|Year : 2018 | Volume
| Issue : 3 | Page : 378-385
Protective effect of captopril on cardiac fibrosis in diabetic albino rats: a histological and immunohistochemical study
Hamdino M Attia1, Medhat Taha2
1 Department of Anatomy, Faculty of Medicine, Al-Azhar University, Damietta, Egypt
2 Department of Anatomy, College of Medicine, Mansoura, Egypt
|Date of Submission||13-Jun-2018|
|Date of Acceptance||12-Aug-2018|
|Date of Web Publication||07-Jan-2019|
Dr. Hamdino M Attia
Department of Anatomy, Faculty of Medicine, Al-Azhar University, Damietta (34518)
Source of Support: None, Conflict of Interest: None
Background Cardiomyopathy is one of the common complications of diabetes mellitus.
Aim The aim of the present study was to investigate the effects of Captopril on myocardial fibrosis in streptozotocin-induced diabetic rats (an animal model of type 1 diabetes mellitus).
Materials and methods Forty albino rats were divided into five groups. Group I (control group), group II (untreated diabetic rats), group III (insulin-treated diabetic rats), group IV (Captopril-treated diabetic rats), and group V (insulin and Captopril-treated diabetic rats) were killed at 4 and 8 weeks, and the samples were collected for histological evaluation using hematoxylin and eosin, Masson trichrome, and immunohistochemical staining with anti-P53 for apoptosis, alpha smooth muscle actin (α-SMA) for collagen deposition.
Results Treatment of streptozotocin-induced diabetic rats with insulin and Captopril (group V) showed marked decrease in myocardial injury and apoptosis, which was confirmed by marked decrease of sarcoplasmic p53 expression. Group V also showed decreased collagen deposition as confirmed by Masson trichrome staining and α-SMA expression. The results were better after 8 weeks compared with those after 4 weeks.
Conclusion The administration of Captopril alone with diabetic rats minimally affected cardiac fibrosis, while the combination of Captopril and insulin markedly improved cardiac fibrosis.
Keywords: Captopril; cardiac fibrosis; diabetes mellitus; diabetic cardiomyopathy
|How to cite this article:|
Attia HM, Taha M. Protective effect of captopril on cardiac fibrosis in diabetic albino rats: a histological and immunohistochemical study. Benha Med J 2018;35:378-85
|How to cite this URL:|
Attia HM, Taha M. Protective effect of captopril on cardiac fibrosis in diabetic albino rats: a histological and immunohistochemical study. Benha Med J [serial online] 2018 [cited 2019 Dec 15];35:378-85. Available from: http://www.bmfj.eg.net/text.asp?2018/35/3/378/249409
| Introduction|| |
Diabetes mellitus (DM) represents a serious public health problem worldwide. It is estimated that the number of diabetic patients will reach 300 million by 2025 . Somaratne et al.  reported that 56% of DM patients have diabetic cardiomyopathy, which is characterized by cardiac fibrosis . Overproduction of extracellular matrix (ECM) proteins in cardiac fibrosis leads to myocardial stiffness and cardiac dysfunction. Diabetes alters the structure and function of the heart. Cardiac fibrosis is multifactorial and the existing evidence suggest that persistent hyperglycemia is a significant contributing factor ,.
One of the links between diabetes and such a high prevalence of cardiovascular disease is renin angiotensin aldosterone system (RAAS) activation. It has been shown that RAAS plays a major role in the development of diabetic cardiac complications  as it promotes cardiomyocyte loss and extensive myocardial fibrosis . Renin angiotensinogen and angiotensin II (AngII) receptor were all present in the heart where they are found upregulated in models of cardiac injury such as myocardial infarction and heart failure .
