| Objective: To establish the cellular model of parenteral nutrition-associated liver disease(PNALD),hepatocyte fattydegeneration model by cultivating normal rat hepatocytes,disturb PNALD cellular model with IRE1? specific inhibitor STF-083010,as IRE1?/XBP1 is one of ERS`s key pathways and probe ERS`s function in PNALD pathogenesis so as to provide some inspirations for clinical precaution and therapy of ameliorating PNALD on cell level,based on previous basic study.Methods:First,hepatocytes were randomly assigned to control group and experimental group.Control group was cultivated in RMPI-1640 medium and experimental group was cultivated in fat emulsion with culture medium dilution.Cells in the same state were added to 0.4% fat emulsion,1.0% fat emulsion,2.0% fat emulsion,4.0% fat emulsion,respectively.In view of obvious lesions and higher cell vitality,we select 1.0% fat emulsion as the best choice for further study.Then,according to the scheme above,the hepatocytes were collected at 0h,12 h,24h,36 h,respectively and cell was viability detected with CCK-8.We observed the accumulation of lipid droplets and morphology of hepatocytes by oil red-o staining;RT-PCR was then performed to quantify the mRNA expression of GRP78,IRE1?,sXBP1,JNK in each group's hepatocytes.Western blot was used to examine protein expression of GRP78,IRE1?,p-IRE1?,sXBP1,JNKand p-JNK.Next,a series of concentrations of STF-083010(0,10,25,50,75,100,125?M)(grouped into A(control group),B,C,D,E,F,G)disturbing PNALD cellular model were added with fat emulsion(1%).Cell viability was determined by CCK-8 assay and the mRNA expression of sXBP1 was performed by RT-PCR at 24 h so as to decide the optimal concentration of inhibitor STF-083010 for PNALD cellular model at which the expression of sXBP1 mRNA can be effectively suppressed and cell viability declined to the minimum extent.For further analysis,cultured cells were treated with RMPI-1640 medium(group CON),soybean-based intravenous lipid emulsions(Group SLE),soybean-based intravenous lipid emulsions plus STF-083010 of the optimal concentration(group SLE+STF)and soybean-based intravenous lipid emulsions plus dimethyl sulfoxide(equal volume with STF)(group SLE+DMSO),respectively.Group CON was cultured in RMPI medium,and other groups was cultured in 1% fat emulsion,and 50?M inhibitor was added.Hepatocytes of every group were collected and we observed the accumulation of lipid droplets by light microscopy after oil red-o staining;RT-PCR was then performed to quantify the mRNA expression of GRP78,IRE1?,sXBP1,JNK in each group's hepatocytes.To analyze whether GRP78,IRE1?,p-IRE1?,sXBP1,JNK and p-JNK proteins were expressed during each group's hepatocytes,western blot was used to examine protein expression.Results:1.To decide concentration of fat emulsion in our model1.1 Check the OD values of each group witha CCK-8 method: there was significantly difference on the OD values among groups of different concentration at 24h(P< 0.05).Compared with group CON,OD values of group 0.4%&1% decreased slightly(P > 0.05),while OD values of group 2%&4% increased obviously(P< 0.05).1.2 Observation on hepatocytes morphology and the accumulation of lipid droplets in each group's hepatocytes:hepatocyte morphology is normal in group CON,the connection among cells is extremely close;lipid droplets began to appear in group 0.4% after 24 hours.Hepatocyte morphology of group 1% is still normal,the connection among cells keeps close.Large amount of lipid droplets can be seen within the cells and large number of steatosis hepatocytes generated.Ring of lipid droplets on the inside of cell membrane and cell swelling were observed in group 2% & 4%.1.3 The level of medium biochemical indexes: there were significant differences in the levels of the ALT,AST,GGT,ALB,TG atfour points of time(P< 0.05).However,concerning the levels of TBIL,DBIL,ALP,LDH,there were no significant differences atfour points of time(P>0.05).Compared with group CON and 0.4%,group 1%,2% and 4% had an obvious