The influence of Anbol to immunological function

of S180 tumor-bearing mouse and it¡¯s tumor-inhibited action

Drug Research Center Of Lonjer

Abstract   This article studied the dynamic change procedure of cellular immunological function of S180 tumor-bearing mouse. The RPI of the mouse on d 2 and d 4 are respective 166% and 390% of their normal level after the mice were inoculated tumor cell; the immunoreactions of cell on d6 decline, RPI is only 21% of normal level, d14 is 4%, Anbol 10mg/ (kg•d ) (total 7d, the following is same ) can make the incorporation value (cpm) of splenic lymphocyte[⁸H ] of the tumor-bearing mouse rise from 139¡À62 to 5581¡À788, RPI from 0.3% of nearly normal level to 24.6%; the tumor-inhibited rate with 10~20md/ (kg•d ) is 31~39%. If Cy: 12.5mg/kg, sc, single dose, the rate is 14%, and the rate is 54% if Anbol 10mg/(kg. d ) is used together, there is evident synergistic action.

Keywords  Anbol; Cyclophosphmide (Cy); S180 tumor; immunocompetence; transform of T lymphocyte

The remedial function of medicine to tumor is other than directly acting on tumor cell, the immunological surveillance of the host plays a very important role too^[1]. Thus, the pharmacological research of anti-tumor medicine have developed from chemotherapy to immunological chemotherapy. It has been know that the effective competent of many beneficial medicine, for example, spicy mushroom polysaccharides, tremella polysaccharides, agaric polysaccharides, have preferable immune reinforcement and inhibition action to tumor^[2-4].

Ai Cao and his colleagues have studied that Anbol extracted from the fruit of Lycium barbarum Linn have the action of reinforcing immunological function of normal mouse cell^[5]. But it has never been reported that the remedial effect of Lycium barbarum polysaccharides (LBP) to tumor and whether LBP can improve the low cellular immunological function of tumor-bearing mouse. This article studied the reinforcement action of Anbol to cellular immunological function of S180 tumor-bearing mouse and its respective tumor-inhibited effect, at the same time, observed the synergistic action with Cy used together.

Materials and methods

Drug  Anbol, provided by plant and chemist chamber of our own graduate school, and its main components are arabinose, glucose, galactose, mannose, xylopyranose, rhamnose; Cyclophosphamide, the product of the 12th pharmaceutical factory in shanghai.

Animal  Swiss little white mouse, female, 30~40 days age, weight of 20¡ÀSD 2g, provided by animal yard of our academy.

Inoculation of S180 tumor cell^[6]   subcultured S180 tumor cell strain is extracted from mouse and diluted to certain concentration ( trypan blue staining£¬live cell counting), 2 10⁵ tumor cell was inoculated into infra-skin of right side axilla, and tumor mass extracted at different time were weighed.

T lymphocyte proliferation response experiment^[7]  TdR incorporated-method was adopted to detect the proliferation ability of splenic T lymphocyte of the tumor-bearing mouse. The mail steps are as follows: in different time after inoculating S180 cell, kill the mouse, and draw spleen to make splenic lymphocyte suspension, use 1640 culture medium(including 15% calf blood serum) to adapt cell concentration to 1 10⁷/ml. Draw 0.1 ml cell suspension into 96 hole cultivation board, at the same time add ConA 0.1ml/hole (10¦Ìg/ml) into the culture medium, foster in 5%CO₂ greenbox for 54 hours in 37⁰C, add [³H]TdR 1.85 kBq/hole (radioactive concentration is 99.9 kBq/ml) again, keep on culturing for 16 Hours, collect cell to fiberglass membrane by multi-head cell collection meter, liquid scintillation counting.

