The Study Of Urotensin Inhibiting Monocytes Secreting Matrixmetalloproteinases

Background: Heart failure is the advanced stage of all sorts of heart disease. With the high morbility, five-year survival rate of it is just as malignant tumor. Professor Braunwald call it as the final battlefield of heart disease on the opening ceremony of annual meeting of AHA. The morbidity of it is 1.5%~2.0% in average humen, but it is ascend to 6%~10% in the people whose age are beyond 65. The death caused by HF were 6 times high in the past 40 years. So HF becoming the most important disease of cadiovascular system. The variation of ECM appearsed as overdeposited or degradation inappropriate of fibrocoUagenous. As non-infarct area in infarct heart, the LVH and non-hypertrophyin RV in hypertensive heart disease all had interstitial fibrosis. Matrix metalloproteinase is the role protease in metabolism of ECM, and it plays a pivotal effects in regulation of heart remodling. Monocyte is the main site of matrix metalloproteinase synthesis. It is secreated in proenzyme fomartand can degradate ECM after being activated. There are many therapeutic measures to cure HF, but some patients have no response. So it is urgent to search new pathogenesy and target of therapy.Urotensin-II was one of these points.lt was vasoactive somatostatin-like cyclic peptide which composed of twelvs amino acids.lt was abstracted from caudal hypophyse in neurosecretory system of teleost primitively, then also was discovered in neurosecretory system of mollusc and mammalian lately. GPR14,the receptor of Urotensin- II was discovered in cardiovasular tissues of human body by Ames firstly in 1999. Urotensin- II was discovered in myocardium, coronary atherosclerotic plaque, lipid sedimentary smooth smuscle cell and macrophage also.Its levating in inflammatory cell was also discovered in advanced stage of heart failure by Douglas.All these findings were showed that Urotensin-II may affect cardiovascular poikilostasis and pathology.Now it was clear that Urotensin-II can contract blood vessel and inhibit the contraction of myocard, it also can promote proliferation of myo-mechanocyte and synthesis and secretion of collagen protein.lt indicate that Urotensin- II may affect the poikilostasis and pathology of cardiovascular, and involved in the structure remodeling of cardiovascular disease. So we study the ralationship between Urotensin- II and ventricular remodeling in cultured monocyte.Objective: To study the effect of Urotensin- II on the proliferation and function of monocyte and elucidate the sense and the possible effects of Urotensin- II on the remodling of cardiovascular ventricles. And to offer the base of the prevention andentrationcure of HF.Methods: l.Monocytes were extracted from venous blood which were drawed from healthy people by PercoU separating medium(relative density 1.076). Washouted by Hanks. Cultured in RPMI1640 medium, 37°C for 2 hours. Lymph fluid were washouted. Monocytes were cultured in RPMI1640 medium which contained 30% autoserum, 37 °C, 5%CO2 for 7 days and had feature of macrophage.2. Monocytes concentration were adjusted to 2xl05 pieces/ml, 0.5ml were plus to each hole of cultural plate. 4 groups were randomly designed(n=6), Cone of Urotensin-II were Ixl0"8mmol/L,lxl0"9mmol/L,lxl0"10mmol/L and Omrnol/L respectively. 3 subgroups were randomly designed in each group. 