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Objective To observe the effect of urantide on the expression of osteopontin (OPN) and α-smooth muscle actin (a-SMA) in the heart tissue of atherosclerosis (AS) rats, and to explore its mechanism of prevention and treatment of myocardial fibrosis injury in rats. Methods Totally 120 3-week-old healthy male Wistar rats in SPF grade were randomly divided into six groups; control group, model group, simvastatin group, urantide (3 days, 7 days, 14 days). HE and Masson trichrome staining were used to observe the morphology of rat heart and the expression of collagen fibers. Immunohistochemistry and Western blotting were used to detect the expression of OPN and α-SMA protein. Results In AS model group, cardiomyocyte hypertrophy or atrophy, a large number of inflammatory cell infiltration and a small amount of foam cells were observed in the heart tissue of rats. The increase of collagen fibers and the expression of OPN and α-SMA protein in cardiac tissue were significantly higher than those in the control group. Compared with the AS model group, after urantide treatment, cardiac injury was significantly improved, and the expression of collagen fiber, OPN and α-SMA protein was decreased. Conclusion Urantide can inhibit the expression of OPN and α-SMA protein in the heart tissue of AS rats to alleviate myocardial fibrosis and play a protective role in the heart tissue of AS rats.
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Objective To observe the effect of the peptide compound urantide on the expression of type I collagen (Col I ) in the heart tissue of rats with atherosclerosis ( As) , and to explore its mechanism of prevention and treatment of heart damage in As rats. Methods Sixty healthy male 3-week-old SPF Wistar rats were selected. The As model was established by intraperitoneal injection of vitamin D3(VD3) to damage the arterial intima and high-fat diet. They were randomly divided into normal group, As model group, simvastatin group and urantide (3 days, 7 days, 14 days) groups. HE staining and Masson trichrome staining were used to observe the morphology and collagen fiber expression of rat hearts. Immunohistochemistry, Western blotting and Real-time PCR were used to detect the expression of Col I protein and gene in rat heart. Results Compared with the normal group, pathological phenomena such As myocardial cell degeneration, intercellular infiltration of a large number of neutrophils, scattered foam cells and hyperemia and hemorrhage were observed in the heart tissues of the As model group. Meanwhile, collagen fibers increased, and the gene and protein expression levels of Col I increased. Compared with the As model group, the cardiac pathological phenomena were effectively alleviated after the treatment with urantide. With the extension of the administration time, the collagen fibers decreased, and the gene and protein expression levels of Col I were gradually down-regulated, especially the effect was the best when the drug was given for 14 days. Conclusion Urantide can inhibit the expression of Col I in As heart to reduce myocardial interstitial damage, and has a protective effect on the heart of As rats.
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AIM:To investigate the effect of urantide on the liver function and histomorphology in the rats with atherosclerosis (AS).METHODS:The AS Wistar rat model was induced by intraperitoneal injection of vitamin D3 (VD3) and feeding with high-fat diet.The rats were randomly divided into normal control group, AS model group, positive medicine group and urantide group.The liver function indexes of the rats were measured by biochemical test, and the pathological changes of the aorta and liver of the rats were observed by hematoxylin-eosin (HE) staining.The mRNA expression of urotensinⅡ (UII) and GPR14 at mRNA and protein levels in rat livers was determined by RT-qPCR and Western blot.RESULTS:The levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , γ-glutamyltransferase (γ-GT) , lactate dehydrogenase (LDH) , total bilirubin (TBIL) , indirect bilirubin (IBIL) and alkaline phosphatase (ALP) in AS model group were significantly increased compared with normal control group (P<0.05).The above indexes in urantide group were remarkably decreased compared with AS model group (P<0.05).No change of the levels of direct bilirubin (DBIL) , total protein (TP) , globulin (GLB) and albumin (ALB) in each group was observed.Urantide postponed hepatocyte fatty degeneration and repaired hepatocyte injury in the AS rats.Compared with normal control group, the mRNA and protein levels of UII and GPR14 in the liver were significantly increased in AS model group (P<0.05).With the prolongation of dosing time, the mRNA and protein levels of UII and GPR14 in the liver were significantly decreased in urantide group compared with AS model group (P<0.05).CONCLUSION:Urantide significantly attenuates the liver damage caused by liver fatty degeneration in AS rats.
