Effects Of Chronic Fluoride Exposure On The Structure And Function Of Small Intestine And Colon In Mice

Epidemiological studies have shown that fluoride exposure could cause gastrointestinal discomfort symptoms,such as loss of appetite,nausea,vomiting,constipation,diarrhea and abdominal pain,and some scholars have suggested that fluoride exposure may be one of the causes of inflammatory bowel disease(IBD).Small intestine,which absorb 70-75% of the total ingested fluorine,is the main site of fluorine absorbed after fluoride was ingested,but it is still not clear whether small intestine could been damaged by excessive fluoride exposure;Colon is the main site of inflammatory bowel disease,however,the effects of fluoride exposure on it and its possible mechanisms are also poorly understood;Intestinal microorganisms,the body's largest micro-ecosystem,play vital roles in the health of the body,and an imbalances of the intestinal flora has been reported could cause inflammatory bowel disease.Therefore,this study was to investigate the effect of long-term excessive fluoride exposure on the small intestine,colon,and intestinal microorganisms.(1)Effect of fluoride exposure on small intestine and the self-recovery study of fluoride-induced impairment of small intestine in mice8-week-old C57BL/6J male mice were treated with distilled water(control group),25,50,and 100mg/L Na F distilled water for 90 days,and the duodenum,jejunum,and ileum tissues were taken for subsequent studies.The results showed that fluoride exposure impaired the morphology and ultrastructure of the small intestine,and significantly reduced the ratio of villi height to crypt depth(VH/CD)in duodenum,jejunum and ileum,suggested that fluoride exposure damaged the absorptive function of small intestine;The m RNA and protein expressions of tight junction related ZO-1 and Occludin were also notably decreased in duodenum,jejunum,and ileum,suggested that fluoride exposure impaired the barrier function of small intestine;The serum DAO,D-LA and ET levels were increased,indicated that the intestinal permeability were increased after fluoride exposure;The levels of pro-inflammatory cytokines IL-1?,IL-18 levels and the m RNA expression of IL-1?,IL-18,IL-6 and TNF? were also remarkably increased in duodenum,jejunum and ileum,indicated that fluoride exposure induced the intestinal inflammation;The m RNA and protein expressions of Caspase-1,NLRP3 and GSDMD in the duodenum,jejunum and ileum,the content of serum LDH,were also significantly increased,suggested that fluoride exposure induce the pyroptosis.In summary,this work found that long-term excessive fluoride exposure markedly disrupted the structure and intestinal epithelial barrier function of small intestine,and pyroptosis induced inflammation may contribute to this disruption.Epidemiological investigations have shown that fluorosis patients with chronic abdominal pain have been relieved from the abdominal pain after transferred to a fluorine safe environment(less than 1.5 mg /L)for a certain period of time.A previous study in our laboratory also showed that the damage of sperm induced by fluoride has a reversible tendency in male rat after recovery for 2 weeks.Therefore,we established a two self-recovery periods(15 and 30 days)model on 90-day fluoride-exposed mice to investigate whether the damage of the small intestine induced by fluoride exposure could restore.There is also 4 groups: control group and 25,50,100 mg/L Na F treatment group.After 90 days of fluoride exposure,the 3 fluoride exposure groups were drinking distilled water instead of Na F water for continuous 15 days and 30 days.The results showed that after 15 days recovery period,the damage of small intestine caused by fluoride exposure was partially relieved.After 30 days recovery period,the damage of small intestine caused by fluoride exposure was almost completely recovered to normal levels.Among the 3 regions of small intestine,duodenum seems to be more vulnerable to the fluoride exposure.We have evaluated 16 indicators in duodenum,jejunum and ileum,except for the 3 serum indicators,and after 15 days recovery,6,8 and 10 indicators have recovered in duodenum,jejunum and ileum,respectively.The histopathological analysis also showned that duodenum but not jejunum and ileum were still remained in seriously damaged condition after 15 days recovery.