Fructus Ligustri Lucidi modulates estrogen receptor expression with no uterotrophic effect in ovariectomized rats

Pentobarbital sodium was purchased from Sigma-Aldrich (St. Louis, USA), Estradiol valerate (EV) tablets were bought from Bayer (Monheim, German). Rat monoclonal anti-ERα antibody (ab3575) and mouse monoclonal anti-ERβ (ab288) were purchased from Abcam (Cambridge, UK). All the other chemicals, except specially identified, were obtained from Beijing Sinopharm Chemical (Beijing, China).

FLL was bought from Beijing TongRenTang (Beijing, China) and authenticated by Professor Zexin Ma (TCM museum at Beijing University of Traditional Chinese Medicine (BUCM)). For preparation of FLL water extracts, 100 g of raw FLL was grinded into powder and dissolved in 1000 ml of distilled water by continuous stirring for 48 h under low temperature. Then the aqueous extracts were collected by centrifugation (4000 rpm at 4 °C for 10 min). And the supernatants were harvested and lyophilized to obtain a powder (20 g).

Female 12-week-old Sprague Dawley rats (200 ± 20 g) were purchased from Beijing SiBeiFu Animal Technology company (license number: SCXK (Beijing) 2014-0037, Beijing, China). The animals were housed in the clean level conditions (certification number: SCXK (Beijing) 2011-0024) at BUCM with the temperature of 22 ± 1 °C, humidity of 55 ± 5%, and a 12 h-light/dark cycle. All rats had free access to tap water and standard chow. All procedures in this study were approved by the Animal Care Committee of BUCM, Beijing, China.

After 1 week of acclimation, the OVX rats were established by removing the bilateral ovaries from the corresponding anesthetized rats. The sham control groups were performed by removing the equal volume of fat surrounding the bilateral ovaries. One week after surgery, the OVX animals were randomly divided into three groups of 9 rats in each, named OVX control, OVX + EV and OVX + FLL, respectively. For the treatment, the rats in the OVX + FLL group were orally administrated with the water extracts of FLL (3.5 g/kg/day). The rats in the OVX + EV group were orally administrated with EV tablets (0.1 mg/kg/day). The rats in OVX control group and Sham control group were orally administrated with the same volume of distilled water.

After 12 weeks of administration, rats were anesthetized by intraperitoneal injection with 1% pentobarbital sodium (0.4 ml/100 g, i.p.). Subsequently, blood was collected from the heart by puncture. Then the rats were sacrificed by cervical dislocation. After that, the uteri, femurs and tibias were harvested for the following experiments.

After trimming off the fat and absorbing the excess surrounding fluid, the wet weight of the uterus was recorded with analytical balance. Then, the uteri were cut into pieces just above the junction with the cervix. Half of the uteri were stored in liquid nitrogen until use. Another half of it was fixed in 10% neutral buffered formalin for histological analysis. Uterus coefficient was determined by uterus wet weight divided by the corresponding body weight of the rat (g/100 g).

E₂, LH and FSH were determined by ELISA (CUSABIO, China) according to the manufacturer’s instructions. All the samples were evaluated in duplicates.

The femurs and uteri were fixed with 10% neutral buffered formalin. Furthermore, the tibias were decalcified in 15% ethylenediaminetetraacetic acid (EDTA) buffer (pH 7.4) for 90 days. After that, the femurs and uteri were dehydrated in graded ethanol, defatted in xylene, and embedded in paraffin. Then, 5 μm sections were deparaffinized in xylene and rehydrated with graded ethanol. Subsequently, the sections were incubated with 3% H₂O₂ and antigen retrieval solution (0.1 M sodium citrate buffer, pH 6.0) followed by incubation with 10% goat serum in phosphate-buffered saline (PBS) for 30 min to block nonspecific binding sites. The sections were then incubated with the primary antibodies (anti-ERα antibody (1:500) or anti-ERβ antibody (1:500)) overnight at 4 °C. The next day, after washing in PBS, the sections were incubated with biotinylated anti-rat secondary antibody for 30 min and with peroxidase for 10 min according to the SP staining system (Beijing ZhongShan JinQiao, Beijing, China). Diaminobenzidine (DAB) was used as the substrate for color development and visualization under the microscope. For controls, the primary antibodies were replaced by non-immunized goat serum. The slides were then taken for histopathological evaluations. The results of immunohistochemical staining were quantified by Image Pro-Plus software (version 6, SPSS Inc., Chicago, IL, USA) and the integral optical density (IOD) values were recorded. The measurements were performed by two investigators who were blinded regarding the animals’ treatment groups.

