| BackgroundOver the past two decades,a striking increase in the number of people with the metabolic syndrome(MS)worldwide has taken place.This increase is associated with the global epidemic of obesity and diabetes.With the elevated risk not only of diabetes but also of cardiovascular disease from the MS,there is urgent need for strategies to prevent the emerging global epidemic.The constellation of metabolic abnormalities includes glucose intolerance,insulin resistance,central obesity, dyslipidaemia,and hypertension.Insulin resistance is considered central to the pathophysiology of this metabolic and cardiovascular syndrome.A proinflammatory state probably contributes to the syndrome.In clinical study,the patients with MS have cardiac diastolic dysfunction, characterized by decreased relaxation and increased stiffness.Cardiac fibrosis is the major cause of cardiac diastolic dysfunction,but the mechanism in MS is still unclear. Many studies have shown that inflammation is the third fibrosis-triggering pathway, not independent of the hormonal control and/or blood pressure,but related to the inflammatory cytokines that invariably infiltrate the overloaded cardiac tissue. Chronic subclinical inflammation is thought to be part of the MS.Serum level of high sensitive-C reactive protein(hs-CRP),interluekin-6(IL-6)and IL-18 are increased.It known that IL-18,a member of the IL-1 family of ligands,induces a variety of responses via JNK pathway.The JNK pathway is a cascade of serine/threonine kinases that transduce the signals from the cell surface to the nucleus in response to growth factors and cellular stress.AP-1,which contains the c-Jun/c-fos protein, occurs downstream of JNK.Activated JNK phosphorylates AP-1 and binds to a specific DNA sequence known as the 'AP-1 binding site'.The gene promoters related to cardiac fibrosis such as collagenâ… ,collagenâ…¢,matrix metalloproteinase-2 (MMP-2)and MMP-9 all have AP-1 binding sites.This suggests that IL-18 promoted cardiac fibrosis is related to the AP-1 complex via JNK activation.In this study,molecular biology,cellular biology,histopathology and echocardiography were used to qualitatively observe and quantitatively analysize the morphology and ultrastructure of cardiac fibrosis in MS rats.The effect of IL-18 and JNK/AP-1 pathway on the pathogenesis of cardiac fibrosis were studied.Objectives(1)To establish an animal model of MS used fructose;(2)To explore the changes of myocardial histopathotogy and ultrastructure;(3)To define the myocardial expressions of key molecules of the IL-18/JNK/ AP-1 signal pathway;(4)To delineate the role of IL-18/JNK/AP-1 signal pathway in cardiac fibrosis used IL-18 adenovirus administered;(5)To explore the changes of cardiac structure and function used echocardiographie and hemodynamic methods.Methods50 male Wistar rats were randomly assigned to two groups:control group(n=12) and MS group(n=38).Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum.Rats in the MS group were given 10%fructose water and standard rat chow ad libitum.After being fed for 32 weeks,rats in the MS group were randomly assigned to three groups:the MS group(n=9)which were given 10%fructose water continuedly;the AdGFP group(n=9)which were administered adenovirus containing GFP through tail vein at a dose of 1×1010PFU and were given 10%fructose water meanwhile;the AdIL-18 group(n=13)which were administered adenovirus containing murine IL-18 through tail vein at a dose of 1×1010PFU and were given 10%fructose water meanwhile.The control and MS group were given an equal volume of normal saline solution administered.After 6-week-treatment,rats were kill and the heart was rapidly excised for further study.Following observations on animals were performed during the whole experiments:(1)Body weight(BW)and systolic blood pressure(SBP)were documented every two weeks;(2)Fast blood glutose,insulin,triglyceride,total cholesterol and serum IL-18 were analysis at baseline,32 weeks after and at the end of the study,with HOMA calculated; (3)Echocardiography was performed at baseline,32 weeks after and at the end of the study to assess cardiac structure and function;(4)In vivo hemodynamie measurements was