elevation of ALT,AST,TG(P<0.05),ALB significantly reduced in group 2% & 4%(P<0.05),GGT significantly increased in group 4%(P < 0.05).2.Dynamic observation of morphology,vitality of hepatocytes and the mRNA and protein expression of ERS-related signaling molecules in PNALD-FD(Parenteral Nutrition Associated Liver Disease-Fatty Degeneration)model2.1 Check the OD values of each group with CCK-8 method: there was significantly difference on the OD values of hepatocytes of group 1.0% at four times(P< 0.05).OD values of hepatocytes at 36 h decreased significantly while compared with hepatocytes at 0h(P< 0.05).There were no significant changes at other points of time(P > 0.05).2.2 Changes on hepatocytes morphology and the accumulation of lipid droplets of group 1.0% : hepatocyte morphology is normal at four points of time(0h,12 h,24h,36h)and the connection among cells is still extremely close;We observed the increasing amounts and size of lipid droplets,and blurring of nuclear edge with the lapse of time.2.3 The dynamic expression of GRP78?IRE1??sXBP1?JNK mRNA of hepatocytes at four points of timeBRL cells were cultured in 1% fat emulsion medium,the expression of GRP78 mRNA significantly increased over time(P < 0.05).The expression of IRE1? mRNA at 12,24,36 h was significantly higher compared with 0h(P<0.05)while there was no significant changes between 36 h and 24h(P > 0.05).The expression of sXBP1 mRNA had a rise at 12 h,reached the peak at 24 h and tended to decline at 36 h,and the difference was statistically significant(P<0.05).The expression of JNK mRNA increased from 24 h and was obviously higher than 12h(P<0.05),The expression of JNK mRNA at 36 h was higher than 24h(P<0.05).2.4 The dynamic expression of GRP78?IRE1??p-IRE1??XBP1s?JNK?p-JNK protein of hepatocytes at four point of timeBRL cells were cultured in 1% fat emulsion medium,the expression of GRP78 protein significantly increased over time(P < 0.05).There was a significant difference in expression of GRP78 protein of hepatocytes at 36 h compared with 12 h & 24h(P<0.05).There was no significant difference in expression of IRE1? protein of hepatocytes at four times(P>0.05),while there was a significant difference in theexpression of p-IRE1? protein at four points of time(P<0.05).Compared to 12 h,the expression of p-IRE1? protein was higher at 24 h & 36h(P<0.05),while there was no significant changes between 36 h and 24h(P > 0.05).The expression of sXBP1 protein also had a rise at 12 h,reached peak at 24 h and tended to decline at 36 h,and the difference was statistically significant(P<0.05).There was no significant difference in the expression of JNK protein of hepatocytes at four point of time(P>0.05),while there werea significant differences in expression of p-JNK protein of hepatocytes at four point of time(P<0.05).The expression of p-JNK protein in PN was higher at 36 hcompared with other points of time(P<0.05).3.To decide concentration of inhibitor STF-083010 in our model3.1 Check the OD values of each group with CCK-8 methodThe difference between group G and F was not statistically significant among the other groups(P > 0.05),comparisons among the other groups were not the case.3.2 Real-time RT-PCR was then performed to quantify the mRNA expression of sXBP1 in hepatocytes: there was a significantly difference on the level of the mRNA expression of sXBP1 among different groups at 24h(P< 0.05).Except for group B,the difference between the other groups and group A was statistically significant(P < 0.05).sXBP1 mRNA in group D,E,F was obviously down-regulated compared with group C(P< 0.05).The results showed that sXBP1 mRNA in group D was obviously down-regulated and still kept the cell activity for further study.4.Effects of STF-083010 inhibitors on NALD-FD model4.1 The level of medium biochemical indexes: on the levels of the ALT,AST,LDH,TG,there were significant differences among four groups(P< 0.05).However,on the levels of TBIL,DBIL,ALB,ALP,GGT,there were no significant differences among four groups(P>0.05).Compared