Ovarian Failure-Resistant Effects of Catalpol in Aged Female Rats

Abstract

Catalpol, an iridoid glycoside obtained from various natural sources, has many biological functions. However, its ovarian failure-resistant effect has scarcely been studied. The present study used senile 14-month-old Sprague-Dawley female rats to examine the in vivo ovarian failure-resistant activity of catalpol. Daily oral graded doses of catalpol (1, 3, or 5 mg/kg/d) for 4 weeks significantly increased the levels of serum 17β-estradiol (E₂) and progesterone (P4) but reduced follicle-stimulating hormone and luteinizing hormone levels. Electron microscopic analysis and flow cytometry showed that catalpol significantly retarded apoptosis of the ovarian granulocytes of the rats. These findings suggest that catalpol works on the sex organs by nourishing ovarian tissues and improving both the quality and quantity of follicles, thus leading to rebalanced E₂ and P4 levels in aged rats so that catalpol has a direct in vivo antiaging effect on the rat ovarian system.

Menopause, a naturally occurring phenomenon in women between the ages of 40 and 60 years, represents the cessation of menstrual cycles due to loss of estrogen and progesterone secretion. Research into the main cellular and molecular aspects of ovarian follicle ageing that lead to menopause include a wide range of important factors. Follicle ageing is characterized by the impairment of specific functions of oocytes and granulosa cells, as well as general cellular dysfunctions including reduced mitochondrial activity, energetic failure, and changes in gene and protein expression. It is speculated that the level of cellular decline is sufficient to make ovarian follicles and ovulated oocytes more susceptible to apoptosis.¹⁾ A significant number of women entering menopause exhibit symptoms, inculding hot flushes, sleep disorders, irritability, depression and anxiety, which can lead to significant disruptions in the couse of daily life. In extreme cases, such symptoms lead women to seek medical attention.²⁾ The possibility of side-effects, including a transitory increased risk of heart disease at the start of treatment^3,4) and increased risk of endometrial or various forms of cancer,^5–7) which cause many women to seek alternatives, has led most doctors to disagree on the utility of hormone replace therapy (HRT) in all menopausal patients. On the basis of the risks and findings from the Women’s Health Initiative, the U.S. Preventive Services Task Force published a recommendation against the routine use of HRT for the prevention of chronic conditions in postmenopausal women.⁸⁾

Recently, herbal medicines origin without hormonal replacement have been attracting a great deal of attention as alternative and supplemental medicines because of their proven therapeutic efficacy and low risk of side-effects.⁹⁾ Many extracts from the herbal agents have been proven to be useful in the treatment of age-associated diseases.^10,11) Rehmanniais an important traditional Chinese herbal medicine which is widely used to replenish vitality, strengthen the liver, kidney, heart, and for treatment of a variety of ailments like diabetes, anemia, and urinary tract problems. There have been growing evidences that the extract from the root of Rehmannia possesses significant neuroprotective activity.^12,13) Catalpol (Fig. 1), an iridoid glycoside, was isolated from the fresh root of Rehmannia with column chromatography method in our laboratory. It exists broadly in many plants all over the world and has many biological functions such as anti-inflammation, protection of liver damage, and reduction of elevated blood sugar, but its ovarian failure resistant effect to our knowledge has been scarcely studied. In this study, we investigated the in vivo ovarian failure resistant activity of catalpol.

Fig. 1. The Chemical Structure of Catalpol

MATERIALS AND METHODS

Animals

A total of 48 Sprague-Dawley (SD) female rats (SPF grade; Sino-British Sippr/BK Lab) were included in the study. Forty of these rats were 14 months old (250–300 g), while the other eight were 4 months old (180–220 g). The animals were maintained under regulated environmental conditions (temperature, 22±2°C; humidity, 45–55%; 12 h light/dark cycle) and provided with a standard diet and water ad libitum and processed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and its associated guidelines. The 14-month-old SD female rats were observed for four continuous estrous cycles using a smear with vaginal exfoliated cells followed after staining with hematoxylin which were obtained between 9:00 and 10:00 h. The ageing female model was established using a vaginal cytological technique involving an extended estrogenic cycle, continuous estrous, and repeated fake pregnancy in cells.

Preparation of Catalpol

Catalpol was separated from the traditional Chinese herbal medicine Rehmannia glutinosa and and its structure was verified via various spectral techniques.¹⁴⁾ The medicinal herb had been bought at Anhui Bozhou medicinal materials market, whose origin is Henan province. The isolated compound was then disolved into diluted in normal saline (1% sodium carboxymethylcellulose (CMC-Na)) for treatment. A HPLC analysis confirmed the purity of catalpol as 95%.

