Effect of sorghum ethyl-acetate extract on benign prostatic hyperplasia induced by testosterone in Sprague–Dawley rats

ABSTRACT

Benign prostatic hyperplasia (BPH) is commonly observed in men > 50 years worldwide. Phytotherapy is one of the many treatment options. Sorghum (Sorghum bicolor L.) contains various health-improving phytochemicals with antioxidant and inhibitory activities on cell proliferation, both in vitro and in vivo. To confirm the effects of Donganme sorghum ethyl-acetate extract (DSEE) on BPH, we induced BPH in Spragye–Dawley rats using exogenous testosterone. We measured prostate weight, examined prostrates histopathologically, and analyzed mRNAs associated with male hormones and proteins associated with cell proliferation in the prostate. DSEE inhibited weight gain of the prostate; decreased mRNA expressions of androgen receptor and 5α-reductase II; and improved histopathological symptoms, the protein-expressed ratio of Bax/Bcl-2, and the oxidative status of BPH induced by testosterone in SD rats. Therefore, DSEE may have potential as a preventive or therapeutic agent against BPH.     

Mutagenic activity of metabolites contained in the plasma of Sprague—Dawley rats treated with cyclophosphamide

In the present study we have tried to add some new results to those data previously obtained by Natarajan et al. (1983) and Darroudi and Natarajan (1985), where they have used in vivo metabolization and cytogenetic testing for in vitro analysis of xenobiotic compounds. Sprague-Dawley rats were treated intraperitoneally with 2.5, 5.0, 10.0 and 20.0 mg/kg b.w. of cyclophosphamide in order to obtain plasma containing active metabolites of the drug. The mutagenic activity was assessed by estimating the frequencies of sister-chromatid exchanges (SCE) in human and rat lymphocytes. No influence of animal age was observed on the metabolism of cyclophosphamide, which could be detected by SCE analysis. The increase in SCE frequencies in both human and rat lymphocytes was dependent on the doses applied. SCE frequencies are highly variable among individuals, showing statistically significant differences. The same effect, but to a lesser extent, was also found in rats. Rat lymphocytes can be assumed to be good biological material for chemical mutagenesis, as the animals can be maintained at almost constant experimental conditions. However, rat lymphocytes do not grow well in in vitro cultures. These data contribute to the preview proposal that combining metabolism in vivo and chromosome SCE analysis in vitro can be regarded as an important and very sensitive system to detect the mutagenic activity of low-dose exposure to chemical compounds requiring metabolic activation.     

Endothelial cell apoptosis in brown adipose tissue of rats induced by hyperinsulinaemia: the possible role of TNF-α

The aim of the present study was to investigate whether hyperinsulinaemia, which frequently precedes insulin resistance syndrome (obesity, diabetes), induces apoptosis of endothelial cells (ECs) in brown adipose tissue (BAT) and causes BAT atrophy and also, to investigate the possible mechanisms underlying ECs death. In order to induce hyperinsulinaemia, adult male rats of Wistar strain were treated with high dose of insulin (4 U/kg, intraperitonealy) for one or three days. Examinations at ultrastructural level showed apoptotic changes of ECs, allowing us to point out that changes mainly but not exclusively, occur in nuclei. Besides different stages of condensation and alterations of the chromatin, nuclear fragmentation was also observed. Higher number of ECs apoptotic nuclei in the BAT of hyperinsulinaemic rats was also confirmed by propidium iodide staining. Immunohistochemical localization of tumor necrosis factor-alpha (TNF-α) revealed increased expression in ECs of BAT of hyperinsulinaemic animals, indicating its possible role in insulin-induced apoptotic changes. These results suggest that BAT atrophy in hyperinsulinaemia is a result of endothelial and adipocyte apoptosis combined, rather than any of functional components alone.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Does estrogen contribute to the hepatic regeneration following portal branch ligation in rats?

The aim of this study was to determine whether estrogen plays any role in the hepatic regeneration of nonligated lobe following portal branch ligation (PBL). Male rats were subjected to PBL on the left and middle lobes. Two and 7 days after PBL, the rats were killed and blood and liver samples were analyzed. Sham animals underwent only laparotomy. The serum estradiol levels were significantly elevated on day 2 following PBL and returned to normal levels on day 7. The expression of estrogen receptors (ER) in the liver evaluated by Western blotting did not show any change in the nonligated lobe compared with shams. Immunohistochemical study for ER showed a predominant ER expression in the hepatocyte nucleus in periportal area (zone 1), although there was no apparent difference in the amount and expression pattern between sham and PBL. However, chronic inhibition of ER by an ER antagonist (ICI 182,780) showed a significantly lower regeneration rate of the nonligated lobe compared with vehicle treatment. Liver regeneration-associated genes also were less activated in the ICI group. Moreover, portal venous flow, determined by fluorescent microsphere injection, was significantly lower in the ICI group compared with vehicle group. These changes correlated with the attenuated expression of endothelial nitric oxide synthase mRNA in both superior mesenteric arteries and veins. In conclusion, these results indicate that the estrogen's contribution on hepatic regeneration following PBL is at least partly mediated through maintaining mesenteric blood flow by mesenteric endothelial nitric oxide synthase upregulation rather than directly activating liver regeneration in the liver.     