performed to evaluate cardiac function;(5)The changes of myocardial ultrastructure and histopathology were observed;(6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining;(7)The mRNA expression of IL-18, collagenâ… and collagenâ…¢were detected by quantification real-time PCR;(8)The protein level of IL-18,collagenâ… and collagenâ…¢were determined by immunohistochemistry;(9)The protein level of JNK and phosphate-JNK were determined by Western-Blot;(10)The activity of AP-1 was determined by electrophoretic mobility shift assay(EMSA).Results1 BW,SBP and biochemical indices measurementMS rats exhibited significant increases in BW(P＜0.01)and SBP(P＜0.001) compared with those of controls;Serum insulin levels(P＜0.001),triglycerides (P＜0.01)and HOMA index(P＜0.001)were also increased in the MS group(P＜0.001). However,there were no differences in glucose,cholesterol levels.Compared with the AdGFP group,serum insulin levels(P＜0.001)and HOMA index(P＜0.05)were also increased in the AdIL-18 group.However,there were no differences in BW,SBP, glucose and cholesterol levels.2 Serum IL-18 levelsBefore IL-18 adenovirus administered,serum IL-18 levels of the MS rats were higher(P＜0.001)compared with those of the controls.On the first week after IL-18 adenovirus administered,serum IL-18 levels in the AdIL-18 group were higher (P＜0.001)compared with those in the AdGFP group.On the second week,serum IL-18 levels in the AdIL-18 group were four times higher(P＜0.001)than those in the AdGFP group.On the third week,serum IL-18 levels in the AdIL-18 group began decreased,but were also higher(P＜0.001)than those in the AdGFP group.On the forth week,the levels of serum IL-18 in the AdIL-18 and AdGFP groups were similar.3 Echocardiographic detectionMitral and tricuspid valvular regurgition detection:none of 12 rats in the control group showed mitral and tricuspid valvular regurgitation.In the MS group,mild mitral valvular regurgitation was occurred in 2 rat,moderate mitral valvular regurgitation was occurred in 1 rat,mild tricuspid valvular regurgitation was occurred in 2 rats,severe tricuspid valvular regurgitation was occurred in 1 rat;2 rats of the MS group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the MS group did not show valvular regurgition.In the AdGFP group,mild mitral valvular regurgitation was occurred in 1 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 1 rats,severe tricuspid valvular regurgitation was occurred in 1 rat,2 rats of the AdGFP group showed both mitral and tricuspid valvular simultaneous regurgition;5 rats of the AdGFP group did not show valvular regurgition.In the AdIL-18 group,mild mitral valvular regurgitation was occurred in 3 rat,moderate mitral valvular regurgitation was occurred in 2 rat,severe mitral valvular regurgitation was occurred in 1 rat;mild tricuspid valvular regurgitation was occurred in 2 rats,moderate tricuspid valvular regurgitation was occurred in 1 rat,severe tricuspid valvular regurgitation was occurred in 1 rat;3 rats of the AdIL-18 group showed both mitral and tricuspid valvular simultaneous regurgition;6 rats of the AdIL-18 group did not show valvular regurgition.There were no rats with valvular regurgitation in the control group.The incidence of valvular regurgitionin the MS group was 44.44%and 53.85%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of valvular regurgitation in the MS group was significantly increased(P＜0.01).Compared with that of the MS and the AdGFP groups,the incidence of valvular regurgitation in the AdIL-18 group was significantly increased(P＜0.05).Compared with the controls,LVEDD(P＜0.01),LAD(P＜0.001),PW and SW (P＜0.05)were greater in the MS rats.E and Ea were lower(P＜0.001)in the MS rats. IVRT(P＜0.01)and Tei index(P＜0.001)were higher in the MS rats.No significant differences in EF,FS,RWth and E/Ea ratio were found between the control group and the MS group.There was no differences in LVEDD,LAD,PW,SW and RWth among the MS, AdGFP and AdIL-18 groups.Compared with the rats in the MS and AdGFP groups, IVRT and Tei index were higher(P＜0.01)in the AdIL-18 group.No significant differences in EF,FS and E/Ea ratio were found among the three groups.4 Electrocardiogram