Results

The influence of inoculation amount of S180 tumor cell to the weight of tumor and incidence

Inoculate different amount S180 tumor cell into infra-skin of the mouse, kill the mouse after 9 days and weigh the tumor. It can be seen from table 1 that when the inoculated tumor cell amount of each mouse exceed 1 10⁵, the tumor occurrence rate is 100%, when the amount is less than the number, there are a few mice that have no tumor, and when the amount of tumor cell is 2 10⁵, the average weight of tumor exceed 1g and this accords with the standard as a control group of tumor. Therefore, every inoculation bar of tumor cell in this article is all 2 10⁵.

Tab 1. Effect of inoculated S180 tumor cell number on tumor

weights and tumor occurrence rate in mice

Tumor weights of S180-bearing mice were examined 9 days after S180 cells were implanted.

The mutual relationship between tumor weight and T lymphocyte immunological function after mice were inoculated S180 tumor cells.    Tumor can be touched on d 6 after mouse was inoculated S180 tumor cells, and the weight is 0.24 SD0.11g, tumor weights on d9 and d14 are respective 1.2 0.4, 4.0 2.1g. Splenic T lymphocyte cell proliferation reactions of tumor-bearing mouse enhance obviously, among which the relative proliferation index(RPI) of splenic T Ly of d2 and d4 are respective 166%, 390%, and RPI of d6 drop to 21%, and almost any T Ly proliferation reaction can be detected after d9. The cellular immunological function of mouse is ascending in the early days after tumor cell inoculated, but cellular immunological reaction quickly descend after the tumor mass can be touched(Table 2).

Tab 2. Relationship between the tumor weights (8 mice/group) and the

proliferation of splenic lymphocyte (4 mice/ group) in S180 bearing mice

All mice were inoculated 2 10⁵ S180 tumor cells on d 0, RPI(Relative proliferation index)= .

The influence of Anbol to cellular immunological function of normal mouse   Anbol: 5mg/(kg•d) for sever days (d0~6), ip, draw spleen to detect T lymphocyte proliferation reaction on d7. The results show that Anbol can obviously enhance splenic T lymphocyte proliferation reaction of normal mouse induced by ConA, the cpm value rise from 28410 3110 in control group to 64870 2571 in administration group.

The influence of Anbol to the immunological function of tumor-bearing mouse and the tumor-inhibited action   After mouse was inoculated S180 tumor cell for 2 days, Anbol 10mg or 20mg/(kg•d) 7, ip, kill the mouse in the 9^th day, weigh the tumor and measure  splenic T lymphocyte proliferation reaction. The results show that the tumor-inhibited rates of Anbol 10mg and 20mg/kg are respective 31%, 39%. At the same time it can be seen that the cpm value of T lymphocyte proliferation reaction of mice in Anbol 10mg group rose from 139 SD of control group to 5581 783 of administration group, and RPI from 0.3% of normal level to 24.6%. This experiment use Cy as positive control, single sc dose is 25mg/kg, the tumor-inhibited rate is 47%, and the cpm value of T lymphocyte proliferation reaction is 165 31. (Tab 3, 4)

Anbol¡¯s anti-inhibition action of Cy to cellular immunological function of normal mouse    Cy: 25mg/kg, sc, begin on d 0 of experiment, Anbol: 5mg/(kg•d) 7, ip, on the same day, draw spleen to examine T lymphocyte proliferation reaction. It can be seen from Tab 5 that RPI of the group of mice treated only by Cy is 33% of normal level, it is 105% after Anbol was added, this show that Anbol completely antagonized the action of low cellular immunological function caused by Cy.

Tab 3. Effect of Anbol on the tumor weights of S180-bearing mice

All mice received 2 10⁵ S180 tumor cells on d 0, and tumor weights were examined on d 10. Cy: 25mg/kg, single dose in d 2, Anbol: 10~20mg/(kg•d) 7, ip, beginning on d 2. ^¡ñP< 0.05, compared with control. , n=23

Tab 4. Effect of Anbol on splenic lymphocyte proliferation in mice bearing S180

The mice were those of table 3, Cy: 25mg/kg, single dose on d 2, Anbol: 10mg/(kg•d) 7, ip, beginning on d 2. ^¡ñP<0.01, compared with control tumor-bearing mice and that of Cy treated mice. , n=8.