Experimental Schedule

The 14-month-old SD female rats as the ageing model were randomly divided into five groups (8 rats/group): vehicle-treated (control group), 17β-estradiol[E₂]-treated (E₂-treated group, 0.18 mg/kg), low dose catalpol-treated (L-catalpol-treated group, 1 mg/kg), medium dose catalpol-treated (M-catalpol-treated group, 3 mg/kg) and high dose catalpol-treated (H-catalpol-treated group, 5 mg/kg). Four-month-old SD female rats as healthy controls were given vehicle (normal group). All samples were suspended in normal saline (CMC-Na). Rats in the control group and normal group were administered the same volumes of normal saline. Rats were all administered the test substances through an oral gavage. The treatments lasted for 4 weeks. The dosing was adjusted according to rat weight on a weekly basis.

Blood samples that were collected from the inferior vena cava before and after administration were centrifuged at 3000×g for 15 min at 4°C and stored at −20°C for serum analyses at the time of diestrus. At the end of the treatment period, all of the rats were sacrificed after taking blood, the brain, bilateral ovaries, bilateral uterine, adrenal gland and pituitary glands were removed and weighed to determine the tissue indices. Thereafter, follicles of the rats dissected from wet ovarian tissues were fixed in 2% glutaraldehyde solution and examined under a transmission electron microscope. The remaining ovarian tissue were rinsed with PBS at 37°C and granulose cells were isolated from medium (2–5 mm diameter) antral follicles by fine-needle aspiration according to the method of Channing and Ledwitz–Rigby (1975) for flow cytometry analysis.

Histological Observation

The brain indices, adrenal indices, uterine indices, ovarian indices and pituitary indices were defined respectively as the weight ratio of these tissues to the rat body to which they belonged (g/100 g body weight).

Determination of Apoptosis

The ovarian cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (1 : 1, v/v, HyClone, local agency of Thermo Fisher Scientific Inc., Beijing, China) at a concentration of 1×10⁶ cells/mL. 1×10⁶ cells were collected and resuspended in 400 µL of annexin V binding buffer containing fluorescein isothiocyanate (FITC)-conjugated annexin V (Mbchem, Shanghai, China). After 15 min of incubation at 37°C, 10 µL of propidium iodide (PI, 25 µg/mL) was added. The cell suspension was immediately analyzed using fluorescence activated cell sorter (FACS) Calibur flow cytometer (BD Biosciences, San Jose, CA, U.S.A.). The excitation wavelength was set at 488 nm with the emission wavelength set at 513 and 675 nm for detecting FITC-annexin V (FL1 channel) and PI (FL3 channel), respectively. Apoptosis rate was calculated by the equation: Apoptosis rate%=the number of apoptosis cells/(the number of apoptosis cells+the number of normal cells)×100%.

Relevant Gynecological Hormones Levels

Progesterone (P₄) was estimated in plasma samples using a commercially available radioimmunoassay (RIA) kit (Beijing North Institute of Biotechnology, Beijing, China). The sensitivity of the assay was 0.25 ng/mL. The mean intra- and interassay coefficients of variation (CVs) were 5.5 and 9.2%, respectively. E₂ was estimated in plasma samples using a commercially available RIA kit (Beijing North Institute of Biotechnology). The sensitivity of the assay was 2.5 pg/mL. The mean intra- and interassay CVs were 4.6 and 11.5%, respectively. Follicle-stimulating hormone (FSH) was estimated in plasma samples using a commercially available RIA kit (MP Biomedicals (Shanghai) Co., Ltd., China). The sensitivity of the assay was 0.28 IU/L. The mean intra- and interassay CVs were 4.8 and 10.2%, respectively. Luteinizing hormone (LH) was estimated in plasma samples using a commercially available RIA kit (Shanghai Kaibo Biochemical Reagent Co., Ltd., China). The sensitivity of the assay was 0.21 IU/L. The mean intra- and interassay CVs were 4.2 and 9.0%, respectively.

Statistical Analysis

Results are presented as mean±standard error of the mean (S.E.M.) of the observations. The statistical analysis was performed using two-way ANOVA and the Tukey test was conducted using Student’s t-test in the SYSTAT 10.2 statistical software program. The values were considered significantly different at values of p<0.05.

RESULTS

Effect on Uterine, Ovarian, and Pituitary Indices

In the present study, the initial body weights of the five groups (except normal group) were similar. The body weights increased continuously in all groups. At the end of the study, the rats in E₂-treated group and the three catalpol-treated groups showed no increases in body weight, which were not different in control group (Fig. 2A; p>0.05). As expected, the brain, ovarian and adrenal indices (g/100 g body weight) were significantly reduced in control group compared to normal group (p<0.01, p<0.01, and p<0.05, respectively), indicating that apolexis resulted in atrophy of the brain, ovary and adrenal. But the the pituitary and uterine indices of control group were not seen to have significantly difference from normal group (p>0.05). 17β-E₂, high dose catalpol, medium dose catalpol, and low dose catalpol administration prevented ovary index loss compared to control group (p<0.05, p<0.01, p<0.01, and p<0.05, respectively) but still resulted in a lower ovary index than that of normal group (p>0.05; Fig. 2B).