recordationElectrocardiograms(ECG)of 12 rats in the control group were all normal.6 rats of the MS group showed characteristic QRS wave interchanges and 1 rats showed complex arrhythmia.The AdGFP group was similar to the MS group.10 rats of the AdIL-18 group showed QRS wave interchanges ECG,and 2 rats showed complex arrhythmia.There were no rats with arrhythmia in the control group.The incidence of arrhythmia was 77.78%in the MS group and 92.31%in the AdIL-18 group.Fisher analysis:compared with that of the control group,the incidence of arrhythmia in the MS group was significantly increased(P＜0.001).Compared with those of the MS group and the AdGFP group,the incidence of arrhythinia in the AdIL-18 group was significantly increased(P＜0.01).5 In vivo hemodynamic measurementsCompared with the controls,LVSP,LVEDP and tau were significantly increased (P＜0.001),and -dp/dt was more negative(P＜0.01)in the MS rats.The load-independent index of relaxation,namely,-dp/dt/LVSP,was significantly lower (P＜0.001)in the MS group.There was no significant difference in HR and +dp/dt.Compared with the rats in the MS group and AdGFP group,LVEDP(P＜0.001) and tau(P＜0.05)were increased,and -dp/dt was more negative in the AdIL-18 group. -dp/dt/LVSP(P＜0.05)was also lower.HR and +dp/dt were similar during invasive hemodynamic study.6 Ultrastructural change observation by transmission electron microscopyThe left ventricular myocytes from the control group arranged regularly.The pericellular membrane was uninterrupted and intact.The thick and thin myofilament arranged regularly.The sarcomere and light dark band were clear.The uniformly sized mitochondrial was abundant and showed round or oval shape.A little fibroblast and collagenous fibers distributed in extra-cellular matrix.The left ventricular myocytes from the MS and AdGFP groups arranged irregularly.The pericellular membrane was interrupted partly.The local myofibril was disintegrated partly.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated. Collagenous fibers proliferated in extra-cellular matrix.The left ventricular myocytes from the AdIL-18 group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibril was disintegrated.The myofilament was distorted and interrupted.The sarcomere was in a bad apposition.The swelling mitochondrial increased and accumulated.A lot of collagenous fibers distributed in extra-cellular matrix.7 Pathological detecionHE staining showed that the myocytes from the control group arranged regularly. The size of the nuclear was uniform.The staining cytoplasm was homogeneous.The myocytes from the MS group arranged irregularly.The nuclear was irregular and interrupted myofibril arranged irregularly.The AdGFP group was same with the MS group.The myocytes from the AdIL-18 group arranged more irregularly.The nuclear was more irregular and interrupted myofibril arranged more irregularly compared with the AdGFP group.8 The detection of collagen content by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue by Masson-staining.The collagen tissue was appropriate arranged among cardiomyocytes in the control group.However,collagen tissue increased markedly, and disrupted in some area in the MS group.The AdGFP group was same with the MS group.Collagen tissue increased and disrupted in the AdIL-18 group compared with the AdGFP group.Quantitative analysis results:the CVF(P＜0.001)and PVCA (P＜0.01)were higher in the MS group compared with the control group.The CVF and PVCA were higher in the AdIL-18 group compared with the other three groups(P＜0.05).Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).9 Immunohiatochemistry detectionIL-18:The positive reaction of IL-18 protein were stained brown and localized in myocytes.Brown granules were not seen in the control group,but thick brown granules in the MS group.A lot of thicker brown granules were observed in the AdIL-18 group.Compared with the control group,the level of IL-18 IOD was increased(P＜0.001)in the MS group.Compared with the the MS and the AdGFP groups,the level of IL-18 IOD was increased(P＜0.001)in the AdIL-18 group.Collagenâ… :The positive reaction of collagenâ… protein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenâ… IOD