BACKGROUND
Tumor formation is related to immunologic inadequacy
in recent year, there are many report on immunologic
inadequacy index which was regarded as an important
index in the screening of antineoplastic. The study
investigated the effect of FRC001 on nonspecific
immunity of mice and GVHR.
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MATERIALS AND METHODS
Materials
1.1 Subject : FRC001.
1.2 Experimental Animal : Mouse, Kunming species,
18~22g, half male and half female, provided by Experimental
Animal Center of Jiangsu Tumor Prophtlactico-therapeutic
Research Institute.
Method
2.1 Phagocytic function of mononuclear macrophage
(mouse carbon clearance test)
2.1.1 Experimental principle :
Experimental mouse was injected with Indian ink
which was used as granular foreign body. The Indian
ink was phagocytized and cleared by mononuclear
macrophage after it went into circulation. Ninety
percent of it was phagocytized by liver Kupffer
cell while the rest was phagocytized by spleen macrophage.
The clearance rate of granular foreign body in circulation
reflected the phagocytic function of mononuclear
macrophage. Within given scope, the clearance rate
of granular foreign body had exponential function
relation with granular dose, i.e. phagocytosis rate
had direct proportion relation with granular concentration
in circulation. They have linear relation in system
of coordinates with the time as the abscissa and
granular concentration as the vertical ordinate.
The slope K of the line is the phagocytosis rate
(or clearance rate).
2.1.2 Manipulation.
Mice were randomly divided into negative control
group, positive control group and different dose
of FRC001 groups, They were fed with distilled water,
elemene milk and different dose of FRC001 for successive
10 days. Each mouse was injected with Indian ink(diluted
to 1~5 times) 0.05ml/1/0g.bw from mouse tail vein
in 30 minutes after the last feeding. In the first
minute (t1) and 5th minute (t5) after injection
20 m 1 blood was drawn from postobital vein with
suction tube in which was moistened with heparin
solution and diluted in 2ml 0.1% sodium carbonate
solution. Then absorbance (A) of blood solution
was assayed at 680nm. To calculate the value of
K by following formula. (lgAl-lgA5)/(t5-t1)=(lgA1/A5)/4
2.2 GVHR test. Two pure line mice C57BL JCR hybridized
to give birth to newborn mouse. Beginning at the
first day of the birth of the newborn mouse, experimental
male parent were fed with FRC001 for 10 days while
control male parent were not given any treatment.
On the 11th day after the birth of newborn mouse,
male parent mice were killed by dislocation to take
out spleens. Spleen cell suspension (1X10 8/ml)
was prepared by aseptic technique. The newborn mice
were divided into experimental group, control group
and normal group. The experimental newborn mice
were inoculated 0.1 ml spleen cell suspension of
experimental male parent, the control newborn mice
were inoculated 0.1 ml spleen cell suspension of
control male parent, and the normal group were not
given any treatment. All these newborn mice were
killed at seventh day after inoculation, and their
spleens were weighed to calculate spleen coefficient
(mg spleen/10g.bw). Then calculation spleen stimulus
index (SI) by following formula : SI = (Average
spleen coefficient of experimental or control) /average
spleen coefficient of normal group.
2.3 Spleen index and thymus index test
Kunming species mice (18~22g) were randomly divided
into negative control group (fed with N>S) positive
control group and FRC001 group. They were fed with
respective subject for successive 10 days, and killed
by orbital bleeding on the second day after the
last feeding. Their spleen and thymuses were stripped
out and weighed accurately with torsion balance.
The results showed with organ index-weight of spleen
or thymus (mg)/10g.bw
2.4 Mouse lymphocyte transformation test-co;orimetry
of mouse splenic MTT
Pure species Kunming mouse was killed by orbital
bleeding and its spleen was stripped out to make
5 X 10 (8) /ml cell suspension with Hank’s
solution and RPM 1640 medium cell suspension 100
m 1 was added to pore of culture dish, and every
pore was added PHA 100 m 1 and experimental drugs,
in the meantime, control pore was set up. They were
cultured in 37~38 °C.5% CO2 incubator for 72
hrs. Every pore was added 50 m 1 MTT 4~5 hours before
the ending of culture, then continue to culture
24h, 36h, 48h, respectively. They were put into
4 C refrigerator for one night. Supernate 150 m
1 was sucked, added 150 m 1 acid isopropanol and
blown to homogeneous solution. Absorbance (A) of
every tube was assayed at 630nm of ELISA photometer.
To see detail in reference[10].
