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Dapagliflozin ve trimetazidin'in tip 1 diyabetli sıçanlarda doksorubusine bağlı kardiyak disfonksyon üzerine endoplazmik retikulum stresi yoluyla kardiyoprotektif etkileri

Cardioprotective effects of dapagliflozin and trimetazidine on doxorubusin-induced cardiac dysfunction in rats with type 1 diabetes through endoplasmic reticulum stress

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  1. Tez No: 905942
  2. Yazar: MUHAMMED MÜRSEL ÖĞÜTVEREN
  3. Danışmanlar: PROF. DR. ÖMER ŞATIROĞLU
  4. Tez Türü: Tıpta Uzmanlık
  5. Konular: Kardiyoloji, Cardiology
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2024
  8. Dil: Türkçe
  9. Üniversite: Sağlık Bakanlığı
  10. Enstitü: Recep Tayyip Erdoğan Üniversitesi Eğitim ve Araştırma Hastanesi
  11. Ana Bilim Dalı: Kardiyoloji Ana Bilim Dalı
  12. Bilim Dalı: Belirtilmemiş.
  13. Sayfa Sayısı: 91

Özet

Giriş ve Amaç: Diyabet Mellitus toplumda sık görülen ve sekonder patofizyolojik süreçleri tetikleyen önemli bir halk sağlığı sorunudur. Diyabet ve komplikasyonlarının tedavisinde birçok ilaç ve tedavi yöntemi kullanılmakla birlikte yeni tedavi yöntemleri için arayışlar sürekli devam etmektedir. Bunun yanında diyabetin vücuttaki tüm doku ve sistemleri etkilediği de bilinmektedir. Bu sebeple diyabetin sekonder komplikasyonlarına karşı koruyucu araştırmalar güncelliğini korumaktadır. Diğer taraftan yapılan çalışmalar diyabetin patofizyolojisi hakkında mekanizmasal süreçlerin de aydınlatılmasına katkı sağlamaktadır. Kalp yetmezliği ise benzer şekilde insidansı ve prevalansı giderek artan ve diyabet ile yakından ilişkili olan bir sağlık sorunudur. Dapaglifozin (DAPA) tip 2 D diyabet tedavisi kalp yetmezliği ve kronik böbrek hastalıgı tedavilerinde kullanılmaktadır. Trimetazidene (TMZ) ilk sitoprotektif ajanlardan olup anti-iskemik etkinliği bilinen bir metabolik ajandır. Sodyum-glukoz ko-transporter 2 inhibitöerleri ve anti iskemik ajanların tip 1 diyabet ve komplikasyonlarının eşlik ettiği kalp yetmezliği üzerine etkisi bilinmemektedir. Bu nedenlerle Dapagliflozin ve Trimetazidinin ratlarda streptozotosin ve Doksorubisin ile indüklenen tip 1 diyabeti olan kardiyak disfonksiyonlu rat modelindeki kalp dokusu üzerindeki etkilerinin ve mekanizmasının gösterilmesini amaçladık.SGLT2 inhibötürünün ve anti iskemik ajanların tip 1 diyabet ile eşlik eden kardiyak disfonksiyonuna karşı olası koruyucu etkilerinin ve endoplazmik retikulum stres mekanizmasının araştırmasını sağlayarak bu konudaki literatür boşuluğunu doldurmak ve toplumda sık görülen hastalıkların başında yer alan tip 1 diyabet ile eşlik eden kardiyak disfonksiyonun tedavisinin yönetiminde alternatif ajan ortaya koymaya çalıştık. Gereç ve Yöntemler: 6-8 haftalık Sprague-Dawley ratları (erkek), yiyecek ve suya sınırsız erişimi ve kontrollü oda sıcaklığı olan 12 saatlik aydınlık-karanlık döngüsüne sahip ortamda barındırıldı ve 8 li kafeslere randomize olarak dağıtıldı. Streptozosin ile Oluşturulan Diyabet Modeli Diyabet intraperitonel (i.p.) streptozosin (STZ) uygulamasıyla oluşturuldu. Tek doz 55-60 mg/kg STZ, taze hazırlanan 0,1 M pH:4,5 olan sitrat tamponunda çözündürülerek uygulandı. STZ enjeksiyonundan 72 saat sonra açlık kan şekeri seviyeleri kuyruk veninden alınan örneklerden glukometre (On call Plus) ile ölçüldü. Açlık kan şekerleri 250 mg/dl'den yüksek çıkan sıçanlar