Potential protective role of chlorogenic acid against cyclophosphamide-induced reproductive damage in male mice

Abstract

Background

Cyclophosphamide (CP) is an anticancer drug; however, clinical utilization of CP is limited, resulting from its considerable toxicities. This research was performed to explore the protective effects of Chlorogenic acid (CGA) on reproductive damage induced by CP in mice.

Methods

Blood samples were collected for analysis of hormone content subsequently; semen samples were evaluated for quality, and testis samples were used for histopathological evaluation and analysis of oxidative stress biomarkers, protein and gene expression levels of steroid regulatory factors, and steroid synthase.

Results

The results noted that CGA increased serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) activity; increased SOD, GPx, and GSH oxidative stress levels in testis tissue; and decreased MDA content in testis tissue. Testicular cells in the CGA treatment group gradually returned to normal morphology, and CYP11A1 and CYP17A1 levels increased after CGA treatment. The mRNA levels of CYP11A1, CYP17A1, StAR, 3β-HSD, and 17β-HSD were significantly raised in the CGA dose group. In the test dose range, CGA can improve sperm quality, quantitative abnormality, and serum T synthesis disorder caused by CP. This mechanism may be correlated with the inhibition of oxidative stress and antioxidation levels.

Conclusions

Therefore, CGA has a protective impact on testicular injuries arising from CP in mice.

Introduction

Cyclophosphamide (CP) is a commonly used immunosuppressive and anticancer drug in clinical settings. This drug is activated and metabolized by CYP450 enzyme in the liver to produce the cytotoxic byproduct acrolein,^1–3 which can cause the consumption of reduced glutathione in cells; produce many free radicals, similar to ROS; destroy the normal standard metabolism of cells; kill spermatogonial stem cells in testicular tissues; cause damage to spermatogenic epithelial cells; and cause spermatogenic cell mutation, which results in different degrees of damage to male testis and epididymis function and can even result in infertility.^4,5 CP is an effective broad-spectrum anti-tumor drug. Its mechanism of action is the action of highly active alkylation groups on DNA, RNA, enzymes, proteins, and so on, causing them to lose their physiological activity, thereby inhibiting cell division and leading to cell death. CP leads to a reduction in body weight and weight of the reproductive organs, reducing sperm count, viability, and motility. However, CP not only produces cytotoxicity against proliferating active tumor cells but also acts on rapidly growing germ cells, thus affecting male fertility. Reproductive toxicity caused by CP has garnered increasing attention from researchers, and many scholars have carried out studies to pursue the mechanisms of reproductive toxicity produced by alkylating agents and corresponding protective measures. In particular, CP can lead to reproductive damage and imbalance of the antioxidant system in testicular tissue, ultimately resulting in an oxidative stress response. Accordingly, it is of extensive sense to find natural antioxidants to alleviate the oxidative stress caused by cyclophosphamide.

Antioxidants include synthetic antioxidants and natural antioxidants. Natural antioxidants are mainly derived from plants and have attracted the attention of many scholars due to their high efficiency, low toxicity, and wide variety of sources.⁶ Chlorogenic acid (CGA) is present in both plants and beverages (e.g. coffee and tea).^7,8 It is a common natural organic acid, existing as a colorless crystalline solid. CGA is easily soluble in water and can dissolve in various organic solvents. It is a weak acid and stable at room temperature. The biological activities of CGA include antimicrobial effects,⁹ antiviral effects,¹⁰ anti-inflammatory effects,¹¹ hepatoprotective effects,¹² hypoglycemic and lipid-lowering effects,¹³ neuroprotective effects,¹⁴ antioxidant effects,¹⁵ scavenging free radical activity,¹⁶ mutation-inhibiting and anti-tumor effects,¹⁷ and a wide range of pharmacological effects. At present, research on the biological activity of CGA has penetrated many fields, including the food, healthcare, medicine, and daily chemical industries.¹⁸ Although the effects and functions of CGA have been studied extensively, to the best of our knowledge, there have been no previous reports on the improvement of CP-induced reproductive injury in mice.

