Molecular Responses And Mechanism Of Radiation-induced Liver Damage In Rats

Objective: By observing the expression levels of related cytokines in the rat model of radiation induced liver injury, ①To find biomarkers for reducing the role of radiation induced liver injury or early diagnosis of damage and the key targets of the related signaling pathways, ②To analyze and initiate the potential mechanisms of RILD and potential targets for treatment of RILD. Methods: Sprague-Dawley rats(6-weeks-old) were irradiated once at a dose of 20 gy to the right upper quadrant of the abdomen. The rats were then sacrificed 3 days and 1, 2, 4, 8 and 12 weeks after irradiation and rats, which were not exposed to irradiation were used as controls. Weight measurements and blood was obtained from the rats and liver tissues were collected for histological and apoptotic analysis. Immunohistochemistry, reverse transcription quantitative polymerase chain reaction(RT-q PCR) and western blot analysis were performed to measure the expression levels of mRNAs and proteins, respectively. Isolate, purify and primary culture of SD rats’ Kupffer cells; immunohistochemistry was used to detect the expression of F4/80, cell surface markers of Kupffer. Cells were randomly divided into control group, radiation group and gadolinium chloride group, respectively. MTT method was used to detect cell proliferation. And Caspase3 apoptosis kit was used to determine cell apoptosis. ELISA and Real-time PCR methods were employed to dectct the cytokines and growth factors secreted by Kupffer cells. Results: Received a total dose irradiation of 20 Gy irradiation, RILD rats body weight decreased, compared with the control group, with significant difference(P<0.05). Compared with the control group, the body weight of radiation induced liver injury in rats of the experimental group from radiation after second weeksdecreased gradually, and continued to twelfth weeks of the end of the study, withsignificant difference(P<0.05). The serum levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were increased significantly in the RILD rats. Histological investigation revealed the proliferation of collagen and the formation of fibrotic tissue 12 weeks after irradiation. Apoptotic cells were observed predominantly 2 and 4 weeks after irradiation. The immunohistochemistry, RT-qPCR and western blot analysis all revealed the same pattern of changes in the expression levels of the molecules assessed. The expression levels of transforming growth factor-β1(TGF-β1), nuclear factor(NF)-κBp65, mothers against decapentaplegic homolog 3(Smad3) and Smad7 and connective tissue growth factor were increased during the recovery period following irradiation up to 12 weeks. The expression levels of tumor necrosis factor-α, Smad7 and Smad4 were only increased during the early phase(first 4 weeks) of recovery following irradiation. The primary cultured Kupffer cells with macrophages characteristics, typical of high puritywas identified by F4/80 with the positive expression. Received radiation can promote the proliferation of Kupffer cells(P<0.05); and the application of Kupffer cell inhibitor gadolinium chloride can inhibit the proliferation of Kupffer cells was inhibited(P<0.05). Accept the irradiation can inhibit the Capase3 activity(P<0.05); while gadolinium chloride can increase Capase3 activity with significant difference(P<0.05). Kupffer cells activated and the expression of TGF-β1, NF kappa Bp65, Smad4, Smad3, Smad7, TNF alpha, CTGF was significantly increased, compared with the control group, with significant difference(P< 0.05). Gadolinium chloridecan inhibit the expression of cytokines and growth factors, compared with the irradiated group, withsignificant difference(P<0.05). Conclusions: In the RILD rat model, the molecular responses indicated that the TGF-β1/Smads and NF-κB/p65 signaling pathways are involved in the mechanism of RILD recovery. Kupffer cells can activate TGF-β1/Smads and NF-κB/p65 signaling pathways after radiation; while Gadolinium chloridecan can inhibit Kupffer cells, and then inhibit TGF-β1/Smads and NF-κB/p65 signaling pathways, which may reduce the damage of RILD. This research provides new strategy and methods for clinical research.