Hemp seed oil can accelerate the virgin mice mature and balance sex hormones, modulates organ atrophy, and restores in ovariectomized mice through upregulates ER𝛼 and ER𝛽 expressions

doi:10.21203/rs.3.rs-242208/v1

Download PDF

Research

Hemp seed oil can accelerate the virgin mice mature and balance sex hormones, modulates organ atrophy, and restores in ovariectomized mice through upregulates ER𝛼 and ER𝛽 expressions

https://doi.org/10.21203/rs.3.rs-242208/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Hemp seed, the dried fruit of Cannabis sativa L., has been well documented as a folk source of food due to its nutritional and functional value. The aim of this study was to evaluate the estrogen-like effect of hemp seed oil (HSO) and analyze its action on the development of ovary and uterus of mice, concentration of sex hormones, endometrial thickness, and ER𝛼 and ER𝛽 expression.

Methods

In the present work, virgin and ovariectomized kunming mice were subject to treat with HSO. Gonad morphous was detected by Hematoxylin-Eosin (HE) staining, serum levels of E2, FSH and LH was determined used ELISA to evaluate the function of hypothalamic-pituitary-ovarian axis, and ERα and ERβ immunohistochemistry were used to evaluate gonad function.

Results

The results showed that HSO can promote the vaginal opening time of virgin mice. There was significant increase in the gonad coefficient. Moreover, the endometrial thickness, number of primary follicles, secondary follicles and mature follicles were significantly increased, the uterus and ovary were well developed, the ERα and ERβ positive cells and intensity were increased in the virgin mouse ovary. The level of serum E2 was significantly increased, and the levels of FSH and LH were decreased. HSO could obviously relieve the endocrine disorders, modulate organ atrophy, and restore ER𝛼 and ER𝛽 expression in the ovariectomized mice model.

Conclusions

Hemp seed oil can accelerate the virgin mice mature and balance sex hormones, modulates organ atrophy, and restores in ovariectomized mice through upregulates ER𝛼 and ER𝛽 expressions. This study demonstrated that HSO activity is mediated through estrogenic components, and provided evidence for HSO treatment for post-menopausal symptoms.

Integrative & Complementary Medicine

Hemp seed oil

Estrogen

endocrine

immunohistochemistry

positive expression

As a sex hormone, estrogen not only plays an important role in regulating the growth and development of the female reproductive system but also affects the normal function of the skeletal system, cardiovascular system and central nervous system [1]. Ovarian function and estrogen levels decline with age [2], which can lead to post-menopausal symptoms, including short-term symptoms such as hot flushes, night sweats, palpitations, irritability and anxiety as well as long-term symptoms such as menstrual changes, vasodilation, emotional instability, memory loss, depression, insomnia and other symptoms, and even cause endothelial cell dysfunction, arteriosclerosis, cardiovascular and cerebrovascular diseases such as early pathological changes, hypertension, heart disease and Alzheimer's disease, which seriously affect women's quality of life. Consequently, hormone replacement therapy (HRT) has often been given to peri and post-menopausal women to treat the somatic and cognitive symptoms of menopause [3]. However, HRT is thought to increase the risk of breast cancer and cause other undesirable side effects, including breast tenderness and uterine bleeding [4]. Studies have found that phytoestrogens in plants have animal hormone-like activity, with partial agonistic or antagonistic effects. Phytoestrogens can effectively supplement estrogen in the body, and their activity is only 1/1000–1/100,000 of endogenous estrogen [5]. Therefore, phytoestrogens have often been used as alternatives in the post-menopausal women, such as natural isoflavonoids from herbal medicines [6]. Phytoestrogens are both structurally and functionally similar to mammalian estrogens, but with reportedly lower side effects compared with synthetic HRT [5, 6].

Traditional Chinese medicines (TCMs) contain multi-interactive compounds, which have been used for centuries in China for the treatment of peri-menopausal syndrome. TCMs have become research hotspots as a new source of phytoestrogens. Hemp seed (Fructus cannabis, the dried fruit of Cannabis sativa L.) has been well documented as a folk source of food [7]. Hemp seed has a broad pharmacological effect in the gastrointestinal system [8], the cardiovascular system [9], the central nervous system [10, 11], and the immune system [12]. Compared with other extracts [13], the cold-pressed hemp seed oil (HSO) [14] was reported to have anti-aging effects [15], and the potential to improve impaired learning and memory induced by chemical drugs in mice. HSO is especially rich in sitosterol, campesterol, phytol, cycloartenol, and γ-tocopherol [13], which are beneficial to women. Therefore, we hypothesized that hemp seed may have an estrogen-like effect, and HSO, as the main compound of hemp seed, may drive the estrogen-like effect of hemp seed. Consequently, this study aimed to systematically investigate whether HSO has an estrogen-like effect, could be potentially used in the treatment of ovariectomy (OVX), and the underlying potential mechanism(s) in mice.

The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study.

