Hepatoprotective Effects of Albumin-Encapsulated Nanoparticles of a Curcumin Derivative COP-22 against Lipopolysaccharide/D-Galactosamine-Induced Acute Liver Injury in Mice

Abstract

Acute liver injury (ALI) is a severe syndrome and can further develop into acute liver failure (ALF) which can lead to high mortality and cause irreversible liver injuries in the clinic. Liver transplantation is the most common treatment; however, liver donors are lacking, and the progression of ALF is rapid. Nanoparticles can increase the bioavailability and the targeted accumulation of drugs in the liver, so as to significantly improve the therapeutic effect of ALI. Curcumin derivative COP-22 exhibits low cytotoxicity and effective anti-inflammatory activity; however, it has poor water solubility. In this study, COP-22-loaded bovine serum albumin (BSA) nanoparticles (22 NPs) were prepared and characterized. They exhibit effective hepatoprotective effects by inhibiting inflammation, oxidative stress, and apoptosis on Lipopolysaccharide/D-Galactosamine-induced acute liver injury of mice. The anti-inflammatory activity of 22 NPs is related to the regulation of the NF-κB signaling pathways; the antioxidant activity is related to the regulation of the Nrf2 signaling pathways; and the apoptosis activity is related to mitochondrial pathways, involving Bcl-2 family and Caspase-3 protein. These three cellular pathways are interrelated and affected each other. Moreover, 22 NPs could be passively targeted to accumulate in the liver through the retention effect and are more easily absorbed than 22.HCl salt in the liver.

Keywords: acute liver injury; apoptosis; bovine serum albumin nanoparticles; curcumin derivative; inflammation; oxidative stress.

Figure 1

Chemical structures of curcumin, EF24, and COP-22.

Figure 2

Particle size, PDI changes (A), and zeta potential changes (B) in 22 NPs. (n = 3).

Figure 3

Powder X-ray diffraction patterns of COP-22, E80, BSA, physical mixture (PM) of COP-22/E80/BSA (1/1/1, w/w/w), and 22 NPs.

Figure 4

COP-22 and E80 in the binding site of BSA (A) and their enlarged pictures (B,C).

Figure 5

The comparison of COP-22, 22 NPs, and 22 HCl salt on the inhibitor activity of NO (A) or ROS (B) production. Data were expressed as means ± SD (n = 3) from independent experiments. *** p < 0.001, compared with the control group. ^### p < 0.001, compared with the model group.

Figure 6

(A) Cellular uptake of 22 NPs and 22.HCl salt in Raw264.7 cells. (B) The representative fluorescence microscopy images (200×) using L02 cells after 4 h incubation with Cy5.5-COOH labeled 22 NPs. Scale bar: 10 μm.

Figure 7

The representative histological microphotographs of H&E-stained liver sections (200×). Scale bar: 10 μm.

Figure 8

Effects of 22 NPs and 22.HCl salt on the levels of AST (A) and ALT (B) in serum of mice. Values were mean ± SD (n = 8). * p < 0.05, *** p < 0.001, compared with the control group. ^## p < 0.01, ^### p < 0.001, compared with the model group.

Figure 9

Effects of 22 NPs and 22.HCl salt on inflammation in LPS/D-GalN-induced mice. (A,B) Levels of serum TNF-α and IL-1β were examined by ELISA. (C,D) Level of TNF-α in the liver of mice was examined by Western blot analysis. (E) The expressions of NF-κB p65 of cytoplasmic and nuclear proteins were assessed by Western blot analysis. Values are mean ± SD (n = 8) (A,B). Data were expressed as means ± SD (n = 3) from independent experiments (D). ** p < 0.01, *** p < 0.001, compared with the control group. ^### p < 0.001, compared with the model group.

Figure 10

Effects of 22 NPs and 22.HCl salt on oxidative stress in liver tissue in LPS/D-GalN-induced mice. (A–D) Levels of CAT, MDA, MPO, and NO in the liver were examined. (E–G) Levels of Nrf-2 and HO-1 in the liver of mice were examined by Western blot analysis. Values are mean ± SD (n = 8) (A,B). Data were expressed as means ± SD (n = 3) from independent experiments (D). * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the control group. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, compared with the model group.

Figure 11

Effect of 22 NPs and 22.HCl salt on apoptosis in LPS/D-GalN-induced mice. (A) The representative fluorescence microscopy images (200×) of TUNEL staining. (B–D) The expressions of Bax and caspase-3 proteins were assessed by Western blot analysis. Data were expressed as means ± SD (n = 3) from independent experiments (C and D). * p < 0.05, *** p < 0.001, compared with the control group. ^## p < 0.01, ^### p < 0.001, compared with the model group. Scale bar: 10 μm.

Figure 12

In vivo tissue biodistribution of COP-22. (A) In vivo images of mice with Cy5.5-labeled 22 NPs at different times by IVIS. (B) COP-22 concentration in liver tissues was determined at different times by HPLC. Data were expressed as means ± SD (n = 3) from independent experiments.

Figure 13

The representative HPLC chromatogram of standard curves and ultra-filtrate of 22 NPs for the evaluation of encapsulation efficiency.

Figure 14

The standard curves of COP-22 in homogenate of liver.