diyabet oldukları kabul edildi. Diyabet tanısı konulan ratlarda daha sonra kardiyotoksisite oluşturmak için intraperitoneal (i.p.) enjeksiyon yoluyla 4 hafta boyunca 5 mg/kg/hafta Dox uygulamasına yapıldı.. Kümülatif doz 20 mg/Kg olacak şekilde Dox uygulamasını takiben 7-8 gün ara verildikten sonra tedavi gruplarına TMZ (10 mg/kg) ve DAPA (10 mg/kg) uygulaması başlanılandı. İlaç uygulamasının 14. gününde tüm denekler 50 mg/kg Ketamin hidroklorik asit ve 10 mg/kg Ksilazin HCl uygulaması ile anestezi altına alınarak yüksek doz anestezik uygulaması ile sakrifiye edilerek gerekli kan ve materyaller toplandı. Oksidatif Stres Markerları Malondialdehit (MDA), redükte glutatyon (GSH) gibi oksidatif stres mediyatörleri incelendi. Dokudan, MDA ve GSH düzeyleri manuel (kimyasallar ile) olarak analiz edildi Histopatolojik analizler Spraque Dawley cinsin erkek sıçanlardan çıkarılan kalp dokusuna ait örnekler 1.5 cm3 hacminde parçalara ayrıldı. Elde edilen kalp dokusuma ait numumneler 10% phosphate-buffered formalin (Sigma Aldrich, Germany) solüsyonunda 24-36 saat boyunca bekletilerek fiksasyon işlemi uygulandı. Fiksasyon işlemini takiben rutin histolojik takip prosedürleri uygulanarak sırası ile dehidrasyon (artan ethanol serisi ile, Merck KGAa, Darmstadt, Almanya), şefaflaştırma aşaması (ksilol, Merck KGAa, Darmstadt, Almanya), yumuşak parafinde embedding (Merck KGAa, Darmstadt, Almanya) ve son olarak sert paraffin ile bloklandı (Merck KGAa, Darmstadt, Germany). Elde edilen paraffin bloklarda bloklardan rotary microtome (Leica RM2525, Lecia, Germany) ile 4-5µm kalınlığında kesitler alınarak Harris hematoxylin and Eosin G (H&E; Merck KGAa, Darmstadt, Almanya) ile boyandı. Kalp dokusu içerek kesitler sırasi ile; Deparafinizasyon işlemi için 56 °C' deki etüv kullanıldı ve tüm kesitler Leica, ST5020 Multistainer Bath Array boyama cihazında aşağıdaki prosedür uygulandı:Çalışmamızda Endoplazmik retikulum stresinin belirlemek için CHOP (ab3392, Abcam, UK) ve GRP-78 primer antikorları kullanıldı (ab21685, Abcam, UK). Bunun yanında primer antikorlar ile uyumlu sekonder antikor (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, UK) kiti kullanıldı.Kalp dokusuna ait kesitlerden 1-2 µm kalınlığında kesitler alınarak üretici firmanın klavuzuna uygun olarak deparafinizasyon işlemi arıdından sırası ile antigen retrival procedure uyguladı. Immunohistokimyasal yöntem Bond Max II IHC/ISH boyama cihazı (Leica Biosystems, Avustralya) kullanılarak primer ve sekonder antikorlar ile 60 dakika boyunca inkube edildi. Bir sonraki aşamada kalp dokusuna ait kesitler diaminobenzidine tetrahydrochloride (Ultraview, Leicaa Biosystems, Almanya) ve Harris hematoksilen ile boyandı (Merck, Germany).Elde edilen preparatlar 2 uzman tarafından değerlendirildi. Farklı analizlerden elde edilen veriler SPSS 18.0 yazılımında (IBM Corp., NY, Armonk, ABD) istatistiksel analize tabi tutulacaktır. Çalışmamızda elde edilen histolojik verilerin parametrik olup olmadığı SPSS programı kullanılarak Shapiro-Wilk, Q-Q grafiği, Skewness-Kurtosis değerleri ve Levene's testleri yapılarak değerlendirilecek. Normallik testlerini takiben yarı niceliksel analizden elde edilen veriler ortalama ve %25 ve %75 çeyrekler arası aralıklar olarak hesaplanacak. Gruplar arası farklılıklar daha sonra Kruskal Wallis testi ve ardından Tamhane's T2 testi kullanılarak değerlendirilecek. p

Özet (Çeviri)

