Part 2:Echinacoside Increases Sperm Quantity in Rats By Targeting The Hypothalamic Androgen Receptor
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Zhihui Jiang1,2, Bo Zhou2, Xinping Li2, Gordon M. Kirby3 & Xiaoying Zhang1,2
Experiment treatment.
Experiment 1. Mice were randomly divided into six groups, seven mice in each group (n = 7). ECH(Echinacoside from cistanche) (CAS: 82854-37-3; Chengdu Preferred Biotechnology Co., Ltd, Sichuan, China) was administered using an intragastric tube, and testosterone propionate (TP, CAS: 57-85-2; KingYork, Tianjin, China) were given by intramuscular injection once daily for 14 days as per the following experimental design: Control group: normal saline (10 mL/kg), ECH(L) group (5 mg/kg), ECH(M) group (20 mg/kg), ECH(H) group (80 mg/kg), TP (15 mg/kg) and enzalutamide (AR inhibitor, once daily every other day for 14 days; 20 mg/kg; CAS: 915087- 33-1; Aladdin, Shanghai, China).
After 2 weeks of treatments, the mice were anesthetized with diethyl ether to collect blood samples for analyses of hormone levels. The mice were then dissected to separate the hypothalamus, encephalon + pituitary, testis and cauda epididymis. Samples of cauda epididymis were put in normal saline with 5% BSA for sperm quality assessment, and the hypothalamus, encephalon + pituitary and testes were frozen in liquid nitrogen and stored at−80 °C for further investigations.
Experiment 2. Mice were randomly divided into 10 groups, five animals per group. ECH (20 mg/kg) was administered orally and the mice in each group were anesthetized with diethyl ether at the time points 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 9 h and 12 h after administration for sample collection.
Plasma was collected and the heart was exposed. Perfusion of normal saline into the left ventricles was conducted by an infusion apparatus until the liver and lungs were blanched. Then the hypothalamus and testis were collected to determine the tissue concentrations of ECH(Echinacoside from cistanche).
Experiment 3. Mice were randomly divided into 4 groups, seven in each group. ECH(Echinacoside from cistanche) and BPA (CAS: 80-05-7; Aladdin, Shanghai, China) were administered using an intragastric tube once daily for 6 weeks as per the follow- ing experimental design: normal group (corn oil, 10 mL/kg, bw/d), model group: BPA group (BPA 200 mg/kg, bw/d, corn oil), and experimental group: BPA + ECH group (BPA 200 mg/kg; ECH 20 mg/kg).
After 42 days of treatment, the mice were anesthetized with diethyl ether and the blood samples were collected for hormone level analyses. The testis and cauda epididymis were separated and collected. The cauda epididymis was used for sperm quality assessment and the testis was frozen in liquid nitrogen and stored at −80 °C for further investigation.
Figure 7. Pathways regulated by selected HPG axis-related hormone and protein in male reproduction as predicted by Pathway Ontology Database and KEGG Pathway Database.
Notes: Regulated processes are represented by backgrounds of different colors and shapes, and regulatory events are displayed using arrows and lines.
The references linked to each protein were based on Pathway Ontology Database and KEGG Pathway Database, and are not listed.
Determination of sperm quality. Sperm suspension preparation. Cauda epididymides were minced into 5 mL of normal saline with 5% BSA and incubated for 5 min at 37 °C to allow their contents to spread into the medium.
Sperm number: As per the method described by Yokoi (2003), the diluted sperm suspension (1:10; v/v; sperm suspension/10% methanol) was transferred to each counting chamber of a hemocytometer and was allowed to stand for 5 min and then counted under a light microscope (Nikon, Instruments Inc., Japan) at X200 magnification.
Sperm viability: A total of 20 μL of sperm suspension was mixed with an equal volume of eosin-nigrosin stain for 2 min; the un-stained sperms were counted under a light microscope at 200x magnification.
Sperm motility: A total of 10 μL of sperm suspension was put onto a glass slide and were recorded as either mobile or immobile under a light microscope at 200x magnification.
Determination of hormone levels. Levels of testosterone (T) and LH were quantified in serum, encephalon + pituitary and testis homogenates using radioimmunoassay (RIA) kits (Beijing Sino-UK Institute of Biological Technology, Beijing, China). Briefly, anti-testosterone or anti-LH IgG antibody (100 μL) and 125-I-conjugated anti-mouse antibody were added to samples or the standard (100 μL), and mixed on a rocker overnight at 4 °C. After washing three times with PBS-Tween 20 (500 μL), the mixtures were centrifuged (3500 rpm/min) at 4 °C for 15 min. The CPM values of the precipitates were assessed by radioimmunoassay instrument (Beijing Sino-western Technology Co. Ltd, CN202M/KZ4GC-1200, Beijing, China) and the concentrations of T and LH were calculated according to the formula of a standard curve. The T and LH coefficient of variation is 2.8% and 3.2% in sample groups, and 2.1% and 2.8% in standard groups, respectively.