In the present experiment, a mouse model of reproductive injury was established with CP treatment followed by CGA treatment. Measured variables included the serum testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels; testicular histomorphology; testicular oxidative stress levels; lipid peroxide content; testosterone synthesis-related genes; protein expression; and sperm quality of mice. These indicators can be used to determine whether CGA has protective effects on the reproductive systems of mice with CP-induced reproductive injuries and to further elaborate the mechanism of CGA effects on CP-induced reproductive injury.

Materials and methods

Chemicals and drugs

CGA (98%), Cyclophosphamide was purchased from Baxter (Shanghai, China). Rabbit anti-Nrf-2, Keap-1 polyclonal antibody, and sheep anti-rabbit IgG secondary antibody were purchased from Bioss (Beijing, Chian); Total RNA rapid extraction kit, cDNA reverse transcription kit, PCR, and SYBR-Green Mix kit were purchased from Accurate (Catalog numbers AG21101, AG11707, and AG11701, respectively; Hunan, China). MDA, ALT, SOD, AST, CAT, GPx, and GSH kits were purchased from Boxbio (Catalog numbers AKFA013M, AKAM006M, AKAO001M, AKAM019M, AKAO003-1 M, AKPR014M, and AKPR008M, respectively; Beijing, Chian). T-Octylphenoxypolyethoxyethanol, formaldehyde, and hydrogen peroxide were purchased from Damao (Tianjin, China); BSA and PBS buffer were obtained from Solarbio (Beijing, China). T, LH, and FSH Elisa test kits were purchased from Enzyme-linked (Catalog numbers ml064301, ml063366, and ml001910, respectively; Shanghai, China). Methyltestosterone tablets were purchased from Lisheng (Tianjin, China).

Animals

Six-week-old SPF male Kunming mice weighing 22 ± 5 g were bought from Dashuo Biotechnology [Chengdu, China; SCXK (Sichuan) 2015–030]. These mice were housed in a clean environment at 22 ± 2 °C under an alternating 12 h light–dark cycle. All animal procedures were performed in accordance with our institution approved protocols.

Experimental design and drug treatment

After acclimatisation for one week, the mice were randomly split into six groups, including ten mice in several groups (n = 10). The dose of CGA was determined on the basis of previously published papers^19,20 and our pre-experiments.

• (1) Normal control (NC) group: injected with 0.9% saline only.

• (2) CP group: administered CP (60 mg/kg, ip) for 5 times on the 1, 3, 5, 7, and 9 days. Each injection is administered with an interval of one day, and the model establishment took a total of 10 days.

• (3) Low CGA (LC) group, Medium CGA (MC) group, High CGA (HC) group, and P group: administered CP (60 mg/kg, ip, same as model group), then followed by CGA (10, 20, 40, and 2 mg/kg, ig) for 23 days.

The mice were euthanized on the 33rd day. Blood samples were collected from the heart, centrifuged (3,000 rpm, 10 min) and then stored at −80 °C for hormone content analysis. The epididymis, testis, prostate, and seminal vesicle samples were collected instantly for further analysis.

Evaluation of serum hormone content

Serum T, LH, and FSH hormone levels were evaluated according to the kit instructions.

Tissue antioxidant assay

The SOD, GSH, GSH-Px, and MDA levels of the testicular tissues were evaluated with commercial kits based on methods previously utilized in our laboratory.²¹

Histopathological testicular identification

Histomorphometry was identified according to the formerly depictive method in our labs.²² Testicular tissue fixed in paraformaldehyde was paraffin-embedded and sliced into 5 μm sections. Slides were prepared and subjected to H&E. The distributions of spermatogonia and spermatogenic cells at all levels of the testicular tissues were observed under a light microscope (100 × ) with pathological section micrographs.

Immunohistochemistry

The expression levels of CYP11A1 and CYP17A1 proteins were examined in testicular tissues by immunohistochemical analysis. Testicular sections were oven-dried at 60 °C for 4 h, followed by dewaxing in xylene and gradient alcohol, and then treated with T-Octylphenoxypolyethoxyethanol (30 min), immersed in 3% hydrogen peroxide (30 min), blocked with 3% BSA (20 min), and then incubated with the first antibody from rabbit source (CYP11A1, CYP17A1) for 2 h and the secondary antibody for 1.5 h. Diaminobenzidine was developed for 5 min. After the membrane was covered, four visual fields were randomly observed under a light microscope (Leica DM3000, Weztlar, Germany, 100 × ), and the expression levels of CYP11A1 and CYP17A1 in the testicular tissues of each group of mice were analyzed with Image Pro Plus 6.0.

