*3.8. Nephroprotective Activity*

Currently, the most often used antimicrobial agents provoke an acute renal injury in 60% of acquired infections in hospitals with noticeable morbimortality rates [96]. Among them, aminoglycosides have been frequently used to treat bacterial infections, and gentamicin is the most commonly used aminoglycoside, given its lower costs and lower rate of antibiotic-acquired resistance; nevertheless, its therapeutic use at 80 mg/kg/day for more than 7 days induces nephrotoxicity in about 30% of patients [97]. Acute renal toxicity characterization is assessed by the sudden decrease of kidney function due to the accumulation of urea, creatinine, and other waste products. Gentamicin-induced nephrotoxicity is related to enhanced oxidative stress levels, which may be a major contributing factor for renal injury [98].

RA led to a decrease in serum blood urea nitrogen and creatinine levels in Sprague Dawley rats and ultimately decreased myeloperoxidase and MDA levels. In fact, the level of incident injury decreased in the RA-treated group. In addition, RA extensively reduced Bowman's capsules dilatation, glomerular necrosis, tubular epithelium degeneration, tubular epithelium necrosis and dilatation, and focal glomerular necrosis [99].

RA at doses of 1, 2, and 5 mg/kg for 2 days significantly increased serum creatinine and blood urea nitrogen levels and reduced cisplatin (CP)-induced histopathological changes. RA also reduced CP-produced oxidative stress and amplified cytochrome P450 2E1 (CYP2E1), HO-1, and renal-4-hydroxynonenal expression. Additionally, RA repressed TNF-α and NF-κB expression, as stated through inflammation inhibition. Moreover, RA reduced p53, phosphorylated p53, and active caspase-3-expression in kidney through exerting antiapoptotic activity [100]. RA also notably reduced MDA, tubular necrosis, urea, and creatinine levels, and increased renal GSH, SOD, CAT, GPS, volume density creatinine clearance, and PCT [101]. Finally, RA could provide protective effects against 6-hydroxydopamine-induced neurotoxicity via its antioxidant activity [102].

## *3.9. Anti-Aging Activity*

RA administration could effectively reverse chaperones- and Pin1-induced abnormal changes and suppress P-tau and insoluble P-tau formation, induced by chronic restraint stress (CRS), particularly in middle-aged mice [103].

AD is a progressive neurodegenerative disorder that causes dementia in older people. Disease indicators include the appearance of plaques and tangles in brain tissues, which progressively kill neurons from brain cortex, amygdala, hippocampus, and other non-regeneratable brain regions. Consequently, acetylcholine (ACh) levels decline, the widely known cholinergic deficit hypothesis for AD. ACh has a significant role in brain functions, such as thinking, reasoning, remembering, and behavioral abilities [104,105].

In AD, stress is an important risk factor, since it induces tau phosphorylation and enhances tau insolubility in the brain. RA application is able to dominantly suppress the increase in tau phosphorylation levels and insoluble P-tau formation, facilitated by chronic resistant stress, and overturn the abnormal changes in middle-aged mice [103]. At 1.6, 16 and 32 mg/kg, RA exerted markedly useful effects on memory and learning and also reduced the levels of protein carbonyls in the hippocampus [106]. In ALS, a neurodegenerative disease, RA significantly delays motor neuron dysfunction in paw grip endurance tests, through attenuating motor neurons degeneration and extending the life span of ALS mice model, detected at later stages, and about 2% patients present an associated mutation in the gene encoding Cu/Zn-SOD [107].

RA also exerted protective effects against 6-hydroxydopamine-facilitated neurotoxicity and prevented 1-methyl-4-phenypyridinium effects in MES23.5 dopaminergic cells. In fact, 1-methyl-4- phenylpyridinium treatment reduces cell viability and dopamine contents, as well as causes apoptotic morphological changes. Additionally, 1-methyl-4-phenylpyridinium precedes mitochondrial dysfunction, easily detected through inhibiting mitochondrial respiratory chain complex 1-associated activity, suggesting mitochondrial transmembrane collapse and ROS generation. Thus, RA pretreatment was able to restore mitochondrial respiratory chain complex 1 activity and to reverse the other MPP positive damaging effects [91,92] partially. In mice, RA improves oxidative stress parameters and mitochondrial respiratory chain activity [108]. RA also proved to be effective in preventing in vitro

amyloid peptide aggregation and in delaying disease progression in animal models [109]. It also provided neuroprotective e ffects against Aβ-induced toxicity by lowering lipid peroxidation, DNA and ROS formation, and inhibiting phosphorylated p38 MAPK levels [110,111]. RA improved antioxidant properties and healthspan via the IIS and MAPK pathways in *Caenorhabditis elegans* [112].

