The aphrodisiac potential of β-cyclodextrin–curcumin via stimulating cAMP-PKA pathway in testicular Leydig cells

The water-soluble β-cyclodextrin–curcumin (CDC) is used in pharmaceutical applications and as a natural food colorant. The previous study revealed that curcumin potentially impacted the reproductive system. The present study investigated the possible roles of the CDC in testosterone secretion in Leydig cells and mice. Primary Leydig cells were treated with the CDC to determine their effect on cell proliferation, testosterone levels, the protein and mRNA expression of the transcription factor, and steroidogenic enzymes. Our data showed that CDC stimulated testosterone production via upregulating transcription factor steroidogenic factor-1 (NR5A1), cAMP-response element-binding protein (CREB), and steroidogenic enzymes steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (CYP11A1), 17-alpha-hydroxylase/17,20-lyase (CYP17A1), 3β-/17β-hydroxysteroid dehydrogenase type 1 (3β/17β-HSD, HSD3b1/HSD17b1). CDC could significantly stimulate H89-suppressed StAR and CREB expression but not reverse melatonin-suppressed StAR expression. We further detected the hormonal activity with transgenic yeast, and CDC showed potential androgenic antagonistic activity. Meanwhile, we investigated its aphrodisiac effect on hydrocortisone-induced mice. Exposure to hydrocortisone decreased the mating ability, reproductive organs, and testosterone level and disrupted testicular histology. However, all of these effects were significantly improved by CDC treatment. In conclusion, these results indicated that mechanisms of CDC in stimulating testosterone production involve upregulating the cAMP-PKA pathway.

www.nature.com/scientificreports/ pyruvate was superior to glucose as an energy source to support steroidogenesis 21 . Then, the dispersed cells were washed with DMEM/F12 and layered over a Percoll gradient (5%, 30%, 58%, and 70%; Biosharp, Wuhan, China). The gradient was centrifuged for 35 min at 800g, and cells localized between Percoll gradient 70 and 58% were isolated (the second layer). After the repeating wash steps of the medium, the Leydig cells were incubated in the DMEM-F12 culture medium. Cell viability determined by the trypan blue test was more than 90%. The purity of Leydig cells was determined by 3β-HSD histochemical staining 22 . Leydig cells were incubated in the 24-well plates with a 0.4 mL/well 3β-HSD staining solution. The staining solution contained 0.01 M PBS supplemented with 0.1 mg/mL nitro-blue tetrazolium (Biosharp, Wuhan, China), 1.0 mg/mL nicotinamide adenine dinucleotide (Shanghai McLean), 0.1 mg/mL dehydroepiandrosterone (Shanghai McLean) and 0.1 mg/ mL niacinamide for 90 min at 34 °C. The positive cells were stained a dark blue, and the purity of the Leydig cells was over 90%.
Purified Leydig cells (3 × 10 4 /mL) were plated into 24-well plates and cultured at 34 °C in a humidified atmosphere containing 5% CO 2 -95% air. Two-day cultured Leydig cells grown in the DMEM/F12 culture medium were washed three times in PBS. Then the cells were cultured in a serum-free medium containing different doses of curcumin or CDC for 24 h. 1 IU/mL of human chorionic gonadotrophin (hCG) was used as a positive control. The cytotoxicity assay of control and treated cells was tested by MTT assay by the reported method 23 .
Measurement of testosterone production. The testosterone concentration in the cell-free culture medium was measured using ELISA assays according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
RT-qPCR (real-time reverse transcription-polymerase chain reaction). The mRNA expression levels of the Star, Nr5a1, Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b1 in Leydig cells were analyzed using RT-qPCR analysis. The total RNA of different treatment groups was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and their quantity and purity were measured by an ultra microspectrophotometer (Thermo Fisher Scientific, USA) based on the absorbance measurement at 260 and 280 nm. The purified RNA was reverse transcribed using a FastQuant RT kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. RT-qPCR was performed in a LightCycler 480 instrument (Roche, Basel, Switzerland) using the TIANGEN SuperReal PreMix Plus kits (Tiangen Biotech, Beijing, China) with specific primers (Table 1). PCR amplification was initiated by 15 min of denaturation at 95 °C, and then followed by 45 cycles of 95 °C for 10 s, 60 °C for 40 s and 72 °C for 32 s, and a final incubation at 75 °C for 5 min. Analyses were performed using the 2-ΔΔCq method 24 , and Gapdh was used as an internal control.