Real-time quantitative PCR analysis

Total RNA was extracted from the testicular samples using a total RNA Isolation Kit. NanoDrop One was used to examine the concentration of total RNA at 260 nm. A cDNA reverse transcription kit (Takara, Beijing, China) was reverse transcribed to generate the first strand of the cDNA (37 °C, 15 min). Then, the reverse transcriptase was inactivated in a water bath at 85 °C for 5 s. The primers of each pair of tested genes were amplified using a StepOnePlus™Real-Time PCR System (ABI, New York, USA) under optimal conditions. The target genes of the relative mRNA expression analysis included StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD genes. The β-actin was used as a reference to normalize the expression of the target genes. All of the aforementioned primers synthesized by Qingke Biotechnology (Beijing, China).

Sperm quality analysis

Preparation of the sperm suspension

One side of the caudate epididymis of each mouse was isolated, cut, and incubated in 1 mL of normal saline at 37 °C for 5 min to obtain a sperm suspension for future use.⁵

Sperm motility assay

Approximately 10 μL of the prepared sperm suspension was placed on a preheated blood cell count plate. Then, the glass slide was covered, placed under a 40 × multiple optical light microscope for observation. Two hundred spermatozoa were then randomly counted according to the motility and quiescence of the sperm. The results of this assay are expressed as percentages.²³

Sperm count determination

A 450 μL formaldehyde fixative (10% formaldehyde in PBS buffer) was added to 50 μL of the aforementioned sperm suspension and then mixed well so that the sperm would die completely, thus reducing the error caused by the swimming activity of the sperm. Sperm diluent (10 μL) was then accurately added to the blood cell count plate. After standing at 25 °C for 5 min, the amount of sperm in the five squares was counted immediately, and the average number was multiplied by the number of sperm per milliliter of semen (4 × 10⁷ number/mL).²³

Sperm abnormality rate determination

Two drops of sperm suspension smears were dried on slides. After drying naturally at 25 °C, these slides were fixed with methanol and stained with 2% eosin for 30 min.

Under the low-power microscope, the background was clear and the sperm overlap was less. The sperm morphology was then examined and photographed in sequence under a high-power microscope. Each chamber was counted with 200 sperms and repeated three times to calculate the percentage of deformities.⁵

Statistical analysis

Experiments were conducted in three independent parallel phases, and the results are presented as means ± standard deviations. SPSS 19.0 (IBM Corporation, USA) was used for analysis, and one-way ANOVA was performed. P < 0.05 was considered statistically significant.

Result

CGA increases mouse body weight

In Table 1, there was no diversity between the weights of the NC, CP, LC, MC, HC, and P groups at the beginning of the test. However, compared with the NC group, the final weights of the LC, MC, HC, and P groups were different markedly. The differences between the final weights of the LC and MC groups and the CP group were also significant.

Table 1.

Body weight of each group of mice (g).

Group	Initial weight (g)	Final weight (g)
NC	24.79 ± 1.39	36.09 ± 1.49
CP	25.51 ± 0.87	31.48 ± 2.83#
LC	25.19 ± 1.45	32.11 ± 1.71#,*
MC	25.39 ± 2.39	32.01 ± 2.65#*
HC	24.72 ± 1.86	31.83 ± 3.11#
P	24.89 ± 1.45	31.10 ± 2.97#

Note: vs. NC, ^#P < 0.05; vs. CP, ^*P < 0.05; n = 10.

Table 2 gives information about the comparison to the NC group. The testis, prostate, and seminal vesicle indexes of the CP group were markedly decreased. In addition, in contrast to the CP group, the testicular and seminal vesicle indexes of the LC were markedly enhanced (P < 0.05). The testis and seminal vesicle indexes of the MC and HC groups were the same as those of the NC group. The epididymis and seminal vesicle gland indexes of the special HC group were higher than those of the NC and P groups.