Animals and Treatment

All animal experiments were performed in accordance with the internationally accepted principles for laboratory animal use and care by the Ethics Committee of Henan University of Chinese Medicine. Forty-eight Female Kunming mice (SPF grade, 21 days old) were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd. [license number: SCXK (Beijing) 2016-0006, experimental unit license number: SYXK (Yu) 2015-0005, animal certificate number: 11400700309668]. The mice were adaptively raised in the SPF animal room of the Research Center of Henan University of Traditional Chinese Medicine. For experiment 1: the mice were randomly divided into four groups: mice intra-gastrically treated with HSO (Guangxi Bama Ivy Life Science and Technology Development Co., Ltd.) at a daily dose of 2.70 g/kg, 5.40 g/kg, treated with 0.17 mg/kg estradiol valerate (EV, Bayer HealthCare Co., Ltd. Guangzhou Branch). For experiment 2: 45 female Kunming mice (SPF grade, 8 weeks old, reared in the same with experiment 1) was randomly ovariectomized to prepare perimenopausal model. After intraperitoneal injection of 10% chloral hydrate (0.03 ml / 10 g), the mice were fixed in the abdominal position, and bilateral oophorectomy was performed (in the sham operation group, the ovaries were not removed, and the adipose tissue as large as the ovaries were cut off at the same position), and about 1-2 cm was made at the position 1 / 3 below the trunk and 1-2 cm away from both sides of the spine. After ligation, the ovaries and other sutures were cut off and the wound was sutured. After the operation, the animals were carefully reared and ampicillin sodium solution was added to the wound to prevent infection. once a day lasting for 5 days. After 5 days of operation, vaginal smears were examined one by one for once a day. The rats must be in diestrus for 5 consecutive days to confirm the success of modeling. The rats with oestrus reaction on the smears were discarded. Then the successful modeling mice were randomly divided into three groups: model group, HSO group (5.40 g/kg), and EV group (0.17 mg/kg). Meanwhile, the other randomly selected 10 mice were sham-operated group. Untreated control mice and sham-operated group received distilled water only, and the administration volume was 20 ml/kg. EV was used as the positive control. Dose calculations followed guidelines correlating dose equivalents between humans and laboratory animals based on ratios of body surface area. All the above groups were given intra-gastric drug at approximately 9:00–11:00 am every day for eight days. All animals were maintained on the 12 h light/dark cycle under constant temperature (22 ± 2°C, 55 ± 5% relative humidity), and allowed free access to food and water.

Monitoring vaginal opening and body mass

The vaginal opening of the mice was recorded every day. Body weight was monitored every week.

Assay for serum levels of E2, FSH and LH

Animals were sacrificed by decapitation after eight days of treatment. Blood was collected from the eyeball for analysis of serum E2, FSH and LH levels by enzyme-linked immunosorbent assay (ELISA) (Shanghai Zibo Bio-Biotechnology Co., Ltd.). The sensitivities of the three ELISAs were 1.0 pmol/L, 1.0 U/L, and 1.0 ng/mL, respectively. The antibody did not cross-react with other estrogen-like substances. The samples were diluted and added according to instructions. Test sample wells and blank wells were setup separately (sample and HRP-conjugate reagent were not added to the blank wells, while all other steps were the same as test sample wells). The plates were incubated for 30 min at 37 ℃, and washed five times by 888-2143B plate washer (Thermo Fisher Scientific Oy Ratastie 2, FI-01620 Vantaa, Finland). Then the plates were incubated and washed again. The chromogen solution was added and the plate was kept in the dark for 10 min at 37 ℃. Then stop solution was added to stop the reaction. The blank well was considered as zero, and the absorbance was read at 450 nm using 1510-00441C plate reader (Zhengzhou Jinyouning Instrument Co., Ltd.).

Hematoxylin-Eosin (HE) staining of tissue

The hippocampus, spleen, uterus and adrenal gland were removed and weighed. The left horns of the uterus and the vagina were fixed with 4% polyoxymethylene (Guangzhou Saiguo Biotechnology Co., Ltd.) for 48 h. Then alcohol was used to dehydrate the tissue block, and was subsequently replaced with xylene. Thereafter, the transparent tissue block was placed in dissolved paraffin, embedded and cooled to solidify into a block. The wax block was cut into about 5 μm thick slices by a microtome. Then the slice was stained in hematoxylin solution for 5 min, and separately treated with acid water and ammonia water for 5 s each. It was rinsed under running water for 1 h, dehydrated in 70% and 90% alcohol for 10 min each, and stained with alcoholic eosin staining solution for 3 min. The stained sections were dehydrated with pure alcohol and made transparent with xylene. The section was finally covered with a cover glass for sealing. Then, the uterine endometrial thickness, number of uterine glands, and vaginal epithelial layer were observed for a selected single slide of each animal.

