Prolyl endopeptidase gene disruption attenuates high fat diet-induced nonalcoholic fatty liver disease in mice by improving hepatic steatosis and inflammation

Abstract

Background: Prolyl endopeptidase (PREP) is a serine endopeptidase that regulates inflammatory responses. PREP inhibitors can reduce hepatocyte lipid accumulation and may participate in the progression of nonalcoholic fatty liver disease (NAFLD). We investigated whether disruption of PREP regulates hepatic steatosis and inflammation in mice with NAFLD.Methods: Wild-type and PREP gene disrupted mice were randomly divided into low-fat diet wild-type (LFD-WT), high-fat diet wild-type (HFD-WT), low-fat diet PREP disruption (LFD-PREP gt ), and highfat diet PREP disruption (HFD-PREP gt ) groups. Animals were euthanized at the endpoint of 32 weeks. The NAFLD activity score and number of inflammatory cells were determined by hematoxylin-eosin staining and immunohistochemical staining of liver tissue. The expression levels of inflammation-and lipid metabolismassociated genes in the liver and serum were detected by quantitative reverse transcription PCR, mass spectrometry, or enzyme-linked immunosorbent assay. Results:The body weight and epididymal fat tissue index of the HFD-PREP gt mice were significantly decreased compared with that of the HFD-WT mice. Moreover, the NAFLD activity score and liver function were attenuated in the HFD-PREP gt mice. Fat accumulation and the level of expression of mRNAs associated with lipid metabolism and proinflammatory responses were improved in the HFD-PREP gt mice.The number of CD68-positive cells in liver tissue and the serum levels of inflammation-associated factors were significantly decreased in the HFD-PREP gt mice compared with those in the HFD-WT mice. Further mechanistic investigations indicated that the protective effect of PREP disruption on liver inflammation was associated with the suppressed production of matrix metalloproteinases (MMPs) and proline-glycine-proline (PGP) and the inhibition of neutrophil infiltration.Conclusions: Loss of PREP lowers the severity of hepatic steatosis and inflammatory responses in a highfat diet-induced nonalcoholic steatohepatitis model. PREP inhibition may protect against NAFLD.

Prolyl Endopeptidase Gene Disruption Improves Gut Dysbiosis and Non-alcoholic Fatty Liver Disease in Mice Induced by a High-Fat Diet

“…Interestingly, factors associated with proinflammation and its related signaling molecules showed dynamic changes in HFD-PREP gt mice upon hepatic inflammation progression. Our previous works demonstrated that PREP could potentially affect the progression of hepatic inflammation, possibly by regulating chemotactic factors (such as PGP and MMPs) ( Jiang et al, 2020 ), and in this study, we found this effect was more important and evident in the later and more severe stage of NAFLD. This observation may be partially explained by the fact that NASH is considered a potentially progressive disorder, as liver inflammation may prompt collagen matrix synthesis and deposition ( Suzuki and Diehl, 2017 ), which PREP and MMP9 hydrolyze to produce PGP (chemotaxis of neutrophils) ( Weathington et al, 2006 ; Gaggar et al, 2008 ).…”

“…We only detected butyrate levels in the colon of 16 weeks mice; however, a previous study reported that sodium butyrate could delay the onset of early signs of NAFLD in mice ( Jin et al, 2016 ). Our previous studies also indicated that PREP is closely related to energy metabolism and the downstream lipid metabolism targets of AMPK ( Jiang et al, 2020 ). The functional consequences of this taxa shift and our previous work provide clues about how PREP inhibition may regulate flora homeostasis, trigger the AMPK signaling pathway, and improve liver lipid metabolism.…”

“…PREP action is quite complex, as it can induce metabolic benefits or proinflammatory damage to the intestinal environment ( Koelink et al, 2014 ; Olivares et al, 2019 ). Our previous study found that PREP disruption plays a beneficial role in NAFLD progression, mainly through decreases in the number of chemokines (such as PGP) and inflammatory cell accumulation ( Jiang et al, 2020 ). Notably, different hepatic pathogenesis mechanisms have previously been described, and complex PREP actions have been reported; therefore, we carried out this study to imitate the different NAFLD stages to uncover PREP’s influence on the mice gut.…”

“…Our previous work found that N-acetylseryl-aspartyl-lysyl-proline (AcSDKP), generated from thymosin β4 (Tβ4) through hydrolysis of meprin-α and PREP, exerts a therapeutic effect on inflammatory bowel disease (Shi et al, 2020). Our studies also indicated that PREP inhibition improves hepatocyte steatosis in vitro and in vivo (Zhou et al, 2016;Jiang et al, 2020). However, the interactions between PREP and the gut environment in HFD-induced NAFLD and their potential multi-organ mechanisms remain unknown.…”

“…Wild-type (WT) C57BL/6J and PREP-disrupted (PREP gt ) mice were obtained from the Shanghai Model Organisms Center, Inc. The details of PREP knockout mice are provided in the Methods section of our previous study ( Jiang et al, 2020 ). Mice were fed a standard chow diet or a HFD (fat 30 kcal%, carbohydrates 52 kcal%, protein 18 kcal%, and cholesterol 2%) for 16 or 24 weeks.…”