was increased(P＜0.001) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenâ… IOD was increased(P＜0.001)in the AdIL-18 group.Collagenâ…¢:The positive reaction of collagenâ…¢protein were stained brown and localized among cardiomyocytes.Weak brown collagen tissue were uniformly and sparsely distributed in the control group,but thick brown collagen tissue in the MS group.A lot of thicker brown collagen tissue were observed in the AdIL-18 group. Compared with the control group,the level of collagenâ…¢IOD was increased(P＜0.05) in the MS group.Compared with the MS and the AdGFP groups,the level of collagenâ…¢IOD was increased(P＜0.05)in the AdIL-18 group. Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenâ… andâ…¢protein(P＜0.05);the IL-18 protein was positively correlated with the ratio of collagenâ… andâ…¢protein(P＜0.05); the IL-18 protein was positively correlated with CVF(P＜0.05)and PCVA(P＜0.05).10 The mRNA experession of key moleculesIn comparison with the controls,the expression of IL-18(P＜0.001),collagenâ… (P＜0.001),collagenâ…¢(P＜0.01)mRNA in the MS rats were significantly higher.In comparison with the MS and the AdGFP groups,the expression of IL-18(P＜0.001), collagenâ… ,collagenâ…¢mRNA(P＜0.01)in the AdIL-18 group were significantly higher.Correlation analysis demonstrated that,in the MS group,serum IL-18 level was positively correlated with the ratio of collagenâ… andâ…¢mRNA(P＜0.05);the IL-18 mRNA was positively correlated with the ratio of collagenâ… andâ…¢mRNA(P＜0.05); the IL-18 mRNA was positively correlated with CVF(P＜0.05)and PCVA(P＜0.01).11 The protein expression of several factorsThe expression of P-JNK protein in the MS group was higher significantly than that of the control group(P＜0.001).The expression of P-JNK protein in the AdIL-18 group was higher significantly than those of the MS and the AdGFP groups(P＜0.01). However,there was no significant differences in the expression of JNK protein among the four groups.12 The activity of AP-1The activity of AP-1 assessed by EMSA was enhanced in the MS group compared with the control group.The activity of AP-1 was enhanced in the AdIL-18 group compared with the MS and the AdGFP groups.Conclusions(1)An animal model of MS was established by high-fructose water.After fed fructose for 32 weeks,the MS rats had higher SBP and BW compared with the controls.And serum triglyceride concentrations,insulin levels and HOMA index were also increased.This model is valuable to investigate cardiovascular and metabolic abnormalities similar to human MS; (2)Echocardiography showed E and Ea were lower,IVRT and Tei index were higher in the MS rats.Hemodynamics showed LVSP,LVEDP and tau were increased, and -dp/dt and -dp/dt/LVSP were lower in the MS rats.Those findings suggested the MS rats had cardiac diastolic dysfunction;(3)The collagen content was increased in the MS rats detected by Masson-staining.The expressions of collagenâ… andâ…¢mRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were increased.Those changes suggested the MS rats had cardiac fibrosis,which plays an important role in the pathogenesis of diastolic dysfunction;(4)Serum IL-18 levels as well as the myocardial expression of IL-18 were increased in the MS rats.Moreover,collagen content was positively correlated with them,which suggested IL-18 may be play an important role in cardiac fibrosis.The expression of P-JNK protein and the activity of AP-1 were increased,which suggested IL-18 could induce cardiac fibrosis through JNK/AP-1 pathway;(5)After IL-18 adenovirus was administered,cardiac fibrosis and diastolic dysfunction were more aggravating.IL-18-induced the upregulation of JNK and activiation of AP-1 represent an important mechanism leading to cardiac fibrosis and diastolic dysfunction. BackgroundMetabolic syndrome (MS) has many major risks for type 2 diabetes and cardiovascular disease. The constellation of metabolic abnormalities includes glucose intolerance, insulin resistance, central obesity, dyslipidaemia, and hypertension. For the WHO and NCEP:ATPIII definitions, the prevalence of MS is 23.7% in the USA and 43.5% for those aged over 60 years. The risk for coronary heart disease and stroke was increased mreefold