Ashwagandha (ASH) is one of the medicinal plants that is used in traditional Indian, Ayurvedic and Unani medicines for their broad range of pharmacological activities including tonic, aphrodisiac, energy stimulant, and counteracting chronic fatigue. Besides, it is used in the treatment of nervous exhaustion, memory related conditions, insomnia, as well as improving learning ability and memory capacity. ASH is preclinically proven to be efficient in hepatoprotection and enhancing cognitive impairment, however, its beneficial effects against hepatic encephalopathy (HE) is still unclear. Therefore, this study aimed at investigating the protective effects of ASH root extract against thioacetamide (TAA)-induced HE and delineate the underlying behavioural and pharmacological mechanisms.
ASH metabolites were identified using UPLC-HRMS. Rats were pretreated with ASH (200 and 400 mg/kg) for 29 days and administrated TAA (i.p, 350 mg/kg) in a single dose. Then, behavioral (open field test, Y-maze, modified elevated plus maze and novel object recognition test), and biochemical (ammonia and hepatic toxicity indices) assessments, as well as oxidative stress markers (MDA and GSH) were evaluated. The hepatic and brain levels of glutamine synthetase (GS), nuclear factor erythroid 2-related factor 2 (Nfr2), heme-oxygenase (HO)-1, inducible nitric oxide synthase (iNOS) were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of p 38/ERK ½ were determined using real-time polymerase chain reaction (PCR). Moreover, histopathological investigations and immunohistochemical (NF-κB and TNF-α immunohistochemical expressions) examinations were performed.
Metabolite profiling of ASH revealed more than 45 identified metabolites including phenolic acids, flavonoids and steroidal lactone triterpenoids. Compared to TAA-intoxicated group; ASH improved locomotor and cognitive deficits, serum hepatotoxicity indices and ammonia levels, besides brain and hepatic histopathological alterations. ASH reduced hepatic and brain levels of MDA, GSH, GS, iNOS, Nfr2 and HO-1. ASH downregulated p38 and ERK ½ mRNA expressions, NF-κB and TNF-α immunohistochemical expressions in brain and hepatic tissues.
Accordingly, our results provided insights into the promising hepato- and neuroprotective effects of ASH, with superiority to 400 mg/kg ASH, to ameliorate HE with its sequential hyperammonemia and liver/brain injuries. This could be attributed to the recorded increase in spontaneous alternation % and recognition index, antioxidant and anti-inflammatory activities as well as downregulation of Nrf2 and MAPK signaling pathways.

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