Royal jelly mediates fibrotic signaling, collagen cross-linking and cell proliferation in cardiac fibroblasts.

Royal jelly (RJ) is a multifunctional bee product with a unique composition and wide-ranging biological properties, including antioxidant, anti-inflammatory and antiproliferative activities. Still, little is known about the possible myocardial protective properties of RJ. Considering that sonication could enhance RJ bioactivity, this study aimed to assess the effects of non-sonicated (NS) and sonicated (S) RJ on fibrotic signaling, cell proliferation, and collagen production in cardiac fibroblasts. S-RJ was produced by ultrasonication at 20 kHz. Ventricular fibroblasts isolated from neonatal rats were cultured and treated with different concentrations of NS-RJ or S-RJ (0, 50, 100, 150, 200, and 250 µg/well). S-RJ significantly depressed the expression levels of transglutaminase 2 (TG2) mRNA across all the concentrations tested and was inversely associated with the expression of this profibrotic marker. S-RJ and NS-RJ displayed distinct dose-dependent effects on mRNA expression of several other profibrotic, proliferation, and apoptotic markers. Unlike NS-RJ, S-RJ elicited strong negative dose-dependent relationships with the expression of profibrotic markers (TG2, COL1A1, COL3A1, FN1, CTGF, MMP-2, α-SMA, TGF-ß1, CX43, periostin), as well as proliferation (CCND1) and apoptotic (BAX, BAX/BCL-2) markers, indicating that RJ dose-response effects were significantly modified by sonification. NS-RJ and S-RJ increased the content of soluble collagen, while decreasing collagen cross-linking. Collectively, these findings show that S-RJ has a greater range of action than NS-RJ for downregulating the expression of biomarkers associated with cardiac fibrosis. Reduced biomarker expression and collagen cross-linkages upon cardiac fibroblast treatment with specific concentrations of S-RJ or NS-RJ suggests putative roles and mechanisms by which RJ may confer some protection against cardiac fibrosis.

Inhibition of transglutaminase 2 (TG2) ameliorates ventricular fibrosis in isoproterenol-induced heart failure in rats.

AIMS: Transglutaminase (TG) inhibitors represent promising therapeutic interventions in cardiac fibrosis and related dysfunctions. However, it remains unknown how TG inhibition, TG2 in particular, affects the signaling systems that drive pathological fibrosis. This study aimed to examine the effect TG inhibition by cystamine on the progression of isoproterenol (ISO)-induced cardiac fibrosis and dysfunction in rats. MATERIALS AND METHODS: Cardiac fibrosis was established by intraperitoneal injection of ISO to rats (ISO group), followed by 6 weeks of cystamine injection (ISO + Cys group). The control groups were administered normal saline alone or with cystamine. Hemodynamics, lipid profile, liver enzymes, urea, and creatinine were assessed in conjunction with heart failure markers (serum NT-proANP and cTnI). Left ventricular (LV) and atrial (LA) fibrosis, total collagen content, and mRNA expression of profibrotic markers including TG2 were quantified by Masson's trichrome staining, LC-MS/MS and quantitative PCR, respectively. KEY FINDINGS: Cystamine administration to ISO rats significantly decreased diastolic and mean arterial pressures, total cholesterol, triglycerides, LDL, liver enzymes, urea, and creatinine levels, while increasing HDL. NT-proANP and cTnI serum levels remained unchanged. In LV tissues, significant reductions in ISO-induced fibrosis and elevated total collagen content were achieved after cystamine treatment, together with a reduction in TG2 concentration. Reduced mRNA expression of several profibrotic genes (COL1A1, FN1, MMP-2, CTGF, periostin, CX43) was also evidenced in LV tissues of ISO rats upon cystamine administration, whereas TGF-ß1 expression was depressed in LA tissues. Cystamine decreased TG2 mRNA expression in the LV of control rats, while LV expression of TG2 was relatively low in ISO rats irrespective of cystamine treatment. SIGNIFICANCE: TG2 inhibition by cystamine in vivo exerted cardioprotective effects against ISO-induced cardiac fibrosis in rats decreasing the LV abundance of several profibrotic markers and the content of TG2 and collagen, suggesting that TG2 pharmacological inhibition could be beneficial to alleviate cardiac fibrosis.

