Effect of probucol on autophagy and apoptosis in the penile tissue of streptozotocin-induced diabetic rats.

Autophagy and apoptosis have been regarded as important processes in the development of diabetic erectile dysfunction (DMED). Probucol is considered to have anti-apoptotic effects, but its relationship with autophagy has not been reported. The aim of this study was to investigate the effects and mechanisms of probucol on erectile function. Thirty Sprague-Dawley (SD) male rats (12 weeks old) were fasted for 12 h. Twenty SD rats were injected with a single intraperitoneal injection of 60 mg kg-1 streptozotocin (STZ). Ten rats were given vehicle only and used as a sham group. After 72 h, 20 STZ-treated rats with random blood glucose concentrations consistently greater than 16.7 mmol l-1 were used as successfully established diabetic rats. The diabetic rats were divided randomly into two groups and treated with a daily gavage of probucol at a dose of 0 or 500 mg kg-1 for 12 weeks. After treatment, the intracavernous pressure (ICP) was used to measure erectile function upon electrical stimulation of the cavernous nerve. After euthanasia, penile tissue was examined using immunohistochemistry and Western blot to assess the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), microtubule-associated protein light chain 3-II (LC3-II), mammalian target of rapamycin (mTOR), and sequestosome 1 (P62). Caspase-3 activity was measured to determine apoptosis using a caspase-3 assay kit. After 12 weeks of treatment, the erectile function of the probucol group was significantly better than that of the DM group (P < 0.05). Bax and LC3-II protein expression and caspase-3 activity were significantly lower in the probucol group than those in the DM group (all P < 0.05), while Bcl-2, mTOR, and P62 protein expression levels were significantly higher than those in the DM group (all P < 0.05). We demonstrated that probucol inhibited apoptosis and autophagy in STZ-induced diabetic rats.

INTRODUCTION

Erectile dysfunction (ED) is characterized by the inability to achieve and maintain erections and can significantly reduce the quality of life of patients.1 ED affects approximately 50% of men with diabetes.2 DM is a major etiological factor of ED, and the mechanism of this effect of diabetes is still under study.

Autophagy and apoptosis are very important biologic phenomena involved in the development, growth, and maintenance of tissue architecture and physiology. Apoptosis is controlled strictly by polygenes and maintains a stable internal environment. This process involves genes such as the Bcl-2 family and the caspase family.3 Studies have shown that the proportion of apoptotic cells is significantly higher in the erectile tissue of diabetic rats than that of normal rats.4 Dysregulated apoptosis may have a direct or indirect relationship with ED in diabetic rats. Autophagy is a basic physiological process that is crucial for maintaining normal cell function. It has also been shown that autophagy is associated with diabetic ED.5 Microtubule-associated protein light chain 3-II (LC3-II) is critical for autophagosome formation. Mammalian target of rapamycin (mTOR) is an important autophagy regulator. P62 is negatively related to autophagy. Autophagy can be assessed by the expression levels of LC3-II, mTOR, and p62 in rat corpus cavernosum.6

Probucol is widely used to prevent coronary atherosclerosis and lower cholesterol levels in the clinic, and it exerts its effects through various mechanisms, such as anti-inflammatory and anti-oxidation activity.7 Our previous studies have confirmed that probucol can improve ED in diabetic rats by reducing oxidative stress.8 The relationship between autophagy and apoptosis under oxidative conditions is complex. This study was designed to investigate the effect of probucol on apoptosis and autophagy in the penile tissue of streptozotocin (STZ)-induced diabetic rats.

