Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.     

Chronic Treatment with Atrial Natriuretic Peptide in Spontaneously Hypertensive Rats: Beneficial Renal Effects and Sex Differences

Objective

The aim of this study was to investigate the effects of chronic treatment with atrial natriuretic peptide (ANP) on renal function, nitric oxide (NO) system, oxidative stress, collagen content and apoptosis in kidneys of spontaneously hypertensive rats (SHR), as well as sex-related differences in the response to the treatment.

Methods

10 week-old male and female SHR were infused with ANP (100 ng/h/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). Systolic blood pressure (SBP) was recorded and diuresis and natriuresis were determined. After treatment, renal NO synthase (NOS) activity and eNOS expression were evaluated. Thiobarbituric acid-reactive substances (TBARS), glutathione concentration and glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in the kidney. Collagen was identified in renal slices by Sirius red staining and apoptosis by Tunel assay.

Results

Female SHR showed lower SBP, oxidative stress, collagen content and apoptosis in kidney, and higher renal NOS activity and eNOS protein content, than males. ANP lowered SBP, increased diuresis, natriuresis, renal NOS activity and eNOS expression in both sexes. Renal response to ANP was more marked in females than in males. In kidney, ANP reduced TBARS, renal collagen content and apoptosis, and increased glutathione concentration and activity of GPx and SOD enzymes in both sexes.

Conclusions

Female SHR exhibited less organ damage than males. Chronic ANP treatment would ameliorate hypertension and end-organ damage in the kidney by reducing oxidative stress, increasing NO-system activity, and diminishing collagen content and apoptosis, in both sexes.     

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.     

Pentadecapeptide BPC 157 Reduces Bleeding and Thrombocytopenia after Amputation in Rats Treated with Heparin,Warfarin, L-NAME and L-Arginine

Background

BPC 157 is a stable gastric pentadecapeptide recently implicated with a role in hemostasis. While NO is largely implicated in hemostatic mechanisms, in tail-amputation-models under heparin- and warfarin-administration, both the NO-synthase (NOS)-blocker, L-NAME (prothrombotic) and the NOS-substrate L-arginine (antithrombotic), were little investigated. Objective. To investigate the effect of L-NAME and L-arginine on hemostatic parameters, and to reveal the effects of BPC 157 on the L-NAME- and L-arginine-induced hemostatic actions under different pathological condition: tail amputation without or with anticoagulants, heparin or warfarin.

Methods

Tail amputation, and/or i.v.-heparin (10 mg/kg), i.g.-warfarin (1.5 mg/kg/day for 3 days) were used in rats. Treatment includes BPC 157, L-NAME, L-arginine, per se and their combination.

Results

After (tail) amputation, with or without i.v.-heparin or i.g.-warfarin, BPC 157 (10 μg/kg, 10 ng/kg, i.p., i.v. (heparin), 10 μg/kg i.g. (warfarin)) always reduced bleeding time and/or haemorrhage and counteracted thrombocytopenia. As for L-NAME and/or L-arginine, we noted: L-arginine (100 mg/kg i.p.)–rats: more bleeding, less/no thrombocytopenia; L-NAME (5 mg/kg i.p.)-rats: less bleeding (amputation only), but present thrombocytopenia; L-NAME+L-arginine-rats also exhibited thrombocytopenia: L-NAME counteracted L-arginine-increased bleeding, L-arginine did not counteract L-NAME-thrombocytopenia. All animals receiving BPC 157 in addition (BPC 157μg+L-NAME; BPC 157μg+L-arginine, BPC 157μg+L-NAME+L-arginine), exhibited decreased haemorrhage and markedly counteracted thrombocytopenia.

Conclusions

L-NAME (thrombocytopenia), L-arginine (increased haemorrhage) counteraction and BPC 157 (decreased haemorrhage, counteracted thrombocytopenia) with rescue against two different anticoagulants, implicate a BPC 157 modulatory and balancing role with rescued NO-hemostatic mechanisms.     

Levosimendan Inhibits Peroxidation in Hepatocytes by Modulating Apoptosis/Autophagy Interplay

Background

Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoK_ATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoK_ATP.