Involvement and possible role of transglutaminases 1 and 2 in mediating fibrotic signalling, collagen cross-linking and cell proliferation in neonatal rat ventricular fibroblasts.

Transglutaminase (TG) isoforms control diverse normal and pathophysiologic processes through their capacity to cross-link extracellular matrix (ECM) proteins. Their functional and signalling roles in cardiac fibrosis remain poorly understood, despite some evidence of TG2 involvement in abnormal ECM remodelling in heart diseases. In this study, we investigated the role of TG1 and TG2 in mediating fibrotic signalling, collagen cross-linking, and cell proliferation in healthy fibroblasts by siRNA-mediated knockdown. siRNA for TG1, TG2 or negative control was transfected into cultured neonatal rat ventricular fibroblasts and cardiomyocytes. mRNA expression of TGs and profibrotic, proliferation and apoptotic markers was assessed by qPCR. Cell proliferation and soluble and insoluble collagen were determined by ELISA and LC-MS/MS, respectively. TG1 and TG2 were both expressed in neonatal rat cardiomyocytes and fibroblasts before transfection. Other TGs were not detected before and after transfection. TG2 was predominantly expressed and more effectively silenced than TG1. Knocking down TG1 or TG2 significantly modified profibrotic markers mRNA expression in fibroblasts, decreasing connective tissue growth factor (CTGF) and increasing transforming growth factor-ß1 compared to the negative siRNA control. Reduced expression of collagen 3A1 was found upon TG1 knockdown, while TG2 knockdown raised α-smooth muscle actin expression. TG2 knockdown further increased fibroblast proliferation and the expression of proliferation marker cyclin D1. Lower insoluble collagen content and collagen cross-linking were evidenced upon silencing TG1 or TG2. Transcript levels of collagen 1A1, fibronectin 1, matrix metalloproteinase-2, cyclin E2, and BCL-2-associated X protein/B-cell lymphoma 2 ratio were strongly correlated with TG1 mRNA expression, whereas TG2 expression correlated strongly with CTGF mRNA abundance. These findings support a functional and signalling role for TG1 and TG2 from fibroblasts in regulating key processes underlying myocardial ECM homeostasis and dysregulation, suggesting that these isoforms could be potential and promising targets for the development of cardiac fibrosis therapies.

Association of vitamin D levels and polymorphisms in vitamin D receptor with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a leading cause of death. The prevalence of T2DM in countries of the Middle East and North Africa (MENA) region, including Jordan, is among the highest worldwide. The reason(s) behind the epidemic nature of T2DM in Jordan are unknown but warrant further exploration. Studies have indicated that T2DM could be influenced by diet and/or genetic background. Evidence suggests that numerous patients with T2DM are deficient in vitamin D. The activity of vitamin D on its target tissues may be influenced by single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR) gene. It was therefore hypothesized that SNPs in VDR could modify the risk of T2DM. To test this hypothesis, 125 patients with T2DM were recruited along with 125 controls. The study subjects were genotyped for variations in rs2228570, rs1544410, rs7975232, and rs731236 SNPs in the VDR. The levels of 25-hydroxyvitamin D [25(OH)D] were measured from the serum. The analysis revealed that reduced 25(OH)D and age were associated with the risk of T2DM (P<0.05). Moreover, under a dominant inheritance model, the GG genotype of rs2228570 was revealed to increase the risk of T2DM in univariate and multivariate analysis (P<0.05). Additionally, a chromosomal block containing the GAAG haplotype of VDR SNPs increased the risk of T2DM (OR=1.909; CI: 1.260-2.891; P=0.0021). Collectively, the present study revealed that low levels of serum 25(OH)D and rs2228570 of the VDR gene are associated with the risk of T2DM.

Correlation between vitamin D and serum brain derived neurotropic factor levels in type 2 diabetes mellitus patients.

