Quercetin ameliorates Di (2-ethylhexyl) phthalate-induced nephrotoxicity by inhibiting NF-κB signaling pathway

Abstract

This study aimed to evaluate the possible protective effects of quercetin, a natural flavonoid, against nephrotoxicity induced by Di (2-ethylhexyl) phthalate (DEHP) in kidney tissue of rats and human embryonic kidney (HEK) 293 cell line. The HEK-293 cells were treated with different concentrations of quercetin 24 h before treatment with monoethylhexyl phthalate (MEHP). Male rats were treated with 200-mg/kg DEHP, 200-mg/kg DEHP plus quercetin (50 and 100 mg/kg), and 200-mg/kg DEHP plus vitamin E (20 mg/kg) for 45 days by gavage. Quercetin treatment reduced cytotoxicity and oxidative damage inducing by MEHP in HEK-293 cells. The in vivo findings showed that 100-mg/kg quercetin significantly suppressed DEHP-induced kidney damage. For exploring the involved mechanisms, the expressions of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor kappa B (NFκB), and tumor necrosis factor alpha (TNFα) genes were determined via real-time Polymerase chain reaction (PCR) assay. High dose of quercetin significantly decreased the gene expressions of NF-κB and TNFα, whereas the alternations of Nrf2 and HO-1 gene expressions were not significant in quercetin groups in compared with DEHP group. These findings suggested that the suppression of DEHP-induced nephrotoxicity via quercetin is correlated, at least in part, with its potential to regulate NF-κB signaling pathway.

Keywords: Di (2-ethylhexyl) phthalate; HEK293 cell line; mitochondria; nephrotoxicity; nuclear factor kappa B; quercetin.

Graphical Abstract

Fig. 1

The cytoprotective effect of quercetin against MEHP-induced cytotoxicity in HEK-293 cells. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^##P < 0.01 versus control group. ^*P < 0.05, ^**P < 0.01 versus MEHP group.

Fig. 2

The effect of quercetin pretreatment on ROS a), MDA b), and GSH c) levels in HEK293 cells exposed MEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^##P < 0.01 versus control group. ^*P < 0.05, ^**P < 0.01 versus MEHP group.

Fig. 3

The effect of quercetin treatment on level of serum BUN, and creatinine in rats received DEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^#P < 0.05, ^##P < 0.01 versus control group. ^*P < 0.05, ^**P < 0.01 versus DEHP group.

Fig. 4

The effect of quercetin treatment on kidney mitochondrial function (by MTT assay) in rats received DEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^#P < 0.05 versus control group. ^*P < 0.05 versus DEHP group.

Fig. 5

The effect of quercetin treatment on kidney mitochondria membrane potential (MMP) in rats received DEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^#P < 0.05 versus control group. ^*P < 0.05 versus DEHP group.

Fig. 6

The effect of quercetin treatment on kidney mitochondria swelling in rats received DEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^#P < 0.05 versus control group. ^*P < 0.05 versus DEHP group.

Fig. 7

The effect of quercetin treatment on kidney mitochondria levels of ROS a), MDA b), and GSH c) in rats received DEHP. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^##P < 0.01 versus control group. ^*P < 0.05, ^**P < 0.01 versus DEHP group.

Fig. 8

The effects of DEHP and 100 mg/kg of quercetin on mRNA expressions of Nrf2 a), HO-1 b), NFκB c), and TNFα d) in kidney tissue of rats. Values are expressed as mean ± SEM for at least three experiments. Data were analyzed by one-way ANOVA multiple comparison test followed by a Tukey’s multiple comparisons test. ^#P < 0.05 versus control group. ^*P < 0.05 versus DEHP group.

Fig. 9

The appearance of kidney was normal in control group a). The severe inflammation (red arrow) and moderate conjunction (blue arrow) were observed in DEHP treatment group b). The kidney tissue in DEHP plus quercetin at dose of 100 mg/kg was near the normal appearance c).