The Involved Mechanism Of H₂ In Radioprotection

Background:For a long time, molecular hydrogen(H2) is regarded as an inert gas, which has no reaction with any substance in human body and shows no biological activity. In 2007, Ohsawa et al. reported that inhaled 2% hydrogen gas(400ppm or 20 u M in blood) could protect brain from IRI injury and reduce infract size in rats. Accumulated evidence in a variety of biomedical fields using clinical and experimental models for many diseases proves that hydrogen has protection properties.Recently, our department demonstrated that H2 pretrement provided protection against ionizing radiation injury. Results of our experiments showed that hydrogen could protect cultured cells, reproductive system, cardiovascular system and hematopoietic system in mice from ionizing radiation.Ohsawa et al. found that H2 showed antioxidant properties by selectively neutralizing hydroxyl radical(?OH) and peroxynitrite(ONOO-). However, as the research of H2 goes further, more and more results couldn’t be explained by selective free radical scavenging. Accumulating evidence indicates that H2 may regulate some critical cellular signal transductions and gene expressions. The underlying mechanism involved in the radioprotection of H2 remains unknown as yet. Clarifying the molecular mechanism of H2 will provide theoretical support for its use and promote its clinical application.Based on these, our study is to explore the underlying mechanism involved in the radioprotection of H2. Following parts are included in our research. 1、Testify the free radical scavenging ability of H2 in vitro. 2、Detect the effect of H2 on the activity of antioxidases 3、Explore the regulation of Nrf2/ARE signal transduction pathway in the radioprotection of H2, choosing L-02 cell as the object.Contents:1、Free radical scavenging ability of H2: Produce different kinds of free radical in vitro and test the level of free radicals with or without H2 by using ESR; 2、Effect of H2 on the antioxidases: Detect the effect of H2 on both purified antioxidases and that in L-02 cells. 3、The role of Nrf2/ARE signal pathway on radiaprotection cell model by H2. Regulation of Nrf2/ARE signal pathway by H2: ①Establish radioprotection cell model by H2 ②Test the levels of Nrf2. Using si Nrf2 to knockdown Nrf2 level in L-02 cell and detect the changes of radioprotection.Methods:1、Produce by irradiated riboflavin and test the level by ESR. 2、Test the level of DPPH by ESR. 3、Produce singlet oxygen by irradiated hematoporphyrin and test the level by ESR. 4、Detect the activity of antioxidases using assay kits. 5、L-02 cell were irradiated with two different doses and detect the apoptosis and cell viability at 24 h after irradiation. 6、Western blot was used to detect the protein level of Nrf2 after irradiation in different groups. 7、Real time-PCR was used to detect the m RNA level of Nrf2 after irradiation in differernt groups. 8、Using lipo2000 to transfer si Nrf2 into cells and detect the effect of si Nrf2 via Western blot and real time-PCR. 9、Using si Nrf2 to knockdown the levels of Nrf2 in cells and detect the levels of antioxidases after irradiation. 10、Using si Nrf2 to knockdown the Nrf2 level in cells and detect the cell apoptosis and cell viability after irradiation. Then the role of Nrf2/ARE signal pathway in radioprotection of H2 was discussed in the group of si Nrf2 and the control.Results:1、Different kinds of free radicals were generated in vitro and the levels in different groups were detected by ESR. The results indicated that there was no difference between H2 group and control group. H2 showed no ability of scavenging free radical. 2、H2 was bubbled into the purified antioxidases and the activity of the enzyme was detected. The results showed that H2 had no effect on the purified antioxidases. 3、The levels of antioxidases(SOD、CAT and GSH-PX) were detected 24 h after 4Gy irradiation. The results showed that H2 could increase the levels of antioxidases 4、L-02 cells were irradiated with a single dose of 4Gy and 8Gy. 24 h after irradiation, cell apoptosis and cell viability were detected. The results showed that H2 could reduce cell apoptosis and increase cell viability after irradiation. 5、L-02 cells were irradiated with a dose of 4Gy. Real time PCR indicated thatthere was no difference between groups after irradiation. 6、Western blot was performed to detected the protein level of Nrf2 after 4Gy irradiation. The results indicated that H2 could increase the protein level of Nrf2. 7、Using si Nrf2 to knockdown the Nrf2 level via lipo2000 transfection, the expression of Nrf2 was decreased obviously. 8、Using si Nrf2 to knockdown the Nrf2 level in L-02 cells. 24 h after irradiation with a single dose of 4Gy, the levels of antioxidases were detected. The results showed no difference between H2 group and other groups. 9、Using si Nrf2 to knockdown Nrf2. Then L-02 cells were irradiated with a single dose of 4Gy and 8Gy. 24 h after irradiation, cell apoptosis and cell viability were detected. The results indicated that the former observed radioprotective effect of H2 was attenuated. Taken together, our data suggest that H2 may perform radioprotection via Nrf2/ARE single pathway.Discussions:Recently, there is an increasing development in H2 research. It has been demonstrated that H2 has protective effects in a variety of biomedical fields. Our department demonstrated that H2 pretreament could protect cultured cells and mice from ionizing radiation. However, researches on H2 mostly focused on phenomenon instead of mechanism. The underlying mechanism is still unclear as many results can not be explained with selective free radical scavenging. Based on this, our study is to explore the involved mechanism in three ways: 1、Detect the ability of H2 to scavenge different free radicals generated in vitro by ESR. 2、Detect the effect of H2 on antioxidases. 3、Explore the role of Nrf2/ARE signal pathway in radioprotection of H2. Our results indicated that H2 had no free radical scavenging ability in vitro. Western blot revealed an increase in Nrf2 protein level in H2 group, suggesting that H2 could activated Nrf2/ARE signal pathway. Furthermore, si Nrf2 was used to knockdown Nrf2 level in cells. We found that the radioprotection effect of H2 was attenuated. Meanwhile, the increased level of antioxidases observed before was reversed as well. Thus, we came into an conclusion that the radioprotection afforded by H2 might result from the regulation of Nrf2/ARE signal pathway.Conclusions:1、The ability of H2 to scavenge free radicals was not observed in vitro. 2、H2 could increase the leves of antioxidases in L-02 cells after irradiation.3、The underlying mechanism of radioprotection of H2 may be the regulation of Nrf2/ARE signal pathway.