Methods and Findings

In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoK_ATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.

Conclusions

Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoK_ATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.     

Alteration in expression of estrogen receptor isoforms alpha and beta, and aromatase in the testis and its relation with changes in nitric oxide during aging in mice

The aim of present study was to investigate the changes in the testicular expression of aromatase, ER alpha, ER beta and iNOS protein and correlate these with serum testosterone and nitric oxide levels, to elucidate the role of estrogen and nitric oxide in the testis during aging. This study showed localization of aromatase and ER alpha mainly in the Leydig cell and showed close correlation of testicular aromatase level with circulating testosterone level suggesting that estrogen may be modulating testicular steroidogenesis. Localization ER alpha mainly in the mitotically active germ cell suggest possible role of estrogen in germ cell proliferation. This study showed basal level of nitric oxide during reproductively active period, whereas increased serum nitric oxide coincides with decreased testicular activity in old age. This study showed inverse correlation between aromatase and NO level. Treatment with either SNP or L-NAME on testicular steroidogenic factor (3-beta HSD/ StAR) or germ cell survival factor (Bcl2) showed that increased NO causes decreased steroidogenesis and increased germ cell apoptosis. In conclusion this study suggest that estrogen modulate steroidogenesis and germ cell survival in reproductively active period whereas in old age decreased estrogen concentration causes increased nitric oxide which in turn decreases testicular steroidogenesis and germ cell apoptosis.     

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

The dose-dependent immunoregulatory effects of the nitric oxide synthase inhibitor n(g)-nitro-L-arginine methyl ester in rats with sub-acute peritonitis

Background

Chronic inflammation accompanied by arginine deficiency, immune dysfunction, and excess nitric oxide (NO) production is a clinical condition found in patients with peritonitis. A previous study showed that the nonselective NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) may facilitate the metabolism of the immune nutrient arginine without altering NO homeostasis in rats with sub-acute peritonitis. Here, we investigated the effects of L-NAME on the immunocytic subpopulation distribution and response.

Materials and Methods

Male Wistar rats with cecal puncture-induced peritonitis were administered parenteral nutrition solutions supplemented with 0 (CPP group), 5 (LNA group), 25 (MNA group) or 50 (HNA group) mg·kg⁻¹·day⁻¹ of L-NAME for 7 days. Parenteral-fed sham-operated rats (TPN group) and orally-fed healthy rats (R group) were included as controls.

Results

The TPN group had significantly increased spleen weights and levels of plasma nitrite/nitrate (NOx), circulating white blood cells (WBC), and splenocytic T cells, as well as significantly decreased levels of cytotoxic T- and B-leukocytes and B-splenocytes compared to the R group. The CPP group had significantly decreased levels of plasma NOx and concanavalin (Con) A-stimulated interferon (IFN)-γ and interleukin (IL)-2 production by leukocytes and significantly increased production of Con A-stimulated tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS)-stimulated IFN-γ in the leukocytes. In addition, the LNA and MNA groups had significantly decreased spontaneous IL-6 and Con A-stimulated TNF-α and IFN-γ production by the leukocytes while the HNA group had significantly increased LPS-stimulated TNF-α and Con A-stimulated IFN-γ and IL-2 production by the splenocytes compared to the CPP group.

Conclusions

Low-dose L-NAME infusion may suppress proinflammatory and T-helper-1 (Th1) response in leukocytes, and high-dose infusion may activate the proinflammatory response in splenic macrophages and Th1 response in T-splenocytes in rats with sub-acute peritonitis.     

Seasonality in expression and distribution of nitric oxide synthase isoforms in the testis of the catfish, Clarias batrachus: Role of nitric oxide in testosterone production