Introduction and Aim: Diabetes Mellitus is a common public health issue that triggers secondary pathophysiological processes. While many drugs and treatment methods are used in the treatment of diabetes and its complications, the search for new treatment methods continues. Additionally, it is known that diabetes affects all tissues and systems in the body. Therefore, research on protective measures against the secondary complications of diabetes remains current. On the other hand, studies contribute to elucidating the mechanistic processes regarding the pathophysiology of diabetes. Similarly, heart failure is a health issue with increasing incidence and prevalence, closely related to diabetes Dapagliflozin (DAPA) is used in the treatment of type 2 diabetes, heart failure, and chronic kidney disease. Trimetazidine (TMZ) is one of the first cytoprotective agents and is known for its anti-ischemic efficacy as a metabolic agent. The effects of SGLT-2 inhibitors and anti-ischemic agents on heart failure accompanied by type 1 diabetes and its complications are not known. Therefore, we aimed to demonstrate the effects and mechanisms of Dapagliflozin and Trimetazidine on heart tissue in a rat model of cardiac dysfunction induced by streptozotocin and doxorubicin in type 1 diabetic rats. By investigating the potential protective effects of SGLT2 inhibitors and anti-ischemic agents against cardiac dysfunction associated with type 1 diabetes and the mechanism of endoplasmic reticulum stress, we aimed to fill the gap in the literature and propose an alternative agent in the management of cardiac dysfunction associated with type 1 diabetes, which is one of the most common diseases in the community Material-Method: Male Sprague-Dawley rats aged 6-8 weeks were housed in an environment with unlimited access to food and water, a controlled room temperature, and a 12-hour light-dark cycle, and were randomly distributed into cages of eight. The diabetes model was induced by intraperitoneal (i.p.) administration of streptozotocin (STZ). A single dose of 55-60 mg/kg STZ was dissolved in freshly prepared 0.1 M citrate buffer at pH 4.5 and administered. Seventy-two hours after the STZ injection, fasting blood glucose levels were measured from samples taken from the tail vein using a glucometer (On Call Plus). Rats with fasting blood glucose levels above 250 mg/dl were considered diabetic. In rats diagnosed with diabetes, cardiotoxicity was induced by intraperitoneal (i.p.) injection of 5 mg/kg/week doxorubicin for 4 weeks. After a cumulative dose of 20 mg/kg doxorubicin, a 7-8 day break was given, followed by the administration of TMZ (10 mg/kg) and DAPA (10 mg/kg) to the treatment groups. On the 14th day of drug administration, all subjects were anesthetized with 50 mg/kg Ketamine HCl and 10 mg/kg Xylazine HCl, and then sacrificed with a high dose of anesthetic to collect the necessary blood and materials.Oxidative stress markers such as malondialdehyde (MDA) and reduced glutathione (GSH) were examined.Tissue levels of MDA and GSH were manually analyzed using chemicals.Histopathological analyses: Heart tissue samples from male Sprague Dawley rats were divided into pieces of 1.5 cm³. The obtained heart tissue samples were processed through certain stages and stained.Sections containing heart tissue were processed as follows: deparaffinization was performed using an oven at 56°C, and all sections were stained using the Leica ST5020 Multistainer Bath Array according to the following procedure: In our study, CHOP (ab3392, Abcam, UK) and GRP-78 primary antibodies (ab21685, Abcam, UK) were used to determine endoplasmic reticulum stress. Additionally, a secondary antibody kit compatible with the primary antibodies (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, UK) was used. Sections of 1-2 µm thickness were taken from the heart tissue and processed according to the manufacturer's instructions, followed by antigen retrieval procedures. Immunohistochemical methods were performed using the Bond Max II IHC/ISH staining device (Leica Biosystems, Australia), with incubation of primary and secondary antibodies for 60 minutes. In the next stage, sections of heart tissue were stained with diaminobenzidine tetrahydrochloride (Ultraview, Leica Biosystems, Germany) and Harris hematoxylin (Merck, Germany). The stained preparations were evaluated by two experts. Male Sprague-Dawley rats aged 6-8 weeks were housed in an environment with unlimited access to food and water, a controlled room temperature, and a 12-hour light-dark cycle, and were randomly distributed into cages of eight. The diabetes model was induced by intraperitoneal (i.p.) administration of streptozotocin (STZ). A single dose of 55-60 mg/kg STZ was dissolved in freshly prepared 0.1 M citrate buffer at pH 4.5 and administered. Seventy-two hours after the STZ injection, fasting blood glucose levels were measured from samples taken from the tail vein using a glucometer (On Call Plus). Rats with fasting blood glucose levels above 250 mg/dl were considered diabetic. In rats diagnosed with diabetes, cardiotoxicity was induced by intraperitoneal (i.p.) injection of 5 mg/kg/week doxorubicin for 4 weeks. After a cumulative dose of 20 mg/kg doxorubicin, a 7-8 day break was