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Objective: To investigate the effects of urantide on the expressions of type IV collagen (Col IV) in thoracic aorta and vascular smooth muscle cells (VSMC) in the rats with atherosclerosis (AS), and to clarify its mechanism of prevention and treatment of AS Methods: A total of 180 Wistar rats were randomly divided into normal control group (n=30) and AS model group (n=150). The rat models of AS were established by feeding on high-fat diet or intraperitoneally injecting vitamin Da (VDa). The AS model rats were randomly divided into AS group, fluvestation (Flu) group and urantide group (3, 7, and 14 d groups). The expression of Col IV in thoracic aorta wall of the rats was detected by immunohistochemistry. The levels of hydroxyproline (HYP) in serum and urine of the rats in various groups were measured by ELISA. The VSMC were randomly divided into normal control group, urotensin E (U II 10~smol • L_ 1) group, Flu group and urantide (10~10to 10~" mol • L_ 1) groups. The levels of Col IV in VSMC of the rats in various groups were determined by ELISA. Results: There was a significant difference in the expression levels of Col IV in the irregular plaques of the thoracic aorta of the rats between various groups (F = 3 5 . 0 9, P < 0 . 01). The expression levels of Col IV in thoracic aorta of the rats in AS group were significantly increased compared with normal control group (P < 0 . 01); the intensity and extent of Col IV positive staining in urantide group were lower than those in AS group (P < 0 . 01). There were significant differences in the serum and urine HYP levels between various groups (F = 2 4 . 38, P < 0 . 01; F=26. 72, P < 0 . 01). Compared with normal control group, the serum HYP level of the rats in AS group were significantly increased (P < 0 . 01), and the urine HYP level was significantly decreased (P < 0 . 01). Compared with AS group, the serum HYP levels in urantide groups were significantly decreased (P < 0 . 01) and the urine HYP levels were significantly increased (P < 0. 01), no less than the level in Flu group. The expression levels of Col IV in the culture supernatant of VSMC in the rats in various groups had significantly difference (F = 3 1 . 04, P < 0 . 01). The expression level of Col IV in the culture supernatant of VSMC of the rats in U II group was significantly increased compared with normal control group (P < 0 . 01); the expression levels of Col IV in the culture supernatant of VSMC of the rats in urantide groups were significantly decreased compared with U II group (P < 0 . 05 or P < 0 . 01). Conclusion: Urantide can inhibit the expressions of Col IV in the thoracic aorta and VSMC of the AS rats and alleviate the degree of AS lesions, which provides the experimental evidence for the clinical application of urantide in the treatment of AS.
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Objective: To investigate the effect of urantide on the levels of serum calcium, blood lipids and indexes of myocardial enzymes in the rats with atherosclerosis (As), and to clarify its mechanism of prevention and treatment of AS. Methods: A total of 180 Wistar rats were selected and randomly divided into normal control group, AS group, positive drug (simvastatin) group and urantide groups (3, 7 and 14 d group), 30 rats in each group. The rat model of AS was established by feeding on high-fat diet or intraperitoneally injecting vitamin D3 (VDs). The body weights of rats were weighed at three time points: pre-experiment, pre-administration and end of experiment. The levels of Ca", total cholesterol (T O, triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), creatine kinase (CK) and lactic dehydrogenase (LDH) in the serum of the rats in various groups were detected by automatic biochemical analyzer. Results: The body weights of the rats in normal control group were gradually decreased with the prolongation of time; compared with normal control group, the body weights of the rats in AS group at the time point of pre-administration was decreased (P < 0 . 01); compared with AS group, the body weights of the rats in different administration groups were increased (P < 0 . 05 or P< 0. 01). The levels of serum Ca2', TG, TC, LDL, CK and LDH of the rats in AS group were significantly higher than those in normal control group, and the level of HDL was decreased significantly (P < 0 . 01). Compared with AS group, the levels of serum Ca2 , TC, TG, LDL, CK and LDH of the rats in urantide group were significantly decreased (P < 0. 05 or P < 0. 01), while the HDL levels were higher (P < 0. 05 or P < 0. 01). Conclusion: Urantide can prevent and treat AS by down-regulating or up-regulating the levels of serum calcium, blood lipids and indexes of myocardial enzymes in the AS rats.
