| Objective:(1) To study the effects of aluminum on mi R29 a, mi R29a*, mi R29b1, mi R29b2, mi R29c1, mi R29c2, BACEm RNA, BACE, amyloid-β(Aβ)1-40 and Aβ1-42 in rats in vivo.(2) To calrify the effect of mi R29 during the deposition of via BACE induced by aluminum in vitro.(3) To explore the effect mechenism upstream of mi R29 which regulated the deposition of Aβthrough BACE experiments in vivo and in vitro.Methods:(1) Forty healthy, male adult Sprague-Dawley(SD) rats were randomly assigned to four groups:control groups with saline treatment and aluminum-maltolate( Al(mal)3) groups with 15μmol/kg BW(low-dose group), 30μmol/kg B(middle-dose group), and45μmol/kg BW(high-dose group). Saline and Al(mal)3were administered to the rats via intraperitoneal injections for eight weeks. After exposure, the rats were anaesthetised, perfused and sacrificed. The brains were rapidly removed from the skulls and dissected into different regions. Aluminium levels in rat blood and brains were detected using graphite furnace atomic absorption spectrometry following that the serum, cortical(Co) and hippocampal(Hi) tissues of the rats were digested using a microwave.Aβ1-40, Aβ1-42 were detected with ELISA.mi R29 a, mi R29a*, mi R29b1, mi R29b2, mi R29c1, mi R29c2, BACEm RNAwere detemined with RT-PCR. BACE, NF-κB were analyzed with Western-blot. Then the relationship between BACE and mi R29 were analysised.(2) PC12-pheochromocytoma(PC12)were selected in vitro for mi R29 mimics tranferation experiments. PC12 were cultured and mi R29 a mimics or mi R29b1 mimics were transfered into PC12 using liposome transfection technology. The experimental group were divided into control group, 100μmol/l Al(mal)3), 200μmol/l Al(mal)3, 400μmol/lAl(mal)3, negative mimics transfection group and mi R29 a mimics or mi R29b1 mimics transfection + phosphate buffered saline(PBS), 100μmol/l Al(mal)3, 200μmol/l Al(mal)3and 400μmol/l Al(mal)3 group. PC12 and the cell medium were collected for further analysis after exposed to aluminum at 24 h. Cell viability was determined with CCK-8.BACEm RNA was detected with RT-PCR. BACE, Aβ1-40 and Aβ1-42 were analysed with ELISA.(3) PC12 was cultured for intervetion experiment. PC12 were divided into control group with PBS, intervetion control group with PDTC100μmol/L, exposed group with 200μmol/l Al(mal)3, andintervervetion group200μmol/l Al(mal)3+PDTC100μmol/l. PC12 and the cell medium were collected for further analysis after exposed to aluminum after 24 h. Cell viability was determined with CCK-8; mi R29 a, mi R29b1, BACEm RNA was detected with RT-PCR; BACE, Aβ1-40 and Aβ1-42 were analysed with ELISA.Results:(1) The results of experiments in vivo:I The serum aluminum levels in low-dose, middle-dose and high-high Al(mal)3-treated groups are(92.05±5.63)μg/l,(111.74±5.88)μg/l with(165.50±9.79) μg/l, respectively, which increased significantly compared with the control groups, that is(47.76±6.58) μg/l(P<0.05). The content of aluminium in rats cotex in low-dose, middle-dose and high-dose Al(mal)3-treated groups are(31.40±5.74) ng/g,(72.36±4.79) ng/g and(93.37±3.04) ng/g, respectively, which increased significantly compared with control groups, that is(12.11±1.42) ng/g(P<0.05). The content of aluminium in rats cotex in low-dose, middle-dose and high-high Al(mal)3-treated groups are(51.72±6.04) ng/g,(86.92±5.38) ng/g,(106.24±6.18) ng/g and(12.11±1.42) ng/g, respectively, which also increased significantly compared with control groups, that is(11.67±1.04) ng/g(P<0.05).II There was no signi?cant difference in the Aβ1-40 content between the Co and Hi of the Al(mal)3-treated rats and control rats(P>0.05).In contrast, a significant increase in Aβ1-42 and the total Aβ contents were observed in both Co and Hi(P<0.05), the content of Aβ1-42 significantly increased in middle-dose Al(mal)3-treated group and high-dose Al(mal)3-treated group in Co(P<0.05), and it increased significantly in all Al(mal)3-treated groups in Hi(P<0.05).The tolal content of Aβ increased in high-dose Al(mal)3-treated group in Co(P<0.05), and increased in middle-dose and high-dose Al(mal)3-treated group in Hi(P<0.05).III Compared with the control group, the expression of BACEmRNA, BACE increased in middle-dose and high-dose Al(mal)3-treated in Co(P<0.05), and increased in all Al(mal)3-treated groups in Hi(P<0.05).IV mi R29 a, mi R29a*, mi R29b1, mi Rb2, mi Rc1, mi Rc2 decreased in Al(mal)3-treated groups. mi R29a*, mi Rb2, mi Rc1, mi Rc2 all decreased in Co and Hi in all Al(mal)3-treated groups, compared to the control group(P<0.01). But there were not correclation