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Aim To evaluate the mechanism by which intermdin(IMD)inhibits lipopolysaccha ride(LPS)-induced polarization in RAW264.7 cells.Methods RAW264.7 cells were divided into control groups, LPS groups, LPS+IMD groups, LPS+IMD+Compound C groups.The mRNA expressions of tumor necrosis factor-α,(TNF-α,), CD86, inducible nitric oxide synthase(iNOS), Arginase-1(Arg-1)and CD206 were detected by Realtime-PCR.The protein expressions of p-AMPK, AMPK, TNF-α, intereukin-6(IL-6)and intereukin-10(IL-10)were detected by Western blot.The proportion of CD86+ M1 type cells was detected by Flow cytometry.In addition, the expression levels of supernatant cytokines, including IL-6 and TNF-α, were detected by ELISA.Results Compared with control and LPS groups, IMD treatment could up-regulate the expression level of p-AMPK and the ratio of p-AMPK/AMPK.LPS promoted M1 polarization, since the expressions of CD86, TNF-α and iNOS increased, while the expressions of CD206 and Arg-1 decreased by LPS induction.The proportion of M1 type cells increased and the secretion of TNF-α, IL-6 in the cell supernatant increased.And IMD treatment could inhibit the polarization of M1 induced by LPS.These effects were reversed by Compound C, an inhibitor of AMPK.Conclusion IMD can inhibit the M1-type polarization of LPS-induced macrophages by activating AMPK signaling pathway.
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Objective:The effect of intermedin (IMD) on ATP-induced activation of inflammatory bodies and pyroptosis of cells and its mechanism were studied using lipopolysaccharide (LPS)-sensitized mouse macrophage line RAW 264.7.Methods:The cells were divided into the control groups, the LPS groups, LPS+IMD groups, and LPS+IMD+LY294002 groups. The expression of interleukin (IL)-1β and IL-18 and the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory cells were detected by real-time PCR and western blotting, and the pyroptosis of cells was detected by propidium iodide (PI) staining. The measurement data was represented by MS± SD, and the inter-group difference was compared with ANOVA calculations, and P<0.05 represented the difference with statistical significance. Results:Compared with the control group [(0.83±0.09) vs (0.49±0.04)], the ratio of phosphorylated phosphatidylinositol-3-kinase, p-PI3K)/phosphatidylinositol-3-kinase (PI3K) (0.44±0.05) and p-Akt/Akt (0.27±0.06) in the LPS group was significantly decreased. The ratios of p-PI3K/PI3K (1.22±0.18) and pAkt/Akt (0.83±0.09) in LPS+IMD group was significantly increased ( F=31.40, P<0.001; F=50.88, P<0.001). Compared with the control group, the mRNA and protein expressions of IL-1β, IL-18 and NLRP3 inflammasome (NLRP3, caspase-1, ASC) in RAW264.7 cells were up-regulated in the LPS group (LPS and ATP). Compared with LPS group, IMD treatment inhibited the expression of inflammatory cytokines IL-1β, IL-18 and NLRP3 inflammasome, which was blocked by LY294002, a blocker of PI3K/Akt pathway. The results of real-time PCR showed that the relative expression of IL-1β mRNA was (1.00±0.11) in the control group, (8.32±0.61) in the LPS group, (8.32±0.55) in the LPS+IMD group, and (7.23±0.41) in the LPS+IMD+LY group ( F=15.42, P<0.001). The relative expression of IL-18 mRNA in the control group was (1.00±0.17), (1.82±0.21) in the LPS group, (1.14±0.15) in the LPS+IMD group, and (1.53±0.11) in the LPS+IMD+LY group respectively ( F=18.16, P<0.001). The relative expression of NLRP3 mRNA in the control group was (1.00±0.13), (2.58±0.18) in the LPS group, (1.07±0.17) in the LPS+IMD group, and (1.33±0.32) in the LPS+IMD+LY group respectively ( F=15.98, P< 0.001); The relative expression of caspase-1 mRNA in the control group was (1.00±0.09), (6.20±0.19) in the LPS group, (3.43±0.06) in the LPS+IMD group, and (5.50±0.45) in the LPS+IMD+LY group respectively ( F=18.39, P<0.001). The relative expression of ASC mRNA in the control group was (1.00±0.21), (4.58±0.48) in the LPS group, (2.07±0.51) in the LPS+IMD group, and (3.33±0.32) in the LPS+IMD+LY group respectively ( F=15.19, P<0.001). Western blotting results showed that the relative expression of IL-1β protein was as follows: (100%) in the control group, [(188±14)%] in the LPS group, [(112±11)%] in the LPS+IMD group, and [(171±27)%] in the LPS+IMD+LY group respectively ( F=21.25, P<0.001). The relative expression of IL-18 protein in the control group was 100%, [(183±16)%] in the LPS group, [(115±19)%] in the LPS+IMD group, and [(179±23)%] in the LPS+IMD+LY group respectively ( F=19.62, P<0.001). The relative expression of NLRP3 protein was 100% in the control group, [(149±15)%] in the LPS group, [(106±10)%] in the LPS+IMD group, and [(144±15)%] in LPS+IMD+LY group respectively ( F=14.35, P<0.001). The relative expression of ASC protein was 100% in the control group, [(188±12)%] in the LPS group, [(110±18)%] in the LPS+IMD group, and [(192±8)%] in the LPS+IMD+LY group ( F=15.79, P<0.001). Conclusion:IMD inhibits the activation of NLRP3 inflammasome and cell pyroptosis by regulating PI3K/Akt activity.
