The Antiemetic Effect Of Xiao-Ban-Xia-Tang Formula Is Related To Regulation Of 5-HT₃R/CaM/CaMK?/ERK1/2 Signaling Pathway In A Chemotherapy-induced Rat Pica Model

Chemotherapy-induced nausea and vomiting?CINV?is the most common gastrointestinal adverse reaction in the process of cancer chemotherapy,which seriously affects the life quality and the compliance of patients with chemotherapy.At present,most of the widely used drugs act on a single target,and are often needed to be combined with other medicines for the treatment of CINV,which not only increase the side effects,but also greatly increase the economic burden of patients.Studies have found that5-HT and 5-HT₃R play a key role in the pathogenesis of CINV.Enterochromaffin?EC?cells in the gastrointestinal tract can be stimulated by cytotoxic chemotherapeutic drugs such as cisplatin to release 5-HT,vomiting occers when the released 5-HT activate the 5-HT₃R located in the center and periphery.XBXT formula is a traditional Chinese medicine prescription,which has a good effect on various nausea and vomiting.Our previous experimental studies and related reports have confirmed that XBXT formula has a significant prevention and treatment effect on CINV,and its mechanism is related to the reduction of 5-HT level and the down-regulation of 5-HT₃R.Ginger and its active substances 6-gingerol?8-gingerol and 10-gingerol play the antiemetic role by blocking 5-HT₃R.The study found in 5-HT₃R selective agonist 2-Methyl-5-hydroxytryptamine excited 5-HT₃R can cause increased intracellular Ca²⁺concentration,launched the calmodulin and 5-HT₃R connection,and lead to calmodulin-dependent protein kinaseII/extracellular signal-regulated kinase 1/2?CaMKII/ERK1/2?dependencies adjust the activation of protein kinase signaling pathways,ultimately lead to the occurrence of vomiting.However,the influence of XBXT formula on the downstream signaling pathway after the action of 5-HT₃R has not been reported.In the present study,two pica models of rats were established by intraperitoneal injection of cisplatin and a selective 5-HT₃R agonist1-Phenylbiguanide hydrochloride?1-PBG?,and the antiemetic mechanism of XBXT formula was explored on the aspect of 5-HT₃R-mediated activation of CaMKII-dependent ERK1/2 signaling pathway.Objectives:To investigate the effect of XBXT formula on pica induced by cisplatin and 1-PBG in rats.The mechanism of preventing CINV by XBXT formula was discussed through the second messenger after 5-HT₃R.Methods:1.Preparing lyophilized powder of XBXT formula and studying the quality of the active ingredients in the lyophilized powder of XBXT formula by high performance liquid chromatography?HPLC?.2.Wistar male rats were randomly divided into four groups:the normal control group?Control??the cisplatin model group?C-Model??the ondansetron-treated cisplatin model group?C-Ondansetron??the XBXT-treated cisplatin model group?C-XBXT?.C-Model,C-Ondansetron and C-XBXT were injected intraperitoneally?i.p.?with cisplatin at the concentration of 6 mg/kg?body weight?to establish the pica models.The kaolin intake?food intake and body weight of rats were measured every 24 h from the time of kaolin introduce until the end of the experiment.The expression and co-localization of CaM and 5-HT₃R in small intestine and brain were detected by immunofluorescence.Western Blot was used to detect the expression levels of CaMKII?pCaMKII?ERK1/2 and pERK1/2 proteins in small intestine and brain.3.Wistar male rats were randomly divided into four groups:the normal control group?Control??the 1-PBG model group?P-Model??the ondansetron-treated1-PBG model group?P-Ondansetron??the XBXT-treated 1-PBG model group?P-XBXT?.P-Model,P-Ondansetron and P-XBXT were injected intraperitoneally?i.p.?with 1-PBG at the concentration of 25 mg/kg?body weight?to establish the pica models.The kaolin intake?food intake and body weight of rats were measured every 24 h from the time of kaolin introduce until the end of the experiment.The expression and co-localization of CaM and 5-HT₃R in small intestine and brain were detected by immunofluorescence.Western Blot was used to detect the expression levels of CaMKII?pCaMKII?ERK1/2 and pERK1/2 proteins in small intestine and brain.Results:1.The relative retention time and relative peak area of 6-gingerol in the freeze-dried powder samples of XBXT powder were?15.285,4.3475?.The results of relative retention time and relative peak area of shogaol were?0.376,0.0984?.The relative retention time and relative peak area of ephedrine were?17.097,43.9961?.The results of relative retention time and relative peak area of succinic acid were?7.025,3.0048?.2.The intake of kaolin in the C-Model was significantly increased at 24 h and72 h compared with the Control?P<0.05?,this indicated that the rat pica models were successfully constructed.The expressions of 5-HT₃R and CaM in the small intestine and brain tissues were significantly increased in C-Model.The expression of CaMKII?pCaMKII?ERK1/2 and pERK1/2 in the small intestine and brain tissues of the C-Model was increased?P<0.05?.Compared with the C-model,the intake of kaolin in the the C-Ondansetron and C-XBXT decreased to different degrees at 24 h and 72 h?P<0.05?.The expression of5-HT₃R and CaM in the small intestine and brain of rats were inhibited and the co-localization of the two proteins was weakened after giving ondansetron and XBXT formula.The expression of CaMKII?pCaMKII?ERK1/2 and pERK1/2 in the small intestine and brain tissues of the C-Model were decreased.3.The intake of kaolin in the P-Model was significantly increased at 24 h and72 h compared with the Control?P<0.05?,this indicated that the rat pica models were successfully constructed.The expression of 5-HT₃R and CaM in the small intestine and brain tissues were significantly increased in P-Model.The expression of CaMKII?pCaMKII?ERK1/2 and pERK1/2 in the small intestine and brain tissues of the P-Model was increased?P<0.05?.Compared with the P-model,the intake of kaolin in the the P-Ondansetron and P-XBXT decreased to different degrees at 24 h and 72 h?P<0.05?.The expression of5-HT₃R and CaM in the small intestine and brain of rats were inhibited and the co-localization of the two proteins was weakened after giving ondansitron and XBXT formula.The expression of CaMKII?pCaMKII?ERK1/2 and pERK1/2 in the small intestine and brain tissues of the P-Model were decreased.Conclusions:1.XBXT formula can inhibit the pica behavior induced by cisplatin and5-HT₃R selective agonist 1-PBG in rats.XBXT formula has a significant prevention and treatment effect on CINV,which is related to blocking 5-HT₃R2.The mechanism of XBXT preventing and controling CINV is related to regulating CaM/CaMKII/ERK1/2 signaling pathway.