The Effects Of HEP On The Apoptosis Of IPEC-J2 Cells Under Oxidative Stress

In this study,the Hericium erinaceus polysaccharide(HEP)was used as the raw material.Hericium erinaceus polysaccharide poly(lactic-co-glycolic acid)nanoparticles(HEP-PLGA-NPs)were prepared by emulsion-solvent evaporation method.The preparation process of HEP-PLGA-NPs was optimized by response surface methodology,and the nano particles with the highest encapsulation rate were obtained.The Caco-2 cells were selected as the research object and the intestinal epithelial models was constructed using the 12-well Transwell.The safety concentration of HEP and HEP-PLGA-NPs was screened by MTT method.The absorption of HEP and HEP-PLGA-NPs with different dosing time and concentration on intestinal epithelial models was compared,and The effect of absorption of HEP-PLGA-NPs on intestinal epithelial cell at different environmental temperatures were investigated.The expression of P-glycoprotein(P-gp)was detected by Western Blot and the influence of Rhodamine 123 was detected by flow cytometry in the Caco-2 cell models after HEP and HEP-PLGA-NPs treatment.The results are as follows:(1)Through response surface methodology to optimize the preparation technology of HEP-PLGA-NPs,the best preparation conditions were obtained:O:W1 was 8.35,W2:PE was 7.24,and the concentration of PLGA was 23.52 mg/mL.Under these conditions,the theoretical encapsulation efficiency of HEP-PLGA-NPs was 91.81%.Scanning electron microscopy and transmission electron microscopy showed that the particles were spherical,smooth and round,and there was not adhesion between particles.The results of Zeta potential and particle size distribution show that the particle size is 184.5±11.98 nm,and the Zeta potential is-28.33± 0.186 mV.(2)After Caco-2 cells were incubated for 21 days,microscopic observation showed that the cells grew tightly,and the morphology was intact.We could preliminarily conclude that the Caco-2 cell model was successfully established.The alkaline phosphatase(AKP)activity on the sides of AP and BL were detected.The results showed that the AKP activity on the AP side was 5.11 times higher than the BL side,indicating that there was a significant difference in the AKP activity between the AP side and the BL side.After Caco-2 cells were incubated for 21 days,TEER was 507±23.07 Ω.The transmittance of the phenol red decreased with the culture time,and the transmittance was 0.60%at 14 days,then decreased slowly,and became stable.Until 21 days,the monolayer structure was considered to be enough tight.(3)The concentrations of HEP and HEP-PLGA-NPs were not cytotoxic in the range of 0.2-100 μg/mL by MTT test.In the safety concentration,25 μg/mL,50 μg/mL,100 μg/mL were selected for a follow-up test.In the time of different concentrations,the transport of HEP and HEP-PLGA-NPs was time dependent.And the Papp of the HEP-PLGA-NPs group at the corresponding time was significantly greater than that of the HEP group.The PDR of 25 μg/mL,50 μg/mL,100 μg/mL HEP and HEP-PLGA-NPs are less than 1.5.And the PDR of the corresponding concentration of HEP is greater than that of HEP-PLGA-NPs group.At 37℃ and 4℃,the absorption of 25μg/mL,50 μg/mL,100 μg/mL HEP-PLGA-NPs on Caco-2 cell model was dose dependent.The Papp was significantly higher than 4℃ at 37℃.(4)The results of Western Blot showed that both HEP and HEP-PLGA-NPs significantly increased the expression of P-glycoprotein(P<0.01).There was a significant positive correlation between the concentration of HEP and the expression of P-glycoprotein(P<0.01),and the concentration of HEP-PLGA-NPs showed a significant negative correlation with the expression of P-glycoprotein(P<0.01).The results of flow cytometry showed that The average fluorescence intensity of HEP-PLGA-NPs group was higher than HEP group,The concentration of HEP-PLGA-NPs showed a significant positive correlation with the mean fluorescence intensity.The above results showed that the HEP-PLGA-NPs were prepared by the emulsion-solvent evaporation method through response surface methodology to optimize.It has small particle size,uniform distribution,high entrapment efficiency,good stability and other advantages.The transport of HEP-PLGA-NPs was time dependent,concentration dependent and energy dependent.The absorption of HEP and HEP-PLGA-NPs is dominated by passive transport,and there is a active transport process by protein mediated.Nanoscale can effectively increase the uptake and transport rate of HEP on the Caco-2 cell model.Both HEP and HEP-PLGA-NPs significantly increased the expression of P-glycoprotein,HEP increased the expression of P-glycoprotein and the efflux function to a greater extent than HEP-PLGA-NPs,indicating that absorption of HEP-PLGA-NPs was better than HEP.