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The Effect of Folic Acid on the Damage of Osteoblasts Induced by Homocysteine

Author: LiQiang
Tutor: TangXuLei
School: Lanzhou University
Course: Internal Medicine
Keywords: Folic acid Rats Osteoblast Proliferation Differentiation Homocysteine Oxidative damage
CLC: R965
Type: Master's thesis
Year: 2009
Downloads: 21
Quote: 0
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Abstract


Objective To investigate the protection of folic acid（FA） on osteoblasts（OB） by observing the effects of FA and homocysteine（Hcy） on the proliferation,differentiation and mineralization function of OB damaged by Hcy in vitro.Methods 1.Neonatal Wistar rat calvarium osteoblasts（OB） were isolated and digested with trypsin and collagenase repeatedly,which were then cultured and observed the morphologic character both in the light and phase-contrast microscopy.OB was identified by cytochemical staining of alkaline phosphatase（ALP） and the mineralization nodes were stained by different staining.2.The second passage of OB were placed in 96-well culture plates and incubated for 24h,then were randomly divided into three groups:the normal control group,various concentrations of folic acid(10-10～10-2 mol/L, FA) groups and homocysteine(1×10-6～5×10-3 mol/L,Hcy) groups.After several days cultured in different mediums,the following study were continued:The OD values on proliferation of OB（from 1 st to 4th days） and expression of ALP（from the 2nd to 8th days after induction and culture） in each group were determined by microplate reader.The mineral nodes of OB（when were induced-cultured two weeks） were dyed in alizarin red and counted.3.After second passage of OB being incubating for 24 hours and then treated with 10-3 mol/L Hcy for 24 hours,treated with different concentration of folic acid as mentioned earlier（Hcy+FA） for 48h,and then assayed by MTT,activities of ALP.The content of malondialdehyde（MDA）,the activity of superoxide dismutase（SOD） and the total antioxidative capability（T-AOC） were assayed in control,FA(10-5 mol/L),Hcy (10-3 mol/L),Hcy(10-3 mol/L) + FA(10-5 mol/L) groups.And the eNOS and iNOS mRNA express were assayed by reverse transcription polymerase chain reaction （RT-PCR） in control,FA(10-5 mol/L),Hcy(10-3 mol/L),Hcy(10-3 mol/L) + FA(10-5 mol/L) groups.Results 1.Compared with the control group,folic acid at the concentration of 10-6～10-4 mol/L increased the proliferation of OB（p＜0.01）,the significant concentration is 10-5 mol/L.At the concentration of 10-9～10-4mol/L increased the activity of ALP（p＜0.01） and the formation of mineralized nodules（p＜0.05）,especially in the concentration of 10-7mol/L.However,10-3～10-2 mol/L folic acid decreased the proliferation of OB and the activity of ALP and the formation of mineral nodes（p＜0.01）.2.When Hcy group at the concentration of 1×10-6,5×10-5 mol/L,the MTT values,ALP activity have no significant change than the control group.When the concentration of Hcy＞5×10-5 mol/L, the MTT values,ALP activity decreased significantly（p＜0.01）.3.Compared with the Hcy group in MTT value（p＜0.01） and ALP activity（p＜0.01）,we found markedly increase at the concentration(10-4～10-3mol/L) folic acid and the concentration(10-6～10-2mol/L) folic acid respectively in Hcy+FA group.The maximum effect was observed at 10-5 mol/L folic acid（p＜0.01）.4.Compared with the control group,the Hcy group showed significantly increase in the content of mMDA,but markedly decrease at the activity of SOD and T-AOC（p＜0.01）.Compared with the Hcy group,the mMDA significantly decrease（p＜0.01）,the activity of SOD（p＜0.01） and T-AOC（p＜0.01） were increased significantly in the Hcy+FA group.5.Compared with the control group,eNOS mRNA expression increased significantly in FA Group（p＜0.01）,decreased significantly in Hcy group（p＜0.01）.Compared with the Hcy group,the eNOS mRNA expression increased in the Hcy+FA group（p＞0.05）.The expression of iNOS mRNA has not significantly changed in all groups.Conclusion 1.Folic acid could pormote the proliferation,early differentiation and mineralization of neonatal rats’ calvarium osteoblasts in vitro.2.Hcy induced oxidative damage and inhibited the proliferation and the differentiation on OB.3.Folio acid could protect osteoblasts from Hcy-induced oxidative damage in vitro.

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