Microbe-induced T cell apoptosis: subversion of the host defense system?

Cells of multicellular organisms are equipped with a self destruction program called apoptosis to ensure homeostasis of the organism. Contraction of the lymphocyte compartment following recovery from an infection is controlled by this mechanism. But apoptosis of lymphocytes might be an Achilles tendon accessible to microbes to subvert the immune system. Evidence is cumulating that microbes use this mechanism to destroy microbe-specific T cells. We present an overview of microbe-induced T cell apoptosis discussing the consequences for the pathogenesis of microbial infection. The conventional role of lymphocytes during infection is to impose apoptotic threat to infected cells, the subject of this review highlights the opposite, lymphocytes as targets of microbe-induced death.     

Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Ralpha protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.     

The induction of male sexual behavior in red deer (Cervus elaphus) by the administration of testosterone to hinds and estradiol-17β to stags

One-gram implants of testosterone were placed subcutaneously in two adult intact and one castrated red deer hinds, and 100-mg implants of estradiol-17β were given to three castrated and two intact red deer stags. Their sexual behavior was then observed in the wild and in captivity for up to 3 years. Testosterone initially produced prolonged and intense estrous behavior in the hinds; this gradually gave way to behavior normally shown only by rutting stags, such as flehmen, herding threats, and roaring. Testosterone-implanted hinds rose in the dominance hierarchy as measured by competition for food. Pregnancy, or the administration of progestagens, suppressed most of this testosterone-induced behavior. Testosterone also induced development of male secondary sexual characteristics such as formation of small antler pedicles (but not antlers), hypertrophy of the clitoris and neck musculature, and development of a neck mane and male rutting odor. Estradiol-17β mimicked the behavioral effects of testosterone when given to castrated stags, stimulating all components of rutting behavior. In intact stags, the only effects were to abolish antler casting and stimulate roaring; normal rutting behavior continued unchanged. In contrast to the effects of testosterone, estradiol-17β did not influence the social status of either intact or castrated stags. The distinctions between the behavioral effects of androgens and estrogens are ill defined and are determined more by the prior sexual differentiation of the brain and the duration of steroid treatment, than by the nature of the steroid.     

Is administration of n-3 fatty acids by mucosal enema protective against trinitrobenzene-induced colitis in rats?

We investigated the protective role of fish oil (FO-source of n-3 FA) enriched diet (in the first protocol) in 20 rats and FO administration intrarectally (in the second protocol) in 40 rats with trinitrobenzene (TNB) colitis. All colonic specimens were pathologically evaluated, myeloperoxidase enzyme activities were measured, leukotriene B4 (LTB4) and LTC4 levels were determined by radioimmunoassay. In the first protocol 10 rats (group A1) were fed with 8% sunflower and cotton oil enriched diet and (group A2) with 8% FO enriched diet for 6 weeks. At the end of this period, TNB (30 mg in 0.25 ml of 30% ethanol) were intrarectally administered. After 2 weeks, rats were sacrificed. MPO activities (2.47 versus 30.17), LTB4 (34.5 versus 903.3) and LTC4 (77.7 versus 456.0) levels were significantly reduced in group A2 compared with group A1 (P<0.005). There was also a significant difference in pathologic scores (1.55 versus 2.12, P<0.002) between two groups. In the first part of the second protocol, 20 male rats were randomized into two equal groups (B1 and B2) and TNB colitis was induced. After 1 day, 1 ml of saline (group B1) or n-3 FA enemas (group B2) were administered every day for 2 weeks. At the end of this period, rats were sacrificed and evaluated as done for previous groups. Although there was no significant difference between the two groups in comparison with MPO enzyme activities and pathologic scores, the LTB4 (130.1 versus 971.0) and LTC4 (126.0 versus 532.0) levels of FO group were significantly reduced (P<0.005). In the second part of the second protocol, 20 male rats were randomized into two groups. One millilitre of saline (group B3) or FO enemas (group B4) were administered to rats every day for 3 days. At the fourth day, TNB-colitis was induced and after 24 h rats were sacrificed. We could not find any significant difference in MPO activities, pathologic scores, LTB4 and LTC4 levels between groups B3 and B4. In conclusion, FO enriched diet decreased both pathologic damage and tissue LT levels. The second protocol of our study revealed that the long-term FO enemas decreased the LTB4 and LTC4 levels; however, did not have any beneficial effect on the tissue lesions. Short periods of FO enemas did not have a protective role in the occurrence of experimental colitis. The present study showed that FO enemas significantly decreased LT levels. The protective effect of FO (oral and enema) in TNB colitis may open a new insight into the treatment of inflammatory bowel disease.     