in subjects with the MS, and cardiovascular mortality was also markedly increased in those subjects. Cardiovascular disease and all-cause mortality are increased in men with the MS, even in the absence of baseline CVD and diabetes. This means that sub-clinical cardiovascular impairments are the fundamental reason for higher cardiovascular complications in those patients. Therefore, to finding the drug which reverses or retards cardiovascular impairments in patients with the MS is of great importance.Calcium channel blockers (CCBs) are widely used to treat patients with hypertension and coronary heart disease. Clinical study demonstrated that CCBs decrease cardiovascular events in patients with coronary heart disease. The results of CAMELOT study were administration of amlodipine to patients with CAD and normal blood pressure resulted in reduced adverse cardiovascular events. For amlodipine, IVUS showed evidence of slowing of atherosclerosis progression. The INSIGHT study showed nifedipine was as effective as diuretic therapy in reducing cardiovascular complications in hypertensive diabetics. Many clinical studies suggest that CCBs could be considered as first-line therapy for hypertensive diabetics. Moreover, CCBs improve ventricular systolic and diastolic function in hypertensive patients. Cell culture studies have shown that CCBs attenuate the AngII-mediated increases in intracellular calcium levels of adult cardiac fibroblasts, thereby abolishing an Angll-mediated increase in collagen synthesis. Although the mechanisms underlying the direct protective effects of calcium channel blockades on cardiovascular injury are not fully understood, many studies revealed that CCBs decrease cardiovascular events is related to its anti-inflammation effects. A recent clinical study has demonstrated that amlodipine seemed to improve IR. and decrease TNF-alpha levels besides reducing BP, but further interventions are needed. Furthermore, amlodipine attenuated arteriosclerosis through inhibition of inflammatory disorders in a rat model of long-term inhibition of NO synthesis. The anti-inflammatory effects of amlodipine may be mediated by the inhibition of local factors, such as MCP-1, TGF-β1, and oxidative stress. Recently many findings suggest that some dihydropyridine-type CCBs, such as nifedipine, nilvadipine, and benidipine, have a direct beneficial effect by inhibiting the nuclear translocation and DNA-binding activity of NF-kappaB, which plays a central role in this process by regulating the expression of several pro-inflammatory genes.From the first part of the study, we found inflammation was a key player in the pathogenesis of the MS, and was related to the ventricular remodeling of MS. IL-18 played an important role in cardiac fibrosis and diastolic dysfunction of MS through 'JNK/AP-1' pathway. Moreover, CCBs has novel anti-inflammation actions beyond blood pressure lowering. So the aim of this study was to observe the effect of felodipine on inflammation in rat model of MS and to study the possible mechanism of felodipine on cardiac structure and function of the MS rats. Objectives(1) To evaluate the effect of felodipine on the body weight, systolic blood pressure and biochemical indices of the MS rats;(2) To evaluate the effect of felodipine on the changes of cardiac structure and function used echocardiographic and hemodynamic methods;(3) To explore the effect of felodipine on the changes of myocardial histopathology and ultrastructure of the MS rats;(4) To explore the effect of felodipine on serum IL-18 levels and cardiac IL-18 expressions;(5) To explore the effect of felodipine on IL-18/JNK/AP-1 signal pathway in cardiac fibrosis and to delineate the pathogenesis of the beneficial effect of felodipine on the cardiovascular diseases.Methods33 male Wistar rats were randomly assigned to two groups: control group (n=12) and MS group (n=21). Rats in the control group were allowed to drink tap water and were fed standard rat chow ad libitum. Rats in the MS group were given 10% fructose water and standard rat chow ad libitum. After being fed for 8 months, rats in the MS group were randomly assigned to two groups: the MS group (n=9) which