| objective: 1.To study the effect of different concentrations of N-acetylcysteine(NAC)and Lipopolysaccharide(LPS)on the proliferation of MC3T3-E1 cells at different time points.2.To observe the effect of NAC and LPS on osteoblast morphology.3.To evaluate the effect of NAC on the mineralization of osteoblasts induced by LPS.4.To evaluate the effect of NAC on the expression of ALP and BGP in osteoblasts induced by LPS.5.To evaluate the effect of NAC on the expression of IL-6 and NF-?B in osteoblasts induced by LPS.Methods:1.Cells were stimulated with various concentrations of NAC(0.5mmol/L,1 mmol/L,2.5 mmol/L,5 mmol/L)and lipopolysaccharide(1 ?g/ml,5 ?g/ml,10 ?g/ml,20 ?g/ml)and then the proliferation of the cells was observed at different time points(24h 48 h and 72h)by CCK-8.2.Experiments were divided into four groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour)and then the cell morphology of MC3T3-E1 was observed with inverted phase contrast microscope observation after 24 hours.3.Experiments were divided into four groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour)and then the formation of mineralized nodules were observed after 30 days by Alizarin red staining.4.Experiments were divided into four groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour).ALP and BGP m RNA expression were determined using quantitative polymerase chain reaction(q PCR).5.Experiments were divided into four groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour).ALP and BGP protein synthesis were determined using enzyme-linked immunosorbent assay analyses(ELISA).6.Experiments were divided into six groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour).The BAY11-7082 and LPS(B+L;in this group MC3T3-E1 cells were treated with BAY11-7082,followed by LPS after one hour)and BAY11-7082(contained only BAY11-7082).IL-6 m RNA and NF-?B m RNA expression were determined using quantitative polymerase chain reaction(q PCR).7.Experiments were divided into six groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour).The BAY11-7082 and LPS(B+L;in this group MC3T3-E1 cells were treated with BAY11-7082,followed by LPS after one hour)and BAY11-7082(contained only BAY11-7082).IL-6 protein synthesis was determined using enzyme-linked immunosorbent assay analyses.8.Experiments were divided into six groups: control(without LPS and NAC),NAC(contained only NAC),LPS(contained only LPS),NAC and LPS(N+L;in this group MC3T3-E1 cells were treated with NAC,followed by LPS after one hour).The BAY11-7082 and LPS(B+L;in this group MC3T3-E1 cells were treated with BAY11-7082,followed by LPS after one hour)and BAY11-7082(contained only BAY11-7082).NF-?B protein synthesis was determined using Western blots analyses.Results:1.Comparison of the proliferation rate on treatment with NAC and LPS in MC3T3-E1 cells.1.1 The difference between the experimental group and the control group was statistically significant(P < 0.05).The proliferation rate increased following treatment with NAC concentrations from 0 to 1 mmol/L,reaching a peak at 1 mmol/L.The effect of NAC on the proliferation rate was attenuated at concentrations over 1 mmol/L.Considering that the maximum value of the proliferation rate for NAC regulated MC3T3-E1 cells was observed at 1mmol/L,1 mmol/L was considered to be the optimal NAC concentration for MC3T3-E1 cells.1.2 The difference between the experimental group and the control group was statistically significant(P < 0.05).Following treatment with LPS,the proliferation rate of MC3T3-E1 cells showed a similar trend.The proliferation rate increased as the LPS concentration was increased,reaching a peak and decreasing after a certain concentration.The peak in the proliferation rate at 24 h,48 h and 72 h occurred at the concentrations of 10?g/ml.A relative weak increase in the cell proliferation was observed at concentrations lower than10?g/ml,and an inhibition in the proliferation rate was observed at concentrations higher than 10?g/ml.Since the increase in the proliferation rate dropped at concentrations higher than 10?g/ml and the proliferation rate at10?g/m L was close to the corresponding peaks at 24 h,48 h and 72 h,and LPS concentration of 10 ?g/ml was considered as the optimal LPS stimulating concentration for