Diabetes Mellitus (DM) currently ranks as the most common endocrine disorder worldwide. Current opinion views DM as a group of heterogeneous metabolic diseases characterized by hyperglycemia triggered by defects in the ability of the body to produce or use insulin in type 1 and 2 DM, respectively. Brain-derived neurotrophic factor (BDNF), one of the neurotrophin family of growth factors, has been linked to the pathogenesis of DM and insulin resistance. Moreover, vitamin D has been associated with insulin resistance and DM. Recently, the interactions between vitamin D and BDNF have been investigated in diabetic rats. However, this correlation has never been investigated in humans. Thus, the aim of the present study was to assess the alterations in serum BDNF and vitamin D levels in T2DM patients in Jordan, prior to and following vitamin D supplementation. A combination of non-experimental case-control and experimental designed studies were utilized to assess the relationship between serum BDNF and vitamin D levels in T2DM patients. The levels of BDNF and vitamin D were measured using commercially available ELISA kits, and fasting blood glucose (FBG) and HbA1c levels were measured in medical labs. The results showed that diabetic patients had lower levels of serum vitamin D and higher levels of BDNF compared with the healthy controls. Moreover, linear regression analysis indicated that BDNF levels were inversely correlated with serum vitamin D levels. Furthermore, vitamin D supplementation significantly increased vitamin D serum levels and decreased BDNF serum levels in diabetic patients. Intriguingly, FBG and HbA1c levels were significantly improved post vitamin D supplementation. These data demonstrate a positive effect of vitamin D supplementation in diabetic patients suggesting the implementation of vitamin D as part of future T2DM treatment plans. However, additional studies are needed to investigate the direct link between vitamin D, BDNF, and T2DM.

Implications of enigmatic transglutaminase 2 (TG2) in cardiac diseases and therapeutic developments.

Cardiac diseases are the leading cause of mortality and morbidity worldwide. Mounting evidence suggests that transglutaminases (TGs), tissue TG (TG2) in particular, are involved in numerous molecular responses underlying the pathogenesis of cardiac diseases. The TG family has several intra- and extracellular functions in the human body, including collagen cross-linking, angiogenesis, cell growth, differentiation, migration, adhesion as well as survival. TGs are thiol- and calcium-dependent acyl transferases that catalyze the formation of a covalent bond between the γ-carboxamide group of a glutamine residue and an amine group, thus increasing the stability, rigidity, and stiffness of the myocardial extracellular matrix (ECM). Excessive accumulation of cross-linked collagen leads to increase myocardial stiffness and fibrosis. Beyond TG2 extracellular protein cross-linking action, increasing evidence suggests that this pleiotropic TG isozyme may also promote fibrotic diseases through cell survival and profibrotic pathway activation at the signaling, transcriptional and translational levels. Due to its multiple functions and localizations, TG2 fulfils critical yet incompletely understood roles in myocardial fibrosis and associated heart diseases, such as cardiac hypertrophy, heart failure, and age-related myocardial stiffness under several conditions. This review summarizes current knowledge and existing gaps regarding the ECM-dependent and ECM-independent roles of TG2 and highlights the therapeutic prospects of targeting TG2 to treat cardiac diseases.

Mechanism of Oxytocin-Induced Contraction in Rat Gastric Circular Smooth Muscle.

Oxytocin produces an excitatory effect on gastric muscle through the activation of receptors present on stomach smooth muscle cells. However, the intracellular mechanisms that mediate oxytocin excitatory effects are still largely unknown. Therefore, we aimed to investigate the signaling pathways involved in oxytocin-induced contractions in gastric smooth muscle, shedding light on phospholipase C (PLC)-ß1 signaling and its downstream molecules, including inositol 1,4,5- trisphosphate (IP3) and myosin light chain kinase (MLCK). The contractions of gastric smooth muscle from male rats were measured in an organ bath set up in response to exogenous oxytocin 10-7 M, in the presence and absence of inhibitors of the indicated signaling molecules. Oxytocin (10-9-10-5 M) induced dose-dependent stomach smooth muscle contraction. Pre-incubation with atosiban, an oxytocin receptor inhibitor, abolished the oxytocin-induced contraction. Moreover, PLC ß1 inhibitor (U73122) and IP3 inhibitor Xestospongin C inhibited oxytocin-induced muscle contraction to various degrees. Verapamil, a calcium channel blocker, inhibited oxytocin-induced contraction, and pre-incubation of the strips, with both verapamil and Xestospongin C, further inhibited the excitatory effect of oxytocin. Chelation of intracellular calcium with BAPT-AM (1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid) significantly inhibited the effect of oxytocin on muscle contraction. Finally, pre-incubation of the strips with the Ca2+/calmodulin-dependent protein kinase selective inhibitor STO-609 significantly inhibited the contraction induced by oxytocin. These results suggest that oxytocin directly stimulates its cell surface receptor to activate PLC ß1, which in turn liberates IP3, which eventually elevates intracellular calcium, the prerequisite for smooth muscle contraction.

