Preventive effects of thymoquinone on glyco-nitro-oxidized human fibrinogen: A comprehensive biophysical study projecting possible therapeutic role in diabetes and associated complications.

Persistence of long-term hyperglycemia results in the glyco-oxidation of plasma proteins, which is considered to be a significant factor in metabolic dysfunction, linking hyperglycemia to the emergence of vascular complications. Methylglyoxal (MGO), a dicarbonyl species formed excessively under diabetes, elevates the oxidative stress, enhancing the generation of superoxide anion, which ultimately reacts with nitric oxide (NO•) to form peroxynitrite (PON). PON, being a powerful nitro-oxidizing agent distorts protein structure, hampering its function. This article describes the binding mechanism of thymoquinone (TQ) to fibrinogen (Fg) and its protective effects under simultaneous glyco-nitro-oxidation. Thermodynamic investigations revealed hydrogen bonding and Vander Waal interactions stabilise the complex, confirming its spontaneity and exothermic nature. TQ-induced micro-environmental and structural alterations in fibrinogen were observed by synchronous, 3-D fluorescence maps, and red edge excitation shift (REES). Molecular docking confirmed the wet lab experiments. Previous studies have shown that glycation, as well as nitro-oxidation, modifies the key residues of fibrinogen, leading to its aggregation. Our findings showed that TQ prevented MGO + PON-induced damage to fibrinogen. The current study analyzed the protective effects of TQ on glyco-nitro-oxidized fibrinogen using various biochemical, spectroscopic, and computational methods. NBT assay and carbonyl content revealed glyco-nitro-oxidation-mediated oxidative stress, which was effectively mitigated by TQ in a concentration-dependent manner. The secondary structural alterations in fibrinogen were prevented by TQ as observed by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR). Moreover, multiple assays and electron microscopy confirmed structural perturbations leading to the development of fibrillar aggregates that were reduced in TQ treated samples. Our findings project TQ as a potent protective agent against hyperglycemia and related human complications.