Effect of single-dose administration of FK-506 on allogeneic limb transplantation in rats: is there an optimal timing of its administration?

Although a number of experimental studies have demonstrated that high-dose administration of FK-506 induces the success of limb allogeneic transplantation in rats, some of them have reported occurrences of lethal side effects. Therefore, a more effective regimen with lower-dose administration of this agent must be developed. The objective of this study was to determine an optimal timing of a single-dose administration of FK-506 in rat limb allografts. In the current study, orthotopic hindlimb transplantations were performed using major histocompatibility mismatched pairs of inbred rats. The rats were classified into five groups on the basis of the different time schedules of FK-506 administration as follows: syngeneic group, Lewis-to-Lewis transplantation; control group, ACI-to-Lewis without any immunotherapy; day 0 group, recipients treated with a single-dose administration of FK-506 (5 mg/kg of body weight, intramuscular injection) at day 0 postoperatively; day 1 group, with a single-dose administration of FK-506 at day 1 postoperatively; and day 2 group, with a single-dose administration of FK-506 at day 2 postoperatively. The median graft survival time in each group (n = 5) was 9 days in the control group, 19 days in the day 0 group, 49 days in the day 1 group, and 42 days in the day 2 group. The values in the day 1 and the day 2 groups significantly increased compared with those in other groups. For prolonged survival of a grafted limb, a single-dose administration of FK-506 is more effective at 24 to 48 hours after transplantation.     

The role of PKCζ in amikacin-induced apoptosis in the cochlea: Prevention by aspirin

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-κB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCζ, a key regulator of NF-κB activation, in response to aminoglycoside treatment. Amikacin induced PKCζ cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-κB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCζ cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCζ acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity. E. Lecain and B. Omri both authors contributed equally.     

Effects of Cadmium Chloride and Sodium Selenite Alone or in Combination on the Liver of Male Sprague–Dawley Rats Assessed by Different Assays

This study assessed the impact of either cadmium chloride (Cd) or sodium selenite (Se) alone or in combination on male Sprague–Dawley rats. For this purpose, body and liver weights, comet and TUNEL assays, histological analysis and levels of lipid peroxidation and antioxidants in liver were determined in four groups of male Sprague–Dawley rats. The rats were given subcutaneous doses of 1 mg/kg body weight (BW) of either normal saline (control = Ct) or Cd or Se or Cd plus Se (Cd + Se) on alternate days for 4 weeks. The Cd group showed increased DNA damage, apoptosis and hepatic levels of lipid peroxidation and altered histology. Conversely, the antioxidant levels in this group were decreased as compared with the control group. The Se group also showed DNA damage, apoptosis and altered histology and reduced catalase activity, but it was less severe than the Cd group. In the Cd + Se group, ameliorating effects of Se on Cd-induced changes were observed. While the Se was able to curtail the toxic effect of Cd, the Cd or Se alone were genotoxic and cytotoxic for rats receiving a high pharmacological but non-fatal dose of 1 mg/kg BW.     

Cell apoptosis in the testis of male rats is elevated by intervention with β-endorphin and the mu opioid receptor

β-endorphin (β-EP) is involved in the regulation of male germ cells; however, little is known about the effect of β-EP on primary germ cells via opioid receptors. In this study, we first revealed significant cell apoptosis in the testis of male rats after β-EP intervention. Subsequently, the expression of the mu opioid receptor (MOR) was detected in both Leydig cells (LCs) and spermatogonia (SGs) by fluorescence colocalization; overlapping signals were also detected in apoptotic cells. In addition, LCs and SGs were separated from the testis of male rats and primary cells were treated with β-EP; this increased the mRNA levels of MOR and was accompanied by acute cell apoptosis. Our findings provide a foundation for the further study of apoptosis in reproductive cells regulated by β-EP and the MOR receptor.     

