Protective Effects of Matricaria chamomilla Extract on Torsion/ Detorsion-Induced Tissue Damage and Oxidative Stress in Adult Rat Testis

Testicular torsion, as an abnormal twisting of the spermatic cord due to rotation of a testes or the mesorchium (i.e. a fold in the area between the testes and epididymis), is one of the dangerous pathologic conditions which leads to severe scrotal pain and further injuries of the testes which is regarded as an emergency condition. In males, it has been reported the incidence of testicular torsion peaks under the age of 25 years old; however, it may be seen in any age group and it is estimated to occur in 1 out of 4000 males (1).

The degree and the duration of torsion are two important predictors of testicular damage (2). If detorsion occurs within 4 to 6 hours after torsion, testis can be saved in 90% of cases. On the other hand, the success rate decreases to 50% after 12 hours and it drops to 10% after 24 hours. Therefore, in order to maintain the testicular tissue and prevent orchiectomy, an immediate correct diagnosis along with essential interventions, are necessary (1, 3). The twist of spermatic cord leads to reduced testicular blood flow; therefore, for reperfusion of the affected testis, an immediate surgery is needed. However, further damage to the testis results from any attempt to reperfuse the ischemic tissue.

Several studies have been reported that disruption of the seminiferous epithelium and disappearance of germ cells may occur after ischemia/reperfusion (IR) injury in the testis (3-5). Reactive oxygen species (ROS) have been reported as a possible cause of IR-induced damage (3). An increase in the level of ROS leads to DNA damage and testicular germ cell apoptosis (3, 4). Thus, to prevent reperfusion injury, combinations of enzymes, chemical drugs, and herbal extracts have been used after testicular torsion/detorsion or ischemic/reperfusion, along with performing histopathological assessments (6-8). These protocols are intended for inhibition of oxidative stress. For example, several studies have been reported that using zinc aspartate reduces IR-induced injury and also increases the activity of antioxidant enzymes (2, 3). Medicinal herbs are cost-effective and less severe side effects than conventional pharmacological drugs. Therefore, nowadays, they have a special place in the treatment of infertility (9, 10).

One of the perennial plants that belongs to Asteraceae family is chamomile (Matricaria chamomile (MC) which grows in the West Europe and North Africa. It has been used as a tea to treat stomach disorders in traditional medicine. Moreover, the antispasmodic effects of chamomile can reduce the possibility of preterm delivery in women and also alleviate menstrual cramps. It is also used to stimulate menstruation. The stimulating effects of MC extract on leukocytes, such as macrophages and B lymphocytes, can be effective in the treatment of skin inflammation and eczema. The soothing effect of MC extract on the central nervous system is useful for the treatment of insomnia. Also, both lipophilic and hydrophilic components of chamomile extract have great therapeutic activities (9, 10).

Unstable oils and flavonoids, including apigenin, rutin, and luteolin, are the most main active compounds of hydroalcoholic extract of chamomile. Flavonoids, as phenyl benzopyrone chemicals, are observed in all vascular plants. Also, it has been reported that the benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products and has aromatase inhibitory activity (9, 10). Several clinical and experimental studies which were performed on M. recutita reported that the majority of its pharmacological actions are dependent on its antioxidant activity that reduces the free radicals and inhibits lipid peroxidation (9-11). Therefore, we decided to investigate the hydroalcoholic extract of MC on oxidative stress and tissue damage caused by torsion/detorsion in the testes of rats.

In this experimental study, all experimental procedures were approved by the animal Ethics Committee of Gonabad University of Medical Sciences, Gonabad, Iran. Twenty-eight male Wistar rats weighing 200-250 g were maintained for 2 weeks on a moderate fiber (MF) diet and had free access to food and water. They were kept in the animal room at a constant temperature (25 ± 2°C) at 30-70% humidity with 12 hour light/12 hour dark cycles. Rats were randomly divided into 4 groups as follows: sham group (G1) that underwent a surgery without induction of torsion; torsion/detorsion group (T/D or G2) in which testicular torsion was induced for 4 hours followed by detorsion for 24 hours; G3 or T/DMC group in which testicular torsion was induced for 4 hours and rats intraperitoneally received 300 mg/ kg of hydroalcoholic extracts of MC, 30 minutes before detorsion then experienced detorsion for 24 hours; and G4 or MC group in which rats intraperitoneally received 300 mg/kg of hydroalcoholic extracts of MC for 24 hours without application of torsion (5-7).