N2A cells' H2O2-induced cytotoxicity is also positively a ffected by RA; in fact, it is able to attenuate LDH, intercellular ROS, and mitochondrial membrane potential disruption. RA also promoted TH and BDNF genes up-regulation and prevented genotoxicity [113,114]. Clovamide in combination with RA, at the rate of 10–100 μM, in SH-SY5Y cells significantly enhanced PPARγ expression and inhibited NF-κB translocation, respectively [115].

## *3.10. Anti-Allergic Activity*

RA significantly decreased murine double minute (MDM) 2 and thymic stromal lymphopoietin (TSLP) expression in induced mast cells proliferation. It also significantly decreased the levels of phosphorylated signal transducer, IL-13, and transcription-6 activation in TSLP-stimulated HMC-1 cells. Moreover, RA triggered an increment of p53 levels, poly-ADP-ribose polymerase cleavage, caspase-3 activation, and a reduction in Bcl2 and procaspase-3 levels. Furthermore, it significantly reduced TNFα, IL-6, and IL-1β production in TSLP-stimulated HMC-1 cells. It also reduced IL-4, immunoglobulin E (IgE), and TSLP levels in short ragweed pollen-induced allergic conjunctivitis mouse model [116].

In murine model of respiratory allergy caused by Bloma tropicalis (Bt) mite, RA led to a considerable decrease in leukocytes or eosinophils numbers in bronchoalveolar lavage (BAL), of mucus presence in the respiratory tract, reduced lung histopathological changes, and eosinophil peroxidase activity and IL-4 changes [117,118]. At a dose of 1 or 5 μM in NC/Nga mice under specific pathogenic free conditions, RA was shown to be the most e ffective treatment against 2,4-dinitrofluorobenzene (DNFB)-induced AD-like skin lesion. Moreover, it suppressed IL-4 and interferon (INF) production by activated CD4+ cells. RA also inhibited skin lesions and ears thickness development and increased total serum IgE levels in DNFB-treated NC/Nga mice [119]. Furthermore, it inhibited IgE levels increase in spleen, nasal mucosa, and serum, inhibited the increase in rubs number, and reduced histamine levels in ovalbumin unsensitized (OVA) mice. RA also inhibited protein levels and mRNA expressions of IL-6, IL-1β, and TNFα in nasal mucosa or spleen tissues in OVA-sensitized mice [120,121]. In a murine model of allergic asthma triggered by house dust mites (HDMs), RA inhibited the boost up of mononuclear, eosinophils, and neutrophils cells levels around airways and in BAL fluid. Moreover, it also significantly inhibited IL-13 expression increased by HDM allergen [122].

RA (200 mg or 50 mg for 21 days) also reduced the number of eosinophils and neutrophils considerably in nasal lavage fluid. Up-regulation of VCAM-1, COX-2, MIP-2, and ICAM-1 by 2-tetradecanoylphorbol 13-acetate (TPA) were significantly reduced with RA pretreatment. ROS production detected as LPO, 8-hydroxy-2deoxyguanosine (8OH-dG) and thiobarbituric acid reactive substance (TBARS) and TPA levels were markedly reduced by RA pretreatment. Furthermore, RA is conceived as a potential agen<sup>t</sup> against seasonal allergic rhino conjunctivitis (SAR), through mediated polymorphonuclear leukocytes (PMNL) infiltration inhibition [123].

RA also inhibited significantly the increases in eosinophils levels in BAL fluid along with murine airways. In the lungs of sensitized mice, RA inhibited the increase in protein expression of IL-5, IL-4, and eotaxin. Thus, RA seems to be a successful intervention for allergic asthma, given its ability to increase chemokines, cytokines, and allergen-specific antibody levels [124]. Additionally, RA reduced inflammation and allergic immunoglobulin responses occurring in mice PMNL. It also noticeably increased the response rates for itchy eyes, watery eyes, and itchy nose and reduced eosinophils and neutrophils levels in nasal lavage fluid of SAR [125].