between mi R29a*, mi Rb2, mi Rc1, mi Rc2 and BACEm RNA or BACE in Co( r=-0.638, 0.661, P>0.05.r=-0.635,-0.638, P>0.05.r=-0.616,-0.697, P>0.05.r=-0.637,-0.696, P>0.05), also there were not correclation between mi R29a*, mi Rb2, mi Rc1, mi Rc2 and BACEm RNA or BACE in Hi( r=-0.732,-0.717, P>0.05.r=-0.723,-0.717, P>0.05.r=-0.804,-0.779, P>0.05.r=-0.770,-0.753, P>0.05). The expression of mi R29 a, mi R29b1 increased in high-dose Al(mal)3-treatedgroup in both Co and Hi(P<0.05). And mi R29 a correclated with BACEm RNA and BACE in Co(r=-0.991,-0.987, P<0.05), mi R29b1 correclated with BACEm RNA and BACE in Co(r=-0.969,-0.992, P<0.05).The correclation between mi R29 a and BACEm RNA and BACE was abserved in Hi(r=-0.963,-0.981, P<0.05), also the correclation between mi R29b1 and BACEm RNA and BACE was abserved in Hi(r=-0.983,-0.991, P<0.05).V Transfer factor NF-κB/P65 increased in Al(mal)3-treated groups in Co and Hi. The expression of NF-κB/P65 increased in middle-dose Al(mal)3-treated group and high-dose Al(mal)3-treated group in Co and Hi(P<0.05).(2) The results of experiments in vitro which mi R29 mimics were tranfered into PC12:the level of mi R29 a mimics,mi R29b1 mimics were highest at 24 h which transferred into PC12, the levels of mi R29 a mimics,mi R29b1 mimics were 500 times than the negative control group. Then the levels of mi R29 a mimics,mi R29b1 mimics decreased gradually, and it were 100 times at 72 h. PC12 were exposed to PBS or Al(mal)3 at 24 h which transferred into PC12, then the following experiments were administed after 24 h when PC12 were exposed to PBS or Al(mal)3.I The morphological changes of cells: the size of cell was consistent, the quantity and junction were normal in the control group, negative control group and positive transfected control group(and PBS were exposed to PC12). A few dead cells, cell junction reduced in the low dose Al(mal)3-treated group that mi R29 a mimics, mi R29b1 mimics were transfected into cells.The number and junction of cells decreased, some cells shrunken in the middle dose Al(mal)3-treated group.The number of cells were reduced significantly,the cell junction reduced significantly, more cells shrunken in high dose Al(mal)3-treated group. The morphological changes of cells were more evident in groups which mi R29 a mimics or mi R29b1 mimics were not transfected into PC12 than which mi R29 a mimics or mi R29b1 mimics were transfected. The morphological changes of cells in Al(mal)3-treated groups were similar that cells were transfected by mi R29 a mimics or mi R29b1 mimics.II There were not difference in Cell viability amomg the control, negative mimics transfection group and the transfer control group which was mi R29 a mimics or mi R29b1 mimics transfection + PBS(P>0.05). Cell viability decreased with the raising of Al(mal)3 dose in the groups which have not tranfered the mi R29 a mimics or mi R29b1 mimics(P<0.05). Cell viability decreased in every groups which tranfered the mi R29 a mimics or mi R29b1 mimics and treated with Al(mal)3 and cell viability decreased significantly in 400μmol/l Al(mal)3 tranfered group(P<0.05). Cell viability significantly increased in 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 tranfered groups compared to 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 groups which mi R29 a mimics or mi R29b1 mimics were not transfered.III There were not difference in the expression of BACEmRNA and BACE amomg the control, negative mimics transfected group group(P>0.05).The expression of BACEm RNA and BACE with the raising of Al(mal)3 dose in the groups which have not tranfered the mi R29 a mimics or mi R29b1 mimics(P<0.05). The expression of BACEm RNA were similar in every groups which mi R29 a mimics or mi R29b1 mimics were transfered into PC12.The expression of BACE decreased in the transfer control and in low-dose Al(mal)3-treated group which mi R29 a mimics or mi R29b1 mimics were transfered into PC12(P<0.05).There were not significant differece in BACE among200μmol/l Al(mal)3group, 400μmol/l Al(mal)3and the transfer control(P>0.05). The expression of BACEm RNA and BACE decreased in 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 tranfered groups compared to the same dose group which mi R29 a mimics or mi R29b1 mimics were not transfered.IV There were notsignificantly differece in the content of total Aβ between the control, negative mimics transfection group(P>0.05), the content of total Aβ increased with the raising of Al(mal)3 dose in the groups which have not tranfered the mi R29 a mimics or mi R29b1 mimics(P<0.05). the