Angiotensin-converting enzyme (ACE) was expressed not only in the cardiomyocytes adjacent to the site of infarct but also in the fibroblast, macrophage, and endothelial cells. Evidence shows that circulating and local RAAS promote the development of myocardial fibrosis in chronic heart failure. In-vitro experimental studies have shown that both angiotensin II and aldosterone stimulate collagen synthesis in a dose-dependent manner when applied to fibroblast cell cultures, while Ang II suppresses matrix metalloproteinase 1, one of the key enzymes of collagen degeneration  that leads to increasing collagen accumulation within the myocardial interstitium .
The local renin angiotensin system is activated by hyperglycemia resulting in the formation of Ang II and stimulation of endogenous cell death pathway . Diabetes is also associated with an increase in oxidative stress damages. A direct link was found between Ang II and reactive oxygen species (ROS) . Dandona et al.  found that Ang II administration markedly increases ROS. This effect is suppressed by AII receptor antagonists.
Captopril is the prototype of the sulfhydryl-containing ACE inhibitors. Sulfhydryl ACE inhibitors induce sustained reduction of systemic oxidative stress . However, it is unknown whether the generation of the ROS constitutes the intermediate event in the transition of death signals to myocytes by AngII or an interaction between AngII and ROS may be required for cell death to occur in diabetic cardiomyopathy .
Daly et al.  stated that in patients with diabetes, RAAS blockage protected against cardiovascular mortality and morbidity. Captopril significantly reduced the rates of death, myocardial infarction, and shock in patients with diabetes. Hence, the present study was designed to investigate the effect of Captopril (ACE inhibitor) in cardiac fibrosis in a diabetic rat model using histological and immunohistochemical techniques.
| Materials and methods|| |
Forty male albino rats (240–280 g) were obtained from and housed in the animal house, Faculty of Pharmacy, Mansoura University, Egypt. The animals were housed according to the NIH guidelines for animal care and use. Standard balanced diet and water were provided ad libitum. All the experiments were conducted according to the rules and regulations of Mansoura University Committee of Animal Experimentation.
Induction of diabetes mellitus
The animal model was established as previously described ,. Briefly, the animal received a single dose of 40 mg/kg body weight of Streptozotocin (STZ) (Sigma, St. Louis, Missouri, USA), after 12 h of fasting. DM was confirmed by measuring the fasting blood glucose (Accu-Chek Active blood glucose meter; Roche Diagnostic, Mannheim, Germany) on days 3 and 5 after the first injection. Rats with an FMS greater than 13.9 mmol (250 mg/dl) for two consecutive measurements were confirmed as diabetic ,.
The rats were divided into five groups as follows. Four rats were killed at 4 weeks and four rats were killed at 8 weeks.
- Group I (control, non-DM): eight rats received a single intraperitoneal (IP) injection of 0.9% NaCl (normal saline) (pH: 7.4).
- Group II (untreated DM): eight diabetic animals that received no treatment.
- Group III (insulin-treated DM): eight diabetic animals received 2 U/kg of an insulin mixture subcutaneously (Egyptian Drug Trading Company, The Egyptian Pharmaceutical Trading Company, 1353, Corniche El-Nil, Shobra, Cairo, Egypt). Insulin doses were adjusted to maintain the blood glucose level at 170–200 mg/dl .
- Group IV (Captopril-treated DM): eight diabetic rats received Captopril 100 mg/kg/day (L.L.C; SmithKline Beecham, Cairo, Egypt) by intragastric administration .
- E. Group V (combined Captopril and insulin-treated DM): eight diabetic rats received a combined treatment of insulin in a dose of 2 U/kg/day  and Captopril 100 mg/kg.
At the time of killing (4 and 8 weeks), the rats received an overdose of pentobarbital (200 mg/kg) injection, followed by midsternal incision and then the hearts were removed and placed in 10% formaldehyde.
All specimens were paraffin embedded, sectioned at 5 μm thin sections, and were stained with hematoxylin and eosin and Masson’s trichrome for staining of collagen fibers .