MC3T3-E1 cells.2.cell morphological observation: In this experiment,the cell morphology of the control group,NAC group,LPS group and NAC+LPS group were basically consistent,and the cells showed spindle,polygonal and triangle,with protrusion and interconnection.3.alizarin red staining results: Each group have formed different degrees of mineralization nodules.Compares with the control group,NAC group significantly have more mineralized nodule formation.But LPS group mineralized nodules are the smallest in these groups.The area of NAC + LPS group mineralized nodules has almost no difference with the control group.4.Effects of NAC on ALP and BGP m RNA expression and protein expression in LPS-stimulated MC3T3-E14.1 Real-time fluorescent quantitative PCR results showed that LPS reduced ALP,BGP gene expression in MC3T3-E1 cells.NAC can promote MC3T3-E1 cells expressed ALP m RNA,BGP m RNA.The down-regulated gene expressions of ALP and BGP induced by 10?g/ml LPS were promoted significantly after treated with 1mmol/L NAC.4.2 results of ELISA showed that compared with the control group,LPS reduced the expression of ALP and BGP protein in MC3T3-E1 cells,and NAC increased the expression of ALP and BGP in MC3T3-E1 cells.Compared with LPS group,the down-regulated protein expressions of ALP and BGP induced by 10?g/ml LPS were promoted significantly after treated with 1mmol/L NAC.5.Effect of NAC on the expression of IL-6 and NF-?B m RNA in MC3T3-E1 cells induced by LPS.5.1 We examined the m RNA expression levels of IL-6 using the q PCR assay.There was a low level of IL-6 m RNA expression in untreated cells.Stimulation with 10?g/ml LPS induced a significant increase in the IL-6 m RNA levels(P < 0.05).Pretreatment with NAC(1 mmol/L)or BAY11-7082(10?mol/L)significantly decreased the level of LPS-induced IL-6 m RNA(P <0.05).5.2 We examined the m RNA expression levels of NF-?B using the q PCR assay.There was a low level of NF-?B m RNA expression in untreated cells.Stimulation with 10?g/ml LPS induced a significant increase in the NF-?B m RNA levels(P < 0.05).Pretreatment with NAC(1 mmol/L)or BAY11-7082(10 ?mol/L)significantly decreased the level of LPS-induced NF-?B m RNA(P< 0.05).6.Effects of NAC on IL-6 and NF-?B protein expression in LPS-stimulated MC3T3-E16.1 We measured the IL-6 protein expression using the ELISA.A similar characteristic was observed in the changes in the protein expression levels.LPS pretreatment significantly increased IL-6 expression in MC3T3-E1 at all time intervals(3,6 and 12h).NAC or BAY11-7082 pretreatment inhibited the increase in IL-6 protein expression.The IL-6 protein expression was significantly lower in the 1 mmol/L NAC treated group than the LPS group.Pretreatment with NAC(1 mmol/L)or BAY11-7082(10 ?mol/L)significantly inhibited the increase in IL-6 protein expression induced by LPS.6.2 To assess the role of NF-?B in the LPS-induced IL-6 expression and to test whether the effects of NAC on IL-6 expression were due to inhibition of the NF-?B pathway,we observed the effects of NAC and the NF-?B inhibitor(BAY11-7082)on LPS-induced NF-?B expression.LPS treatment significantly increased NF-?B activity in MC3T3-E1.Pretreatment with NAC(1 mmol/l)or BAY11-7082(10 ?mol/l)significantly inhibited the increase in NF-?B activity induced by LPS.Conclusion:1.The proliferation rate increased following treatment with NAC concentrations from 0 to 1 mmol/L,reaching a peak at 1 mmol/L.1 mmol/L was considered to be the optimal NAC concentration for MC3T3-E1 cells.A relative weak increase in the cell proliferation was observed at concentrations lower than 10 ?g/ml,and an inhibition in the proliferation rate was observed at concentrations higher than 10 ?g/ml.LPS concentration of 10 ?g/ml was considered as the optimal LPS stimulating concentration for MC3T3-E1 cells.2.NAC and LPS had no obvious effect on the morphology of osteoblasts.3.NAC can promote the formation of MC3T3-E1 cell mineralized nodules,while NAC can promote the mineralization of MC3T3-E1 cells under the action of LPS.4.Expressions of ALP and BGP induced by LPS were promoted significantly after treated with NAC.5.NAC could decrease the expression of IL-6 induced by LPS through the NF-?B signaling. |
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