| Objective:To investigate the role of trimethyl histone H3 lysine 4(H3K4me3) and MLL in aluminum induced cognitive impairment in vivo and in vitro. Methods:In vivo study: 160 specific pathogen free healthy male SD rats were randomly divided into 4 batches of 16 groups by body weight with 10 rats in each group. Each batch consist of a control group(0 mg/kg Al3+), low-(2 mg/kg Al3+), medium-(12 mg/kg Al3+) and high-dose(72 mg/kg Al3+) exposure groups. Each group was treated with its designated dose of Al3+ in drinking water for designated time. The four batches of rats were exposed to aluminum for 90,180,270 and 360 days, respectively. After the exposure, Open field test and Morris water maze test were performed to assess the autonomous activities, exploration activities and spatial learning and memory abilities of the rats. MLL activity of hippocampus was detected using histone methyltransferase activity/inhibition assay kit. Expression of H3K4me3 of hippocampus was determined using enzyme-linked immunosorbent assay.In vitro study: The same batches of SH-SY5 Y cells which were in the logarithmic growth phase were divided into blank control group,1mM Al3+, 2mM Al3+ and 4mM Al3+ exposure groups. The control group was not given any intervention, other groups were treated with designated doses of Al3+, respectively. The treated SH-SY5 Y cells were cultured for 48 h. Cell viability was determined using CCK-8 assay, and cell apoptosis was evaluated by flow cytometry(FCM). The MLL activity was detected using histone methyltransferase activity/inhibition assay kit, the relative expression of MLL was measured by qRT–PCR, and the relative expression of MLL protein was determined by immunofluoresence. The expression of H3K4me3 of hippocampus was determined using enzyme-linked immunosorbent assay. Results:1.In vivo studyThe results of Open field test:(1) There was a significant interaction between aluminum exposure dose and exposure time for central quadrant residence time(P﹤0.05), and it was prolonged with the increasing of aluminum exposure dose at 270 th and 360 th day(P﹤0.05), central quadrant residence time of each group was increased with the exposure time extension(P﹤0.05), analyzed by multiple linear regression analysis procedure, the regression equation was Y=-10.211+0.234X1+4.558X2(X1:aluminum exposure dose, X2:aluminum exposure time)(P﹤0.01).(2) There was non-significant interaction between aluminum exposure dose and exposure time for standing number(P﹥0.05), it tended to decrease with the increasing of aluminum exposure dose and time, the regression equation were Y=21.525-0.095X1, Y=35.004-2.145X2(X1:aluminum exposure dose, X2:aluminum exposure time), which were analyzed by general linear regression analysis(P1﹤0.01, P2﹤0.01).(3) Modification number of rats had no interaction between aluminum exposure dose and time(P﹥0.05), and it tended to decrease with the increasing of aluminum exposure dose and time, Y=-10.899-0.053X1, Y=-18.105-1.154X2(X1:aluminum exposure dose, X2:aluminum exposure time) were the regression equation, which analyzed by general linear regression(P1﹤0.01, P2﹤0.01).The results of Morris water maze test:(1) There was no significant interaction of aluminum exposure dose and time for the escape latency(P﹥0.05), it tended to increase with the increasing of aluminum exposure dose and time, analyzed by general linear regression analysis procedure, the regression equation were Y=-35.464+0.148X1, Y=-26.075+1.738X2(X1:aluminum exposure dose, X2:aluminum exposure time)(P1 ﹤0.01,P2﹤0.01).(2) Target quadrant residence time had a significant interaction between aluminum exposure dose and exposure time(P﹤0.05), target quadrant residence time of each exposure period was decreased with the increasing of aluminum exposure dose(P﹤0.05), and it was also decreased with the exposure time extention in low-, medium-, and high-dose groups(P﹤0.05), the regression equation was Y=25.002-0.098X1-0.723X2(X1:aluminum exposure dose, X2:aluminum exposure time), which analyzed by multiple linear regression analysis procedure(P﹤0.01).(3) There was no significant interaction of aluminum exposure dose and exposure time for passing through the original platform site times(P﹥0.05), and it tended to decrease with the increasing of aluminum exposure dose and exposure time, analyzed by general linear regression analysis procedure, the regression equation were Y=-4.134-0.023X1, Y=-4.583-0.130X2(X1:aluminum exposure dose, X2:aluminum exposure time)(P1﹤0.01, P2﹤0.01).The results of MLL enzyme activity of hippocampus: There was no interaction between aluminum exposure dose and exposure time for MLL enzyme activity index(P﹥0.05), it tended to decrease with the increasing of aluminum exposure dose and exposure time, analyzed by general linear regression analysis procedure, the regression equation were Y=-4.134-0.023X1, Y=-4.583-0.130X2(X1:aluminum exposure dose, X2:aluminum exposure time)(P1﹤0.01, P2﹤0.01).H3K4me3 expression in hippocampus was tested by ELISA, and it had a significant interaction between aluminum exposure dose and exposure time(P﹤0.01). H3K4me3 expression was decreased with the increasing of aluminum exposure dose at 270 th and 360 th day(P﹤0.05), and it was also decreased with the exposure time extention in low-, medium-, and high-dose groups(P﹤0.05), the regression equation was Y=29.376-0.095 X1-1.212X2(X1:aluminum exposure dose, X2:aluminum exposure time), which was analyzed by multiple linear regression(P﹤0.01).Correlation between cognitive ability indexs and MLL enzyme activity and the H3K4me3 expression: Central quadrant residence time and escape latency were negatively correlated with the decline of MLL enzyme activity(P﹤0.05), stand number, modification number and target quadrant residence time were positively correlated with the decline of MLL enzyme activity(P﹤0.05). The correlation between cognitive ability indexs and H3K4me3 was the same as cognitive ability indexs and MLL enzyme activity.2.In vitro studyCell viability investigated by CCK-8 assay: SH-SY5 Y cell viability tended to decline with the increasing of aluminum exposure dose(P﹤0.05).Apoptosis rate tested by FCM: With the increasing of aluminum exposure dose, apoptosis rate increased(P﹤0.05).MLL enzyme activity of SH-SY5 Y cell: MLL enzyme activity tended to decrease with the increasing of aluminum exposure dose(P﹤0.05), analyzed by general linear regression analysis, the regression equation was Y=-100.344-20.605X(X:aluminum exposure dose)(P﹤0.01).MLL relative expression measured by qRT-PCR, and showed no difference among four groups(P﹥0.05).MLL protein expression tested by immunofluoresence: MLL fluorescence intensity tended to decrease with the increasing of aluminum exposure dose(P﹤0.05), the regression equation:Y=-986.115-110.753X(X: aluminum exposure dose)(P﹤0.01).H3K4me3 protein expression measured by ELISA: H3K4me3 protein expression tended to decrease with the increasing of aluminum exposure dose(P﹤0.05), the regression equation:Y=59.575-7.089X(X: aluminum exposure dose)obtained by general linear regression analysis(P﹤0.01). Conclusions:1.Chronic aluminum exposure results in impairment of exploring, learning and memory ability in rats, exposure dose and time dependently.2.Aluminum can induced MLL enzyme activity decrease, exposure dose and time dependently, and it may affect the post-transcriptional modification of MLL.3.Aluminum enables to cause the decline of H3K4me3 protein level, exposure dose and time dependently.4.Cognitive dysfunction related to the decrease of MLL enzyme activity and H3K4me3 expression.In summary, aluminum may lead to cognitive impairment due to reduced H3K4me3 amount which caused by decreased MLL activity. |
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