The sections were stained with anti-p53 immunoperoxidase stain for the evaluation of anti-p53 expression of the nuclei, which is considered as positive apoptotic myofibril . Other sections were stained with α-SMA immunohistological stain to evaluate interstitial collagen and collagen deposition in the adventitia of blood vessels .
| Results|| |
Light microscopic examination
Hematoxylin and Eosin-stained sections: semiquantitative assessment of the histopathological findings is illustrated in [Figure 1]. The hearts of the control animal showed that myocardial fibers were well arranged in regular rows, the myocardial nuclei were clear, and the myocardial gap was normal. The hearts of the diabetic animal showed myocardial injury within the 4 weeks represented by myocardial large vacuolation, fragmentation, and lysis, which appeared to be focal and were associated with myocardial fibers atrophy and separation from each other. In 8 weeks, the myocardial lesion was increased and was associated with severe nuclear damage. In the diabetic animal treated with insulin, the myocardial damage was obviously ameliorated compared with the diabetic group in 4 and 8 weeks. In the diabetic animals treated with Captopril, the myocardial damage was mildly ameliorated with minute vacuolation and disordered arrangement of myocardial fibers. The vacuolation and the disarrangement of myocardial fibers increased in 8 weeks. The combination of both insulin and Captopril in group V showed a marked decrease in myocardial injury compared with the diabetic animal (group II). Minute myolysis was observed within 4 weeks which was completely abolished within the 8 weeks.
|Figure 1 Heart specimens of the studied groups stained with H&E, (X 200) (A) Heart of control animals (2 months) showing normal myocardial fibers (arrow). (B and C) Heart of diabetic animal after (1 month) showing myolysis (arrowhead) associated with separation and atrophy of myocardial fibers (arrow) while after (2 months) showing severe degree of diffuse myolysis (arrowheads) associated with nuclear karyolysis. (D and E) Heart of diabetic animal treated with insulin (1 and 2 month) showing focal myolysis (arrows) while (F and G) Heart of diabetic animal treated with captopril (1 month) showing focal myolysis (arrow) nuclear karyolysis (arrowhead) while in 2nd month showing large vacuolation of cardiac muscle fibers (arrow). (H and I) Heart of diabetic animal treated with insulin an d captopril (1 month) showing mild degree of myolysis (arrow) while in 2 months showing mild degree of loss of cardiac striation and mostly within normal.|
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Masson trichrome-stained sections: Masson’s trichrome staining shows collagen fibers (blue) and cardiomyocytes (red) as illustrated in [Figure 2]. The hearts of control animals showed very small collagen fibers mostly perivascular and interstitial between the myocardial fibers. The hearts of diabetic animals showed collagen fibers proliferation within 4 weeks, which markedly increased at 8 weeks. Diabetic animals treated with insulin showed decreased collagen proliferation compared with diabetic animals especially after 8 weeks of treatment. Diabetic animals treated with Captopril revealed increased interstitial and perivascular fibrosis in a time-dependent manner compared with the insulin-treated group. A combination of both insulin and Captopril markedly decreased collagen fibers deposition within 4 and 8 weeks.
|Figure 2 Heart specimens of the studied groups stained with Masson trichrome stain (X 200) (A) Heart of control animals (2 months) showing mild interstitial collagen fibers in between myocardial fibers (arrowhead). (B and C) Heart of diabetic animal after (1 month) showing myolysis associated with collagen fibers proliferation (arrowhead) while after (2 months) showing severe myolysis accompanied with advanced collagen fibers proliferation (arrowhead). (D and E) Heart of diabetic animal treated with insulin (1month) showing focal collagen deposition (arrowheads) while in 2 month showing perivascular and interstitial collagen fibers proliferation (arrowhead). (F and G) Heart of diabetic animal treated with captopril (1 month) showing focal collagen fibers proliferation especially in myomalacic areas (arrowhead) while in 2nd month showing increased collagen fibers (arrowhead) (H and I). Heart of diabetic animal treated with insulin and captopril (1 month) showing decrease collagen fibers between myocardial fibers (arrowhead) while in 2 months marked decrease collagen fibers between myocardial fibers (arrowhead).|
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α-SMA immuno-histochemical staining: the histopathological results were confirmed by the immunohistochemical staining with α-SMA expression illustrated in [Figure 3]. Myocardium of control animals showed only mild expression especially within the adventitia of small blood vessels. The diabetic animals showed marked increase of α-SMA expression interstitially and perivascularly. The expression of α-SMA expression was decreased within the treated groups with insulin as compared with the diabetic group, while in Captopril-treated animals α-SMA expression increased compared with the insulin-treated group at 4 and 8 weeks. The diabetic animals treated with a combination of insulin and Captopril revealed a marked decrease in α-SMA expression compared with diabetic animals.