Fig. 2. Effect of Catalpol on the (A) Body Weight and (B) Tissue Indices of Aged Senile Rats (n=8)

Normal group, non-aged (treated with vehicle); control group, aged (treated with vehicle); E₂-treated group, treated with 17β-estradiol 0.18 mg/kg; H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group, treated with catalpol 5, 3, and 1 mg/kg, i.g., respectively. Results are presented as mean±S.E.M. * p<0.05, ** p<0.01, control group, E₂-treated group, H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group versus normal group; ^# p<0.05, ^## p<0.01, E₂-treated group, H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group versus control group.

Effect of Catalpol on the Ovarian Ultrastructure of Aged Female Rats

Healthy Normal Rats (Fig. 3)

Electron microscopy with low resolution disclosed that the granulocytes in normal group of reproductive potential were 12–15 µm in diameter, with irregular oval shape and arrayed next to each other. The granulocytes possessed one or two reticular nucleoli with normal chromatin. The cytoplasm of the granulocytes was found to contain many dark and round secretory granules with a diameter of 0.3–0.8 µm that could be electronically stained. Under electron microscopy with high resolution, a large quantity of utricular smooth endoplasmic reticulum (ER) was observed in the cytoplasm of the granulocytes, and the mitochondria dispersed inside showed clear tabulate cristae in a regular array; the Golgi complexes were normal in shape; no apoptotic cells were found.

Fig. 3. The Ovarian Ultrastructure of Normal Group

Model Rats (Fig. 4)

Compared with normal group, the granulocytes size of control group became small-diameter of 8 µm or so. The numbers of organelles and secretory granules decreased, and secretory granules became vacuoles; the quantity of smooth ER and mitochondria in the cytoplasm was significantly reduced; the size of mitochondria was smaller and cristae were not visible; the Golgi complexes were found in disordered state. The sinusoidal gap widened among the granulocytes and myelination-like modifications appeared.

Fig. 4. The Ovarian Ultrastructure of Control Group

Catalpol-Treated Rats (Fig. 5)

Compared with control group, the organelles in the cytoplasm of catalpol-treated group which treated with catalpol 3 mg/kg were more abundant, and the structure normal. Secretory granules were seen. The size of the granulocytes rejuvenated to that of normal group, and the nucleoli of the cells became apparent with X chromatin. The quantity of smooth ER increased and they looked like round theca. The mitochondria were abundant, and showed clear transverse cristae. The Golgi complexes looked normal and secretory granules were rare. The appearances are similar to other catalpol-treated groups.

Fig. 5. The Ovarian Ultrastructure of Catalpol-Treated Group, Treated with Catalpol 3 mg/kg

Estrogen-Treated Rats (Fig. 6)

Compared with control group, the size of the granulocytes increased, averaging 8–12 µm in diameter. Large amounts of mitochondria, smooth ER and Golgi bodies existed in the cytoplasm. The structure of the organelles looked normal. Secretory granules were seen. Vacuous granules and sinusoidal gaps among granulocytes were abundant.

Fig. 6. The Ovarian Ultrastructure of E₂-Treated Group

Effect of Catalpol on Apoptosis of Ovary Granule Cell in Aged Female Rats

Cell apoptosis was determined by a double staining method using flow cytometry. In apoptotic cells, the phosphatidyl serine (PS), which is confined to the inner layer of the plasma membrane in healthy cells, is exposed to the outer plasma membrane.¹⁵⁾ Annexin V, a Ca²⁺-dependent protein with high affinity for PS, was used to detect apoptotic cells. Meantime, the viable cells were distinguished from the dead ones using PI. As shown in Fig. 7, UL represent the proportions of dead cells; UR represent the proportions of late apoptotic cells; LL represent the proportions of living cells; LR represent the proportions of early apoptotic cells. After administration, the proportions of total apoptotic cells (late and early apoptotic cells) for the ovary granule cell were 25.70%, 86.65%, 47.04%, 29.28%, 46.12%, and 63.28% in normal group (Fig. 7A), control group (Fig. 7B), E₂-treated group (Fig. 7C), H-catalpol-treated group (Fig. 7D), M-catalpol-treated group (Fig. 7E), L-catalpol-treated group (Fig. 7F), respectively. The samples of each group repeat that three more times. As shown in Fig. 8, control group had the highest levels of apoptosis rate (p<0.01), which were coincident with the apoptosis of ovarian granulosa cell. It was also evident that three doses of catalpol (p<0.01) and 17β-E₂ (p<0.01) administration restored the apoptosis of ovarian granulosa cell, compared with those of control group. Moreover, this adjustment of catalpol is superior to that of 17β-E₂.

