Effects Of PAMP On The Multiplication, Collagen And NO Synthesis In Rat Cardiac Fibroblasts Induced By Angâ…¡

Background: Cardiac muscle reconstitution is a common procedure in proceeding of cardiac disease, so we should explore rule of cardiac muscle reconstitution. It is proved that cardiac fibroblasts (CFs) prolifelate and produce collagen notablely in proceeding of cardiac muscle reconstitution by experiment. CFs can produce nitrogen oxide(NO),which inhibit CFs proliferation and collagen synthesis. CFs is object and CFs prolifetion, collagen and NO sythesis are observed in the experiment.Cardiac muscle reconstitution is affected by many factor, such as haemodynamics, cardiac cell death, adherence factor, humoral factor. AngⅡ, which is important molecule in RAS system, stimulate cardial cell and cardiac fibroblasts (CFs) to proliferate directly or by increasing blood pressure indectly. Angll accepter have two main type, AT1 and AT2. AT1 is correlated with growth of cardiac cell and CFs; AT2 is concerned with apoptosis of cardiac cell and CFs. The two affect each other. Conglutination between CFs and collagen can be inforced by Angll. It is said that Angll play an important part in proceeding of cardiac muscle reconstitution on ground of Angll database .Adrenomedullin (ADM), which is a new kind of bioactive peptide, was found inhuman phaeochromocytoma by Kitamura. Its cDNA, which have 1293 bp, and recode 185 amino acids, was cloned already. The amino acide, which is named as preproadrenomedullin (prepro-ADM), is disintegrated into 5 parts by endogenous enzyme, (l)signal peptide; (2)proADM22-41(PAMP) ; (3)proADM45-92 ; (4)pro-ADM95-146; (5) proADM153-185 (Adrenotensin, ADT). PAMP is as analogously effective as ADM, but the artical on PAMP is not as much as ADM.. Lippton found that PAMP can drpress blood vessel pressure, after injecting massive PAMP into paralysed rat, and increase heart rate and activity of adrenergic nerve at the same time. PAMP level of cardiac and aorta tissue in spontaneously hypertensive rat is higher than in normal rat. PAMP level in hypertensive patient is higher than nomal blank. Concentration of PAMP directly correlate with ADM, NA, AD and mean arterial pressure. Mechanism that PAMP depress blood pressure is a secret. It is still uncertain that PAMP directly affect cardiac muscle reconstitution or indirectly affect it by depressing blood pressure.Objective: Ang II ,PAMP and both of them, which is differant concentration,are added into rat cardiac fibroblasts cultured. We measure NO and collagen synthesis of rat cardiac fibroblasts and proliferation of it in order to explore that how do PAMP effect cardiac fibroblasts and cardiac muscle reconstitution.Methods: Cardiac muscle of 3-days-old SD rat that is digged by trypsogen is cultured in the culture flask. We can gain cardiac fibroblasts after throwing over the tissue which is not adherence two hours later. CFs of the 3~8th generation, which purity quotient is 98%, is be used in the experiment.Experiment group: (Dblank gronp (2)10'9> 10"8, 10"\ 10"6mol/L AngH group-, (3)10 ^ 10~8> 10"\ 10'6mol/L PAMP group; @10'7 mol/L AngH + PAMPCIO'9, 10"\10~7> lO^mol/Pgroup. there are 6 hollow in every level.Growing CFs are inoculated into culture 3 board of 96 hollow that there are5000 cells in every hollow. When growing CFs are getting touch each orther, nutrient solution is replaced by one inclunding 0.5% FCs in order to let CFs stand still. After CFs is cluturd for 24 hours, drugs that is pared is added into the every hollow.Quantity of CFs is measured with the MTT colorimetric method. After culturing it for 72 hours, drip 20ul MTT into every hollow, then culture it for 4 hours again. We drip 150ul DMSO into every hollow and oscillate it for 10 min after imbibing the solution of every hollow. The A490 value is measured by enzyme marks machine.We can measure quantity of collagen synthesis by measuring 3H- praline in collagen. After drugs are added into the CFs for we drip 3H- praline and vitam C into the solution for 4 hours, and stop it. CFs are digested intosuspion by 0.125% trypsogen and are engrafted into solitary EP. EP is centrifugated for 20 minutes with 50000r/h and is rinsed by PBS for 2 times. After immigrating it into the counter tuber and add scintillation fluid to the tuber, we measure quantity of radioactivity.Quantity of NO is measured with nitric acid reduction. The method of measurement is that NO3" is transformed into NO2" by nitrate reductase, and absorbance is