MATERIALS AND METHODS

Experimental animals

Thirty 12-week-old Sprague–Dawley male rats were obtained from the Animal Center of Shandong University (Jinan, China). The present experimental design was approved by the Animal Care and Use Committee of Shandong University (Jinan, China). DM was induced by a single intraperitoneal injection of 60 mg kg−1 STZ (Sigma-Aldrich, St. Louis, MO, USA) in 20 Sprague–Dawley rats. Ten rats were given vehicle only (0.1 mol l−1 citrate–phosphate buffer, pH 4.5) and used as a sham group. Animals with blood glucose levels consistently greater than 16.7 mmol l−1 were considered diabetic after 72 h. Twenty diabetic rats were established successfully, that is, 20 rats were all diabetic rats. After 12 weeks, the diabetic animals were divided randomly into two treatment groups: the diabetic control group (DM group, n = 10) and the experimental group (probucol group, n = 10). The experimental group received a daily gavage of probucol (Sigma-Aldrich) at a dose of 300 mg kg−1 for 12 weeks. The diabetic control group received physiological saline only. The body weights and blood glucose levels of all rats were recorded weekly.

Erectile function assessment

After treatment, all rats were anesthetized with 5% sodium pentobarbital. Systemic blood pressure was measured via aortic cannulation. Cavernous nerves were isolated bilaterally and stimulated with a bipolar electrode at 5 V, 15 Hz, and 1.2 ms to induce erection. The maximal intracavernous pressure (max ICP) and mean arterial pressure (MAP) were measured continuously by a BL-420 V pressure transducer system (Chengdu Implement Company, Chengdu, China).9

Western blot analysis

Ice-cold whole penile tissues were homogenized by a bead-beater for protein extraction. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal concentrations of protein lysate for each sample were loaded on 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) at 60 V for 30 min and 110 V for 60 min. Proteins were transferred electrophoretically to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA) and blocked for 1 h with 1% blocking milk. After blocking, the polyvinylidene fluoride membranes were then incubated with primary antibodies against β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ), B-cell lymphoma-2 (Bcl-2; 1:1000, Cell Signaling Technology, BSN, MA, USA), BCL2-associated X (Bax; 1:1000, Abcam, Cambridge, UK), LC3-II (1:500, Affinity Biosciences, Cincinnati, OH, USA), mTOR (1:1000, Cell Signaling Technology), and P62 (1:5000, Abcam) at 4°C overnight. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The protein bands were detected using an enhanced chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL, USA) and quantified by densitometry (Quantity One Analysis Software; Bio-Rad, Hercules, CA, USA).

Immunohistochemical staining

After euthanasia, the penises were harvested. The midshaft segments were fixed in 10% formalin and embedded in paraffin. Paraffin-embedded specimens were stained with Bax (1:250, Abcam) and Bcl-2 (1:500, Abcam) antibodies. Then, the sections were washed and incubated with secondary antibodies (1:100 dilution, Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Thereafter, the sections were incubated with 3,3-diaminobenzidine, and the cell nuclei were stained with hematoxylin. Primary antibodies were replaced with normal serum from the host species and incubated with the secondary antibody as a negative control. Sections were examined under a light microscope. Slides were examined by an observer blinded to the treatment group. Computerized densitometric analyses of Bax and Bcl-2 expression in cavernous tissue in the images were performed using Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Bethesda, MD, USA).

Caspase-3 activity assay

Caspase-3 activity in the penile tissue was determined by a Caspase-3 Activity Assay Kit (Biovision K106; Biovision, Milpitas, CA, USA) according to the manufacturer's instructions. Tissues were ground and then incubated with cold lysis buffer on ice for 15 min. The lysed tissues were centrifuged for 10 min at 16 000 g at 4°C; then, the supernatants were collected, and the protein concentrations were calculated. The supernatants were transferred to a 96-well plate containing detection buffer, and Ac-DEVD-pNA was added. After incubation at 37°C for 2 h, absorbance was measured at 405 nm with a microplate reader (Thermo Fisher Scientific). The caspase-3 activity of each sample was calculated according to the standard curve and normalized to the protein concentration.

Statistical analysis

The research results are expressed as the mean ± standard deviation (s.d.). Statistical analysis was performed with a one-way analysis of variance test with Bonferroni multiple comparison posttest. All statistical analyses were performed using SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). Statistical significance was considered at P < 0.05.

