Effects Of Different Tones Of Music On The Intestinal Barrier And Intestinal Flora Of Mice During The Growth Period

Enriched environments can improve the welfare of domesticated animals and enhance organismal immune function.Music is one of the means of environmental enrichment that enhances gut barrier function in humans,non-human primates,and rodents.However,it needs to be clarified whether this effect is related to the pitch of the music.In this experiment,we investigated the effects of different music tones on intestinal function in mice from the perspectives of the intestinal barrier and intestinal flora.Six hundred female Kunming White mice were selected and divided into four groups: control group(no additional sound added,ambient sound less than 40 d B),D-tone music group(group D,65 d B),G-tone music group(group G,65 d B)and A-tone music group(group A,65 d B),which were housed in four isolated rearing rooms.From 1 day of age,the treatment groups were musically stimulated for 2 h(20:00-22:00)daily for6 weeks using Mozart K.448 music in the keys of D,G,and A.Duodenal,jejunal and ileal tissues and ileal contents were collected from 42-and 63-day-old mice and changes in intestinal tissue morphology and its structure were observed by HE staining.The expression levels of intestinal tissue tight junction proteins and mucins were detected by immunofluorescence,and real-time fluorescence quantitative PCR was performed to detect the expression levels of tight junction proteins and mucins,heat shock proteins,immunoglobulin A,immunoglobulin G,inflammation-related factors in intestinal tissues.The expression levels of endotoxin,DAO,and D-lactate in serum were measured by Elisa,and the effects of different tones of music on the intestinal barrier and intestinal flora of mice were analyzed by 16 S r DNA sequencing and analyzing the structure of ileal intestinal flora.The experimental results showed that:(1)The tissue sections of jejunum and ileum of mice showed that on the 42 nd and 63 rd days of music stimulation,intestinal villi were distributed on the mucosal surface of both music and control groups,with neat distribution of villi and normal morphological structure,and no significant abnormalities were observed.(2)Analysis of m RNA expression results of intestinal tight junction proteins and mucins showed that the expression levels of Claudin-1,Claudin-12,Occludin,ZO-1,ZO-2,and Mucin2 m RNA were significantly increased in duodenal,jejunal,and ileal,tissues on day 42 of music stimulation in group D and group A(P < 0.05),and group G Occludin m RNA expression levels were significantly elevated(P < 0.05).On day 63 of music stimulation,the m RNA expression levels of Claudin-1,Claudin-12,Occludin,ZO-1,ZO-2,and Mucin2 were significantly increased in duodenal,jejunal,and ileal tissues in group D(P < 0.05),Claudin-12,Occludin and ZO-2m RNA expression levels in group A were significantly Claudin-12,Occludin and ZO-2 m RNA expression levels were significantly increased in group A(P < 0.05)and Claudin-1 m RNA expression levels were significantly increased in group G(P < 0.05).Immunofluorescence results were similar to the PCR results.On day 42 of music stimulation,the expression levels of Claudin-1,Mucin2,Occludin,and ZO-1 proteins were significantly increased in duodenal,jejunal,and ileal tissues in group D(P < 0.05),and Claudin-1,Occludin,and ZO-1 proteins were significantly increased in groups A and G(P < 0.05).On day 63 of music stimulation,the expression levels of Claudin-1,Mucin2,Occludin,and ZO-1 proteins were increased in duodenal,jejunal,and ileal tissues in group D(P < 0.05),Claudin-1,Occludin,and ZO-1 proteins in group A(P < 0.05),Mucin2,Occludin,and ZO-1 protein expression levels were significantly increased in group G(P < 0.05).(3)On days 42 and 63 of music stimulation,the m RNA expression levels of IFN-γ and IL-1βwere significantly increased in the duodenum,jejunum,and ileum in groups D,A,and G(P <0.05),the m RNA expression levels of NF-κB and IKK-α were significantly decreased in groups D,A and G(P < 0.05),and the m RNA expression levels of TLR4 and TLR2 in group D were significantly lower(P < 0.05).(4)The results of HSPs m RNA expression showed that the m RNA expression levels of HSP90 in the duodenum of group D,group A,and group G were significantly reduced(P < 0.05).The m RNA expression levels of HSP60 and HSP70 in group D were significantly reduced(P < 0.05)on days 42 and 63 of musical stimulation.The m RNA expression levels of HSP60,HSP70,and HSP90 in group D’s jejunum were significantly lower(P < 0.05).In the ileum,the m RNA expression levels of HSP60 and HSP90 in group D were significantly reduced(P < 0.05).(5)Immunoglobulin-related genes showed that the m RNA expression levels of Ig A and Ig G in duodenal,jejunal,and ileal tissues were significantly increased in group D on days 42 and 63 of musical stimulation(P < 0.05).(6)Serum intestinal permeability indexes showed that the serum levels of DAO,D-lactate,and Endotoxin were significantly reduced on days 42 and 63 of musical stimulation(P < 0.05).(7)Ileal contents were analyzed by 16 S r DNA sequencing.It was found that music of different tones did not significantly affect the abundance and diversity of mouse intestinal flora,i.e.,there was no significant difference in Chao1 and Shannon index after 63 days of music treatment.Pcoa results showed no significant separation between the three groups.At the phylum level,music reduced the abundance of the phylum Firmicutes(P < 0.05),but the abundance of the phylum Actinobacteria and the phylum Bacteroidota increased both(P < 0.05).At the genus level,music exposure increased the abundance of Lactobacillus and decreased the abundance of Enterorhabdus(P < 0.05).In conclusion,(1)Music did not significantly affect the response of intestinal mucosa in terms of its integrity;the results of real-time quantitative PCR and Western Blot showed that Mozart music reduced the inflammatory response of the intestine by decreasing the expression of intestinal NF-κB pathway genes.(2)Music can significantly increase the expression levels of intestinal Claudin-1,Claudin-12,Occludin,ZO-1,ZO-2,Mucin2 and immunoglobulins,and decrease the expression levels of serum DAO,D-lactate and Endotoxin,indicating that music as an enriched environment can significantly improve the barrier function of the intestine and decrease intestinal permeability.(3)Musical stimulation did not affect the diversity of intestinal flora,but increased the abundance of beneficial bacteria and decreased the abundance of harmful bacteria at the phylum and genus levels.