Immunohistochemistry

The paraffin sections were dewaxed by routine method and immersed in sodium citrate antigen retrieval solution (pH 6.0) for complete antigen retrieval. Thereafter, they were incubated for 25 min with 3% hydrogen peroxide (H₂O₂). Each section was incubated with blocking serum (Vectastain ABC kit) at room temperature for 30 min, followed by primary mouse anti-ERα antibody (dilution 1:100, C-311, Santa Cruz Biotechnology Co., Ltd.) and mouse anti-ERβ (dilution 1:100, B1, Santa Cruz Biotechnology Co., Ltd.), respectively, overnight at 4 °C. Sections incubated in phosphate-buffered saline (PBS) without antibody served as negative controls. Then objective tissue was covered with secondary antibody (m-IgGκ BP-HRP, dilution 1/50, Santa Cruz Biotechnology Co., Ltd.) to appropriately respond to primary antibody in species, and incubated at room temperature for 50 min. The sections were stained with 3,3’-diaminobenzidine (DAB) (Sigma). The nucleus was stained, and the section was mounted with resin mounting medium. The Image-Pro Plus 6.0 image analysis system was used for quantitative analysis.

Statistical analysis

Data analysis was performed using SPSS 19.0 statistical software, and the results were expressed as `x±s. One-way ANOVA was used for comparison between groups. When the variance was equal, the LSD method was used for comparison among groups. If the variance was not uniform, Dunnett's T3 test method was used for comparison. A p < 0.05 indicated significant difference among the data, and p < 0.01 indicated statistically significant difference among the data.

Experiment 1: HSO can accelerate maturity of virgin mice by upregulating the expression of ERα and ERβ

Animal body mass

There was no significant difference in body mass between different groups (one-way ANOVA, F_3,35 =1.728, p>0.05, Fig 1).

Sexual maturity time, gonad wet weight and gonad coefficient

HSO could advance the vaginal opening time of mice by varying degrees (Table 1). There were significant differences in the gonad wet weight (F_3,36 =13.561, p<0.01, Fig 2A) and gonad coefficient (gonad coefficient = uterus and ovarian wet weight/body weight, F_3,36 =14.006, p<0.01, Fig 2B) between different groups. Compared with the control group, the gonad wet weight and gonad coefficient of the high-dose HSO group were significantly increased (p<0.05).

Uterine and ovarian tissue morphology

In the control group, the uterus was small, the endometrium was thinner and discontinuous, and the uterine cavity was narrow, with sparse glands. Compared with the control group, there was no significant difference in the low-dose HSO group. In the high-dose HSO group, the uterine volume and cavity were significantly increased, the endometrium was thickened, with more glands, and the degree of curvature was high, almost filling the entire uterine cavity (Fig 3). There was a significant difference in endometrial thickness between the different groups measured by microscopic scale (F_3,36 =8.204, p<0.01, Table 2). Compared with the control group, the endometrial thickness of the high-dose HSO group was significantly increased (p<0.01), there was no significant difference between the low-dose groups (p>0.05).

The ovarian growth and development in the control group was normal, and the structure of the follicle was complete and clear. Compared with the control group, the ovary was significantly enlarged in the high-dose HSO group. The number of primordial follicles, primary follicles and secondary follicles were also higher in the high-dose groups. The corpus luteum shape was clear in each group (Fig 4). There were significant differences in the number of primary follicles (F_3,14 =5.629, p<0.05) and secondary follicles (F_3,11 =6.063, p<0.05) between different groups, but the area of ovary (measured with endometrial thickness) was not significant (F_3,20 =2.902, p>0.05, Table 3). Compared with the control group, the number of primary follicles (p<0.01), secondary follicles (p<0.01) and ovary area (p<0.05) in the high-dose HSO groups were significantly increased.

ERα and ERβ expression

The background of immunohistochemical staining sections was colorless or light blue. The immunoreactive substances of ERα and ERβ were brownish yellow and mainly distributed in the cytoplasm, and the nuclei were stained blue by hematoxylin. The control group had weaker brown-yellow staining than the drug-administered group, indicating that the immunohistochemical method had specificity of ERα and ERβ immunoreactivity. For statistical description, according to follicular development, it was divided into early follicles (primordial follicles and primary follicles), intermediate follicles (secondary follicles), and advanced follicles (mature follicles and atresia follicles).

In the control group, there was no positive reaction between ERα (Fig 5) and ERβ (Fig 6) in primordial follicle oocytes; primary follicular oocytes and granulosa cells (GC) showed weak positive reaction (-~+), and membrane cells (TC) showed almost no expression (-); grade follicular oocytes showed a weak positive reaction (-~+), GC showed a positive reaction (+), TC had almost no expression (-); mature follicular cumulus GC stained weakly (-~+), more brown-yellow particles (+), TC showed a weak positive reaction (-~+), and the degenerated GC in the atresia follicle showed a certain positive reaction (+~+++). Compared with the control group, the positive expressions of ERα and ERβ in oocytes and GC cytoplasm of different doses of HSO were increased significantly in the high-dose groups; positive expression in TC cytoplasm showed no significant changes.