Immunoreactivity to glial cell line-derived neurotrophic factor and its receptors in the trout pancreas: a further endocrine-exocrine relationship?

Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting the survival of several neuronal populations in the central, peripheral and autonomous nervous system. Outside the nervous system, GDNF functions as a morphogen in kidney development and regulates spermatogonial differentiation. GDNF exerts its roles by binding to glial cell line-derived neurotrophic factor receptor (GFR) a1, which forms a heterotetramic complex with rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor. In this study we report the presence of GDNF-, RET- and GFRa1-like immunoreactivity in the pancreas of juvenile trout. GDNF immunoreactivity was observed in the islet cells, while GFRa1- and RET- immunoreactivity was observed in the exocrine portion. These findings suggest a paracrine role of GDNF towards exocrine cells showing GDNF receptors GFRa1 and RET. The relationship could reflect physiological interactions, as previously indicated in mammalian pancreas, and/or a trophic role by endocrine cells on exocrine parenchyma, which shows a conspicuous increase during animal growth.     

Effect of glucocorticoid administration on the rate of muscle protein breakdown in vivo in rats, as measured by urinary excretion of Nτ-methylhistidine

The role of glucocorticoids in regulating the rate of muscle protein breakdown was evaluated by measuring excretion of N(tau)-methylhistidine during administration of various doses of corticosterone to adrenalectomized rats. Groups of rats received daily subcutaneous injections of 0, 0.2, 0.5, 1.0, 5.0 or 10.0mg of corticosterone/day per 100g body wt. for 7 days, followed by 3 days without hormone treatment, after which they were killed. A group with intact adrenal glands served as an additional control. All animals were pair-fed with the untreated adrenalectomized group. No significant differences were noted in growth rate or N(tau)-methylhistidine excretion between the intact or adrenalectomized control groups, or those given 0.2, 0.5 and 1.0mg of corticosterone, whereas growth ceased and N(tau)-methylhistidine excretion rose markedly in the groups receiving 5 and 10mg of corticosterone. After these two high doses of corticosterone, but not after lower doses, there was a loss of weight of the gastrocnemius muscle per 100g of final body wt., but not of the soleus and extensor digitorum longus muscles. The two highest doses of corticosterone also resulted in an increase in liver weight per 100g of final body wt. Lower doses of corticosterone did not cause these changes. Plasma corticosterone concentrations, measured on the final day of injection and again at the time of killing, were decreased to near zero by adrenalectomy and were little raised by doses of 0.2 and 0.5mg daily, but were increased to within the normal range by the 1mg dose. At 5 and 10mg doses, plasma corticosterone concentrations were sustained at 2-3 times those of intact rats, and thus in the range reported for rats exposed to severe stress. Rats given 5 and 10mg doses of corticosterone had glycosuria, and showed considerably elevated concentrations of insulin in the plasma. It is concluded that plasma concentrations of glucocorticoids within the normal range do not regulate the rate of muscle protein breakdown, whereas excessive plasma concentrations of corticosteroids, equivalent to those observed in severe stress, can accelerate muscle protein breakdown.     

Persistent mucus accumulation: a consequence of delayed bronchial mucous cell apoptosis in RAO-affected horses?

This study examined the contribution of delayed apoptosis of bronchial mucous cells to mucus accumulation in equine recurrent airway obstruction (RAO). In pilot studies, Bcl-2, an apoptosis inhibitor, was detected in airway mucous cells of RAO-affected horses in remission and during acute disease, when most mucus was secreted. To study whether delayed apoptosis results in an increase in the number of mucous cells during disease recovery, six RAO-affected and six control horses were fed hay for 5 days to induce inflammation and then pellets for 7 days to partially resolve RAO before euthanasia. RAO-affected horses had more airway obstruction and luminal mucus than control horses under both management systems. At the time of euthanasia, RAO-affected horses had more inflammation and Bcl-2-positive bronchial mucous cells than control animals. In horses with >10 and <10 neutrophils per microliter of bronchoalveolar lavage fluid, >50% and <10% of mucous cells stained positive for Bcl-2, respectively. No differences in mucous cell number or amount of stored mucosubstance were observed between RAO-affected and control horses, but in RAO-affected animals, the amount of stored mucosubstance decreased as the number of neutrophils in bronchoalveolar lavage fluid increased. Because the number of mucous cells was similar in both groups of horses but only mucous cells of RAO-affected horses expressed Bcl-2 during recovery from acute disease, a conclusive role for Bcl-2 in prolonging bronchial mucous cell life could not be determined. Future studies are needed to compare horses that are kept in remission for prolonged periods when all mucous cells are fully developed.