Effects Of ResolvinD1 On Endoplasmic Reticulum Stress-Induced Apoptosis Of Intestinal Epithelial Cells

This study mainly investigated the effects of ResolvinD1 on the apoptosis induced by endoplasmic reticulum stress(ERS)of intestinal epithelial cells,including two parts in vitro and in vivo as follows.Experiment one: The vitro experiment aims to investigate the effects of ResolvinD1 on apoptosis of intestinal porcine epithelial cells(IPEC-J2)induced by ERS.Firstly,the IPEC-J2 cells were stimulated for the different duration(0,6,9,12,18 h)and different concentrations of tunicamycin(0,0.5,1,2 μg/mL)for screening a suitable stimulation time and concentration to build ERS model by detecting the expression of GRP-78(an ER stress marker gene).Meanwhile,The cytotoxicity of different concentrations of ResolvinD1(1、10、20、50 nM)and its impact on cell viability/proliferation were determined to screen the suitable concentration of ResolvinD1 for the following experiment.Finally,the effects of ResolvinD1 on apoptosis rate,ER stress genes(GRP78,IRE1α,PERK,ATF-6,and CHOP),and apoptosis pathway marker genes(Caspase3,BAX,Bcl-2)of IPEC-J2 cells treated with tunicamycin were furtherly explored by flow cytometry analysis,AO/EB staining,RT-qPCR,and western blotting.The results showed that the ERS model of IPEC-J2 cells could be successfully built by stimulating the cells for 9 h with 1 μg/mL tunicamycin,and the increased apoptosis and cell viability/proliferation inhibition also appeared under the ER stress condition.ResolvinD1 had no cytotoxicity and cell viability/proliferation inhibition.What’s more,1 nM ResolvinD1 significantly reduced the apoptosis and proliferation inhibition of IPEC-J2 cells induced by ER stress,and the early apoptosis rates decreased by 21.67%,the late apoptosis rates decreased by 29.67%,and the total apoptosis rates were reduced by 26.03%(P < 0.05).It also reduced the expression of GRP78 and apoptosis gene Caspase-3,increased the expression of Bcl-2(an anti-apoptotic gene),and up-regulated Bcl-2/Bax.Conclusion: ResolvinD1 can alleviate apoptosis triggered by ERS in IPEC-J2 cells.Experiment two: The present study aims to investigate the effects of ResolvinD1 on ERSinduced intestinal epithelial cells apoptosis in mice in vivo.Mice were treated with 1mg/kg tunicamycin for the different duration(8,16,24 h)for screening a suitable stimulation time to build ER stress model by detecting the expression of GRP78 and CHOP.The effects of ResolvinD1 on ER stress and associated apoptosis and intestinal barrier were furtherly explored by Tunel staining,HE staining,ELISA,immunohistochemical analysis,and RT-qPCR.The results showed that the ERS model of epithelial cells of mouse jejunum could be successfully built by stimulating the mice for 16 h with 1 mg/kg of tunicamycin.The food intake of mice decreased,the depth of jejunal recess deepened,the tight junction protein(ZO-1 and Claudin-12)decreased,the apoptosis rate of jejunum epithelial cells increased,and the result of immunohistochemical staining showed that the expression of Caspase-3 was upregulated.Tunicamycin-induced mice treated with ResolvinD1 increased feed intake by 10.05%(P < 0.05),decreased jejunum crypt depth by 15.21%(P < 0.05),and and decreased jejunal epithelial cell apoptosis by 32.28%(P < 0.05).ResolvinD1 also decreased the apoptosis rate of mouse jejunal epithelial cells and the expression of GRP78,CHOP,and Caspase-3 in mouse jejunum epithelial cells.Conclusion: ResolvinD1 can alleviate the apoptosis of mouse jejunum epithelial cells induced by ER stress.In summary,ResolvinD1 can effectively alleviate the apoptosis of enteric epithelial cells induced by er stress.