RESULTS

Body weight and blood glucose assessment

Body weights and blood glucose levels are shown in . Compared with the sham group rats, the DM group rats showed significantly higher blood glucose levels, but significantly lower body weights (all P < 0.05). Probucol treatment improved the weights of the diabetic rats without improving their blood glucose levels.

Assessment of erectile responses

The max ICP/MAP ratios are shown in . Compared with those in the sham group, the max ICP/MAP ratios were markedly lower in all diabetic rat groups (P < 0.05), whereas the diabetic rats treated with probucol exhibited greater max ICP/MAP ratios than DM rats (P < 0.05).

Western blot analysis of Bax, Bcl-2, LC3II, mTOR, and P62

The protein expression levels of Bax and Bcl-2 in rat penile tissue obtained by Western blot are shown in . LC3-II, mTOR, and P62 protein expression levels are shown in . Compared with the DM group, the probucol group had significantly increased protein expression levels of Bcl-2, mTOR, and P62 (all P < 0.05). Probucol treatment significantly decreased the protein expression levels of Bax and LC3-II compared with DM induction (both P < 0.05).

(a) Evaluation of Bax and Bcl-2 protein expression by Western blot. (b) Representative Western blot analysis of Bax and Bcl-2 in the corpus cavernosum among the groups. Each bar depicts the mean values (mean ± s.d.); n = 10 per group. *P < 0.05, the indicated group versus sham group. #P < 0.05, the indicated group versus DM group. Bax: BCL2-associated X; Bcl-2: B-cell lymphoma-2; s.d.: standard deviation; DM: diabetes mellitus.

(a) Evaluation of LC3-II, mTOR, and P62 protein expression by Western blot. (b) Representative Western blot analysis of LC3-II, mTOR, and P62 in the corpus cavernosum among the groups. β-actin was used as a loading control. Each bar depicts the mean values (mean ± s.d.); n = 10 per group. *P < 0.05, the indicated group versus sham group. #P < 0.05, the indicated group versus DM group. LC3-II: microtubule-associated protein light chain 3-II; mTOR: mammalian target of rapamycin; P62: sequestosome 1; DM: diabetes mellitus.

Immunohistochemical analysis of Bax and Bcl-2 expression

The protein expression levels of Bax and Bcl-2 in rat penile tissue endothelial cells obtained by immunohistochemistry are demonstrated in . Immunohistochemical analysis revealed that the protein expression level of Bcl-2 was significantly lower in diabetic ED rats than that in sham group (P < 0.05). After probucol treatment, the protein expression of Bcl-2 was significantly higher than that after DM induction (P < 0.05). The protein expression of Bax was significantly higher in the DM group rats than that in the sham group (P < 0.05), and 12 weeks of probucol administration significantly decreased the protein immunoreactivity of Bcl-2 in the probucol group (P < 0.05).

(a) Evaluation of Bax and Bcl-2 protein expression by immunohistochemistry (×400). (b) Representative immunohistochemistry of Bax and Bcl-2 in the corpus cavernous endothelial cells among the groups. Scale bars = 100 μm; *P < 0.05, the indicated group versus sham group (n = 10). #P < 0.05, the indicated group versus DM group (n = 10). Bax: BCL2-associated X; Bcl-2: B-cell lymphoma-2; s.d.: standard deviation; DM: diabetes mellitus.

The caspase-3 activity analysis of rat penile tissue is shown in . The caspase-3 activity was higher in the DM group rats than that in the sham group rats, and probucol reduced caspase-3 activity in the DM group rats (P < 0.05).

Caspase-3 activity in the corpus cavernous among the groups. *P < 0.05, the indicated group versus sham group (n = 10). #P < 0.05, the indicated group versus DM group (n = 10). DM: diabetes mellitus.