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OBJECTIVE: To investigate intermedin protects against at rat renal tubular epithelial cells(NRK-52E) hypoxia-reoxygenation injury through suppresses endoplasmic reticulum stress(ERS)-related apoptosis. METHODS: The NRK-52E cells were divided into 4 groups. One of them was control group; the other three model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector and highly expressed was used IMD vector, respectively. The cell survival rate was detected by MTT; annexin V-FITC/PI double-stained cell apoptosis detection kit for detection of apoptosis; real-time PCR and Western-blot were used to detect expression of ERS molecules such as GRP78, CHOP and Caspase 12. ER specific resident fluorochrome dapoxyl was used to observe ER morphological changes. RESULTS: MTT RESULTS: showed that the survival rate of NRK-52E cells in H/R group was significantly decreased compared with the control group, and the cell survival rate was increased significantly in IMD group. The apoptosis rate of H/R increased significantly, and the mRNA and protein expressions of GRP78, CHOP and Caspase 12 in H/R group were all up-regulated significantly, IMD gene transfer markedly decreased the apoptosis index compared with that of H/R. The mRNA and protein expressions of GRP78, CHOP and Caspase 12 in IMD group were all down-regulated significantly. Dapoxyl staining demonstrated that the structure of the ER was severely destroyed in H/R, the fluorescence density showed nonuniform distribution, accompanied by notable vacuoles in the ER; Over-express IMD decreased the injury of the ER. CONCLUSION: During rat renal tubular epithelial cells NRK-52E H/R injury may occur activation of ERS apoptotic pathway, IMD inhibites ERS-related apoptosis, reduces renal tubular epithelial cell apoptosis and reduces renal IRI.
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Intermedin/adrenomedullin-2 (IMD/AM2), a member of the calcitonin gene-related peptide/AM family, plays an important role in protecting the cardiovascular system. However, its role in the enhanced sympathoexcitation in obesity-related hypertension is unknown. In this study, we investigated the effects of IMD in the paraventricular nucleus (PVN) of the hypothalamus on sympathetic nerve activity (SNA), and lipopolysaccharide (LPS)-induced sympathetic activation in obesity-related hypertensive (OH) rats induced by a high-fat diet for 12 weeks. Acute experiments were performed under anesthesia. The dynamic alterations of sympathetic outflow were evaluated as changes in renal SNA and mean arterial pressure (MAP) in response to specific drugs. Male rats were fed a control diet (12% kcal as fat) or a high-fat diet (42% kcal as fat) for 12 weeks to induce OH. The results showed that IMD protein in the PVN was downregulated, but Toll-like receptor 4 (TLR4) and plasma norepinephrine (NE, indicating sympathetic hyperactivity) levels, and systolic blood pressure were increased in OH rats. LPS (0.5 µg/50 nL)-induced enhancement of renal SNA and MAP was greater in OH rats than in obese or control rats. Bilateral PVN microinjection of IMD (50 pmol) caused greater decreases in renal SNA and MAP in OH rats than in control rats, and inhibited LPS-induced sympathetic activation, and these were effectively prevented in OH rats by pretreatment with the AM receptor antagonist AM22-52. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor U0126 in the PVN partially reversed the LPS-induced enhancement of SNA. However, IMD in the PVN decreased the LPS-induced ERK activation, which was also effectively prevented by AM22-52. Chronic IMD administration resulted in significant reductions in the plasma NE level and blood pressure in OH rats. Moreover, IMD lowered the TLR4 protein expression and ERK activation in the PVN, and decreased the LPS-induced sympathetic overactivity. These results indicate that IMD in the PVN attenuates SNA and hypertension, and decreases the ERK activation implicated in the LPS-induced enhancement of SNA in OH rats, and this is mediated by AM receptors.