Effect of cytokine treatment on the expression and secretion of brain derived neurotrophic factor in the smooth muscle of the rat colon.

The production of pro-inflammatory cytokines and chemokines is increased during inflammatory bowel disease (IBD). Previously, it was demonstrated that brain derived neurotrophic factor (BDNF) expression is increased in experimental models of colitis. BDNF is partially responsible for the structural and functional changes that take place during IBD. However, the exact mechanisms underlying the upregulation of BDNF during gut inflammation are unknown. The aim of the present study was to determine the effects of direct treatment of smooth muscle cells with inflammatory cytokines on the synthesis and secretion of BDNF. BDNF expression and secretion levels were measured using ELISA kits on tissue lysates and on incubation media used to culture the rat colon smooth muscle tissues treated for 24 h with either tumor necrosis factor (TNF)-α or interleukin (IL)-1ß. Compared with the control tissue samples, treatment with TNF-α and IL-1ß resulted in a significant increase in the protein expression levels of BDNF in the incubated smooth muscle tissue. TNF-α and IL-1ß also stimulated the secretion of BDNF. Chelation of intracellular Ca2+ with BABTA-AM prevented the TNF-α and IL-1ß-induced increase in BDNF protein expression and secretion levels. Furthermore, inhibition of protein kinase A (PKA) significantly reduced BDNF expression levels when treated with cytokines but not secretion. In conclusion, proinflammatory cytokines that are upregulated during IBD, directly stimulated BDNF expression and secretion in a Ca2+ dependent manner. Considering the ability of BDNF to enhance smooth muscle contraction and pain sensation, this autocrine loop may partially explain the characteristic hypercontractility and hypersensitivity associated with IBD.

Erratum: Scavenging of lipid peroxyl radicals protects plasma lipids and proteins from peroxynitrite.

[This corrects the article DOI: 10.3892/br.2018.1144.].

Erratum: Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells.

[This corrects the article DOI: 10.3892/br.2018.1161.].

Erratum: Gender differences in the regulation of MLC20 phosphorylation and smooth muscle contraction in rat stomach.

[This corrects the article DOI: 10.3892/br.2018.1053.].

Insomnia among Medical and Paramedical Students in Jordan: Impact on Academic Performance.

BACKGROUND: Insomnia is a problem that is common in all societies and age groups. However, its importance is increasing between students especially with the highly competitive and demanding environment surrounding them even after their graduation. In spite of the deep understanding of its health and social consequences, the frequency of insomnia among medical students in Jordan was not determined. AIM: To determine the prevalence of sleep disturbances among college students and to look for any association between sleep disturbances and students' academic achievement. METHODS: This is a cross-sectional self-administered questionnaire-based study. The participants were college students of the medical and paramedical specialties. Insomnia Severity Index (ISI) was used and the academic performance was assessed using students' Cumulative Grade Point Average (CGPA). RESULTS: There were 977 responses. Prevalence of clinical insomnia was 26.0%. Students who self-reported good sleep quality had significantly lower ISI scores compared with those who self-reported bad quality of sleep. Students who slept >7 hours had significantly less ISI scores than students who slept <6 hours. Students who had a CGPA more than or equal to 3 had significantly lower ISI scores compared with those who had a CGPA less than 2.5. Self-reported sleep quality was associated with the CGPA. CONCLUSION: A high prevalence of insomnia was found in this group of students. Academic performance was significantly associated with ISI scores and self-reported sleep quality. These results might be useful for future research into the development of interventional strategies to help students get enough sleep quality and quantity.

Changes in Gastric Smooth Muscle Cell Contraction during Pregnancy: Effect of Estrogen.