Table 2.

Organin indexes of each group.

Group	Testicular	Epididymis	Prostate	Seminal vesicle
NC	0.89 ± 0.04	0.36 ± 0.02	0.18 ± 0.05	0.73 ± 0.10
CP	0.48 ± 0.06#	0.29 ± 0.03	0.07 ± 0.01#	0.36 ± 0.11#
LC	0.66 ± 0.04#,*	0.35 ± 0.04	0.13 ± 0.01	0.50 ± 0.05#,*
MC	0.73 ± 0.11*	0.41 ± 0.07*	0.13 ± 0.02	0.63 ± 0.05*
HC	0.90 ± 0.04*	0.40 ± 0.02*	0.17 ± 0.03*	0.76 ± 0.05*
P	0.81 ± 0.10*	0.37 ± 0.02	0.18 ± 0.02*	0.65 ± 0.06*

Note: vs. NC, ^#P < 0.05; vs. CP, ^*P < 0.05; n = 10.

CGA increases testosterone and LH levels in the serum

Figure 1 demonstrates that the expression of T, LH, and FSH in the CP was lower by 65.15%, 61.47%, and 48.68%, severally, in comparison to those of the NC; thus, the reproductive injury model of CP was successfully established. However, after CGA treatment, the increase in T, LH, and FSH levels was related to the dose. In the HC group, LH and FSH activity levels significantly increased by approximately 92.41% and 51.77%, respectively (P < 0.05). The P group experienced an inhibitory effect on the decline in serum T, LH, and FSH activity expression.

Fig. 1.

Effects of CGA on serum hormone (T, LH, and FSH) levels in mice.

CGA mitigates testes oxidative stress in mice

As shown in Fig. 2, SOD, GSH, and GSH-Px levels were markedly reduced in the testes of the CP (Fig. 2). It appears that CGA improved the defense abilities of the antioxidant system, prevented CP from breaking the redox balance of the testes, and significantly alleviated CP-induced oxidative stress injury. Relative to the CP group, SOD and GPx activity levels were markedly heightened in the LC, MC, and HC groups. GSH activity was positively associated with the dose of CGA. The MDA content was markedly improved (82.92%) in the CP than in the NC, reflecting that CP induced oxidative stress in the testes (Fig. 2D). In the LC, MC, and HC groups, CGA inhibited the increase in MDA.

Fig. 2.

Testicular antioxidant enzyme activity levels. The levels of SOD (A), GSH (B), GSH-Px (C), and MDA (D) in the testicular homogenate of mice in the NC, CP, P, LC, MC, and HC groups were determined using commercial kits.

CGA improves testes tissue pathological changes

As shown in Fig. 3, spermatogenic cells in the seminiferous tubules were closely arranged at all levels in the NC, and apoptotic spermatogenic cells were rare in this group. Testicular seminiferous epithelium was thinner in CP group, several spermatogenic cells and interstitial cells were depressed, the basement membrane was missing, the lumen was enlarged, and the wall cells were shed, resulting in atrophic images. All CGA groups exhibited significantly increased layers of spermatogenic cells across all levels of the seminiferous tubules as well as decreased apoptotic cells. All treatment groups exhibited significantly thicker seminiferous epithelia and increased numbers of spermatogenic and stromal cells.

Fig. 3.

Histopathological changes of mouse testis in each group (HE, × 400). NC group (A), CP group (B), LC group (C), MC group (D), HC group (E), P group (F). Notes: A, decrease in the number of cells in the seminiferous tubules; b, increased apoptosis; c, basement membrane detachment; d, lumen enlargement.

CGA increases CYP11A1 and CYP17A1 protein expression in mice testes

Compared with the NC group, we observed that the expression levels of CYP1A1 and CYP17A1 proteins in the CP were markedly decreased by 52.40% and 77.00%, respectively (Fig. 4). The decrease in CYP11A1 and CYP17A1 protein expression levels was inhibited after CGA treatment, and the levels of CYP11A1 and CYP17A1 enhanced in the LC, MC, and HC groups.

Fig. 4.

