**4. Discussion**

Our study showed that a gradual increment of NADPH Oxidase activity with the pathogenesis and severity of DR (HC < DNR < MNPDR< HRPDR). However there is no direct evidence regarding gradual increment and association of NADPH Oxiadse activity with different graded of DR. An in-Vitro study, Meng et al. [20] demonstrated that NADPH Oxidase augment insulin-induced VEGF expression and angiogenesis. In another study by Ushio-Fukai, [21] also showed that VEGF expression is augmented through ROS, produced by NADPH Oxidase.

Vitreous level of vitamin C was also found to be declined with pathogenesis and severity of DR (HC > DNR > MNPDR > HRPDR). However there is lack of evidence between vitreous level of Vitamin C and different stages of DR. Vitamin C suppress the VEGF gene expression through HIF-1αpathway [22].

Vitreous PCC level was found to be increase gradually with progression of DR (HC < DNR < MNPDR< HRPDR). Nevertheless there is no such evidence of vitreous level of PCC and different stages of DR. Loukovaara et al. [23] shown that amount of protein carbonylation and HIF-1α elevated in vitreous of PDR subjects.

The present study showed a gradual increment of LPO products like MDA and HNE and ALE like HLY both in plasma and vitreous sample towards the development and progression of DR. The studies by Chatziralli et al. [24], Mondal et al., [4] also showed plasma MDA level increases towards the DR pathogenesis and progression. Another study by Mancino et al. [25] showed that vitreous MDA level increases among NPDR and PDR subjects compared to nondiabetic HC subjects. Researchers have demonstrated that the MDA compound is associated with protein modification in a pH-dependent fashion. At the physiological pH, it rapidly forms enolates, which are of lower reactivity and do not react as avidly with nucleophilic species as other aldehydes [26]. However, at a lower pH, MDA exists as b-hydroxyacrolein form, exhibiting a higher reactivity, readily reacting with Lys residues of proteins to form the enaminal type MDA adduct, N ϵ -(2-propenal) lysine, and the fluorescent product, dihydropyridine (DHP) lysine and thereby alters proteins structure and functions [27]. On the other hand, the role of HNE in diabetes and its complications is not well understood [28]. Clinical studies have reported elevated levels of HNE in the blood of diabetic patients with retinopathy compared to those without retinopathy and healthy controls [29]. There is also evidence showing that HNE and HNE-derived ALEs increase in the retinas of rats rendered diabetic for 4-6 weeks [30]. Another study confirmed these findings and showed that HNE may contribute to the pathogenesis of DR by activating the WNT signaling pathway through stabilization of the WNT co-receptor LRP6 [31]. Other animal studies have linked HNE to retinal hemodynamics changes during DR. Retinal perfusion deficits during early diabetes are thought to be mediated, at least in part, through the reduced activity of large-conductance Ca2 + −activated K+ (BK) channels on the retinal vascular smooth muscle cells, causing vasoconstriction [32, 33]. HNE impairs BK channel function in rat retinal arterioles, as demonstrated by reduced vasodilatory responses to the BK channel opener, BMS-191011 [34]. HNE exposure is reported to result in endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in cultures of human retinal capillary pericytes and Müller glia [35].

The ALE component like HLY also found to be increased with the pathogenesis and severity of DR in the present study. A previous study by [36] reported a significant elevation of ALE levels among DNR and MNPDR subjects compared to HCs. Moreover, significant elevation of HLY in the vitreous and serum of patients with PDR was also observed by Izuta et al. [37], which is following our findings.

The study showed a significant negative correlation of VEGF with Vitamin-C level and positive correlations with PCC, MDA, HNE, and HLY. Decrease vitamin C level with increased NADPH oxidase activity may turn oxidative stress, which further damages protein and lipids subsequently causes endothelial dysfunction induced VEGF secretion.
