Abstract:

Objective To discuss the role of angiopoietin-like protein 6 （ANGPTL6） in liver fibrosis， and to analyze the improving effect of engineered exosome（Exo）-delivered ANGPTL6 mRNA on liver fibrosis. Methods A total of 12 C57BL/6 mice were randomly divided into olive oil group （OIL group） （intraperitoneally injected with olive oil） and carbon tetrachloride （CCl₄） group （intraperitoneally injected with a mixture of olive oil and CCl?）， with 6 mice in each group； another 12 C57BL/6 mice were randomly divided into control group （fed a with methionine-choline sufficient diet） and methionine-choline deficient （MCD） group （fed a with MCD diet）， and two kinds of mouse liver fibrosis models were established. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting method were used to detect the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in various groups. A total of 30 mice were randomly divided into olive oil + phosphate buffered saline （PBS） group （OIL+PBS group） （intraperitoneally injected with olive oil twice a week for 8 weeks， then injected with PBS buffer by tail vein twice a week for 6 weeks）， CCl₄+Exo-green fluorescent protein （GFP） mRNA group （established liver fibrosis model by intraperitoneal injection of CCl₄ mixture and were injected by tail vein with engineered Exo loaded with GFP mRNA for 6 weeks）， and CCl?+Exo-ANGPTL6 mRNA group （established liver fibrosis model by intraperitoneal injection of CCl₄ mixture and were injected by tail vein with engineered Exo loaded with ANGPTL6 mRNA for 6 weeks）， with 10 mice in each group. The mice in CCl₄+Exo-GFP mRNA group and CCl₄+Exo-ANGPTL6 mRNA group were injected with engineered Exo twice a week， 20 μg per mouse each time （volume 100 μL）. ELISA method was used to detect the serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） activities in the mice in various groups； Masson staining and Sirius red staining were used to observe the collagen deposition in liver tissue of the mice in various groups； immunohistochemistry method was used to detect the α-smooth muscle actin （α-SMA） expression levels in liver tissue of the mice in various groups； RT-qPCR method was used to detect the expression levels of α-SMA， collagen type Ⅰ alpha 1 chain （Col1a1）， transforming growth factor β1 （TGF-β1）， and tissue inhibitor of metalloproteinase 1 （TIMP-1） mRNA in liver tissue of the mice in various groups. Results The bioinformatics analysis results showed that ANGPTL6 expression was significantly down-regulated in activated hepatic stellate cell（aHSC）. The ultrasound examination results showed that the liver surface of the mice in OIL group was fine and smooth； compared with OIL group， the liver section of the mice in CCl? group was rough and uneven. The RT-qPCR and Western blotting results showed that compared with OIL group， the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in CCl? group were significantly decreased （P<0.05）. The engineered Exo extracted from the supernatant of HEK293T cells had intact structure and could be largely enriched in the fibrotic liver after tail vein injection， with GFP protein being largely expressed in the liver. The ELISA assay results showed that compared with OIL+PBS group， the ALT and AST activities in CCl₄+Exo-GFP mRNA group were significantly increased （P<0.05）； compared with CCl₄+Exo-ANGPTL6 mRNA group， the serum ALT and AST activities in CCl₄+Exo-GFP mRNA group were significantly decreased （P<0.05）. The Masson staining and Sirius red staining results showed that compared with OIL+PBS group， the collagen deposition in liver tissue of the mice in CCl?+Exo-GFP mRNA group was significantly increased， and the relative collagen area was increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the collagen deposition in tissue liver of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased， and the relative collagen area was decreased （P<0.05）. The immunohistochemistry results showed that compared with OIL+PBS group， the α-SMA protein expression level in liver tissue of the mice in CCl?+ Exo-GFP mRNA group was significantly increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the α-SMA protein expression level in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased （P<0.05）. The RT-qPCR results showed that compared with OIL+PBS group， the expression levels of Col1a1， α-SMA， TGF-β1， and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-GFP mRNA group were significantly increased （P<0.05）； compared with CCl₄+Exo-GFP mRNA group， the expression levels of Col1a1， α-SMA， TGF-β1， and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group were significantly decreased （P<0.05）. Conclusion Engineered Exo-delivered ANGPTL6 mRNA injected via the tail vein in the mice is mainly enriched in the liver， and engineered Exo delivery of ANGPTL6 mRNA has an improving effect on liver fibrosis in the mice.

Key words: Angiopoietin-like protein 6, Engineered exosome, Liver fibrosis, Liver injury, Disease model， animals