|Figure 3 Heart specimens of the studied groups stained with α-SMA immunostaining 200 (A) Heart of control animals (2 months) showing minimal α-SMA expression within the adventitia of small cardiac capillaries (arrows). (B and C) Heart of diabetic animal after (1 month) showing increased α-SMA expression within the interstitial tissue between fibers (arrows) while after (2 months) showing marked α-SMA expression (arrow). (D and E) Heart of diabetic animal treated with insulin (1 month) showing mild α-SMA expression within the adventitia of blood vessels (arrow) and interstitial tissue (arrowhead) while in 2 month showing α-SMA expression within the adventitia of blood capillaries (arrowhead). (F and G) Heart of diabetic animal treated with captopril (1 month) showed moderate α-SMA expression within the adventitia of cardiac capillaries (arrowhead) while in 2nd month showing α-SMA expression either perivascular (arrow) and interstitium (arrowhead). (H and I) Heart of diabetic animal treated with insulin and captopril showing minimal α-SMA expression within the adventitia of small cardiac capillaries (arrows) in the 1st and 2nd months.|
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P53 immunohistochemical staining: the expression of P53 of the studied groups is illustrated in [Figure 4]. The hearts of control animals showed a mild expression of P53 within the myocardial fibers. Diabetic animals revealed a marked increase of sarcoplasmic P53 expression, and the expression was markedly increased after 8 weeks from the induction of diabetes. Diabetic animals treated with insulin showed a moderate decrease of p53 immunostaining as compared with the diabetic group. Diabetic animals treated with Captopril revealed a mild decrease of p53 immunostaining compared with diabetic animals. Interestingly, the diabetic animals treated with both insulin and Captopril showed a marked decrease of sarcoplasmic p53 expression compared with diabetic rats.
|Figure 4 Heart specimens of the studied groups stained with P53 immunostaining 200 (A) Heart of control animals (2 months) showing minimal sarcoplasmic expression of P53 (arrow). (B and C) Heart of diabetic animal after (1 month) showing increased sarcoplasmic expression of P53 (arrow) while after (2 months) showing marked sarcoplasmic expression of P53 (arrow). (D and E) Heart of diabetic animal treated with insulin showing moderate decrease of the sarcoplasmic expression of P53 (arrow) in 1st and 2nd month. (F and G) Heart of diabetic animal treated with captopril (1 month) showing mild decrease of the sarcoplasmic expression of P53 in 1st and 2nd month. (H and I) Heart of diabetic animal treated with insulin and captopril showing marked decrease P53 staining within the myocardial fibers (arrow) in 1st and 2nd month.|
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| Discussion|| |
Diabetic cardiomyopathy is defined as the ventricular dysfunction in diabetic patients independent of cause, such as coronary artery disease or hypertension . Diabetes leads to myocardial, structural, and functional disturbances, cardiac inflammation, fibrosis, and apoptosis . Captopril is widely used in the management of increased blood pressure, which is commonly associated with DM. Therefore, the present study aimed at investing the effects of Captopril on cardiac fibrosis in an STZ-induced diabetic rat model.