Nitric oxide (NO) is a well-recognized versatile signaling molecule. It is produced by catalytic action of nitric oxide synthase (NOS) on L-arginine in a variety of animal tissues. Existence of different isoforms of NOS has been shown in mammalian testis, but report on their presence in the testis of ectothermic vertebrates is non-existent. This study demonstrates the differential expressions of two isoforms of nitric oxide synthase (neuronal-nNOS and inducible-iNOS) like molecules in different cell types in the testis of seasonally breeding catfish, Clarias batrachus through immunohistochemistry. Positive immunoprecipitation of nNOS and iNOS like molecules were detected in germ cells as well as interstitial cells only in the recrudescing and fully mature fish. The immunoreactions differed in intensity and varied with changing reproductive status. Treatment of adult male fish with NO donor, sodium nitroprusside, and a NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) increased and decreased the total nitrate and nitrite concentration in the testis, respectively. Sodium nitroprusside and L-NAME also induced simultaneous decline and rise in the testicular testosterone level, respectively. These findings, thus, suggest that NOS isoforms are expressed variedly in different cell types in the testis of reproductively active fish. This investigation also suggests that NO inhibits testosterone production in the testis.     

Expression and Localization of Lung Surfactant Proteins in Human Testis

Background

Surfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions.

Objective

The aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated.

Results

Surfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig.

Conclusion

Surfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP''s was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.     

Distribution of histopathology and Ia positive cells in actively induced and passively transferred experimental autoimmune orchitis

Histopathology in testes from mice with actively induced experimental orchitis (EAO) (active EAO) and those from recipients of testis-sensitized lymphocytes (passive EAO) had different distributions. In passive EAO, maximum orchitis existed in the straight tubules, rete testis, and ductus efferentes, obstruction of which led to extreme dilatation of seminiferous tubules. Unusual intralymphatic granulomata also resulted in dilated testicular lymphatics. In active EAO, maximum orchitis affected seminiferous tubules under the testicular capsule, away from the rete testes. Vasitis was common and occurred in both active and passive EAO. In normal testes, IA+ F4/80+ cells were sparse but formed a cuff around the straight tubules. After immunization with testis in adjuvant or with adjuvant alone, the number, size, and staining intensity of IA+ cells increased dramatically beginning on day 5, 7 days before disease onset. Simultaneously, epithelial cells confined to the ductus efferentes became Ia+. Although recipients of sensitized lymphocytes also developed epithelial Ia in the ductus efferentes, they did not show changes in testicular interstitial Ia+ cells. Our findings indicate that testicular autoantigens are not completely sequestered, but are accessible to and can react with passively transferred immune lymphocytes in well-defined regions of the germ cell compartment. These regions coincided to a large extent with maximum expression of periductal or epithelial Ia. Changes in Ia+ cells in the testis, which are inducible by adjuvants and precede orchitis, may account in part for the different distribution of histopathology of active EAO.     

Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1

Background

A distinctive feature of type 2 diabetes is inability of insulin-secreting β-cells to properly respond to elevated glucose eventually leading to β-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of β-cell dysfunction.

Principal Findings

We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon.

Conclusion

The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.     

Opposite Effect of Mast Cell Stabilizers Ketotifen and Tranilast on the Vasoconstrictor Response to Electrical Field Stimulation in Rat Mesenteric Artery

Objectives

We analyzed whether mast cell stabilization by either ketotifen or tranilast could alter either sympathetic or nitrergic innervation function in rat mesenteric arteries.

Methods

Electrical field stimulation (EFS)-induced contraction was analyzed in mesenteric segments from 6-month-old Wistar rats in three experimental groups: control, 3-hour ketotifen incubated (0.1 αmol/L), and 3-hour tranilast incubated (0.1 mmol/L). To assess the possible participation of nitrergic or sympathetic innervation, EFS contraction was analyzed in the presence of non-selective nitric oxide synthase (NOS) inhibitor L-NAME (0.1 mmol/L), α-adrenergic receptor antagonist phentolamine (0.1 µmol/L), or the neurotoxin 6-hydroxydopamine (6-OHDA, 1.46 mmol/L). Nitric oxide (NO) and superoxide anion (O₂ ^.-) levels were measured, as were vasomotor responses to noradrenaline (NA) and to NO donor DEA-NO, in the presence and absence of 0.1 mmol/L tempol. Phosphorylated neuronal NOS (P-nNOS) expression was also analyzed.

