School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China.
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China.
Phytomedicine. 2022 Jul;101:154070. doi: 10.1016/j.phymed.2022.154070. Epub 2022 Mar 22.
Asperuloside is a natural compound extracted from various herbs with several bioactivities. Its effects on anti-inflammation and anti-tumor indicated that asperuloside might prevent colorectal cancer developing from inflammatory bowel diseases (IBD). But there were few reports about the efficacy and mechanism of asperuloside on improving colorectal cancer. It has been reported that vitamin D receptor (VDR) could regulate the expression of SMAD3. In previous study, asperuloside could significantly improve the expression of VDR and reduced Smad3 mRNA in IEC-6 cell.
The present study was aimed to investigate the potential mechanism of asperuloside on inhibiting epithelial-mesenchymal transition (EMT) in colitis associated cancer.
First, in LPS-injured IEC-6 cell, asperuloside inhibited phosphorylated p65 (p-p65) level, improved VDR expression and reduced Smad3 mRNA. Second, we wonder the relationship between VDR signaling and nucleus factor-kappaB (NF-κB) signaling during asperuloside on reducing Smad3 mRNA. And then, the effect of asperuloside on inhibiting EMT development through VDR/Smad3 was investigated. Finally, we testified the effect of asperuloside on protecting against colitis associated cancer (CAC) by inhibiting EMT development through VDR/Smad3.
Pyrrolidinedithiocarbamate ammonium (PDTC) was used for established NF-κB-inhibited IEC-6 cell. This cell was applied for investigating the relationship between NF-κB and VDR of asperuloside on inhibiting Smad3. VDR-inhibited cell was established by small interfering RNA (siRNA) of VDR and was employed to investigate the role of VDR for asperuloside on decreasing Smad3. Transforming growth factor β1 (TGFβ1) was used for inducing EMT/fibrosis in IEC-6 cell. TGFβ1-stimulated cell was used for testifying the effect of asperuloside on inhibiting EMT development. AOM/DSS-induced CAC was established to investigate the effect of asperuloside on suppressing cancer development.
Asperuloside inhibited the level of p-p65 which was up-regulated by LPS. Asperuloside could up-regulate VDR signaling and reduce Smad3 mRNA in NF-κB-knockdown IEC-6 cells. Asperuloside failed to reduce Smad3 mRNA due to VDR knockdown, which implied that asperuloside might down-regulate Smad3 mRNA dependently on activation of VDR signaling and independently on inhibiting NF-κB signaling. Asperuloside exhibited significant prevention of EMT development in TGFβ1-induced IEC-6 cell (EMT cell) and mice CAC. Asperuloside reduced the transform of epithelial phenotype into motile mesenchymal phenotype in EMT cell along with decreasing levels of EMT markers by inhibiting Smad3 mRNA via activation of VDR. In mice with CAC, expression of VDR in colon was improved by asperuloside. Symptoms of colitis, tumor number and tumor size were significantly inhibited by asperuloside. Suppressed EMT development was determined by reduced α-SMA expression and decreased mRNAs of several EMT markers.
Asperuloside might prevent CAC through inhibiting EMT development via regulation of VDR/Smad3 pathway.
山柰酚是一种从多种草药中提取的天然化合物，具有多种生物活性。其抗炎和抗肿瘤作用表明，山柰酚可能预防从炎症性肠病（IBD）发展而来的结直肠癌。但是，关于山柰酚改善结直肠癌的疗效和机制的报道很少。据报道，维生素 D 受体（VDR）可以调节 SMAD3 的表达。在之前的研究中，山柰酚可以显著改善 IEC-6 细胞中 VDR 的表达，并降低 Smad3 mRNA 的水平。
本研究旨在探讨山柰酚抑制结肠炎相关癌症上皮-间充质转化（EMT）的潜在机制。
首先，在 LPS 损伤的 IEC-6 细胞中，山柰酚抑制磷酸化 p65（p-p65）水平，提高 VDR 表达并降低 Smad3 mRNA。其次，我们想研究山柰酚降低 Smad3 mRNA 过程中 VDR 信号与核因子-κB（NF-κB）信号之间的关系。然后，通过 VDR/Smad3 研究山柰酚对抑制 EMT 发展的作用。最后，通过 VDR/Smad3 抑制 EMT 发展，验证山柰酚对预防结肠炎相关癌症（CAC）的作用。
用吡咯烷二硫代氨基甲酸盐铵（PDTC）建立 NF-κB 抑制的 IEC-6 细胞。该细胞用于研究山柰酚抑制 Smad3 过程中 NF-κB 和 VDR 之间的关系。用小干扰 RNA（siRNA）抑制 VDR 建立 VDR 抑制细胞，用于研究 VDR 对山柰酚降低 Smad3 的作用。转化生长因子β1（TGFβ1）用于诱导 IEC-6 细胞 EMT/纤维化。用 TGFβ1 刺激细胞，验证山柰酚抑制 EMT 发展的作用。用 AOM/DSS 诱导 CAC，研究山柰酚抑制癌症发展的作用。
山柰酚抑制了 LPS 上调的 p-p65 水平。山柰酚可上调 NF-κB 敲低的 IEC-6 细胞中的 VDR 信号，并降低 Smad3 mRNA。由于 VDR 敲低，山柰酚未能降低 Smad3 mRNA，这表明山柰酚可能依赖于 VDR 信号的激活，独立于抑制 NF-κB 信号来下调 Smad3 mRNA。山柰酚在 TGFβ1 诱导的 IEC-6 细胞（EMT 细胞）和小鼠 CAC 中表现出显著的 EMT 发展抑制作用。山柰酚通过抑制 Smad3 mRNA，通过激活 VDR，显著降低 EMT 标志物的水平，从而抑制 EMT 细胞的上皮表型向运动性间充质表型的转化。在 CAC 小鼠中，山柰酚改善了结肠中的 VDR 表达。山柰酚显著抑制了结肠炎、肿瘤数量和肿瘤大小的症状。通过减少α-SMA 表达和降低几种 EMT 标志物的 mRNA，证实 EMT 发展受到抑制。
山柰酚可能通过调节 VDR/Smad3 通路抑制 EMT 发展，从而预防 CAC。
