Purpose: Colorectal cancer (CRC) remains the third most common tumor worldwide. Ulcerative colitis (UC) could cause chronic inflammation and ulcers in the colon and rectum. UC is a risk factor for a high incidence of CRC, and the incidence of UC-associated CRC (UC-CRC) is still increasing. Chinese medicine prescription, Xian-Lian-Jie-Du decoction (XLJDD), has been proven its efficacy in some UC-CRC patients. However, the mechanism of XLJDD in treating UC-CRC remains unknown. This study aimed to investigate the mechanism of XLJDD in treating UC-CRC. Methods: We constructed an AOM/DSS mouse model that could simulate the various stages of UC-CRC in humans. XLJDD and its 5 main components are used to treat the AOM/DSS model, respectively. With the power of high-throughput sequencing technology, we described the mechanism of XLJDD from transcriptomics, proteomics, and single-cell transcriptomics. Results: Our results showed that XLJDD could effectively suppress the occurrence and development of colorectal tumors. Using the weighted correlation network analysis (WGCNA), several mRNA and protein modules that respond to XLJDD have been identified. Moreover, two essential genes, Mfsd2a and Ccdc85c, were caught our attention. They were prognostic markers in CRC patients, and their expression could be significantly modulated by XLJDD, showing their potential as effective targets of XLJDD. In addition, we also discovered that XLJDD could affect the cell composition of the colorectal tumor environment, especially in the infiltration of B cells. Conclusion: We demonstrated that XLJDD could prevent the initiation and development of colorectal tumors by modulating the expression of Mfsd2a and Ccdc85c and reducing the infiltration of B cells in the tumor microenvironment of colorectal tumor.

Background: Our previous study found that deletion of Sorting nexin 10 (SNX10) can protect against colonic inflammation and pathological damage induced by dextran sulfate sodium (DSS). This inspired us that modulation of SNX10 expression in colonic epithelial cells might represent a promising therapeutic strategy for inflammatory bowel disease (IBD). Methods: Effective delivery of siRNA/shRNA to silence genes is a highly sought-after means in the treatment of multiple diseases. Here, we encapsulated SNX10-shRNA plasmids (SRP) with polylactide-polyglycolide (PLGA) to make oral nanoparticles (NPs), and then applied them to acute and chronic IBD mice model, respectively. The characteristics of the nanoparticles were assayed and the effects of SRP-NPs on mouse IBD were evaluated. Results: High-efficiency SNX10-shRNA plasmids were successfully constructed and coated with PLGA to obtain nanoparticles, with a particle size of 275.2 ± 11.4mm, uniform PDI distribution, entrapment efficiency of 87.6 ± 2.5%, and drug loading of 13.11 ± 1.38%, displayed dominant efficiency of SNX10 RNA interference in the colon. In both acute and chronic IBD models, SRP-NPs could effectively reduce the loss of mice body weight, relieve the intestinal mucosal damage and inflammatory infiltration, inhibit the expression of inflammatory cytokines IL-1β, IL-23, TNF-α, and down-regulate the expression of toll-like receptors (TLRs) 2 and 4. Conclusion: Oral nanoparticles of SNX10-shRNA plasmid displayed dominant efficiency of SNX10 RNA interference in the colon and ameliorate mouse colitis via TLR signaling pathway. SNX10 is a new target for IBD treatment and nanoparticles of SNX10-shRNA plasmid might be a promising treatment option for IBD.

Background: Non‐apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α‐hederin (α‐hed) induces caspase‐independent paraptotic modes of cell death.Purpose: The present study is aimed to investigate the role of α‐hed induces paraptosis and the associated mechanism of it.Methods: The cell proliferation was detected by CCK‐8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca²⁺) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α‐hed. Molecular docking methods were used to evaluate binding abilities of α‐hed with targets. The expressions of genes and proteins were analyzed by RT‐qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects in vivo.Results: α‐hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg‐2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α‐hed. Besides, both Swiss prediction and molecular docking showed that the structure of α‐hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)‐β3/ inositol 1,4,5‐trisphosphate receptor (IP3R)/ Ca²⁺/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α‐hed. After blocking the GPCR signaling, α‐hed could not elevate Ca²⁺ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α‐hed activated MAPK cascade by elevating Ca²⁺ flux. Since non‐apoptotic cell death is presently emerging as a potential direction to overcome chemo‐drug resistance, we then found α‐hed also induced paraptosis in 5‐fluorouracil‐resistant (5‐FU‐R) CRC cells, and it reduced the growth of 5‐FU‐R CRC xenografts.Conclusions: Collectively, our findings proved α‐hed as a promising candidate for inducing non‐apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic‐based chemo‐resistance in CRC.

Protein homeostasis and quality control are crucial for normal cellular activities, and a severe imbalance in protein homeostasis can lead to cell death. α-Hederin, a pentacyclic triterpenoid saponin isolated from Fructus Akebia, has a clear role in promoting colorectal cancer (CRC) cell apoptosis and has been recently used for CRC therapy. However, whether the pro-apoptotic activity of α-hederin in CRC cells involves disturbing protein homeostasis remains unknown. Here, we aimed to uncover the underlying molecular mechanism of α-hederin-induced apoptosis in CRC cells. Cell viability and proliferation were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) and 5-ethynyl-2'-deoxyuridine (EdU) assays, respectively. Apoptosis was detected using flow cytometry and western blotting. Autophagic flux was examined by western blotting and AdPlus-mCherry-GFP-LC3B adenovirus infection assays, and western blotting and immunofluorescence staining were performed to detect the expression of proteins in related pathways. The results showed that α-hederin significantly inhibited the growth and promoted the apoptosis of human CRC cells. Furthermore, α-Hederin induced endoplasmic reticulum (ER) stress, but inhibited proteasomal degradation. Also, the autophagic flux was blocked by α-hederin although this drug promoted autophagosome formation, and the lysosomal degradation was inhibited. Expression of p-JNK and p-p38 were increased by α-hederin. So, our findings provide strong evidence that α-hederin disrupts protein homeostasis by blocking ER-associated degradation (ERAD) and autophagic flux, thereby contributing to apoptosis. PERK-eIF2α-ATF4-CHOP and IRE1-ASK1-JNK/p38 signal pathway may be involved in those regulation. Our results make it a promising alternative or adjunct therapeutic candidate for CRC.