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Objective: To investigate the effects of Urantide on the expressions of c-Jun N-terminal kinase (JNK) mRNA and protein in the thoracic aorta tissue of the rats with atherosclerosis (AS) • and to elucidate the molecular mechanism and significance of prevention and treatment of AS. Methods: The AS rat models were established by intraperitoneal injection of Vitamin D combined with high-fat diet and control group was set up at the same time. A total of 150 AS model rats were divided into model group, simvastatin group. IJrantide 3 d group. Urantide 7 d group and Urantide 14 d group ( n= 30). The rats in simvastatin group were adminstrated with simvastatin by gavage for 14 d. and the rats in Urantide groups were injected with Urantide by caudal vein for 3. 7. and 14 d. The serum markers of the rats in various groups were detected by automatic biochemical analyzer; the morphology of rat thoracic aorta tissue was observed by HE staining; the expression levels of JNK mRNA and protein in the rat thoracic aorta tissue were detected by immunohistochemical staining. qRT-PCR and Western blotting methods. Results: Compared with control group, the serum levels of triglyceride (TG). total cholesterol (TC) and low density lipoprotein (LDL) of the rats in model group were increased (P<0. 05). and the level of high-density lipoprotein (HDL) was decreased ( P<.0. 05). The HE staining results showed the formation of bubbling cells in the thoracic aorta tissue, rupture of medullary elastic fibers and calcification of the rats in model group; compared with model group, the pathological symptoms of the thoracic aorta tissue of the rats in simvastatin group and Urantide groups were improved. The immunohistochemistry results showed that the JNK positive particles were weakly expressed in the rat thoracic aorta tissue in control group; the expression intensity of JNK in the rat thoracic aorta tissue in model group was increased ( P<0. 05); compared with model group, the expression intensities of JNK in the rat thoracia aorta tissue in Urantide groups were significantly decreased (P∗C0. 05). The qRT-PCR and Western blotting results showed that the expression levels of JNK mRNA and protein in the rat thoracic aorta tissue in model group were significantly increased ( P
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Objective To investigate the effects of urotensin Ⅱ/urotensin Ⅱreceptor ( UⅡ/UT) system on the expression of inflammatory signal molecules p 38 mitogen-activated protein kinase ( p38 MAPK) and nuclear factor-κB ( NF-κB ) in lipopolysaccharide ( LPS )-stimulated Kupffer cells ( KCs ) . Methods Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifuga-tion.The isolated cells were randomly divided into six treatment groups including group 1:UⅡ(-) urantide (-)LPS(-), group 2:UⅡ(+)urantide(-)LPS(-), group 3: UⅡ(-)urantide(+)LPS(-), group 4:UⅡ(-)urantide(-)LPS(+), group 5:UⅡ(+) urantide(-) LPS(+) and group 6:UⅡ(-)urantide(+) LPS(+) .Western blot assay was performed to detect p 38 MAPK/p-p38 MAPK protein and NF-κB p65 sub-unit.The DNA-binding activity of NF-κB was tested by electrophoretic mobility shift assay (EMSA).Re-sults There was no significant difference with the expression of p 38 MAPK protein in KCs among the six groups (P>0.05).The expression of p65 protein and p-p38 MAPK and the DNA-binding activity of NF-κB were significantly enhanced in LPS-stimulated KCs from groups 4, 5 and 6 in comparison with those in group 1 (P0.05), but that were decreased in group 6 than those in group 4 (all P<0.01).Conclusion UⅡ/UT system participated in the activation of p38 MAPK and NF-κB signaling pathways in LPS-stimulated primary Kupffer cells .
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Objecitve:To investigate effects of urotensin Ⅱ( UⅡ)/UT system on innate immune inflammatory signal pathway TLR4-IRF3 in the lipopolysaccharide (LPS)-stimulated Kupffer cells (KCs).Methods: Rat KCs were isolated and cultured.Pro-in-flammatory cytokines including IL-6,IFN-βand IFN-γwere assayed by ELISA in culture supernatant of KCs.Cell surface TLR4 were tested with flow cytometry technique.Expression of IRF3 were tested with real-time PCR and Western blot.Results: Significant increases were showed in IL-6, IFN-βand IFN-γsecretion, TLR4-expressed positive rates and IRF3 mRNA levels in KCs after stimulated by LPS,but were inhibited via urantide pretreatment.In addition,LPS induced upregulation of nuclear IRF3 protein and downregulation of cytoplasm IRF3 protein in KCs,which were blocked by urantide pretreatment.Conclusion:UⅡ/UT system mediates immune inflammatory response in part through activating TLR 4-IRF3 pathway in LPS-stimulated KCs.