were given 10% fructose water continuedly; the felodipine group (n=9) which were given a daily dose of 5mg/kg felodipine by intubation with a stomach tube and were given 10% fructose water meanwhile. The control and MS group were given an equal volume of normal saline solution. After 6-week-treatment, rats were kill and the heart was rapidly excised for further study. Following observations on animals were performed during the whole experiments: (1)Body weight (BW) and systolic blood pressure (SBP) were documented every two weeks; (2)Serum IL-18 level, fast blood glutose, insulin, triglyceride and cholesterol were analysis at baseline, 32 weeks after and at the end of the study, with HOMA calculated; (3)Echocardiography was performed at baseline, 32 weeks after and at the end of the study to evaluate cardiac function; (4)In vivo hemodynamic measurements was performed to evaluate cardiac function; (5)The changes of mvocardial ultrastructure and histopathologv were observed: (6)Cardiac fibrosis was evaluated quantified and qualitatively by Masson staining; (7)The mRNA expression of IL-18, collagen I and collagen III were detected by quantification real-time PCR; (8)The protein level of IL-18, collagen I and collagen III were determined by immunohistochemistry, (9)The protein level of JNK and P-JNK were determined by western blot; (10)The activity of AP-1 was determined by electrophoretic mobility shift assay (EMSA). Results1 BW, SBP and biochemical indices measurementThe SBP of the rats treated with felodipine significant decreased (P<0.001) compared with those in the MS group. However, there were no differences in BW. Compared with the MS group, serum insulin levels and HOMA index (P<0.001) were decreased in the felodipine group. However, there were no differences in glucose and cholesterol levels among the three groups.2 Serum IL-18 levelAfter six-week treatment of felodipine, serum IL-18 levels in the felodipine group were lower (P<0.05) compared with those in the MS group, however, were higher significantly (P<0.001) compared with those in the control group.3 Echocardiographic detectionMitral and tricuspid valvular regurgition detection: In the felodipine group, mild mitral valvular regurgitation was occurred in 1 rat, moderate mitral valvular regurgitation was occurred in 0 rat, severe mitral valvular regurgitation was occurred in 0 rat; mild tricuspid valvular regurgitation was occurred in 2 rats, moderate tricuspid valvular regurgitation was occurred in 0 rats, severe tricuspid valvular regurgitation was occurred in 0 rat; 1 rats of the felodipine group showed both mitral and tricuspid valvular simultaneous regurgition; 7 rats of the MS group did not show valvular regurgition. The incidence of valvular regurgitionin the felodipine group was 22.22%. Fisher analysis: compared with that of the MS group, the incidence of valvular regurgitation in the felodipine group was significantly decreased (P<0.01).Compared with the MS rats, LVEDD (P<0.01), LAD (P<0.001), PW and SW (P<0.05) decreased in the rats treated with felodipine. E, Ea (P<0.001) were higher and IVRT (P<0.01), Tei (P<0.001) index were lower in the rats treated with felodipine.4 Electrocardiogram recordationElectrocardiograms (ECG) of 5 rats in the felodipine group were normal. 3 rats of the felodipine group showed characteristic QRS wave interchanges and none rat showed complex arrhythmia. The incidence of arrhythmia was 33.33% in the felodipine group. Fisher analysis: compared with that of the MS group, the incidence of arrhythmia in the felodipine group was significantly decreased (P<0.01). Compared with those of the control group, the incidence of arrhythmia in the felodipine group was significantly increased (P<0.001).5 In vivo hemodynamic measurementsCompared with the MS rats, LVSP, LVEDP and tau were significantly decreased (P<0.001), and -dp/dt was more positive (P<0.01) in the rats treated with felodipine. The load-independent index of relaxation, namely, -dp/dt/LVSP, was significantly higher (P<0.001) in the rats treated with felodipine. HR and +dp/dt were similar during invasive hemodynamic study.6 Ultrastructural change observation by transmission electron