Determination of gene expressions by real-time quantitative PCR. Total RNA was isolated from frozen testicular and encephalon + pituitary tissues using an RNA Simple Total RNA kit (Tiangen, Beijing, China). Quantitative real-time PCR (q RT-PCR) was carried out for the amplification of cDNA using 2 × SYBR Green I PCR Master Mix (Vazyme, Nanjing, China). The PCR procedure consisted of 95 °C for 30 seconds followed by 35 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds and 72 °C for 30 seconds. The melting curve analysis was performed on the PCR products to verify primer specificity and product purity. A dissociation curve was performed for each plate to confirm the production of a single product. The relative abundance of each mRNA was calculated. The PCR primers used for the study are shown in Table 2.
Western blot. For AR expression analysis, the extraction and isolation of cytoplasmic and nuclear protein were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. The concentrations of cytoplasmic and nuclear proteins were assessed using a Bradford Protein Assay Kit (Beyotime, Jiangsu, China). Protein samples (80 μg) were run on a 12% and 5% SDS-PAGE gel and transferred onto PVDF membranes. After blocking, the membranes were incubated with anti-AR IgG antibodies (1:1,000; Bioss; Beijing China), and mouse polyclonal anti-GAPDH antibodies (1:1,000; Wuhan Boster Biological Technology, Wuhan, China) or mouse polyclonal anti-Lamin b1 antibodies for 2 h. The membrane was washed three times with TBST and incubated with an HRP-conjugated rabbit anti-mouse IgG antibody (1:5,000; Bioss; Beijing China). The signal was visualized using the ChemiDoc Imaging System (Tanon-3,500, Shanghai, China).
High-Performance Liquid Chromatography (HPLC) assay. Blood was collected in heparinized glass tubes. The plasma was separated by immediate centrifugation at 6,000 rpm for 10 min and stored at −20 °C for further experiment. The hypothalamus and testis samples from each time point were pooled and homogenized with methanol. After centrifugation at 10,000 rpm at 4 °C for 10 min, the supernatant was concentrated by N2 and the residue was dissolved in 50 μL of methanol and filtered through a 0.45 μm flter. Ten μL of the sample filtered liquid was injected into the HPLC system for analysis.
The standard curve consisted of samples containing 50, 100, 250, 500 and 750 ng/mL of the ECH(Echinacoside from cistanche) (Chengdu Pufei De Biotech Co., Ltd, Sichuan, China). Plasma quality control samples spiked with 75ng/mL (low), 150 ng/ mL (medium) and 300 ng/mL (high) of the ECH were accordingly prepared to measure the accuracy and precision of the method.
Chromatography was performed with an HPLC system (D2000 Elite series, Hitachi, Japan) coupled to a UV detector (L-2400, Hitachi, Japan). Separation was performed on a Thermo-C18 (250 mm × 4.6 mm i.d., 4.6 μm particles) column kept at 25 °C. The mobile phase was a gradient prepared from 0.1% phosphoric acid containing 0.04% trimethylamine (component A) and methanol (component B). The linear gradient was as follows: 70–90% A over 0-2 min, 60–70% A over 2–6 min, 55–60% B over 6–8 min and then returned to 90% A at 8 min immediately. The flow rate was 0.8 mL/min. The UV detector was operated at 332 nm. The Peak area was evaluated as the analytical measurement.
Pharmacokinetic studies in mice. The penetration of ECH(Echinacoside from cistanche) to the hypothalamus and testis was subjected to pharmacokinetic analysis with Drug and Statistics software (Drug and Statistics, Mathematical Pharmacology Professional Committee of China). Pharmacokinetic parameters were determined using the non-compartmental method based on statistical moment theory. The preliminary pharmacokinetic parameters including the time to peak constant (Tmax), peak concentration (Cmax), elimination rate constant (Ke), elimination half-life (T0.5), the area under the curve (AUC0–12), apparent distribution volume (Vc), and clearance (CL) were obtained for further analysis. Brain/plasma ratios were calculated based on AUC0-t values for plasma and brain.
ECH-Ovalbumin (ECH-OVA) synthesis and identification. A total of 9.8 mg of ECH(Echinacoside from cistanche) and 1.0 mg of butanediol anhydride were dissolved in 2 mL pyridine, mixed for 12 h with stirring at room temperature. The mixture was concentrated by N2 and the residue was combined with 10.8 mg of N-hydroxysuccinimide (NHS) and 19.3 mg of dicyclohexylcarbodiimide (DCC) and dissolved in 4 mL N, N-dimethylformamide (DMF) for 12 h with stirring at room temperature. After centrifugation at 2,000 g for 5 min at 4 °C, the supernatant was added to 5 mL of PBS in which 14.4 mg ovalbumin (OVA) was dissolved. The new mixture was stirred for 24 h at 4 °C. The reaction solution was then dialyzed against PBS for three days. The presence of ECH-OVA was confirmed by UV spectra (Shimadzu Scientifc Instruments, Inc. Columbia, MD USA) at a wavelength ranging from 190 to 400 nm as well as by denaturing PAGE.