2.5 Serum hemolysin test
1.Mice (Kunming species, 18~22g) were randomly
divided into nonimmunologic group, immunologic group
immunology and drug (low, middle and high dose)
group. Every mouse of the last two groups was injected
introper itoneally 0.2 ml sheep red blood cell (SRBC)
which diluted to 3:5(V/V) with N.S Mice of drug
group were given drug before or after immunization.
Four days after immunization. 1 ml blood was taken
to get serum. To see the detail in reference [8].
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RESULTS AND DISCUSSION
1. The effect of FRC001 on phagocytic function
of mononuclearmacrophage in mice (Figure 1)
Figure 1. The effect curve of FRC001 on Phagocytic
Function of Mononuclearmacrophage in mice.
The increase of carbon clearance index (K) reflects
the enhancement of phagocytic function of mononuclearmacrophage
and nonspecific immunity. According to Figure 1,
low, middle and high dose of FRC001 increased K
value significantly (p<0.01). The result showed
FRC001 can enhance the phagocytic function of mononuclearmacrophage
and nonspecific immunity.
2. The effect of FRC001 on spleen index and thymus
index. (Table 1)
|
| (n)
|
| ±SD(mg/10g.bw)
|
|
|
|
|
| |
|
|
|
| ±25.59
| ±9.01 |
|
|
| X 10(mg/kg X d)
| ±35.91*
| ±13.03* |
| Milk
|
| X 10(mg/kg Xd)
| ±22.82*
| ±2.60 ***
| |
Compared with control. * P>0.05; *** P<0.01
Table 1. The effect of FRC001 on spleen index (SI)
and thymus index (T1)
Spleen and thymus are important immunologic organs.
The degeneration and atrophy of them will influence
their normal function. According to Table 1, FRC001
had nonsignificant effect on SI and TI (P>0.05).
The result showed Edfrann had no evident influence
on immunologic organs. The authors thought it may
be related to the shot time.
3. The effect of FRC001 on splenic lyphocyte transformation
in mouse. (Figure 2)
Figure 2. The effect of FRC001 on splenic lyphocyte
transformation in mouse.
Splenic lymphocyte transformation reflects cellular
immunologic function. From Figure 2, FRC001 had
evident influence on splenic lymphocyte transformation.
Splenic lymphocyte transformation went up as the
increasing of time. The increasing amplitude between
26h and 48h was the biggest. Compared with control,
splenic lymphocyte transformation at 24h, 36h, and
48h increased significantly (P<0.01). The result
showed FRC001 can enhance cellular immunologic function.
4. The effect of FRC001 on serum hemolysin (Figure
3)
Figure 3. The effect curve of FRC001 on serum hemolysin
Formation.
Hemolysin (IgM) reflects humoral immunity. Increasing
of absorbance at the time half hemolysin takes place
(HC50) shows the increasing of hemolysin and enhancement
of humoral immunity. According to Figure 3, HC50
went up gradually as the increasing of FRC001. The
difference between experiment group and control
group was significant (P<0.01). The result showed
Edfrann had enhance humoral immunity.
5. The effect of FRC001 on GVHR in mice (Table
2).
|
|
| way
| (mg/kg Xd)
| Reaction Index (mg spleen/g.w)
| Index (SI) |
|
|
|
|
| ±1.14 ***
| |
|
|
|
| X 10
| ±1.36 **
| |
|
|
|
| X 10
| ±1.88
| | |
Compared with FRC001 group, *P>0.05; **P<0.05;
***P<0.01
Table 2. The effect of FRC001 on GVHR in mice.
GVHR reflects cellular immunologic function by
host reaction index and stimulus index. According
to Table e, host reaction index and stimulus index
of FRC001 group was obviously higher than normal
group (without any treatment) and control group
(fed with distilled water only) (P<0.05 or P<0.01).
The result shows Edfrann can increase and enhance
cellular immunologic function.
All of these results shows FRC001 had some influence
on nonspecific immunity of experimental mice. The
regulation function of FRC001 to immunity may be
related to its components. It is reported that Chinese
drugs with the function of invigorating Qi, blood
and warming Yang, etc. contain many bioactive components
which can enhance the function of reticuloendothelial
system, antibody formation, specific and nonspecific
immunity. In addition, free radical scavenges in
Chinese drugs have cooperative function in protecting
immulogic organs, regulating immunity, enhancing
immunologic factors, etc. FRC001 contains components
which can invigorate Qi and blood, warm blood and
scavenge free radicals then the components which
play roles in regulating immunity and their action
intensity, best bioeffect are waiting for future
study.
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