Study Of The Effect Of Zuogui Pill Plus HuangQi、S1P On Chemotherapy-induce Ovarian Function Damage And On The Antitumor Effect In S180Tumor Bearing Mice

Part One:Study of the effect and its mechanism of SIP on chemotherapy-induce ovarian function damage and on the antitumor effect in S180bearing tumor miceObjectiveK To investigate the effects and mechanism of inhibitor of apoptosis Sphingosine-1-Phosphate (SIP) on cyclophosphamid (CTX) combined with cisplatin (DDP) induce ovarian function damage in mice inoculate with S180murine sarcoma cells and on the anticancer efficacy of the treatment of cyclophosphamid and cisplatin in combination.2^To investigate the effects mechanism of SIP on ovarian function damage induced by chemotherapy and antitumor effectMethods1.Eighty C57BL/6female mice were divided randomly into four groups:normal group(fourteen mice), model group(twenty-two mice), CTX+DDP group(twenty-two mice), SlP+CTX+DDP group(twenty-two mice). The mice of groups were killed before medication, in the eleventh day, in the twenty-sixth day of medication of medication during diestrus stage, respectively to compare the weight of the ovaries and the number of the primordial follicles and the growth fol1icles, weight of the tumor and efficacy of antitumor, levels of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH)and estradiol (E2) which were measured successively by the enzyme-linked immunosorbent assay (ELlSA) method.Todiscuss the possible protect mechanism of inhibitor of apoptosis-Shingosine-1-Phosphatc (SIP) on cyclophosphamid (CTX) combined with cisplatin (DDP) induce ovarian function damage in mice inoculate with S180murine sarcoma cells and on the anticancer efficacy by the apoptosis of follicles by transmission electron microscope and the express level of Bcl-2and Bax in the follicles tested by immunohistochemistry. The apoptosis rate of follicles was observed by transmission electron microscope. Two mice were selected from every group respectively and lived with one male mouse in one cage at the30th day after injection. The reproduction was studied.2, Data are presented as mean±S. E. M, and was analyzed by SPSS10.0. Experiments among multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test, or test by Dunnett T3if variance is not equal. a=0.05.Results1,hrough the experiment, in the eleventh and twenty-sixth day after injection the levels of serum FSH-. E2, LH in the CTX+DDPgroup were different significantly from the others (P<0.01), except the levels of serum LH in the eleventh day after medication treatment (P>0.05). Levels of serum FSH, E2, LH in the S1P+CTX+DDP group were not different significantly from the control groups in the eleventh and twenty-sixth day after injection (P>0.05).2> The number of different classified follicles in the CTX+DDP group was significantly different among the other groups in the eleventh and twenty-sixth day after injection (P<0.01), expect the number of growth follicles in the eleventh day after injection (P<0.01). The number of primodial follicles, and growth follicles in the S1P+CTX+DDP group was close to that in the control groups in the twenty-sixth day after injection (P>0.05).3> The expression of Bax> Bcl-2in the primodial-primary follicles was no significant difference among all groups (P>0.05). The expression of Bax in the growth follicles was significantly high comparing with other groups in the groups of CTX+DDP (p<0.01). The expression of Bcl-2in the growth follicles was significantly high comparing with the CTX+DDP groups in the groups of S1P+CTX+DDP (P<0.01), no significant difference between the group of CTX+DDP and two control groups (p>0.05).4n The weight of tumors in the groups of CTX+DDP and S1P+CTX+DDP were lower than that of the control groups (P<0.01).The inhibition rate of tumors in the groups of CTX+DDP and SIP+CTX+DDP was higher than60%.5v Ultrastructural organizationof granulocyte cellsand oocyte colls:The apoptosis of oocyte of early follicle and granulocyte cell of growing follicle was observed by electron microscope. The apoptosis of granulocyte cell of growing follicle was typical particularly.ConclusionSIP could reduce the chemotherapy-induce ovarian function damage and be ensure in antitumor effect, and its mechanism mybe is relevant with apoptosis related protein Bcl-2/Bax. Part Two:Study of the effect and its mechanism of Zuogui pill plus HuangQi on chemotherapy-induce ovarian function damage and on the antitumor effect in S180tumor bearing miceObjectiveI To investigate the effects and mechanism of Zuogui pill plus HuangQi on cyclophosphamid(CTX) combined with cisplatin(DDP)-induce ovarian reproductive endocrinology function damage in mice inoculate with S180murine sarcoma cells and on the anticancer efficacity of the treatment of cyclophosphamid and cisplatin in combination, based on the theory of traditional Chinese medicine syndrome differentiation.2> To investigate the effects mechanism of Zuogui pill plus HuangQi on ovarian function damage induced by chemotherapy and antitumor effectMethodsU Eighty C57BL/6female mice were divided randomly into four groups.