Article, Urology

Berberine inhibits the ischemia-reperfusion induced testicular injury through decreasing oxidative stress

a b s t r a c t

Purpose: The aim of this study was to investigate the effect of berberine (BBR) on oxidative stress in an experimental testicular I/R injury model.

Methods: Eighteen rats were divided into three groups: control group, torsion-detorsion (T/D) group, and BBR + T/D group. In the pre-treatment of the BBR group, 200 mg/kg BBR was given intraperitoneally 30 min before detorsion. Tissue malondialdehyde , total oxidant status (TOS), and total antioxidant status (TAS) levels were determined using colorimetric methods. Histological evaluation of the tissue samples was evaluated using hematoxylin-eosin staining.

Results: In T/D group, tissue MDA, TOS, and oxidative stress index levels were higher than control group. These increases were significantly reversed with BBR pre-treatment. Although Johnsen scores were lower in T/D group than the control group, BBR pre-treatment recovered the Johnsen scores.

Conclusion: These results suggest that BBR can inhibit I/R-induced testicular injury by suppressing oxida- tive stress. Further studies may prove that BBR is a useful agent as an adjunctive treatment in surgical repair in human cases.

(C) 2019

Introduction

testicular torsion (TT) is a urological emergency situation espe- cially in adolescents and young men [1]. The incidence of TT is esti- mated to be around 1 in 4000 per year. The time intervals between torsion and detorsion, and the degree of torsion are the main fac- tors determining the severity of testicular injury. When testicular torsion occurs, blood flow is disrupted and edema, bleeding, arte- rial obstruction, and hypoxia developed. Because the blood flow in the testes is limited, they are particularly susceptible to ischemic conditions. With this loss of function, there is a decrease in fertility in the ipsilateral testis and testicular atrophy is developed in sev- ere cases [2]. Surgical intervention is therefore usually necessary for reperfusion of the testis. However, testicular atrophy that may occur after reperfusion is common [1]. Surgical detorsion

* Corresponding author.

E-mail address: [email protected] (S. Demir).

has a testicular salvage rate of 42-88%, but it is still unknown whether these testicles have actually been saved [3]. It is therefore clinically important to find therapeutic applications as an adjunc- tive treatment in surgical repair [1].

The main pathophysiology of testicular torsion/detorsion (T/D) is Ischemia-reperfusion injury. I/R induces the production of Reactive oxygen species thought to play a critical role in tis- sue injury [4]. ROS are produced in all mammalian cells due to acti- vation of normal cellular metabolism oxidant-producing enzymes and in response to exogenous stimuli. The balance between ROS production and antioxidant defense determines the degree of oxidative stress [5,6]. ROS has negative effects on the structure and function of macromolecules, such as DNA, carbohydrates, lipids, proteins, and enzymes in cells [7,8]. Mammalian testes are also highly susceptible to oxidative free radical damage [1]. Several pharmacological agents, such as phosphodiesterase inhibitors, vitamin C and E, selenium, flavonoids, NSAIDs, Ethyl pyruvate, and N-acetyl cysteine, have been investigated for their potential

https://doi.org/10.1016/j.ajem.2019.04.001

0735-6757/(C) 2019

as an adjunctive therapy in the surgical repair of TT in animal mod- els. These chemicals usually contain anti-inflammatory, antioxi- dant, or ROS scavenging properties [9-12]. However, none of these agents have been used as adjunctive therapy in torsion repair in humans [1].

Although various pharmacological agents have been developed for the treatment of diseases, the use of Medicinal plants continues to increase [13]. In the modern medical system, standard drugs are not available to protect the testicular tissue against various dam- ages. Generally, some bioactive compounds found in plants may help to protect cells against oxidative stress by the prevention or

Table 1

A summary of the procedures in the experimental groups.

Control

T/D

BBR + T/D

Torsion 0 min

210 min after torsion

240 min after torsion (detorsion + 2 h)

+

+

+

200 mg/kg

+

Tissue samples + 6 h

+

+

+

T/D, torsion/detorsion; BBR, berberine.