There was a significant difference in the average optical density of ERα and ERβ (Tables 4) in early follicle (F_3,12 =33.986, p<0.01, F_3,12 =18.589, p<0.01), intermediate follicle (F_3,12 =12.439, p<0.01, F_3,12 =8.358, p<0.01) and late follicle (F_3,12 =10.032, p<0.01, F_3,12 =12.672, p<0.01) between different groups. Compared with the control group, the average optical density of ERα and ERβ in early follicles, intermediate follicles and late follicles of the high-dose HSO groups were significantly increased, but there was no significant change in the low-dose HSO group.

Serum E₂, FSH and LH content

There were significant differences in E₂ (F_3,24 =10.270, p<0.01), FSH (F_3,26 =5.576, p<0.01) and LH (F_3,24 =4.855, p<0.01) levels among different groups. Compared with the control group, the levels of E₂ (p<0.01) increased significantly and FSH (p<0.05) decreased significantly in the high-dose HSO group (Fig 7).

Experiment 2: HSO can upregulate expression of ERα and ERβ in ovariectomized mice

Body mass

There was no significant difference in the body mass levels of mice between different groups (F_3,37=2.001, p>0.05, Fig 8).

Uterine wet weight and coefficient

There were significant differences in the uterine wet weight (F_3,36=26.029, p<0.01) and gonadal coefficient (F_3,36=31.665, p<0.01) of mice among different groups (Fig 9). Compared with the sham-operation group, the uterine wet weight (p<0.01) and gonad coefficient (p<0.01) of the model group were significantly reduced. Compared with the model group, the uterine wet weight (p<0.01) and gonad coefficient (p<0.01) of the HSO group was significantly increased.

Analysis of uterine morphology

The sham operation group had normal uterine morphology including developed glands, high degree of curvature, and large uterine cavity. However, the model group had smaller uterine volume, almost no uterine gland development, thin endometrium, and narrow uterine cavity. The uterine volume of the HSO group was increased, the glands were increased and developed, and the endometrium was significantly thickened (Fig 10). The endometrial thickness of mice was measured with a ruler under a microscope. The results of univariate analysis of variance showed significant differences in endometrial thickness of mice between different groups (F_3,37=19.491, p<0.01). Compared with the sham operation group, the endometrial thickness of the model group was significantly reduced (p<0.05). Compared with the model group, the endometrial thickness of the HSO group was significantly increased (p<0.05, Table 5).

Analysis of pathological results of spleen

There was no difference in spleen morphology among the groups (Fig 11A), however, there was a significant difference in the spleen wet weight of mice between different groups (F_3,34=4.369, p<0.05, Fig 11B), and a significant difference in the number of Langerhans cells in the spleen (F_3,28=11.902, p<0.01, Fig 11C). Compared with the sham operation group, the spleen wet weight (p <0.01) and the number of Langerhans cells (p <0.01) in the spleen of the model group were significantly reduced. Compared with the model group, the spleen wet weight (p <0.05) and the number of Langerhans cells (p <0.01) in the spleen of the HSO group were significantly increased.

Analysis of hippocampal pathological results

The morphology of the granular cells in the dentate gyrus area of the sham operation group in the 400X visual field was observed to be regular and orderly. Compared with the sham operation group, the dendritic gyrus of the hippocampus in the model group generally showed shrinking of solid cells and deep staining, and the cell body was deformed. Compared with the model group, the granule cells of the dentate gyrus in the HSO group were generally deformed. The layers were arranged neatly, the pyramidal cells were solidified and shrunk, and there was less deep staining. The granular cells of the hippocampal dentate gyrus in the estradiol valerate group were arranged neatly, and the cells were full and round. There was almost no deep staining and shrinkage.

The cells in the hippocampal CA3 area were arranged in the shape of an arc. When the cells were damaged, solidification, shrinkage and deep staining were observed, but the arrangement was loose and divergent toward the radian direction. In the sham operation group, the cells in the hippocampal CA3 region of the mice were arranged neatly and tightly, and the cell bodies were full. Compared with the sham operation group, the cells in the hippocampal CA3 area of the model group had more deep staining, solidification and shrinkage, and the arrangement was loose and irregular. Compared with the model group, the cell body in the HSO group was fuller and arranged, tight, with almost no shrinkage. The estradiol valerate group was full and tightly arranged, with almost no shrinkage (Fig 13).