measured by measuring density of NC>2~. We manipulate it by description. Results:1. Effects of AngI I on CFs cell number, collagen and nitric oxide(NO) synthesis Effects of A ng I I on CFs number: After 10'9,10"8,10"7,10" 6mol/L Angll are added into every hollow for 72h, it is measured. Quantity of every MTT chromaticity is respectively 1.0613±0.0042, 1.2413±0.0037, 1.4600±0.0033, 1.6607 ±0.0037, and quantity of blank one is 0.9513±0.0046o while level of Angll is increasing quantity of their MTT chromaticity is also increasing at the same time. Each group is differential notablely(P<0.01). It mean that proliferation of CFs is stimulated by AngllEffects of Ang I I on CFs collagen synthesis: Radioactive quantity of 10~9, 10"8, 10"7and 10'6mol/L Angll groups is respectively 1587.45±22.72, 1581.96±28.42, 1939.82±51.94, 2599.02±81.27dpm, and radioactive quantity of blank group is 1400.35 ± 45.75dpm (P<0.01)o While level of Angll is increasing, radioactive quantity of groups is also increasing at the same time. Each group is differential notablely (P<0.01). It mean that collagen synthesis of CFs is stimulated by AngllEffects of A ng I I on CFs NO synthesis: After CFs is cultured for 24h, NO level of 10"9> 10'8> 10~\ 10"6umol/L Angll group is respectively 73.88 ±2.23s 64.34 ±3.02> 54.12 + 2.82, 40.21 + 1.45umol/L. Each group is differential notablely (P<0.05).there isn't differential notably between blank group and 10"9umol/L Angll group (P>0.05) c It's mean that NO synthesis is inhibited by Angll.. 2. Effects of PAMP on CFs cell number, collagen and nitric oxide(NO) synthesisEffects of PAMPon CFs cell number: After CFs is stimulated by 10'^ 10"8> 10"7> 10" 6mol/L PAMP for 72h, quantity of their MTT chromaticity is respectively 0.9512±0.0053, 0.9525±0.0039, 0.9510±0.0036> O.95O8±O.OO57, and quantity of blank group is 0.9513±0.0046(P>0.05). It mean that proliferation of CFs is't affected by PAMP.Q ftEffects of PAMP on CFs collagen synthesis: radioactive quantity of 10" s 10" , 10"7^P10"6mol/L PAMP groups is respectively 1405.36±44.42, 1403.39±35.49> 1433.49±49.56. 1419.36±43.05cpm, and radioactive quantity of blank group is 1400.35±45.75cpm ( P>0.05), Each goup is't differential notablely. It mean that CFs collagen synthesis is't affected by PAMP.Effects of PAMP on CFs NO synthesis: NO level of 10"9x 10"\ 10"\ 10"6umol/L PAMP groups is respectively 74.40±3.42 > 74.91±2.66 > 75.77±3.31 > 74.23±2.43umol/L.and blank group is 74.57±2.49fimol/L. there isn't notably differential among groups (P>0.05) . It mean that CFs NO synthesis is't affected byPAMP.3. Effects of PAMP and Ang II on CFs cell number, collagen and nitric oxide(NO) synthesis:Effects of PAMP and Angll on CFs cell number: After CFs is stimulated by 10'9> 1O'\ 10"\ 10" 6mol/L PAMP and 10"7mol/LAng E for 72h, quantity of their MTT chromaticity is respectively 1.4542±0.0039 ^ 1.2293±0.002^ 1.1333±0.0038 ^ 1.0315±0.0033, quantity of blank group is 0.9513±0.0046, and quantity of 10"7mol/L Angll group is 1.4600±0.0033. while concentration of PAMP is increasing quantity of their MTT chromaticity is variational notablely.(P<0.01). there isn't differential notably between 10~7mol/LAngII group and 10"7mol/L AngII+10~9mol/L PAMP group(P>0.05,)- there is differential notably among other groups(P<0.01). It mean that proliferation effect of Angll on CFs cell number is inhibited by PAMPEffects of PAMP and Angll on CFs collagen synthesis: radioactive quantity of 1(T9, 10"8, 10~7, 10"6mol/L PAMP + 10"7mol/L Angll groups is respectively 1.4542±0.0039^ 1.2293±0.0029, 1.1333±O.OO38> 1.0315±0.0033dpm. While level of Angll is unchangeable and level of PAMP is increasing, radioactive quantity of groups is decreasing, there is differential notably among groups(P<0.05). It mean that augmented effect of AngH on CFs collagen synthesis is inhibited by PAMP.Effects of PAMP and Ang II on NO synthesis: NO level of 10"7 umol/L Angn + (10~\ 10'\ 10"\ 10~6umol/L) PAMP groups is respectively 66.15±2^ 80.58±3^ 88.67±1.46> 96.22±2.96umol/L (P<0.05.) there is differential notably among other groups(P<0.05), while level of PAMP is increasing, NO synthesis of groups is also increasing at the same time. It mean that augmented effect of AngH on CFs NO synthesis is inhibited by PAMP.Conclusion: 1. AngH stimulate CFs to mulitplicate and sysnthesize collagen, and inhabit CFsto sysnthesize NO.2. PAMP can't make CFs to more propagate, more produce collagen and NO.3. PAMP make CFs to sysnthesize collagen and multiplicate by increasing NO sysnthesise...