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OBJECTIVE To investigate the protective effect of urantide on myocardial apoptosis in rats induced by ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injury and explore the underlying mechanism. METHODS ① In vivo test A rat myocardial I/R injury model was induced by ligating and untying the left anterior descending coronary artery with occlusion 30 min/reperfusion 60 min. Urantide 3, 10 and 30 μg·kg-1 was iv given 10 min before ischemia. TUNEL labeling was used for apoptosis measurement in myocardium. Immu-nohistochemical assay was used for Bcl-2 and Bax proteins expression detection. ② In vitro test An H/R cell model was set up by 3 h hypoxia/3 h reoxygenation. Urantide 0.1,1 and 10 nmol·L-1 was added just before hy-poxia, respectively. Hoechst33258 assay and flow cytometric techniques were used to detect apoptotic cells. RESULTS ① In vivo test Compared with sham group, the number of TUNEL-positive cells in I/R model group significantly increased (P<0.01) ; Bcl-2 protein expression slightly increased with no significant difference, Bax protein expression markedly increased ( P < 0. 01 ) , while Bcl-2/Bax ratio in I/R model group significantly decreased (P <0.01). Compared with I/R model group, the number of TUNEL-positive cells in urantide 10 and 30 μg·kg-1 groups was significantly decreased by about 36.6% and 57. 2% (P<0.05) ; Bax protein expression markedly decreased ( P <0.05 ) , while Bcl-2/Bax ratio was significantly augmented ( P <0.05 ). Urantide 30 u,g-kg1 also markedly increased Bcl-2 protein expression(P <0.05). ② In vitro test Compared with normal control group, the apoptosis rate in H/R model group significantly increased (P<0. 01). Hoechst33258 assay revealed that urantide 0.1, 1 and 10 nmol·L-1 reduced H/R-induced apoptotic nuclei by about 27.9% , 59.0% and 75. 4% , respectively (P <0.05). Flow cytometric techniques showed that the apoptosis rate was significantly reduced by about 32.8% and 64. 7% with administration of urantide 1 and 10 nmol·L-1 (P < 0. 01). CONCLUSION Urantide exerts an inhibitory effect on I/R or H/R-induced apoptosis by increasing Bcl-2 protein expression and decreasing Bax protein expression.
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Aim To investigate the protective effect of the potent UT receptor(urotensin Ⅱ receptor,UTS2R)antagonist-urantide against myocardial ischemia-reperfusion(I/R)injury and its probable mechanisms in rats.Methods Rat myocardial I/R injury was induced by ligating and untying the left anterior descending coronary artery.The rats were randomly assigned into 6 groups:sham group,model group,urantide 3 ?g?kg-1 group,urantide 10 ?g?kg-1 group,urantide 30 ?g?kg-1 group and Ver(Verapamil)1.6 mg?kg-1 group.All animals except sham group were subjected to 30 min of occlusion and 60 min of reperfusion.Urantide or Ver was given ten minutes before occlusion through intravenous drug perfusion.Heart rate(HR)and the ST segment change of electrocardiogram(ECG)were recorded.The content of malondialdehyde(MDA)and nitric oxide(NO),and the activity of lactate dehydrogenase(LDH)and nitric oxide synthase(NOS)in blood serum were measured.Infarct size(IS),as a percentage of the area at risk(AAR),was determined by Evans blue and TTC double staining.The expression of iNOS protein was detected by western blotting.Results The results demonstrated that during the process of I/R,HR decreased significantly whereas ST segment of ECG markedly elevated.After I/R,MDA content and LDH activity in blood serum increased significantly,while total NO content and total NOS activity decreased sharply.Urantide(10,30 ?g?kg-1)had no significant effect on HR changes,but could markedly inhibit the elevation of ST segment of ECG,MDA content and LDH activity,and inhibit the decline of total NO content and total NOS activity,at the same time decrease I/R induced IS/AAR.But 3 ?g?kg-1 urantide had no significant effect on above indexes.Except for this,urantide(10,30 ?g?kg-1)down-regulated the I/R induced expression of iNOS.Conclusions Our findings indicate that urantide has a protective effect against myocardial I/R injury in rats.The cardio-protective involves the inhibition of lipid peroxidation and the stimulation of NO release.