microscopyThe myocytes from the felodipine group arranged more regularly than the MS group. The phenomenon that the local myofibril was disintegrated was decreased. The mitochondrial was more regular than the MS group. The nuclear shape was more regular than the MS group. The collagenous fibers in extra-cellular matrix decreased obviously.7 Pathological detecionHE staining showed that the myocytes from the felodipine group arranged more regularly. The size of the nuclear was more uniform. The staining cytoplasm was more homogeneous compared with the MS group.8 The content of collagen detection by Masson-stainingThe myocyte was red or yellow and the collagen was green or blue. The collagen tissue was more appropriate arranged among cardiomyocytes in the felodpine grouptissue was more appropriate arranged among cardiomyocytes in the felodpine group compared with the MS group. Quantitative analysis results: the CVF and PVCA were lower in the felodipine group compared with the MS group(P<0.01).9 Immunohiatochemistry detectionIL-18: The positive reaction of IL-18 protein were stained brown and localized in myocytes. Weak brown granules were uniformly and sparsely distributed in the felodipine group, but thick brown granules in the MS group. Compared with the control group, the level of IL-18 IOD was increased (P<0.01) in the felodipine group. Compared with the MS group, the level of IL-18 IOD was decreased (P<0.001) in the felodipine group.Collagen I: The positive reaction of collagen I protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen I IOD was increased (P<0.001) in the felodipine group. Compared with the MS group, the level of collagen I IOD was decreased (P<0.001) in the felodipine group.Collagen III: The positive reaction of collagen III protein were stained brown and localized among cardiomyocytes. Weak brown collagen tissue were uniformly and -sparsely distributed in the felodipine group, but thick brown collagen tissue in the MS group. Compared with the control group, the level of collagen III IOD was increased in the felodipine group. Compared with the MS group, the level of collagen III IOD was decreased (P<0.01) in the felodipine group.10 The mRN A experession of key moleculesIn comparison with the controls, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.0l) in FFRs treated with felodipine were higher. In comparison with the MS group, the expression of IL-18 (P<0.001), collagen I, collagen III mRNA (P<0.001) in the felodipine group were significantly lower.11 The expression of several factorsThe expression of P-JNK protein in the felodipine group was lower significantly than that of the MS group (P<0.01). However, there was no significant differences in the expression of JNK protein among the three groups.12 The activity of AP-1 The activity of AP-1 assessed by EMSA was enhanced in the felodipine group compared with the control group. The activity of AP-1 was weakened in the felodipine group compared with the MS group. Conclusions(1) Felodipine treatment decrease SBP and the insulin level and improve insulin resistance;(2) Echocardiography showed E and Ea were higher, IVRT and Tei index were lower in the rats treated with felodipine. Hemodynamics showed LVSP, LVEDP and tau were decreased, and -dp/dt and -dp/dt/LVSP were higher in the rats treated with felodipine. Those findings suggested felodipine treatment ameliorates diastolic dysfunction in the MS rats;(3) The collagen content detected by Masson-staining was decreased in the rats treated with felodipine. The expressions of collagen I and III mRNA as well as protein detected by real-time RT-PCR and immunohistochemistry were also decreased. Those changes suggested felodipine had beneficial effects on cardiac fibrosis;(4) Serum IL-18 levels as well as the myocardial expressions of IL-18 were decreased in the felodipine group, which suggested felodipine had anti-inflammation effect;(5) The expression of P-JNK protein and the activity of AP-1 were decreased in the felodipine group, which suggested felodipine could decrease cardiac fibrosis and ameliorate diastolic dysfunction through IL-18/JNK/AP-1 pathway.
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