Recombinant Lactococcus lactis Expressing Ling Zhi 8 Protein Ameliorates Nonalcoholic Fatty Liver and Early Atherogenesis in Cholesterol-Fed Rabbits

Abstract

Atherosclerosis is an inflammatory disease characterized by lipid deposits in the subendothelial space leading to severe inflammation. Nonalcoholic fatty liver disease (NAFLD) shares several risk factors with atherosclerosis, including dyslipidemia, type 2 diabetes mellitus, and metabolic syndrome, all of which lead to lipid deposition in the liver causing inflammation and fibrosis. Several clinical trials have shown that certain Chinese herbal medicines with anti-inflammatory effects can be used as adjuvant therapy to prevent the development of cardiovascular events and liver disease. Ling Zhi 8 (LZ8) is an immunomodulatory protein isolated from a medicinal mushroom and has been well documented to possess a broad range of pharmacological properties. This study aimed to evaluate the protective effects of recombinant Lactococcus lactis expressing LZ8 protein on NAFLD and atherogenesis in a cholesterol-fed rabbit model. Twelve rabbits were divided into three groups and fed with syrup only, L. lactis vehicle, or recombinant L. lactis-LZ8 once a day on weekdays for five weeks, respectively. The gene expression of IL-1β in the aorta was significantly suppressed after oral administration of L. lactis-LZ8. Moreover, in hematoxylin and eosin staining of the aorta, the intima-medial thickness was decreased, and foam cells were significantly reduced in the subendothelial space. LZ8 also inhibited the expression of IL-1β in the liver, decreased fat droplet deposits and infiltration of inflammatory cells, and improved liver function by decreasing liver enzymes in an animal model. Our results suggest that the Lactococcus-expressing LZ8 appears to be a promising medicine for improving both NAFLD and early atherogenesis owing to its anti-inflammatory effect. Furthermore, it is available as a low-cost food-grade product.

Figure 1

Experimental scheme of oral recombinant Lactococcus lactis in rabbits fed with a high cholesterol diet. Twelve male New Zealand white rabbits were divided into three groups and fed 3 ml of the prepared fructose syrup as indicated once a day on weekdays. Blood samples were collected weekly via the marginal ear vein and all rabbits were sacrificed on day 35.

Figure 2

(a) Expression analysis of recombinant LZ8-producing L. lactis after nisin induction for 1–20 hours. Immunoblotting of bacterial lysate was performed with lab-made rabbit anti-LZ8-specific antibody. E. coli-derived recombinant LZ8 was used as positive control. Lane M, prestained protein markers. (b) Correlation curve of the induction times and the intensity levels of band signals by video densitometer.

Figure 3

Histological changes in the livers of cholesterol-fed rabbits by H&E staining. Under higher magnification, hepatocyte ballooning and steatosis were observed throughout the sections in the rabbits from the sham and VO groups. H&E staining showed that steatohepatitis was attenuated in rabbits of LZ8 L. lactis treatment group. CV, central vein. Scale bar = 100 μm. Magnification ×400. (a) Sham group.(b) VO group. (c) LZ8 group.

Figure 4

Histological images of aortic arches from three groups of rabbits after 5 weeks of a high cholesterol diet. (a) Sudan IV staining, arrowhead indicates small plaques in fatty streaks were observed, mostly in the aortic arch from sham group. (b) H&E staining, bracket indicates thickness of the intimal layer and arrow indicated foam cell deposition. Scale bar = 100 μm.

Figure 5

Recombinant LZ8 L. lactis vaccine reduced IL-1β mRNA expression. (a) Aortas; and (b) livers of high cholesterol-fed rabbits.RT-PCR data were normalized to β-actin and presented as mean ± SEM of intensity. ^∗∗p < 0.01; ^∗∗∗p < 0.001, by one-way analysis of variance with the Bonferroni multiple range test.