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Animals , Male , Adrenomedullin , Metabolism , Blood Pressure , Physiology , Hypertension , Lipopolysaccharides , Pharmacology , Neuropeptides , Metabolism , Obesity , Rats, Sprague-Dawley , Receptors, Adrenomedullin , Metabolism , Sympathetic Nervous System , Metabolism , Toll-Like Receptor 4 , MetabolismABSTRACT
Objective To investigate the predicting value of intermedin (IMD) for the prognosis of elderly sepsis patients.Methods A retrospective analysis was conducted. Forty-one patients with sepsis, aged ≥65 years, and admitted to geriatrics intensive care unit of Aerospace Center Hospital from April 2015 to December 2016 were enrolled. Thirty healthy patients were studied as control during the same time. The expression of C-reactive protein (CRP), procalcitonin (PCT) and IMD were tested within 24 hours during hospitalization, and the acute physiology and chronic health evaluation Ⅱ(APACHE Ⅱ) score and prognosis was evaluated. According to APACHE Ⅱ score, patients were divided into 3 groups, 10-20 score, 21-30 score, and > 30 score group. And based on the prognosis, the patients were divided into death group and survival group. The differences of expression levels of CRP, PCT and IMD in each group were assessed. The relationship of IMD and infection index was analyzed by Pearson correlation. Receiver operating characteristic curve (ROC) was used to evaluate the prognostic value of CRP, PCT and IMD in patients with sepsis.Results Compared with the control group, the levels of CRP, PCT and IMD were significantly higher in the sepsis patients [CRP (mg/L): 114.71±40.08 vs. 4.03±2.68, PCT (μg/L): 1.338±0.812 vs. 0.007±0.001, IMD (ng/L):43.03±9.67 vs. 16.77±2.06, allP 30 score groups, PCT (μg/L) were 0.397±0.129, 1.164±0.326, and 1.999±0.888, respectively (F = 19.392,P = 0.000); IMD (ng/L) were 29.12±5.60, 40.48±4.40,52.75±4.73, respectively (F = 33.310,P = 0.000). There was no significant difference in CRP among APACHE Ⅱ score groups (F = 2.137,P = 0.132). The level of IMD was positively correlated with CRP and PCT (r1 = 0.351,P1 = 0.024;r2 = 0.617,P2 = 0.000), and there was no correlation with temperature and white blood cell count (r1 = 0.063,P1 = 0.697;r2 = 0.064,P2 = 0.692). The expression of PCT and IMD in the death groups were significantly higher than the survival group [PCT (μg/L): 1.547±0.883 vs. 1.043±0.608, IMD (ng/L): 47.44±8.23 vs. 36.80±8.13, bothP < 0.05], while CRP was not significantly different. The area under the ROC curve [AUC (95% confidence interval, 95%CI)] of IMD was larger than that of PCT and CRP [0.809 (0.675-0.943) vs. 0.680 (0.511-0.849), 0.664 (0.490-0.838)]; when cut-off value of IMD was 41.58 ng/L, the sensitivity was 75.0% and the specificity was 82.4%.Conclusions The levels of CRP, PCT and IMD were increased in elderly sepsis patients, and IMD and PCT can better reflect the severity of sepsis. IMD is more valuable in predicting the prognosis of sepsis patients.
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Objective To explore the effect of intermedin ( IMD ) and adrenomedullin ( ADM ) on cerebral microcirculation in rats with cerebral ischemia. Methods Rat cerebral ischemia ( CI) model was established by middle cerebral artery occlusion. 40 SPF male adult Sprague-Dawley ( SD) rats were randomly divided into three groups:CI+NS ( normal saline) group, CI+ADM group and CI+IMD group, which were used to observe the changes of brain surface microcirculatory perfusion with a laser Doppler flowmeter. Results The differences of brain surface microcirculatory perfusion were statistically significant among the CI+NS group, CI+ADM group and CI+IMD group ( F=53. 426, P<0. 05 ) . Multiple comparison showed that the brain surface microcirculatory perfusion in the CI+IMD group was higher than that of the CI+NS group and CI+ADM group. Conclusions Intermedin can improve the cerebral microcirculation in rats with cerebral ischemia, and its therapeutic effect is better than adrenomedullin.
