Yangjing capsule attenuates cyclophosphamide-induced deficiency of testicular microcirculation in mice

Purpose: To explore the protective effects of Yangjing capsule (YC) on testicular microcirculation in a mouse model of deficiency of testicular microcirculation. Methods: Immunohistochemistry was applied to determine the effects of YC on microvascular density of mice. The protein level of CD34 and vascular endothelial growth factor A (VEGF A) was measured by western blot. The viability of Testicular cell line (TM4 cells) was examined by CCK-8 assay. Results: Histopathological changes demonstrated that CP-induced decrease of microvascular density of the mice was rescued by YC dose-dependently (p < 0.5). Western blot data showed that the protein levels of CD34 and VEGF A in CP group were significantly decreased, but dose-dependently increased by YC, respectively, following co-administration of CP + YC, compared with those in CP group (p < 0.5). The results from CCK-8 assay showed that the cell viability of TM4 cells increased with the amount of YC administered, and that high concentrations of YC (0.1 and 1 mg/mL) showed significant effects (p < 0.5). Moreover, YC showed little effect on VEGF A mRNA and protein expression in TM4 cells. Conclusion: YC may be considered an alternative therapeutic agent for the management of testicular microcirculation disease. However, further studies are required to ascertain this.


INTRODUCTION
Testicular microcirculation is featured with a high-amplitude vasomotion and a low capillary pressure [1]. It possesses a series of temporal variation for complicated physiologic or pathophysiologic disturbances [1][2][3]. Testicular microcirculation play key roles in transporting nutritive compounds and removal of cellular waste products in the testis [4][5]. Testicular tissue microcirculation had a close relationship with sperm production and sperm function [6][7][8]. Abnormal testicular microcirculation might cause male infertility, especially in those with weak sperm [9]. Vascular endothelial growth factor (VEGF) is crucial regulator of phenotypic behaviors of vascular endothelial cells [10]. VEGF participates in angiogenesis, and could stimulate growth and development of vascular endothelial cells [11]. VEGFA, as the most efficient angiogenic factor in the VEGF family, has a crucial effect on embryonic lethality [12]. The downregulation of VEGFA in vascular endothelial cells induced postnatal mortality for vascular degeneration, which suggested an important role of VEGFA in vascular homeostasis [13]. Moreover, depletion of VEGFA isoforms mediates the expression of apoptotic testicular genes in mice, induces subfertility, decreased the number of sperms [14]. Meanwhile, it is known that VEGFA is involved in the chemotherapy of breast cancer cells [15]. However, whether VEGFA participates in the effects of YC on CP induced deficiency of testicular microcirculation is still lacking.
Recently, traditional Chinese medicine (TCM) was shown to have a huge advantage in the treatment of male infertility [16]. Yangjing capsule (YC) is a type of TCM [17]. It plays key roles in stimulating kidney activity [18]. YC could increase the number of sperm and sperm vitality for patients with male infertility [19]. YC promotes testosterone progression via steroidogenic enzymes and mediates male infertility and sexual dysfunction [20]. In previous research, it was indicated that YC could promote sperm concentration and motility in infertile males [17]. In addition, the YC might enhance androgen synthesis and hormonal balance [18]. However, whether YC affect the expression of VEGFA protein and testicular microcirculation remains unclear.
In the present study, the roles of YC in CPinduced deficiency of testicular microcirculation and expression of VEGFA was investigated. These findings may provide a novel strategy for CP-mediated deficiency of testicular microcirculation.

Animals and treatments
Adult male (8 -10 weeks old) Balb/c mice (weighing 20 ± 2 g) were acquired from Model Animal Research Center of Nanjing University (Nanjing, China Finally, the testes tissues were rapidly excised and weighed followed by storing in liquid nitrogen or applying for the isolation of RNA and protein.

YC extraction and cell culture
YC extract was obtained as described previously [21,22]. YC was extracted from Yinyanghuo, Muli, Wangbuliuxing ect., and diluted with 333 mL of distilled water. Then the compounds were ultrasonicly extracted for 45 min. The supernatants were collected at 13,000 g at 4 °C for 30min to 100 mg/mL of the crude herb.
TM4 cell lines were provided by ATCC (Rockville, MD, USA). Cells were incubated with DMEM, supplemented with 10 % FBS and 1% penicillin/streptomycin in 5% CO2 at 37°C for 24 h. Cells were subjected to 0, 0.01, 0.1, and1mg/mL YC in 5% CO2 at 37°C for 24 h. After the above different treatments, TM4 cells were collected for the use of the following experiment.

Immunohistochemistry
Testis were embedded in paraffin. The sections were cultured a citrate buffer. The sections were subjected to primary antibodies against CD34 (Dako, Glostrup, Denmark) and then to the secondary antibody. Thereafter, the slices were incubated with DAB. Immunostaining was confirmed with a light microscope (K5007, Dako).

Cell viability
CCK-8 was conducted to determine the cell viability after treatment with YC (0.01, 0.1 and 1 mg/mL). Cells (2x10 3 cells/well) were plated into 96-well plates. After that, CCK-8 kit was supplemented with 10 μL of CCK8 solution. The absorbance rate at the wavelength of 450 nm was determined with a microplate reader (Bio-Rad Laboratories, Inc.).

