Betulin

Betulin is a naturally occurring triterpene holding great promise as a lead for developing new antitumor agents. Given that the design, synthesis and anticancer activity of betulin-succinate derivatives remain unexplored, we herein reported the synthesis and cytotoxicity evaluation of such 22 new derivatives against A172, HT29, SW579, and TCA8113 cancer cell lines in vitro using the CCK8 (Cell Counting Kit-8) assay. The results revealed that most compounds showed potent antitumor activity relative to the positive control and betulin. Particularly, compound 21 also exhibited superior activities in inducing the early apoptosis of HT29 cells and blocking the cell cycle in the G2 phase than the chemotherapeutic agent cisplatin. In vitro kinase screening results from the 207 tested kinases demonstrated that MARK2 (Microtubule Affinity-Regulating Kinase 2), significantly inhibited by compound 21, may be the potential target of betulin-succinate derivatives. Hence, the affinity and interactions between compound 21 and MARK2 were obtained by molecular docking simulations. Compound 21, which prevented tumor cell metastasis and induced tumor cell apoptosis, exhibited potent antitumor activity in a zebrafish tumor model with xenografted HT29 cells. Furthermore, the histopathological evaluation demonstrated the ability of compound 21 to reduce the proliferation and improve the atypia of tumor cells in the abdominal cavity of zebrafish.

Enhanced apoptosis of intestinal epithelial cells during sepsis results in impaired barrier function, facilitating the influx of bacteria and endotoxins into the bloodstream, which worsens the organism's damage. Therefore, addressing intestinal injury in sepsis may represent a novel approach to treatment. Shenling Baizhu Powder (SLBZP), a classical traditional Chinese medicine formula, has been widely used for the treatment of Inflammatory diseases including sepsis. However, its potential mechanisms of action in sepsis-induced intestinal injury remain unclear.

This study aimed to investigate whether SLBZP and its betulin ameliorate intestinal barrier function and cellular death in sepsis mice and LPS-induced IEC-6 cells through GADD45B/TAOK1/p38 MAPK pathway.

Network pharmacology and Gene Expression Omnibus (GEO) database were used to identify the potential active ingredients and epigenetic regulators of SLBZP. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was used to measure the betulin present in SLBZP. Besides, the animal model of sepsis was developed by using a cecal ligation and puncture (CLP) to investigate the protective roles of SLBZP and betulin on intestinal injury in sepsis. Furthermore, the determination of cell viability, inflammation, and apoptosis of LPS-induced IEC-6 cells treated by betulin was performed by Cell counting Kit-8 (CCK-8), ELISA, Tunel staining, and flow cytometry assays. Meanwhile, the underlying mechanism was investigated through IHC, qMSP, and Western blot assays, respectively.

Through network and GSE202261 analysis, three epigenetic regulators including GADD45B, MAP3K7, and PRKAA1 were screened from the "drug-component-target" network. The betulin and the GADD45B had a good binding ability in molecular docking. Animal experiments indicated that SLBZP and betulin could inhibit inflammation, ameliorate intestinal injury, and reduce cell apoptosis in mice. Moreover, the intestinal cytotoxicity of LPS-treated IEC-6 cells was significantly inhibited after betulin treatment, as accompanied by an increase in DNA methylation level in the TAOK1 promoter. Importantly, we found that the overexpression of GADD45B and TAOK1, or p38 MAPK inhibitor reversed the anti-apoptosis effect induced by the betulin.

SLBZP and betulin may exert anti-inflammatory and anti-apoptosis effects against sepsis-associated intestinal barrier injury, possibly via the GADD45B/TAOK1/p38 MAPK pathway.