Akut ve subakut dönem travmatik beyin hasarı sonrası pde1b'in etkilediği protein profilinin proteomik açıdan incelenmesi
Proteomic investigation of protein profile affected by pde1b after acute and subacute traumatic brain injury
- Tez No: 929554
- Danışmanlar: DOÇ. DR. NEVİN ÖZTEKİN
- Tez Türü: Doktora
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2025
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Lisansüstü Eğitim Enstitüsü
- Ana Bilim Dalı: Kimya Ana Bilim Dalı
- Bilim Dalı: Kimya Bilim Dalı
- Sayfa Sayısı: 88
Özet
Travmatik beyin hasarı (TBH), nörodejeneratif rahatsızlıklar arasında en yüksek insidansa sahip olan intrakraniyal yaralanma olarak da bilinen yaygın bir yaralanma çeşididir. TBH sonucunda kan beyin bariyeri, sinir ve damar hücreleri zarar görür ve bu durum uzun vadede kronik baş ağrısı, vertigo, konuşma zorluğu, anksiyete, depresyon ve demans gibi çeşitli nörodejeneratif hastalıklara neden olma ihtimali bulunur. Ayrıca; teknolojik gelişmeler, artan nüfus ve motorlu araç kullanımının artması günden güne daha fazla insanın TBH'a uğramasına neden olduğu için, halk sağlığı yükü oluşturur ve dolayısıyla etkin tedavi yöntemlerine ihtiyaç duyulur. Tedavi olarak çeşitli yöntemler kullanılmaktadır, ancak soğuk kaynaklı TBH'ın etkileri kapsamlı bir şekilde incelenmedi. Halkalı nükleotitler hücre için sinyal basamaklarının düzenlenmesinde önemli role sahip olan ikincil mesajı olarak adlandırılan bileşenlerdir. Hücre içi sinyal basamaklarının düzeni nörojeneratif rahatsızlıklar açısından önemli oldukları için TBH sonrası etkilerinin araştırılması önemlidir. Halkalı adenozin monofosfat (cAMP) ve halkalı guanozin monofosfat (cGMP)'ın düzenlenmesinde rol alan fosfodiesteraz 1B (PDE1B) enziminin inhibisyonunun incelenmesi gerekir, bu işlem için nöroprotektif etkiye sahip olduğu bilinen vinpocetine (VPN) farelere uygulandı. VPN, vinca minörden elde edilen, anti-inflammatuar ve antioksidan özelliklere sahip bir maddedir. Bu tezin amacı da soğuk kaynaklı TBI sonrasında VPN'in farklı dozlarda (5 mg/kg veya 10 mg/kg) nöroprotektif etkilerini araştırmaktır. Bu etkileri ayrıntılı olarak inceleyebilmek adına öncelikle BALB/c erkek fareler kullanılarak fizyolojik koşullarda VPN etkisi incelendi, daha sonra ise sırasıyla akut ve subakut dönemde soğukla indüklenmiş VPN'in TBH'a olan etkisi ayrıntılı olarak incelendi. Sonuçlar, farelere VPN uygulamasının beyin hasar hacmini, beyin ödemini ve DNA fragmantasyonunu doza bağlı bir şekilde önemli ölçüde azalttığını gösterdi. Ek olarak, VPN ipsilezyonel kortekste nöronal sağkalımı arttırdı. Uzun vadede, VPN tedavisi (5 mg/kg/gün veya 10 mg/kg/gün, TBI'dan 48 saat sonra başlanır) lokomotor aktiviteyi iyileştirdi. Fizyolojik, akut ve subakut koşullarında VPN tedavisinden etkilenen proteinleri tespit etmek için altta yatan mekanizmaları aydınlatmak amacıyla sıvı kromatografi-tandem kütle spektrometrisi (LC-MS/MS) uygulandı. Proteomik analiz sonrasında subakut döneme ait verilere yolak analizleri uygulanarak, etkilenen nörolojik yolaklar tespit edildi. Bunun sonucunda, elde edilen protein profilinin VPN tedavisi ile önemli ölçüde değiştiğini tespit ettik, bu bulgu da iyileştirici hedeflerin geliştirilmesi için daha fazla araştırma yapılması gereken bir konudur. Tez çalışması sonucunda, VPN'in soğuk kaynaklı TBH'nda nöroprotektif bir role sahip olabileceği gösterildi.
