Protective Effect And Mechanism Of Melatonin On Intestinal Inflammation In Mice With Circadian Rhythm Disorder

Objectives:The purpose of this study was to observe the changes of intestinal flora diversity and abundance after circadian rhythm disorder in mice,to explore the effects of circadian rhythm disorder on intestinal morphology and the release of peripheral inflammatory factors,and to further observe whether the above changes were significantly improved after melatonin supplementation in the normal group and antibiotic treated group under continuous light,so as to reveal the effect of melatonin on circadian rhythm disorder The protective effect and mechanism of intestinal function in disordered model mice.Method:Adult C57BL/6J mice aged 6 weeks were randomly divided into five groups,10 individuals per group:Group I(LD group): L: D = 12:12hours,mice received basic diet and normal drinking solution for 4 weeks;Group II(LL group): continuous light,mice received basic diet and normal drinking solution for 4 weeks;Group III(LL-MT): continuous light,mice received basic diet and MT(0.2mg /ml)drinking solution for 4 weeks;Group IV(LL-ant): continuous light,mice receiving basic diet and drinking liquid containing streptomycin(1g / L),ampicillin(1g / L),gentamicin(1g / L)and vancomycin(0.5g / L)to remove intestinal bacteria for 4 weeks;Group V(MT-ant): continuous light,mice receiving basic diet and streptomycin(1g/L),ampicillin(1 g / L),gentamicin(1 g / L),vancomycin(0.5 g / L)and MT(0.2mg / ml)for 4 weeks.The mice were killed at 9:00 a.m.at the end of the experiment.The expression level of IL-6 in colon tissue and MT in serum were measured by Elisa method.The changes of intestinal morphology were observed by hematoxylin eosin staining(HE).The changes of intestinal flora were measured by 16 S r DNA sequencing and analysis.Results:1.The results of melatonin Enzyme-linked immunosorbent assay(Elisa)showed that:compared with LD group,the serum MT content of LL group decreased significantly(P < 0.05);compared with LL group,the MT expression of LL-MT group increased significantly(P < 0.05);compared with MT-ant group,the MT content of LL-ant group had no significant difference(See Table 1 and Figure 2).2.The results of hematoxylin eosin staining(HE)showed that compared with LD group,the lymphocyte infiltration in colon of LL group was obvious after 4 weeks of continuous illumination,After oral administration of MT to mice,there was no significant inflammatory change in colon of LL-MT group(See Figure 3).3.Compared with LD group,the expression of IL-6 in LL group increased significantly(P < 0.05),Compared with LL group,IL-6 in LL-mt group and LL-ant group decreased,but there was no significant difference(See Table 2 and Figure 4)..4.16 S r DNA sequencing and analysis showed that: The β diversity of LD group and LL group was significantly different in the horizontal and vertical principal coordinates,and the R value = 0.422,P < 0.05,indicating that the difference between the groups was significantly greater than that within the group,indicating that there was a significant difference between the two groups in species(See Figure5).Compared with LD group,the relative abundances of Corynebacterium,Enterobacteriaceae and Anaerobes in LL group were significantly higher,while those of Lactobacillus,Ruminococcus,Alloprevotella and Butyricimonas were also significantly higher(See Figure 6).There was a significant difference between LL group and LL-MT group in the abscissa,and R = 1,P < 0.05,indicating that the difference between the groups was significantly greater than that within the group,indicating that there was a significant difference between the two groups in species(See Figure 7).Compared with LL group,the relative abundances of Prevotella,Cladosporium,Bacteroides,Akkermansia,Parabacteroides increased,the relative abundance of Corynebacterium,Anaerobe decreased(See Figure 8).The difference of beta diversity between LL-ant group and MT-ant group was significant on the ordinate,and R = 0.379,P < 0.05,indicating that the difference between the groups was significantly greater than that within the group,indicating that there was significant difference between the two groups in species(See Figure 9).In MT ant group,the relative abundances of harmful bacteria,Pseudomonas and Aeromonas increased,while the relative abundances of beneficial bacteria,Acetatifactor increased,and the relative abundances of Prevotella decreased(See Figure 10);MT supplementation for antibiotic treated mice did not significantly improve the phase to relative abundances of Bacteroides,Akkermansia and Parabacteroides,nor significantly reduce the phase to relative abundances of Corynebacterium and Anaerobes.Conclusions:1.After the disturbance of circadian rhythm caused by continuous light,the content of serum MT decreased significantly.the serum MT could be significantly increased by continuous light plus oral MT supplementation,but the MT content was not significantly increased in antibiotic treated mice.Therefore,we proved that circadian rhythm disorder affected MT secretion,and oral MT supplementation could be absorbed and utilized to increase serum MT level,and the absorption of MT may be mediated by intestinal bacteria2.After the disorder of circadian rhythm,the lymphocyte infiltration in the local colon of mice was obvious.After MT supplementation,there was no obvious inflammatory change in the colon pathology of mice.Therefore,we speculated that circadian rhythm disorder could cause the inflammatory reaction of mice colon,and oral MT supplementation could improve the inflammatory reaction.3.The content of IL-6 in colon tissue of mice increased significantly after the disorder of circadian rhythm,therefore,we speculated that circadian rhythm disorder could promote the occurrence of colitis in mice.4.After the disorder of circadian rhythm,the abundance of colonic contents was increased,and that of harmful bacteria Ruminococcus,Corynebacterium,Enterobacter and Anaerobe were increased,After MT supplementation,the abundance of beneficial bacteria,such as Prevotella,Cladosporium,Bacteroides,Akkermansia,Parabacteroides increased significantly;the relative abundance of Corynebacterium,Anaerobe decreased,but that of Escherichia/Shigella increased;MT supplementation for antibiotic treated mice did not significantly improve the phase to reletive abundances of Prevotella,Cladosporium,Bacteroides,Akkermansia and Parabacteroides,nor significantly reduce the phase to relative abundances of Corynebacterium and Anaerobes.Therefore,it can be inferred that the intestinal flora disorder of mice after circadian rhythm disorder,oral MT supplementation can partially improve the symptoms of intestinal flora disorder,and the intestinal flora of mice played a part in the process of melatonin playing its beneficial effect.In conclusion,the inflammatory response of colon in mice was aggravated after circadian rhythm disorder.Oral administration of MT can regulate intestinal flora of mice,and MT may play a part of its beneficial role through intestinal flora,so as to provide new ideas for clinical treatment.