| Objective:To study the toxicity and differentiation effect of nicotine on osteoclast precursor RAW264.7 and osteoclasts,and to further explore the effect of nicotine on the differentiation of osteoclasts and the influence of MAPK(mitogen-activated protein kinase)signaling pathway to understand the molecular biological mechanism of nicotine on the differentiation process of osteoclasts..Methods:1.Induced differentiation of RAW264.7 cells:The mouse RAW264.7 cell line was induced to differentiate into osteoclasts at a concentration of 50ng/m L RANKL,and was identified as osteoclasts by TRAP staining.2.CCK-8 detection to determine the experimental concentration of nicotine:select different concentrations of nicotine to act on RAW264.7 cells.After a certain period of time,observe the difference in their morphology and the proliferation under the electron microscope.At the same time,CCK-8 measures the proliferation activity of the cells.Determine the appropriate nicotine concentration for subsequent experiments.3.The effect of nicotine on osteoclast differentiation of RAW264.7 cells:RNA was extracted from RAW264.7 cells under the action of different concentrations of nicotine with 50ng/m L RANKL were cultured for 6 days,and the expression of TRAP,CTSK,ITG?V and ITG?3 of each group were detected by q-PCR technology.4.The role of MAPK signaling pathway in the effect of nicotine on the osteoclast formation of RAW264.7 cells:RAW264.7 cells with different concentrations of nicotine with 50ng/ml RANKL were cultured for 6 days to extract proteins,and the expression of key proteins was detected by Western blot.Result:1.Osteoclasts appeared in mouse RAW264.7 cells treated with 50ng/m L's RANKL induction medium for four days,and reached the peak of osteoclast differentiation on the seventh day of culture.2.The nicotine culture media of 10-2,10-3,10-4,10-5mol/L were used to treat the mouse RAW264.7 cells.One day later,the cell activity was detected by CCK-8.The results showed that 10-2mol/L nicotine culture medium had obvious toxic effect on RAW264.7 cells because of its high concentration,but there was no significant difference among the other three groups.Then 10-3,10-4,10-5mol/L nicotine culture medium was selected to act on mouse RAW264.7 cells,and the cell activity was detected after 5 days.There was no significant difference among the three groups,so10-3,10-4,10-5mol/L nicotine culture medium was selected in the follow-up experiment.3.The control group was induced by 50ng/m L's RANKL,and the nicotine treated group was divided into three groups(nicotine solution of 10-3,10-4 and10-5mol/L+50ng/ml RANKL).On the 6th day of culture,the expression levels of CTSK,TRAP,ITG?V and ITG?3 were detected by q-PCR technique.The results showed that the expression of CTSK decreased with the increase of nicotine concentration.The nicotine mixture of 10-3mol/L and 10-4mol/L was significantly different from the control group(P<0.05).However,the expression of TRAP,ITG?V and ITG?3 in 10-5mol/L nicotine mixture was higher than that in the control group,and the expression of ITG?V in 10-5mol/L nicotine mixture was different from the control group.Then the expression of TRAP,ITG?V and ITG?3 decreased gradually with the increase of nicotine concentration,and the expression of 10-3mol/L nicotine mixture was different from that of the control group(P<0.05).4.The control group was induced by 50ng/m L's RANKL,and the nicotine treatment group was divided into three groups(10-3,10-4,10-5mol/L nicotine solution+50ng/ml RANKL),).On the 6th day of culture,there was no significant difference in the expression of MAPK signal pathway proteins ERK1/2,p38 and JNK.Compared with the control group,the phosphorylation level of ERK1/2 and p38 in10-3mol/L nicotine mixture group was inhibited,while JNK was promoted compared with the control group(P<0.05).Conclusion:The change of nicotine concentration will affect the proliferation,differentiation,absorption,apoptosis and adhesion of osteoclasts.Nicotine concentration of10-5mol/L may promote the differentiation,absorption and adhesion of osteoclasts.High concentration of nicotine will have inhibitory effect.Through the detection of key proteins in the MAPK signaling pathway,it is found that p-p38 and p-erk decrease with the increase of nicotine concentration.High concentration of nicotine may inhibit the proliferation and differentiation of osteoclasts and promote their apoptosis.Nicotine may pass The MAPK signaling pathway affects the differentiation and apoptosis of osteoclasts. |
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