| Objective:Clinical and animal studies have demonstrated the efficacy of 6-gingerol in the control of chemotherapy-induced nausea and vomiting(CINV),and its mechanism is related to the blockade of 5-hydroxytryptamine 3 receptors(5HT3R).Since 5-HT3R-mediated emesis is mainly regulated by the Ca2+/calmodulin kinase II(CaMKII)/extracellular regulated kinase 1/2(ERK1/2)signaling pathway,the present work investigated the mechanism of 6-gingerol in the prevention and treatment of CINV by focusing on the 5HT3R-mediated Ca2+/CaMKII/ERK1/2 signaling pathway in vivo and ex vivo experiments,using a cisplatin-induced rat model and an NG108-15 cell model.Methods:(1)Chemotherapy-induced rat pica model:Male Wistar rats were randomly divided into normal control group,cisplatin model group,6-gingerol treated group and positive drug treated group,6 rats in each group.At 8:00 AM on the day of modeling,all rats were injected intraperitoneally with 6 mg/kg of cisplatin,except for the normal control group,which was injected intraperitoneally with an equal volume of saline,to establish rat pica model.One hour before modeling(7:00 AM),6-gingerol treated group was gavaged with 6-gingerol 50 mg/kg,positive drug treated group was gavaged with ondansetron 1.3 mg/kg,normal control group and cisplatin model group were gavaged with equal volume of vehicle medium(3%Tween 80),and the above doses were repeated once at 7:00 PM.The amount of kaolin intake,food intake and body weight of the rats were recorded daily.After 24 h of moulding,the ileum and medulla oblongata of the rats were removed after cervical dislocation.The levels of 5-HT3R,calmodulin(CaM),CaMKII and its phosphorylation(p-CaMKII),ERK1/2 and its phosphorylation(p-ERKl/2)and β-arrestin 2 in ileum and medulla were measured by western blot(WB)technique.The expression and distribution of 5-HT3R and p-ERK1/2 proteins in ileal and medullary tissues were examined by Immunofluorescence(IF).(2)Cellular experiments:NG108-15 cells are an ideal cellular model for studying 5-HT3R and 5-HT3R-mediated signaling pathways because there with 5-HT3R on them.The selective 5-HT3R agonist 2-Me-5-HT was used as a tool drug to investigate the regulation of 5-HT3R-mediated Ca2+/CaMKII/ERK1/2 signaling pathway by 6-gingerol at the cellular level.The intracellular Ca2+level([Ca2+]i)was measured by flow cytometry using Fluo-3/AM as fluorescent indicator;the expression levels of 5-HT3R,CaM,CaMKII,pCaMKII,ERK1/2,p-ERK1/2 and β-arrestin 2 in cell lysates were measured by WB technique.The expression and distribution of 5-HT3R and p-ERK1/2 proteins were measured by immunofluorescence technique.Results:(1)Behavioral resultsCompared with control group,kaolin intake of cisplatin model group was significantly increased(P<0.001),both food intake and body weight were significantly decreased(P<0.001);compared with cisplatin model group,kaolin intake of 6-gingerol treated group was significantly decreased(both P<0.001)and food intake was significantly increased(P<0.05,while there was no significant improvement on body weight(P>0.05).(2)[Ca2+]ilevels of NG108-15 cells2-Me-5-HT could significantly increase[Ca2+]i levels in NG108-15 cells(P<0.001);6-gingerol could inhibit the 2-Me-5-HT-mediated increasement of[Ca2+]ilevels in NG108-15 cells(P<0.001).(3)Results of WB detectionCompared with control group,the expression of 5-HT3R,CaM,pCaMKII,p-ERK1/2 and β-arrestin 2 protein in ileum and medulla oblongata of rats of cisplatin model group was significantly increased(all P<0.05).Compared with cisplatin model group,the expression of 5-HT3R,CaM,pCaMKII,p-ERK1/2 and β-arrestin 2 proteins of 6-gingerol treated group was significantly decreased(all P<0.05).The expression of 5-HT3R,CaM,p-CaMKII,p-ERK1/2 and β-arrestin 2 proteins was significantly increased in NG108-15 cells after administration of 2-Me-5-HT(P<0.05).6-gingerol reversed the higher expression of 5-HT3R,CaM,p-CaMKII,p-ERK1/2 and β-arrestin 2 protein in NG108-15 cells treated with 2-Me-5-HT(P<0.05).(4)Immunofluorescence results of ileal and medullary tissues detectionCompared with the normal control group,the fluorescence intensity of 5HT3R and p-ERK1/2 was significantly higher in the ileum and medulla of the rats in the cisplatin model group(both P<0.05).5-HT3R was mainly expressed in the ileal mucosa,ileal submucosa and the area postrema and nucleus of the solitary of the medulla,and p-ERK1/2 was mainly expressed in the ileal mucosa,ileal submucosa and the raphe nuclei of the medulla.Compared with the cisplatin model group,the fluorescence intensity of 5HT3R and p-ERK1/2 protein was significantly decreased in the ileum and medulla oblongata of the 6-gingerol-treated group(both P<0.05).(5)Immunofluorescence results of NG108-15 cell detection2-Me-5-HT significantly increased the fluorescence intensity of 5-HT3R and p-ERK1/2 protein in NG108-15 cells(P<0.001).6-gingerol reversed the increasement of 5-HT3R and p-ERK1/2 protein fluorescence intensity in NG108-15 cells treated with 2-Me-5-HT(P<0.01).Conclusion:The present study has again demonstrated that 6-gingerol could improve cisplatin-induced pica in rats,suggesting its definite effect against CINV.The results of this study suggest that the definite effect against CINV of 6-gingerol is associated with the modulation of the 5-HT3R-mediated Ca2+/CaMKII/ERK1/2 signaling pathway. |
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