The Effects Of Interleukin-1β And Ox-LDL On The Ventricular Remodeling And Its Being Interfered By Statin

Ventricular remodeling in the process of post- myocardial infarction is the major mechanism of heart failure.It contributes to ventricular dilation and dysfunction, patients' disability and death.Ventricular remodeling is a complex dynamic process including cardiomyocyte hypertrophy,apoptosis,necrosis and extracellular matrix (ECM) remodeling,especially dynamic equilibrium of synthesis and decomposition of collagen,the major composition of ECM.Pressure overload,inflammations and rennin- angiotensin-aldosterone systems(RAS) and some other factors are involoved in the process and cause collagen synthesis decreasing,degradation and denaturation. Those changes promote the ventricular dilation and rupture.The 75%cells of heart are non-cardiocyte and cardiac fibroblasts(CFs) are the major composition.So we can see that CFs play important role in the process of post-MI remodeling.Most collagen and skeleton protein were synthesized by CFs, especially the typeⅠand typeⅢcollagen,which induced the collagen network structure formation and to maintain the fuction of heart.The ordered degradation of ECM is the key point of heart grow,remodeling and repair.Collagen,the major componant of ECM,whose degradation is regulated by the balance between MMPs and its inhibitor called TIMPs in ECM.MMPs are a class of zinc-dependent enzymes that secreted in a proenzyme form and require proteolytic cleavage for activation and have a high specificity for components of the ECM.It participated in ECM turnover and vascular wall remodeling,angiogenesis,and atherosclerosis.MMPs selectively degrade extracellular proteins such as the fibrillar collagens.MMP-2 and MMP-9 are the important composition of MMPs family which can degrades the denatured collagen fibrosis and elastin.The inbalance of MMPs/TIMPs after AMI caused dynamic equilibrium of synthesis and decomposition of collagen and promoted the adverse ventricular remodeling.Atherosclerosis(AS) is presently one of the most harmful human diseases with highest morbidity.It has been proved that AS has complex factors at multiple levels and infection,inflammation,and immunity were involoved in the process of initiation and progress.Oxidized low density lipoprotein(ox-LDL),one of the initiations which could be englobed by macrophage,formed foam cells and plaque.AMI was happened after the unstable plaque was ruputure.Interleukin-1β(IL-1β),Interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) are pro-inflammatory cytokines that have been found to be elevated in the serum of patients who underwent MI.This initiates pro-inflammatory cytokines which may play important role in the process of post-MI remodeling.Previous study showed that IL-1β,TNF-αdecreased collagen synthesis without change on cell number or total protein synthesis,its also increased total MMPs activity,causing specific increases in the bands corresponding to MMP-13, MMP-2,and MMP-9 and the expression of proMMP-2 and proMMP-3 mRNA.But the effects of ox-LDL on the post-MI ventricular remodeling and the interaction with the elevating of pro-inflammatory cytokines are not very clear.From the above,we can see the post-MI ventrivular is a complicated process and ox-LDL,IL-1β,IL-6 and TNF-αis all involoved in.They caused imbalance between the synthesis and degradation of collagen,damaged the integrity of ECM,initiated the ventricular remodeling and dysfuction.The aim of anti-remodeling is to prevent, restrict and reverse the adverse remodeling in order to protect the ruction of cardiac ventricle.One aspect is to recorve the integrity of ECM.The onset and duration of theraphy is the key point in the treatment of ventricular remodeling because it was originated in early time after AMI and maintained for several months to years.ACEI, ARB,aldosterone antagonist and beta blosks were used to anti-remodeling theraphy. Statin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA(HMG CoA) reductase,a key enzyme in the cholesterol synthesis pathway.Recently,Statins were founded can reduce adverse ventricular remodeling independently of their cholesterol-lowering ability.So we used IL-1β,one of the pro-inflammation cytokines,combined with ox-LDL to work on the cardiac fibroblast of neonatal SD rat and then interfere with Simvastatin,observed the effects on collagen synthesis and the activity of matrix metalloproteinases.In this study,we firstly used ~3H-thymidine(~3H-TdR) incorporation,~3H-proline (~3H-Pro) incorporation,gelatinase zymography,fluorescence microscope, Western-blot and reverse transcription PCR(RT-PCR) to measure the effect of IL-1βon the collagen synthesis,decomposion and the expression of Matrix Metalloproteinases(MMPs) in cardiac fibroblasts respectively.Then LDL was separated from human serum and oxidized in vitro and CFs was treated with different concentration of it.The proliferation of CFs,the collagen synthesisi and expression of MMP-2 and MMP-9 were observed as before.At last,we combined IL-1βwith ox-LDL to work on the CFs and pretreated with Simvastatin respectively.The effect of Simvastatin on the proliferation of CFs,the collagen synthesisi and decomposition were studied.We got the result as follows:PartⅠ:Interleukin-1βinhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.Methods:Cardiac fibroblast of neonatal Sprague-Dawley rat were cultured in vitro and treated with IL-1β(0.01ng/mL,0.1ng/mL,1ng/mL,10ng/mL 100ng/mL) for 24h.Cell proliferation was measured by MTT assay.Cell DNA synthesis was measured by ~3H-TdR incorporation and collagen synthesis was measured by ~3H-Pro incorporation.MMPs activity was measured by gelatinase zymography.MMP-2, MMP-9 protein levels were measured by Western-blot.