Abstract:

Objective To investigate the protective effect of quercetin against 5-fluorouracil （5-FU）- induced damage in the human immortalized keratinocytes （HACAT）， and to elucidate its possible mechanism. Methods The HACAT cells were divided into control group （normal cultured cells）， 5-FU group （treated with 7.5 mg·L^-1 5-FU for 24 h）， and low， medium， and high doses of quercetin groups （HACAT cells treated with 25， 50， and 75 μmol·L^-1 quercetin combined with 7.5 mg·L^-1 5-FU for 24 h）. Cell counting kit-8（CCK-8） assay was used to detect the survival rates of HACAT cells treated with different doses（0，10，25，50，75 and 100 μmol·L^-1） of quercetin in various groups. The fluorescent probe of reactive oxygen species（ROS） was used to detect ROS levels in the HACAT cells in various groups. Annexin Ⅴ-FITC/PI double staining was used to detect the apoptosis of HACAT cells in various groups. Western blotting method was used to detect the expression levels of B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax）， Cleaved Caspase-3， cyclooxygenase-2 （COX-2）， interleukin-1β （IL-1β）， and interleukin-6（IL-6） in the HACAT cells in various groups. Results The CCK-8 assay results showed that compared with 0 μmol·L^-1 quercetin group， the survival rates of HACAT cells in 10， 25， 50 and 75 μmol·L^-1 quercetin groups showed no significant differences （P>0.05）， while the survival rates of HACAT cells in 100 μmol·L^-1 quercetin group was significantly decreased （P<0.05）. Compared with 5-FU group， the survival rates of the HACAT cells in low， medium and high doses of quercetin group were significantly increased （P<0.05）. Compared with 5-FU group， the ROS levels in low， medium， and high doses of quercetin groups were significantly decreased （P<0.05）. Annexin Ⅴ-FITC/PI double staining assay showed that compared with 5-FU group， the apoptotic rates in low， medium and high doses of quercetin groups were significantly decreased （P<0.05）. The Western blotting results showed that compared with 5-FU group， the expression levels of Bcl-2 protein in medium and high doses of quercetin groups were significantly increased （P<0.05）， the expression levels of Bax and Cleaved Caspase-3 proteins in low， medium and high doses of quercetin groups were significantly decreased （P<0.05）， the expression levels of COX-2 protein in medium and high doses of quercetin groups were significantly decreased （P<0.05）， and the expression levels of IL-1β and IL-6 proteins in medium dose of quercetin group were significantly decreasd （P<0.05）. Conclusion Quercetin has protective effect on 5-FU-induced damage in the HACAT cells， and its mechanism may be related to the reduction of the expression of ROS and inflammatory factor COX-2 which attenuate the apoptosis.

Key words: Quercetin, Hand-foot syndrome, Human immortalized keratinocytes, Apoptosis, Cyclooxygenase-2