The Effect Of Intestinal Epithelial CellsHIF-1α On Cholestatic Liver Injury

Objective Cholestasis is a common pathological manifestation in the course of chronic liver disease.Impaired bile acid metabolism is one of the causes of the cholestasis.Studies have shown that intestinal epithelial cells play an important role in the enterohepatic circulation of bile acids.Moderate exercise intervention is apllied to alleviate metabolic liver diseases such as fatty liver.Exercise induces the blood redistribution.The blood perfusion and oxygen concentration are decreased in the intestine and liver area during exercise.The transcription factor hypoxia inducible factor-1α（HIF-1α）signaling pathway is involved in the function maintenance of intestinal epithelial cells（IECs）.Therefore,this study exams the effects of moderate exercise intervention on cholestatic liver injury,observing the distribution of HIF-1α in the enterohepatic region,and exploring the possible mechanisms of HIF-1α in intestinal epithelial cells on cholestasis liver injury.Methods1 Male 8-10 weeks old C57BL/6J mice（n= 6/group）were used in the first part of study to establish cholestatic liver injury model.The experimental group was fed with 0.1%3,5-diethoxycarbonyl-1,4-dihydrotrimethylpyridine（DDC）for 5 weeks.The control group（CON）was fed with common chow.A 30-minute swimming exercise without weights was applied as the exercise intervention（DDC+SWIM and CON+SWIM）.The body weight,liver-to-weight ratio,serum liver function related miomarkers and liver structure were analyzed to evaluate the cholestasis liver injury model,and to observe the effect of the exercise intervention.2 In the second part of the study,male 8-10 weeks old HIF-1α luciferase reporter ROSA26 ODD-Luc/⁺ mice（n=3/group）were was fed with 0.1% DDC to establish the cholestasis liver injury model（ODD-Luc DDC）.Control group（ODD-Luc CON）was fed with common chow.Bioluminescence in vivo imaging was used to observe the distribution of HIF-1α in the colon,small intestine and liver of mice.3 Prolyl hydroxylase 2（PHD2）is a key enzyme in HIF-1α degradation.In the third part of the study,we constructed the IECs-specific Hif1 a knockout mice(HIF-1α^VKO,control strain HIF-1α^fl/fl,n=6/group),and Phd2 knockout mice(PHD2^VKO,control strain PHD2^fl/fl,n =6/group),through the Cre-Lox P system.The body weight,liver-to-weight ratio,serum liver function related miomarkers and liver structure were examed to explore the role of IECs PHD2/HIF-1α signaling pathway in cholestatic liver injury.4 According to the results of the third part of study,the HIF-1α^VKO and control HIF-1α^fl/fl mice（n = 6 / group）were selected to explore the possible mechanisms.The composition of their bile acids in the liver was analyzed through metabolomics.Quantitative PCR was used to detect the m RNA expression of genes related to bile acid metabolism（Bsep,Ntcp,Fxr and Shp-1 detected in the liver,Ostα,Ostβ,Fxr,Asbt,I-babp and Mrp3 detected in the small intestine）.Results1 Compared with CON,the body weight of DDC was dropped sharply,and the relative body weight detected at the end of experiment was 66.8 ± 15% of the baseline（P<0.01）.The TBIL,DBIL,IBIL,TBA,ALT,AST and ALP were significantly increased in the DDC mice（P <0.001）.HE staining revealed the extensive bile duct reactions,porphyrin crystals blocked the bile ducts,in the liver of DDC mice.The results indicate that we constructed the cholestatic liver injury mouse model.However,compared with the DDC group,no significant improvement was found in the DDC+SWIM group,except a slight increase in the body weight.2 Compared with ODD-Luc CON,the amount of photons significantly increased in the small intestine and the liver of ODD-Luc DDC mice.The amount of photons increase in the small intestine was more obvious,indicating that cholestasis-induced liver injury mainly resulted in increased expression of HIF-1α in the small intestine.3 The relative body weight percentage of HIF-1α^VKO mice was significantly higher than that of HIF-1α^fl/fl mice（P <0.05）after fed DDC for 5 weeks.The ALP,IBIL,TBA and AST were significantly reduced（P <0.05）.The bile duct hyperplasia and inflammatory cell infiltration was alleviated in the HIF-1α^VKO group.However,no significant differences were observed between the PHD2 ^VKO and PHD2^fl/fl mice.4 Compared with HIF-1α^fl/fl,we found altered bile acid composition in the HIF-1α^VKO group,with significantly increased TDCCA content（P <0.05）.Bsep in the liver（P<0.05）and I-babp in the small intestine（P <0.01）were significantly up-regulated.Conclusion This study shows that moderate exercise dose not significantly resolve the symptoms of the cholestatic liver injury.The level of HIF-1α is increased in the small intestine and liver of cholestatic liver injury mice.Intestinal epithelial cells HIF-1α is involved in the process of cholestatic liver injury by regulating the bile acid metabolism related genes expression.