Tab 5. Effect of Anbol on splenic lymphocyte

proliferation in cyclophosphamide treated mice

Cy: 25mg/kg, sc, single dose on d 0, Anbol: 5 mg/(kg•d), ip, beginning on d 0, and splenic lymphocyte proliferation was assayed on d 7. ^¡ñP<0.01, compared with that of Cy alone. , n=8.

The inhibition action of Anbol and Cy used together to tumor    Cy: 25mg/kg, sc, after mouse was inoculate S180 cell into infra-skin 2 days, the tumor-inhibited rate is 31%, Anbol: 10mg/(kg•d) 7, ip at the same time, then the rate rise to 47%, but there is no significant difference between the two rates(P>0.05). when Cy: 12.5mh/kg, sc, the inhibition rate is 14%, and tumor weights have no significant difference compared with that of the control group, if Anbol 10mg/(kg•d) 7 used together, ip, then the rate rise to 54%, (P<0.01). This shows that Anbol can enhance the inhibition action of Cy to tumor (Tab 6). The cpm of the group treated by Cy 12.5mg/kg alone is 245 146, and it is 222 155 in the group treated by Cy and Anbol together. This suggests that there is no significant difference for T lymphocyte proliferation reaction between Cy alone and Anbol added.

Tab 6. Effect of Anbol combined with Cy on tumor weights in S180-bearing mice

 Anbol: 10mg/(kg•d) 7, ip, beginning on d 2; Cy: 25 or 12.5 mg/kg, sc, single dose on d2. ^¡ñP<0.01, compared with control tumor-bearing mice and that of Cy alone. .

Discussion

It has been reported by documents ^{[1, 8]}, the reason that the enhancement of body immunologic reaction in the early growth stage of tumor is caused by the stimulation of many antigens transplanted by tumor. This kind of immunologic reaction is similar to reject reaction of body to allotransplant, and they are both guided by specific T lymphocyte ^[9]. This experiment has also observed that immunological function of T lymphocyte in the early stage rise gradually, and RPI from 100% of normal level, rise gradually to 166~390%.

Immunologic reaction caused by tumor has its ascending and extinct change procedure ^[1.10], immunologic reaction of host begins to decline with the gradual growth of cell in the body of host. This experiment has also observed that cellular immunologic reaction of tumor-bearing declined quickly when the tumor has grown big enough to be touched, and RPI is 20% of normal level. The immunologic reaction of body almost vanished with the gradual growth of the tumor. The reason that the descent of body¡¯s immunologic reaction in terminal growth period of the tumor is mainly because that the body fluid suppressive-factor produced by tumor inhibited the receptor in the membrane of lymphocyte^[11], and the tumor antigen inhibited body¡¯s immunologic reaction through activating T-inhibited cell^[12].

Cy is cytotoxicity drug for chemotherapy, at the time of kill tumor cell, it kills body immunologic cell, which restricts its application. Therefore, scholars domestic and overseas begin to apply immunologic chemotherapy complex treatment to combat with tumor. It is well known that anti-tumor immunopotentiating drug can not only assist medicine for chemotherapy by killing tumor cells, but also reduce its immunosuppressive action. Medlar is the traditional herb of our country, this experiment had ever proved that operative component--- Anbol has the action of enhancing cellular immunological function of normal mouse, Anbol 10mg/kg can increase cellular immunological function of S180 tumor-bearing mouse, has at the same time the action of inhibiting tumor, and can assist medicine for chemotherapy---Cy by killing tumor cell, especially when Cy doesn¡¯t reach the amount of inhibiting tumor obviously, this type of synergistic action is more obviously. But, Anbol and Cy used together can¡¯t improve cellular immunological function of tumor-bearing mouse obviously. It is guessed that maybe when Cy is applied to tumor-bearing mouse, the cellular immunological function was more inhibited, and it¡¯s difficult for Anbol to recovery the cellular immunological function of tumor-bearing mouse in this condition.