基于NF-κB信号通路研究加味阳和汤对破骨细胞活性的影响

Study on the Effect of Jiawei Yanghe Decoction on Osteoclast Activity Based on NF-κB Signaling Pathway

目的:研究加味阳和汤对破骨细胞活性的影响,并基于NF-κB信号通路探讨其作用机制。方法:制备加味阳和汤含药血清,用RAW264.7细胞诱导培养破骨细胞,CCK8法筛选出实验给药浓度,干预破骨细胞,收集细胞培养上清,检测并计算各组TRAP酶活力值,Western Blot法检测破骨细胞细胞质IκB-α和细胞核p65蛋白表达,计算各组蛋白灰度值,分析各组数值差异有无统计学意义。结果:CCK8结果显示5%、10%、20%浓度的加味阳和汤血清对RAW264.7细胞活性无明显影响(P>0.05)。细胞上清TRAP酶活力值显示:与诱导组比,加味阳和汤血清组酶活力值下降,20%浓度组下降更为显著,差异有统计学意义(P<0.01)。WB结果显示:与生理盐水组比,诱导组细胞核p65蛋白表达量明显增加;与诱导组比,含药血清组p65蛋白表达量下降,且含药血清浓度越高,p65蛋白表达量越低,差异有统计学意义(P<0.01);与生理盐水组相比,诱导组细胞质IκB-α蛋白表达量下降,与诱导组相比,含药血清组IκB-α蛋白表达量增高,差异有统计学意义(P<0.01)。结论:加味阳和汤可以抑制破骨细胞的活性,降低破骨细胞分泌的抗酒石酸酸性磷酸酶活性,其作用可能与其抑制NF-κB信号通路有关。

Objective:To study the effect of Jiawei Yanghe Decoction on osteoclast activity,and to explore its mechanism based on NF-κB signaling pathway.Methods:Prepare Jiawei Yanghe Decoction-containing serum,induce and culture osteoclasts with RAW264.7cells,screen out the experimental administration concentration by CCK8method,intervene with osteoclasts,Collect the cell culture supernatant,detect and calculate the TRAP enzyme activity value of each group.Western Blot method was used to detect the expression of osteoclast cytoplasmic IκB-αand nuclear p65 protein,calculate the gray value of each histone,and analyze whether the difference between the values of each group was statistically significant.Results:CCK8 results showed that the serum of Jiawei Yanghe Decoction at 5%,10%,and 20% concentration had no significant effect on the viability of RAW264.7 cells(P>0.05).The TRAP enzyme activity value of the cell supernatant showed that compared with the induction group,the enzyme activity value of the Jiawei Yanghe Decoction serum group decreased,and the decrease in the 20% concentration group was more significant,and the difference was statistically significant(P<0.01).WB results showed that:compared with the normal saline group,the expression of p65 protein in the nucleus of the induction group was significantly increased;compared with the induction group,the expression of p65 protein in the drug-containing serum group decreased,and the higher the concentration of drug-containing serum,the lower the expression of p65 protein.The difference was statistically significant(P<0.01);compared with the normal saline group,the cytoplasmic IκB-αprotein expression in the induction group decreased,and compared with the induction group,the drug-containing serum group IκB-αprotein expression increased.The difference was statistically significant(P<0.01).Conclusion:Jiawei Yanghe Decoction can inhibitthe activity of osteoclasts and reduce the activity of anti-tartrate acid phosphatase secreted by osteoclasts.Its effect may be related to its inhibition of NF-κB signaling pathway.