*3.5. Antioxidant Activity*

RA exhibited free radicals scavenging activity in hepatic stellate cells (HSCs) as a result of its antioxidant e ffects by boosting GSH synthesis and participating in NF-κB-dependent inhibition of MMP-2 activity. It also has the ability to reverse activated HSCs to quiescent cells and ultimately inhibits MMP-2 activity. RNA interference-imposed knockdown of NF-κB abolished MMP-2 down-regulation by RA. NF-κB inactivation mediated by RA could be blocked by the diphenyleneiodonium chloride, a potent inhibitor of NADH/NADPH oxidase. Moreover, transfection of dominant-negative (DN) mutant JNK1, p38 α kinase, or extracellular signal-regulated kinases 2 (ERK2) had no such e ffect. At once, RA suppresses lipid peroxidation (LPO) and ROS generation, whereas in HSC-T6 cells it increases cellular GSH. Additionally, it significantly increases Nrf2 translocation and catalytic subunits from glutamate cysteine ligase (GCLc) expression but was not able to modulate GCL (GCLm) subunits and antioxidant response element (ARE)-mediated luciferase activity. GClc up-regulation mediated by RA is inhibited by shRNA-induced Nrf2 knockdown. The knocking down of Nrf2 abolished RA-mediated inhibition of ROS [16,62].

On the other hand, lycopene and RA administration reduced elevated blood urea nitrogen, renal malondialdehyde (MDA), proapoptotic protein (Bax) immuno-expression, serum creatinine, inducible nitric oxide synthase (iNOS), and autophagic marker protein (LC3/B) levels induced by gentamicin. This combination also increased the reduced SOD, an antiapoptotic protein (Bcl2) immuno-expression, GPx, and GSH levels and ameliorated gentamicin-induced histopathological changes. Moreover, it also evidenced a greater protective e ffect than corresponding monotherapy [63].

The in vivo antioxidant defense system consists of antioxidant enzymes including CAT, GPx, SOD, and nutritional antioxidants. Any disturbance in normal antioxidant defense system triggers several diseases, including diabetes, cancer, atherosclerosis, and degenerative diseases [64]. In experimental animals, carbon tetrachloride (CCl4) induced neurotoxicity, whereas CCl4 skin absorption, inhalation, and ingestion increased lipid peroxidation and reduced protein and antioxidant enzymes contents. This molecule produces free radicals in the lungs, heart, blood cells, and kidney. Under aerobic conditions, CCl4 is converted into highly reactive trichloromethyl radicals through the action of the cytochrome P450 system [65].

Brain amyloid-β (Aβ) accumulation is a hallmark of Alzheimer's disease (AD) and has an important role in cognitive dysfunction [66]. At a dose of 0.25 mg/kg, RA significantly enhanced cognitive function and object discrimination and recognition test. Furthermore, RA decreased the time to reach the platform and increased the number of crossings over the removed platform, when compared with Aβ25-35-induced group in Morris water maze test; moreover, it reduced NO and MDA levels in kidney, brain, and liver [67]. RA also suppresses AD development by reducing amyloid β aggregation by increasing monoamine secretion in mice [68].

RA also prevented oxidative stress in C6 glial cells by increasing cell viability and inhibiting lipid peroxidation. It also decreased H2O2-induced COX-2 and iNOS expression at the transcriptional level and down-regulated COX-2 protein expression and iNOS in C6 glial cells treated with RA [67]. It significantly reduced oxidative stress and increased antioxidant status in Wistar rats post-spinal cord injury (SCI). RA also facilitated the inflammatory process through pro-inflammatory post-SCI and down-regulated NF-κB [69]. It also exerted a significant cytoprotective effect through covering the intercellular ROS in HaCat keratinocytes. RA also increased CAT, SOD, heme oxygenase-1 (HO-1), and transcription factor Nrf2 expression and activity, markedly reduced by UVB radiation [70].

On the other hand, in G93A-SOD1 transgenic mice with amyotrophic lateral sclerosis (ALS), RA application at a daily dose of 400 mg/kg significantly increased their survival by reliving function motor neurons deficits. These types of systematic changes were closely correlated with a decrease in neuronal loss and oxidative stress in ventral horns of G93A-SOD1 mice [71]. RA also increased SOD, GSH and GPx activities and decreased MDA levels in kidney and liver of sepsis-induced rats [72].

RA also reduced threshold shift, attenuated noise-induced hearing loss, and promoted hair cells survival. Moreover, it enhanced the endogenous antioxidant defense system by decreasing SOD production and up-regulation and decreased 4-HNE expression [73,74]. SOD, CAT, and GPx activities were increased through RA application at a dose of 50, 100, and 200 mg/kg. Furthermore, it induced structural changes in the kidney and liver at a dose of 200 mg/kg [75,76].
