The Immunomodulatory Mechanism Of Brain Injury Induced By Homocysteine In H Type Hypertensive Rats

Objective:(1)To measure the body weight、SBP and plasma Hcy level of WKY and SHR at different time points by diverse ways of interventions;(2)To investigate plasma cytokines, m RNA/protein expression and histopathological change of brain in WYK and SHR;(3)To investigate the Th17 and Tregs cells within the peripheral blood in WKY and SHR;(4)To investigate the brain change by using MRI scan. Methods:(1) 60 WKY and 60 SHR were randomly assigned into WKY-C/M/T and SHR-C/M/T, given common food 16 weeks, 2% methionine(2mg/kg/d)16 weeks,2% methionine(2mg/kg/d) 8weeks and Vit B12 12mg/kg/d, folate 4mg/kg/d, Vit B6 0.09mg/kg/d 8 weeks respectively, measured the body weight、SBP and plasma Hcy level of WKY and SHR at 0, 4, 8,12, 16 weeks after interventions;(2)Using ELISA to measure the levels of plasma cytokines(IL-17,IL-10,TGF-β,IL-6);using QT-PCR method to investigate the m RNA expression of RORγt,IL-17,Foxp3 and IL-10 gene; using western blot to value the expression of Foxp3 and RORγt protein; and finally observing the histopathological change of brain in WYK and SHR;(3)Flow cytometry was used to detect the Th17 and Tregs cells within the peripheral blood in WKY and SHR at different time points;(4)By using high field strength MRI machine(7.0 T) on live animals to find the ischemic change in the brain of WKY-C/M/T and SHR-C/M/T groups at the time of 24 weeks old.Result:(1)The body weight of 6 groups were on the rise with WKY-C most obviously. SBP and plasma Hcy level of SHR-C/M/T were higher the WKY-C/M/T respectively, level of SBP and plasma Hcy were higher in M/T groups compared to the C group no matter in WKY or SHR, after intervened with Vit B12,Vit B6 and folate for 8 weeks, the level of SBP and plasma Hcy were down regulated in the SHR-T and WKY-T compared to the other 4 groups;(2)The plasma level of IL-6 in WKY remained stable during the 16 weeks and there were no significant difference within the WKY-C/M/T groups, the plasma level of IL-6 in SHR-C were raising during the 16 weeks, but kept falling in SHR-T, at the time of 16 weeks, the IL-6 was much higher than in the SHR-C/M. The plasma level of IL-17 in WKY-M kept raising during the 16 week and SHR-C/M had the same tendency, at the first 12 weeks in SHR-T the level of IL-17 were raising and declining in the next 4 weeks. The level of IL-10 in WKY-C/SHR-C remained stable during the 16 week, WKY-M and SHR-M declined, but in WKY-T/SHR-T at the first 12 weeks in SHR-T the level of IL-17 were raising and declining in the next 4 weeks. The level of TGF-β in WKY-C/SHR-C remained stable during the 16 weeks, in WKY-M/SHR-M it performed declining trend, and the M groups had the same trend with IL-10 during the experiment procedure. The WKY-C/SHR-C RORγt m RNA level kept stable during the 16 weeks, while in WKY-M/SHR-M kept declining, and in WKY-M/SHR-M it showed the raising trend; the WKY-C/SHR-C IL-17 m RNA level kept stable during the 16 weeks, while in WKY-M/SHR-M kept raising, and in the T groups it showed the declining trend, just like the phenomenon in the expression of RORγt and the opponent trend in IL-10 and Foxp3 m RNA expression. After the observation of histopathological change in the brain, we found Th17+ cell in WKY-M were much higher than in WKY-C, and after intervention of folate, Vit B6, Vit B12 the positive cells in T group reduced, and we got opposite result with Treg cells, and we got the same phenomenon in the SHR-C/M/T as in WKY-C/M/T. We also found vascular proliferation slightly in the M groups, neuron increased in SHR-M but declined in WKY-M;(3)The Tregs cells in WKY-C and SHR-C had the trend of dropping during the 16 weeks of intervention while WKY-T and SHR-T showed the trend of raising, but the Th17 had the opposite trend in WKY-C, SHR-C and WKY-T, SHR-T compared to the trend mentioned above. In the groups of WKY-M and SHR-M, the Tregs reduced in the first 8 weeks while raised right after intervention by the Vit B6, Vti B12 and folate in the following 8 weeks, and the trend of Th17 showed the opponent phenomenon;(4)No obviously ischemic phenomenon were found in the 6 groups.Conclusion:(1)Methionine can raise the the level of SBP and plasma Hcy of WKY and SHR, and taking Vit B12,Vit B6 and folate can an effective way for regulating SBP and plasma Hcy level;(2)The pathogenic affection of Hcy was proofed in different ways such as in m RNA level, protein level, plasma levels and tissue level, after given the folate, Vit B6 and Vit B12 its pathogenic effect was decreased;(3)Methionine can up-regulate the ratio of Th17 cells in WKY and SHR, and taking Vit B12,Vit B6 and folate can be an effective way for down-regulate the Th17 cells after intervention, while Tregs cells showed the opposite trends mentioned above in the 6 groups;(4)No obviously ischemic phenomenon were found in the 6 groups regarding on the species, age, and the ways of establishing the models.