Fig. 7. Effect of Catalpol on Apoptosis of Ovary Granule Cells in Aged Female Rats by Flow Cytometry

(A) Normal group, treated with vehicle; (B) control group, treated with vehicle; (C) E₂-treated group, treated with 17β-estradiol 0.18 mg/kg; (D) H-catalpol-treated group, treated with catalpol 5 mg/kg; (E) M-catalpol-treated group, treated with catalpol 3 mg/kg; (F) L-catalpol-treated group, treated with catalpol 1 mg/kg.

Fig. 8. Effect of Catalpol on Apoptosis Rate of Ovary Granule Cells in Aged Female Rats (n=3)

Apoptosis rate %=the number of apoptosis cells/(the number of apoptosis cells+the number of normal cells)×100%. Normal group, non-aged (treated with vehicle); control group, aged (treated with vehicle); E₂-treated group, treated with 17β-estradiol 0.18 mg/kg; H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group, treated with catalpol 5, 3 and 1 mg/kg, i.g., respectively. Results are presented as mean±S.E.M. ** p<0.01, control group versus normal group; ^# p<0.05, ^## p<0.01, E₂-treated group, H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group versus control group.

Effect of Catalpol on Hormone Levels of Aged Female Rats

In the present study, the initial hormone levels of the five groups (except normal group) were similar. The effects of the different diet supplements on plasma mean levels of E₂, P4, FSH, and LH are shown in Fig. 9. It can be seen control group had the lowest levels of E₂ and P4, and the highest levels of FSH and LH (p<0.01), which were coincident with the gradual failure of ovulation and hyposecretion or deficiency of sex hormones in aged rats. Catalpol significantly increased the levels of E₂ (p<0.01) and P4 (p<0.01) and reduced the levels of FSH (p<0.05) and LH (p<0.05) in the aged rats compared with control group. Moreover, this adjustment of catalpol on the levels of P4 and LH are superior to those of 17β-E₂.

Fig. 9. Effect of Catalpol on Hormone Levels in the Sera of Aged Senile Rats (n=8)

Normal group, non-aged (treated with vehicle); control group, aged (treated with vehicle); E₂-treated group, treated with 17β-estradiol 0.18 mg/kg; H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group, treated with catalpol 5, 3 and 1 mg/kg, i.g., respectively. Results are presented as mean±S.E.M. ** p<0.01, control group versus normal group; ^# p<0.05, ^## p<0.01, E₂-treated group, H-catalpol-treated group, M-catalpol-treated group, and L-catalpol-treated group versus control group.

DISCUSSION

Catalpol, separated from Rehmannia glutinosa has shown protective effects in many experimental models of neurodegeneration. As is well known, the pituitary-adrenal gland-ovary system in the rat is a good tool for the investigation of emergent responses in neuroendocrinology. The present study focused on the pharmacological effects of catalpol on the ovary system of aged rats. From this experimentation results, it is found that use of catalpol resulted in significant increases of serum E₂ and P4 levels in the aged rats, and restores of serum FSH and LH levels. Specifically, the adjustment of catalpol over P4 and LH is superior to that of 17β-E₂. The increased ovarian indices in the catalpol-treated group perhaps correspond to neurobiological factors and the greater nutrition that ovarian tissues obtained, confirmed that catalpol’s mechanism is not estrogenic.

Moreover, the electronic microscopy analysis revealed that new follicles were formed after the administration of catalpol. Follicles are associated with the generation of internal hormonal agents. If catalpol had only estrogenic activities, the levels of P4, FSH, and LH should not have undergone significant changes. Rejuvenation of the granulocytic ultrastructure is further evidence to support that catalpol nourishes and harmonizes ovarian tissues to regain hormonal balance. From the electron microscopic studies, it was seen that catalpol inhibits the apoptosis of cells by modulating granule synthesis and producing endogenous estrogens. From the flow cytometry studies, it was seen that catalpol reduces apoptosis of cells by modulating the apoptotic mechanism of granulocytes. All the results revealed that catalpol reinvigorates the morphogenesis and function of organelles, such as ER and mitochondria, and granulocytes, and reduce the apoptotic rate of granules.

In summary, catalpol works on the sex organs by nourishing ovarian tissues and improving both the quality and quantity of follicles, thus leading to rebalanced E₂ and P4 levels in the aged rats. But it is unclear that catalpol has a regulating activity of ovarian tissues via expression of apoptosis-related molecules or other signal transduction pathways, which in turn are coupled to catalpol’s differential pharmacological activity in a quantitative and temporal fashion, are involved in the production of endogenous E₂ rather than the action of FSH and LH. This puzzle is being addressed in another ongoing study we are conducting by means of molecular biological techniques.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (Grant No. 30973927).

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