3H-Leu, 3H-TdR and 3H-UR luCi/ml were treated respectively.3. Determination of DNA expression, RNA expression and protein synthesis. After being cultured for 24 hours, medium were spilled. Monocytes were washouted by Hanks, digested by 0.25%trypsogen which contained 0.04%EDTA and transferred to filter-paper. After being eluted, fixed, dehydrated and torrefied, Monocytes were put into xylene scintillation fluid which contained 0.03%POPO and then assayed by liquid scintillator.Another 3 groups were randomly designed(n=6), 3H-Leu, 3H -TdR and3H -UR luCi/ml were treated respectively. DNA expression, RNA expression and protein synthesis were assayed in 1st day, 4th day and 7th day.4. Cell protein assay: 4 groups were randomly designed(n=2). After being cultured for 3 days, cells were treated with Urotensin- II with the concentration of lxl0"8mmol/L,lxl0"9mmol/L,lxl0"10mmoiyL and Ornmol/L respectively for another 4 days. Cell protein were assayed by Lowery's methods.5.Extraction of total RNA and RT-PCR: Total RNA were extract from lxlO7HMDM with lml TRIZOL in one-step methods. Oligonucleotide primer of MMP-2 and MMP-9 were synthesised according to reports and humen P-actin as the reference. OPTDM were measured by Image processing system.6.Assays of interest protein: WesternBlot was the methods. Firstly, the sample of protein were separated by SDS-PAGE. Electrophoresised with 200mA and 100V for 6 hours, trarsmembraned, recorded the location of marks, washed, blocked, rinsed, hybridisated with buffer solution which contained dicho-antibody, rinsed again, washed by substrate buffer solution, colorated, photosensitised, shooted and scaned with photosensitise.7. Assays of MMPs: It were assayed by Zymography methods. Put some gelatin into the 12% separating gel until the concentration were lg/L in SDS-PAGE electrophoresis. The ratio of cell supernatant and earring buffer was 3:1. Put 20ul into well in 4% condensed gel without boiling. Electrophoresised with 4°C and 200V. Then the jel was rinsed and incubated in buffer for one night. It was dyed by CoomassieBlueR-250,then bleached. Density and area of degradation belt wereassaied. The cell supernate of each control group as the reference. The activity of MMPs were indicatied by multiplication of density and area of degradation belt.Results: 1.Being cultured in RPMI1640 medium which contained 30% autoserum, Monocytes were in good condition. Synthesises of DNA, RNA and protein were intense, especialy treated with 3H-UR. Cell morphology weren't change after being treat with Urotensin- II for 24 hours. Intakes of 3H-TdR and 3H-UR were significantly inhibited by Urotensin- II in dose-depedent way.Compared with the synthesises of DNA, the synthesises of RNA were significantly inhibited by Ixl0~8mmol/L and Ixl0~9mmol/L Urotensin- II. Ixl0"10mmol/L Urotensin- II had no effect on the synthesises of protein. lxl0-8mmol/L and lxlO-9mmol/L Urotensin- II can significantly inhibited the synthesises of protein.2. Compared with control group, more brightness, less satiation and particles were observed in Urotensin- II group after being incubated with Urotensin- II for 4 days. Protein content were significantly decresed in dose-dependent way also. No effect were observed on cell viability.3. Effect of Urotensin II on the expression of mRNA and protein of MMP-2 and MMP-9 in HMDM. Compared with control group, the expression of mRNA of MMP-2 and MMP-9 were significantly decreased in lxl0~8,lxl0~9 and 1x10" 10mmol/L Urotensin II (average RZ were 0. 85 ± 0. 05 , 0. 86 ± 0. 06 , 0. 89 ± 0. 04vs0. 99±0. 05, n=4, P<0. O5andO. 84 + 0. 05, 0. 85±0. 04, 0. 88 + 0. 04vs0. 99± 0. 05, n=4, P<0. 05 respectively). The expression of protein of MMP-2 and MMP-9 were also significantly decreased.4. Eeffect of Urotensin II on the activity of MMP-2 and MMP-9 in HMDM. Compared with control group, the activity of MMP-2 and MMP-9 were significantly decreased .( average RZ were 98. 35 ±11. 15, 102. 29 ± 10. 45 ? 108.45 + 11.25vsl27. 93±13. 61, n=4, P<0. 05and40. 01 + 8.96, 45. 38 + 9. 32, 52.19 + 10. 05vsll5. 95 + 11. 03, n=4, P<0. 05 .respectively)Conclusion: 1. The expression of DNA and RNA, the synthesis of protein in Monocytes can be inhibited by Urotensin- II.2. The secretion of protein including matrix metalloproteinase(MMP) can be inhibited by Urotensin- II. So it affects degradation of collagen protein, participated in the development of ventricular remodeling.