Yeast estrogen screen (YES) and yeast androgen screen (YAS) assay. The XenoScreen YES/YAS
test was performed on 96-well plates to determine hormonally active substances [25][26][27] . The oestrogenic, antioestrogenic, androgenic, and anti-androgenic activities were measured with the XenoScreen XL YES/YAS Assay kit according to the manufacturer's protocol (Xenometrix, XenoScreen XL YES/YAS, Instructions for Use Version 3.04). Growing yeast cells (Saccharomyces cerevisiae) were stably transformed with either hER or hAR and a β-galactosidase reporter system and were exposed to different concentrations of compounds. 17β-Estradiol (E2) and 4-hydroxytamoxifen (4-HT) standards were used as the estrogen agonist and antagonist controls, respectively. For androgen activity, 5α-dihydrotestosterone (DHT) was used as agonist control and flutamide (FL) as . Female mice were brought into estrous by administering estradiol valerate (0.4 mg/kg) 48 h and 6 h before the copulatory study. After 30 daily doses, the female was then introduced into the chamber, and the following sexual behavior parameters were recorded: mount frequency (MF), the number of mounts without intromission from the introduction of the female to ejaculation; intromission frequency (IF): the number of intromissions from the introduction to ejaculation; mount latency (ML): the interval between the introduction and the first mount by the male; intromission latency (IL): the interval from the introduction to the first intromission by the male 28 . Under ethyl ether anesthesia, blood samples were collected from the orbital plexus, and the major organs were then collected. Meanwhile, the testes were fixed in 10% neutral buffered formalin, and the slide sections were stained with hematoxylin and eosin (HE) for light microscopic examination. The testosterone concentration in the mouse serum was detected using the ELISA kits.
Statistical analysis. Data were presented as the mean ± standard error of the mean. Differences between means were assessed using the one-way analysis of variance followed by Dunnett's t-test with GraphPad Prism 8. P < 0.05 or P < 0.01 were considered to indicate a statistically significant difference.