Immunohistochemical analysis of CYP11A1 and CYP17A1 protein expression in testicular tissue. Positive-staining area of CYP11A1 in each group (A). Positive-staining area of CYP17A1 in each group (B). Expression levels of CYP11A1 protein (C); expression levels of CYP17A1 protein (D).

CGA increases testosterone synthesis-related factor gene expression

The mRNA expression levels of steroid regulatory factors and steroid synthase were detected in mouse testes with RT-qPCR (Fig. 5). In contrast to the NC group, the mRNA expression quantities of StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD in the testis of mice in the CP group were decreased. After CGA treatment, the mRNA expression of StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD were significantly upregulated. The mRNA expression levels of target genes in the MC and HC groups showed a marked discrepancy in the model group, then followed a dose-dependent increase.

Fig. 5.

The mRNA expression levels of StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD in mice testes as detected with RT-qPCR.

CGA enhances sperm quantity

The number of spermatozoa, sperm motility, and sperm malformation rate are important indicators of reproductive health. The number of spermatozoa determines the normal fertilization probability of the body. Sperm motility usually reflects the strength of sperm motility. The rate of sperm malformation affects the fertilization ability of sperm to some extent. In Fig. 6, compared with the NC, the number and motility of spermatozoa in the CP were markedly decline, and the deformity rate of the CP group was markedly growth. In contrast to CP, CGA treatment significantly improved the number and vitality of sperm and significantly inhibited and reduced the rate of sperm malformation.

Fig. 6.

Sperm quality and morphology.

The sperm morphology of the NC exhibited very few abnormalities and was basically normal with a hooked head, no tail folding, and a linear shape (Fig. 6). In the CP group, most of the spermatozoa were deformed; specifically, most of these spermatozoa exhibited tail folding, and a few had no head hooks. In contrast to the CP group, the sperm malformations of the CGA groups improved, the number of sperm with tail-fold malformations decreased, and the quantity of sperm with normal morphology increased in the CGA group.

Discussion

Many experimental studies have demonstrated that CGA has a variety of biological functions.²⁴ Previous studies have shown that injections of saffron and curcumin can effectively protect against CP-induced reproductive damage.²⁵ In order to construct the spermatogenesis disorder model in mice in a short time, some studies used intraperitoneal injection of CP for 5 days to achieve the damage effect.¹⁹ In keeping with the results of antecedently reports, our study indicated that in comparison with the NC, the CP group exhibited markedly decreased sperm concentration and motility after 5 days of intraperitoneal injection with CP (60 mg/kg), indicating that the CP-induced reproductive damage model was successfully replicated.

The fertility of male mice is closely related to the quality and quantity of sperm. Sperm is produced in the testes and matures in the epididymides. Therefore, the weight of the testes, prostate, epididymides, and the seminal vesicle glands, as well as the number, vitality, and malformation rate of sperm, can reflect the influence of CP on the reproductive function of mice. The calculation of testis, epididymis, prostate, and seminal vesicle gland indexes can exclude the effect of body weight differences on the testes, epididymides, and anterior. Excluding this effect, CP could still have caused weight loss in the testes and the seminal vesicles but had no marked impact on the weight of the epididymides and prostate, possibly due to the fact that the weight loss of the mice was more significant than weight loss in the testes and the epididymides in this experiment. At the same time, in this experimental condition, CP harm to the reproductive system mainly achieved by testicular damage.

Previous reports have noted that CP can result in decreases in sperm count, increases in malformed sperm, and decreases in sperm survival in male rats.⁴ Our results suggested that after intraperitoneal injection with CP, sperm count and activity were decreased markedly, and the proportion of abnormal sperm was raised compared with the NC group markedly. Sperm morphology is controlled by multiple genes, and changes in sperm morphology can reflect DNA damage. The percentage of abnormal sperm increased after CP treatment. In the present experiment, we found that CGA treatment after CP injection could significantly improve the abnormalities in sperm quality and quantity caused by CP, thus indicating that CGA has the potential to improve reproductive damage caused by CP in mice.