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Objective To investigate the pharmacological effect and mechanism of mouse intermedin (IMD)for antagonizing cardiac hypertrophy by using isoproterenol (ISO)induced mouse cardiomegaly model.Methods The mouse cardiomegaly model was constructed by small dose of ISO subcutaneous injection.The mouse blood dynamic indexes and heart weight/body weight rati-o were investigated.Then the cardiomyocyte cross-sectional area,apoptosis and cardiac tissue fibrosis were evaluated by using the cardiac tissue section.ANP,BNP,mRNA expression levels of endogenous IMD and its receptors system in cardiac tissues were de-tected by using real time fluorescence PCR method.Results Compared with the ISO induced mouse cardomegaly model group,the intraperitoneal injection of IMD significantly decreased the heart weight/body weight ratio and cardiomyocyte cross-sectional area increased by ISO treatment,cardiomegaly marker ANP and BNP mRNA level,cardiomyocyte apoptosis and cardiomyocyte fibrosis, improved the cardiac function,left ventricular pressure,maximal and minimal rate of LV pressure increase and decrease during the isovolumetric contraction phase,stroke volume,cardiac output and ejection fraction,but significantly decreased the left ventricle end-diastolic pressure.In addition,the ISO treatment significantly induced the expressions of endogenous IMD and its receptors in heart tissues.Conclusion ISO induces the expressions of endogenous IMD and its receptors in cardiac tissues,and exogenous IMD sup-plementation therapy realizes the pharmacological protective effect for antagonizing cardiomegaly by ISO activated IMD receptor system.
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Aim To evaluate the effect of intermedin (IMD)on cell proliferation and regeneration in rat tu-bular epithelial cell line (NRK-52E)that was subjec-ted to hypoxia-reoxygenation (H/R)injury.Methods The NRK-52E cells were divided into control group and three model groups (H/R,H/R +primitive vec-tor,H/R +IMD vector).The content of LDH was de-tected to observe the influence of IMD on H/R injury. The cell proliferation was detected by MTT.The cell cycle was detected by flow cytometry.Real-time PCR and western blotting were used to determine mRNA and protein levels.Results ① In comparison to the con-trol,H/R treatment decreased the cell viability and in-creased LDH activity (P <0.01 );in contrast,com-pared to H/R,IMD treatment ameliorated cell viability (79.1 5 ±1 .421 % vs 61 .22 ±1 .63%,P <0.05)and decreased LDH activities by 33.85% (P <0.01 ).②The proliferation of NRK-52E cells was significantly in-hibited by H/R treatment.In comparison to the con-trol,H/R treatment of NRK-52E cells increased the proportion of cells in the G0 /G1 phase but decreased the proportion of cells in the S and G2 /M phases. Moreover,the over-expression of IMD resulted in S and G2 /Mphase redistribution and the accumulation of G2 /M-phase cells.The real-time PCR and western blotting results indicated that the mRNA and protein expression levels of cyclin D1 ,CDK4 and p57 were increased in H/R-treated cells.IMD further stimulated this up-reg-ulated expression of cyclin D1 ,CDK4 and decreased the expression of p57 in NRK-52E cells.④Cyclin D1 had a predominantly nuclear localization in NRK-52Ecells,although cytoplasmic localization was also ob-served.Conclusion The study shows that the over-expression of IMD may promote renal cell proliferation and regeneration after renal tubular cell H/R injury via the up-regulation of cyclin D1 ,CDK and the down-reg-ulation of p57.
