Green Tea Derivative EGCG Protects Against Radiation Induced Intestinal Injury By Modulating Nrf2 Signaling Pathway

Abstract

Intestine is one of the most radio-sensitive tissues, and radiation induced intestinal injury (RIII) remains one of the most prevalent dose-limiting toxicities in radiotherapy for pelvic and abdominal cancers. The failure of synthetic compounds for efficient prevention or treatment of RIII led to growing interest in the study of natural derivatives from nontoxic plants. Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a free radical scavenger and potent antioxidant agent, and has been widely studied in cancer chemoprevention. However, whether EGCG has the therapeutic effect on RIII remains unknown. Male C57BL/6J mice were treated with 9Gy of total body irradiation (TBI). 25mg/kg of EGCG were intraperitoneally injected before (once a day for 5 days) and 30minutes after TBI. Then, the survival rate and body weight were recorded. Meanwhile, jejunum tissues were collected and the severity of RIII was measured by HE staining, immunohistochemistry (IHC), immunofluorescence (IF) and TUNEL. Next, human intestinal epithelial cells (HIEC) were treated with EGCG and irradiated. Then, colony formation assay, flow cytometry, IF were conducted, and the level of reactive oxygen species (ROS) was assessed. Further, the expression of pivotal anti-oxidative response regulator nuclear factor erythroid 2 related factor 2 (Nrf2) was detected by IF and western blotting. Compared to the control, EGCG treatment significantly improved the survival time (from 6.1days to 10.4days, P<0.05) and increased the body weights of mice after TBI. HE staining of jejunum tissues showed that EGCG mitigated RIII, as reflected by the dramatic attenuation of crypt-villi architecture destruction. IHC results showed that EGCG treatment remarkably increased the Ki67-positive (from 15.7 to 30.1, P<0.001) and Lgr5-positive (from1.5 to 3.0, P<0.001) cells per crypt, indicating that EGCG promoted proliferation and survival of intestinal stem cells after TBI. Meanwhile, in EGCG treatment group the number of TUNEL-positive cells (from 4.9 to 1.0, P<0.001), γH2AX foci (from 2.8 to 0.4, P<0.001) and 8-OHdG-positive cells (from 3.5 to 0.6, P<0.001) significantly decreased per crypt, indicating that EGCG inhibited radiation-induced apoptosis and DNA damage in intestine. Consistently, the EGCG treatment remarkably improved the colony forming ability, while decreased apoptosis ratio, γH2AX foci and 8-OHdG foci of irradiated HIEC cells. For the mechanism study, EGCG treatment diminished the radiation-induced production of ROS, and promoted the nuclear translocation of key anti-oxidative response regulator Nrf2 of irradiated HIEC cells, indicating that EGCG scavenged radiation-induced ROS, and subsequently attenuated ROS-regulated intestinal oxidative DNA damage. Collectively, our findings suggested that EGCG protects against RIII by reducing ROS-induced DNA damage, and might be a novel therapeutic option for the clinical treatment of RIII.