The Effects of Vitamin D on mRNA Expression of IL-1? and TNF-? in Hippocampus of Transient Global Brain Ischemic Attack Model in Rat

Almas Larasati, dr. Nur Arfian, Ph.D; dr. Eryna Ayu Nugra Desita, M.Biomed; dr. Junaedy Yunus, M.Sc., Ph.D.

2024 | Skripsi | PENDIDIKAN DOKTER

Latar Belakang: Cedera iskemik otak global transien menyebabkan proses inflamasi di hipokampus dengan peningkatan regulasi regulator proinflamasi seperti tumor necrosis factor-alpha (TNF-?) dan interleukin-1? (IL-1?). Vitamin D pada penelitian sebelumnya menunjukkan efek antiinflamasi. Namun, pengaruh vitamin D pada model cedera reperfusi iskemik belum diteliti. Penelitian ini menyelidiki pengaruh Vitamin D pada hipokampus tikus dengan model cedera reperfusi iskemik dengan mengevaluasi ekspresi mRNA IL-1? dan TNF-?.

Tujuan: Untuk mengetahui pengaruh pemberian vitamin D pada tikus hipokampus dengan model transient ischemic attack menggunakan ekspresi mRNA TNF-? dan IL-1?.

Metode: Penelitian ini menggunakan tikus Wistar jantan (n=25, BB=150-300 gram) dan dibagi menjadi empat kelompok. Kelompok pertama (SO) terdiri dari tikus yang menjalani operasi tanpa penjepitan karotis communis (n=6). Kelompok tikus kedua menjalani oklusi arteri karotis komunis bilateral (BCCAO) tanpa vitamin D (n=6). Kelompok ketiga (VD1) menerima BCCAO dengan 0,125µg/kgBB vitamin D (n=6), dan kelompok terakhir (VD2) tikus menjalani BCCAO dengan 0,5µg/kgBB vitamin D (n=6). Suplementasi Vitamin D disuntikkan secara intraperitoneal sekali sheari selama sepuluh hari sampai tikus diterminasi. RNA diekstraksi dari jaringan hipokampus dan diproses lebih lanjut menjadi cDNA dengan metode Polymerase Chain Reaction (PCR) atau reverse-transcriptase PCR. Hasil Polymerase Chain Reaction dinyatakan melalui pita putih setelah dielektroforisasi. Ekspresi mRNA IL-1? dan TNF-? diperoleh melalui pengukuran densitometri menggunakan aplikasi ImageJ, dan normalisasi dilakukan dengan ekspresi GAPDH yang berfungsi sebagai gen housekeeping.

Hasil: Ekspresi interleukin-1? (IL-1?) pada model tikus transient global ischemic attack dengan suplementasi vitamin D 0,125 µg/kgBB (p=0,119) dan 0,5 µg/kgBB (p=0,044) lebih rendah dibandingkan pada hipokampus. model tikus cedera iskemik global sementara. Selain itu, ekspresi mRNA tumor necrosis factor-alpha (TNF-?) pada model tikus serangan iskemik global transien dengan suplementasi vitamin D 0,125 µg/kgBB (p=0,000) dan 0,5 µg/kgBB (p=0,001) secara signifikan lebih rendah dibandingkan dalam hipokampus model tikus cedera iskemik global sementara.

Kesimpulan: Ekspresi mRNA IL-1? dan TNF-? pada model tikus transient global ischemic attack dengan dosis suplementasi vitamin D 0,125 µg/kgBB dan 0,5 µg/kgBB lebih rendah dibandingkan pada hippocampus model tikus transient global ischemic attack tanpa suplementasi vitamin D dosis tinggi. suplementasi vitamin D.

Background: Transient global brain ischemic injury ensues inflammatory processes in the hippocampus with upregulation of proinflammatory regulators such as tumor necrosis factor-alpha (TNF-?) and interleukin-1? (IL-1?). Vitamin D in previous studies show anti-inflammatory effects. However, the effect of vitamin D on ischemic reperfusion injury models have not been investigated. This study investigates the effect of Vitamin D on rats’ hippocampus with ischemic reperfusion injury models by evaluating mRNA expression of IL-1? and TNF-?.

Aim: To elucidate the effect of vitamin D in rats hippocampus with transient ischemic attack’s model using mRNA expression of TNF-? and IL-1?.

Methods: In this study, Male Wistar rats (n=25, BW=150-300 grams) were used and divided into four groups. The first group (SO) consisted of rats that underwent an operation without carotid communis clamping (n=6). The second group of rats underwent bilateral common carotid artery occlusion (BCCAO) without vitamin D (n=6). The third group (VD1) received BCCAO with 0.125µg/kgBW of vitamin D (n=6), and the last group (VD2) of rats underwent BCCAO with 0.5µg/kgBW of vitamin D (n=6). Supplementation of Vitamin D was injected intraperitoneally once a day for ten days until the rats were terminated. RNA was extracted from hippocampal tissue and further processed into cDNA for the Polymerase Chain Reaction (PCR) or reverse-transcriptase PCR method. The Polymerase Chain Reaction results were expressed through white band after electrophorized. The mRNA IL-1? and TNF-? expressions were acquired through densitometry measurements using the ImageJ application, and normalization was performed with GAPDH expression serving as the housekeeping gene.

Result: The expression of interleukin-1? (IL-1?) in the transient global ischemic attack rat models with 0.125 µg/kgBW (p=0.119) and 0.5 µg/kgBW (p=0.044) vitamin D supplementation is lower than in the hippocampus of transient global ischemic injury rat models. Moreover, the expression tumor necrosis factor-alpha (TNF-?) mRNA in the transient global ischemic attack rat models with 0.125 µg/kgBW (p=0.000) and 0.5 µg/kgBW (p=0.001) vitamin D supplementation is significantly lower than in the hippocampus of transient global ischemic injury rat models.

Conclusion: The mRNA expression of IL-1? and TNF-? in the transient global ischemic attack rat models with 0.125 µg/kgBW and 0.5 µg/kgBW doses of vitamin D supplementation is lower than in the hippocampus of transient global ischemic attack rat models without vitamin D supplementation.

Keywords: Vitamin D, Transient global brain ischemic injury, IL-1?, TNF-?, Inflammation

Kata Kunci : Vitamin D, Transient global brain ischemic injury, IL-1?, TNF-?, Inflammation