To Explore The Mechanisms Of Xiao-Ban-Xia-Tang Decoction Against Chemotherapy-induced Nausea And Vomiting Based On Transcriptomics

Objectives:RNA sequencing(RNA-seq)was used to explore the potential mechanisms of Xiao-Ban-Xia-Tang decoction(XBXT)against chemotherapy-induced nausea and vomiting(CINV)and to locate the core genes and signaling pathways.The further experiments were performed to verify the sequencing results.Methods:(1)Grouping and drug treatment of animals: Male Wistar rats were randomly divided into normal control group,cisplatin model group and XBXT-treated group.Rats in XBXT-treated group were given XBXT 1.6 g crude drug/kg,normal control group and cisplatin model group were given the same volume of distilled water twice a day for three consecutive days.1h after the first intragastric administration,the rats in cisplatin model group and XBXT-treated group were intraperitoneally injected with cisplatin 6mg/kg to establish chemotherapeutic rat pica model,and the normal control group was injected with an equal volume of normal saline.The daily kaolin consumption,food intake and body weight of rats were recorded.Gastric antrum and ileum tissues were collected 1h after the last intragastric administration.The histopathological changes of gastric antrum and ileum were observed by hematoxylin-eosin(HE)staining.(2)RNA-Seq: the ileums of rats were taken for RNA-seq.Basic difference analysis,Venn diagram analysis,GO function annotation,KEGG pathway enrichment analysis and protein-protein interaction network analysis were used to locate the key genes and biological signaling pathways.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to verify the results of transcriptomics sequencing analysis.(3)To observe the protective effects of 6-gingerol and 6-shogaol,the active ingredients in XBXT,on chemotherapeutic damage of rat intestinal epithelial cells(IEC-6 cells)induced by cisplatin,and preliminary to explore the working mechanisms of XBXT by signaling pathway of ROS/NF-κB/NLRP3/Caspase-1/GSDMD mediated pyroptosis: based on the results of RNA-seq and laboratory previous work,cisplatin was used to establish a chemotherapeutic injury model of IEC-6,and CCK-8 method was used to detect cell viability;DCFH-DA fluorescent probe was used to detect the level of intracellular reactive oxygen species(ROS);Hoechst33342/PI staining was used to observe cell morphology changes;Western Blot was used to detect the expression of related protein about pyroptosis mediated by ROS/NF-κB/NLRP3/Caspase-1/GSDMD pathway;q RT-PCR was used to detect the levels of genes(such as Il18,Nlrp3,Gsdmd,and Hmgb1)in IEC-6 cells.Results:(1)Compared with normal group,kaolin intake of rats in cisplatin model group was significantly increased at 24h and 72h after modeling;The kaolin intake of rats in XBXT-treated group at 24 h and 72 h after cisplatin treatment was significantly lower than in model group,indicating that XBXT could significantly inhibit the excessive consumption of kaolin induced by cisplatin in pica rats during the acute and delayed phases.(2)HE staining results showed that obvious inflammatory pathological damage could be seen in the gastric antrum and ileum of cisplatin-induced pica rats.XBXT could effectively ameliorate the cisplatin-induced gastrointestinal tissue damage and reduce inflammation response.(3)RNA-seq revealed that there were total of 619 differentially expressed genes co-regulated by cisplatin and XBXT,including 343 genes that were significantly up-regulated by cisplatin and reversed by XBXT and75 genes that were significantly down-regulated by cisplatin and reversed by XBXT.XBXT could significantly inhibit the overexpression of cisplatininduced tryptophan hydroxylase(5-HT synthesis rate-limiting enzyme)corresponding gene Tph1.(4)GO function and KEGG enrichment analysis results showed that XBXT could inhibit the overexpression of inflammatory signaling pathways induced by cisplatin,such as TRP channel regulation,cytokine-cytokine receptor interaction,NF-κB signaling pathway,IL-17 signaling pathway and NOD-like receptor signaling pathway and the overexpression of inflammation-related genes such as Nlrp3,Il1 b,Tnf,and Ptprc.(5)6-gingerol and 6-shogaol,the active components of XBXT,could significantly increase the viability of IEC-6 cells damaged by cisplatin,reduce intracellular ROS level and cell necrosis;could reverse the hyperphosphorylation of NF-κB protein,the overexpression of NLRP3 inflammasome related proteins(NLRP3,ASC,and Caspase-1)and the overproduction of N-terminal fragments of GSDMD(GSDMD-NT);and could reverse the excessive increase of Il18,Nlrp3,Asc,Caspas-1,Gsdmd and Hmgb1 genes.Conclusions:Inhibition of inflammation and amelioration of gastrointestinal inflammatory damage might be important mechanisms of XBXT’s prevention and treatment aganist CINV.The specific mechanisms might be involved in inhibiting excessive activation of ROS/NF-κB/NLRP3/Caspase-1/GSDMD mediated pyroptosis signaling pathway and other inflammationrelated signal pathways.