六味地黄方含药血清对H_(2)O_(2)诱导的氧化损伤MC3T3-E1细胞的干预作用

Intervention effect of medicated serum of Liuwei Dihuang Decoction on oxidative damaged MC3T3-E1 cells induced by H_(2)O_(2)

目的 观察六味地黄方含药血清对H_(2)O_(2)诱导的氧化损伤小鼠胚胎成骨细胞前体细胞(MC3T3-E1)增殖、分化、矿化的干预作用,并从无翅型MMTV整合位点家族成员/β-连环蛋白(Wnt/β-Catenin)、护骨素/核因子-κB受体活化因子配体(OPG/RANKL)通路及线粒体凋亡途径初步探讨其作用机制。方法 制作六味地黄方汤剂,对成年Wistar大鼠进行灌胃,获取六味地黄方含药血清。将MC3T3-E1细胞分为正常组、模型组、N-乙酰半胱氨酸(NAC)组、对照含药血清组、低剂量含药血清组、中剂量含药血清组及高剂量含药血清组。除正常组外,其余各组先用1.0 mmol/L过氧化氢(H_(2)O_(2))预处理MC3T3-E1细胞6 h,随后模型组更换为正常培养基,NAC组更换为含有2.5 mmol/L NAC的培养基,药物血清组更换为含有10%药物血清的培养基,各组均处理24 h,然后根据检测指标的不同给予进一步处理。采用CCK-8法检测培养24 h、48 h、72 h后MC3T3-E1细胞的增殖情况,诱导培养7 d后检测碱性磷酸酶(ALP)活性,21 d后通过茜素红染色检测矿化结节。采用RT-PCR检测以下通路中关键基因表达:Wnt/β-Catenin基因通路中Wnt2、低密度脂蛋白受体相关蛋白5(Lrp5)、β-Catenin mRNA表达;OPG/RANKL通路中OPG、RANKL mRNA表达,计算OPG/RANKL比值;线粒体凋亡途径中B细胞淋巴瘤2(Bcl-2),半胱氨酸天冬氨酸蛋白酶3(Caspase-3),半胱氨酸天冬氨酸蛋白酶9(Caspase-9)mRNA表达。结果 与正常组比较,模型组细胞增殖、ALP活性均降低(P<0.01),矿化结节数量减少;Wnt/β-Catenin基因通路中Wnt2、Lrp5、β-Catenin mRNA表达降低(P<0.01);在OPG/RANKL通路中OPG mRNA表达降低,RANKL mRNA表达升高,OPG/RANKL比值降低(P<0.01);在线粒体凋亡途径中,Bcl-2 mRNA表达降低,Caspase-9、Caspase3 mRNA表达升高(P<0.01)。与模型组比较,六味地黄方含药血清各剂量组在24 h、48 h、72 h均能促进MC3T3-E1细胞增殖(P<0.01),促进ALP活性(P<0.05,P<0.01),增加矿化结节数量。在Wnt/β-Catenin基因通路中,与模型组相比,除低剂量含药血清组外,中、高剂量含药血清组Wnt2 mRNA表达均高于模型组(P<0.01)。六味地黄方含药血清各剂量组Lrp5 mRNA表达均高于模型组(P<0.01),β-Catenin mRNA表达亦高于模型组(P<0.05,P<0.01)。在OPG/RANKL基因通路中,除低剂量含药血清组外,中、高剂量含药血清组OPG mRNA表达均高于模型组(P<0.05,P<0.01),RANKL表达均低于模型组(P<0.01),但含药血清各剂量组OPG/RANKL比值均高于模型组(P<0.01),并呈剂量依赖性升高。在线粒体凋亡途径中,含药血清各剂量组Bcl-2 mRNA表达均高于模型组(P<0.05,P<0.01),Caspase-9、Caspase-3 mRNA表达均低于模型组(P<0.01)。结论 六味地黄方含药血清可以降低H_(2)O_(2)对MC3T3-E1细胞的损伤,促进MC3T3-E1细胞的增殖、分化、矿化,其保护作用可能与调控Wnt/β-catenin信号通路、OPG/RANKL、线粒体凋亡途径中的关键靶点有关。