-normal control group(fourteenmice), model control group(twenty-twomice), CTX+DDP group(twenty-two mice), herbs+CTX+DDP group(twenty-two mice). Levels of serum follicle-stimulating hormone(FSH), luteinizing hormone(LH)and estradiol(E2) were measured successively by the enzyme-linked immunosorbent assay (ELISA) method, and mice of groups were killed before medication, in the eleventh day, in the twenty-sixth day of medication, respectively to compare weight of the tumor and efficacity Of antitumor> the weight of the ovaries and mice. To discuss the possible protect mechanism of Zuogui pill plus HuangQi on cyclophosphamid (CTX) combined with cisplatin (DDP) induce ovarian function damage in mice inoculate with S180murine sarcoma cells and on the anticancer efficacy by the apoptosis of follicles by transmission electron microscope and the express level of Bcl-2and Bax in the follicles tested by immunohistochemistry. The apoptosis rate of follicles was observed by transmission electron microscope. Two mice were selected from every group respectively and lived with one male adultmouse in one cage at the30th day after injection. The reproduction was studied.2^Data are presented as mean+S. E. M, and was analyzed by SPSS10.0. Experiments among multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test, or test by Dunnett T3if variance is not equal. a=0.05.Results1-. Through the experiment, levels of serum FSHE2, LH in the four groups were not different significantly from the others before medication (P>0.05).In the eleventh day the levels of serum FSH, E2in the CTX+DDP group was higher significantly from the others (p<.01);but the herbs+CTX+DDP group was not between the two control groups (P>0.05). The result of LH was the similar with that of the FSH;but the herbs+CTX+DDP group was not different significantly from the CTX+DDP group (P>0.05). In the twenty-sixth day the levels of serum FSH^LH in the CTX+DDP group was higher significantly from the others (P<0.01);but the herbs+CTX+DDP group was not between the two control groups (P>0.05). The result of E2was the similar with that of the FSPL H(p<0.1);but the herbs+CTX+DDP group was not not different significantly from the CTX+DDP group (P>0.05)2> The number of different classified follicles in the CTX+DDP group was significantly different among the other groups in the eleventh and twenty-sixth day after injection (P<0.01). The number of primodial foilicles, and growth follicles in the S1P+CTX+DDP group was close to that in the control groups in the twenty-sixth day after injection (P>0.05), expect the number of primodial follicles in the eleventh day after injection (P<0.01)3^The expression of Bax> Bcl-2in the primodial-primary follicles was no significant difference among all groups (P>0.05). The expression of Bax in the growth follicles was significantly high comparing with other groups in the groups of CTX+DDP(P<0.01). The expression of Bcl-2in the growth follicles was significantly high comparing with the CTX+DDP groups in the groups of S1P+CTX+DDP (P<0.01), no significant difference between the group of CTX+DDP and two control groups p<O.05).4> The weight of tumors in the groups of CTX+DDP and herbs+CTX+DDP were lower highly than that of the control groups (p <0.01), and the two groups were equal (P>0.05). The inhibition rate in the groups of CTX+DDP and herbs+CTX+DDP was higher than60%.5, Ultrastructural organizationof granulocyte cellsand oocyte cells:The apoptosis of oocyte of early follicle and granulocyte cell of growing fol licle was observed by electron microscope. The apoptosis of granulocyte cell of growing follicle was typical particularly.ConclusionZuogui pill plus HuangQi could reduce the chemotherapy-induce ovarian function damage and be sure in antitumor effect, and its mechanism mybe is correlation with the express of apoptosis related protein Bcl-2/Bax.