Statistical analysis
Data are presented as mean ± standard deviation (SD, n = 3). Difference between various groups were determined with one-way ANOVA. P < 0.05 was deemed statistically significant.

Effect of YC on microvascular density in mice
Testicular micro vessels were closely distributed in microtubules. As shown in Figure 1, compared with control group (Figure 1 A), the intensity of CD34 staining in CP group was decreased (Figure 1 B); compared with CP group, YC dosedependently (630, 1260 and 2520 mg/kg) increased the intensity of CD34 staining in CP + YC groups ( Figure 1C-E). In addition, the intensity of CD34 staining in CP + YC group (2520 mg/kg) was similar with that of control group. A previous study suggested that immunoexpression of CD34 was used to analyze testicular microvascular density [23]. To further clarify the effects of YC on the microvascular density of the mice, we examined the expression of CD34 protein levels in each group of testicular tissues as well. The western blot result showed that CD34 protein level in control group was significantly lower; compared with CP group, YC dose-dependently (630, 1260 and 2520 mg/kg) increased the CD34 protein level in CP + YC groups; in addition, the CD34 protein level in CP + YC group (2520 mg/kg) was similar with that of control group (Figure 2 A), which showed a protective role of YC in CP induced decrease of microvascular density of mice.

Effect of YC on expression of VEGFA in mice testis
The qRT-PCR results indicated that the expression of VEGFA in the CP group and control group was of no significant difference, which was paralelled with YC (630, 1260, and 2520 mg/kg) treatment group (Figures 3 A). Interestingly, western-blot results suggested that compared with control group, CP significantly decreased VEGFA protein levels, which were dose-dependently reversed by YC treatment, and the protein level of VEGFA in CP + YC group (2520 mg/kg) was similar with that of control group (Figure 3 B and C).

YC increased TM4 cell viability
CCK-8 assay was performed to examine TM4 cell viability. At 48-hour post treatment of YC extract (0.01, 0.1 and 1 mg/mL), the cell viability of TM4 cells was increased in a dose-dependent manner, and high doses (0.1 and 1 mg/mL) of YC significantly increased the cell viability ( Figure 4).

Effect of YC on expressions of VEGFA in TM4 cells
When TM4 cells were treated with YC at different concentrations (0.01, 0.1 and 1 mg/mL), we examine the expression of VEGFA in TM4 cells. The mRNA expression level of VEGFA was not changed even with gradually increased concentration of YC (Figure 5 A). Similarly, western blotting results indicated that VEGFA protein expressions were also not changed with the increase of the YC concentration (Figures 5 B and C).

DISCUSSION
So far, low sperm count or poor motility and quality of sperm induced male infertility still affected male (24). Testicular microcirculation induced change of spermatogenesis is a common cause for male infertility [25]. Usually, infertile males are treated with hormones or empiric medical treatment, but the corresponding therapeutic effect is often not satisfactory [8]. Traditional Chinese formulations have achieved great advantages in the treatment of male infertility [17]. However, their molecular mechanisms of action on testicular microcirculation in infertile males remain unclear. In this study, we studied the roles of YC in testicular microcirculation in a mouse model whose dysregulation of testicular microcirculation was induced by CP.
Vascularization is an important part of microenvironment for the testis [4]. Increased microvessel density is a crucial factor in many physiologic and pathologic conditions [4]. In this study we found that administration of CP significantly decreased micro vessel density compared with the control group which was evidenced by decreased IHC staining for CD34 as well as decreased CD34 mRNA and protein levels. Interestingly, compared with CP group, YC significantly dose-dependently increased CD34 staining as well as in decreased CD34 mRNA and protein levels in testis sections of mice, indicating the protective effect of YC on micro vessel density and the development of blood vessels to improve testicular microcirculation in mice exposed to CP. The results are paralleled with Arena et al study [23].
In the current study, results indicated that VEGFA protein but not mRNA level was significantly decreased by CP, which was significantly upregulated by YC in mice testis, suggesting the involvement of VEGFA in testicular microcirculation, which is consistent with Zhao et al study [20].
In addition, in this study, YC concentrationdependently increased the TM4 cell viability, suggesting that YC could promote the viability of TM4 cells in a concentration-dependent manner. Moreover, we found that VEGFA expression did not significantly changed the mRNA or protein level after treatment with different concentrations of YC in TM4 cells, indicating that YC alone had little effect on VEGFA expression in TM4 cells. Therefore, the YC exerted a beneficial effect on TM4 cells. The findings of YC in cells are consistent with Gu et al study, which revealed that YC stimulates testosterone synthesis [17].
Traditional Chinese medicine is drug mixture plays key role in in the treatment of male infertility via suppressing the progression of teratospermia and improving the sperm acrosin activity [16]. YC consisted of 11 traditional Chinese drugs [17]. Therefore, additional studies will be required to understand the pharmacological mechanisms of action of YC on the testes microenvironment in our future work.
In conclusion, the present study suggested that YC could rescue CP-induced decrease in the density of testicular microcirculation in male mice, which is likely due to the enhancement of TM4 cell viability. In addition, high expression of CD34 and VEGFA might be involved in the process of micro circulation in testis tissues, but YC had little effect on VEGFA expression in TM4 cells. Therefore, YC might be regarded as an alternative therapeutic treatment for testicular microcirculation in the future.