Antioxidant activity of Coridius chinensis extracts on manganese‐induced testicular damage in rats

ABSTRACT Coridius chinensis (C. chinensis) is a traditional Chinese medicine that has been used to treat pain, erectile dysfunction, and other diseases. Our previous study demonstrated that manganese‐induced reproductive damage was partially rescued by a medium dose of C. chinensis treatment in rat. However, the underlying mechanism is unknown. In this study, we found that the weight of reproductive organs and the sperm count in manganese‐exposed rat were partially rescued by C. chinensis extracts (CcE) treatment. The number of apoptotic cells was significantly decreased and the expression of malondialdehyde, cytochrome c, and caspase‐3 in manganese‐exposed rats was significantly decreased after high dose of CcE treatment. Further studies revealed that the activity of superoxide dismutase, total antioxidant capacity, and glutathione peroxidase enzymes was significantly increased in testis tissues and serum of manganese‐exposed rats with high dose of CcE treatment. Taken together, the results of this study suggest that CcE inhibits the Mn2+‐induced apoptosis in testes by inducing the activity of antioxidants.


| INTRODUCTION
Manganese (Mn 2+ ) is an essential ion that is required for normal immune function, bone growth, reproduction, and blood sugar regulation, 1 but extreme excess intake of manganese causes serious neurotoxicity, reproductive toxicity, and even death. 2,3 It has been reported that high dose of manganese exposure causes decreased testis weight, sperm concentration, and serum testosterone level in animal models. In human, high manganese level was associated with increased risk of low sperm motility and concentration. 4 Manganese exposure also causes decreased activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and increased apoptosis of spermatogenic cells and malondialdehyde (MDA) levels. [5][6][7][8][9][10] Coridius chinensis (C. chinensis) is a traditional Chinese medicine, which has been used to treat pain, erectile dysfunction, and kidney diseases. 11 Our previous study found that Mn 2+ -induced reproductive damage in rats was partially rescued by C. chinensis treatment. The testosterone levels and sperm concentration were increased and sexual behavior indexes including capturing time and ejaculation ability were recovered. 12 These studies indicate that manganese exposure-induced reproductive damage can be intervened by C. chinensis. In this study, we further investigate the underlying mechanism of C. chinensis in protecting Mn 2+ -induced damage in rat testis. We find that C. chinensis

| Tissue collection and histological analysis
The testes and epididymis+vas deferens of Mn 2+ -treated and control SD rats were dissected and weighed immediately after euthanasia on the day after termination of the Mn-CcE coadministration. Testes were fixed in 4% (v/v) paraformaldehyde for up to 24 hours, stored in 70% (v/v) ethanol, and embedded in paraffin. The 5-μm-thick sections were prepared and mounted on glass slides. 14 After deparaffinization, the sections were stained with hematoxylin-eosin (H&E) for histological analysis.
2.6 | Activity assays of MDA, T-AOC, T-SOD, and GSH-Px in serum and testis All male SD rats were sacrificed to expose the left ventricle of the heart. Blood was collected by puncture of the left ventricular using 1 mL of heparin to prevent blood clotting and centrifuged at 5000 rpm at 4 C for 10 minutes to separate the serum from blood cells. The testis was homogenized in cold normal saline (tissue weight:normal saline = 1 g:9 mL), centrifuged at 2500 rpm at 4 C for 10 minutes, and then the supernatant was collected. The activity assays of MDA, T-AOC, T-SOD, and GSH-Px of the serum and testicular tissue extraction were performed with MDA assay kit, total antioxidant capacity assay kit, T-SOD assay kit, and GSH-Px assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions, respectively.

| Western blot analysis
The testis homogenate (100 μg) was separated on a 10% SDS-PAGE gel and transferred to a methanol-activated PVDF membrane T A B L E 1 Treatment on each group of rats (Millipore) by electroblotting. The membrane was then blocked with 5% nonfat milk powder in 10 mM PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ) for 2 hours at 37 C. The Chemicon, Proteintech) at 37 C for 2 hours. After washing three times, the membranes were exposed to the chemiluminescence substrate (ECL; 7Sea Biotech Co., Shanghai, China) according to the manufacturer's instructions.

| Immunohistochemistry
After deparaffinization and rehydration, the paraffin-embedded sections were performed using a Vectastain ABC (avidin-biotin-peroxidase) kit (Vector Laboratories, Burlingame, CA) as recommended and using the primary rabbit antibodies MVH (1:1000, Abcam, ab13840) and SOX9 (1:500, Millipore, AB5535), and these were followed by staining with HRP-conjugated secondary antibody. After rinsing with PBS, the sections were stained with 3,3 0 -diaminobenzidin. 15 Images were captured using a Nikon microscope with a CCD camera.

| Protective effect of CcE on Mn 2+ -induced testicular damage
To further investigate the effects of CcE on Mn 2+ -induced testicular damage in SD rats, the histology of testis was examined by H&E staining. As shown in Figure 1  Note: Data were expressed as mean ± SD (n = 10). Abbreviation: CcE, Coridius chinensis extracts. *significant differences (P < .01) compared with the Mn group; **significant differences (P < .05) compared with the control group, ***Significant differences (P < .01) compared with the control group, respectively.

| Number of apoptotic cells decreases with C. chinensis treatment
The apoptotic cells in Mn 2+ -treated testes were examined by TUNEL assay. As shown in Figure 2     To further explore the potential mechanism of CcE in protecting Mn 2+ -induced apoptosis, the expression of cytochrome c (cyt c) and caspase-3 was examined by Western blotting analysis. The results showed that the protein level of cyt c and cleaved caspase-3 was dramatically increased in the Mn 2+ -treated group compared to that of the control group. By contrast, the expression of these two proteins was significantly reduced after administration of CcE ( Figure 3).

| DISCUSSION
With wide applications of Mn 2+ and its products, it has become a major environmental pollutant that affects male reproductive health.
Previous studies have shown that testis is an organ with high Mn 2+ sensitivity, and Mn 2+ can be stored in testicular tissues via the blood- ptosis by releasing cyt c from the mitochondria. 22 In this study, we found that the number of apoptotic cells and the expression of MDA, cyt c, and caspase-3 were significantly increased, but the activity of SOD, T-AOC, and GSH-Px was significantly decreased in Mn 2 + -exposed rats. These results indicate that oxidative stress is most likely the major reason that causes testicular damage in rat upon Mn 2+ exposure. Our recent study demonstrated that Mn 2+ -induced reproductive damage was partially rescued by a medium dose of C. chinensis treatment in rat. 12 22 Flavonoids can also form a chelate with metal ions to inhibit the generation of free radicals and have the ability to scavenge free radicals and antioxidation. 25 In a recent study, we tested the free radical scavenging activity and also proved that flavonoids possess obvious free radical scavenging activity. 26 Moreover, we also confirmed that the intervention of C. chinensis can repair the reproductive system injury caused by acute Mn 2+ impregnation in rats, reduce the MDA level of serum and testicular tissue, improve the SOD activity and T-AOC level, and significantly improve the morphological structure of testicular tissues in rats with acute Mn 2+ impregnation. 12,27 In the present study, we found that the activity of SOD, T-AOC, and GSH-Px was significantly increased in testis tissues after high dose of CcE treatment.
These results indicate that CcE may inhibit the ROS level by increasing antioxidant enzyme activity, thereby inhibiting the mitochondriamediated cyt c/caspase-3 signaling cascade apoptosis pathway.
In summary, our study demonstrated that Mn 2+ -induced testicular damage could be repaired by CcE, and the main factors responsible for the testicular protective effect were the antioxidant activity of CcE.