ERα and ERβ expression

The background of immunohistochemical staining positive sections was colorless or light blue, and the ERα (Fig 13) or ERβ (Fig 14) immunoreactive substances were yellow or brownish yellow. The cytoplasm of positive cells contained yellow or brownish yellow reaction particles, and the nuclei of the cells were stained light blue or blue. The staining of the control group was negative, the background was colorless or light blue, and there was no yellow or brownish yellow staining, indicating that the immunohistochemical method had the specificity of ERα and ERβ immune response. In the sham operation group, obvious positive expression of ERα and ERβ (Tables 6) was observed in mouse uterine glandular epithelial cells, and the positive expression of ERα was stronger than that of ERβ. There was no significant difference in the average optical density of ERα (F_3,31=0.254, p>0.05) in mouse lamina propria cells between different groups. However, there was significant difference in the average optical density of ERβ in mouse lamina propria cells between different groups (F_3,32=3.077, p<0.05). There was significant difference in the average optical density of ERα (F_3,30=16.443, p<0.01) and ERβ (F_3,27=3.366, p<0.05) in uterine gland epithelial cells between different groups. Compared with the sham operation group, the average optical density of ERα (p <0.01) and ERβ (p <0.05) of uterine gland epithelial cells in the model group was significantly reduced. Compared with the model group, the average optical density of ERα (p <0.01) and ERβ (p <0.05) of uterine gland epithelial cells in the HSO group and the estradiol valerate group was significantly increased.

E2, FSH and LH content in serum

There were significant differences in E2 (F_3,28=21.615, p<0.01), FSH (F_3,27=10.588, p<0.01) and LH (F_3,28=6.702, p<0.01) levels among the different groups. Compared with the sham operation group, the E2 level in the model group was significantly reduced, and the FSH and LH levels in the model group were significantly increased. Compared with the model group, the E2 level in the HSO group was significantly increased, and FSH and LH levels were significantly reduced. E2 level in the estradiol valerate group was significantly increased, and FSH and LH levels were significantly reduced (Fig 15).

Phytoestrogens are structurally and functionally similar to estrogens, but have lower side effects compared to synthetic estrogens, so identifying an effective phytoestrogen is important for the prevention and treatment of postmenopausal syndromes [16]. In the present study, we evaluated the estrogenic activity of HPO using an immature and OVX mouse model. HPO showed potent estrogenic activity by stimulating vaginal proliferation, antagonizing target tissue atrophy and estrogen decline in circulation caused by ovariectomy. In addition, HPO could relieve weight increase induced by estrogen decline. The estrogenic activity of HPO may be mediated by stimulating biosynthesis of estrogen and increasing the quantity of ERs in the uterus and vagina.

Natural product such as soybean extract was reported to proliferate the vagina of adult rats [17], and could lower the cancer risk compared to estrogen treatment [18]. The present study provides new evidence suggesting that HSO might be an effective supplement for estrogen in mice.

E2 is a reproductive hormone that plays a vital role in ovary development [19]. In the present study, serum E2 levels were also elevated by HSO (Fig 7), implying that HSO might exert estrogenic effects by increasing the level of E2 and improving the uterine and vaginal tissue nutrition. Moreover, HSO could increase the weight of the uterus and reduce the weight of the adrenal gland in OVX mice (Fig 9). HSO could also improve vaginal wall thickening, mucosal thickening, and relieve atrophy, which was similar to EV groups (Fig 10). Furthermore, HSO could regulate the disturbance in hormonal secretion. The onset of menopause is associated with a dramatic change in hormonal levels, decrease in estrogen, and increase in FSH and LH hormones, which causes permanent amenorrhea [20]. Under physiological condition, estrogen is mainly regulated by the hypothalamic-pituitary-ovarian axis (HPOA) [21]. Hypothalamic secretion of Luteinizing Hormone Releasing Hormone (LHRH) stimulates the pituitary gland to secrete FSH and LH, which in turn promote the secretion of estrogen (E2) from the ovary [22]. Meanwhile, E2 regulates the pituitary secretion of FSH and LH by negative feedback [23]. The increased release of LHRH results in increased secretion of FSH and LH. Hence, the levels of FSH and LH in serum were higher in menopausal female rats [24]. Indeed, we found that HSO could reduce the levels of FSH and LH in serum of OVX mice, while increasing E2 levels (Fig 16). Thus, HSO might relieve the disorders of menopausal syndrome by the HPOA. In addition, the restoration of ER𝛼 and ER𝛽 expression might account for the protective effect of HSO. Estrogen binds to estrogen receptors and exerts its impact on the reproductive system [25]. Meanwhile, ER𝛼 and ER𝛽 modulate the physiological functions of estrogenic compounds by regulating transcription of specific target genes [4]. These two ERs share common physiological roles, for example, in the development and function of the ovaries. Herein, we found that the expressions of ERs were greatly decreased in OVX mice, and HSO could increase the expression of ER𝛼 and ER𝛽 in the uterus and vagina (Fig 14 and 15). ER𝛼 is mainly present in the uterus and ovary, with a more prominent role in the uterus [26]. Hence, the increased vaginal epithelial thickness might result from increased ER𝛼 expression by HSO. However, since ER𝛽 apparently has a more profound effect on the central nervous and immune systems [27], its increased expression might account for the protective effect of HSO in our study.

