| Objective:Based on phosphatidylinositol 3-kinase（PI3K）/protein kinase B（AKT）/cysteine aspartic specific protease 9（Caspase-9）signaling pathway to explore the role and mechanism of hydrogen-rich water in acute and chronic radiation brain injury.Methods:1、Forty male SD rats were randomly divided into Control group,IR group,IR+HHRW group and IR+LHRW group,with 10 rats in each group.Except the control group,animals in other groups received a single 20 Gy whole brain irradiation.Animals in all groups were given intragastric administration once a day from 3 d before irradiation to7 d after irradiation.Control group and IR group were given purified water(20 m L·kg-1),and IR+HHRW group and IR+LHRW group were given hydrogen-rich water(20 m L·kg-1,10 m L·kg-1).After 7 days of exposure,5 rats in each group were selected for Morris water maze test to detect behavioral changes.The remaining animals were killed after anesthesia,blood samples were collected（to observe the changes of RBC,HGB and WBC of rats）,brain tissues were taken for TUNEL staining to observe the apoptosis of nerve cells,hematoxylin and eosin（HE）staining to observe the histopathological changes,enzyme linked immunosorbent assay（ELISA）to detect the changes of oxidative stress index.The m RNA and protein expressions of PI3K,AKT,Cytc and Caspase-9 were detected by real-time quantitative PCR and Western blotting.2、Animal grouping and intervention methods are as above.Continuous gavage for 30days after irradiation.The behavioral changes of rats after 30 days of hydrogen-rich water intervention were evaluated by behavioral experiments.ELisa was used to detect the expression of protein factors related to inflammation,oxidative stress and antioxidant reaction in brain.Real-time quantitative PCR was used to detect the gene expression of PI3K/AKT/Caspase-9,and Western Blotting was used to detect the protein expression.Results:1、The weight of rats in each group receiving irradiation on the 7 th day after irradiation was lower than that of the control group（P<0.05）,and symptoms such as arched back and malaise occurred to varying degrees,and the symptoms of rats in the IR+HHRW group were significantly reduced compared with those in the IR group.The behavioral test results showed that from the second day,the evasion latency of rats in the IR+HHRW group and the IR+LHRW group was shorter than that in the IR group（P<0.05）,and the time spent by the rats in the IR+HHRW group to reach the original platform position after removing the platform on the 6th day was reduced（P<0.05）.The results of hematological tests showed that red blood cells（RBC）,hemoglobin（HGB）and white blood cells（WBC）were reduced in the IR group（P<0.05）,and the changes of RBC,HGB and WBC in the IR+LHRW group and IR+HHRW group were improved,among which the changes in the IR+HHRW group were statistically significant（P<0.05）。The results of HE staining showed that the number of abnormal nerve cells and ruptured and lyzed nuclei in the IR+HHRW group were significantly reduced compared with the IR group,and the degree of damage was mild.The results of the oxidative stress index showed that the ability of IR group to inhibit free radicals decreased,the level of malondialdehyde（MDA）increased（P<0.01）,and the MDA level decreased（P<0.05）after LHRW intervention.SOD activity was elevated after HHRW intervention（P<0.05）.The results of TUNEL staining showed that the apoptosis signal in the IR+HHRW group was sparser than that in the IR group.The real-time PCR results showed that compared with the IR group,the m RNA expression levels of PI3K and AKT in the IR+HHRW group and the IR+LHRW group increased（P<0.05）,while the m RNA expression levels of Cytc and Caspase-9 decreased（P<0.05）.The Western blotting results showed that compared with the IR group,the expression of PI3K and p AKT proteins in the IR+HHRW group and the IR+LHRW group tended to increase,among which the PI3K changes in the IR+HHRW group were statistically significant（P<0.05）,while the expression of Caspase-9 and Cytc proteins decreased（P<0.05）.2、Within 30 days after radiation exposure,hair loss occurred on the top of the head of the irradiated rats,and the IR group showed the most obvious hair loss,and the IR+HHRW group showed the least hair loss.Two weeks after irradiation,the irradiated rats experienced reduced food intake and body weight,during which the rats in the IR+HHRW group had lower food intake and weight loss than those in the IR group and the IR+LHRW group.In the third week,the food intake and body weight of the rats in each group began to recover one after another,and the rats in the IR+HHRW group recovered faster.The hematological test showed that BRC and HGB of IR group rats re decreased after radiation irradiation,and RBC and HGB levels of IR+HHRW group rats were increased compared with IR group（P<0.01）.The results of the new object recognition（NOR）experiment showed that compared with IR group,IR+HHRW group significantly increased the preference index for new objects（P<0.05）.Morris water maze experiment showed that compared with IR group,the escape latency of IR+LHRW group and IR+HHRW group was significantly shortened,and on day 5,IR+HHRW group had a significant difference（P<0.01）;On day 6,compared with Control group,IR group rats took significantly longer time to reach the platform for the first time（P<0.01）;Compared with IR group,IR+HHRW group took significantly shorter time to reach the original platform（P<0.05）,The Times of crossing the platform and the residence time in the original platform quadrant were prolonged（P<0.05）.HE staining and TUNEL staining showed that the apoptosis of hippocampal neurons in IR group was more serious than that in Control group,and the apoptosis of hippocampal neurons in IR+HHRW group was less severe than that in IR group.Free radical detection of brain tissue showed that ROS,MDA and IL-6 levels were significantly decreased in IR+HHRW group compared with IR group（P<0.05）;SOD and GSH increased significantly（P<0.01,P<0.001）.PCR results:Compared with IR group,the expressions of PI3K and AKT in IR+HHRW group were significantly increased（P<0.05）,the apoptotic factors Caspase-9 and Cytc were significantly decreased（P<0.05）.Western-Blot results:Compared with IR group,the expressions of PI3K and p AKT in IR+HHRW group were significantly increased,and the apoptotic factors Caspase-9 and Cytc were significantly decreased（P<0.05）.Conclusions:1、Hydrogen-rich water intervention can significantly reduce inflammation and oxidative stress caused by acute radiation brain injury,and reduce apoptosis of nerve cells,and the mechanism may be related to the reguthe lation of PI3K/AKT/Caspase-9 signaling pathway.2、Hydrogen-rich water improves chronic radiation brain injury in rats caused by ionizing radiation through anti-inflammatory and antioxidant and modulates the PI3K/AKT/Caspase-9 pathway,and there is a certain dose-effect relationship. |
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