Network pharmacology and experimental verification-based strategy to explore the underlying mechanism of Liu Jun An Wei formula in the treatment of gastrointestinal reactions caused by chemotherapy for colorectal cancer.

Abstract

Background: Liu Jun An Wei formula (LJAW), derived from “Liu Jun Zi Decoction”, is a classical prescription of Tradition Chinese Medicine and has been used for the treatment of gastrointestinal reactions caused by chemotherapy for colorectal cancer (CRC) for many years. Its molecular mechanism remains to be further explored. Objective: To clarify the mechanism of LJAW in attenuating gastrointestinal reactions caused by chemotherapy for CRC. Methods: The 5-fluorouracil (5-FU) induced mouse and intestine organoid models were established to observe the effect of LJAW. The ingredients of LJAW were analyzed and identified by UPLC-Q-TOF-MS technology. Targets of LJAW and chemotherapy-induced gastrointestinal reactions were collected from several databases. “Ingredient-target” network and protein-protein interaction network were constructed based on network pharmacology. Then, gene ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Subsequently, molecular docking method was used to verify the interaction between the core ingredients and key targets. The results were validated by both in vivo experiments and organoid experiments. Western Blot was used to analyze the influence of LJAW on key targets including PI3K, AKT1, MAPK1, MAPK14 proteins and their phosphorylated proteins. RT-qPCR and Western Blot were used to detect the mRNA and protein levels of apoptosis-related gene PUMA. Results: Compared with the 5-FU group, the LJAW group had better morphology in mouse small intestine and intestine organoids. In total, 18 core ingredients and 19 key targets were obtained from 97 ingredients and 169 common targets. KEGG analysis showed that the common targets were involved in PI3K/Akt, MAPK, apoptosis and other signal pathways, which are closely related to gastrointestinal injury. Experiments confirmed that LJAW lowered the expressions of phosphorylated proteins including p-PI3K, p-AKT1, p-MAPK1, and p-MAPK14 and reduced the mRNA and protein levels of PUMA. Conclusion: LJAW shows protective effect on 5-FU induced small intestine and intestinal organoids injury. LJAW attenuates gastrointestinal reactions caused by chemotherapy for CRC probably by regulating apoptosis-related genes through PI3K/AKT and MAPK signaling pathways.