with the other three groups,group SLE+STF had an obvious elevation of ALT,ASTand the difference was statistically significant(P<0.05).While On the levels of LDH,TG,group CON significantly decreased(P < 0.05)compared with the other three groups.4.2 The expression of GRP78,IRE1?,XBP1 s,JNK mRNA of hepatocytes among four groups at 24h4.2.1 The dynamic expression of GRP78 mRNA of hepatocytes in four groups at 24h: there was a significant difference in expression of GRP78 mRNA of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of GRP78 mRNA was significantly higher in other three groups(P<0.05),while there was no obvious differences between every two groups when the three groups were compared pairwise.4.2.2 The dynamic expression of IRE1? mRNA of hepatocytes in four groups at 24h: there was a significant difference in expression of IRE1? mRNA of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of IRE1? mRNA was significantly higher in other three groups(P<0.05),while there was no obvious differences between every two groups when the three groups were compared pairwise.4.2.3 The dynamic expression of sXBP1 mRNA of hepatocytes in four groups at 24h: there was significant difference in the expression of sXBP1 mRNA of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of sXBP1 mRNA was significantly higher in other three groups(P<0.05).In addition,sXBP1 mRNA in group SLE+STF was obviously down-regulated compared with group SLE+DMSO(P<0.05).4.2.4 The dynamic expression of JNK mRNA of hepatocytes in four groups at 24h:there was significant difference in the expression of JNK mRNA of hepatocytes in four groups at 24h(P<0.05).Compared to group SLE+DMSO,the expression of JNK mRNA in group SLE+STF was higher at 24h(P<0.05).4.3 Western blot was used to examine protein expression of GRP78,IRE1? p-IRE1?,sXBP1,JNK,p-JNK4.3.1 The dynamic expression of GRP78 protein of hepatocytes in four groups at 24h: there was significant difference in expression of GRP78 protein of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of GRP78 protein was significantly higher in other three groups(P<0.05),while there was no obvious differences between every two groups when the three groups were compared pairwise.4.3.2 The dynamic expression of IRE1?,p-IRE1? protein of hepatocytes in four groups at 24h: there was no significant difference in expression of IRE1? protein of hepatocytes among four groups or within each group at 24h(P>0.05),while there were significant difference in expression of p-IRE1? protein of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of p-IRE1? protein was significantly higher in other three groups(P<0.05),while there was no obvious differences among the other three groups.4.3.3 The dynamic expression of sXBP1 protein of hepatocytes in four groups at 24h: there was a significant difference in the expression of sXBP1 protein of hepatocytes in four groups at 24h(P<0.05).Compared to group CON,the expression of sXBP1 protein was significantly higher in other three groups(P<0.05).In addition,sXBP1 protein in group SLE+STF was obviously down-regulated compared with group SLE+DMSO(P<0.05).4.3.4 The dynamic expression of JNK,p-JNK protein of hepatocytes in four groups at 24h:there was no significant difference in expression of JNK and p-JNK protein of hepatocytes in four groups at 24h(P>0.05).Conclusion:1.This experiment has successfully established the cellular model on rat of PNALD-FD(Parenteral Nutrition Associated Liver Disease-Fatty Degeneration)of hepatocytes,and founded a basis for further study of its pathogenesis.2.During the process of PNALD-FD model,the expression of the related molecules GRP78,p-IRE1?,XBP1 s,p-JNK protein and IRE1?,XBP1 s,JNK mRNA in the IRE1?/XBP1 s /JNK signaling pathway of ERS increased,and with prolonged modeling,stimulation showed a generally progressive increase.It suggests that IRE1?/XBP1 s /JNK signaling pathway directly related to the occurrence and development of PNALD. |
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