Results

EFS-induced contraction was increased by ketotifen and decreased by tranilast. L-NAME increased the vasoconstrictor response to EFS only in control segments. The vasodilator response to DEA-NO was higher in ketotifen- and tranilast-incubated segments, while tempol increased vasodilator response to DEA-NO only in control segments. Both NO and O₂ ^- release, and P-nNOS expression were diminished by ketotifen and by tranilast treatment. The decrease in EFS-induced contraction produced by phentolamine was lower in tranilast-incubated segments. NA vasomotor response was decreased only by tranilast. The remnant vasoconstriction observed in control and ketotifen-incubated segments was abolished by 6-OHDA.

Conclusion

While both ketotifen and tranilast diminish nitrergic innervation function, only tranilast diminishes sympathetic innnervation function, thus they alter the vasoconstrictor response to EFS in opposing manners.     

Tim-4 Inhibits NO Generation by Murine Macrophages

Objective

T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation.

Methods

Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot.

Results

Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages.

Conclusion

These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.     

Caffeic Acid,a Phenol Found in White Wine,Modulates Endothelial Nitric Oxide Production and Protects from Oxidative Stress-Associated Endothelial Cell Injury

Introduction

Several studies demonstrated that endothelium dependent vasodilatation is impaired in cardiovascular and chronic kidney diseases because of oxidant stress-induced nitric oxide availability reduction. The Mediterranean diet, which is characterized by food containing phenols, was correlated with a reduced incidence of cardiovascular diseases and delayed progression toward end stage chronic renal failure. Previous studies demonstrated that both red and white wine exert cardioprotective effects. In particular, wine contains Caffeic acid (CAF), an active component with known antioxidant activities.

Aim of the study

The aim of the present study was to investigate the protective effect of low doses of CAF on oxidative stress-induced endothelial injury.

Results

CAF increased basal as well as acetylcholine—induced NO release by a mechanism independent from eNOS expression and phosphorylation. In addition, low doses of CAF (100 nM and 1 μM) increased proliferation and angiogenesis and inhibited leukocyte adhesion and endothelial cell apoptosis induced by hypoxia or by the uremic toxins ADMA, p-cresyl sulfate and indoxyl sulfate. The biological effects exerted by CAF on endothelial cells may be at least in part ascribed to modulation of NO release and by decreased ROS production. In an experimental model of kidney ischemia-reperfusion injury in mice, CAF significantly decreased tubular cell apoptosis, intraluminal cast deposition and leukocyte infiltration.

Conclusion

The results of the present study suggest that CAF, at very low dosages similar to those observed after moderate white wine consumption, may exert a protective effect on endothelial cell function by modulating NO release independently from eNOS expression and phosphorylation. CAF-induced NO modulation may limit cardiovascular and kidney disease progression associated with oxidative stress-mediated endothelial injury.     

Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice

Background

Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model.

Methods

Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits.

Results

Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group.

Conclusion

Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the activation of the mitochondrial apoptosis signaling pathway in the mouse testis.     

Using Gelatin Nanoparticle Mediated Intranasal Delivery of Neuropeptide Substance P to Enhance Neuro-Recovery in Hemiparkinsonian Rats

Purpose

Intranasal administration of phospholipid-based gelatin nanoparticles (GNP) was prepared to investigate the neuro-recovery effects of neuropeptide Substance P (SP) on hemiparkinsonian rats.

Methods

The SP-loaded gelatin nanoparticles (SP-GNP) were prepared by a water-in-water emulsion method and possessed high stability, encapsulating efficiency and loading capacity. PC-12 cells were used to examine the growth enhancement of SP-GNP in vitro by MTT assays and flow cytometry (FCM). The therapeutic effects of SP-GNP on 6-hydroxydopamine (6-OHDA) induced hemiparkinsonian rats were assessed by quantifying rotational behavior and the levels of tyrosine hydroxylase (TH), phosphorylated c-Jun protein (p-c-Jun) and Caspase-3 (Cas-3) expressed in substantia nigra (SN) region of hemiparkinsonian rats.

Results

PC-12 cells under SP-GNP treatment showed better cell viability and lower degree of apoptosis than those under SP solution treatment. Hemiparkinsonian rats under intranasal SP-GNP administration demonstrated better behavioral improvement, higher level of TH in SN along with much lower extent of p-c-Jun and Cas-3 than those under intranasal SP solution administration and intravenous SP-GNP administration.