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Objective To observe the effect of intermedin(IMD) on microvascular injury of renal fibrosis in unilateral ureteral obstruction (UUO) rat model.Methods Seventy-two male Wistar rats were randomly divided into two groups:the sham-operation group (n=24) underwent the left ureteral dissection,the other 48 rats were made as unilateral ureteral obstruction models and subdivided into model group(UUO,n=24) and IMD group (n=24).At the 7,14,21,28 day after the operation,6 randomly-selected rats from each of the three groups respectively were blooded by abdominal arotic and their obstructive kidneys were taken out.The renal histopathological changes were observed through HE and Masson staining,the contents of BUN,Scr and cystatin C (CysC) of the obstructive kidneys were determined,the expressions of transforming growth factor-β1 (TGF-β1),α-SMA,bone morphogenetic protein-7 (BMP-7),E-cadherin,thrombospondin 1 (TSP-1) and vascular endothelial growth factor (VEGF) were detected by RT-PCR and immunohistochemistry.Results Compared with the sham-operated group,the pathological changes of kidney in the model group showed that the degree of fibrosis was obvious,tubular interstitial damage aggravated,the levels of BUN,Scr,CysC in the model group increased (P ＜ 0.05),the mRNA expression and protein content of TGF-β1,oα-SMA,TSP-1 increased (P ＜ 0.05),while the levels of BMP-7,E-cadherin and VEGF decreased (P ＜0.05).Compared with the UUO group,renal tubular damage,interstitial fibrosis in the IMD group were lighter,the levels of BUN,Scr,CysC in the IMD group were lower (P ＜ 0.05),the mRNA expression and protein content of TGF-β1,α-SMA,TSP-1 were down-regulated (P ＜ 0.05),while the levels of BMP-7,E-cadherin and VEGF were up-regulated (P ＜ 0.05).Conclusion IMD can ameliorate the renal interstitial fibrosis,and the mechanism may be related to the fact that VEGF mediated by IMD can reduce vascular injury.
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Objective To analyze the relationship between serum intermedin ( IMD ) , CystatinC ( CysC ) and fibro-blast growth factor23(FGF23) with hypertensive left ventricular hypertrophy (HLVH). Methods Serum IMD,Cy-sC and FGF23 levels of 30 patients with essential hypertension(EH group),30 hypertensions with LVH(HLVH group)and 30 healthy subjects(control group)were detected by enzyme-linked immunosorbent assay(ELISA). All the subjects did UCG for LVMI. Results LVMI and serum IMD,CysC,FGF23 levels were significantly higher in EH group than in control(P<0. 05),higher in HLVH group than in EH and control groups(P<0. 05). In the pa-tients with hypertension LVMI and serum IMD,CysC,FGF23 levels were increasing with increased blood pressure levels. LVMI level was positively correlated with serum IMD,CysC and FGF23 levels(r=0. 769,0. 517,0. 700;P<0. 01). Line to LVMI level as the dependent variable,multiple stepwise regression analysis showed that:systolic blood pressure( SBP) ,IMD,CysC,homocysteine( Hcy) ,FGF23 entered regression equation. They were independent risk factors for LVMI. Conclusion Serum IMD, CysC and FGF23 levels are closely associated with EH and HLVH. They may participate in the development of EH and affect the process of HLVH. Combined-detection of ser-um IMD,CysC and FGF23 can be used as a reference index for the condition and prognosis of HLVH.
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Objective To investigate collagen metabolism modulation of Intermedin 1-53 ( IMD1-53 ) on angiotensin Ⅱ( AngⅡ)-induced rat cardiac fibroblasts .Methods SD neonatal rat cardiac fibroblasts were cultured and divided them into control group, AngⅡ +different concentrations IMD1-53 groups.ⅠandⅢcollagen expression in cardiac fibroblasts were measured by Westem blot , IMD1-53 receptor-like receptor ( calcitonin receptor-like receptor , CRLR) and transforming growth factor-β( TGF-β) mRNA expression were exammed by real-time PCR.Results IMD1-53 significantly inhibited AngⅡ-induced collagen synthesis in cardiac fibroblasts , and this effect was more ob-vious with the increase of IMD 1-53 ( P<0.01 ,P<0.05 ) .Similar phenomenon was recorded in TGF-βexpression in cardiac fibroblasts ( P<0.05 ) .Conclusions IMD1-53 inhibited collagen synthetic in cardiac fibrosis induced by AngⅡ, down-regulated TGF-βexpression.These may relate to IMD1-53 anti myocardial fibrosis.
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[ ABSTRACT] AIM:To explore the regulatory effect of intermedin ( IMD) on pulmonary collagen synthesis and ac-cumulation in rats with pulmonary hypertension induced by high pulmonary blood flow.METHODS: Healthy male SD rats (n=20) were randomly divided into control group (n=7), shunt group (n=7) and shunt with IMD group (n=6).The shunting of abdominal aorta and inferior vena cava was produced in rats of shunt group and shunt with IMD group.After 8 weeks, IMD was administered into the rats of shunt with IMD group subcutaneously by mini-osmotic pump for 2 weeks.Mean pulmonary artery pressure (mPAP), relative medial thickness (RMT) of pulmonary arteries, contents of hydroxyproline, collagen type I and III, bone morphogenetic protein-2 (BMP-2), and the mRNA expression of procollagen I and III in lung tissues were measured and compared.RESULTS:Compared with control group, mPAP and RMT of medium and small pul-monary arteries in the rats of shunt group were significantly increased.Meanwhile, the lung hydroxyproline, collagens I and III and BMP-2 contents, and the mRNA expression of lung procollagen I and III were all significantly increased compared with control group.However, IMD significantly decreased mPAP, alleviated the changes of pulmonary vascular micro-struc-ture, decreased the collagen accumulation and pulmonary tissue homogenate BMP-2 contents, and inhibited the mRNA ex-pression of procollagen I and III in the lung tissue of shunting rats.CONCLUSION:IMD plays a protective role in the de-velopment of pulmonary hypertension and pulmonary vascular structural remodeling induced by high blood flow by inhibiting pulmonary collagen synthesis and accumulation, possibly in association with the BMP-2 pathway.
