Role of NF-κB p65 and related cytokines in rats with liver function injury induced by dibutyl phthalate and benzo (a) pyrene

Abstract

Objective: To explore the role of nuclear factor-κB (NF-κB) p65 and related cytokines in rats with liver function injury induced by dibutyl phthalate (DBP) and benzo (a) pyrene (BaP) , in order to provide support for enriching the mechanism of liver injury induced by DBP and BaP. Methods: In September to December of 2019, a total number of 160 specific pathogen free Sprague Dawley rats were numbered in order of sex and body weight, then using the statistical table of random numbers, they were randomly divided into eight groups and each group consists of twenty animals (10 male and 10 female rats) , including blank control group, vehicle control group (given corn oil) , DBP 50 mg/kg (DBP(50)) group, DBP 250 mg/kg (DBP(250)) group, BaP 1 mg/kg (BaP(1)) group, BaP 5 mg/kg (BaP(5)) group, DBP 50 mg/kg plus BaP 1 mg/kg (DBP(50)+BaP(1)) group and DBP 250 mg/kg plus BaP 5 mg/kg (DBP(250)+BaP(5)) group, then DBP and BaP were administered to rats as a homogenous mixture in corn oil by gavage. After exposure for 90 days, liver was separated to test the mRNA and protein expression levels of NF-κB p65 by Real-time fluorescence quantitative polymerase chain reaction and Western blotting. Then serum of rats was collected to detect the levels of CXCL-13, interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) by Enzyme-Linked Immunosorbent Assay, and the levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , albumin (ALB) and total protein (TP) were detected by Reitman-Frankel assay. Results: The protein expression of NF-κB p65 in BaP(1) group was not statistically significant, but the mRNA and protein expression levels of NF-κB p65 in the liver tissues of rats in other exposure group were higher than those in the blank control group (P<0.05) , and the expression levels of NF-κB p65 increased more obvious in the DBP and BaP co-exposed groups than those in the low and high dose groups that single-exposed to DBP and BaP (P<0.05) . The serum levels of CXCL-13 and IL-6 of rats in other group were obviously higher than those of the blank control group except for the BaP(1) group, and the increase was more obvious in the high-dose group that co-exposed to DBP and BaP (P<0.05) . While the level of TNF-α in each exposure group was higher than those in the blank control group and the levels of TNF-α in the DBP and BaP co-exposed groups were strongly augmented compared to those in the low and high dose groups that single-exposed to DBP and BaP (P<0.05) . What's more, compared with the blank control group, the level of ALT in each exposure group was increased significantly. Except for the BaP(1) group, the levels of AST in other exposed groups were increased (P<0.05) , and the levels of ALT and AST in the DBP and BaP co-exposed groups were significantly elevated in comparison to the low and high dose groups that single-exposed to DBP and BaP (P<0.05) . On the contrary, the level of ALB in each exposure group was significantly lower than that in the blank control group, especially decreased significantly in the DBP and BaP co-exposed group (P<0.05) . The level of TP decreased only in the high-dose group that single and co-exposed to DBP and BaP, and the decrease was more significant in the DBP and BaP co-exposed group (P<0.05) . When DBP exposed alone, Pearson correlation analysis showed that NF-κB p65 protein expression level was positively correlated with IL-6, TNF-α and ALT (r=0.762, 0.951, and 0.924, P<0.05) . After BaP exposed alone, the NF-κB p65 protein expression level was positively correlated with TNF-α and ALT (r=0.911 and 0.910, P<0.05) . When DBP and BaP exposed together, NF-κB p65 protein expression level was positively correlated with CXCL-13, IL-6, TNF-α, ALT and AST (r=0.711, 0.764, 0.955, 0.903 and 0.827, P<0.05) . In addition, Pearson correlation analysis showed a positive correlation between TNF-α and ALT (r=0.833 and 0.894, P<0.05) when DBP or BaP exposed alone. Furthermore, when DBP and BaP exposed together, CXCL-13, IL-6 and TNF-α were positively correlated with ALT (r= 0.871, 0.925 and 0.942, P<0.05) , and also positively correlated with AST (r=0.910, 0.892 and 0.890, P<0.05) . Conclusion: Single and co-exposed to DBP and BaP may regulate the abnormal secretion of related cytokines by upregulating the expression level of NF-κB p65 in rat liver tissue, thus leading to hepatocyte injury in rats, and the damage effect may be enhanced when DBP and BaP are exposed together.