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR.Results: 1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR and ~3H-Pro was decreased in high dose IL-1β(≥0.1ng/ml) but not in low dose(0.01ng/ml).2.0.01ng/ml IL-1βhad no effects on the collagen synthesis of CFs. 0.1～100ng/ml IL-1βmarked inhibites ~3H-Pro incorporation in concengtration dependent way.3.IL-1βcould significantly increased MMP-2 activities in concentration dependent way and MMP-9 activities were increased also.But it was different with MMP-2 levels,the extent of elevation between 0.01ng/ml to 1ng/ml had no significant difference.4.IL-1βalso dose dependently increased the protein levels of MMP-2(fold change,0.01ng/mL,1.35;0.1ng/mL,1.56;1ng/mL,2.15;10ng/mL,2.34;100ng/mL, 2.41) and MMP-9(fold change,0.01ng/mL,1.38;0.1ng/mL,2.37;1ng/mL,2.56; 10ng/mL,2.60;100ng/mL,2.67) respectively.5.Similar results were observed in the mRNA expression of MMP-2(fold change,0.01ng/mL,1.81,0.1ng/mL,2.17;1ng/mL,2.18;10ng/mL,2.27;100ng/mL, 2.31) and MMP-9(fold change,0.01ng/mL,1.36;0.1ng/mL,1.49;1ng/mL,1.50; 10ng/mL,1.51;100ng/mL,1.52).Conclusion:These results suggest that IL-1βdecreases collagen synthesis and activates MMPs through transcriptional and posttranslational processes that degrade collagen in dose dependent way.Elevation of IL-1βafter infarction myocardial is possibly involved in the process of post-myocardial infarction ventricle remodeling, and that the concentration of IL-1βmay the major factor which affects the degree of ventricle remodeling.PartⅡ:ox-LDL inhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.Methods:ox-LDL was seprated and oxidated in vitro.Cardiac fibroblast of neonatal SD rat were cultured and treated with ox-LDL(10,20,50,100μg/ml) for 24h.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.Results:1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR were increased in ox-LDL treated group in concentration way.2.The effect of ox-LDL on ~3H-Pro incorporation were associated with its concentration.10μg/ml ox-LDL could decrease the ~3H-Pro incorporation and 100μg/ml ox-LDL had the most power.3.ox-LDL could significantly increased MMP-2 activities in concentration dependent way,MMP-9 activities were increased also in high dose of ox-LDL. 10μg/ml ox-LDL could slightly promate the MMP-9 activities,but had no significant difference with control group.4.ox-LDL also dose dependently increased the protein levels of MMP-2 (foldchange,1.17,1.32,1.42,1.53 respectively).10μg/ml ox-LDL have no effect on the protein level of MMP-9.20μg/ml～100μg/ml could significantly enhance the protein expression of MMP-9(fold change,1.17,1.39,1.52,respectively).5.10μg/ml and 20μg/ml ox-LDL had no effect on the mRNA expression of MMP-2 and MMP-9.50μg/ml and 100μg/ml ox-LDL could significantly increase the mRNA expression of MMP-2(foldchange 1.21 and 1.33)and MMP-9(foldchange 1.18 and 1.22).Conclusion:ox-LDL decreases collagen synthesis with the cell proliferation in the same time.It activates MMPs through transcriptional and posttranslational processes that degrade collagen in high concentration.But in low dose,it only could through posttranslational processes to increase the MMPs only.These results suggest that hyperlipemia may not only initiate the coronay artery disease,hut involvded in the whole course from onset to ventricle remodeling.PartⅢ:Simvastatin prevent the effect of IL-1βand ox-LDL on the collagen synthesis and decomposition in cultured cardiac fibroblast.Methods:ox-LDL was seprated and oxidated as partⅡ.Cardiac fibroblast of neonatal SD rat were cultured and treated with IL-1β(10 ng/ml),Simvastatin (10μmol/L)+IL-1β(10 ng/ml),ox-LDL(50μg/ml),Simvastatin+ox-LDL(10μmol/L and 50μg/ml),IL-1β+ox-LDL(10 ng/ml and 50μg/ml),Simvastatin+IL-1β+ox-LDL (10μmol/L,10 ng/ml and 50μg/ml) respectively for 24h.In all study,Simvastatin were pretreated for 30min.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.Results:1.The same effects on the DNA synthesis of IL-1βand ox-LDL were observed as before when treated solo and pretreated with Simvastatin could prevent those effects.The synthesis of DNA was decreased when cotreated with IL-1βand ox-LDL. Simvastatin could inhibite those effects.2.The same effects on the collagen synthesis of IL-1βand ox-LDL were observed as before when treated single and pretreated with Simvastatin could reverse those effects.After cotreated with IL-1βand ox-LDL,the collagen synthesis was highly decreased than every single treatment.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.3.Pretreated with Simvastatin could significantly inhibit the effects of IL-1βand ox-LDL single on the MMPs activity to the normal levels.After cotreated with IL-1βand ox-LDL,the MMPs activity was slightly increased,but had no significant difference with each single factor.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.4.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the protein content of MMP-2 and MMP-9.After cotreated with IL-1βand ox-LDL,the protein levels of MMP-2 and MMP-9 were significantly higher than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels.Although it could significantly decreased those protein levels in the combined group,it still was higher than control group.5.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the mRNA expression of MMP-2 and MMP-9,especially in the treating of IL-1β.And those effects were significantly higher after cotreated with IL-1βand ox-LDL than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels,but not in the combined group as protein leves.Conclusion:Combine IL-1βwith ox-LDL could decreases DNA synthesis and collagen synthesis in the same time.They could modulate the activity of MMPs through transcriptional and posttranslational processes together which promote the decomposion of collagen.Simvastatin could prevent those effects through transcriptional and posttranslational processes also.These results suggest that hyperlipemia and inflammation were all involove in the post-MI ventricular remoding. And interfere with Simvastatin early may inhibit those adverse effects.