Conclusions

With the advantages of GNP and nose-to-brain pathway, SP can be effectively delivered into the damaged SN region and exhibit its neuro-recovery function through the inhibition on JNK pathway and dopaminergic neuron apoptosis.     

Proinflammatory effects of exogenously administered IL-10 in experimental autoimmune orchitis

We studied the effects of exogenously administered recombinant murine interleukin (IL)-10 on the development of experimental autoimmune orchitis (EAO) in C3H/He mice. IL-10 significantly augments histological signs of EAO when administered for 6 consecutive days from days 15 to 20 after primary immunisations with testicular germ cells. These data demonstrate that IL-10, in addition to its well-known antiinflammatory property, also has proinflammatory functions capable of up-regulating testicular immunoinflammatory processes in vivo.     

Deregulation of Hepatic Insulin Sensitivity Induced by Central Lipid Infusion in Rats Is Mediated by Nitric Oxide

Background

Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis.

Methodology

We investigated here whether hypothalamic nitric oxide (NO) could mediate deleterious peripheral effect of central lipid overload. Thus we infused rats for 24 hours into carotid artery towards brain, either with heparinized triglyceride emulsion (Intralipid, IL) or heparinized saline (control rats).

Principal Findings

Lipids infusion led to hepatic insulin-resistance partly related to a decreased parasympathetic activity in the liver assessed by an increased acetylcholinesterase activity. Hypothalamic nitric oxide synthases (NOS) activities were significantly increased in IL rats, as the catalytically active neuronal NOS (nNOS) dimers compared to controls. This was related to a decrease in expression of protein inhibitor of nNOS (PIN). Effect of IL infusion on deregulated hepatic insulin-sensitivity was reversed by carotid injection of non selective NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and also by a selective inhibitor of the nNOS isoform, 7-Nitro-Indazole (7-Ni). In addition, NO donor injection (L-arginine and SNP) within carotid in control rats mimicked lipid effects onto impaired hepatic insulin sensitivity. In parallel we showed that cultured VMH neurons produce NO in response to fatty acid (oleic acid).

Conclusions/Significance

We conclude that cerebral fatty acid overload induces an enhancement of nNOS activity within hypothalamus which is, at least in part, responsible fatty acid increased hepatic glucose production.     

Chronic Testosterone Replacement Exerts Cardioprotection against Cardiac Ischemia-Reperfusion Injury by Attenuating Mitochondrial Dysfunction in Testosterone-Deprived Rats

Background

Although testosterone deficiency is associated with increased risks of heart disease, the benefits of testosterone therapy are controversial. Moreover, current understanding on the cardiac effect of testosterone during cardiac ischemia-reperfusion (I/R) periods is unclear. We tested the hypothesis that testosterone replacement attenuates the impairment of left ventricular (LV) function and heart rate variability (HRV), and reduces the infarct size and arrhythmias caused by I/R injury in orchiectomized (ORX) rats.

Methodology

ORX or sham-operated male Wistar rats (n = 24) were randomly divided and received either testosterone (2 mg/kg, subcutaneously administered) or the vehicle for 8 weeks. The ejection fraction (EF) and HRV were determined at baseline and the 4^th and 8^th week. I/R was performed by left anterior descending coronary artery ligation for 30 minutes, followed by a 120-minute reperfusion. LV pressure, arrhythmia scores, infarct size and cardiac mitochondrial function were determined.

Results

Prior to I/R, EF and HRV were impaired in the ORX group, but were restored in the testosterone-treated group. During I/R, arrhythmia scores and the infarct size were greater, and cardiac mitochondrial function was impaired, whereas the time to 1^st VT/VF onset and the LV end-systolic pressure were decreased in the ORX group when compared to the sham group. Testosterone replacement attenuated the impairment of these parameters in ORX rats during I/R injury, but did not show any benefit or adverse effect in non-ORX rats.

Conclusions

Testosterone replacement restores cardiac function and autonomic regulation, and exerts cardioprotective effects during the I/R period via mitochondrial protection in ORX rats.