The uteri were placed in a 1.5 ml Eppendorf tube and washed with PBS twice, and then were cut with scissors and grounded. The tibias were prepared by lyophilizing and grinding. After that, the samples were lysed in a buffer containing 20 mM Tris–HCl, pH 7.5, 0.1% (v/v) Igepal, 6 mM sodium deoxycholate, 150 mM NaCl, 2 mM ethyleneglycoltetraacetic acid (EGTA), 2 mM EDTA, 0.1 mM Na₂SO₄, 20 mM NaF, and a protease inhibitor cocktail tablet (Roche, German). The lysates were centrifuged at 10,000 g for 15 min at 4 °C, and protein concentrations in the supernatants were determined by BCA protein assay kit (Applygene, China). Then 50 μg/lane of proteins were loaded into 10% polyacrylamide gel, and transferred onto nitrocellulose membrane, and then incubated with the primary antibody (anti-ERα or anti-ERβ) and the corresponding HRP labeled secondary antibody. The membranes were developed using enhanced chemiluminescence solution. The images were captured with Bio-Rad bioimaging system. The gray values of the blots were quantified using the Image J software (NIH, Bethesda, MD), and normalized with the corresponding β-actin (1:2000) as the internal control.

The uterus coefficient of the rats were shown in Table 1. As expected, ovariectomy resulted in a significant reduction in the relative uterus weight of the rats. The uterus coefficient in the OVX control group was only around 16% of that in the Sham control group. EV treatment for 12 weeks significantly increased the uterus coefficient in OVX rats (P < 0.05). By contrast, FLL treatment did not increase the uterus coefficient in OVX rats.

As shown in Table 2, serum E₂ level was decreased, and serum LH and FSH levels were increased in the OVX control group rats as compared to those of rats in the Sham control group. The administration of EV and FLL to OVX rats for 12 weeks significantly decreased serum LH and FSH levels (P < 0.05). However, FLL treatment did not increase serum E₂ in OVX rats (P > 0.05).

The effects of FLL on ERα and ERβ expressions in the femurs were assessed by immunohistochemical staining. As shown in Figs. 1 and 2, ERα and ERβ expressions in the femurs of the OVX control group were significantly decreased (P < 0.01), when compared with those of rats in the Sham control group. Both FLL and EV treatment significantly increased ERα and ERβ expressions in the femurs of the OVX rats (P < 0.05 or 0.01) when compared to those in the OVX control group.

Furthermore, the effects of FLL on ERα and ERβ expressions in the tibias of the rats in different groups were also evaluated by western blot. As shown in Fig. 3, FLL treatment did markedly increase ERα expression in response to ovariectomy (P < 0.01). By contrast, FLL treatment showed a trend toward increasing ERβ expression in OVX rats, but the differences did not reach the statistically significant level when compared to those of rats in the OVX control group.

As shown in Figs. 4 and 5, ERα and ERβ were mainly located in the endometrium and glandular epithelia cells, and were highly expressed in the uteri of rats in sham control group and EV treatment group compared to those of rats in the OVX control group as evaluated by immunohistochemical staining (P < 0.01). Further, FLL treatment did not increase ERα expression but did obviously increase ERβ expression in the uteri of the OVX rats (P < 0.05). These results were also further confirmed by western blot (Fig. 6).