Groups

detoxification of free radicals and to prevent various dysfunctions [14]. Berberine (C20H19NO5, BBR) is an isoquinone quaternary alka- loid which can be isolated from many medicinal plants, such as Hydrastis canadensis, Berberis aristata, Coptis chinensis, Coptis rhi- zome, Coptis japonica, Phellodendron amurense [15]. It is widely used in the treatment of various diseases, such as gastrointestinal disorders, inflammation, hyperlipidemia, and insulin resistance in traditional medicine [16]. Several reports demonstrated that it has many biological activities, such as antihypertensive, anti- arrhythmic, antihyperglycemic, anticancer, antidepressant, anxi- olytic, neuroprotective, antioxidant, anti-inflammatory, analgesic, and hypolipidemic [15,17]. Nowadays, the berberine is getting more interest from researchers because of its excellent biological activity. Therefore, scientific studies on the biological activities of berberine are increasing every year [18].

In recent years, some scholars have done a lot of experimental studies on protective effect of BBR on I/R injury in various organs, including brain, heart, intestine, and liver. Chen et al. demonstrated that BBR exhibits a protective effect against intestinal I/R injury model by decreasing the levels of malondialdehyde (MDA), Myeloperoxidase , and Diamine oxidase in rats [19], while Liu et al. reported that BBR protects brain tissue against I/R injury through its antioxidant activity [20]. Recently, Zheng et al. reported that BBR improves renal functions in a rat renal I/R injury model by inhibiting apoptosis and oxidative stress [21]. However, there is no study investigating the effect of BBR on the testicular I/R injury model. This study was aimed to determine the role of BBR in pro- tecting testicular tissue against I/R injury and to explore the main mechanisms.

Materials and methods

Chemicals

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA.) unless otherwise noted. Berberine chloride was dis- solved in 10% dimethyl sulfoxide (DMSO), then suspended in nor- mal saline before use (the final concentration of DMSO never exceeded 1%).

Study design

This protocol was a randomized, controlled, and interventional animal study approved by the Karadeniz Technical University Ani- mal Care and Ethics Committee. Eighteen mature male Sprague- Dawley rats (weighing 250-300 g) were used. Animals were kept in steel cages at a Room temperature of 22 ?C and water and fed on a standard chow pellet diet and with ad libitum access to tap water. Eighteen rats were randomly divided into three groups of six individuals each: Group 1 (control group): Only laparotomy was performed, and testis tissue samples were taken after 6 h. Group 2 (T/D group): Laparotomy was performed and the left testis was rotated 720 degrees clockwise to establish torsion. After 4 h of torsion, detorsion was performed and maintained for 2 h. Group 3

(BBR + T/D group): The same T/D procedures were performed in this group, but 200 mg/kg BBR was administered intraperitoneally 30 min before detorsion [19,22]. Experimental interventions are summarized in Table 1. General anesthesia was induced by intra- muscular injection of 10 mg/kg xylazine and 50 mg/kg ketamine. After detorsion time, testis tissue samples were taken. The tissue specimens were divided into small volume tubes and stored at

–80 ?C until measurements.

Biochemical analysis

Sections weighing approximately 30 mg were taken from all tis- sues. The tissues were homogenized at 9500 rpm in 1 mL of phos- phate buffer (PBS, pH:7.4) using a homogenizer (IKA, T25 Ultra- Turrax, Staufen, Germany). The homogenates were then cen- trifuged at 1800 xg for 10 min. The supernatant sections were

carefully separated and used for the measurement of biochemical

parameters.

Tissue malondialdehyde measurement was performed using the method described by Mihara and Uchiyama [23]. This method is based on measuring the color of the molecule formed by MDA with thiobarbitUric acid (TBA) in an acidic environment at an absorbance of 532 nm. Homogenates were centrifuged at 1800 xg for 10 min. Supernatants obtained by centrifugation were

diluted with PBS at ratio of 1:10 and MDA measurement was per-

formed. 500 lL of homogenate was mixed 3 mL of 1% H3PO4 and 1 mL of 0.672% TBA. After mixing, this was incubated for 60 min in a boiling water bath. Next, the tubes were left to cool at room

temperature and were then centrifuged at 1800 xg for 10 min. 200 lL of specimens were taken from the resulting supernatant and placed into 96-well plate. Absorbances were then read in a

microplate reader spectrophotometer (Versamax, Molecular Devices, CA, USA) at 532 nm. 1,1,3,3-Tetramethoxypropane was used as a standard and a concentration vs. absorbance graph was plotted. Tissue MDA levels were calculated as nmol MDA/g wet tis- sue using this graph.

