Effects Of Boron On Proliferation,Apoptosis,Barrier Function And Tissue Structure Of Rats’ Small Intestinal Epithelial Cells

To study the effects of different concentrations of Boron on the proliferation and apoptosis,barrier function and tissue structure of rat intestinal epithelial cells.In this study,IEC-6 cell line was selected as the experimental object.CCK-8 and flow cytometry were used to detect the toxic effect of Boron on IEC-6 cells,and the effects of boric acid concentration of 0.8 mmol/L and 40 mmol/L boric acid were selected as the appropriate addition amount for IEC-6 cells in vitro.Then,the PI3K/Akt signaling pathway was blocked by boron and specific blockers.The apoptosis rate,cell cycle,mRNA expression of proliferation and apoptosis related genes and the protein expression levels of SIG a,occludin and ZO-1 were detected before and after PI3K and Akt blocking respectively.On the basis of previous studies,80 3-month-old clean SD rats were randomly divided into 8 groups(n=10):blank control group and test groupⅠ,Ⅱ,Ⅲ,Ⅳ,Ⅴ,Ⅵ,Ⅶ.The control group(Boron concentration is 0 mg/L)was drinking distilled water,and the test group was drinking distilled water containing 10,20,40,80,160,320,640 mg/L Boron for 90 days.At the end of the experiment,HE staining,immunohistochemistry,ELISA and RT-PCR were used.The test results are as follows:1.Toxic effect of Boron on IEC-6 cells and its effect on proliferation and apoptosisWhen the concentration of boric acid was 0.8 mmol/L,the proliferation rate and apoptosis rate of IEC-6 cells increased significantly;when the concentration of boric acid was 40 mmol/L,IEC-6 cells had significant toxic effect,apoptosis rate increased significantly,proliferation rate decreased.2.Effects of Boron on barrier function,proliferation and apoptosis of IEC-6 cells before and after PI3K/AKT blockadeWhen IEC-6 cells were cultured in vitro,different concentrations of Boron were added to the cells.Boric acid concentration of 0.8 mmol/L could promote the proliferation of IEC-6 cells,inhibit apoptosis and improve the immune barrier function of IEC-6 cells.On the contrary,adding 40 mmol/L boric acid could inhibit the proliferation of IEC-6 cells,promote apoptosis and reduce the barrier function,resulting in obvious damage to the cells.However,the addition of PI3K/AKTblocker can not only reduce cell activity and barrier function,but also change the effect of Boron on IEC-6 cells.This indicates that Boron can affect the proliferation,apoptosis and barrier function of IEC-6 cells through PI3K/AKT signaling pathway.3.Effects of different doses of Boron added to drinking water on the morphological structure and immune function of duodenum in ratsIn the 40 and 80 mg/L groups,the height of the duodenal villi,the depth of the crypt and the V/C ratio increased,while in the 320 mg/L group,the height of the duodenal villi,the depth of the crypt and the V/C ratio decreased.In terms of histological structure,the low-dose group had complete histological structure and developed well,while the high-dose group(640 mg/L)had irregular morphology of midgut villi,and the epithelial cells at the top of villi were swollen,damaged and obviously detached.The contents of intraepithelial lymphocytes,goblet cells,SIg A,ZO-1 and Occludin in the intestinal tract increased in the low dose group,among which the contents of intraepithelial lymphocytes,goblet cells,SIg A,ZO-1 and Occludin increased significantly in the 40 and 80 mg/L groups,while the contents of gut related immune cells and gut related immunoglobulin decreased significantly in the high dose group,especially in the 640 mg/L group.The results showed that the appropriate concentration of Boron(40 and 80 mg/L)could promote the development of intestinal tract and enhance the immune function of intestinal mucosa,while the high concentration of Boron(640 mg/L)could inhibit the development of intestinal tract and even produce toxic effects on it.4.Effects of different doses of Boron added to drinking water on proliferation and apoptosis of duodenal cells in ratsThe proliferation and apoptosis of duodenal cells induced by Boron were observed by immunohistochemistry.PCNA positive cells were distributed mainly in the intestinal crypt,caspase-3 positive cells were mainly distributed in the intestinal villi.With the increase of Boron addition,the number of PCNA positive cells increased gradually,while the number of Caspase-3 positive cells decreased first and then increased.According to the mRNA expression level of proliferating gene PCNA and apoptosis key gene Caspase-3,PCNA mRNA of 20,40,80 and 160 mg/L groups was significantly higher than that of the control group,while that of 640 mg/L group was significantly lower;Caspase-3 mRNA of 20 and 40 mg/L group was significantly lower,while that of 320 and 640 Caspase-3 was significantly higher.