(2)Effect of fluoride exposure on colon and the mechanism of IL-17 A on colon injury caused by fluoride exposureEight-week-old C57BL/6J male mice were treated with distilled water(control group),25,50,and100 mg/L Na F distilled water for 90 days,and colon were taken for subsequent experiments.The results showed that fluoride exposure reduced the levels of s Ig A and mucin2,suggested that fluoride exposure damaged the colonic mucosal barrier;The m RNA and protein expressions of tight junction related ZO-1and Occludin were also notably decreased,suggested that fluoride exposure impaired the colonic physical barrier function;The levels of pro-inflammatory cytokines IL-1?,IL-18 levels and the m RNA expression of IL-1?,IL-18,IL-6 and TNF? were also remarkably increased in colon,indicated that fluoride exposure induced the intestinal inflammation in colon;The serum IL-17 Levels were also increased,the m RNA and protein expressions IL-17 A,NF-?B(p65),Caspase-1(p20),NLRP3 and GSDMD in colon were also increased,indicated that IL-17 A play a vital role in the colonic inflammation.In summary,this work suggested that fluoride exposure disrupted the colonic function and induced colonic inflammation involved in the IL17A-NF?B-GSDMD signaling pathway.To further explore the mechanism of IL-17 A in fluoride-induced colon damage,we established IL-17 A knockout mice and WT mouse models.In detail,twenty 8-week-old IL-17 A knockout mice and twenty WT mice were respectively treated with distilled water(control group)and 50 mg/L Na F distilled water,that is,totally 4 groups: WT C,WT F,KO C,KO F.After 180 days,colon tissue was taken for subsequent testing.The results showed that the levels of s Ig A and mucin2,m RNA and protein expressions of tight juction related cytokines ZO-1 and Occludin were significantly reduced in colon of WT mice,but not in IL-17 A knockout mice.Comparing the WT F group,the s Ig A and mucin2 levels,protein expressions of tight juction related cytokines ZO-1 and Occludin were significantly decreased in IL-17A-/-F group.Protein expression levels of IL-1? and IL-18,m RNA expression levels of IL-1?,IL-18,IL-6 and TNF?,m RNA and protein expression levels of IL-17 A,NF-?B(p65),Caspase-1(p20),NLRP3 and GSDMD were significantly increased in WT mice,but not in IL-17A-/-mice.And Comparing the WT F group,there pro-inflammatory cytokines and IL17A-NF?B-GSDMD signaling pathway related genes and proteins were also significantly decreased in IL-17A-/-F group.In summary,this part of work showed that IL-17 A knockout could alleviate the fluoride-induced colonic impairment,which may through the IL17A-NF?B-GSDMD signaling pathway.In order to further verify the mechanism of IL-17 A in fluoride-induced colon damage,we performed an in vitro study with human normal colon epithelial cells NCM460.Briefly,NCM460 cells were treated with 0,1,2,4 mg/L Na F for 24 hours,and the results showed that fluoride exposure significantly reduced the cell viability,damaged the colon epithelial tight junctions,secreted more pro-inflammatory cytokines,and increased the expressions of IL17A-NF?B-GSDMD signaling pathway related proteins.To explore the role of NF-?B in IL-17 A relieved fluoride-induced colon damage,NF-?B inhibitor Bay 117082 were treated on NCM460 cells,and the result showed that the protein expressions of Caspase-1(p20),NLRP3,GSDMD,IL-1? and IL-18 were significantly reduced,suggesting that IL-17 A works through NF-?B and then activates NLRP3 and Caspase-1,which lead to the GSDMD-N domain binds to cell membranes and form pores,and mature IL-1? and IL-18 cleaved by caspase1 were largely released through the membrane pore,and eventually result in excessive inflammation and colon epithelial cells damage.(3)Effects of fluoride exposure on intestinal microorganisms in WT mice and IL-17A-/-mice16S r RNA sequencing technology were used to detect the intestinal microbial diversity in WT mice and IL-17A-/-mice.The result showed that compared with the control group,fluoride exposure reduced the richness(Chao and Ace index)and diversity(Shannon index and Simpson index)of intestinal microorganisms in WT mice,but not in IL-17A-/-mice.At the phylum level,fluoride exposure reduced the ratio of Firmicutes and Verrucomicrobia,increased the ratio of Bacteroides and proteobacteria in both WT and IL-17A-/-mice.At the genus level,fluoride exposure increased the ratio of Muribaculaceae and reduced the ratio of Lactobacillus,lachnospiraceae,Dubosiella and AKKermansia in WT mice,but just increased the ratio of Muribaculaceae and reduced the ratio of Lactobacillus,lachnospiraceae in IL-17A-/-mice.Compared with WT mice,Firmicutes were increased,Bacteroides and Verrucomicrobia were decreased in IL-17A-/-mice at the phylum level,and at the genus level,Muribaculaceae were decreased,Lactobacillus were increased in IL-17A-/-mice.This work suggesting that fluoride exposure disturbed the homeostasis of intestinal microorganisms in WT mice,but IL-17 A knockout could alleviate this disturbance caused by fluoride exposure in some extent.In conclusion,chronic excessive fluoride exposure damaged the structure and function of small intestine and colon,disrupted the homeostasis of intestinal microorganisms in mice.And the impairment of small intestine caused by fluoride could be relieved after transfer to the fluoride safe(< 1.5 mg/L)environment for a certain period of time.The damage of colon induced by fluoride were involved in IL17A-NF?B-GSDMD signaling pathway and IL-17 A knockout could relieved this colon injure.Moreover,fluoride exposure reduced the richness and diversity of intestinal microflora,altered the component of intestinal microorganisms,but IL-17 A knockout could alleviate this disturbance in some extent.