Results
Leydig cell viability. The 3β-HSD and trypan blue staining showed that Leydig cells were successfully isolated from testes ( Fig. 1), with an average of 96.23% viability and an approximate purity of 90%. MTT results showed that 50 μM and 100 μM curcumin and CDC significantly reduced the cell viability compared to untreated control. However, there were no apparent differences at lower concentrations, with ≤ 25 μM CDC or curcumin (Fig. 1). Therefore, the subsequent experiments were performed in concentrations ranging from 0.1 to 25 μM.
Testosterone production. Exposure to 1 IU/mL hCG resulted in a significant increase in levels of testosterone production in rat Leydig cells. Similarly, the CDC played an excellent role in increasing testosterone secretion ranging from 0.2 to 25 μM (Fig. 2), and 5 μM CDC showed the highest effect on testosterone secretion. Besides, curcumin had a somewhat inhibitory impact on steroidogenesis, but there was no significant difference between control and regular curcumin treatment (1-25 μM).
Changes in mRNA expression levels of transcription factor and steroidogenic enzymes. The above results suggested that the CDC might regulate the expression levels of steroidogenic enzyme genes to promote testosterone secretion in Leydig cells. Therefore, the mRNA expression levels of Nr5a1 and steroidogenic enzymes (Star, Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b1) were analyzed by RT-qPCR. As shown in Fig. 3, cells exposed to hCG and CDC (1 μM or 5 μM) demonstrated significantly higher expression levels of Nr5a1 and steroidogenic enzymes compared to controls (P < 0.05 or P < 0.01). Therefore, the results suggested that the CDC could promote testosterone synthesis by regulating the transcription of the Nr5a1 gene and activating the expression of steroidogenic enzyme genes.
Changes in transcription factors and steroidogenic proteins. We next tested if the CDC could affect transcription factors and steroidogenic proteins. Figure 4 and Supplementary Figures showed that hCG www.nature.com/scientificreports/ and CDC (1 μM or 5 μM) increased the protein expression of NR5A1, CREB, 3β-HSD, and StAR compared to controls (P < 0.05 or P < 0.01). H89 is a selective and strong inhibitor of cAMP-activated protein kinase A (PKA) via competitive antagonists of the ATP site on the PKA catalytic subunit 29 . Melatonin, an indolamine neurohormone, can decrease testicular androgen synthesis and the size of testes through melatonin subtype 1a (mel1a) receptors and the local corticotrophin-releasing hormone system 30 . These data indicate that 1-h pretreatment with H89 resulted in a significant diminution in the protein expression of CREB and StAR compared with their expressions in control cells (P < 0.01) (Fig. 5) and Supplementary Figures. Treatment with hCG or 0.2-5 μM CDC restored the H89-inhibited CREB and StAR levels (P < 0.05 or P < 0.01). Similarly, the addition of melatonin to Leydig cells prevented the StAR level (P < 0.05) compared to control. HCG could recover melatonin-inhibited StAR protein expression (P < 0.01), but the CDC had no significant effect on it (P > 0.05).
XenoScreen YES/YAS assay. The CDC interacting with hER or hAR was assessed through XenoScreen XL YES/YAS assay. The results revealed no apparent cytotoxicity under the testing concentrations of 100 μM. As shown in Fig. 6, CDC showed androgenic antagonistic activity as flutamide. The IC50 values of flutamide and CDC were 1.05 × 10 -5 M and 7.00 × 10 -8 M, respectively. However, no estrogenic agonistic, androgenic agonistic, and estrogenic antagonistic potential of CDC was observed below 100 μM ( Table 2).
Effect of CDC on sexual behavior, hormone levels, and organ coefficient. Compared with the blank control group, hydrocortisone treatment decreased the copulatory performance of sexually experienced male mice: mount and intromission latencies were significantly increased (P < 0.05), and mount and intromission frequency were significantly reduced (P < 0.05) (Fig. 7). Compared with the model animal group, CDC (in medium and high doses) and JKSQP groups reduced the mount and intromission latencies while increased mount and intromission frequency significantly (P < 0.05 or P < 0.01). The hydrocortisone administration exhibited a decrease in serum testosterone concentration (P < 0.01), while after CDC or JKSQP resulted in a significant increase (P < 0.01) (Fig. 8).  www.nature.com/scientificreports/ The weight of testes and epididymis showed a significant decrease in the hydrocortisone-treated mice compared to the control group (P < 0.05) (Fig. 9). A significantly increased weight of testes and epididymis was observed in CDC groups compared with hydrocortisone-treated mice (P < 0.05 or P < 0.01).

Effect of CDC on histological changes in testicular tissue.
Histological changes in mouse testis are shown in Fig. 10. The control mice exhibited a normal process of spermatogenesis with a regular arrangement of the spermatogenic epithelium in the seminiferous tubules. The model group showed various testicular changes, including the loss, and disorder of the spermatogenic cells, the decrease of spermatogenesis, as well as sloughing in the cytoplasm of Sertoli cells. However, the tubules in the CDC (in medium and high doses) and JKSQP groups restored their regular shape with comparatively well-organized spermatogenic layers and a moderate amount of sperm. These groups also showed histologic characteristics similar to those of the control group.  www.nature.com/scientificreports/

