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Background Flurochloridone (FLC) is toxic to male reproduction and can induce apoptosis of testicular tissue and supporting cells under oxidative stress. Of particular concern is whether nuclear factor-erythrocyte 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway and nuclear factor kappa B (NFκB) signaling pathway participate this process. Objective To observe apoptosis of testicular tissue and sertoli TM4 cells and alterations of Nrf2/HO-1 and NFκB signaling pathways in mice treated with FLC in vivo/in vitro. Methods (1) Animal experiment. Testis samples were harvested from male C57BL/6 mice after 28-day FLC (0, 3, 15, 75, and 375 mg·kg−1 per day) exposure via oral route. Malondialdehyde (MDA) and superoxide dismutase (SOD) in homogenate of testicular tissue were measured by colorimetry. Apoptosis of testicular tissue was evaluated by TUNEL staining. Expression and distribution of Nrf2 and NFκB were detected by immunohistochemistry. Protein expression levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), NFκB, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and phosphorylated recombinant inhibitory subunit of nuclear factor kappa-B alpha (P-IκBα) in testicular tissue homogenate were determined by Western blotting. (2) Cell experiment. TM4 cell lines were treated with 40, 80, 120, 160, and 200 μmol·L−1 FLC for 6 h, and cell viability was detected by CCK-8. After 6 h exposure to 40, 80, and 160 μmol·L−1 FLC, the apoptosis rate was detected by flow cytometry, and the protein expression levels of Nrf2, HO-1, NQO1, NFκB, IKKβ, and IκBα were detected by Western blotting. Results (1) Animal experiment. Apoptosis occurred in the interstitial and basal parts of spermatogenic tubules in male C57BL/6 mice after 28 days of oral FLC exposure. Compared with the control group, the MDA level in testicular tissue of the 375 mg·kg−1 FLC-treated group was significantly increased (P<0.05), and the SOD activity was significantly decreased (P<0.05). After 375 mg·kg−1 FLC exposure, apoptosis occurred in the interstitial and basal parts of spermatogenic tubules. The results of immunohistochemistry showed the expression of Nrf2 and NFκB in the interstitium and basal part of spermatogenic tubules of the treated groups. Compared with the control group, the protein levels of Nrf2, NQO1, P-IκBα, NFκB, and IKKβ in the 15, 75, and 375 mg·kg-1 groups were significantly increased (P<0.001), and the HO-1 protein level was significantly increased in the 375 mg·kg−1 group (P<0.001). (2) Cell experiment. Compared with the control group, the TM4 cell viabilities in the 40, 80, 120, 160, and 200 μmol·L−1 FLC-treated groups significantly decreased (P<0.01). The apoptosis rates were significantly increased (P<0.05), and the apoptosis rates increased from 5.7% in the control group to 7.4%, 9.4%, and 11.7% in the 40, 80, and 160 μmol·L−1, respectively. The Nrf2 protein level in the 40 μmol·L−1 group was significantly increased (P<0.01), while the levels significantly decreased in the 80 and 160 μmol·L−1 groups (P<0.01). The HO-1 protein levels in the 40, 80, and 160 μmol·L−1 groups were significantly increased (P<0.01). The level of NQO1 protein in the 40 μmol·L−1 group was significantly increased (P<0.01). The NFκB protein levels were significantly increased in the 80 and 160 μmol·L−1 groups (P<0.001). The IκBα protein levels were significantly decreased in all treated groups (P<0.001). The IKKβ protein had no significant change. Conclusion FLC induces testicular tissue apoptosis, and the process affects Nrf2/HO-1 signaling pathway and NFκB signaling pathway. The in vitro study confirms that FLC could induce apoptosis of TM4 cells and activate Nrf2/HO-1 and NFκB signaling pathways.
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Objective To investigate the protective effect of resveratrol on γ-ray radiation induced small intestinal injury in mice.Methods Sixty-four C57BL/6 male mice were divided into 7.2 Gy irradiation group,7.2 Gy irradiation+resveratrol group,15 Gy irradiation group,15 Gy irradiation+resveratrol group,17 Gy irradiation group,17 Gy irradiation +resveratrol group,resveratrol group and control group according to the method of radiation and administration.Radiation irradiation was performed with a single dose of 0.99 Gy/min.The control and single irradiation groups were given 14.4％ of ethanol solution by intragastric administration.The resveratrol group and irradiation+resveratrol groups were given a resveratrol mixed solution at a dose of 60 mg/kg.The 30-day survival rate of the mice was observed.The morphological changes of the small intestine at 96 hours after the irradiation,and the expression of the intestinal crypts at 4 hours after irradiation was examined.Results Compared with the single irradiation groups,resveratrol can significantly increase the 30-day survival rate of the mice after 7.2 Gy total body irradiation and 17 Gy abdominal irradiation (all P＜0.05).In addition,resveratrol can significantly improve the small intestinal crypt-villus structure damage in mice after 15 Gy abdominal irradiation,down-regulated the expression of p21 and Puma by 0.71 and 0.52 times,respectively (all P＜0.05),and up-regulated the expression of Nrf2,Ho1,iNos and Nqo1 by 3.03,1.62,3.94,and 4.13 times,respectively (all P＜0.05).Conclusion Resveratrol can effectively alleviate the radiation-induced small intestine injury in mice,which may be achieved by regulating anti-apoptotic and anti-oxidant gene expression.