| [Background and objective]Coronary heart disease (CHD) is one of the main diseases which is a hazard to human health.Myocardial ischemia and hypoxia result in damage, cell necrosis and repair, leading to ventricular remodelling (VR). Myocardial fibrosis is one of the main pathological characteristics. Tissue fibrosis irreversible structural pathological changes, Therefore, it is crucial to gain a better understanding of the mechanism causing myocardial fibrosis.Multiple studies indicated that myocardial ischemia caused increase of IL-6 release, and IL-6 pays an important role in cardiac dysfunction induced by myocardial ischemia reperfusion injury. This suggests that IL-6 may be an important factor in the organ fibrosis. Transforming growth factor-βs (TGF-βs) are multifunctional cytokines. Among them, TGF-β1 has a leading position in cardiac fibrosis. It induces myocardial remodelling and fibrosis via Smads-dependent pathways. IL-6 can mediate the TGF-β1 signalling pathway to regulate and control fibrosis remodelling process in many diseases. However, how IL-6/TGF-β1 signaling affects ischemic myocardium remodeling remains fewer investigation.Therefore, in our present study, we firstly conducted a clinical observation and analysis of ischemic cardiomyopathy in coronary artery stenosis patients receiving complete revascularization(CR), incomplete revascularization (IR) and drug therapy alone. Follow up on cardiac function, cardiac remodelling index and the degree of myocardial ischemia was observed at 6 months and 12 months. We observed the dynamic changes of serum IL-6 at all time points, and analysed its correlation to the changes of cardiac function and cardiac remodelling index.To further confirm the link between IL-6 and TGF-01 increase and the ventricular remodelling after myocardial infarction, we investigated the mechanisms of IL-6 induced myocardial remodelling and fibrosis under the aspect of whether TGF-β1/Smad3 signalling pathway was involved. We established a rat model of an acute myocardial infarction (AMI), determined the inflammation response, myocardial fibrosis and serum levels of IL-6, TGF-β1, MMP2 and MMP9 after stimulation or inhibition of IL-6. In addition, myocardial structure and function were analysed. In vitro apoptosis, proliferation, inflammation and fibrosis were determined in cultured cardiac fibroblasts (CFs) after stimulation or inhibition of IL-6. Our study aimed to provide more insight into the underlying processes and thus suggest treatment options for myocardium remodelling after AMI.[Methods]Clinical part:66 patients with ischemic cardiomyopathy, associated with severe heart failure, were selected, according to the patient’s willingness to accept complete revascularization (CR)+standard drug therapy (D), incomplete revascularization (IR)+standard drug therapy (D), and standard drug therapy (D). Three groups were further divided into CRD group (n=14), IRD group (n=34), and D group (n=18). Observation of heart function and cardiac remodelling index was performed at 6 months and 12 months, including the dynamic changes of IL-6 in serum at different time points. The relationship between IL-6 dynamic changes and cardiac function and cardiac remodelling index were analysed.In vivo:AMI model was established by ligation of the left anterior descending coronary artery in rats. The rats were divided into 4 groups-sham operation group (Sham group), AMI model with saline (LINS group), AMI model with IL-6 injection (LIIL group) and AMI model with injection of IL-6 receptor inhibitor (LIILR group). Infarcted animals were randomized to receive salt, IL-6 or IL-6R inhibitor, which was equally injected into the infarct and peri-infarct area. Heart function was determined by echocardiography. Left ventricular end systolic diameter (LVESD), left ventricular end diastolic diameter (LVEDD) and left ventricular ejection fraction (EF) were measured at lw and 2w post-MI. Two weeks later, the rats were sacrificed, and the heart was embedded in paraffin and paraffin section. The pathological changes and infarct size were observed by H&E staining. Myocardial collagen was detected by Masson staining. Myocardial expression of IL-6 and TGF-β1 was evaluated by immunohistochemistry. ELISA was performed to measure expression of cytokines including inflammation index MPO, oxidative stress index ROS, and fibrosis indexes IL-6, TGF-β1 and MMP2/9 in the supernatant of the infarcted tissues.In vitro:cardiac fibroblasts (CFs) were isolated and cultured from the hearts of neonatal Lewis rats. Hypoxic culture was established (cultivation conditions 37 ℃,5% CO2,2.5%O2). CFs were then divided into 4 groups:normoxic culture (CFs group) (cultivation conditions 37℃,5% CO2,20% O2), hypoxic culture alone (H-CFs group), hypoxic culture plus IL-6 intervening (H-CFs+IL-6 group) and hypoxic culture plus adding IL-6 receptor inhibitor (2.5umol/L) (H-CFs+anti-IL6r group). The CFs were cultivated for 72h. Vimentin stain was detected by immunofluorescence, cell proliferation by CCK-8 assay and cell apoptosis by flow cytometer. mRNA and protein levels of IL-6, TGF-β1, Smad3 and MMP2/9 were detected by RT-PCR and Western blot.