It is well known that pregnancy is associated with frequent gastrointestinal (GI) disorders and symptoms. Moreover, previous reports have shown that estrogen, which changes in levels during pregnancy, participates in the regulation of GI motility and is involved in the pathogenesis of various functional disorders in the stomach. The aim of the current study was to explore the changes in the expression of estrogen receptors (ERs) and examine the effect of estrogen on nitric oxide- (NO-) cyclic guanosine monophosphate (cGMP) pathway and thus relaxation in gastric smooth muscle cells (GSMC) during pregnancy. Single GSMC from early-pregnant and late-pregnant Sprague-Dawley rats were used. Protein and mRNA expression levels of ERs were measured via specifically designed enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), respectively. NO and cGMP levels were measured via specifically designed ELISA kits. Effect of estrogen on acetylcholine- (ACh-) induced contraction of single GSMC was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, N-nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Estrogen increased both NO and cGMP levels and their levels were greater in early compared to late pregnancy. Expression of ERs was greater in early compared to late pregnancy. ACh induced greater contraction of GSMC in late pregnancy compared to early pregnancy. Estrogen inhibited ACh-induced contraction in both periods of pregnancy. Importantly, pretreatment of GSMC with either L-NNA or ODQ abolished estrogen inhibitory action on muscle contraction. In conclusion, GSMC contractile behavior undergoes drastic changes in response to estrogen during pregnancy and this might explain some of the pregnancy-associated gastric disorders.

The role of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptors-α in mediating the antinociceptive effects of palmitoylethanolamine in rats.

Palmitoylethanolamine (PEA) is a ligand at peroxisome proliferator-activated receptors-α (PPARα), a nuclear receptor that has anti-inflammatory effects. Herein, complete Freund's adjuvant (CFA)-induced inflammatory pain model in rats and in-vitro calcium imaging studies were used to evaluate the mechanisms that underlie the antinociceptive effects of PEA, including modulating the activity of the transient receptor potential vanilloid receptor 1, which is a key receptor involved in the development of inflammatory pain. Adult male Sprague-Dawley rats (180-250 g) received subcutaneous injections of CFA (0.1 ml) into the plantar surface of the left hind paw. Von Frey filaments were used to determine the paw withdrawal threshold. PEA (50 µg), WY14643 (50 µg, a selective PPARα agonist) were injected into the plantar surface of the left hind paw at day 7 after CFA injection, and behavioral tests were repeated 6 h after drug administration. Rats were killed and dorsal root ganglia neurons were dissected and prepared for calcium imaging. Neurons were loaded with the calcium-sensitive ratiometric dye Fura-2AM. Changes in [Ca]i were measured as ratios of peak florescence at excitation wavelengths of 340 and 380 nm and expressed as a percentage of the KCl (60 mM) response. Both PEA and WY14643 significantly restored the paw withdrawal threshold in a PPARα-dependent fashion (P<0.01). Capsaicin of 15 nM produced 63.9±13.4% of KCl response. Preincubation of dorsal root ganglia neurons with PEA 6 h before stimulation with capsaicin, significantly reduce capsaicin-evoked calcium responses (42.9±6.4% of KCl response, n=54, P<0.001). In conclusion, modulating transient receptor potential vanilloid receptor 1 activity could provide the mechanism that underlies PEA antinociceptive effects observed in vivo.

Effect of progesterone on nitric oxide/cyclic guanosine monophosphate signaling and contraction in gastric smooth muscle cells.

Previous studies have shown that progesterone could inhibit muscle contraction in various sites of the gastrointestinal tract. The underlying mechanisms responsible for these inhibitory effects of progesterone are not fully known. The aim of the current study was to investigate the effect of progesterone on the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway and muscle contraction in the stomach. Single gastric smooth muscle cells from female Sprague-Dawley rats were used. The expression of progesterone receptor (PR) mRNA was analyzed by reverse transcription polymerase chain reaction. NO and cGMP levels were measured via specific ELISAs. Acetylcholine (ACh)-induced contraction of single gastric muscle cells preincubated with progesterone was measured via scanning micrometry in the presence or absence of the NO synthase inhibitor, Nω-Nitro-L-arginine (L-NNA), or guanylyl cyclase inhibitor, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and expressed as percent shortening from resting cell length. PR expression was detected in the stomach muscle cells. Progesterone inhibited ACh-induced gastric muscle cell contraction. Furthermore, progesterone increased NO and cGMP levels in single gastric muscle cells. Most notably, pre-incubation of muscle cells with either L-NNA or ODQ abolished the inhibitory action of progesterone on muscle contraction. These present observations suggest that progesterone promotes muscle cell relaxation in the stomach potentially via the NO/cGMP pathway.