Ischemia-reperfusion (IR) is the main phenomenon that occurs following testicular torsion and causes testicular damage, apoptosis, and even infertility. The histological damage caused by IR injury in testis has been shown in several studies with different time period and degree of torsion and different time period of detorsion. As in this study, 4-hour torsion and 24-hour reperfusion caused damage to the testicles (6, 12, 13). Ischemia and reperfusion can lead to tissue damage through several mechanisms including increasing ROS levels and production and secretion of inflammatory factors (6). Former research has shown that the severity of ischemic histological damage depends on two important factors namely, the duration and degree of torsion (14).

Yulug et al. (6) showed that 4-hour ischemia followed by 24-hour reperfusion could cause testicular tissue damage. Previous studies have also shown that torsion of 720 degrees is enough to stop the testicular blood flow in a rat model (6, 7, 12-16). In the present study, according to previous studies, we induced 4-hour ischemia following by 24-hour reperfusion.

Furthermore, our present study showed that torsion of 720 degrees for 4 hours and a consecutive reperfusion for 24 hours led to edema. Moreover, histological features such as degeneration of germ cells layer and decreases in the seminiferous tubule diameter, Johnson’s score and the number of germ cells were observed. Spermatogenesis is an extremely regulated process which is mainly controlled by testosterone and gonadotropins (17). In a study, Moghimian et al. (1, 18) showed that 5-hour ischemia followed by 24-hour reperfusion reduced serum levels of testosterone.

As a fact, the half-life of testosterone in the blood is 24 hours. Also, IR in testicles results in damages in testis tissue such as Leydig cells, which act as the source of testosterone secretion. In the present study, a statistically significant difference in serum levels of testosterone was observed. It was significantly decreased in the T/D group. One study reported that 30 minutes of ischemia followed by reperfusion leads to decreased levels of GPx but increased levels of SOD level, 24 hours after the procedure (19, 20). These findings show that the antioxidant defense against oxidative stress is activated after the testicular ischemia and reperfusion. On the other hand, Ozkan et al. (2) reported that 4-hour torsion followed by detorsion led to decreased levels of SOD but increased levels of MDA 4 hours after the procedure.

In the present study, the serum and tissue levels of SOD and GPx in the T/D group significantly decreased while the serum and tissue levels of MDA increased. In agreement with our results, Ozbek et al. (21) and Ozturk et al. (22) in separated studies showed that testicular torsion for 4 hours and detorsion increase tissue levels of MDA and reduce SOD and GPx levels. According to the previous studies, it can be concluded that the effects of chamomile on serum testosterone levels act in a dose-dependent manner so that low doses can reduce serum testosterone levels while high doses increase serum testosterone levels (23).

In an experimental study, Johari et al. (23) showed that an intraperitoneal injection (10, 20, and 40 mg/kg) of M. chamomile flower extract reduced the serum level of testosterone in male rats. Another study has reported that testosterone levels decrease in rats which received M. chamomile extract (400 mg/kg) for 8 weeks (24). Moreover, Hatami and Estakhr (25) reported that MC 100 mg/kg increases serum testosterone levels, the function of the hormonal pituitary- testis axis, and spermatogenesis. The present study showed that 300 mg/kg of MC extract can significantly increase the serum levels of testosterone in the T/DMC and MC groups as compared to the T/D group. Therefore, MC extract by preventing Leydig cells damage and its components, increases the serum levels of testosterone.

On the other hand, in the present research, we observed that the serum level of testosterone in MC group was higher than that of sham group. Possibly, chamomile extracts exert its effect via its flavonoids, phenolic compounds, and alpha-bisabolol content and also through its antioxidant potentials which result in neutralization free radicals (9, 10). One study has reported that hydroalcoholic extract of MC and its compounds such as flavonoids increase the serum levels of testosterone (26). Antioxidants are compounds that prevent the formation of free radicals and inhibit lipid peroxidation; therefore, they can be effective in treatment of infertility induced by oxidative stress (27, 28). The enzymatic antioxidants, such as SOD and catalase have an important role in the prevention of cells insults induced by oxidative conditions (29).

Several studies have reported that extract of MC reduced the lipid peroxidation (as reflected by MDA levels) and increased the serum level of SOD, catalase, and glutathione (10, 30). In addition, one study reported that MC extract decreased the level of MDA in the brain tissue and increased the tissue level of SOD and GPx (5). Finally, our study showed that the dose of 300 mg/kg of MC extract decreased the level of MDA while increased the levels of SOD and GPx.

According to the results of the present study, the extract of Matricaria chamomile could change the level of testosterone and protect the tissue against damage and oxidative stress following testicular torsion/detorsion.