Zhang Peng, He Jiahui, Gan Yanchi, Shang Qi, Chen Honglin, Zhao Wenhua, Shen Gengyang, Jiang Xiaobing, Ren Hui
Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
The Affiliated TCM Hospital of Guangzhou Medical University, Guangzhou 510130, China.
Pharmaceuticals (Basel). 2023 Oct 17;16(10):1482. doi: 10.3390/ph16101482.
(PT), a widely used traditional Chinese medicine, exerts protective effects against bone diseases such as intervertebral disc degeneration (IDD). Despite its effectiveness, the molecular mechanisms underlying the effects of PT on IDD remain unclear. In this study, we used a comprehensive strategy combining bioinformatic analysis with experimental verification to investigate the possible molecular mechanisms of PT against IDD. We retrieved targets for PT and IDD, and then used their overlapped targets for protein-protein interaction (PPI) analysis. In addition, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to investigate the anti-IDD mechanisms of PT. Moreover, in vivo and in vitro experiment validations including hematoxylin-eosin (HE) and safranine O-green staining, senescence-associated β-galactosidase (SA-β-gal) assay, cell immunofluorescence staining, intracellular ROS measurement and Western blot analysis were performed to verify bioinformatics findings. We identified 342 and 872 PT- and IDD-related targets (32 overlapping targets). GO enrichment analysis yielded 450 terms related to oxidative stress and inflammatory response regulation. KEGG analysis identified 48 signaling pathways, 10 of which were significant; the TNF-α signaling pathway had the highest -value, and prostaglandin G/H synthase 2 (), endothelin-1 (), TNF-α, and were enriched in this pathway. Histopathological results and safranin O/green staining demonstrated that PT attenuated IDD, and SA-β-gal assay showed that PT ameliorated nucleus pulposus cell (NPC) senescence. An ROS probe was adopted to confirm the protective effect of PT against oxidative stress. Western blot analyses confirmed that PT downregulated the protein expression of , , TNF-α, and in the TNF-α signaling pathway as well as cellular senescence marker p16, proinflammatory cytokine interleukin-6 (), while PT upregulated the expression of NPC-specific markers including and in a concentration-dependent manner. To the best of our knowledge, this study is the first to report that PT alleviates IDD by downregulating the protein expression of , , TNF-α, and in the TNF-α signaling pathway and upregulating that of and , thus suppressing inflammatory responses and oxidative stress in NPCs.
丹参（PT）是一种广泛应用的中药，对椎间盘退变（IDD）等骨疾病具有保护作用。尽管其疗效显著，但PT对IDD作用的分子机制仍不清楚。在本研究中，我们采用生物信息学分析与实验验证相结合的综合策略，研究PT抗IDD的可能分子机制。我们检索了PT和IDD的靶点，然后使用它们的重叠靶点进行蛋白质-蛋白质相互作用（PPI）分析。此外，我们使用基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析来研究PT的抗IDD机制。此外，还进行了体内和体外实验验证，包括苏木精-伊红（HE）和番红O-固绿染色、衰老相关β-半乳糖苷酶（SA-β-gal）检测、细胞免疫荧光染色、细胞内活性氧（ROS）测量和蛋白质印迹分析，以验证生物信息学研究结果。我们鉴定出342个和872个与PT和IDD相关的靶点（32个重叠靶点）。GO富集分析产生了450个与氧化应激和炎症反应调节相关的术语。KEGG分析确定了48条信号通路，其中10条具有显著性；肿瘤坏死因子-α（TNF-α）信号通路的P值最高，前列腺素G/H合酶2（PTGS2）、内皮素-1（ET-1）、TNF-α和环氧合酶-2（COX-2）在该通路中富集。组织病理学结果和番红O/固绿染色表明PT减轻了IDD，SA-β-gal检测表明PT改善了髓核细胞（NPC）衰老。采用ROS探针证实了PT对氧化应激的保护作用。蛋白质印迹分析证实，PT下调了TNF-α信号通路中PTGS2、ET-1、TNF-α和COX-2的蛋白表达以及细胞衰老标志物p16、促炎细胞因子白细胞介素-6（IL-6），而PT以浓度依赖的方式上调了包括波形蛋白（VIM）和Ⅱ型胶原（COL2A1）在内的NPC特异性标志物的表达。据我们所知，本研究首次报道PT通过下调TNF-α信号通路中PTGS2、ET-1、TNF-α和COX-2的蛋白表达以及上调VIM和COL2A1的表达来减轻IDD，从而抑制NPC中的炎症反应和氧化应激。