The testes not only produce sperm but also secrete testosterone. Testosterone is mainly produced by Leydig cells, which are not only related to male sexual function but also closely related to spermatogenesis.⁵ Thus, testosterone synthesis serves as a crucial indicator of male reproductive health. Testosterone synthesis in the body is regulated through two mechanisms: rough regulation of the testicular gonadal axis and fine regulation of steroid synthase. The testicular gonadal axis pathway stimulates the pituitary release of LH through the release of gonadotropin secreted by the hypothalamus and LH receptors on the testicular surface and then promotes the expression of StAR through the action of the cAMP-pKA signaling pathway to promote related groups. Steroid synthase regulation mainly catalyzes the conversion of cholesterol to testosterone by four steroid synthases. In the testicular tissue, cholesterol enters the testis with the assistance of StAR-regulated transporters. Cholesterol is initially catalyzed by CYP11A1 to produce pregnenolone, a pivotal rate-limiting enzyme in steroid synthase regulation. The synthesized secreted pregnenolone is then catalyzed by 3β-HSD and CYP17A1 to produce androstenedione. Finally, androstenedione is catalyzed by 17β-HSD to produce testosterone. A previous study documented that ethyl acetate extract from Humulus lupulus (AHE) can increase the levels of the serum hormones T, LH, and FSH in male rats with reproductive injuries induced by CP. Our study revealed that in contrast to the NC group, the levels of serum T, LH, and FSH in the CP were dimmish markedly (P < 0.05); these decreased hormone levels led to spermatogenesis dysfunction, which may be one of the reasons for the decrease in sperm count observed in this experiment. After CGA treatment, T, LH, and FSH levels enhanced markedly (P < 0.05), indicating that CGA can improve the decrease in testicular endocrine function that occurs in mice with CP-induced reproductive damage. Whereas, the possibility mechanisms of this impact require to study in the future.

The results of our findings also revealed that compared to the NC group, the activities of SOD, GSH, and GSH-Px in the CP were markedly reduced, while the MDA content of the CP was markedly heightened, supported that CP induces oxidative stress in the testes. The use of CGA improved the defense abilities of the antioxidant system, prevented CP from breaking the redox balance of the testes, and inhibited the rise of MDA. These results were confirmed by those of previous studies that revealed that resveratrol can reverse the oxidative stress damage caused by CP and reduce lipid peroxide content.²⁰

A previous study has documented that Cistanche deserticola can increase the expression of steroid synthase proteins (CYP11A1 and CYP17A1) in a reproductive injury model.²⁶ Our study findings unveiled that the expression of CYP1A1 and CYP17A1 proteins in the CP was reduced than that of the NC markedly. After CGA treatment, the expression of CYP1A1 and CYP17A1 proteins was enhanced in the LC, MC, and HC groups, which was related to the dose of CGA. The consequences of RT-qPCR detection of the mRNA levels of steroid regulatory factors and steroid synthase revealed that the mRNA levels of StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD in the testes of mice in the CP were markedly decreased in comparison to the NC group. After CGA treatment, the mRNA levels of StAR, CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD were markedly increased. Furthermore, ECH increased the mRNA levels of CYP11A1, CYP17A1, 3β-HSD, and 17β-HSD in rats with oligospermia.²⁷

Conclusions

In conclusion, CGA can improve sperm quality and quantity abnormalities as well as serum hormone synthesis disorders caused by CP and can upregulate the gene and protein expression of steroid synthase. The mechanism of these effects may be accounted for by the fact that CGA can inhibit oxidative stress and decrease antioxidant levels in mice with CP-induced reproductive injury and may therefore play a role in the cAMP-pKA signaling pathway. This discovery lays a theoretical basis for the clinical application of CGA in patients with reproductive injuries caused by the use of CP chemotherapy for malignant tumors, but its specific mechanism of action requires further research.

Table S1. List of abbreviations

The abbreviations mentioned in this article can be found in Table S1.

Funding

This study was sponsored by the Shaanxi Province Key Research and Development Plan (grant number 2023-XCZX-14 and 2024NC-YBXM-182), the Incubation Project on State Key Laboratory of Biological Resources and Ecological Environment of Qinba Areas (grant number SXC-2301).

 

Conflict of interest statement. There are no conflicts of interest to declare.

Data availability statement

The data supporting the findings of this study can be obtained from the corresponding author upon reasonable request.