Date on Master's Thesis/Doctoral Dissertation
Physiology and Biophysics
Physiology and Biophysics, PhD
Committee Co-Chair (if applicable)
glucose metabolism; inflammation; retinal degeneration; vision; JSH-23; Akita
Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus (DM). Its pathology is consisted of the breakdown of the blood-retinal barrier (BRB), particularly the inner blood-retinal barrier (iBRB). The exact mechanism that leads to the iBRB impairment and the development of DR has not been elucidated yet. However, chronic inflammation that is hallmark of the type I DM has recently become a key focus in DR study. Hyperglycemia causes dysregulation of nuclear factor-kappa B (NF-kB); and its constant activation increases the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1) in the retina and vitreous of DR patients and animal models. This increase in inflammatory mediators leads to the degradation of junctional molecules, occludin, connexin-43, death of retinal cells via pyroptosis, and thus disruption of the iBRB. Derailment of the iBRB induces fluid accumulation into the retina of DR patients, which eventually progresses into vision impairment. The purpose of this study is to investigate whether chronic inflammation in DM induces disruption of iBRB; and whether inhibition of the expression and activity of NF-kB by Methyl-N1-(3-phenyl propyl) benzene-1, 2-diamine (JSH-23) would mitigate this iBRB disruption. Methods: Type 1 diabetic mouse model (Akita) on a C57BL/6J background along with non-diabetic littermate were used in the experiments. All mice were 22-24 weeks of age at the start of the experiments. The experimental and the control animals were treated for four weeks on alternate days with JSH-23 @5mg/Kg body weight. The intraocular pressure (IOP), blood pressure, blood glucose, and body weights of all animals were measured before and after treatment. Fluorescence angiography (FA), optical coherence tomography (OCT), and dark-adapted electroretinogram were performed before and after the treatment. Also, intraperitoneal glucose tolerance test (GTT) were performed in the animals before and after the treatment. Then, the retinas and plasma were collected. Western blotting and real-time polymerase chain reaction were performed to analyze protein expression and the mRNA levels of NF-kB p65, iNOS, COX-2, ICAM-1, occludin, connexin-43, NLRP3, caspase-1, and IL-1b. Additionally, plasma insulin levels were also analyzed via ELISA kit. Results: The body weight of Akita mice was significantly lower than the C57BL/6J mice. The treatment with JSH-23 did not have any effect on body weight. The blood glucose of Akita mice was significantly higher than the blood glucose of non-diabetic littermate control mice. The treatment substantially decreased blood glucose in Akita mice. Also, the plasma insulin level for the Akita mice was significantly lower compared to non-diabetic littermate control mice. The intraperitoneal GTT showed that the Akita mice were unable to lower their blood glucose levels. The total and phosphorylated protein expression levels of NF-kB p65 were significantly decreased after treatment with JSH-23 in Akita mice, and the gene expression of NF-kB p65 also reduced. The protein and gene expression levels of iNOS, COX-2, and ICAM-1 were reduced after treatment in Akita mice, and the treatment significantly lowered the expression of pyroptosis marker NLRP3. The gene expression of other proteins in the pyroptosis pathway were also reduced, including caspase-1 and IL-1b. On the other hand, the protein and gene expression levels of junctional molecules occludin, connexin-43 were significantly increased in Akita mice after treatment with JSH-23. The blood pressure of the animals was normal and the treatment did not have any effect on the blood pressure. FA depicted an increase in retinal capillary permeability in Akita mice, and the treatment significantly reduced that permeability. The OCT showed retinal layers thickening in the retina of Akita mice, and treatment increased the retinal layers thickness. Electroretinogram (ERG) showed an increase in the amplitude of the A-wave and the B-wave in Akita mice. Conclusions: The results showed that inhibitor (JSH-23) improved visual function by decreasing the retinal vascular permeability, IOP, and the retinal inflammation in Akita mice.
Petit Homme, Rubens, "Modulation of inflammatory pathway by inflammatory inhibitor JSH-23 mitigates diabetic retinopathy." (2020). Electronic Theses and Dissertations. Paper 3479.