Study On The Intestinal Cytoskeleton Remodeling And Zonula Occludens 1 Impairment Induced By High Fluoride

In recent years,with the rapid development of industry and agriculture,fluorine pollution in the natural environment has become increasingly severe.Fluorine is one of the trace elements necessary for the growth and development of livestock and poultry,which enters the body of livestock through the digestive tract,respiratory tract or skin.Fluoride is beneficial for the growth and development of bones at low concentrations,while excessive fluoride intake triggers various organ injuries and functional disorders.Over 60%fluoride is absorbed through the digestive tract,and the impairment to intestinal barrier caused by high fluoride has received extensive attention,however,the underlying mechanism is still obscure.In this study,Caco-2 cell line was used to construct the intestinal epithelial barrier model in vitro,validated the toxic effect of high fluoride using CCK-8 assay and flow cytometry,and optimized the experimental conditions;combined with transmembrane resistance technology,dextran method,calcium ion fluorescence probe technology,Western blot,multiple fluorescent immunostaining and other technical means,to reveal the impaction of fluoride on tight junction（TJ）and the actin cytoskeleton,and further explored possible regulatory pathways as well as the improvement effect of Lactobacillus johnsonii BS15 on intestinal barrier damage induced by high fluoride.The main research findings are as follows:1.The results showed that the cell survival rate gradually decreased with the increase of Na F dose according to the CCK-8 assay.The cell survival rate in the 3.2 m M dose group was significantly reduced（P<0.01）,but still maintained a relatively high cell viability（0.8328±0.061）.Inverted microscopy observation revealed cell swelling and rounding with the appearance of vacuoles in the cytoplasm.Flow cytometry data showed a significant increase in cell apoptosis rate in the Na F group（P<0.01）.2.Barrier permeability results showed that TEER of Na F group was significantly decreased compared to the control group（P<0.01）.The permeability of FD-4 in the 1.6 m M Na F group was not significantly different from the control group（P>0.05）,which was significantly increased（P<0.01）after 3.2 and 6.4 m M Na F treatment.Immunoblotting results showed that the expression of ZO-1 in the 3.2 and 6.4 m M Na F groups was significantly decreased compared to the control group（P<0.01）.Confocal microscopy observed that the intracellular localization of ZO-1 was altered and its continuity was wrecked.Considering the results in（1）,3.2 m M Na F was the optimal dose.3.Western blot results showed that the expression level of p-MLC2 in the 3.2 m M group was significantly increased compared to the control group（P<0.01）.Blebbistatin reversed Na F-induced TEER reduction and FD-4 permeability elevation（P<0.001）,restoring them to control levels（P>0.05）,while Ionomycin caused a comparative increase in paracellular permeability（P<0.0001）.The F-actin was neatly arranged around plasma membrane in control group by Immunofluorescence.After Na F or Ionomycin treatment,the fluorescence intensity of F-actin was significantly increased（P<0.001）,accompanied by the formation of stress fibers.Meanwhile the continuity of the ZO-1 structure was disrupted.Blebbistatin inhibited the polymerization of F-actin and restored the linear connection of ZO-1.4.Compared with the control group,the expression levels of Rho A,ROCK,and MLCK were strikingly elevated（P<0.01）in the 3.2 m M group.The immunofluorescence results were consistent with Western blot.In the control group,the fluorescence signals of these proteins showed irregular diffuse distribution.After Na F treatment,the fluorescence intensity of Rho A,ROCK,and MLCK significantly increased（P<0.001）.The addition of inhibitors restored the linear coherent distribution of ZO-1,inhibited the aggregation of stress fibers,and the fluorescence intensity of F-actin in inhibitor groups did not differ significantly from that of the control group（P>0.05）,but was strikingly lower than that of the Na F group（P<0.0001）.Barrier permeability results showed that inhibiting Rho/ROCK or MLCK could significantly ameliorate the Na F-induced TEER reduction and FD-4 permeability increase（P<0.05）.5.Confocal microscopy revealed a significant increase in Ca²⁺fluorescence intensity,consistent with the results of flow cytometry.The Ca²⁺fluorescence intensity in the Na F group was significantly higher than that in the control group（P=0.002）.6.Flow cytometry results indicated that the fluorescence intensity of[Ca²⁺]_i in the BAPTA-AM group was dramatically decreased compared to the Na F group（P=0.0115）,while there was no obvious discrepancy between the EGTA group and the Na F group（P=0.4132）.TEER and FD-4 experiments demonstrated that BAPTA-AM could reverse Na F-triggered barrier permeability increase（P<0.0001）and restore it to the control group level（P>0.1）.In contrast,the barrier function after EGTA pretreatment was even worse than that of the Na F group（P<0.05）.Confocal images indicated that the BAPTA-AM promoted normal connections of ZO-1 and regular arrangement of F-actin,impeding stress fibers formation.The F-actin fluorescence intensity in BAPTA-AM group had no apparent discrepancy compared to the control group（P>0.05）,which was noticeable lower than the Na F group（P<0.0001）.7.The TEER results showed that the addition of Lactobacillus johnsonii BS15 in vitro at concentrations of 1×10⁶,1×10⁷ and 1×10⁸ CFU/m L was not effective in improving the elevated intestinal barrier permeability caused by Na F.Collectively,this study demonstrated that high fluoride exposure activated the Rho A/ROCK pathway and MLCK by releasing intracellular calcium ions,which synergistically phosphorylating MLC2 to trigger F-actin and ZO-1 remodeling,eventually inducing epithelial barrier impairment.