Protective Effect Of Adrenomedullin Research Articles

Protective effect of adrenomedullin on hyperoxia-induced lung injury.

To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. HPMECs were randomly divided into an air group and a hyperoxia group (n=3 each).The HPMECs in the hyperoxia group were cultured in an atmosphere of 92% O2 (3 L/minute) +5% CO2. RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB. Other HPMECs were divided into a non-interference group and an interference group (n=3 each), and the mRNA and protein expression levels of ADM, ERK1/2, and PKB were measured after the HPMECs in the interference group were transfected with ADM siRNA. Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (P<0.05). Compared with the non-interference group, the interference group had significant reductions in the mRNA and protein expression levels of ADM, ERK1/2, and PKB (P<0.05). ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.

Adrenomedullin protects rat dorsal root ganglion neurons against doxorubicin-induced toxicity by ameliorating oxidative stress.

Objective(s):Despite effective anticancer effects, the use of doxorubicin (DOX) is hindered due to its cardio and neurotoxicity. The neuroprotective effect of adrenomedullin (AM) was shown in several studies. The present study aimed to evaluate the possible protective effects of AM against DOX-induced toxicity in dorsal root ganglia (DRGs) neurons. Materials and Methods:Rat embryonic DRG neurons were isolated and cultured. The effect of various concentrations of DOX (0.0 to 100 µM) in the absence or presence of AM (3.125 -100 nM) on cell death, apoptosis, oxidative stress, expression of tumor necrosis-α (TNF-α), interleukin1- β (IL-1β), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP) 3 and 13, and SRY-related protein 9 (SOX9) were examined. Results:Based on MTT assay data, DOX decreased the viability of DRG neurons in a dose and time-dependent manner (IC50=6.88 µm) while dose-dependently, AM protected DRG neurons against DOX-induced cell death. Furthermore, results of annexin V apoptosis assay revealed the protective effects of AM (25 nm) against DOX (6.88 µM)-induced apoptosis and necrosis of DRG neurons. Also, AM significantly ameliorated DOX-induced oxidative stress in DRG neurons. Real-time PCR results showed a significant increase in the expression of TNF-α, IL-1β, iNOS, MMP 3, and MMP 13, and a decrease in the expression of SOX9 following treatment with DOX. Treatment with AM (25 nM) significantly reversed the effects of DOX on the above-mentioned genes expression.Conclusion:Our findings suggest that AM can be considered a novel ameliorating drug against DOX-induced neurotoxicity.

Protective Effect of Adrenomedullin on Rat Leydig Cells from Lipopolysaccharide-Induced Inflammation and Apoptosis via the PI3K/Akt Signaling Pathway ADM on Rat Leydig Cells from Inflammation and Apoptosis.

This study was carried out to investigate whether ADM can modulate LPS-induced inflammation and apoptosis in rat Leydig cells. Leydig cells were treated with ADM before LPS-induced cytotoxicity. We determined the concentrations of ROS, MDA, GSH, LDH, and testosterone and the MMP. The mRNA levels of IL-1, IL-6, iNOS, and COX-2 were obtained, and the concentrations of IL-1, IL-6, NO, and PGE2 were determined. Apoptosis was assessed by TUNEL and detection of DNA fragmentation. The levels of mRNA and protein were determined for Bcl-2, Bax, caspase-3, and PARP. The protein contents for total and p-Akt were measured. ADM pretreatment significantly elevated the MMP and testosterone concentration and reduced the levels of ROS, MDA, GSH, and LDH. ADM pretreatment significantly decreased the mRNA levels of IL-1, IL-6, iNOS, and COX-2 and the concentrations of IL-1, IL-6, NO, and PGE2. LPS-induced TUNEL-positive Leydig cells were significantly decreased by ADM pretreatment, a result further confirmed by decreased DNA fragmentation. ADM pretreatment decreased apoptosis by significantly promoting Bcl-2 and inhibiting Bax, caspase-3, and PARP expressions. The LPS activity that reduced p-Akt level was significantly inhibited by ADM pretreatment. ADM protected rat Leydig cells from LPS-induced inflammation and apoptosis, which might be associated with PI3K/Akt mitochondrial signaling pathway.

Adrenomedullin improves intestinal epithelial barrier function by downregulating myosin light chain phosphorylation in ulcerative colitis rats.

