Effects Of Different Concentrations Of D-valine On Proliferation And Apoptosis Of L929 Cells

Background and Objective:The peri-implant and peri-implant mucositis,as a common implant complication,has attracted great concern to clinicians.As we all know,the initiating factor of peri-implantitis is the accumulation of biofilm on the neck surface of implants.In recently studies,significant data showed that D-amino acids possessed great potential in controlling the infection of biofilms as it could directly or indirectly inhibit bacteria occurrence and destroy biofilms.This study was a part of a NSFC project of our team.In previous study,our experimental group have reported that D-valine at specific concentration(50-150mM)could effectively inhibit the formation of Porphyromonas gingivalis biofilm.However,the cytotoxicity of these concentrations has not been studied or been clarified.Therefore,it is of great significance to define its biological safety and screen the safe concentration range of D-valine,which may be used as a new preparation to treat peri-implantitis through inhibiting the formation of biofilm or/and dispersing the formed biofilm.Methods:1 Mice fibroblast L929 cells were cultured.The morphology and growth state of L929 cells were observed histologically,and the growth curve was drawn.2、The experimental groups were added with different concentrations of D-val,and the control group was added with the same volume of DMEM medium.The effects of different concentrations of D-valine on the proliferation of fibroblasts L929 were detected by CCK-8 on day 1,3 and 5 respectively.3、The apoptosis was measured through Flow Cytomete after Annexin V FITC/PI double staining at 5th day after D-val was added.4、Quantitative Real-time PCR was used to detect the relative expression of apoptosis-related genes Bax,Bcl-2 and Caspase-3.Using SPSS 17.0 statistical packages for statistical analysis.The data was expressed as the standard deviation of the mean number,and the t-test method was used to analyze the data.Results:1、CCK-8 proliferation test showed that the absorbance value of the experimental groups decreased compared with the contral group.With the increase of D-valine concentration,the decrease of absorbance value was more obvious.When D-valine concentrations were within the range of 100-150mM,the inhibitory effects on L929 cell proliferation were observed in all 1,3,and 5d respectively,and the results were statistically different(P<0.05).2、The result showed that the apoptosis rate of L929 cells cultured with D-valine was lower than that of contral group cells.3、The results of qRT-PCR showed that the expression of Bax gene in the 150,100,75,50mM groups on the first day was lower than that in the control group,and had statistical difference(P<0.05).On the 5th day,the expression of Bax gene in the experimental group was lower than that in the control group at 150,125,100 and 50mM groups,and the expression level of Bax gene in the experimental group was lower than that in the control group on the 5th day.The results showed statistical difference(P<0.05).On the first day,the expression of anti-apoptotic gene Bcl-2 in the 150mM group was slightly higher than that in the control group(P<0.05),while in the other experimental groups,the expression of Be in 125,100,75 and 50 mM groups were slightly higher than that in the control group.Compared with the control group,there was no significant difference between the two groups(P>0.05).On the 5th day,the expression of Bcl-2 in 150mM group was higher than that in control group(P<0.05).On the 1st and 5th day,the expression of caspase-3 in the 150mM group was lower than that in the control group,and the results were statistically significant.The Bcl-2/Bax ratio showed that the ratio of Bcl-2/Bax in the high concentration groups(150,125,100mM)were also higher than that in the control group on the 5th day.In the 50,75mM groups,the ratio were lower than that in the control group,but the results were not statistically significant(P>0.05).Conclusions:1、When the concentration of D-valine was≥100mM,the proliferation activity of mouse fibroblasts L929 was significantly inhibited.The safy conceration range of D-valine is 50-75mM.2、D-valine may play an anti-apoptotic role by upregulating the ratio of Bcl-2/Bax and decreasing the expression of Bax and caspase-3 apoptosis genes in L929 cells.