Haematococcus pluvialis Carotenoids Enrich Fractions Ameliorate Liver Fibrosis Induced by Thioacetamide in Rats: Modulation of Metalloproteinase and Its Inhibitor

Abstract

Hepatic fibrosis is a consequence of chronic liver diseases. Metalloproteinase and its inhibitor have crucial roles in the resolution of liver fibrosis. The current relevant study is aimed to evaluate the therapeutic effect of Haematococcus pluvialis (H. pluvialis) extract, astaxanthin-rich fraction, astaxanthin ester-rich fraction, and β-carotene-rich fraction as well as their mechanisms of action in curing hepatic fibrosis induced by thioacetamide (TAA). Liver fibrosis was induced using TAA (intraperitoneal injection, two times a week for 6 weeks), in a rat model and H. pluvialis extract (200 mg/kg), and other fractions (30 mg/kg) were orally administered daily for 4 weeks after the last TAA injection. Based on HPLC analysis, H. pluvialis extract contains β-carotene (12.95 mg/g, extract) and free astaxanthin (10.85 mg/g, extract), while HPLC/ESI-MS analysis revealed that H. pluvialis extract contains 28 carotenoid compounds including three isomers of free astaxanthin, α or β-carotene, lutein, 14 astaxanthin mono-esters, 5 astaxanthin di-esters, and other carotenoids. H. pluvialis and its fractions reduced liver enzymes, nitric oxide, collagen 1, alpha-smooth muscle actin, and transforming growth factor-beta as well as elevated catalase antioxidant activity compared to the TAA group. Also, H. pluvialis extract and its fractions exceedingly controlled the balance between metalloproteinase and its inhibitor, activated Kupffer cells proliferation, and suppressed liver apoptosis, necrobiosis, and fibrosis. These findings conclude that H. pluvialis extract and its fractions have an antifibrotic effect against TAA-induced liver fibrosis by regulating the oxidative stress and proinflammatory mediators, suppressing multiple profibrogenic factors, and modulating the metalloproteinase and its inhibitor pathway, recommending H. pluvialis extract and its fractions for the development of new effective medicine for treating hepatic fibrosis disorders.

Figure 1

HPLC analysis of all-trans-astaxanthin and β-carotene in H. pluvialis dichloromethane: methanol (3 : 1, V/V) crude extract.

Figure 2

HPLC analysis of β-carotene in β-carotene enriches fraction of H. pluvialis.

Figure 3

HPLC analysis of astaxanthin esters in astaxanthin esters enriches fraction of H. pluvialis.

Figure 4

HPLC analysis of all-trans-astaxanthin in astaxanthin enriches fraction of H. pluvialis.

Figure 5

LC/ESI-MS chromatogram in the positive-ion of crude extract of H. pluvialis.

Figure 6

ESI-MS positive-ion spectra of (a) astaxanthin, (b) α or β-carotene, (c) lutein or zeaxanthin, (d) astaxanthin monoester, C22:4, and (e) astaxanthin diester, C16:4/C8:0.

Figure 7

Effects of crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) on hepatic contents of (a) TGF-β, (b) SMA-α, (c) collagen 1, (d) TIMP-1, and (e) MMP9. Data are presented as the mean ± S.E. of (n = 8) for each group. Statistical analysis was carried out by one-way analysis of variance followed by Fisher's LSD comparisons test. A Statistically significant from the control group. B Statistically significant from the TAA group. C Statistically significant from the silymarin group. D Statistically significant from the crude extract group. E Statistically significant from the astaxanthin ester group at P < 0.05.

Figure 8

Effects of crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) on hepatic contents of (a) IL-6 and (b) TNF-α. Data are presented as the mean ± S.E. of (n = 8) for each group. Statistical analysis was carried out by one-way analysis of variance followed by Fisher's LSD comparisons test. A Statistically significant from the control group. B Statistically significant from the TAA group. C Statistically significant from the silymarin group. D Statistically significant from the crude extract group. E Statistically significant from the astaxanthin ester group at P < 0.05.

Figure 9

Effects of crude extract and its fractions (astaxanthin, astaxanthin ester, and β-carotene) on histopathological alteration. Data are presented as the mean ± S.E. of (n = 8) for each group. Statistical analysis was carried out by one-way analysis of variance followed by Fisher's LSD comparisons test. A Statistically significant from the control group. B Statistically significant from the TAA group. C Statistically significant from the silymarin group. D Statistically significant from the crude extract group. E Statistically significant from the astaxanthin ester group at P < 0.05.

Figure 10

Liver section of the central vein and surrounding hepatocytes. (a) Liver section of the rat from the control normal rat showed no histopathological alteration and the normal histological structure of the central vein and surrounding hepatocytes in the parenchyma were recorded. (b) Liver section of rats from the TAA-treated group showed fine fibroblastic cell proliferation was dividing the degenerated and necrobiotic changes hepatocytes into lobules. (c) Liver section of rat from the silymarin-treated group there was few pigmented cell infiltration surrounding and adjacent to the dilated central vein (H&E X 400). (d) Liver section of rats from crude extract showed diffuse Kupffer cell proliferation was detected in between the degenerated hepatocytes. (e) Liver section of rats from astaxanthin ester showed Kupffer cell proliferation was detected in between the hepatocytes. (f) Liver section of the rat from astaxanthin showed degeneration in the hepatocytes adjacent to the congested central vein. (g) Liver section of rats from β-carotene showed dilatation in the central vein associated with focal hemorrhage in the parenchyma. Fine fibroblastic cell proliferation was dividing the degenerated and necrobiotic changed hepatocytes into lobules. (H&E X 400 bar scale: 50 μm).

Figure 11

Liver sections of the portal area. (a) Liver section of the portal area of rats from the TAA-treated group showed fibrosis with inflammatory cell proliferation in between the congested portal vein and hyperplastic bile ducts. (b) Liver section of rats from the silymarin-treated group of the portal area showed inflammatory cell infiltration. (c) Liver section of the rat from crude extract showed the portal area showed few inflammatory cell infiltration. (d) Liver section of the rat from the astaxanthin ester showed inflammatory cell infiltration in the portal area. (e) Liver section of rat from astaxanthin focal hemorrhage was detected in the parenchyma adjacent to the portal area. (f) Liver section of rats from β-carotene of the portal area showed congestion in the portal vein and mild fibrosis with inflammatory cell infiltration in between. (H&E X 400 bar scale:50 μm).