Scavenging of lipid peroxyl radicals protects plasma lipids and proteins from peroxynitrite.

Peroxynitrite can be produced in the vasculature from a superoxide anion reaction with nitric oxide. A surplus of peroxynitrite in the intravascular compartment is a common feature of several chronic diseases. The development of pharmacological modalities that interfere with the formation of peroxynitrite or inhibit its oxidative damage may be of utility for the prevention and/or treatment of several pathologies. Our previous investigations showed that catalytically inactivating peroxynitrite-derived free radicals with tempol or scavenging reactive aldehyde species with phenelzine protects the blood plasma and platelets from the oxidative damage of peroxynitrite. However, the degree of inhibition of the cytotoxic effects of peroxynitrite using tempol or phenelzine was modest. In the present study, the aim was to examine if scavenging lipid peroxyl radicals with U-83836E can achieve superior protection from peroxynitrite. This was assessed by treating blood plasma or platelets with 100 µM peroxynitrite alone or in combination with U-83836E, and then measuring the levels of thiobarbituric acid reactive substances (TBARS) and protein carbonyl formation as indices of lipid peroxidation and protein oxidation, respectively. It was observed that scavenging lipid peroxyl radicals with 75-100 µM U-83836E increasingly reversed protein carbonylation induced by peroxynitrite in blood plasma and platelets, in addition to TBARS formation in blood plasma. These findings are further discussed in the context of the mechanisms by which U-83836E may protect against the cell-damaging effects of peroxynitrite.

Association of Adiponectin and rs1501299 of the ADIPOQ Gene with Prediabetes in Jordan.

Type 2 diabetes mellitus (T2DM) is a worldwide health problem caused by resistance to insulin action. This chronic debilitating diseaseis preceded by a stage, known as prediabetes, in which a healthy lifestyle can delay the disease. The discovery of biochemical changes in prediabetes is important to identify individuals at risk of developing T2DM and in explaining disease pathogenesis. Adiponectin is secreted by fat cells and is linked with insulin resistance. Adiponectin levels are dysregulated in prediabetic subjects. This relationship had not been tested in Jordan. We recruited 130 subjects with prediabetes and 130 control subjects. We measured serum levels of adiponectin and genotyped subjects for three single nucleotide polymorphisms (SNPs) in the ADIPOQ gene; rs266729, rs1501299 and rs2241766. In multivariate analysis, we found that serum adiponectin lowers the risk of prediabetes (p = 0.002; odds ratio (OR), 0.764; 95% confidence interval (CI), 0.646⁻0.905). The rs1501299 SNP of the ADIPOQ gene was associated with prediabetes in our population (p = 0.041). Specifically, in multivariate analysis, the GT genotype of rs1501299 increased the risk of prediabetes (p = 0.010; OR, 2.350; 95% CI, 1.231⁻4.486) as well as the TT genotype (p = 0.006; OR, 4.774; 95% CI, 1.551⁻14.693). Our findings indicate that serum adiponectin and SNPs in the ADIPOQ gene are associated with prediabetes in Jordan.

Estrogen relaxes gastric muscle cells via a nitric oxide- and cyclic guanosine monophosphate-dependent mechanism: A sex-associated differential effect.