Indirect ELISA (iELISA). A microtiter plate was coated with the ECH-OVA (2 μg/100 μL) and incubated overnight at 4 °C. The plate was washed three times with PBST and two times with PBS; and 5% PBSM (PBS containing 5% skimmed milk) was added to block the unbound sites at 37 °C for 2 h. After the plates were washed with PBST and PBS, wells were divided into 4 groups; experimental group, 1 μg AR total protein/100 μL; positive group, rabbit anti-OVA antibody (1:1,000); negative group, 1 μg of bovine serum albumin; and a blank group containing PBS. After incubation for 1.5 h, the plates were washed, and 100 μL/well of rabbit HRP-conjugated IgG (1:1,000) was added to the positive group, 100 μL/well of anti-AR was added (1:1,000) to the other groups. After incubation for 1.5 h, rabbit HRP-conjugated IgG (1:5,000) dilution was added to the plates. After incubation at 37 °C for 1 h, the plates were washed three times with PBST and two times with PBS. TMB was then added and incubated for 10 min in the dark at room temperature and 2 M H2SO4 was added to stop the reaction. The absorbance was read at a wavelength of 450 nm.
Molecular docking. A molecular docking study was performed to investigate the binding mode of the compound ECH(Echinacoside from cistanche) to the human androgen receptor (AR) using Autodock vina 1.1.2 (http://vina.scripps.edu). The three-dimensional (3D) structure of AR (PDB ID: 2YHD) was downloaded from Protein Data Bank (http://www. rcsb.org/pdb/home/hone.do). The 3D structure of ECH was obtained by ChemBioDraw Ultra 14.0 and ChemBio 3D Ultra 14.0 software. The AutoDockTools 1.5.6 package (http://mgltools.scripps.edu) was employed to generate the docking input files. The search grid of AR was identified as center x: 36.141, center y: 8.513, and center z:
21.304 with dimensions size x: 15, size y: 15, and size z: 15. The value of exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMOL 1.7.6 software (http://www.pymol.org).
Data analysis and statistical methods. The data were analyzed using statistical software SPSS 19.0 (SPSS Inc., Chicago, IL, USA). A one-way ANOVA was employed for comparison among the groups. Tukey’s comparison tests of significant differences among groups were determined. The results were expressed as mean ± standard deviation (SD) using Graph Pad Prism software v.7 (GraphPad Software, Inc, California, USA).
Ethics approval and consent to participate. Ethical approval for this study was obtained from the Ethics Committee of Anyang Institute of Technology.
References
1. Cano, S. N., Misra, M. & Ackerman, K. E. Exercise, training, and the hypothalamic-pituitary-gonadal axis in men and women.Front. Horm. Res. 47, 27–43 (2016).
2. Papadopoulos, V. & Miller, W. L. Role of mitochondria in steroidogenesis. Best Pract. Res. Cl. En. 26, 771–790 (2012).
3. Rone, M. B. et al. Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to a steroid hormone. Mol. Endocrinol. 26, 1868–1882 (2012).
4. Shibari, S., Ylonen, H. & Palme, R. Excretion and measurement of corticosterone and testosterone metabolites in bank voles (Myodes glareolus). Gen. Comp. Endocrinol. 243, 39–50 (2017).
5. O’Hara, L. et al. Pituitary androgen receptor signaling regulate prolactin but not gonadotrophins in the male mouse. PloS one. 10, e0121657 (2015).
6. Amador, A. G., Parkening, T. A., Beamer, W. G., Bartke, A. & Collins, T. J. Testicular LH receptors and circulating hormone levels in three mouse models for inherited diseases (Tfm/y, lit/lit and hyt/hyt). Endocrinol. Exp. 20, 349–58 (1986).
7. Bulldan, A., Dietze, R., Shihan, M. & Scheiner-Bobis, G. Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells. Cell. Signal. 28, 1075–85 (2016).
8. Walker, W. H. Testosterone signaling and the regulation of spermatogenesis. Spermatogenesis. 1, 116–20 (2011).
9. Cao, C. & Kindscher, K. The Medicinal Chemistry of Echinacea Species (ed. Kindscher, K.) 127–145 (Springer, 2016).
10. Jiang, Z. H., Jian, W., Li, X. P. & Zhang, X. Y. Echinacoside and Cistanche tubulosa (Schenk) R. Wight ameliorate bisphenol A-induced testicular and sperm damage in rats through gonad axis regulated steroidogenic enzymes. J. Ethnopharmacol. 193, 321–328 (2016).