Tissue TAS and TOS levels were determined using commercial colorimetric kits (Rel Assay Diagnostics, Gaziantep, Turkey) according to manufacturer’s recommendations. TOS and TAS levels were expressed as lmoL H2O2 equivalent/L and mmoL trolox equivalent/L, respectively. The TOS/TAS ratio was used as the OSI and was calculated using the formula [2]:

OSI = TOS, lmoL H2O2 equivalent/L/[(TAS lmoL trolox equivalent/L) x 10].

Histological analysis

Testis tissues were incubated in Bouin solution at room temper- ature for 72 h. The fixed tissue samples were dried through 70%, 90%, 96% and 100% alcohol series. They were then passed through the xylene solution and made transparent. After the preparation of the paraffin blocks, 5 lm thick sections were taken using an auto- mated microtome. The sections were stained with hematoxylin- eosine (H&E) for general morphology and analyzed under a light

microscope (Olympus BX 51, Tokyo, Japan) at high magnification by a histologist blinded to the particular animal groups. The find- ings obtained were photographed with a camera attachment adapted to a light microscope (Olympus DP71, Tokyo, Japan). The

Table 3

A comparison of histopathological scores in the groups.

Parameters Control T/D BBR + T/D

Johnsen scores 10.0 +- 0.001 7.0 +- 0.817a 9.25 +- 0.500b

histological sections were graded for testicular injury and sper- matogenesis using the Johnsen score (JS). At least 50 tubules were evaluated and each tubule was scored from 1 to 10. Ten points expressed complete spermatogenesis with regular tubules; 9

Diameter of seminiferous tubules (lm)

Thickness of germinal epithelium (lm)

263 +- 3.28 222 +- 11.9a 256 +- 5.88b

92.6 +- 1.71 64.8 +- 11.7a 88.4 +- 4.39b

points, many spermatozoa and irregular germinal epithelium; 8 points, presence of few spermatozoa; 7 points, no spermatozoa, many spermatids; 6 points, no spermatozoa, few spermatids; 5 points, no spermatozoa or spermatids; 4 points, few spermato- cytes; 3 points, presence of spermatogonia; 2 points; sertoli cells only; and 1 point, complete absence of germ cells and spermatogenesis [24].

Statistical analysis

The results were expressed as arithmetic mean and standard deviation (X +- SD). Statistical analysis of the results were done using SPSS 23.0 statistical package program. The conformity of the data to the normal distribution was checked with Kolmogorov-Smirnov test. One-way ANOVA was used to compare more than two independent groups with normal distribution. The Tukey test was used for post-hoc evaluations within the group. p < 0.05 was considered statistically significant.

Results

In this study, the effects of BBR on I/R induced oxidative stress, and histopathologic scores were evaluated.

As shown in Table 2, in T/D group tissue MDA level was higher than the control group, and BBR pre-treatment decreased the enhanced MDA level by I/R injury. Although there are numerical changes, these changes are not statistically significant (p > 0.05). Tissue TOS, and OSI levels were significantly higher in the T/D group compared to the control group (p = 0.004, and p = 0.001), but the levels of these parameters were significantly reduced by BBR pre-treatment (p = 0.021 for both). In T/D group TAS level was significantly lower than the control group (p = 0.012), and BBR pre-treatment significantly enhanced the decreased TAS level by I/R injury (p = 0.039).

As shown in Table 3, the histopathological score was signifi- cantly lower in the T/D group compared to the control group (p = 0.0001), but the level of histopathological score was signifi- cantly restored by BBR pre-treatment (p = 0.0001). Similarly, diam- eter of seminiferous tubules, and thickness of germinal epithelium was significantly lower in the T/D group compared to the control group (p = 0.0001 for both), but these parameters were signifi- cantly restored by BBR pre-treatment (p = 0.0001, and p = 0.002). In the control group, regular seminiferous tubular morphology with normal spermatogenesis was detected. In the T/D group, degeneration and cell loss in the seminiferous tubule epithelium

Table 2

A comparison of Biochemical parameters in the groups.