In summary, the present study provides novel insights into the protective effect of HSO on menopausal syndrome, at morphological and behavioral levels. The results indicated that HSO could be a new food choice for such patients. We would investigate the effects of HSO on the expression of relevant genes and signaling pathways in the future. If the traditional use of HSO for relieving menopausal syndrome or other female aging disorders could be scientifically supported, and progress is made in the analysis techniques, it would increase the potential clinical use of HSO in the future.

Authors’ Contributions

Su Hui Wu and Han-bing Li conceived and designed the study. Chen Yang Guo, Lin Lin Wang and Juan Zhang collected the data and performed the statistical analysis. Gen Lin Li revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are pleased to thank all the teachers of Henan Zhong-jing Recipe and Health Aging Industry Engineering Research Center for technical support.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflicts of interest.

Disclosure

The founding sponsors had no role in the preparation or design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Consent for publication

All of authors consent to publication of this study in journal of Chinese Medicine.

Ethics approval and consent to participate

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (no. 81473435) to Han-Bing Li

Xu Y, Ma XP, Ding J, Liu ZL, Song ZQ, Liu HN, et al. Treatment with qibaomeiran, a kidney-invigorating Chinese herbal formula, antagonizes estrogen decline in ovariectomized rats. Rejuvenation Res 2014; 17 (4): 372-381. doi: 10.1089/rej.2014.1557.
Zhang X, Chen Q, Chen B, Wang F, Chen XH. Herb Formula ZhenRongDan Balances Sex Hormones, Modulates Organ Atrophy, and Restores ERalpha and ERbeta Expressions in Ovariectomized Rats. Evidence-based complementary and alternative medicine : eCAM 2018; 2018 5896398. doi: 10.1155/2018/5896398.
Grady D. Postmenopausal hormones--therapy for symptoms only. N Engl J Med 2003; 348 (19): 1835-1837. doi: 10.1056/NEJMp030038.
Amitay EL, Carr PR, Jansen L, Alwers E, Roth W, Herpel E, et al. Postmenopausal hormone replacement therapy and colorectal cancer risk by molecular subtypes and pathways. Int J Cancer 2020; 147 (4): 1018-1026. doi: 10.1002/ijc.32868.
Rietjens I, Louisse J, Beekmann K. The potential health effects of dietary phytoestrogens. British journal of pharmacology 2017; 174 (11): 1263-1280. doi: 10.1111/bph.13622.
Al-Maharik N. Isolation of naturally occurring novel isoflavonoids: an update. Nat Prod Rep 2019; 36 (8): 1156-1195. doi: 10.1039/c8np00069g.
Wang S, Luo Q, Fan P. Cannabisin F from Hemp (Cannabis sativa) Seed Suppresses Lipopolysaccharide-Induced Inflammatory Responses in BV2 Microglia as SIRT1 Modulator. Int J Mol Sci 2019; 20 (3): DOI:10.3390/ijms20030507. doi: 10.3390/ijms20030507.
Cheng CW, Bian ZX, Zhu LX, Wu JC, Sung JJ. Efficacy of a Chinese herbal proprietary medicine (Hemp Seed Pill) for functional constipation. Am J Gastroenterol 2011; 106 (1): 120-129. doi: 10.1038/ajg.2010.305.
Richard MN, Ganguly R, Steigerwald SN, Al-Khalifa A, Pierce GN. Dietary hempseed reduces platelet aggregation. J Thromb Haemost 2007; 5 (2): 424-425. doi: 10.1111/j.1538-7836.2007.02327.x.
Chen NY, Liu CW, Lin W, Ding Y, Bian ZY, Huang L, et al. Extract of Fructus Cannabis Ameliorates Learning and Memory Impairment Induced by D-Galactose in an Aging Rats Model. Evidence-based complementary and alternative medicine : eCAM 2017; 2017 4757520. doi: 10.1155/2017/4757520.
Yousofi M, Saberivand A, Becker LA, Karimi I. The effects of Cannabis sativa L. seed (hemp seed) on reproductive and neurobehavioral end points in rats. Dev Psychobiol 2011; 53 (4): 402-412. doi: 10.1002/dev.20534.
Wang S, Luo Q, Zhou Y, Fan P. CLG from Hemp Seed Inhibits LPS-Stimulated Neuroinflammation in BV2 Microglia by Regulating NF-kappaB and Nrf-2 Pathways. ACS Omega 2019; 4 (15): 16517-16523. doi: 10.1021/acsomega.9b02168.
Montserrat-de la Paz S, Marin-Aguilar F, Garcia-Gimenez MD, Fernandez-Arche MA. Hemp ( Cannabis sativa L.) seed oil: analytical and phytochemical characterization of the unsaponifiable fraction. J Agric Food Chem 2014; 62 (5): 1105-1110. doi: 10.1021/jf404278q.
Teh SS, Morlock GE. Effect-directed analysis of cold-pressed hemp, flax and canola seed oils by planar chromatography linked with (bio)assays and mass spectrometry. Food Chem 2015; 187 460-468. doi: 10.1016/j.foodchem.2015.04.043.
Frassinetti S, Moccia E, Caltavuturo L, Gabriele M, Longo V, Bellani L, et al. Nutraceutical potential of hemp (Cannabis sativa L.) seeds and sprouts. Food Chem 2018; 262 56-66. doi: 10.1016/j.foodchem.2018.04.078.
Li F, Yang X, Bi J, Yang Z, Zhang C. Antiosteoporotic activity of Du-Zhong-Wan water extract in ovariectomized rats. Pharm Biol 2016; 54 (9): 1857-1864. doi: 10.3109/13880209.2015.1133657.
Carbonel AA, Baracat MC, Simoes RS, Simoes MJ, Baracat EC, Soares JM, Jr. The soybean concentrated extract proliferates the vagina of adult rats. Menopause 2011; 18 (1): 93-101. doi: 10.1097/gme.0b013e3181e5ee25.
Carbonel AA, Calio ML, Santos MA, Bertoncini CR, Sasso Gda S, Simoes RS, et al. Soybean isoflavones attenuate the expression of genes related to endometrial cancer risk. Climacteric 2015; 18 (3): 389-398. doi: 10.3109/13697137.2014.964671.
Gupte AA, Pownall HJ, Hamilton DJ. Estrogen: an emerging regulator of insulin action and mitochondrial function. J Diabetes Res 2015; 2015 916585. doi: 10.1155/2015/916585.
Merrheim J, Villegas J, Van Wassenhove J, Khansa R, Berrih-Aknin S, le Panse R, et al. Estrogen, estrogen-like molecules and autoimmune diseases. Autoimmun Rev 2020; 19 (3): 102468. doi: 10.1016/j.autrev.2020.102468.
Uchida S. Sympathetic regulation of estradiol secretion from the ovary. Auton Neurosci 2015; 187 27-35. doi: 10.1016/j.autneu.2014.10.023.
Christensen A, Bentley GE, Cabrera R, Ortega HH, Perfito N, Wu TJ, et al. Hormonal regulation of female reproduction. Horm Metab Res 2012; 44 (8): 587-591. doi: 10.1055/s-0032-1306301.
Berga S, Naftolin F. Neuroendocrine control of ovulation. Gynecol Endocrinol 2012; 28 Suppl 1 9-13. doi: 10.3109/09513590.2012.651929.
Butler L, Santoro N. The reproductive endocrinology of the menopausal transition. Steroids 2011; 76 (7): 627-635. doi: 10.1016/j.steroids.2011.02.026.
Rettberg JR, Yao J, Brinton RD. Estrogen: a master regulator of bioenergetic systems in the brain and body. Front Neuroendocrinol 2014; 35 (1): 8-30. doi: 10.1016/j.yfrne.2013.08.001.
Hamilton KJ, Hewitt SC, Arao Y, Korach KS. Estrogen Hormone Biology. Current topics in developmental biology 2017; 125 109-146. doi: 10.1016/bs.ctdb.2016.12.005.
Fan X, Xu H, Warner M, Gustafsson JA. ERbeta in CNS: new roles in development and function. Prog Brain Res 2010; 181 233-250. doi: 10.1016/S0079-6123(08)81013-8.