Adrenomedullin (AM) is a pivotal endogenous vasoactive peptide, which can maintain epithelial barrier function in inflammatory bowel disease. Myosin light chain kinase (MLCK)‑dependent phosphorylated myosin light chain kinase (p‑MLC) is a key regulator of intestinal barrier function. The aim of the present study was to investigate the effect and mechanism of AM on the intestinal epithelial barrier in a rat model of ulcerative colitis (UC) induced by 2,4,6‑trinitro‑benzene‑sulfonic acid (TNBS). A total of 21 male Sprague‑Dawley rats were randomly divided into the following three groups and administered different agents for 7 days: The normal group (water and saline), model group (TNBS and saline) and the AM group (TNBS and AM; 1.0 µg). The weight of rats was recorded every day. Serum tumor necrosis factor‑α (TNF‑α) and interleukin‑6 (IL‑6) levels were detected using ELISA kits. Colon tissue was collected for the assessment of histological alterations. The protein expression of MLCK, p‑MLC and zonula occludens‑1 (ZO‑1) was examined by western blot analysis. Intestinal epithelial tight junctions were examined using transmission electron microscopy. The results demonstrated that in colitis model rats, the expression of TNF‑α, IL‑6, MLCK and p‑MLC significantly increased compared with normal rats. In addition, the expression of ZO‑1 decreased (P<0.05) and intestinal epithelial cell permeability increased. Following AM administration, TNF‑α, IL‑6, MLCK and p‑MLC expression significantly decreased compared with the model rats, the expression of ZO‑1 increased (P<0.05) and intestinal epithelial cell permeability reduced. These data indicate a protective effect of AM on intestinal epithelial barrier dysfunction via suppression of inflammatory cytokines and downregulation of MLCK‑p‑MLC in TNBS‑induced UC. In conclusion, AM/MLCK‑p‑MLC may be an important signaling pathway in the occurrence and development of UC.

The Protective Effect of Adrenomedullin on Renal Injury, in a Model of Abdominal Aorta Cross-Clamping

Renal injury induced by aortic ischemia-reperfusion (IR) is an important factor in the development of postoperative acute renal failure following abdominal aortic surgery. The aim of this study was to examine the effect of adrenomedullin (AM) on kidney injury induced by infrarenal abdominal aortic IR in rats. Thirty-two Wistar Albino rats were randomized into four groups (eight per group) as follows: Control group, IR group (120-minute ischemia and 120-minute reperfusion), IR + AM group (a bolus intravenously of 0.05 µg/kg/min AM), and control + AM group. At the end of the experiment, blood and kidney tissue specimens were obtained for biochemical analysis. Immunohistological evaluation of the rat kidney tissues was also done. IR significantly increased (p < 0.05 vs control group) and AM significantly decreased (p < 0.05 vs. IR group) all of the biochemical parameters. Immunohistological evaluation showed that AM attenuated morphological changes as apoptosis associated with kidney injury. The results of this study indicate that AM attenuates both biochemically and immunohistopathologically kidney injury induced by aortic IR in rats.

Adrenomedullin protects against fructose-induced insulin resistance and myocardial hypertrophy in rats

Adrenomedullin (ADM) has been recognized as a multipotent multifunctional peptide. To explore the pathophysiological roles of ADM in insulin resistance (IR), we studied the changes in ADM mRNA level in the myocardium and vessels and the effect of ADM supplementation on rats with IR induced by fructose feeding. Rats were fed 4% fructose in drinking water for 8 weeks, and ADM was administered subcutaneously in pure water through an Alzet Mini-osmotic Pump at 300ng/kg/h for the last 4 weeks. Compared with controls, rats with IR showed increased levels of fasting blood sugar and serum insulin, by 95% and 67%, respectively (all P<0.01), and glycogen synthesis and glucose transport activity of the soleus decreased by 54% and 55% (all P<0.01). mRNA level and content of brain natriuretic peptide (BNP) in myocardial were all increased significantly. Fructose-fed rats showed increased immunoreactive-ADM content in plasma by 110% and in myocardia by 55% and increased mRNA level in myocardia and vessels (all P<0.01). ADM administration ameliorated the induced IR and myocardial hypertrophy. The glycogen synthesis and glucose transport activity of the soleus muscle increased by 41% (P<0.01) and 32% (P<0.05). ADM therapy attenuated myocardial and soleus lipid peroxidation injury and enhanced the antioxidant ability. Our results showed upregulation of endogenous ADM during fructose-induced IR and the protective effect of ADM on fructose-induced IR and concomitant cardiovascular hypertrophy probably by its antioxidant effect, which suggests that ADM could be an endogenous protective factor in IR.

The effect of sepsis serum on intercellar adhesion molecule-1 of rat intestinal mucosa microvascular endothelial cells and the intervention of Adrenomedullin

：Objective To observe thelevels of intercellar adhesion molecule-1(ICAM-1)of rat intestinal mucosa microvascularendothelial cells in sepsis rat,and to investigate the possible protective effect ofadrenomedullin on the expression of intercellar adhesion molecule-1(ICAM-1)of ratintestinal mucosa microvascular endothelial cells,and to confirm whether adrenomedullincan decrease released inflammatory factors to slow down or inhibit the occurrence anddevelopment of sepsis.Methods The rat intestinal mucosa microvascular endothelial cellswere isolated and cultured in vitro,and it was divided into sham operated(sham operatedserum),sepsis serum(with addition of sepsis serum)and ADM serum(with 0.lng/kgadrenomedullin 6h after sepsis treatment)groups.The soluble ICAM-1(sICAM-1)content inRIMMVECs culture supernatant was measured by ELISA at the 24th hour after serumstimulation.Results The ICAM-1 content in the supernatant of sepsis group [(0.33 ±0.04)ng/L]was obviously higher than that in sham operated [(0.15 ± 0.02)ng/L] and ADM [(0.17 ±0.04)ng/L] group(P ＜ 0.05),but ADM group was nosignificantly different compared with sham operated group(P ＞0.05).Conclusions ADM could decrease theexpression of ICAM-I in RIMMVECs,and the low level of ICAM-1 could suppress its downstreaminflammation factors and inhibit the occurrence and development of sepsis. Key words: Sepsis/BL/ME/DT; Intercellular adhesion molecule-1/ME; Endothelium,vascular/ME; Adrenomedullin/PD

