Sulforaphane-Mediated Nrf2 Activation Prevents Radiation-Induced Skin Injury through Inhibiting the Oxidative-Stress-Activated DNA Damage and NLRP3 Inflammasome

Abstract

This article mainly observed the protective effect of sulforaphane (SFN) on radiation-induced skin injury (RISI). In addition, we will discuss the mechanism of SFN's protection on RISI. The RISI model was established by the irradiation of the left thigh under intravenous anesthesia. Thirty-two C57/BL6 mice were randomly divided into control group (CON), SFN group, irradiation (IR) group, and IR plus SFN (IR/SFN) group. At eight weeks after irradiation, the morphological changes of mouse skin tissues were detected by H&E staining. Then, the oxidative stress and inflammatory response indexes in mouse skin tissues, as well as the expression of Nrf2 and its downstream antioxidant genes, were evaluated by ELISA, real-time PCR, and Western blotting. The H&E staining showed the hyperplasia of fibrous tissue in the mouse dermis and hypodermis of the IR group. Western blotting and ELISA results showed that the inflammasome of NLRP3, caspase-1, and IL-1β, as well as oxidative stress damage indicators ROS, 4-HNE, and 3-NT, in the skin tissues of mice in the IR group were significantly higher than those in the control group (p < 0.05). However, the above pathological changes declined sharply after SFN treatment (p < 0.05). In addition, the expressions of Nrf2 and its regulated antioxidant enzymes, including CAT and HO-1, were higher in the skin tissues of SFN and IR/SFN groups, but lower in the control and IR groups (p < 0.05). SFN may be able to suppress the oxidative stress by upregulating the expression and function of Nrf2, and subsequently inhibiting the activation of NLRP3 inflammasome and DNA damage, so as to prevent and alleviate the RISI.

Keywords: NLRP3; Nrf2; oxidative stress; radiation-induced skin injury; sulforaphane.

Figure 1

Results of skin injury and repair in mice. (A): Photographs of skin wound in 4 groups of mice at week 1, week 4, and week 8 after irradiation exposure. Scale bars = 4 cm. (B): H&E staining results of mouse skin tissue. Black arrows represent fibrous connective tissue. Yellow arrows represent proliferating blood vessels. (H&E staining, original magnifications × 200, bar = 50 μm.) (C): Skin damage scores of mice at week 1, week 4, and week 8 after irradiation exposure were expressed as the mean ± S.D. (D): Mean weights ± S.D. of 4 groups of mice at week 1, week 4, and week 8 after irradiation exposure (n = 3 at least in each group) (* p < 0.05 vs. CON).

Figure 2

SFN prevents DNA damage caused by radiation. (A): The expression of 8-OHdG in skin blood serum was examined by ELISA. (B): The expression of γ-H2AX in skin tissues was examined by Western blotting. Data were expressed as the mean ± S.D. (n = 3 at least in each group) (* p < 0.05 vs. CON; ^& p < 0.05 vs. IR).

Figure 3

Prevention and treatment of SFN on radiation-induced skin inflammation. (A): The expressions of inflammatory factors, including NLRP3, caspase-1, and IL-1β mRNAs, were examined by real-time PCR. (B): The protein expressions of inflammatory factors, including NLRP3, caspase-1, and IL-1β, in skin tissues were examined by Western blotting. Data were expressed as the mean ± S.D. (n = 3 at least in each group) (* p < 0.05 vs. CON; ^& p < 0.05 vs. IR).

Figure 4

Prevention and treatment of SFN on radiation-induced skin oxidative stress. (A): The expression of ROS in serum was examined by ELISA. (B): The expressions of 3-NT and 4-HNE were measured by Western blotting due to the oxidative damage in the skin tissues. Data were expressed as the mean ± S.D. (n = 3 at least in each group) (* p < 0.05 vs. CON; ^& p < 0.05 vs. IR).

Figure 5

Activation of Nrf2 and expressions of its downstream genes in the skin induced by SFN. (A): The Nrf2 transcription function was examined by the expression of its downstream antioxidant gene (CAT, HO-1) mRNAs by real-time PCR. (B): The expression of Nrf2 in mouse skin tissues was detected by Western blotting. Nrf2 transcription function was examined by the expressions of its downstream antioxidant (CAT, HO-1) proteins with Western blotting. Data were expressed as the mean ± S.D. (n = 3 at least in each group) (* p < 0.05 vs. CON; ^& p < 0.05 vs. IR).

Figure 6

Illustration of the mechanism of radiation-induced skin injury prevented and treated by SFN. SFN can upregulate antioxidant enzymes, including HO-1 and CAT, through the activation of Nrf2 and inhibit the NLRP3 inflammatory pathways by suppressing the ROS production, alleviating radiation-induced skin injury (RISI) in mice. In addition, inhibition of NLRP3 inflammasome may have a positive effect on DNA damage repair, thereby promoting the repair of RISI.