given, followed by the administration of TMZ (10 mg/kg) and DAPA (10 mg/kg) to the treatment groups. On the 14th day of drug administration, all subjects were anesthetized with 50 mg/kg Ketamine HCl and 10 mg/kg Xylazine HCl, and then sacrificed with a high dose of anesthetic to collect the necessary blood and materials. Oxidative stress markers such as malondialdehyde (MDA) and reduced glutathione (GSH) were examined. Tissue levels of MDA and GSH were manually analyzed using chemicals. Histopathological analyses: Heart tissue samples from male Sprague Dawley rats were divided into pieces of 1.5 cm³. The obtained heart tissue samples were processed through certain stages and stained. Sections containing heart tissue were processed as follows: deparaffinization was performed using an oven at 56°C, and all sections were stained using the Leica ST5020 Multistainer Bath Array according to the following procedure: In our study, CHOP (ab3392, Abcam, UK) and GRP-78 primary antibodies (ab21685, Abcam, UK) were used to determine endoplasmic reticulum stress. Additionally, a secondary antibody kit compatible with the primary antibodies (Goat Anti-Rabbit IgG H&L (HRP), ab205718, Abcam, UK) was used. Sections of 1-2 µm thickness were taken from the heart tissue and processed according to the manufacturer's instructions, followed by antigen retrieval procedures. Immunohistochemical methods were performed using the Bond Max II IHC/ISH staining device (Leica Biosystems, Australia), with incubation of primary and secondary antibodies for 60 minutes. In the next stage, sections of heart tissue were stained with diaminobenzidine tetrahydrochloride (Ultraview, Leica Biosystems, Germany) and Harris hematoxylin (Merck, Germany). The stained preparations were evaluated by two experts. Data obtained from different analyses will be subjected to statistical analysis using SPSS 18.0 software (IBM Corp., NY, Armonk, USA). The histological data obtained in our study will be evaluated for parametric properties using the SPSS program with Shapiro-Wilk, Q-Q plot, Skewness-Kurtosis values, and Levene's tests. Following normality tests, data obtained from semi-quantitative analysis will be calculated as mean and interquartile ranges (25% and 75%). Differences between groups will then be evaluated using the Kruskal-Wallis test followed by Tamhane's T2 test. Values of p < 0.05 will be considered statistically significant. Additionally, biochemical data will be presented as mean±SD for normally distributed continuous variables, median (interquartile range, IQR) for non-normally distributed continuous variables, and percentages for categorical variables. Normality will be tested using the Kolmogorov-Smirnov test. Differences between the values of study subgroups will be evaluated using one-way analysis of variance (ANOVA) with Least Significant Difference (LSD) or Kruskal-Wallis with Mann-Whitney post hoc tests. Comparisons between categorical variables will be made using the chi-square test or Fisher's exact test as appropriate. Group differences with a p-value < 0.05 were considered significant. Results: When the study groups were histopathologically evaluated based on parameters of degenerative cardiomyocytes, vascular congestion, and edema, it was shown that both trimetazidine and dapagliflozin, whether applied alone or in combination, reduced damage in heart tissue. Streptozotocin administration caused significant degeneration in cardiomyocytes, which can be considered a histological indicator of diabetes-related cardiomyopathy. With the addition of doxorubicin (Stz+Doxo), cardiomyocyte degeneration became more severe. This suggests that doxorubicin-induced cardiotoxicity is more pronounced under diabetic conditions and that the two agents may synergistically increase heart damage. Both TMZ and Dapa reduced cardiomyocyte damage, and their combination provided the lowest level of damage. These results suggest that TMZ and Dapa can protect cardiomyocytes and prevent cardiotoxicity. Trimetazidine is known to exert cardioprotective effects by regulating energy metabolism and reducing oxidative stress. Dapagliflozin functions as an agent that reduces cardiac load and balances glucose levels. The combination of these two agents helped minimize the observed degeneration in heart tissue. GRP78 is a molecule that increases in response to endoplasmic reticulum stress and indicates protein folding stress in the cell. CHOP is a molecule that triggers the apoptotic pathway upon activation in the later stages of ER stress. In this study, streptozotocin and doxorubicin administration significantly increased GRP78 and CHOP levels, indicating that cellular stress associated with diabetes and cardiotoxicity is increased via the ER pathway. TMZ and Dapa alleviated ER stress by reducing both GRP78 and CHOP positivity. However, TMZ and the combination treatment provided a stronger reduction compared to Dapa alone. This suggests that TMZ may provide more effective protection against ER stress. When tissue MDA and GSH levels were examined, no statistically significant findings were detected between the control group and the Stz, Stz+Doxo, Stz+Doxo+TMZ, Stz+Doxo+Dapa, Stz+Doxo+TMZ+Dapa groups. (p=0.05

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