DISCUSSION

Although ED is not life-threatening, it can seriously affect patient's quality of life and psychology. The incidence of ED in patients with diabetes mellitus (DM) is higher than that in men without diabetes.10 The mechanism of diabetic erectile dysfunction (DMED) is largely unknown and is still under investigation. An increasing number of studies have shown that autophagy and apoptosis play important roles in the development of DMED.11 Our previous studies have confirmed that probucol can improve erectile function in diabetic rats.12 However, the impact of probucol on apoptosis and autophagy in the corpus cavernosum of diabetic rats has not been studied until now.

Apoptosis is a basic biological process regulating programmed cell death. The Bcl-2 family includes the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax, both of which play an important role in the process of apoptosis. The Bcl-2/Bax ratio is considered to be a marker of the level of apoptosis. Bcl-2 is an apoptosis-inhibiting gene, and Bax can antagonize the inhibitory effect of Bcl-2 on apoptosis and promote apoptosis. Bax plays a key role in apoptosis induced by mitochondrial stress. Bax is localized mostly in the cytoplasm. Bax can be transferred from the cytoplasm to the mitochondrial membrane to form a multimer with Bcl-2, which enhances mitochondria permeability and finally leads to the release of cytochrome c. In addition, Bax can promote Ca2+ release, activate caspase-3, and cause apoptosis.1314 Therefore, a decrease in the Bcl-2/Bax ratio further leads to an upregulation of caspase-3, which is considered to be an indicator of increased levels of apoptosis. In our experiments, it was also found that compared with that in the control group, the protein expression level of Bcl-2 in the penis tissue of STZ-induced diabetic rats was significantly decreased, and the Bax protein level and caspase-3 activity level were increased. After treatment, the protein level of Bcl-2 in the penile tissue of the probucol group increased significantly, and the protein level of the proapoptotic factor Bax and the caspase-3 activity in the penile tissue decreased significantly. These results indicate that probucol can effectively reduce the level of apoptosis in the cavernous tissues of diabetic ED rats. What is the mechanism? First, the strong antioxidant ability of probucol is a likely mechanism. Our previous study demonstrated that probucol could alleviate oxidative stress in the penile tissue of diabetic rats by activating the Nrf2 pathway. On the one hand, reduced levels of oxidative stress can reduce apoptosis induced by external or intrinsic signals, reducing caspase-3 activation and ultimately reducing apoptotic chromatin condensation and cell shrinkage. On the other hand, a decrease in the level of oxidative stress can reduce modifications of the key apoptosis regulator Bcl-2 family.15 Second, the anti-inflammatory property of probucol is a likely mechanism. Tumor necrosis factor-alpha (TNF-α) and nuclear factor kappa B (NF-κB) are believed to be the main inflammatory mediators that induce apoptosis in DM. Increasing evidence has shown that probucol can effectively reduce inflammation by inhibiting the NF-κB pathway and reducing the release of inflammatory cytokines, such as TNF-α.161718

Autophagy is a basic physiological process that helps maintain cell homeostasis, which has been shown to be involved in the pathological process of diabetic erectile dysfunction. However, the exact mechanism remains unclear. In this experiment, we observed that the LC3-II protein level in the penile tissue of DM rats was significantly decreased after probucol treatment, while the protein expression of mTOR and P62 was upregulated in penile tissue. Because the LC3-II level is directly proportional to the number of autophagic vacuoles, the level of autophagy can be reflected to some extent by this measurement. The expression of mTOR and P62 was negatively correlated with autophagy. Therefore, we conclude that probucol inhibits the level of autophagy in the penile tissue of diabetic rats induced by STZ. The mechanism of probucol in autophagy in the penile tissue of STZ-induced diabetic rats may be related to the regulation of oxidative stress and inflammation. In the corpus cavernosum of the diabetic penis, increased levels of both oxidative stress and inflammatory factors lead to increased production of reactive oxygen species (ROS). An increasing number of studies have demonstrated that ROS act as upstream modulators of autophagy induction,1920 which could induce autophagy by regulating the mTOR and mitogen-activated protein kinase (MAPK) pathways.2122 Therefore, probucol could downregulate autophagy by activating the Nrf2 pathway. Recent studies have shown that promoting Keap1 degradation by P62-dependent autophagy could in turn activate Nrf2 to prevent oxidative stress and inhibit inflammation.232425

