基于转录组学补肾通络丸经MAPK通路保护软骨细胞的作用

Role of Bushen Tongluo Wan in the protection of chondrocytes via MAPK pathway based on transcriptomics

目的探究补肾通络丸(BSTLW)对软骨保护作用的分子机制。方法从新生C57BL/6J乳鼠肋软骨中获得原代软骨细胞,加入不同浓度(0,0.1,0.2,0.4,0.6,0.8,1.0,1.2 mg/ml)的BSTLW提取物培养,CCK-8检测细胞活力。将软骨细胞分为BSTLW组和对照组,分别用BSTLW提取物和空白DMEM/F12培养基作用24 h后,提取各组总RNA进行RNA测序和转录组学分析。将差异基因进行GO生物学分子功能富集和KEGG通路富集分析,选择Dusp8、Bdnf、Egf、Fgfr4进行qRT-PCR验证。结果CCK-8检测结果显示,BSTLW能够提高软骨细胞的增殖活性,浓度为0.8 mg/ml时,促进软骨细胞增殖的能力最强(P<0.01)。RNA-Seq分析结果显示,与对照组比较,BSTLW组软骨细胞中差异基因共709个,其中上调344个,下调365个(P<0.01)。KEGG富集分析显示BSTLW对MAPK通路关键因子的调控丰度最高,整体呈下调趋势(P<0.01)。qRT-PCR验证结果提示BSTLW可以抑制MAPK通路中Dusp8、Bdnf、Fgfr4、Egf关键基因的表达(P<0.01),这与RNA-Seq测序结果一致。结论补肾通络丸能够对MAPK通路上Bdnf等多基因进行调控发挥软骨保护作用。

Objective To explore the protective effect and its molecular mechanism of Bushen Tongluo Wan(BSTLW)on cartilage.Methods Primary chondrocytes were obtained from the costal cartilage of C57BL/6J newborn mice,and the effect of BSTLW extracts(0,0.1,0.2,0.4,0.6,0.8,1.0,1.2 mg/ml)on cell viability was detected by CCK-8 method.The chondrocytes were divided into BSTLW group and blank group,and treated with BSTLW and blank DMEM/F12 medium for 24 h,respectively.Then the total RNA was extracted for RNA-Seq detection and transcriptome analysis.Differentially expressed genes(DEGs)were enriched by GO biological molecular function and KEGG pathway,and Dusp8,Bdnf,Egf and Fgfr4 were selected for qRT-PCR verification.Results CCK-8 results showed that BSTLW significantly increased the proliferation activity of chondrocytes(P<0.01),and the proliferation activity was the highest at the concentration of 0.8 mg/ml.RNA-Seq analysis showed that compared with blank group,709 DEGs were obtained in chondrocytes in BSTLW group,including 344 up-regulated genes and 365 down-regulated genes(P<0.01).KEGG pathway enrichment analysis showed that the regulation abundance of BSTLW on key factors of MAPK pathway was the most,showing a significant down-regulation(P<0.01).The results of qRT-PCR showed that BSTLW inhibited the expression of key genes Dusp8,Bdnf,Fgfr4 and Egf in MAPK pathway(P<0.01),which was consistent with the results of RNA-Seq sequencing.Conclusion BSTLW plays a protective role in cartilage by regulating Bdnf and other genes in the MAPK pathway.