Various gastrointestinal (GI) disorders have a higher prevalence in women than in men. In addition, estrogen has been demonstrated to have an inhibitory effect on the contractility of GI smooth muscle. Although increased plasma estrogen levels have been implicated in GI disorders, the role of gastric estrogen receptor (ER) in these sex-specific differences remains to be fully elucidated. The present study was designed to investigate the sex-associated differences in the expression of the two ER isoforms, ERα and ERß, and the effect of estrogen on gastric muscle contraction via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. Experiments were performed on single gastric smooth muscle cells (GSMCs) isolated from male and female Sprague Dawley rats. The effect of acetylcholine (ACh), a muscarinic agonist, on the contraction of GSMCs was measured via scanning micrometry in the presence or absence of 1 µM 17ß-estradiol (E2), an agonist to the majority of ERs, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), an ERα agonist, or diarylpropionitrile (DPN), an ERß agonist. The protein expression levels of ER subtypes in GSMCs were measured using a specifically designed ELISA. GSMCs from female rats had a higher expression of ERα and ERß protein compared with GSMCs from males. ACh induced less contraction in female that in male GSMCs. Pre-treatment of GSMCs with E2 reduced the contraction of GSMCs from both sexes, but to a greater extent in those from females. PPT and DPN inhibited ACh-induced contraction in GSMCs from females. Furthermore, E2 increased NO and cGMP levels in GSMCs from males and females; however, higher levels were measured in females. Of note, pre-incubation of female GSMCs with Nω-nitro-L-arginine, a NO synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylyl cyclase inhibitor, reduced the inhibitory effect of estrogen on GSMC contraction. In conclusion, estrogen relaxes GSMCs via an NO/cGMP-dependent mechanism, and the reduced contraction in GSMCs from females by estrogen may be associated with the sex-associated increased expression of ERα and ERß, and greater production of NO and cGMP, compared with that in GSMCs from males.

The 4-hydroxynonenal mediated oxidative damage of blood proteins and lipids involves secondary lipid peroxidation reactions.

Lipid peroxidation is associated with several metabolic diseases. Lipid peroxidation causes cellular damage through reactive aldehyde species such as 4-hydroxyonenal (4-HNE). The exact mechanism(s) by which 4-HNE causes damage in the intravascular compartment is not yet exactly understood. Using an in vitro system, the damage induced by 4-HNE on the blood was investigated by measuring protein carbonyl groups and thiobarbituric acid reactive substances (TBARS) following 4-HNE treatment. The findings demonstrated that treatment with 4-HNE increased the carbonylation of protein and the formation of TBARS in the blood plasma. It was also tested whether phenelzine, a scavenger of aldehyde species, or U-83836E, a scavenger of lipid peroxy radicals, attenuated the damage caused by 4-HNE. It was demonstrated that phenelzine or U-83836E both mitigated the effects of 4-HNE on the proteins and the lipids of the blood plasma. The findings of the current study suggest that phenelzine, U-83836E or functionally similar therapeutics may prevent or treat diseases that involve an increased production of 4-HNE in the intravascular compartment.

Metformin exerts anti-inflammatory effects on mouse colon smooth muscle cells in vitro.

Inflammatory bowel disease (IBD) is a chronic incurable condition characterized by relapsing inflammation of the gut. Intestinal smooth muscle cells (SMCs) are affected structurally and functionally during IBD due to excessive production of different inflammatory mediators. Metformin is a widely used antidiabetic agent known to exert several anti-inflammatory effects in different tissues independently from its hypoglycemic effect. The aim of the present study was to investigate the effect of metformin on expression and secretion of different cytokines and chemokines from mouse colon SMCs (CSMCs) following induction of inflammation with lipopolysaccharide (LPS) in vitro. CSMCs from male BALB/c mice were isolated and cultured in Dulbecco's modified Eagle's medium and treated with LPS (1 µg/ml) and 0, 5, 10 or 20 mM metformin for 24 h. Expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), macrophage colony stimulating factor (M-CSF), T cell activation gene-3 (TCA-3) and stromal cell-derived factor-1 (SDF-1) was evaluated by ELISA. LPS-treated CSMCs demonstrated significantly increased expression of TNF-α, IL-1α, M-CSF, TCA-3 and SDF-1 when compared with the control group (P<0.05). Co-treatment with metformin (5 and 10 mM) significantly reduced their expression by ~20-40% when compared with LPS treatment alone (P<0.05). Furthermore, secretion of TNF-α, IL-1α, M-CSF and TCA-3 into the conditioned media was significantly decreased by metformin (5 and 10 mM; P<0.05). In addition, metformin decreased levels of LPS-induced nuclear factor-κB phosphorylation. These data suggest that metformin may provide beneficial anti-inflammatory effects on CSMCs and it may be utilized as an adjunct therapy for patients suffering from IBD.