Parameters Control T/D BBR + T/D

MDA (nmol/g tissue)

335 +- 52.6

438 +- 140

301 +- 72.2

TAS (mmol trolox Eq/L)

8.82 +- 0.631

7.19 +- 0.699a

8.54 +- 0.707b

TOS (lmol H2O2 Eq/L)

51.2 +- 10.9

96.2 +- 26.8a

48.6 +- 9.57b

OSI

0.591 +- 0.139

1.35 +- 0.426a

0.569 +- 0.099b

p values according to One way ANOVA test, post hoc Tukey test. Data were expressed as mean +- SD.

a p < 0.05 compared with control group.

b p < 0.05 compared with T/D group.

p values according to One way ANOVA test, post hoc Tukey test. Data were expressed as mean +- SD.

a p < 0.05 compared with control group.

b p < 0.05 compared with T/D group.

was observed. In the BBR pre-treatment group, seminiferous tubule structure in normal morphology and mature sperm cells in lumen of seminiferous tubule were observed (Fig. 1).

Discussion

Testicular torsion is a urological emergency situation, especially in adolescent and young men, which is estimated to occur in one in 158 men under 24 years of age. Surgical detorsion should be per- formed immediately to prevent loss of function in ipsilateral testes. Approximately 25% of men with a history of torsion will experience adult-onset infertility [1]. Although testicular recovery with surgi- cal detorsion rates have been reported to be between 42 and 88%, it is unclear whether the functions are fully conserved [9]. Therefore, it is clinically important to find therapeutic applications as addi- tional treatment in surgical repair [1]. Current clinical and experi- mental data suggest that unilateral testicular ischemia induced by torsion of the spermatic cord resulted in specific and irReversible changes in the histology of the ipsilateral and contralateral testis; damage is proportional to the duration of ischemia. These irre- versible changes can be associated with a subsequent reduction in fertility [25]. Torsion in the testes is a cause of ischemic damage. The goal of treatment is to correct blood flow in ischemia and improve tissue perfusion. After detorsion, excessive amounts of oxygen are introduced into the tissues and this causes the overpro- duction of ROS. Increased number of neutrophils in the testicular circulation and overproduction of ROS cause tissue damage by damaging the cellular membranes. This process, called ”I/R injury”, is characterized by oxidative stress, which is the main cause of organ damage. Cellular damage after ischemia-reperfusion results from the imbalance between ROS and antioxidant defense mecha- nisms [26]. The testis is particularly vulnerable to oxidative stress due to the abundance of highly unsaturated fatty acids. Oxidative stress decreases sperm functions due spermatozoa membranes are very sensitive to oxidative damage. It has been reported that after testicular detorsion process, oxidative stress impairs testicu- lar functions by disrupting the normal structure of seminiferous tubules and diminishing the number of germ cells. biochemical markers of oxidative stress are more sensitive indicators of tissue damage and can be detected much earlier than histological changes [27]. Therefore, various substances, including anti-inflammatory and antioxidant drugs, are used to prevent I/R injury [26].

The use of plants with pharmacological properties is the main

medical resource in Developing countries. Approximately 80% of the population from the developing countries of Africa and Asia uses traditional medicine for diagnosis, treatment, Disease prevention, and health protection [15]. Berberine is an isoquinoline alkaloid found in a number of important medicinal plants [28]. It has many biological activities, such as antitumor, anti-HIV, anti- fungal, cardioprotective, immunoregulative, antimalarial, anti- inflammatory, antioxidant, anxiolytic, and analgesic [29]. BBR has been widely used in many studies related to I/R injury due to its

Fig. 1. Photomicrographs of hematoxylin and eosin stained sections of testicle of rats (200x). (A) Seminiferous tubule epithelial structure (“) was observed in normal morphology in control group and there were many matured spermium (?) in lumen. (B) In T/D group, the seminiferous tubule epithelial structure (“) was disrupted in some places. Loss was observed in germinal cells forming the tubule epithelium. Germinal epithelial cells were observed intensively in the tubular lumen (w). (C) In BBR + T/D group, the seminiferous tubule epithelium structure (“) was close to normal. Mature sperm cells (?) were observed in the lumen of the seminiferous tubule.

