Effects of alpha-lipoic acid on skeletal muscle ischemia-reperfusion injury in mice

Abstract

Aim: The main problem in the treatment of cardiovascular diseases, which is the most common cause of death in the world today, is reperfusion injury. In our study, we investigated the effects of alpha-lipoic acid on lower limb ischemia-reperfusion injury.
Methods: In this study, 30 male Swiss breed albino mice weighing 45-65 g were used. Mice were randomly divided into 5 groups (Control group (C), Dimethyl Sulfoxide (DMSO) group, ischemia-reperfusion (IR) group, alpha-lipoic acid control group (ALA), alpha-lipoic acid - IR group (ALA+IR)), 6 mice in each group. All groups were administered 100 IU/kg intravenous heparin bolus 30 minutes before the procedure. Lower limb ischemia (2 hours) and reperfusion (2 hours) protocols were applied to the IR and ALA+IR groups by clamping the main femoral artery. Alpha-lipoic acid was administered to ALA and ALA+IR groups 1 hour before the experimental procedure (100 mg/kg intraperitoneal). After the experiment protocol, the lower limb gastrocnemius muscles were collected from the sacrificed mice and total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), Myeloperoxidase (MPO) and Malondialdehyde (MDA) levels were determined in the tissue. In addition, edema, necrosis, and inflammatory cell infiltration levels in the skeletal muscle were determined by histologic grading, and apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method.
Results: TAS measurements of ALA group subjects were higher than the control group (P=0.01). The TOS level of the ALA+IR group was lower than the IR group (P=0.01), but higher than the C, ALA, and DMSO groups. The OSI index of the IR group was significantly higher than the ALA+IR group (P=0.01). It was observed that the IR group was at higher levels than the ALA+IR group (P=0.01). MDA measurements of IR group subjects were higher than that of the C, ALA, DMSO, ALA+IR groups (P=0.01). Edema of the IR and ALA+IR group subjects were higher than the C, ALA, DMSO groups (P=0.01). Necrosis measurements of IR and ALA+IR group subjects were higher than C, ALA, DMSO groups (P=0.01). Inflammatory cell infiltration measurements of IR and ALA+IR group subjects were higher than C, ALA, DMSO groups (P=0.01). Apoptosis level was significantly lower in the ALA+IR group than in the IR group. (P=0.01).
Conclusion: The results showed us that alpha-lipoic acid has a protective effect against oxidative damage, inflammation, and apoptosis in the tissue in lower limb ischemia-reperfusion. 

Keywords

• Alpha-lipoic acid
• Ischemia-reperfusion injury
• Skeletal muscle

Farelerde iskelet kasında iskemi reperfüzyon hasarında alfa lipoik asidin etkileri

Öz

Amaç: Günümüzde dünya üzerinde en sık ölüm nedeni olan kardiovasküler hastalıkların tedavisnde temel sorun reperfüzyon hasarıdır. Çalışmamızda alfa lipoik asid’in alt ekstremite iskemi reperfüzyon hasarında etkilerini araştırdık.
Yöntemler: Çalışmada ağırlıkları 45-65 gr arasında değişen 30 adet, erkek Swiss cinsi albino fare kullanıldı. Fareler her grupta 6 tane olmak üzere, rastgele 5 gruba ayrıldı (Kontrol grubu (C), Dimethyl Sulphoxyde (DMSO) grubu, iskemi- reperfüzyon (İR) grubu, alfa-lipoik asid kontrol grubu (ALA), alfa-lipoik asid - İR grubu (ALA+İR)). Tüm gruplara işlemden 30 dakika önce 100 IU/kg heparin intravenöz bolus olarak uygulandı. İ-R ve ALA+İR gruplarına ana femoral arter klemplenerek alt ekstremite iskemisi (2 saat) ve reperfüzyon (2 saat) protokolü uygulandı. ALA ve ALA+İR gruplarına deney prosedüründen 1 saat önce alfa lipoic asid uygulaması (100 mg/kg intraperitoneal) yapıldı. Deney protokolü sonrası farelerin alt ekstremite gastroknemus kası sakrifiye edildi ve dokudan totalantioksidan status (TAS), total oksidan status (TOS), oksidatif stress indexi (OSI), Myeloperoxidase (MPO) ve Malondialdehyde (MDA) düzeyleri bakıldı. Ayrıca iskelet kasından histolojik gradeleme ile ödem, nekroz ve inflamatuar hücre infiltrasyon düzeyleri ile Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) yöntemi ile apoptozis tayini yapıldı.
Bulgular: ALA grubu deneklerinin TAS ölçümlerinin kontrol grubunda göre daha yüksek düzeylerde olduğu tespit edilmiştir (P=0,01). ALA+İR grubunun TOS seviyesi IR grubuna göre daha düşük çıkmış (P=0,01) ancak C, ALA ve DMSO gruplarına göre yüksek olarak tespit edilmiştir. IR grubunun OSI indeksi, ALA + İR grubunda anlamlı derecede yüksek çıkmıştır (P=0,01). İR grubun ALA+İR grubundan daha yüksek düzeylerde olduğu görülmüştür (P=0,01). İR grubu deneklerinin MDA ölçümlerinin C, ALA, DMSO, ALA+İR gruplarına göre daha yüksek düzeylerde olduğu tespit edilmiştir (P=0,01). İR ve ALA+İR grubu deneklerinin ödem ölçümlerinin C, ALA, DMSO gruplarına göre daha yüksek düzeylerde olduğu tespit edilmiştir (P=0,01). İR ve ALA+İR grubu deneklerinin nekroz ölçümlerinin C, ALA, DMSO gruplarına göre daha yüksek düzeylerde olduğu tespit edilmiştir (P=0,01). İR ve ALA+İR grubu deneklerinin inflammatory cell infiltration ölçümlerinin C, ALA, DMSO gruplarına göre daha yüksek düzeylerde olduğu tespit edilmiştir (P=0,01). Apoptozis düzeyi ALA+İR grubunda İR grubuna göre anlamlı olarak düşük çıkmıştır (P=0,01).
Sonuç: Sonuçlar bize alfa lipoik asid’in alt ekstremite iskemi reperfüzyonunda dokuda oluşan oksidan hasara ve inflamsyona ve apoptozise karşı koruyucu etkinliğinin olduğunu gösterdi. 

Anahtar Kelimeler

• Alfa lipoik asit
• İskemi reperfüzyon hasarı
• İskelet kası

Kaynakça

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