Icariin Regulates EMT and Stem Cell-Like Character in Breast Cancer through Modulating lncRNA NEAT1/TGFß/SMAD2 Signaling Pathway.

Metastases and drug resistance are the major risk factors associated with breast cancer (BC), which is the most common type of tumor affecting females. Icariin (ICA) is a traditional Chinese medicine compound that possesses significant anticancer properties. Long non-coding RNAs (lncRNAs) are involved in a wide variety of biological and pathological processes and have been shown to modulate the effectiveness of certain drugs in cancer. The purpose of this study was to examine the potential effect of ICA on epithelial mesenchymal transition (EMT) and stemness articulation in BC cells, as well as the possible relationship between its inhibitory action on EMT and stemness with the NEAT1/transforming growth factor ß (TGFß)/SMAD2 pathway. The effect of ICA on the proliferation (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony assays), EMT (Western blotting, immunofluorescence, and wound healing), and stemness (mammosphere formation assays, Western blotting) of BC cells were examined. According to the findings, ICA suppressed the proliferation, EMT, and stem cell-like in MDA-MB-231 cells, and exerted its inhibitory impact by downregulating the TGFß/SMAD2 signaling pathway. ICA could significantly downregulate the expression of lncRNA NEAT1, and silencing NEAT1 enhanced the effect of ICA in suppressing EMT and expression of different stem cell markers. In addition, silencing NEAT1 was found to attenuate the TGFß/SMAD2 signaling pathway, thereby improving the inhibitory impact of ICA on stemness and EMT in BC cells. In conclusion, ICA can potentially inhibit the metastasis of BC via affecting the NEAT1/TGFß/SMAD2 pathway, which provides a theoretical foundation for understanding the mechanisms involved in potential application of ICA for BC therapy.

Indirubin regulates T cell differentiation by promoting αVß8 expression in bone marrow-derived dendritic cells to alleviate inflammatory bowel disease.

Inflammatory bowel disease is a disease that can invade the whole digestive tract and is accompanied by immune abnormalities. Immune dysfunction involving dendritic cells (DCs) and T cells is recognized as a key factor in diseases. Indirubin (IDRB) exerts antiinflammatory effects and can help in treating immune diseases. This study aimed to isolate bone marrow-derived dendritic cells (BMDCs) using lipopolysaccharide (LPS) to obtain mature DCs (mDCs). The expression of CD80, CD86, CD40, and MHC-II was detected using flow cytometry after treatment with IDRB. αVß8 siRNA was used to knock down αVß8 in mDCs, and the expression of CD80, CD86, CD40, and MHC-II was detected. Meanwhile, DCs were co-cultured with T cells. Then, T cell differentiation was detected using flow cytometry, and the cytokine levels were detected using enzyme-linked immunosorbent assay. The animal model of dextran sulfate sodium (DSS)-induced inflammatory bowel disease was established in mice. After intervention with IDRB and αVß8 shRNA, the intestinal tissues were evaluated using H&E staining, disease activity index (DAI) score, and histological damage index, and the corresponding factors and cytokines to regulatory T cells (Treg) and Th17 were measured. The results showed that αVß8 was expressed in immature DCs and mDCs. CD80, CD86, CD40, and MHC-II expression decreased after IDRB treatment in mDCs. Meanwhile, the expression of TNF-α and TGF-ß also decreased after IDRB treatment. The effect of IDRB on the expression of CD80, CD86, CD40, MHC-II, TNF-α, and TGF-ß in mDCs was reversed by αVß8 siRNA. The Treg differentiation increased after IDRB treatment, while the differentiation of Th17 cells was inhibited. This effect of IDRB was reversed by mDCs after treatment with αVß8 siRNA. In vivo experiments showed that IDRB alleviated the symptoms of inflammatory bowel disease in animals. Enteritis significantly reduced, and the effect of IDRB was reversed by αVß8 shRNA. The results suggested that IDRB regulated the differentiation of T cells by mediating the maturation of BMDCs through αVß8. This study confirmed the therapeutic effect of IDRB in inflammatory bowel disease and suggested that IDRB might serve as a potential drug.

Xi Lei San Attenuates Dextran Sulfate Sodium-Induced Colitis in Rats and TNF-α-Stimulated Colitis in CACO2 Cells: Involvement of the NLRP3 Inflammasome and Autophagy.

OBJECTIVE: Inflammatory bowel disease (IBD) is a chronic nonspecific inflammatory bowel disease with an unclear etiology. The active ingredients of traditional Chinese medicines (TCMs) exert anti-inflammatory, antitumor, and immunomodulatory effects, and their multitarget characteristics provide them with a unique advantage for treating IBD. However, the therapeutic effects and underlying mechanisms of Xi Lei San in treatment of IBD remain unknown. This study was designed to investigate whether Xi Lei San exerted an anti-inflammatory effect in IBD via a mechanism involving NLRP3 inflammasomes and autophagy. METHODS: We successfully established a rat model of dextran sulfate sodium- (DSS-) induced colitis as well as a cellular model of TNF-α-induced colitis. Xi Lei San and indirubin were identified by HPLC analysis. Rats were treated with Xi Lei San or alum crystals, and their body weights and morphology of intestinal tissues were examined. A western blot analysis was performed to determine the expression levels of inflammasome-related proteins and autophagy-related proteins, ELISA was performed to analyze IL-1ß, IL-18, and IL-33 concentrations, and flow cytometry was used to monitor cell apoptosis and ROS levels. RESULTS: Xi Lei San and indirubin were identified by HPLC analysis. We found that Xi Lei San could significantly increase the weights of rats and improve the structure of the intestinal tissues in DSS-induced colitis model rats. We also found that Xi Lei San significantly inhibited NLRP3 inflammasome activity, reduced the levels of inflammatory cytokines, and suppressed autophagy in DSS-induced colitis model rats. In vitro experiments revealed that Xi Lei San could repress apoptosis as well as ROS and inflammatory cytokine production in TNF-α-induced CACO2 cells by reducing the activity of NLRP3 inflammasomes and autophagy. CONCLUSIONS: Our findings showed that Xi Lei San significantly ameliorated IBD by inhibiting NLRP3 inflammasome, autophagy, and oxidative stress.