Antioxidant properties [19-21]. However, there is no study to determine the protective effect of BBR in the testicular I/R injury. The main aim of our study was to evaluate the potential preven- tive effect of BBR on the I/R injury after testicular T/D using the histopathological and biochemical methods. In this study, we therefore examined the histological changes, and oxidative stress status in the testicular tissue after I/R injury. The MDA, TOS, and OSI levels in the T/D group are significantly higher than control group and this situation was also consistent with our hypothesis. BBR pretreatment decreased the enhanced level of oxidative stress by I/R injury. Histological evaluation using a microscopic scoring system has been accepted as a good standard in the assessment of I/R injury [30]. In this study, the decrease in Johnsen scores seen in T/D group showed that I/R damage was performed successfully. According to our results, when T/D was applied, significant I/R associated histopathological damage developed and BBR showed

a protective effect against this injury.

Consistent with our results, Sheng et al. reported that BBR dramatically restored liver function, reduced histopathological damage, the level of oxidative stress and apoptosis compared to the I/R group in a rat model. It is demonstrated that BBR treatment effectively blocked the increase of MDA with an elevation of super- oxide dismutase (SOD) activity in the same study [16]. Visnagri et al. demonstrated that berberine exhibits a protective role on kid- ney tissue against I/R injury by increasing the levels of SOD and glutathione (GSH) and decreasing the levels of MDA and MPO [31], while Yu et al. reported that BBR protects heart tissue against I/R injury through decreasing MDA, MPO levels, and increasing SOD activity [32]. Zhao et al. demonstrated that BBR pre- treatment reduces I/R-induced tissue damage via suppresses myocardial apoptosis and oxidative damage [33], while Chu et al. reported that BBR prevents brain tissue against I/R injury damage through decreasing MDA and nitric oxide levels, and increasing SOD activity [34]. Recently, Zhu et al. demonstrated that BBR pro- tects brain tissue against I/R injury through its anti-inflammatory effects mediated by suppressing the activation of HMGB1/TLR4/ NF-jB signaling [35]. In studies investigating the effects of BBR on oxidative stress in testicle tissue, Dkhil et al. reported that BBR protects testicular tissue against Schistosoma mansoni– induced tissue damage through decreasing the level of MDA, and increasing the level of SOD, Glutathione peroxidase (GPx), glu- tathione reductase (GR), and catalase (CAT) [29], while Saleh et al. demonstrated that BBR protects testicles against gossypol- induced damage by reducing the levels of inflammation and oxida- tive stress [36].

Seconder metabolites, including alkaloids, are an important component of medicinal plants. The antioxidant effect of these compounds is attributed to their ability to donate electrons to ROS, chelating metal ions, and stimulating antioxidant and detox- ifying enzymes [37-39]. We therefore think that the protective effect of BBR against testicular I/R injury may derive from its pow- erful antioxidant activity. Our study was not without limitations. First, we applied BBR only 200 mg/kg. In a future study, a dose- dependent assay should be performed to assess the likelihood of an additional testicular protective effect. Second, because this was an experimental study, our conclusions regarding the effec- tiveness of BBR on I/R induced testicular damage and reducing associated tissue injury also need to be supported by clinical research. We believe that further studies are needed to include these findings in clinical use.

Conclusion

Based on these results, early treatment with BBR after testicular torsion is found to have local and systemic antioxidant effects and is protective against oxidative stress related tissue damage. The testicular protective effect of BBR may be related to its antioxidant effect.

Ethics committee approval

Ethics committee approval was received for this study from the ethics committee of Ethical Committee for Experimental Research on Animals.

Sources of support

There was no source of support.

Conflict of interest

None declared.

References

  1. Payabvash S, Salmasi AH, Kiumehr S, Tavangar SM, Nourbakhsh B, Faghihi SH, et al. Salutary effects of N-acetylcysteine on apoptotic damage in a rat model of testicular torsion. Urol Int 2007;79(3):248-54.
  2. Tusat M, Mentese A, Demir S, Alver A, Imamoglu M. Medical ozone therapy reduces oxidative stress and testicular damage in an Experimental model of testicular torsion in rats. Int Braz J Urol 2017;43(6):1160-6.
  3. Aktas BK, Bulut S, Bulut S, Baykam MM, Ozden C, Senes M, et al. The effects of N-acetylcysteine on testicular damage in experimental testicular ischemia/ reperfusion injury. Pediatr Surg Int 2010;26(3):293-8.
  4. Huang KH, Weng TI, Huang HY, Huang KD, Lin WC, Chen SC, et al. Honokiol attenuates torsion/detorsion-induced testicular injury in rat testis by way of suppressing endoplasmic reticulum stress-related apoptosis. Urology 2012;79

    (4):967 (e5-11).

