Aging increases the susceptibility of hepatic inflammation, liver fibrosis and aging in response to high-fat diet in mice

Abstract

We aimed to investigate whether aging increases the susceptibility of hepatic and renal inflammation or fibrosis in response to high-fat diet (HFD) and explore the underlying genetic alterations. Middle (10 months old) and old (20 months old) aged, male C57BL/6N mice were fed either a low-fat diet (4 % fat) or HFD (60 % fat) for 4 months. Young (3 months old) aged mice were included as control group. HFD-induced liver and kidney injuries were analyzed by serum and urine assay, histologic staining, immunohistochemistry, and reverse-transcription real-time quantitative polymerase chain reaction. Total RNA sequencing with next-generation technology was done with RNA extracted from liver tissues. With HFD feeding, aged was associated with higher serum alanine aminotransferase levels, marked infiltration of hepatic macrophages, and increased expression of inflammatory cytokines (MCP1, TNF-α, IL-1β, IL-6, IL-12, IL-17A). Importantly, aged mice showed more advanced hepatic fibrosis and increased expression of fibrogenic markers (Col-I-α1, αSMA, TGF-β1, TGF-β2, TGFβRII, PDGF, PDGFRβII, TIMP1) in response to HFD. Aged mice fed on HFD also showed increased oxidative stress and TLR4 expression. In the total RNA seq and gene ontology analysis of liver, old-aged HFD group showed significant up-regulation of genes linked to innate immune response, immune response, defense response, inflammatory response compared to middle-aged HFD group. Meanwhile, aging and HFD feeding showed significant increase in glomerular size and mesangial area, higher urine albumin/creatinine ratio, and advanced renal inflammation or fibrosis. However, the difference of HFD-induced renal injury between old-aged group and middle-aged group was not significant. The susceptibility of hepatic fibrosis as well as hepatic inflammation in response to HFD was significantly increased with aging. In addition, aging was associated with glomerular alterations and increased renal inflammation or fibrosis, while the differential effect of aging on HFD-induced renal injury was not remarkable as shown in the liver.

Keywords: Aging; Fibrosis; High-fat diet; Liver.

Fig. 1

Hepatic steatosis and inflammation with aging and HFD feeding. a Representative images of H&E stain (original magnification ×100) and immunohistochemical staining for anti-F4/80 and anti-Ly6G antibodies (×200). b Image analysis for hepatic steatosis, anti-F4/80- and anti-Ly6G-positive cells. c Serum ALT levels. Ycon, young control; Mlfd, middle-aged low-fat diet; Olfd, old-aged low-fat diet; Mhfd, middle-aged high-fat diet; Ohfd, old-aged high-fat diet. *P < 0.05

Fig. 2

Changes of mRNA expressions of hepatic inflammatory cytokines. Expressions of all genes were analyzed by real-time PCR. *P < 0.05

Fig. 3

Hepatic fibrosis with aging and HFD feeding. a Representative images of Sirius red stain (original magnification ×100) and immunohistochemical staining for anti-desmin and anti-αSMA antibodies (×200). b Sirius red positive area, anti-desmin and anti-αSMA-positive cells were analyzed by image J. *P < 0.05

Fig. 4

Changes of mRNA expressions of hepatic fibrogenic markers. Expressions of all genes were analyzed by real-time PCR. *P < 0.05

Fig. 5

Hepatic oxidative stress and expression of ROS production related genes, TLR4, and Bambi. a Representative images of immunofluorescent staining for anti-4-HNE antibody (original magnification ×100). b Image analysis for anti-4-HNE-positive area and real-time PCR analysis for expression of ROS production related genes, TLR4, and Bambi. *P < 0.05

Fig. 6

Gene ontology analysis. Gene ontology analysis and clustering were performed using HOMER and Cluster3.0/Java Tree View, respectively

Fig. 7

Glomerular changes and renal fibrosis with aging and HFD feeding. a Representative images of PAS stain (original magnification ×400), Sirius red stain (×100), and immunohistochemical staining for anti-αSMA and anti-F4/80 antibodies (×200). b Image analysis for glomerular tuft area, mesangial area, Sirius red-positive area, anti-αSMA- and anti-F4/80-positive cells. c Urinary albumin creatinine ratio (UACR). d Expression of renal fibrogenic genes analyzed by real-time PCR. *P < 0.05