Mechanism Of ω-3Fish Oil Improving Parenteral Nutrition-Associated Liver Disease And Its Endoplasmic Reticulum Stress At Cellular Level

Objective：By cultivating newborn rabbit primary hepatocytes, and establishing the cellularmodel of parenteral nutrition-associated liver disease (PNALD)—hepatocyte fattydegeneration model, to study the expression changes of endoplasmic reticulum stresschaperone protein-glucose regulatory protein94(GRP94) in newborn rabbit hepatocytes,to explore the role of GRP94in the pathogenesis of PNALDand try to reveal themechanism on the-3fish oil how to improve PNALD at cellular level, and so as toprovide new ideas and experimental basis for clinical prevention and treatment of PNALD.Materials and Methods:Eight newborn New Zealand rabbits were randomly picked without genderdiscrimination., breast-fed for7days, raised in constant temperature warm box withtemperature between26-28℃and relative humidity between40%-60%. Liver tissues wereremoved under general anesthesia, liver cells were acquired by type I collagenase digestion for theisolation of hepatocytes for primary culture, and then sub-cultured. Hepatocytes wererandomly divided into five groups: cultivated with DMEM medium (group1)、0.2%fatemulsion (group2)、0.4%fat emulsion (group3)、1%fat emulsion (group4) and2%fatemulsion(group5) respectively. We then observed the accumulation of lipid droplets ineach group’s hepatocytes by light microscopy after oil red-o staining and tested cellsactivity with MTT at different times (24h,48h,72h,96h), and in order to choose theoptimal concentration of fat emulsion for PNALD cellular model.Furthermore, hepatocytes were randomly divided into five groups: control group(CON group)、1%-3fish oil fat emulsion group(A group)、0.4%-3fish oil fatemulsion group (B group)、1%fat emulsion group (C group)、0.4%fat emulsion group (D group)，and five groups were cultivated with DMEM medium、1%-3fish oil fatemulsion、0.4%-3fish oil fat emulsion、1%fat emulsion and0.4%fat emulsionrespectively. Hepatocytes of every group were collected and we observed the accumulationof lipid droplets by light microscopy after oil red-o staining, cells activity with MTT atdifferent times （24h,48h,72h,96h）respectively; biochemistry indicators of the everygroup cultured fluid were determined using automatic biochemistry analysis apparatus at atdifferent times (0h,24h,48h,72h); GRP94mRNA were detected by semi-quantitativereverse transcription polymerase chain reaction (RT-PCR).; immunohistochemistry wasused to test GRP94protein.Results:1.Hepatocytes in primary culture:8.82×105hepatocytes were acquired,and the cellsurvival rate was92.11%with trypan blue staining. The cultured cells began to grewagainst the wall of flask after4hours,90%of the cells were adhered after96hours,andformed a monolayer after8~10days.we could see large amounts of dyed red glycogenparticles by light microscopy after PAS staining..2.Concentration of fat emulsion in our modle: Examination of the OD values withMTT method showed that on the level of MTT OD values there were significantdifferences among the five groups (P <0.01) at each time point (24h,48h,72h,96h).Compared with group1, OD values of group5decreased significantly at every timepoints (24h,48h,72h,96h)(P<0.01); group4decreased significantly only at96h (P<0.01). there were no significant changes in the other three time points (P>0.05); eithergroup2or group3decreased significantly at every time points (24h,48h,72h,96h)(P>0.05).3.Changes on hepatocytes morphology, vitality and ultrastructure:Examination of theOD values with MTT method showed that there were significant differences among thefive groups only at96h(P <0.01), Compared with CON、A、B and D group, group Cdecreased significantly (P<0.01). lipid droplets began to appear in group A after24hours,and then increased over time; endoplasmic reticulum expaned minorly, the number ofmitochondria decreased, which still have a lot of crests; lipid droplets began to appear ingroup B after96hours,while there no obvious changes in ultrastructure; in group C,thereare a lot of lipid droplets and obvious change in ultrastructure after24hours:endoplasmicreticulum expansion, mitochondria vacuoles change, its crest was obvious minus. lipid droplets began to appear in group D after24hours, and then increased over time;endoplasmic reticulum expaned minorly.4.The level of medium biochemical indexes: on the levels of the TBIL,DBIL,ALT,AST,γ-GT,ALBand LDH, there were significant differences among the fivegroups (P <0.01) at every time point (24h,48h,72h). However, on the levels of TP,TGand PA, there were no significant differences among the five groups (P>0.05).Compared with CON、A、B and D group, group C had an obvious elevation of TBIL,DBIL,ALT,AST,γ-GT,ALBand LDH, and the difference was statistically significant(P<0.01). While, the ALB significantly reduced, the difference was statistically significant(P<0.01).In group CON, A, B, and D, On the levels of TBIL,DBIL,ALT,AST,γ-GT,LDH,TP,TG,PA there were no significant differences (P>0.05) at every time point (0h,24h,48h,72h). While on the levels of TBIL,DBIL,ALT,AST,γ-GT,LDH, group C hadsignificant differences (P <0.01).While On the levels of TP, TG, PA,there was nosignificant differences (P>0.05). In group C the level of ALT,AST, TBIL,DBIL,γ-GT,LDH and ALB significantly increased (P <0.01) over time, the trend of ALBsignificantly decreased (P <0.01).5.Expression of Endoplasmic reticulum stress marker protein-GRP94mRNA: Theresults in RT-PCR showed that the expression of GRP94mRNA in group C significantlyincreased compared with group CON，A, B and D (P <0.01). There were no significantdifferences among groups A, CON, B and D (P>0.05). The expression of GRP94mRNAin group CON、A、B and D had no significant difference (P>0.05). In group C theexpression of GRP94mRNA significantly increased (P <0.01) over time.6.Expression of Endoplasmic reticulum stress marker protein-GRP94protein: Theimmunohistochemistry results showed that the expression of GRP94protein in group Csignificantly increased compared with group CON，A, B and D (P <0.01). There were nosignificant differences among group A, CON, B and D (P>0.05). In group C theexpression of GRP94mRNA significantly increased (P <0.01) over time.Conclusion:1.The experiment cultivated the original generation of the newborn rabbit liver cellssuccessfully;2.This experiment develop liver cells by different concentrations of fat emulsion,screened the optimal concentration of fat emulsion,and laid a foundation for further study of cell experiment;3.The experiment established the cellular model on newborn rabbit of parenteralnutrition-associated liver disease successfully-fatty degeneration of hepatocytes, and laid afoundation for further study of its pathogenesis.4.The expression of GRP94mRNA and protein significantly increased when comparedwith group CON in hepatocytes cultivated with concentration of1%fat emulsion, revealedthat ERS were related with the occurrence and development in PNALD;5.Replaced ordinary fat emulsion by-3fish oil lipid emulsion could effectivelyimprove the PNALD, and it might play a role by inhibiting the protein-GRP94involved inendoplasmic reticulum stress in liver cells.