Discussion
Curcumin administration is hampered by its low solubility and stability in water. The water-soluble curcumin complex with β-CD is used in pharmaceutical applications and as a natural food colorant 31 . Although the hydrophilic curcumin from different companies varied in curcumin content, we found they had a similar effect on rat testicular Leydig cells. However, the regular curcumin was not as sensitive as CDC, and it had no significant effect on Leydig cells. These results were similar to the reported that curcumin had no impact on the basal steroidogenesis in MA-10 cells, but it could inhibit the steroidogenesis in primary mouse Leydig cells 32 . Meanwhile, the 5 μM CDC showed higher testosterone levels, and the subsequent experiments were performed in concentrations ranging from 0.1 to 5 μM.
To explore the aphrodisiac effect of CDC in vivo, we used hydrocortisone to establish the Kidney-Yang deficiency model. The metabolic pathways and endocrine and reproductive systems were disturbed by hydrocortisone 33 . Compared with the control group, the subcutaneous hydrocortisone injection in mice could lead to Kidney-Yang deficiency, evidenced by prolonged sexual arousal, reducing the number of sexual encounters, the reproductive organs' indexes, and testosterone concentration, as well as pathological damage to the testicular tissue structure. After the administration of the CDC, mice mating ability with impotence, reproductive organ weights, testosterone level, and pathological damage was significantly improved. These indicated that the CDC had an aphrodisiac effect on a hydrocortisone-induced mice model of Kidney-Yang deficiency.
Testosterone is synthesized from cholesterol in a multi-step enzymatic response to pituitary hormones. Our data showed that CDC stimulated testosterone production via cAMP/PKA signaling pathways, upregulating transcription factors (NR5A1 and CREB), and steroidogenic enzymes (StAR, CYP11A1, CYP17A1, and 3β/17β-HSD). Steroidogenesis is mediated in cAMP/PKA-dependent and -independent signaling pathways 34 . H89 could down-regulate StAR expression via selective inhibiting PKA activity, while melatonin has a significant protective and regulatory role in Leydig cells. The effects of melatonin on the levels of reproductive hormones depend   www.nature.com/scientificreports/ on physiological conditions and species of animals 35 . Melatonin inhibits StAR, GATA-4/SF-1, or other protein expressions through specific binding sites by blocking StAR protein expression without altering the activity of the P450 enzyme [36][37][38] . Meanwhile, melatonin promotes male reproductive performance and increases testosterone synthesis in mammalian Leydig cells 39 . The previous study reported that the effect of melatonin on oxytocin and vasopressin release could not entirely be blocked by H89 40 , and it regulated CRE-dependent gene transcription underlying osteoblast proliferation via activating Src and PKA in parallel 41 . Our results demonstrated that the CDC could significantly upregulate H89-suppressed StAR and CREB expression and could not reverse melatonin-suppressed StAR expression. This result was strengthened by previous evidence that determined H89 abolished the effect of curcumin treatment to increase phosphorylation of LKB-1 and CREB 42 . Thus, the CDC seemed to stimulate steroidogenesis via the cAMP/PKA signaling pathway, not affected by the cAMP synthesis inhibitor. The present study further showed that the CDC had androgenic antagonistic activity on transgenic yeast without estrogenic agonistic, androgenic agonistic, and estrogenic antagonistic potential. This means that the CDC might exert two-way regulation in the biological effects of hormones in the body. It is an androgen receptor antagonist with potential as an anti-prostate cancer agent 43 and could also stimulate androgen production through the steroidogenic pathway.

Conclusion
The in vitro study showed that the CDC stimulated testosterone production via the cAMP/PKA signaling pathway. In vivo, CDC had an aphrodisiac effect on a hydrocortisone-induced mouse model of Kidney-Yang deficiency, evidenced by significantly improving the sexual abilities of mice with impotence, reproductive organ weights, testosterone level, and pathological damage. Besides, CDC showed potential androgenic antagonistic activity on transgenic yeast. These results mean that the CDC might exert two-way regulation in the biological effects of hormones in the body (Fig. 11). www.nature.com/scientificreports/

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.