content of total Aβ decreased in the transfer control group and 100μmol/l Al(mal)3 tranfered group(P<0.05). There were not significant differece in the content of total Aβ among200μmol/l Al(mal)3group, 400μmol/l Al(mal)3and the transfer control groups(P>0.05).The content of total Aβ decreased in 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 tranfered group compared to the same dose group which mi R29 a mimics or mi R29b1 mimics were not transfered.There were notsignificant differece in the content of Aβ1-40 between the control and negative mimics transfection group(P>0.05).The content of Aβ1-40 increased in the groups of 200μmol/l Al(mal)3 and 400μmol/l Al(mal)3 which have not tranfered the mi R29 a mimics or mi R29b1 mimics(P<0.05). the content of Aβ1-40 decreased in the transfer control group and 100μmol/l Al(mal)3 tranfered group(P<0.05). There were not significant differece in the content of Aβ1-40 among200μmol/l Al(mal)3group, 400μmol/l Al(mal)3and the transfer control(P>0.05).The content of Aβ1-40 decreased in 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 tranfered group compared to the same dose group which mi R29 a mimics or mi R29b1 mimics were not transfered(P<0.05).There were notsignificant differece in the content of Aβ1-42 betweem the control and negative mimics transfection group(P>0.05), the content of Aβ1-42 decreased in the transfer control group(P<0.05).The content of Aβ1-42 increased in every groups exposed to Al(mal)3 which have not tranfered the mi R29 a mimics or mi R29b1 mimics(P<0.05). The content of Aβ1-42 decreased in the transfer control group and 100μmol/l Al(mal)3 tranfered group(P<0.05). There were not significantly differece in the content of total Aβ1-42 among200μmol/l Al(mal)3group, 400μmol/l Al(mal)3and the transfer control(P>0.05).The content of Aβ1-42 decreased in 100μmol/l Al(mal)3, 200μmol/l Al(mal)3, and 400μmol/l Al(mal)3 tranfered groups compared to the same dose group which mi R29 a mimics or mi R29b1 mimics were not transfered.(3) The results of intervetion experiment:I The morphological changes of cells: the size of cell was consistent, the quantity and junction of cells, were normal, the protuberances of cells were plentiful in the control group andthe group of 100μmol/l PDTC.But some cells were dead, the number of cells decreased, cell junction reduced and some cells shrunken in 200μmol/l Al(mal)3. the number and junction of cells increases,while the protuberances of cells increased in 200μmol/l Al(mal)3+100μmol/l PDTC group.II There were not difference in Cell viabilitybetween control group and 100μmol/L PDTC group. Cell viability significantly decreased in 200μmol/l Al(mal)3 group(P<0.05),butcontrodictarly, increased in 200μmol/l Al(mal)3+PDTC100μmol/l group compared to 200μmol/l Al(mal)3 group(P<0.05).(3) The expression of mi R29 a or mi R29 b increased significantly in 100μmol/L PDTC group(P<0.05, but in 200μmol/l Al(mal)3 group decreased significantly(P<0.05), and increased in 200μmol/l Al(mal)3+PDTC100μmol/l group compared to 200μmol/l Al(mal)3 group(P<0.05).III There were not difference between control group and100μmol/L PDTC group The expression of BACEm RNA, BACE in 200μmol/l Al(mal)3 group increased significantly(P<0.05),but not significantly different with that in 200μmol/l Al(mal)3+PDTC100μmol/l group which showed a little higher expression level(P>0.05). BACEdecreased in Al(mal)3+PDTC100μmol/l group than that in 200μmol/l Al(mal)3 group.(P<0.05).IV The content of total Aβ decreased in 100μmol/l PDTC group(P<0.05), and it increased significantly in 200μmol/l Al(mal)3group(P<0.05), and it decreased in 200μmol/l Al(mal)3+PDTC100μmol/l group compared to 200μmol/l Al(mal)3 group(P<0.05).The decreasing of the content of Aβ1-40 did not have statistical significance in PDTC100μmol/l group(P>0.05, butincreased significantly in 200μmol/l Al(mal)3group(P<0.05). The content of Aβ1-40 decreased in 200μmol/l Al(mal)3+PDTC100μmol/l group compared to 200μmol/l Al(mal)3 group(P<0.05). The content of Aβ1-42 decreased in 100μmol/l PDTC group(P<0.05), but increased significantly in 200μmol/l Al(mal)3group(P<0.05). While decreased again in 200μmol/l Al(mal)3+PDTC100μmol/l group compared to 200μmol/l Al(mal)3 group(P<0.05)Conclusions:Al(mal)3increases the serum aluminum levels and the content of aluminium in rats Co and Hi, induces the deposition of Aβ1-42 in rats Co and Hi,which are regulated by mi R29 a, mi R29b1 via BACEm RNA and BACE,and regulates mi R29 through effecting the expression of NF-κB. |
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