Table 1 The effect of HSO on the Opening rate of vagina mice

Group	Dosage (g/kg)	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8
Control		0	0	16.67%	25%	41.67%	58.33%	75.00%	100%
HSO	2.70	0	0	18.18%	54.55%	72.73%	81.82%	90.91%	100%
HSO	5.40	0	0	33.33%	58.33%	83.33%	100%	100%	100%
EV	0.17 mg/kg	0	8.33%	45.45%	81.82%	100%	100%	100%	100%

Table 2 The effect of HSO on Endometrial Thickness and Uterus Gland Development in vagina mice

Group	Dosage (g/kg)	endometrial thickness (μm)	uterine glands
Control		881.81±164.30	+
HSO	2.70	958.38±126.51	++
HSO	5.40	1196.90±237.77^**	+++
EV	0.17mg/kg	1330.30±268.20^**	+++

Note: Data are expressed as mean ± SD. One-way ANOVA, ^**P<0.01 versus control. Use the number of + to represent the degree of development of the uterus glands.

Table 3 Effect of HSO on follicular development in vagina mice

Group	Dosage (g/kg)	Number of primordial and Primary follicle	Number of secondary follicle	Area of Ovarian section (mm²)
Control		7.60±4.16	4.00±2.00	2.96±0.44
HSO	2.70	13.67±3.21	7.67±1.53	3.01±0.12
HSO	5.40	17.83±5.42^**	10.20±2.28^**	3.55±0.62^*
EV	0.17 mg/kg	11.00±2.16	8.50±1.91^*	3.62±0.50^*

Note: Data are expressed as mean ± SD. One-way ANOVA, ^*P<0.05,^**P<0.01 versus control.

