Effects Of Prolyl Endopeptidase Intervention On The Development Of NAFLD In Mice Fed With High Fat Diet

Background and objective:Nonalcoholic fatty liver disease（NAFLD）has been increasing in the world.Nonalcoholic steatohepatitis（NASH）is a serious type.NAFLD/NASH is a risk factor of cardiovascular and cerebrovascular diseases,metabolic syndrome and so on.It also leads to liver cirrhosis and liver cancer,which causes a heavy disease burden to the society.At present,the pathogenesis of NAFLD is largely unknown.Prolyl endopeptidase（PREP）,a serine protease composed of about 700 amino acid residues,has high activity in hepatocytes despite its low density.Our previous in vivo and in vitro experiments have confirmed that hepatocytes with steatosis have increased PREP activity.Inhibition of PREP activity can inhibit steatosis of hepatocytes in vitro.These research indicated protective effect of PREP inhibition on NAFLD.Rosmarinic acid is a kind of valuable natural multifunctional phenolic acid compound,which has antioxidant,anti-inflammatory,anti-tumor and hepatoprotective effects.However,some studies have suggested that rosmarinic acid may have certain cytotoxicity.Valproic acid（VPA）and its sodium salt are the most commonly used antiepileptic drugs in the world at present,but many reports say that long-term use of VPA has obvious damage to human liver function.Rosmarinic acid and valproic acid have significant inhibitory activities on PREP.Whether and how they affect the development of NAFLD has not been reported.This study attempts to use prep gene knockout mice(PREP^gt mice,C57BL/6J mice as background),NAFLD model induced by high fat diet and rosmarinic acid and valproic acid with PREP inhibition function to observe the effect of PREP inhibition on the development of NAFLD,so as to provide a preliminary theoretical basis for the development and utilization of PREP inhibitors in NAFLD.Methods:The study included 24 wild type（WT）C57BL/6J mice and 21 PREP^gtmice.The mice were randomly divided into the normal diet（low fat diet,LFD）wild type group（LFD-WT,10 mice）,the high fat diet（HFD）wild type group（HFD-WT,14 mice）,normal Diet PREP knockout group(LFD-PREP^gt,9)and high-fat diet PREP knockout group(HFD-PREP^gt,12).For 1week of adaptive feeding,mice in the LFD group were fed with standard chow diet,and mice in the HFD group were fed with high-fat diet.The mice were kept for 16weeks.Record the average body weight,liver index,and epididymis index of mice in each group,and detect changes in serum biochemical indexes,liver tissue fatty changes,inflammation.For Rosmarinic acid and VPA intervention experiment,after one week of adaptive feeding,male C56BL/6J mice were divided into control group,model group and treatment group.The control group was fed with standard diet,the model group was fed with high-fat diet,and the treatment group was fed with rosmarinic acid（rosmarinic acid 60mg/kg,once a day）or VPA（0.4%sodium valproate in drinking water）eight weeks later.The animals were killed after continuous intervention.The changes of average body mass and liver index,serum biochemical indexes,steatosis of liver tissue,inflammation and the expression and activity of PREP were recorded.Results:（1）Compared with LFD-WT group and LFD-PREP^gt group,HFD-WT group and HFD-PREP^gt group were significantly higher in body weight,testis fat index and liver index,ALT,AST,TC,TG,LDL,FBG and insulin（P<0.05）.Compared with LFD-WT group and LFD-PREP^gt group,NAS scores of HFD-WT group and HFD-PREP^gt group were significantly higher,the difference was statistically significant（P<0.05）.Compared with the HFD-WT group,the NAS score of the HFD-PREP^gtgroup was significantly lower and the difference was statistically significant（P<0.05）.The number of F4/80 positive expression cells in high-power field of view of HFD-WT group and HFD-PREP^gt group was significantly more than that of LFD-WT group and LFD-PREP^gt group,and the number of positive expressions in HFD-PREP^gt group was significantly reduced compared to HFD-WT group.（2）In the rosmarinic acid intervention experiment,the RQ of PREP m RNA was（2.01±0.35）and the protein activity was（470.10±50.86）PM/Mg*min in the high fat group,which was significantly higher than that in the control group.After rosmarinic acid intervention,PREP m RNA was（1.21±0.22）and PREP activity was（269.75±34.32）PM/Mg*min,which was significantly lower than that in the high fat group,but there was no significant difference in protein content among the three groups.The liver index of rosmarinic acid group was（4.51±0.30）%,which was not significantly different from that of high-fat group,while the epididymis index was（3.88±1.10）%,which was higher than that of high-fat diet group（2.94±1.30）%,and the relative weight of epididymal fat was increased;the LDL,ALT,AST,ALP levels of high-fat group in rosmarinic acid group were significantly decreased;the liver pathology of high-fat group in mice after rosmarinic acid intervention was not improved,and there was no significant difference between rosmarinic acid group and the high fat group in the NAS score.（3）In the VPA intervention experiment,the protein content of PREP in the high fat group was（0.67±0.08）times of that in the control group,which was lower than that in the control group;the protein activity in the high fat group was（467.94±75.69）PM/Mg*min,which was higher than that in the control group.After VPA intervention,the PREP activity was（379.55±42.33）PM/Mg*min,which was lower than that in the high fat group,but there was no significant difference between the two groups.But the PREP m RNA of the VPA intervention group was（0.61±0.09）,which was significantly lower than that of the control group and the high fat group.The liver weight of VPA group was significantly lower than that of high-fat group,and the liver index of VPA group was significantly lower than that of the high-fat group.The LDL of the high fat group was（0.54±0.08）mmol/L,ALT was（35.30±12.52）U/L,which was significantly higher than that of the control group;the LDL of the high fat group was（0.63±0.08）mmol/L,but ALT was（26.29±10.59）U/L,which was lower than that of the high fat group.The steatosis and lobule inflammation of the liver in the high fat group was obvious,but there were attenuated after the intervention of VPA.When NAS score was used for evaluation,there was significant improvement in steatosis and lobule inflammation and total NAS score in the VPA group when compared with the high fat group.Conclusion:PREP gene knockout can effectively prevent liver inflammation and steatosis in NAFLD model mice fed with high fat;Rosmarinic acid inhibited PREP activity and had limited improvement on liver function and lipid metabolism in NAFLD model mice,while inhibition of PREP activity by VPA alleviate liver steatosis and lobule inflammation of NAFLD in mice.