The aim of the present study was to investigate the potential protective effects of new Wenshen Shengjing Decoction (new WSSJD; including
Cyclosporine A (CsA) is a commonly used immunosuppressive agent that specifically targets T cells and inhibits the secretion of interleukin-2, and dramatically increases the success rate of liver, kidney and cornea transplantations, as well as the survival rate of recipients (
Previous studies have demonstrated that CsA decreased the levels of serum testosterone by influencing testosterone biosynthesis and secretion in the testes. He
Wenshen Shengjing Decoction (WSSJD) consists of 15 herbal medicines, primarily including
In the present study, the mechanism underlying the protective effect of new WSSJD in testicular tissues was investigated to determine the therapeutic efficacy of the modified medicine. Elucidating the protective mechanism may provide a basis for the use of new WSSJD in ameliorating CsA-induced spermatogenic damage.
A total of 90 male Kunming mice (8-weeks-old; 35–40 g) were provided by the Changchun Institute of Biological Products Co., Ltd. (Changchun, China). The present study was conducted with approval from the Ethics Committee of Jilin Medical University (Changchun, China). Mice were housed at 21±3°C and 55–65% relative humidity under a 12 h dark-light cycle. Mice were given free access to a standard laboratory mouse diet and sterile water.
The 15 Chinese medicine components of WSSJD, including
The concentrations of crude drug extracts were determined as follows: Weight of the crude material/final volume. This calculation method is widely used in the study of traditional Chinese medicine (
Mice were randomly divided into 6 groups (15 mice per group) and were administrated with medicine intragastrically. The groups were as follows: Control (normal saline); dimethylsulfoxide (DMSO); CsA; CC; WSSJD; and new WSSJD. Mice in the CsA, CC, WSSJD and new WSSJD groups were intraperitoneally (i.p.) injected with 15 mg/kg/day CsA (Shanxi Powerdone Pharmaceutics Co., Ltd., Datong, China) for 30 days, as described previously (
At the end of the experimental period (at day 31), mice were placed in sealed cages, which were subsequently infused with 10–30% CO2. When mice ceased to move for 5 min, it was confirmed that mice had succumbed to CO2 exposure.
Mice testes were harvested and immediately fixed in 4% paraformaldehyde (pH=7.2) at room temperature for 24 h, followed by ethanol dehydration, xylene treatment to remove the ethanol, wax embedding and sectioning. Histological sections (5 µm) were obtained using a rotary microtome. These were affixed to glass slides for H&E staining. Testicular sections were observed using highlight histopathological microscopy to evaluate the development of seminiferous tubules and obtain histometric data. The development of seminiferous tubules was assessed using the Johnsen scoring system (
On day 30 of treatment, the mice were anesthetized with 10% chloral hydrate (Shanghai Guoyao Chemical Reagent Co., Ltd) at 0.004 ml/g of body weight i.p. prior to euthanasia with CO2. Trunk blood was harvested and the serum was separated and stored at −20°C. Briefly, blood was collected into 1.5-ml tubes and stored at 4°C overnight. The tube was then centrifuged at 1,300 × g at 4°C for 6 min and the serum was collected. ELISA kits (Shanghai Elisa Biotech Co., Ltd., Shanghai, China) were used to determine the serum contents of testosterone (cat. no. EIA-2380) and LH (cat. no. EIA-2385) according to the manufacturer's protocol. The optical density (OD) was determined using a Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the levels of testosterone and LH were determined based on the standard curve.
Testicular tissues were fixed in 4% formaldehyde at room temperature for 24 h and embedded in paraffin. Paraffin sections were cut into 5 µm sections and were subsequently dewaxed. Antigen retrieval was achieved by incubating sections in 0.01 M citrate buffer (pH=6.0) at 95–98°C for 5 min. The sections were subsequently blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; A3675) at room temperature for 1 h. Following blocking, sections were incubated with rabbit polyclonal antibodies against LHR (cat. no. L6792; Sigma-Aldrich; Merck KGaA; 1:200) or P450 side chain cleavage (P450scc; cat. no. ab75497; Abcam, Cambridge, UK; 1:200) at 4°C overnight in the dark. The streptavidin-biotin complex (SABC) method was used to detect the expression of LHR and P450scc using an SABC kit (SA2010; Boster Biological Technology, Pleasanton, CA, USA), according to the manufacturer's protocol. For the tissues isolated from negative control mice, the primary antibody was replaced with PBS. Leydig cells with yellow or brown staining on the membrane or plasma were considered to be LHR-positive cells. Five slides were obtained from each sample and five fields in the Leydig tissue areas were selected at random and assessed under a fluorescence microscope (magnification, ×400). The mean OD of positive cells in the Leydig tissue areas was obtained using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The expressions of LHR and P450scc were proportional to the OD values, with higher OD values representing higher protein expressions.
