葛根素基于内质网蛋白核心/信号分子/C/EBP同源蛋白通路对脂多糖诱导成骨细胞骨保护素及核因子-κB受体mRNA表达的影响

Study on effects of puerarin on expressions of OPG and RANKL mRNAs in lipopolysaccharide-induced osteoblasts based on GRP78/PERK/CHOP pathway

目的探讨基于内质网蛋白核心/信号分子/C/EBP同源蛋白(GRP78/PERK/CHOP)通路研究葛根素对脂多糖(LPS)诱导成骨细胞骨保护素(OPG)和核因子-κB受体(RANKL)mRNA表达的影响。方法体外培养小鼠胚胎成骨细胞前体细胞MC3T3-E1,采用随机数字表法分为对照组、模型组、4-苯基丁酸钠盐(内质网应激抑制剂,4-PBA)组、葛根素组、葛根素+4-PBA组,除对照组,其余各组以LPS处理MC3T3-E1细胞建立成骨细胞炎症模型,以茜素红染色、碱性磷酸酶(ALP)试剂盒分别检测各组细胞矿化结节相对比例、ALP活性,判定细胞成骨分化情况;以酶联免疫吸附(ELISA)试剂盒检测各组细胞培养基上清液中肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)水平;以实时荧光定量(qRT-PCR)检测各组细胞成骨相关基因(Runx2)、骨桥蛋白(OPN)、骨钙素(OCN)mRNA水平及OPG、RANKL mRNA水平;以免疫印迹检测细胞GRP78、PERK、CHOP蛋白表达。结果与对照组相比,模型组MC3T3-E1细胞矿化结节相对比例[(8.23±1.18)%比(100.00±0.00)%]、ALP活性[(0.49±0.12)U/mgprot比(1.57±0.35)U/mgprot]、细胞Runx2[(0.41±0.05)比(1.01±0.13)]、OPN[(0.39±0.04)比(1.02±0.15)]、OCN[(0.40±0.03)比(0.99±0.14)]及OPG mRNA[(0.41±0.11)比(1.03±0.17)]水平明显降低(P<0.05),细胞培养基上清液中TNF-α[(602.43±20.01)pg/mL比(381.86±18.16)pg/mL]及IL-6水平[(483.63±16.61)pg/mL比(280.15±11.18)pg/mL]、细胞RANKL mRNA[(3.03±0.62)比(0.99±0.13)]水平及GRP78[(1.31±0.24)比(0.19±0.05)]、PERK[(1.23±0.27)比(0.33±0.08)]、CHOP蛋白[(1.21±0.28)比(0.31±0.07)]表达明显升高(P<0.05)。与模型组相比,4-PBA组、葛根素组、葛根素+4-PBA组MC3T3-E1细胞矿化结节相对比例、ALP活性、细胞Runx2、OPN、OCN及OPG mRNA水平均升高(P<0.05),细胞培养基上清液中TNF-α及IL-6水平、细胞RANKL mRNA水平及GRP78、PERK、CHOP蛋白表达均降低(P<0.05)。与4-PBA组、葛根素组分别相比,葛根素+4-PBA组MC3T3-E1细胞矿化结节相对比例、ALP活性、细胞Runx2、OPN、OCN及OPG mRNA水平均升高(P<0.05),细胞培养基上清液中TNF-α及IL-6水平、细胞RANKL mRNA水平及GRP78、PERK、CHOP蛋白表达均降低(P<0.05),4-PBA组与葛根素组上述各项指标比较均差异无统计学意义(P>0.05)。结论葛根素可上调OPG mRNA水平,下调RANKL mRNA水平,降低LPS诱导的成骨细胞炎症反应,拮抗LPS对成骨细胞成骨分化的抑制作用,可能是通过下调GRP78/PERK/CHOP通路实现的。

Objective To investigate the effects of Puerarin on the expressions of OPG and RANKL mRNAs in osteoblasts induced by lipopolysaccharide(LPS)based on GRP78/PERK/CHOP pathway.Methods MC3T3-E1 cells,the mouse embryonic osteoblast precursor cells,were cultured in vitro and randomly divided into control group,model group,4-PBA group,puerarin group and puerarin+4-PBA group.Except control group,other groups were treated with LPS to establish osteoblasts inflammation model.Alizarin red staining and alkaline phosphatase(ALP)kit were used to detect the relative proportion of mineralized nodules and ALP activity in each group.The differentiation of osteoblasts was determined,the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA),quantitative real-time fluorescence(qRTPCR)was used to detect the expressions of osteogenesis-related genes Runx2,OPN,OCN mRNAs and OPG,RANKL mRNAs in each group,and the expressions of GRP78,PERK and CHOP proteins were detected by Western blotting.ResultsCompared with the control group,the relative proportion of mineralized nodules of MC3T3-E1 cells[(8.23±1.18)%vs.(100.00±0.00)%],ALP activity[(0.49±0.12)U/mgprot vs.(1.57±0.35)U/mgprot],Runx2[(0.41±0.05)vs.(1.01±0.13)],OPN[(0.39±0.04)vs.(1.02±0.15)],OCN[(0.40±0.03)vs.(0.99±0.14)]and OPG mRNAs[(0.41±0.11)vs.(1.03±0.17)]levels in the model group were significantly lower(P<0.05),while the levels of TNF-α[(602.43±20.01)pg/mL vs.(381.86±18.16)pg/mL]and IL-6[(483.63±16.61)pg/mL vs.(280.15±11.18)pg/mL],RANKL mRNA[(3.03±0.62)vs.(0.99±0.13)]and GRP78[(1.31±0.24)vs.(0.19±0.05)],PERK[(1.23±0.27)vs.(0.33±0.08)]and CHOP protein[(1.21±0.28)vs.(0.31±0.07)]in the supernatant of cell culture medium were significantly increased(P<0.05).Compared with the model group,the relative proportion of mineralized nodules of MC3T3-E1 cells,ALP activity,Runx2,OPN,OCN and OPG mRNAs levels in 4-PBA group,puerarin group and puerarin+4-PBA group increased(P<0.05),while the levels of TNF-αand IL-6,RANKL mRNA and GRP78,PERK and CHOP protein in supernatant of cell culture medium were all decreased(P<0.05).Compared with 4-PBA group and puerarin group,the relative proportion of mineralized nodules of MC3T3-E1 cells,ALP activity,Runx2,OPN,OCN and OPG mRNAs levels in puerarin+4-PBA group increased(P<0.05),while the levels of TNF-αand IL-6,RANKL mRNA and GRP78,PERK and CHOP protein in supernatant of cell culture medium were all decreased(P<0.05).There was no significant difference in the above indexes between 4-PBA group and puerarin group(P>0.05).Conclusion Puerarin can up-regulate OPG mRNA level,down-regulate RANKL mRNA level,reduce LPS-induced osteoblast inflammation,antagonize the inhibitory effect of LPS on osteogenic differentiation of osteoblasts,which may be achieved by down-regulating GRP78/PERK/CHOP pathway.