The present results demonstrated that STZ injection successfully induced diabetes and the hearts of diabetic animals (group II) showed myocardial injury represented by myocardial fragmentation and lysis associated with severe nuclear damage. Also diabetic animals revealed a marked increase of sarcoplasmic P53 expression especially within the myofibril. These results agree with the previous reports  that p53 has a significant role in initiating programmed cell death (apoptosis) in hyperglycemic rats. Fiordaliso et al.  revealed that the apoptotic effect of hyperglycemia could be in part due to P53 glycosylation, Ang II synthesis, and ROS overproduction.
In the current study, Captopril (in group III) mildly ameliorates the apoptotic effect of P53 expression in hyperglycemia. These results agree with Birben et al.  who stated that Captopril showed antioxidant characteristics which can inhibit the detrimental effect of ROS. Also, Tamba and Torreggiani  stated that Captopril prevents the increased superoxide flux associated with the renin angiotensin system.
On the other hand, administration of insulin to diabetic rats (group IV) markedly improved cardiomyocyte myolysis and P53 expression which agree with Frustaci et al. , who stated that cardiomyocyte apoptosis is controlled with blood glucose. Additionally, the present results showed that Captopril treatment combined with insulin can significantly ameliorate myocardial damage and apoptosis in group V, which is in agreement with Singh et al. .
The results of the present study showed that collagen expression using the Masson trichrome technique and α-SMA expression were significantly increased in the diabetic group (group II) compared with the control group (group I), which indicated that inflammatory reaction and myocardial fibrosis were significantly aggravated in diabetic rats. These results agree with those of Singh et al.  who stated that hyperglycemia induces myocardial oxidative stress which may be related to glucose metabolism or activation of cytokines and other hormones. Also, Yang et al.  stated that excess ECM proteins, which is caused by an imbalance between collagen synthesis and degradation, plays an important role in cardiac fibrosis; therefore, the content of ECM proteins can be used to assess the degree of myocardial fibrosis.
Normalization of the glucose level in insulin-treated diabetic rats (group III) significantly decreased the collagen and α-SMA expression compared with the diabetic group (group II), which agree with previous studies , that suggested that most of the observed effects in the diabetic rats were secondary to hyperglycemia.
Captopril (ACE inhibitor) alone in diabetic rats (group IV) mildly ameliorates the collagen and α-SMA expression while a combination of Captopril and insulin (group V) markedly decreases collagen and α-SMA expression. These results are consistent with those of Singh et al.  who used benazepril as an ACE inhibitor.
Activation of the RAS (renin angiotensin system) appears to be a major event in hyperglycemia because of increased oxidative stress . Yang and colleagues observed a dramatic activation of the intracellular RAS (iAngII) which is correlated with the development of the pathology associated with DM. Other studies ,, also reported that high glucose promoted accumulation of renin and iAng II intracellularly resulting in a dramatic rise in AngII concentration without affecting the extracellular AngII levels. They added that AngII synthesis in cardiac myocytes induced by high glucose is catalyzed by chymase and not by ACE. Li et al.  reported that as iAngII synthesis is chymase dependent and the ACE inhibitors do not block the intracellular RAS which is activated in DM. The above findings may explain the weak protective effect of Captopril (ACE inhibitor) in combating myocardial fibrosis in diabetic rats. Also, Norton et al.  stated that oral Captopril did not reduce stiffness in diabetic rat’s heart.The improvement of cardio-protective effect of insulin by combination with captopril in group V may be explained by mechanisms other than ACE inhibition such as the anti-inflammatory and antioxidative stress of Captopril . Captopril also increases the sensitivity of insulin .
In conclusion, Captopril mildly attenuates fibrosis in type I diabetes and renin inhibitors may be more effective than ACE inhibitors in combating cardiac fibrosis as they can block both intracellular and extracellular Ang II syntheses.
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Conflicts of interest
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