Özet (Çeviri)
Traumatic brain injury (TBI), also referred as intracranial injury, is a common type of injury and the most common among neurodegenerative disorders. It is identified as a result of an external mechanical force such as crushing, impact or explosion. In conclusion of TBI, the blood-brain barrier, nerve, and vascular cells are damaged, which in the long term may lead to various neurodegenerative diseases such as chronic headaches, vertigo, speech difficulties, anxiety, depression, and dementia. There are two types of TBI injuries named primary and secondary brain injuries. Primary brain injury is the first tissue damage that occurs immediately with the impact of the injury and results in the death of affected tissue. Secondary brain injury follows primary brain injury in the long term and occurs through biomolecular and cellular changes around the area where the primary injury is located. In this thesis, secondary brain injury was the main point, and as a TBI model, cold-induced TBI was performed. It has the capability to limit the extent and location of damage better than other TBI models. For example, fluid percussion and controlled cortical impact can highly re-induce cortical damage. Furthermore, technological advances, a growing population, and increased use of motorized vehicles mean that more people are suffering from TBI day by day. Besides, TBI poses a significant public health burden for which effective treatment methods are necessary. There are various treatment methods, however the effects of cold-induced TBI have not been extensively studied. Cyclic nucleotides are called secondary message components that have an important role in the regulation of signaling pathways for the cells, which is essential for neurodegenerative disorders, so it is important to investigate their effects after TBI. Secondary message components are regulated by the PDE enzyme family, especially cAMP and cGMP, which are the part of main signal pathways, and are under the control of PDE1 enzymes, that have three isoforms :PDE1A, PDE1B and PDE1C. Considering this situation, the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), need to be studied and investigated with inhibition of phosphodiesterase 1B (PDE1B) enzyme, that is considered to have a significant role for the process of neurodegenerative conditions. The effects of vinpocetine (VPN) treatment, which is known for its neuroprotective effects and inhibits PDE1B enzyme, on TBI need to be studied in detail. VPN is a substance obtained from vinca minor and has anti-inflammatory and antioxidant properties. VPN has the ability to cross the blood brain barrier easily and due to the fact that, it dilates the cerebral blood vessels and also enhances the blood flow mechanism in the brain to use more glucose and oxygen. Proteomic analysis, one of the omics studies that have gained an important place in scientific studies with the developing technology, has an important place in this thesis. Proteomics investigates the properties of proteins post-translational modification, function in tissues/cells, such as localization, amount, post-translational modification, function in tissues/cells, and interaction with other proteins and macromolecules. One of the important proteomic methods, which is performed in the thesis, is performed by mass spectrometry, which has an ionization part, separation of ions part by magnetic and electric field, and signal detection part based on mass over charge ratio. The process of protein identification, from protein digestion to peptide analysis and characterized peptides using a protein database is called 'bottom characterized peptides using a protein database is called 'bottom-up' proteomics. If bottom-up proteomics analysis is performed from a protein mixture, it is called shotgun proteomics. This method generally has 3 steps: Preparation of sample, which includes protein isolation and digestion to peptide; mass spectrometry analysis of peptides to create and collect the data, including fragmentations from peptides and bioinformatic step to process data to have protein expressions and affected pathways. This thesis aims to investigate the neuroprotective effects of VPN at different doses (5 mg/kg and 10 mg/kg) following cold-induced TBI. To achieve this, three experimental sets were designed, which are an investigation of the effect of VPN at physiological conditions as the first set, TBI after treatment of VPN at acute term as the second set, and VPN treatment after TBI for subacute term as the third set. As a first step, the effects of VPN were studied under physiological conditions using BALB/c male mice. In this context, changes in the amount of cAMP response element binding (CREB) protein determination by Western blot protocol and proteome profile analyses were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to use brain tissue samples, collected after the intraperitoneal injection and sacrification. As a result of these analysis, the significantly increasing of CREB protein between vehicle and dose groups confirmed the decreasing of the amount of PDE1 isoforms, including PDE1B, and proved the general effect of VPN. Besides, significant differentiation of some neurological and signaling-related proteins were investigated like guanine nucleotide-binding protein-like 1 (Gnl1), plasma membrane calcium-transporting ATPase 4 (Atp2b4), and protein piccolo (Pclo). Then, the impact of cold-induced VPN treatment on TBI was examined in detail for the acute and subacute phases. For the studies of the thesis, the cold-induced TBI model was applied to the mice. During the acute phase study, another set was prepared. Firstly, intraperitoneal injections were applied to the mice, and after 30 minutes, a cold-induced TBI model was performed and waited for 48 hours for reperfusion, then animals were sacrificed, and their brain tissues were collected for DNA fragmentation, neuronal survival, brain infarct volume, brain swelling, and proteomic analysis. To perform DNA fragmentation analysis, TUNEL and DAPI staining protocols were applied to coronal sections of brain tissue. For neuronal survival analysis, the NeuN staining protocol was used. Stained cells from both protocols were then observed and counted using a confocal laser microscope. Brain infarct volume and brain swelling were analyzed using the cresyl violet staining protocol. After staining the coronal brain sections, the ImageJ program was used to determine infarct volume and edema. Also, proteomic analysis was performed for this set as it was done for the first set. The results show that VPN administration to mice significantly reduced brain infarct volume for both VPN doses. Moreover, brain swelling and DNA fragmentation also significantly decreased effects with only a high VPN dose. Otherwise, VPN increased neuronal survival in the ipsilesional cortex. The last analysis step of the acute phase set, the proteomic analysis, resulted in 27 statistically significant proteins. Some neurologically interested proteins, such as cell division control protein 42 homolog (Cdc42), disks large homolog 5 (Dlg5), and cell adhesion molecule 3 (Cadm3), had an altered expression level by increasing. In the subacute term, the last experimental set of study, VPN treatments (5 mg/kg/day or 10 mg/kg/day, started 48 hours after TBI) improve the locomotor activity. For behavioral testing, the open field test was performed on the mice the day before the TBI operation and on days 2, 7, 14, and 28 after the operation. During the test, mobility, immobility, and distance parameters were evaluated. In conclusion, mobility and immobility showed significant differences on day 28, and distance showed statistically significant differences on days 2, 7, and 28 compared to the vehicle group. Furthermore, to discover proteins differentiated by VPN treatment under subacute conditions, LC-MS/MS method was performed to express the underlying mechanisms, and 192 proteins were detected, which had Anova ≤0.05 and max fold change ≥1.4. It was found that the protein profile obtained was significantly altered by VPN treatment, a finding that warrants further investigation for the development of therapeutic targets. As an essential point, pathway analyses were performed on the protein profile obtained to find out its relationship with TBI. PANTHER program were used to find some neurological-related pathways. In addition, the DAVID online tool was utilized to identify some pathways to use some databases, which are KEGG, REACTOM and Wikipathways. In conclusion, 9 pathways from PANTHER, 24 pathways from KEGG, 151 pathways from REACTOM, and 5 pathways from Wikipathway were achieved with p
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