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Objective To observe the effects of intermedin (IMD) on nitric oxide synthetase (NOS) in renal ischemia-reperfusion (IR) rat models and the action mechanism.Methods A total of 24 rats were divided into four groups (n =6 each).Group Ⅰ underwent right nephrectomy one week prior to the exposure of left renal pedicles,but did uot receive any I/R.Group Ⅱ underwent right nephrectomy one week prior to left renal I/R surgery.Group Ⅲ underwent right nephrectomy and left renal IMD-pCDNA3.1 ( + ) transfection by ultrasound-mircobubbles and renal I/R surgeries were performed one week after gene transfection.Group Ⅳ was treated with the same way as group Ⅲ except that empty control vector was transfected.All the animals were killed at the end of 24 h of reperfusion.The expression and site of IMD were determined by using immunohistochemistry.Serum levels of BUN and creatinine were determined.The kidney formaldehyde-fixed and paraffin-embedded sections were stained with HE and PAS by standard methods and then histological changes were analyzed semiquantitatively.The mRNA expression levels of endothelial NOS (eNOS),inducible NOS (iNOS) and neuronal NOS (nNOS) in the kidneys of the four groups were detected by using RT-PCR.The protein expression levels of the three NOS mentioned above in the kidneys were semiquantitatively analyzed by Western blotting.Results IMD was weakly expressed in the plasma of tubulointerstitial cells in sham-operated group; whereas IMD expression in the kidneys subject to I/R was increased.Moreover,as compared with I/R group,IMD expression levels were obviously increased (P＜0.01 ).The degree of morphological changes as well as renal dysfunction in group Ⅲ was obviously lessened as compared with group Ⅱ.The mRNA and protein expression levels of eNOS in group Ⅲ were notably increased as compared with group Ⅱ,while the mRNA and protein expression levels of iNOSin group Ⅲ were obviously reduced as compared with I/R group not transfeeted with IMD (P＜0.05).Meanwhile,there were no significant differences in the mRNA and protein expression levels of nNOS among groups Ⅱ,Ⅲ and Ⅳ.Conclusion IMD gene in the kidneys of rats can promote the expression of eNOS and attenuate over-expression of iNOS in the kidneys following I/R,thus protecting against tubulointerstitial damages and renal dysfunction in rat I/R models.
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Objective To investigate the expression and significance of intermedin (IMD) and its.receptors CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues of patients with non-small cell lung cancer.Methods The mRNA gene expressions of IMD,CRLR,RAMP1,RAMP2 and RAMP3 were detected by realtime quantitative RT-PCR in cancerous and para-cancerous tissues from 27 patients with lung cancer.Results Real-time quantitative PCR detection results showed that the expression of IMD,CRLR,RAMP1,RAMP2 and RAMP3 in cancer tissues were [(59±7.9)×10-8,(96±2.7)×10-6,(29±3.9)×10-9,(14±2.6)×10-6,(65±1.1)×10-6]which were higher than those in adjacent tissues[(40±4.7)×10-10,(21 ±3.9)×10-6,(53±7.8)×10-10,(64±1.9)×10-8,(36±1.3)×10-9] to some extent (all P ＜ 0.05); the higher expression of RAMP3 was found is higher expressions than RAMP1 and RAMP2 in cancer tissues (all P ＜ 0.05).Conclusion The expressions of IMD and its receptors in cancer tissues are higher than those in paracancerous tissues.IMD may play an important role in the development of cancer by activate RAMP3 which is the most high expressed receptor in cancer tissues.Therefore,it might be helpful for the investigation of new gene thereapy in non-small cell lung cancer.