Mice testes were dissected, embedded in wax and sectioned. The sections (5-µm thick) were then stained using the TUNEL assay kit (MK1024; Boster Biological Technology), according to the manufacturer's protocol. In brief, testicular tissues form control (normal saline), DMSO, CsA, CC, WSSJD and new WSSJD groups were stained using a 1:100 dilution of biotin labeled digoxin antibody for 30 min at 37°C. Stained rat interstitial epithelial tissue (provided in the kit) was used as a positive control, and samples incubated with PBS instead of biotin labeled digoxin antibody were used as a negative control. The frequency of TUNEL-positive cells exhibiting green nuclear staining was evaluated using a laser scanning confocal microscope. A total of 10 random fields were assessed under high-magnification (×400) and positive cells were counted. The mean number of positive cells per field was calculated.
The cauda epididymidis was harvested following sacrifice and the epidermis was cut with a blade to release the sperm from the epididymis into 2 ml PBS at 37°C to obtain epididymis suspensions. The suspensions were subsequently incubated at 37°C for 10 min to allow sperm to swim out. Aggregations of sperm were discarded and the remaining sperm samples were isolated and resuspended in PBS to give a concentration of 106 cells/ml. Sperm apoptosis was analyzed using a Annexin V-FITC Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. In brief, 1 ml of sperm suspension was stained with 10 µl PI or with 500 µl binding buffer and 5 µl Annexin V-FITC in the dark for 10 min at room temperature. Cells were immediately analyzed by Epies XL flow cytometry (Beckman Coulter, Inc.) and using a TetraONE™ System (6915050; Beckman Coulter, Inc.). Cells in the early stages of apoptosis were identified by Annexin V-positive staining and necrotic cells were identified by PI-positive staining.
Data analysis was performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and expressed as the mean + or ± standard deviation as indicated. Differences between groups were evaluated for significance using one-way analysis of variance followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
To investigate the potential protective effect of new WSSJD on the development of mouse testicular seminiferous tubules, the morphology of H&E-stained mouse testes were compared under light microscopy. The seminiferous tubules of mice from the CsA and CC groups exhibited shrinkage of the tubule fringe, decreased tubule diameters and reduced layers of testicular seminiferous epithelium compared with the control. In addition, spermatogenic cells exhibited a disordered arrangement, the number of spermatogenic cells was reduced and few mature sperm were visible in the lumen (
The levels of testosterone and LH in the serum were subsequently measured. Serum levels of testosterone and LH were unaffected following DMSO administration. By contrast, serum testosterone was significantly downregulated and LH was significantly upregulated in CsA-treated mice, relative to controls (P<0.05;
The expressions of P450scc and LHR were assessed in testicular Leydig cells by immunohistochemistry (
The apoptosis of spermatogenic cells in the mouse testes was analyzed by a TUNEL assay. The nuclei of apoptotic cells were principally observed in the spermatogonia and primary spermatocytes (
To verify the protective effects of new WSSJD against CsA-induced sperm apoptosis, the survival and early apoptosis of sperm in the epididymis were determined. Epididymal sperm were stained with PI or Annexin V and analyzed by flow cytometry (
It has previously been documented that long-term use of CsA as an immunosuppressive agent affects reproductive capacity (
The specific microenvironment of the testis promotes spermatogenesis, and thus impairment of testicular structure and function may lead to spermatogenic arrest (
The hypothalamic-pituitary-testicular axis serves an important role in the regulation of genital activity (
CsA-induced oxidative stress and testis damage induce the dysplasia of sperm and spermatogenic cells (
In the present study, the effects of new WSSJD on CsA-induced impairment of testosterone synthesis and spermatogenic apoptosis were investigated, and it was observed that new WSSJD significantly decreased the apoptotic rates of spermatogenic cells and sperm, and thus repaired damage to the testicular seminiferous epithelium. The morphology of testicular seminiferous tubules and the apoptosis of spermatogenic cells and sperm were investigated using histochemistry and flow cytometry. Although cell cycle distribution and daily sperm production were not investigated, they will be the focus of future studies by our group.
In conclusion, compared with traditional WSSJD, new WSSJD significantly increased testosterone levels in the testes and decreased the apoptosis of spermatogenic cells and sperm, which effectively repaired CsA-induced testicular damage. These results indicate that new WSSJD may be a useful pharmacological agent in the treatment and prevention of CsA-induced testicular damage.