Objective To investigate the effect of medicated serum of Liuwei Dihuang Decoction on the proliferation,differentiation and mineralization of H_(2)O_(2)-induced oxidatively damaged MC3T3-E1cells,and to preliminarily explore its mechanism from the Wnt/β-Catenin,OPG/RANKL pathway,and mitochondrial apoptosis pathway.Methods The Liuwei Dihuang Decoction was prepared and given to the adult Wistar rats by intragastric administration,and then the medicated serum of Liuwei Dihuang Decoction was collected.The MC3T3-E1 cells were divided into normal group,model group,N-acetylcysteine(NAC)group,control medicated serum group,low-dose medicated serum group,medium-dose medicated serum group and high-dose medicated serum group.Except for the normal group,the other groups of MC3T3-E1 cells were first pretreated with 1.0 mmol/L H_(2)O_(2) for 6 h.Then the culture medium was replaced with normal culture medium in the model group,and was replaced with 2.5 mmol/L NAC in the NAC group and replaced by 10%medicated serum with different concentrations in the medicated serum groups.Each group was treated for 24 hours,and then further treatment was given according to the different detection indicators.The proliferation of MC3T3-E1 cells was detected by CCK-8 after incubated for 24 h,48 h and 72 h respectively.The alkaline phosphatase(ALP)activity was detected 7 days after the induction culture,and the mineralized nodules were detected by Alizarin Red staining 21 days later.RT-PCR was used to detect the expression of key genes such as the mRNA expression of Wnt2,Lrp5,β-Catenin in the Wnt/β-Catenin gene pathway,the mRNA expression of OPG and RANKL in the OPG/RANKL pathway,the mRNA expression of Bcl-2,Caspase-9 and Caspase-3 in the pathway of mitochondrial apoptosis,and the OPG/RANKL ratio was calculated.Results Compared with the normal group,the cell proliferation and ALP expression of the model group were decreased(all P<0.01),and the number of mineralized nodules was decreased.The mRNA expressions of Wnt2,Lrp5 andβ-catenin in Wnt/β-catenin gene pathway were decreased(all P<0.01).In the OPG/RANKL gene pathway,the mRNA expression of OPG was decreased,while the mRNA expression of RANKL was increased,and the ratio of OPG/RANKL was decreased(all P<0.01).In the mitochondrial apoptosis pathway,the mRNA expression of Bcl-2 was decreased,while the mRNA expressions of Caspase-9 and Caspase-3 were increased(all P<0.01).Compared with the model group,the medicated serum of Liuwei Dihuang Decoction in each dose group could promote the proliferation of MC3T3-E1 cells(all P<0.01)and the activity of ALP(P<0.05,P<0.01)at 24 h,48 h,and 72 h,and increase the number of mineralized nodules.In the Wnt/β-Catenin gene pathway,when compared with the model group,except for the low-dose medicated serum group,the mRNA expression of Wnt2 in the medium-and high-dose group was higher than that in the model group(all P<0.01).The mRNA expression of Lrp5 in each dose group of Liuwei Dihuang Decoction was higher than that of the model group(all P<0.01),and the mRNA expression ofβ-Catenin was also higher than that of the model group(P<0.05,P<0.01).In the OPG/RANKL gene pathway,except for the low-dose medicated serum group,the mRNA expression of OPG in the medium-and high-dose group was higher than that in the model group(P<0.05,P<0.01),while the expression of RANKL was significantly lower than that in the model group(all P<0.01),and the ratio of OPG/RANKL in each medicated serum group was higher than that in the model group(all P<0.01)with dose-dependent manner.In the mitochondrial apoptosis pathway,the mRNA expression of Bcl-2 in each dose medicated serum group of Liuwei Dihuang Decoction was significantly higher than that in the model group(P<0.05,P<0.01),while the mRNA expressions of Caspase-9 and Caspase-3 were significantly lower than those in the model group(all P<0.01).Conclusion The medicated serum of Liuwei Dihuang Decoction can reduce the damage of MC3T3-E1 caused by H_(2)O_(2) and generally promote the proliferation,differentiation and mineralization of MC3T3-E1.Its protective effect may be related to the regulation of key targets in the Wnt/β-catenin signaling pathway,OPG/RANKL and mitochondrial apoptosis pathway.