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The aim of this study was to evaluate the effects of bush sophora root polysaccharide (BSRPS) on the aflatoxin B1 (AFB1)-induced hepatotoxicity and to explore the underlying mechanisms. The primary chicken hepatocytes were used as the model in the present experiment. The results showed that AFB1 induced hepatotoxicity of chicken hepatocytes in a dose dependent manner as demonstrated by decreasing cell viability and increasing LDH activity, ALT and AST levels. AFB1 at 0.16 μM significantly increased the levels of hepatic cytochrome P450 1A5 (CYP450 1A5) mRNA and malondialdehyde (MDA) and decreased the activity and mRNA level of manganese superoxide dismutase(SOD2) and the glutathione peroxidases (GSH-Px) activity in the hepatocytes compared with the blank control. BSRPS at 8.93 μM, 17.86 μM, and 35.72 μM supplementation could significantly reverse the above-mentioned changes induced by AFB1, and 17.86 μM of BSRPS has the largest effects on protecting the AFB1-induced hepatocytes damage. Knock-down of SOD2 by SOD2-specific siRNA significantly eliminated the protective effects of BSRPS on AFB1-induced the increase of CYP450 1A5 mRNA levels and hepatotoxicity. These results suggested that the BSRPS has protective effects on AFB1-induced hepatotoxicity by down-regulating CYP450 1A5 mRNA level via up-regulating SOD2 expression in the primary chicken hepatocytes.BSRPS could help prevent AFB1-induced hepatocytes damage in the primary chicken hepatocytes.BSRPS can reverse the decreases of SOD2 expression and the increases of CYP450 1A5 expression induced by AFB1.Knock-down of SOD2 by SOD2-specific siRNA eliminated the preventing effects of BSRPS on AFB1-induced hepatocytes damage.