Both autophagy and apoptosis affect cell death. These two important physiological roles can be coordinated. In some cases, autophagy inhibits apoptosis and is a pathway for cell survival. In some cases, autophagy also induces cell death or interacts with apoptosis to induce cell death as a backup mechanism in the absence of apoptosis. Many important signaling pathways, such as the PI3K/Akt pathway, can simultaneously inhibit or promote autophagy and apoptosis in cells. Key apoptosis regulators also interact with autophagy regulators. On the one hand, Bcl-2 can inhibit autophagic vesicle formation through its interacting protein Beclin 1. On the other hand, Bcl-2 blocks mTOR expression by preventing the release of Ca2+ from the receptor, thereby blocking the autophagy signaling pathway dependent on mTOR activation.26 Therefore, probucol can reduce the level of autophagy in the penile tissue of diabetic rats through its anti-apoptotic effects.

Probucol consists of two butyl-hydroxy toluene fractions that can be used as single-electron donors and singlet oxygen scavengers to remove oxygen-free radicals from cells.27 In our previous study, we demonstrated that probucol ameliorates erectile function in DMED rats. In this study, we demonstrated that autophagy and apoptosis were suppressed by probucol in STZ-induced diabetic rats. Studies have demonstrated that ROS induce apoptosis and activate autophagy in certain conditions. In our study, we summarize the mechanism of probucol in autophagy and apoptosis in the penile tissue of STZ-induced diabetic rats, which may include the following: (1) probucol inhibits apoptosis and autophagy by reducing oxidative stress and inflammation in the penile tissue of STZ-induced diabetic rats and (2) probucol indirectly inhibits autophagy through the interaction between apoptosis and autophagy. It should be noted that STZ-induced diabetes in this study is type 1 diabetes, while type 2 diabetes is more common. Although there are many similarities in the pathogenesis of DMED between type 1 and type 2 diabetes, the role of probucol in type 2 diabetes remains to be verified in future experiments.

AUTHOR CONTRIBUTIONS

KQZ and TT performed the molecular genetic studies and participated in the Western blot analysis and erectile function assessments via stimulating the cavernous nerves. LLH and HRW conducted the immunoassays and performed the statistical analyses. HRW conducted the immunoassays and performed the statistical analyses. QF conceived the study, participated in its design, helped draft the manuscript, and gave final approval of the version to be published. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.

Table 1

Comparisons of body weights and blood glucose levels in experimental animals

	Sham (n=10)	DM (n=10)	Probucol (n=10)
Initial
Body weight (g)	293.48±21.77	307.43±20.27	307.60±19.60
Glucose (mmol l−1)	6.25±1.87	6.37±1.05	6.75±1.08
After 12 weeks
Body weight (g)	592.21±44.74	280.32±14.02*	339.21±18.88*,#
Glucose (mmol l−1)	6.17±1.67	20.05±2.54*	20.33±1.70*,#

The data are expressed as the mean±standard deviation; n=10 per group. *P<0.05, the indicated group versus sham group; #P<0.05, the indicated group versus DM group. DM: diabetes mellitus

Table 2

Comparisons of mean systemic arterial pressure and maximal intracavernous pressure

	Sham (n=10)	DM (n=10)	Probucol (n=10)
Max ICP (mmHg)	98.41±11.15	65.16±6.59	73.21±5.89
MAP (mmHg)	114.01±6.58	105.53±11.16	102.57±10.30
Max ICP/MAP	0.86±0.07	0.60±0.08*	0.72±0.07*,#

The data are expressed as the mean±standard deviation; n=10 per group. *P<0.05, the indicated group versus sham group; #P<0.05, the indicated group versus DM group. MAP: mean systemic arterial pressure; Max ICP: maximal intracavernous pressure; DM: diabetes mellitus