Table 4 Image analysis of ERα and ERβ positive substance distribution and change of staining intensity in follicles of different maturity treated with HSO in vagina mice

Ovarian Follicle Development		Dosage (g/kg)	Positive cell amount（C±S_c）	Mean optical density（`x±s）	Staining intensity
Ovarian Follicle Development		Dosage (g/kg)	Positive cell amount（C±S_c）	Mean optical density（`x±s）	Oocytes	Granular cells	Theca cells
ERα	Early follicular phase
	Control		35.00±13.02	0.09±0.03	-～+	-～+	-
	HSO	2.70	37.90±7.43	0.14±0.02	-～+	-～+	-
	HSO	5.40	64.50±14.50^*	0.20±0.04^*	-～+	+	-～+
	EV	0.17 mg/kg	72.00±16.15^**	0.36±0.06^**	++	++	+
	Intermediate follicular phase
	Control		196.00±24.98	0.18±0.04	-～+	+	-
	HSO	2.70	247.67±22.37	0.24±0.05	-～+	+	-
	HSO	5.40	397.00±25.60**	0.33±0.08^*	++	++	-
	EV	0.17 mg/kg	390.33±30.48**	0.46±0.08^**	++	+++	+
	Late follicular phase
	Control		356.67±31.78	0.22±0.04	-～+	+	-～+
	HSO	2.70	397.00±17.09	0.23±0.03	-～++	+	-～+
	HSO	5.40	440.33±25.19*	0.34±0.06	++	++	-～+
	EV	0.17 mg/kg	443.33±23.52*	0.48±0.12^**	+++	+++	++
ERβ	Early follicular phase
	Control		51.29±19.30	0.10±0.03	-～+	-～+	-
	HSO	2.70	54.00±20.06	0.09±0.03	-～+	-～+	-
	HSO	5.40	93.38±23.16^**	0.30±0.06^**	-～+	++	-～+
	EV	0.17 mg/kg	96.67±22.30^**	0.38±0.09^**	+～++	+++	-～+
	Intermediate follicular phase
	Control		443.00±173.03	0.16±0.03	-～+	+	-～+
	HSO	2.70	434.75±210.82	0.17±0.01	-～+	+	-～+
	HSO	5.40	685.25±186.01	0.30±0.08^**	-～++	++	-～+
	EV	0.17 mg/kg	782.33±128.88^*	0.31±0.04^**	++～+++	+++	-～++
	Late follicular phase
	Control		357.33±137.23	0.24±0.07	-～+	+	-～+
	HSO	2.70	364.00±149.08	0.23±0.06	-～++	+	-～+
	HSO	5.40	498.00±188.05	0.42±0.06^**	++～+++	+++	+
	EV	0.17 mg/kg	574.67±62.93	0.40±0.04^**	+++	+++	+

Note: Data are expressed as mean ± SD. One-way ANOVA, ^*P<0.05,^**P<0.01 versus control. Use the number of “+” signs represents the degree of development of different follicular cells.

Table5 The effect of HSO on Endometrial Thickness and Uterus Gland Development in Ovariectomized mice

Group	Dosage (g/kg)	Endometrial Thickness (μm)	Uterine Glands
Sham		1061.63±210.84^*	++～+++
Model		759.30±193.79	+
HSO	5.40	1036.64±175.15^*	++～+++
EV	0.17 mg/kg	1320.15±94.78^**	+++

Note: Data are expressed as mean ± SD. One-way ANOVA, ^*P<0.05,^**P<0.01 versus control. Use the number of “+” signs represents the degree of development of the uterus glands.

Table 6 The effect of HSO on the Mean optical density of ERα and ERβ in Endometrial Cells of Ovariectomized mice

Group	Dosage (g/kg)	Mean optical density of Lamina propria	Mean optical density of Uterine glands
ERα
Sham		0.21±0.04	0.31±0.05^**
Model		0.20±0.06	0.21±0.03
HSO	5.40	0.21±0.03	0.30±0.01^**
EV	0.17 mg/kg	0.20±0.03	0.28±0.03^**
ERβ
Sham		0.19±0.04	0.15±0.03^*
Model		0.20±0.04	0.11±0.03
HSO	5.40	0.23±0.05	0.14±0.02^*
EV	0.17 mg/kg	0.24±0.03	0.15±0.02^*

Note: Data are expressed as mean ± SD. One-way ANOVA, ^**P<0.01 versus control.

reportingchecklist.pdf

Download PDF

Version 1

posted

You are reading this latest preprint version

Hemp seed oil can accelerate the virgin mice mature and balance sex hormones, modulates organ atrophy, and restores in ovariectomized mice through upregulates ER𝛼 and ER𝛽 expressions

Status:

Version 1

Abstract

Figures

Background

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Supplementary Files

Status:

Version 1