    Aliyazicioglu Y, Demir S, Turan I, Cakiroglu TN, Akalin I, Deger O, et al. Preventive and protective effects of Turkish propolis on H2O2-induced DNA damage in foreskin fibroblast cell lines. Acta Biol Hung 2011;62(4):388-96.

  5. Turan I, Deger O, Aliyazicioglu Y, Demir S, Kilinc K, Sumer A. Effects of Turkish propolis on expression of hOGG-1 and NEIL-1. Turk J Med Sci 2015;45 (4):804-11.
  6. Mentese U, Dogan OV, Turan I, Usta S, Dogan E, Oztas Mentese S, et al. Oxidant- antioxidant balance during on-pump Coronary artery bypass grafting. Sci World J 2014:. https://doi.org/10.1155/2014/263058263058.
  7. Yalcin CO, Aliyazicioglu Y, Demir S, Turan I, Bahat Z, Misir S, et al. Evaluation of the radioprotective effect of Turkish propolis on foreskin fibroblast cells. J Cancer Res Ther 2016;12(2):990-4.
  8. Turkmen S, Mentese A, Karaguzel E, Karaca Y, Kucuk A, Uzun A, et al. A comparison of the effects of N-acetylcysteineand ethyl pyruvate on experimental testicular ischemiareperfusion injury. Fertil Steril 2012;98:626-31.
  9. Karaguzel E, Kadihasanoglu M, Kutlu O. Mechanisms of testicular torsion and potential protective agents. Nat Rev Urol 2014;11:391-9.
  10. Turkmen S, Mutlu A, Sahin A, Karaca Y, Mentese A, Demir S, et al. Effects of N- acetylcysteine and ethyl pyruvate on ischemia-reperfusion injury in experimental electrical burn model. Am J Emerg Med 2016;34(7):1217-24.
  11. Turkmen S, Cekic Gonenc O, Karaca Y, Mentese A, Demir S, Beyhun E, et al. The effect of ethyl pyruvate and N-acetylcysteine on ischemia-reperfusion injury in an experimental model of ischemic stroke. Am J Emerg Med 2016;34 (9):1804-7.
  12. Turan I, Demir S, Kilinc K, Arslan Burnaz N, Ozer Yaman S, Akbulut K, et al. Antiproliferative and apoptotic effect of Morus nigra extract on human prostate cancer cells. Saudi Pharm J 2017;25(2):241-8.
  13. Gholampour F, Karimifard F, Owji SM. Berberine improves kidney injury following renal ischemia reperfusion in rats. Int J Zool Res 2015;11(1):9-18.
  14. Tillhon M, Guaman Ortiz LM, Lombardi P, Scovassi AI. Berberine: new perspectives for old remedies. Biochem Pharmacol 2012;84(10):1260-7.
  15. Sheng M, Zhou Y, Yu W, Weng Y, Xu R, Du H. Protective effect of berberine pretreatment in hepatic ischemia/reperfusion injury of rat. Transplant Proc 2015;47(2):275-82.
  16. Kumar A, Ekavali Chopra K, Mukherjee M, Pottabathini R, Dhull DK. Current knowledge and pharmacological profile of berberine: an update. Eur J Pharmacol 2015;761:288-97.
  17. Wang K, Feng X, Chai L, Cao S, Qiu F. The metabolism of berberine and its contribution to the pharmacological effects. Drug Metab Rev 2017;49 (2):139-57.
  18. Chen T, Lv B. Protective effects of berberine on intestinal ischemia and reperfusion injury in rats. Arch Biol Sci 2017;69(1):45-51.
  19. Liu H, Ren X, Ma C. Effect of berberine on angiogenesis and HIF-1/VEGF signal transduction pathway in rats with cerebral ischemia-reperfusion injury. J Coll Physicians Surg Pak 2018;28(10):753-7.
  20. Zheng H, Lan J, Li J, Lv L. Therapeutic effect of berberine on renal ischemia- reperfusion injury in rats and its effect on Bax and Bcl-2. Exp Ther Med 2018;16(3):2008-12.
  21. Ma YG, Zhang YB, Bai YG, Dai ZJ, Liang L, Liu M, et al. Berberine alleviates the cerebrovascular contractility in streptozotocin-induced diabetic rats through

    modulation of intracellular Ca2+ handling in smooth muscle cells. Cardiovasc Diabetol 2016;15:63.

    Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978;86(1):271-8.

  22. Johnsen S. Testicular biopsy score count-a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1970;1:2-25.
  23. Quintaes IP, Tatsuo ES, Paulo DN, Musso C, Boasquevisque PC. Decompressive fasciotomy in testicular torsion of the spermatic cord in rats. Acta Cir Bras 2013;28(6):423-9.
  24. Caglayan EK, Caglayan K, Gocmen AY, Cinar H, Seckin L, Seckin S, et al. Protective effect of ethyl pyruvate on ischemia-reperfusion injury in rat ovary: biochemical and histopathological evaluation. Eur J Obstet Gynecol Reprod Biol 2014;182:154-9.
  25. Cvetkovic T, Stankovic J, Najman S, Pavlovic D, Stokanovic D, Vlajkovic S, et al. Oxidant and antioxidant status in experimental rat testis after testicular torsion/detorsion. Int J Fertil Steril 2015;9(1):121-8.
  26. Hasanein P, Ghafari-Vahed M, Khodadadi I. Effects of isoquinoline alkaloid berberine on lipid peroxidation, antioxidant defense system, and liver damage induced by lead acetate in rats. Redox Rep 2017;22(1):42-50.
  27. Dkhil MA, Abdel Moneim AE, Al-Quraishy S. Berberine protects against Schistosoma mansoni-induced oxidative damage in renal and testicular tissues of mice. Pak J Zool 2014;46(3):763-71.
  28. Ayvaz S, Aksu B, Inan M, Uzun H, Aydin S, Bilgi S, et al. The effects of N- acetylcysteine on intestinal ischemia/reperfusion injury in rats. Saudi Med J 2009;30(1):24-9.
  29. Visnagri A, Kandhare AD, Bodhankar SL. Renoprotective effect of berberine via intonation on apoptosis and mitochondrial-dependent pathway in renal ischemia reperfusion-induced mutilation. Ren Fail 2015;37(3):482-93.
  30. Yu L, Li Q, Yu B, Yang Y, Jin Z, Duan W, et al. Berberine attenuates myocardial ischemia/reperfusion injury by reducing oxidative stress and inflammation response: role of silent information regulator 1. Oxidative Med Cell Longev 2016:. https://doi.org/10.1155/2016/16896021689602.
  31. Zhao GL, Yu LM, Gao WL, Duan WX, Jiang B, Liu XD, et al. Berberine protects rat heart from ischemia/reperfusion injury via activating JAK2/STAT3 signaling and attenuating endoplasmic reticulum stress. Acta Pharmacol Sin 2016;37 (3):354-67.
  32. Chu X, Zhou Y, Zhang B, Xue B, Zhao Y. Berberine attenuates cerebral ischemia- reperfusion injury via activating PI3K-Akt signaling in a rat model of type 2 diabetes. Int J Clin Exp Med 2017;10(12):16196-202.
  33. Zhu JR, Lu HD, Guo C, Fang WR, Zhao HD, Zhou JS, et al. Berberine attenuates ischemia-reperfusion injury through inhibiting HMGB1 release and NF-jB nuclear translocation. Acta Pharmacol Sin 2018;39(11):1706-15.
  34. Saleh SR, Rana R, Ghareeb DA. The ameliorating effect of berberine-rich fraction against gossypol-induced testicular inflammation and oxidative stress. Oxidative Med Cell Longev 2018:. https://doi.org/10.1155/2018/ 10561731056173.
  35. Demir S, Turan I, Aliyazicioglu Y. Selective cytotoxic effect of Rhododendron luteum extract on human colon and liver cancer cells. J BUON 2016;21 (4):883-8.
  36. Demir S, Turan I, Demir F, Ayazoglu Demir E, Aliyazicioglu Y. Cytotoxic effect of Laurocerasus officinalis extract on human cancer cell lines. Marmara Pharm J 2017;21(1):121-6.
  37. Aliyazicioglu R, Demir S, Badem M, Sener SO, Korkmaz N, Ayazoglu Demir E, et al. Antioxidant, antigenotoxic, antimicrobial activities and phytochemical analysis of Dianthus carmelitarum. Rec Nat Prod 2017;11(3):270-84.