The present study was supported by the Key Research Project of the Scientific and Technological Development Program of Jilin Province (grant no. 20140204033YY), the College Science and Technology Program of Shandong Province (grant no. J13LL04) and the Undergraduate Training Programs for Innovation and Entrepreneurship of Jilin Province (grant no. 2014024).
Hematoxylin and eosin staining of mouse testicular tissue. Mice were administered with CsA, followed by CC, WSSJD, new WSSJD or DMSO. Untreated mice were used as a negative control. The testes were harvested 30 days later and stained with hematoxylin and eosin. CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. The length of arrows represented the thickness of spermatogenic tubule, and the ratio of their thickness was 1.4:1.4:1.2:1.22:1.3:1.4 in control, DMSO, CsA, WSSJD and new WSSJD group, respectively. Magnification, ×200; Scale bar, 100 µm.
ELISA analysis of serum testosterone and LH. Mice were administered with CsA and treated with CC, WSSJD, new WSSJD or DMSO. Serum was harvested 30 days later. Levels of (A) testosterone and (B) LH in the serum were determined by ELISA analysis. Untreated mice were used as a negative control. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. LH, luteinizing hormone; CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
Immunohistochemistry of LHR protein in Leydig cells. (A) Immunohistochemistry identified an accumulation of Leydig cells positive for LHR (brown) between the seminiferous tubules. Negative tissues were treated using PBS instead of antibodies against LHR. Magnification, ×400; Scale bar, 50 µm. (B) The mean OD of LHR-positive cells in each group. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. LHR, luteinizing hormone receptor; OD, optical density; CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
Immunohistochemistry of P450scc protein in Leydig cells. (A) Immunohistochemistry identified positive staining for p450scc (brown) in the cytoplasm of Leydig cells. Negative sections were treated with PBS instead of antibodies against P450scc. Magnification, ×400; Scale bar, 50 µm. (B) The mean OD of P450cc-positive cells in each group. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. OD, optical density; P450scc, P450 side chain cleavage; CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
TUNEL staining of mouse testes. (A) TUNEL staining (green) and Hoechst 33342 immunostaining (blue) of sections derived from the testes of mice administered with CsA followed by CC, WSSJD, new WSSJD or DMSO. Untreated mice were used as a control. Spermatogoniums and primary spermatocytes were observed. Fine arrows indicate spermatogonia, which were close to the basal membrane of spermatogenic tubules. Thick arrows indicate primary spermatocytes, which were close to spermatogonia/inside spermatogonia. Interstitial tissue of the testis provided by the TUNEL kit were used as positive sections. Negative sections were treated with PBS instead of biotin-labeled digoxin antibody. Magnification, ×400. (B) Quantification of apoptosis in spermatogenic cells. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
Flow cytometry analysis of live sperm. Mice were administered with CsA followed by CC, WSSJD, new WSSJD or DMSO. Untreated mice were used as a control. (A) Sperm were isolated from the epididymis, stained with propidium iodide and analyzed using flow cytometry. Percentages displayed in the lower left quadrant represent the PI-negative (live) cells. (B) Percentages of live sperm. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
Flow cytometry analysis of early apoptotic sperm. Mice were administered with CsA followed by CC, WSSJD, new WSSJD or DMSO. Untreated mice were used as a control. (A) Sperm were isolated from the epididymis and stained using an Annexin V-fluorescein isothiocyanate Apoptosis Detection kit. Annexin V-positive staining was used to identify early apoptotic sperm. (B) Percentages of early apoptotic sperm. *P<0.05 vs. control; #P<0.05 vs. CsA group; ΔP<0.05 vs. CC group; ▲P<0.05 vs. WSSJD group. CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction; DMSO, dimethylsulfoxide. Data are presented as the mean ± standard deviation.
Effect of new WSSJD on Johnsen scoring of the testes.
Groups | Johnsen score (≤10) |
---|---|
Control | 9.75±0.25 |
DMSO | 9.50±0.25 |
CsA | 7.25±0.43 |
CC | 7.33±0.29 |
WSSJD | 9.00±0.25 |
New WSSJD | 9.25±0.25 |
Data are expressed as the mean ± standard deviation.
P<0.05 vs. control
P<0.05 vs. CsA group
P<0.05 vs. CC group. DMSO, dimethylsulfoxide; CsA, cyclosporine A; CC, clomifene citrate; WSSJD, Wenshen Shengjing Decoction.