Protective Effects Of Basic Fibroblast Growth Factor(BFGF) Against Gentamicin-Induced Injury On The Cells Of Hippocampus Tissue

Objective:The present study aims to establishing the gentamicin-induced damage model of the cells in hippocampus tissue and observing the differences of damage with different concentrations of gentamicin in the cells of hippocampus tissue.To discuss the antagonistic effect of bFGF on the damage of the cells in hippocampal tissue and clarify the treatment mechanism of bFGF on the damage of the cells in hippocampus tissue at the same time. It is expected that the findings could provide certain theoretical basis for clinical application of FGFs to treat the nervous tissue which is damaged by aminoglycoside antibiotics.PartⅠ.The preventive role of bFGF on the cells of hippocampus tissue damaged by gentamicin.Objective: To observe the characteristic of damage and preventive effect on the cells of hippocampus tissue induced by gentamicin.Methods: The cells of hippocampal tissue being cultured for seven days were divided into five groups randomly. The cells of every group were incubated with DMEM-F12 culture medium containing GM respectively and the final concentrations were followed by 0 g/L,0.4 g/L,0.8 g/L,1.2 g/L ,1.6g/L.Then were cultured for 24 hours placed in 5% CO2, 37℃incubator ,and measured the livability of the cells in hippocampal tissue by MTT . Besides,the group containing 0.8 g/LGM in culture medium was added culture medium containing 2.0μg/L bFGF at the same time,and compared the difference of cell morphology in the group with bFGF and not.Results: According to the resuits of the MTT method, The A490nm average values were 0.65±0.04, 0.55±0.04, 0.46±0.06, 0.29±0.05 in 0 g/L,0.4 g/L,0.8 g/L,1.2 g/L,1.6g/L gentamicin treated groups respectively. The A490nm average values of every treated groups were lower than the value (1.04±0.04) of the blank control group,there were significant difference (P<0.05). Moreover,the extent of damage was strengthened with the increase of concentration.The damage of the cells were obviously in the group with 0.8g/LGM in culture medium ,the processes of the cells were shorten than the normal group, The network between the processes was reduced and the sense of three-dimensional was not strong . there were multiple cells fusing into a mass, the black particles were increased within the cytoplasm of some cells,the cells appeared the phenomenon of senescence. and even some cells were suspended in culture medium; while in the group adding bFGF, the extent of damage in the cells was reduced and the outline of the cells was clear, the cell fusion was not obvious and the black aging particles were rarely seen in the cytoplasm of the cells.Conclusion:①the extent of damage in the cells of hippocampal tissue is associated with the concentration of gentamic in a dose-dependent relationship from 0.4 g/L to 1.6 g/L.②bFGF may have a preventive effect on the cells in hippocampal tissue damaged by GM.PartⅡNeuroprotective effect and the mechanisms of bFGF against gentamicin -induced injury on the cell of hippocampus tissueObjective: To study the effect of bGFF on gentamicin–induced injury in primary hippocampal neuron culture and explore the mechanism by measurements of cell viability (MTT),and content of NO, MDA and SOD .Methods: The cells cultured for 7 d were removed from their medium before treatment. After rinsed twice with PBS solution,the cells were randomly divided into 6 groups:⑴control group: only add 10% FBS containing DMEM-F12 medium;⑵experimental group: containing 10% FBS in DMEM-F12 culture medium add 0.8g/L GM;⑶Prevention group:bFGF2.0μg/L and 0.8g/L in DMEM-F12;⑷low concentration of bFGF group (treatment groupⅠ): 0.8g / L after 12 h treatment, add bFGF to make final concentration of 1.0μg/L;⑸bFGF concentration group (treatment groupⅡ): 0.8 g/L after 12 h treatment, add bFGF to make final concentration of 2.0μg/L;⑹high concentration of bFGF group (treatment groupⅢ): 0.8 g/L after 12 h treatment, add bFGF to make final concentration of 3.0μg/L. The cells incubated at 5%CO2 and 37℃for 24 h were used to measure the cell viability by MTT and the content of NO,MDA and SOD according to the methods of kits respectively.Results:⑴Cell viability was evaluated by MTT assay,and the absorbance was measured at 490 nm, which was called A490nm. A490nm value in model group was0.52±0.03, which was markedly lower in control group(0.98±0.07 P<0.01);A490nm value was 0.91±0.2 in bFGF 2.0μg/L group, which was markedly higher than that in model group(P<0.01); A490nm value was 0.73±0.04 in 3μg/L group,which was markedly higher than model group(P<0.01); A490nm value was 0.66±0.03in 2μg/L group,which was markedly higher than that in model group(P<0.01); A490nm value was 0.53±0.07 in bFGF 1μg/L group, which showed no statistical significance compared with model group(P>0.05); A490nm value in Prevention group 2.0μg/L group was 0.91±0.2, which was markedly higher than model group(P<0.01).⑵Content of MDA: The content of MDA in culture media was measured after the cell membrane being permeated by Triton X-100.The content of MDA was 5.48±0.32μmol/L in control group and 9.36±0.46μmol/L in model group. Obviously, the content of MDA in model group was higher than control group (P<0.01). The content of MDA was 6.78±0.42μmol/L in bFGF 2.0μg/L group and 6.72±0.24μmol/L in bFGF 3.0μg/L group, which were both significantly lower than model group (P<0.01).The content of MDA in bFGF 1.0μg/L group, 8.94±0.54μmol/L,showed no significant difference compared with model group (P>0.05).⑶Activity of SOD: Activity of SOD in cell culture media was measured after the cell membrane being permeated by Triton X-100. The activity of SOD in control group was 89.5±0.7 U/ml, which was 74.6±3.0 U/mL in model group(P<0.05). The activities of SOD in bFGF 1.0μg/L, 2.0μg/L and 3.0μg/L groups were 79.1±2.0 U/mL (P<0.05), 81.2±1.0 U/mL (P<0.01) and 81.3±1.0 U/mL (P<0.01), respectively.⑷Content of NO: The content of NO in control group was 16.1±0.3μmol/L,which increased to 21.4±2.2μmol/L in model group (P<0.01). The content of NO in Prevention group 2.0μg/L group was 12.6±0.5μmol/L, and those in bFGF 1.0μg/L, 2.0μg/L and 3.0μg/L groups were 17.3±1.7μmol/L, 13.0±1.2μmol/L and 16.8±1.2μmol/L,which all showed obvious difference compared with model group(P<0.05 or P<0.01).Conclusion: bFGF 1.0μg/L and 3.0μg/L could lighten gentamicin -induced injury of cultured hippocampal neurons. bFGF holds neuroprotective effect on gentamicin -induced injury in hippocampal neuron cultures,which was possibly due to the antagonism on lipid peroxidation.