[Results]Clinical part:The survival rate of each group was significantly varying in relation to changes of ischemia degree. It was 94% in CRD group,91%, in IRD group and 86% in the D group. After 6 months follow-up, the NYHA heart functton classification in CRD group and IRD group improved from preoperative III to IV into II to III grade, and LVEF increased (P< 0.01), which were significantly higher than that in D group (P< 0.01); Heart function further improved at 12 months with a higher improvement rate in CRD group.Consistent with the changes of heart function, serum biochemical parameters of quantitative heart failure marker NT-proBNP decreased after the heart function improved, more significantly in PCI group than that in the D group. In CRD group, it dropped the most at 6 months (P< 0.01 compared with admission); followed by the IRD group; group D decreased substantially (P< 0.01),no statistical significance at 12 months compared with 6 months (P> 0.05).The ventricular wall motion score index (WMSI) in the D group showed no significant change at 6 months and 12 months follow-up when compared with admission. The WMSI in CRD group was significantly decreased at 6 months follow-up (P< 0.01), and sustained at 12 months (P< 0.01). The WMSI at 6 months follow-up was significantly decreased (P< 0.01) in the IRD group compared with admission, but revealed no significant difference from 12 months (P>0.05).Heart structural parameters (LVEDD,LVESD,LVEDV,LVESV) were significantly decreased (P< 0.01) in the patients receiving revascularization at 6 months, and 12 months follow-up. The improvement was more significant in the CRD group than in the IRD group (P< 0.01). The LVEDD and LVEDV were reduced in the D group at 6 months compared with at admission(P<0.01), but did not sustain in the 12 months follow-up (P>0.05)Serological detection showed that the expression levesl of IL-6 and myocardial necrosis index hs-cTnT were significantly higher in these three groups than those in the normal limits (P< 0.01). These indexes in the CRD group gradually dropped to the normal value at 6 month later and remained at 12 months. Serum IL-6 and hs-cTnT in the IRD group were significantly decreased at 6 months (P< 0.01), but slightly higher at 12 months, while IL6 and hs-cTnT in the D group had a lower trend after regular medicine treatment, and there was no significant difference between 6 months and 12 months (P>0.05).Correlation analysis showed that the IL-6 level was positively correlated with the change of heart function index (HYHA 0.6614, NT-proBNP 0.8356), while it was negatively correlated with LVEF (-0.7604) and positively correlated with the change of the index of cardiac remodelling (LVEDD 0.8335, LVESD 0.7987,LVEDV 0.8323,LVESV 0.8365). Positive correlation was seen with WMSI (0.8393)In vivo:We successfully established a stable model of AMI. The rate of survival was:Sham:100%, LINS:65%, LIIL:50% LIILR:90%(p<0.01). Cardiac function was evaluated by echocardiography. LVEF was decreased in all the MI group compared with the SHAM group at 1w post-MI, and further declined at 2w post-MI. The LVEF in the LIIL group revealed the lowest, and it was higher in LIILR group than in the LINS group (P< 0.05). H&E staining showed that the animals in the LIIL group had the most serious inflammatory reaction among the four groups, followed by those in the LINS group and LIILR group. Consistent with inflammatory response assessed by HE staining under microscope, the MPO and ROS levels in the tissue homogenate showed the same change trend in the four groups. Masson staining showed disturbed arrangement of myocardium, increased interstitial collagen fibers and increased myocardial fibrosis within the infarct border zone in the LINS group when compared with sham group. IL-6 injection caused further significant increase in the LIIL group in the conten of the collagen fibers stained as blue and green and collagen deposition in the infarcted myocardium, and injection of IL-6 receptor inhibitor significantly inhibited collagen formation in the LIILR group. ELISA showed that the homogenate levels of IL-6, TGF-β1 and MMP2/9 in the infarcted myocardium were significantly higher in the LIIL group than in the other groups (P< 0.05), and injection of IL-6 receptor inhibitor led significant decrease in the LIILR group compared with the LIIL group and LINS group (P< 0.05). Immunohistochemistry showed that IL-6 and TGF-β1 expressed in both LIIL group and LIILR group. The semi-quantitative analysis of staining revealed that the expressions of IL-6 and TGF-β1 were seen the highest in the LIIL group, and followed by the LINS group and LIILR group (P< 0.05).In vitro:CFs were successfully isolated from neonatal rats, revealed fusiform shape under the microscope, and were confirmed by immunofluorescence detection of Vimentin. The cell density of fibroblasts progressively increased in the CFs group at 24h,48h, and 72h post-normoxic culture (P<0.05), hypoxia significantly decreased fibroblast proliferation in the H-CFs group. After the addition of IL-6, the proliferation of CFs was further decreased in the LIIL group, and showed no significant difference in each point of culture. However, the proliferation of CFs was significantly increased after usage of IL-6 receptor inhibitor, and the cell proliferation rate of CFs was significantly higher in the H-CFs+anti-IL-6r group than in the H-CFs group and the H-CFs+IL-6 group (P< 0.05) at 24h,48h, and 72h post-hypoxic culture (P< 0.05). Cell apoptosis was measured by flow cytometry. The apoptosis rate was only about 0.2% in the CFs group, significantly increased after hypoxia culture, further increased by adding anti-IL-6r, but signifcantly decreased by adding IL-6, revealing the highest apoptosis rate in the H-CFs+IL-6 group (P< 0.05). The mRNA expressions of IL-6. TGF-β1, Smad3, MMP2 and MMP9 in H-CFs+IL-6 group were much higher than those in the H-CFs group. However, the expression of these cytokines were much lower in H-CFs+anti-IL6r group (P< 0.05). The protein expressions of IL-6, TGF-β1 Smad3 and MMP2/9 assessed by Western blot showed the similar trend of the mRNA expression.[Conclusion]1. The clinical ischemic heart function and remodelling index were ameliorated with the improvement degree of myocardial ischemia after percutaneous coronary intervention; serum IL-6 was also improved; Changes of IL-6 level were significantly correlated with the changes of cardiac function and remodelling index.2. IL-6 could promote inflammation reaction of myocardial infarction, and increased interstitial collagen fibers, promote myocardium fibrosis it thus may be an important signal molecule in ventricular remodelling.3. IL-6 mediated ventricular fibrosis and remodellingare processes that might be conducted through TGF-β1/Smad3/MMP2/9 signalling pathway. |
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