基于BMP2/Smad1/Runx2/Osterix信号通路探讨健骨颗粒氯仿萃取部位对体外成骨细胞分化的影响

Effect of Chloroform Extraction Site of Jiangu Granule on Obteoblast Differentiation in Vitro via BMP2/Smad1/Runx2/Osterix Signaling Pathway

目的:通过观察健骨颗粒氯仿萃取部位对体外培养成骨细胞的影响,研究其调控BMP2/Smad1/Runx2/Osterix信号通路影响成骨细胞分化的可能机制。方法:选用年龄<3 d的BALB/c小鼠,从其颅盖骨提取原代成骨细胞,并采用碱性磷酸酶染色法对所提取的细胞进行鉴定;取生长状态良好的第3代成骨细胞作为实验对象,将健骨颗粒氯仿萃取部位稀释至不同的浓度梯度,即设置0、2、4、8、16、32、64 ng/mL 7个药物浓度组对细胞进行干预,采用MTT法筛选健骨颗粒氯仿萃取部位促进成骨细胞增殖的最佳浓度,作为最佳药物干预浓度进行后续实验。取第3代细胞,将其分为空白组、抑制剂组(BMP2抑制剂Noggin干预)、药物组(健骨颗粒氯仿萃取部位干预)和抑制剂加药物组(Noggin加健骨颗粒氯仿萃取部位干预),按组别干预后进行后续实验。采用RT-PCR检测各组BMP2、Smad1、Runx2、Osterix及成骨细胞分化相关基因ALP、CollagenⅠmRNA的表达水平;采用Western blot法检测各组BMP2、Smad1、Runx2、Osterix及ALP、CollagenⅠ的蛋白表达,观察健骨颗粒氯仿萃取部位对成骨细胞分化的影响。结果:经碱性磷酸酶染色法鉴定,所提取的原代细胞阳性率高达90%为成骨细胞;采用MTT法检测发现健骨颗粒氯仿萃取部位在药物浓度为32 ng/mL时,成骨细胞增殖速度最快,在干预2 d时达到增殖高峰,故选用32 ng/mL干预2 d作为健骨颗粒氯仿萃取部位最佳干预浓度进行后续实验;RT-PCR与Western blot检测结果显示,与空白组相比,抑制剂组BMP2、Smad1、Runx2、Osterix及ALP、CollagenⅠ各指标基因表达水平显著降低,蛋白相对表达量明显降低(P<0.01或P<0.05);与空白组相比较,药物组6个指标基因表达水平显著升高,蛋白相对表达量也明显升高(P<0.01或P<0.05);抑制剂加药物组与抑制剂组比较,各指标基因表达水平和蛋白相对表达量均显著升高(P<0.01或P<0.05)。结论:(1)使用Noggin抑制细胞内BMP2的表达后,Smad1、Runx2、Osterix的基因和蛋白的表达明显下降,与此同时成骨细胞分化相关指标ALP、CollagenⅠ基因和蛋白表达也明显下降,提示BMP2通路与成骨细胞分化密切相关。(2)健骨颗粒氯仿萃取部位干预原代成骨细胞后能够升高细胞内BMP2、Smad1、Runx2、Osterix与成骨细胞分化相关指标ALP、CollagenⅠ基因和蛋白的表达,促进成骨样细胞分化;提示健骨颗粒氯仿萃取部位可以通过调控BMP2/Smad1/Runx2/Osterix通路,促进成骨细胞分化,从而达到防治绝经后骨质疏松症的作用。

Objective:To investigate whether the chloroform extraction site of Jiangu Granule could affect the differentiation of osteoblast in vitro via BMP2/Smad1/Runx2/Osterix signaling pathway.Methods:Primary osteoblasts were extracted from the calvaria of BALB/c mice born less than 3 days old,and the extracted cells were identified by alkaline phosphatase staining;the thirdgeneration osteoblasts in good growth condition were selected as experimental subjects.The chloroform extraction site of Jiangu Granule was diluted to different concentration gradients,which included 7 drug concentration groups of 0,2,4,8,16,32,and 64 ng/mL set for cell intervention.MTT method was used to screen the optimal concentration of the chloroform extraction site of Jiangu Granule to promote the proliferation of osteoblasts,which was used as the optimal drug intervention concentration for follow-up experiments.The third-generation cells were extracted and divided into 4 groups,namely blank group,inhibitor group(intervened by BMP2 inhibitor Noggin),drug group(intervened by Jiangu Granule chloroform extraction site)and inhibitor plus drug group(intervened by Noggin plus Jiangu Granule chloroform extraction site).After cell interventions in each experiment group,RT-PCR was used to detect the mRNA expression levels of BMP2,Smad1,Runx2,Osterix and osteoblast differentiation-related genes ALP and CollagenⅠin each group.Western blot was used to detect the protein expressions of BMP2,Smad1,Runx2,Osterix,ALP and Col-l agenⅠin each group,and the effect of chloroform extraction site of Jiangu Granule on osteoblast differentiation was observed.Re-s ults:The alkaline phosphatase staining showed the positive rate of osteoblasts in the collected primary cells was as high as 90%.The results of MTT method showed that when the concentration of chloroform extracted from Jiangu Granule was 32 ng/mL,the osteoblasts showed the fastest proliferation,and reached the peak of proliferation after 2 days of intervention.Therefore,32 ng/mL concentration gradient of Jiangu Granule was selected as the plan for 2 days intervention.The optimal intervention concentration of the chloroform extraction site was used for follow-up experiments.RT-PCR and Western blot detection showed that,compared with the blank group,the expression levels of each index gene in the inhibitor group significantly decreased,and the relative protein expression significantly decreased(P<0.01 or P<0.05);compared with the blank group,the expression levels of 6 index genes in the drug group significantly increased,and the relative protein expression also significantly increased(P<0.01 or P<0.05);compared with the inhibitor group,the expression levels of index genes and proteins in inhibitor plus drug group significantly increased(P<0.01 or P<0.05).Conclusion:1)The gene and protein expressions of Smad1,Runx2,and Osterix significantly decreased when Noggin was used to inhibit the expression of intracellular BMP2.At the same time,osteoblast differentiation-related indicators ALP and CollagenⅠalso significantly decreased,suggesting that the BMP2 pathway could be closely related to osteoblast differentiation.2)After thec hloroform extraction site of Jiangu Granule intervention in osteoblasts,the expression of BMP2,Smad1,Runx2,Osterix and osteoblast differentiation-related indexes ALP and CollagenⅠgenes and proteins increased in the cells,which promoted the differentia-t ion of osteoblast-like cells.It suggested that the chloroform extraction site of Jiangu Granules could promote the differentiation of osteoblasts by regulating the BMP2/Smad1/Runx2/Osterix pathway,so as to prevent and treat postmenopausal osteoporosis.