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Objective To examine the effects of exogenously administered intermedin (IMD,adrenomedullin-2) on arterial blood pressure,cardiac function and the cardiovascular IMD receptor system in spontaneously hypertensive rats (SHRs) as well as to investigate the associated mechanisms.Methods Thirteen week-old male rats were divided in Wistar Kyoto (WKY) group (n =12),SHR group (n =12),IMD group (SHRs infused with IMD 1-47 500 ng/kg per hour,n =12),and ADM group (SHRs infused with adrenomedullin 500 ng/kg per hour,n =12).Results A two-week continuous administration of low dose IMD 1-47 via mini-osmotic pumps markedly reduced blood pressure,the maximal rates of increase and decrease of left-ventricle pressure development (LV ± dp/dtmax),left ventricular systolic pressure and heart rate in SHRs.Furthermore,IMD also inhibited protein over-expression of cardiovascular IMD receptors,myocardial Receptor Activity-Modifying Proteins (RAMP1 and RAMP2),aortic RAMP1,RAMP2,RAMP3,and calcitonin receptor-like receptor (CRLR);suppressed up-regulation of aortic RAMP1,RAMP2,RAMP3 and CRLR gene expression; and markedly elevated the mRNA abundance of myocardial atrial natriuretic peptide (ANP) and myocardial brain natriuretic peptide (BNP).Additionally,IMD 1-47 administration in SHRs increased aortic cAMP concentration and reduced myocardial cAMP concentration.Conclusion These findings support the speculation that IMD,as a cardiovascular active peptide,is involved in blood pressure reduction and cardiac function amelioration during hypertension.The mechanism underlying this effect may involve IMD binding of a receptor complex formed by RAMPs and CRLR,and consequential regulation of cAMP levels and other cardiovascular active factors,such as ANP and BNP.
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Objective To investigate the effect of intermedin (IMD) on renal ischemia/ reperfusion (I/R) injury after the up-regulation of IMD. Methods A total of 24 healthy Wistar male rats were randomly divided into four groups,sham-operated group,I/R group,IMD gene transfection +I/R group and empty plamid +I/R group.All the animals were killed at the end of 24 h of reperfusion.Histological changes and renal function were estimated.The expression and site of IMD were determined by Immunohistochemistry method,semi-quantitative RT-PCR and Western blotting.The protein expressions of endothelin 1 (ET-1),tumor necrosis factor αt (TNF-α) were detected by Western blotting. Results Compared with sham-operated group,tubulointerstitial pathological injury was significant aggravated in I/R group (7.6±2.3) and empty plamid +I/R group (7.0±1.8),and such injury was improved in IMD+I/R group (1.5±0.8) (P＜0.05).Compared with I/R group and empty plamid +I/R group,the renal dysfunction of IMD +I/R group was obviously lessened [BUN:(7.73±1.03) mmol/L vs (10.13±2.14) mmol/L,(9.77±1.92) mmol/L; Scr:(58.50±3.27) μmol/L vs (80.33±7.15) μmol/L, (75.67±7.58) μmol/L,all P＜0.05].IMD expression was weak in the plasma of tubulointerstitial cells in sham-operated group,and was up-regulated in I/R group. Compared with I/R group, immunohistochemical IMD expression increased obviously (262.03±67.89 vs 175.57±48.06,P＜0.01).The mRNA expression of IMD in IMD+I/R group was up-regulated significantly by 60.7％,66.1％ and the protein expression of IMD in IMD+I/R group increased significantly by 51.4％,55.9％ as compared to I/R and empty plasmid +I/R group.Meanwhile,the protein expressions of ET-1 and TNF-αt in IMD+I/R group were obviously lower compared with those in I/R group (ET-1:0.08±0.02 vs 0.17±0.02; TNF-α:0.21±0.04 vs 0.35± 0.02,all P＜0.05). Conclusion IMD gene transfected into kidneys of rats prior to I/R surgery can attenuate the over-expressions of both ET-1 and TNF-o in I/R injured rat kidneys as well as the damages to the structure and function of the kidneys.