Pre-treatment of adrenomedullin suppresses cerebral edema caused by transient focal cerebral ischemia in rats detected by magnetic resonance imaging

Recent studies suggest the protective effects of adrenomedullin (AM) on ischemic brain damage. The present study was aimed at investigating the effects of AM and its receptor antagonist, AM 22–52, on ischemia-induced cerebral edema and brain swelling in rats using magnetic resonance imaging. Rats were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Intravenous injection of AM (1.0 μg/kg), AM 22–52 (1.0 μg/kg), or saline was made before MCAO. Effects of AM injection just after reperfusion were also investigated. One day after ischemia, increases in T 2-weighted signals in the brain were clearly observed. Total edema volume, as well as brain swelling, was greatly and significantly reduced by pre-treatment of AM (reduced by 53%). Extent of brain swelling was significantly correlated with the volume of cerebral edema. The protective effect of AM against edema was more clearly observed in the cerebral cortex (reduced by 63%) than the striatum (reduced by 31%). Increased T 2 relaxation time in the cortex was recovered partially by pre-treatment of AM. Post-treatment of AM had no effects. Pre-treatment of AM 22–52 tended to exacerbate the edema. In another line of experiment, cocktail administration of AM with melatonin, a pineal product having neuroprotective potential as a free radical scavenger, failed to enhance the protective effects of AM alone. The present study clearly suggests the prophylactic effects of AM against cerebral edema, especially the cortical edema, in a rat stroke model.

Adrenomedullin limits reperfusion injury in experimental myocardial infarction

Adrenomedullin (AM) is a vascular-derived polypeptide that exerts numerous actions in cardiovascular homeostasis. Recent studies have demonstrated a cytoprotective action of exogenously applied or genetically over-expressed AM in experimental myocardial infarction. The present studies were undertaken to test the hypothesis that AM exerts its effects through direct augmentation of NO generation in the myocardium during early reperfusion. Rat isolated hearts underwent 35 min left coronary artery occlusion followed by 120 min reperfusion. Infarct size (as percentage of ischaemic riskzone) was determined by Evans' blue and tetrazolium double staining. AM 1 nM administered 5 min prior to and during the first 15 min of ischaemia did not significantly influence infarct size. However, the same concentration of AM given during the last 5 min ischaemia and first 15 min of reperfusion significantly limited infarct size (AM reperfusion 15.9 +/- 3.5% vs control 31.4 +/- 2.1%, P < 0.01). AM at reperfusion improved coronary flow and LV contractility. The protective effects of adrenomedullin were abolished in the presence of the NO synthase inhibitor, L-NAME 100 microM (infarct size 24.6 +/- 5.7%, P > 0.05 vs control). AM treatment at reperfusion was associated with augmented phosphorylation of the pro-survival kinase, Akt, determined by immunoblotting of tissue sampled 30 min following reperfusion. These studies provide the first evidence that AM exerts its cytoprotective action specifically during early reperfusion through a NO-dependent mechanism.

Protective effect of adrenomedullin in mannitol-induced apoptosis.

Mannitol therapy is widely used for reducing brain edema, and ischemic brain swelling. However, mannitol at clinical concentrations induces apoptosis in endothelial cells. Because apoptosis may be a pathogenic mechanism in vascular injury, antiapoptotic agents may have a protective role in mannitol-induced apoptosis. In this study, we examined whether adrenomedullin (AM) prevents mannitol-induced apoptosis and also evaluated the associated signaling pathway of AM in human umbilical vein endothelial cells. AM prevented mannitol-induced apoptosis in a dose-dependent manner. Pretreatment with wortmannin blocked the AM-induced antiapoptotic effect. AM stimulated Akt at Ser473, and wortmannin inhibited the AM-induced Akt phosphorylation. These findings indicate that phosphatidylinositol 3'-kinase/Akt pathway transmits the survival signal from AM. The potency of antiapoptotic effect of AM is stronger than that of vascular endothelial growth factor and angiopoietin-1 in mannitol-induced apoptosis. AM can have a protective role not only in umbilical vein, but also in pulmonary, coronary, and aortic endothelial cells. These findings indicate that AM has a potent protective role in mannitol-induced apoptosis, through phosphatidylinositol 3'-kinase/Akt pathway. Therefore, pretreatment with AM might help to maintain normal endothelial integrity during systemic mannitol therapy.