Effect Of Angiogenesis Inhibitor Nordihydroguaiaretic Acid On Embryo And Ovarian Function In Rat

Nordihydroguaiaretic acid (NDGA) is the major chemical component of a shrub named L.tridentata growing widely in Southwest America and North of Mexican. It has been used in traditional therapy for wound healing, easing pain and driving out parasites by oral or compressing the plant. NDGA can be synthesised. Generally, it is also used as an anticorrosive for food. Many studies about the biological activities of NDGA have been performed. One of the most representative activities of NDGA is that it can inhibit the activity of lipoxygenase. Some studies have found that NDGA not only has antihyperglycenic activity in both rat and mouse models of Type Ⅱdiabetes, inhibits the accumulation of Ca2+ induced byβ-amyloid (Aβ) in seahorse neuron, inhibits the secretion of casein stimulated by prolactin and also non-competitively inhibits the binding of both estradiol and testosterone to the human sex hormone binding protein (SHBP). Further studies showed that it has antitumor activity. Most researchers suggested that new growing vessels in tumor tissue could promote the growth and metastasis of tumor cells. NDGA has anti-angiogenesis activity by inhibiting the proliferation of vascular endothelial cells; decreasing the expression of vascular endothelial growth factor receptor 2 (Flk-1/KDR). In the female reproductive system, phenomena of angiogenesis can be observed widely in the processes of periodical proliferation of endometrium, follicle growth, ovulation, corpus luteum formation and decidualization. The main aim of this research is to observe the effect of NDGA on the ovarian function and angiogenesis in early embryo and explore the related pathogenesis. We have investigated the morphological changes of ovary and uterus, detected the expression of vascular endothelial growth factor(VEGF), proliferation cell nucleus antigen (PCNA) and factor Ⅷrelated antigen (FⅧRAg) and measured the serum concentrations of estradiol (E2), progesterone (P), follicle stimulating hormone (FSH) and luteinizing hormone (LH) by methods including immunohistochemistry (IHC) stain, HE stain, image analysis and chemiluminoimmunoassay analysis(CLIA). The main results are as follows: Part 1. Effect of NDGA on the ovarian function in rat After were injected subcutaneously with NDGA and PBS respectively every other day for twenty days, some female rats that have regular estrus cycle mated up with the male rats and the others were killed and have an ovariohysterectomy. The uterus and ovary resected fixed in 10% neutral formaldehyde fixative, and embedded in paraffin. Sections were cut at 4～6μm and stained with HE and IHC with antibody PCNA and FⅧRAg. The serum concentrations of E2, P, FSH and LH were detected. (1) The female rats injected with NDGA subcutaneously couldn't mate up with the male rats successfully and the smear of the vaginal desquamated cells displayed that these rats were in diestrus. (2) The most mature corpora lutea and the glands of endometrium in female rats treated with NDGA were less than that of PBS in HE stained sections, while atresic follicles were more than the control group(p<0.01). (3) Expression of the PCNA and FⅧRAg in the endometrium stromal cells, epithelial cells of glands and vascular endothelial cells in female rats treated with NDGA were weakly positive in IHC stain sections(p<0.01) and the count of microvessel density(MVD) has decreased(p<0.05). There was a significant difference between group NDGA and group PBS. (4) In NDGA group, both the secretion of E2 and P were decreased(p<0.01)but that of FSH was increased(p<0.05). There was a significant difference between before and after injection. The secretion of LH was irregular(p>0.05). Part 2. Effect of NDGA on the angiogenesis of early embryo by subcutaneous injection in rat Pregnant rats were injected with NDGA and PBS subcutaneously with the dose of 30mg/kg on the first embryo day (E1)and on E7. Some rats were killed on E9; the others were permitted to labor. After fixed in 10% neutral formaldehyde fixative, embedded in paraffin and cut, the sections of uterus resected stained with HE and IHC with antibody VEGF, PCNA and FⅧRAg. The serum concentration of P was detected. The body weights and number of newborn litters was observed.(1) Effect on the embryos: The number and diameter of embryos decreased when NDGA was injected on the first day of embryo (E1) (p<0.01). While NDGA was injected on E7, the number of embryos has no change(p>0.05), but the diameter decreased(p<0.01). (2) Effect on the newborn litters: In NDGA group, the number of litters was less than that of the PBS group(p<0.05), but body weigh has no significant difference between the two groups who injected on E1 and E7(p>0.05). (3) The serum concentration of progesterone: There was no difference between the NDGA group and PBS group(p>0.05). (4) IHC stains and image analysis: In NDGA group, the expression of VEGF and PCNA in the decidua reduced. The average optic density (AOD) of positive area and the count of MVD were reduced than that of PBS group and there was a significant difference between the two groups(p<0.01). Part 3. Effect of NDGA on the angiogenesis of early embryo by intrauterine perfusion in rat Pregnant rats were injected with NDGA and PBS by intrauterine perfusion with the dose of 30mg/kg on the first embryo day (E1)and on E7. Some rats were killed on E9; the others had been a cesarean section before labor. After fixed in 10% neutral formaldehyde fixative, embedded in paraffin and cut, the sections of uterus resected stained with HE and IHC with antibody VEGF, PCNA and FⅧRAg. The serum concentration of P was detected. The body weights and number of mature fetus was observed. (1) Effect on the embryos: In NDGA group, both of the number and diameter of embryos on the side of perfusion on E1 were less than the other side and the diameter was less than that of the PBS group and blank group. But only the diameter of embryos has a significant difference between the two groups(p<0.05). The number of embryos did not decrease on the side of perfusion on E7, but the mean diameter of embryos was less than that of the two control groups. The change of diameter has significant difference between two perfusion phases(p<0.05). (2) Effect on the mature fetus: On E1, the number of mature fetus on the side of perfusion has significant difference among the groups of NDGA, PBS and blank(p<0.05). On E7, the number of fetus on the side of perfusion was no difference among these three groups. The weight of fetus in the three groups has no difference on either E1 or E7.(3) The serum concentration of progesterone: There was no difference among the three groups(p>0.05). (4) IHC stains and image analysis: In NDGA group, the expression of VEGF and PCNA in the decidua decreased. The AOD of positive area and the count of MVD were reduced than that of the controls and there was a significant difference among the three groups(p<0.01). The main conclusions are as follows: 1. NDGA has anti-angiogenesis activity by inhibiting the proliferation of endothelial cells in the uterus and ovary. It can interfere the secretion of ovary and confuse the estrus cycle of female rat if treated for a long term. 2. NDGA can inhibit the expression of VEGF in placeta and decidua; inhibit the angiogenesis of microvessels and proliferation of cells by either subcutaneous injection or intrauterine perfusion. NDGA couldn't affect the secretion of progestrone directly. This research has explored the different avenues of medical treatment and provided an experimental base for studying the biological activities of NDGA as an anti-imbed drug. Particularly, it would be more interesting to have further studies of the angiogenesis inhibitor effecting on the diseases of female reproductive system.