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Objective To observe the effect of intermedin on renal interstitial fibrosis of unilateral ureteral obstruction rats. Methods Forty male Wistar rats were randomly divided into two groups:the sham-operation group (n=10) underwent the left ureteral dissection,the other 30 rats were made as unilateral ureteral obstruction models and divided into the model group (UUO),the losartan group,the IMD group (each group n=10).At the day 14,21 after operation,randomly 5 rats from each group were blooded by abdominal arotic and obstructive kidneys were taken out.The renal histopathological changes were observed by HE and Masson staining.The BUN,Scr,and hydroxyproline (Pro) of the obstructive kideys were measured by colorimetry. The expression of TGF-β1 and intermedin was observed by immunohistochemisty staining. Results Compared with the sham-operated group,the levels of BUN,Scr,Pro,TGF-β1 and IMD in the model group increased (P＜0.05).Compared with the UUO group,the levels of Scr,BUN,Pro,TGF-β1 and IMD in the losartan group were lower (P＜0.05).However,IMD in the IMD group was significantly up-regulated (P＜ 0.05),the others were down-regulated (P＜0.05). Conclusion IMD can improve the renal interstitial fibrosis,and the mechanism maybe antagonizes the TGF-β1.
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Objective To observe the effect of intermedin (IMD) preconditioning on cyclin D1, cyclin E and CDKs expression, and explore its role in.promoting kidney tissue regeneration after renal ischemiareperfusion injury. Methods One hundred and forty-four healthy male Wistar rats were randomly divided into four groups: sham operation (S) group, ischemia-reperfusion injury (IR) group, empty plasmid (EP) group and IMD group. In the IR group, after the right kidney was excised, the aorta abdominalis and left renal artery were bluntly dissected in EP and IMD group, empty plasmid and IMD plasmid were transfected into the left kidney using ultrasound-micro-bubble (SonoVue) mediated system, respectively. One week later, renal IRI model was made by clasping the left renal artery for 45 min. After 1, 2, 3, 4, 7 and 14 day of reperfusion, the kidney in each group was collected to detect the expression of cyclin D1, cyclin E, CDK4 and CDK2 by western blot analysis or enzyme-linked immunosorbent assay (ELISA). Results Compared with S group, the expression of cyclin D1, cyclin E, CDK4 and CDK2 was significantly up-regulated in day 1, 2,3, 4, 7 and 14 in IR group. And the above index increased gradually after reperfusion, and reached the peak at day 7 (F=54.92, 69.60, 61.28, 77.38, P＜0.05). While in IMD group, these indexes reached the peak at day 1, then progressively declined, and could not be detected at day 14 (compared with the IR group, F=54.92, 69.60, 61.28, 77.38, P＜0.05). Conclusion IMD preconditioning can up-regulate the expression of cyclin D1, cyclin E, CDK2 and CDK4 in the early phase of renal ischemia-reperfusion injury that may accelerate repair of renal tissue, at least by part, by enhancinge cell proliferation.
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Objective To investigate the effects of intermedin (IMD) pretreatment on associated repairing genes of the rats with injured kidneys by ischemia reperfusion injury (IRI)during the repair and regeneration process.Methods A total of 144 healthy male Wistar rats were randomly divided into 4 gourps:sham opration group,IRI group,empty plasmid group (EP)and IMD group.After resection of right kidneys of the rats,plasmid was transfected into the left kidneys by using ultrasonic microbubble technology.After one week,the renal IRI models were programmed.The samples of renal tissues after 1 d,2 d,3 d,4 d,7 d and 14 d of reperfusion were harvested respectively,then the mRNA expressions of the Pax-2,ZO-1,Ncam,Wt-1 and vimentin in renal tissues were detected by RT-PCR; the protein expressions of Pax-2,Wt-1 and vimentin were analyzed by Western blotting and the protein expressions of ZO-1 and Ncam were measured by ELISIA.Results (1) Compared with the sham group,the mRNA and protein expression of Pax-2,ZO-1,Ncam,Wt-1 and vimentin of the rats in IRI group increased significantly from day 1 to day 3 (P＜0.05),which peaked at day 2.After day 4,the above expressions in IRI group returned to normal level.(2) In IMD group,the mRNA and protein expressions of Pax-2,Zo-1,Ncam,Wt-1 and vimentin were significantly higher than those in other 3 groups (P＜0.05) at the same time after IRI day 1 to day 4,with a maximum in day 2.(3) The above expressions in IRI group,EP group,IMD group had no significant differences compared with sham group after day 7 or day 14 respectively (P＞0.05),and either between IRI group and EP group,though the expressions of the genes in IRI group and EP group increased compared with sham group after day 4.(4) The above expressions between IRI group and EP group also had no significant difference (P＞0.05).(5) Changes of ZO-1 and Ncam protein expression detected by ELISA were similar to those abore mRNA and protein expression by RT-PCR and Western blotting.Conclusion IMD pretreatment plays an important role in up-regulation of the expressions of associated repair genes during the process of repair and regeneration after renal ischemia reperfusion injury.
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Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.