Protective Effects And Mechanisms Of Sympathetic Nerve On 5-Fluorouracil-Induced Intestinal Stem Cells Damage

Objective:To explore the protective effects and mechanisms of sympathetic nerve on 5-flurouracil-induced intestinal stem cells damage,and to study the application value of sympathetic neuroamines in gastrointestinal syndrome treated by 5-FU.Methods:Eight-week old male C57BL/6 mice were randomly divided into 4 groups:Control group,5-fluorouracil(5-FU)group,isoproterenol(ISO)group and5-FU+ISO group.The changes in the number of Olfm4~+intestinal stem cells,lysozyme~+penney cells and PCNA~+proliferating cells in jejunum were observed and analyzed by immunohistochemistry.TUNEL method was used to detect the distribution and number of apoptotic cells in intestinal crypt in different groups.On day 3.5 and 7 after 5-FU/ISO treatment,jejunum was taken and intestinal stem cells were separated.RNA and protein were measured through real-time fluorescent quantitative PCR and western blot to analyze apoptotic genes(Bcl-xl,Puma and Bax,etc.),PI3K/Akt signaling pathway(phosphorylated Akt,etc.)and the NF-?B signaling pathways.Results:1.Compared with the control group,the body weights of mice were significantly decreased after 5-FU treatment,which reached the lowest point on day3.5(P<0.01),and returned to the normal level on the 7th day after treatment.2.Compared with the control group,intestinal mucosa in the 5-FU group showed congestion and hemorrhage,inflammatory cell infiltration,villus shortening and decaying,epithelial cell necrosis and shedding,and lamina propria exposure,etc.The damage was obviously observed on day 3.5 and alleviated on day 7.Compared with the 5-FU group,the mucosal injuries induced by 5-FU treatment were attenuated under ISO administration.3.Compared with the normal group,the 5-FU group significantly reduced the number of intestinal stem cells(P<0.01)and the density of tyrosine hydroxylase(TH)positive protein staining(P<0.05),and the intestinal stem cells returned to normal level after ISO treatment(P<0.01).Compared with the 5-FU group,the density of TH positive protein staining in the 5-FU+ISO group was significantly increased(P<0.05).4.Compared with the control group,the 5-FU group could significantly reduce the number of Paneth cells,which was the most obvious on day 3.5(P<0.01).Under ISO treatment,the numbers of Paneth cells were significantly recovered and returned to the normal levels on day 7 after the treatment(P<0.05).5.Compared with the normal group,the 5-FU treatment group significantly increased the number of PCNA positive cells in the jejunal crypt(P<0.01)and the number of apoptotic cells(P<0.05),and the number of proliferative cells and apoptotic cells significantly decreased after ISO treatment(P<0.05).6.Compared with the normal group,the expressions of pro-apoptotic genes PUMA and Bax in the 5-FU group were significantly increased(P<0.05),and the mRNA expressions of these two genes were significantly decreased after ISO treatment(P<0.01).Compared with the normal control group,the relative expression of PGES1 in the 5-FU treatment group was significantly increased(P<0.01)and significantly decreased after ISO treatment(P<0.05).Compared with the normal control group,the relative expression levels of PGES2 and PGES3 in the ISO group were significantly higher(P<0.05).Compared with the 5-FU group,the relative expression of PGES2 in the 5-FU+ISO group was significantly lower(P<0.05).7.The expression level of p-p65 protein in the intestinal crypt was detected by immunohistochemistry and Western Blotting.Compared with the normal control group,the expression level of p-p65 protein in the 5-FU group was significantly increased(P<0.01),and the expression level of p-p65 protein was significantly decreased after ISO treatment(P<0.01).8.The p-Akt protein expression in the intestinal crypt was detected by immunohistochemistry and Western Blotting.Compared with the normal control group,p-Akt protein expression was significantly increased in the 5-FU group(P<0.01),and significantly decreased in the ISO treatment group(P<0.01).Conclusion:1.Chemotherapy drug 5-FU causes intestinal sympathetic nerve injury and decreases numbers of intestinal stem cells along with intestinal mucosal damage.2.Sympathetic nerve regulates the proliferation and differentiation of intestinal stem cells to repair intestinal damage.3.The damage of 5-FU on intestinal stem cells and the protective effects of isoproterenol may be achieved by regulating the signal pathway of NF-?B and PI3K-Akt to affect the apoptosis and proliferation of intestinal stem cells.