Abstract:

Objective To investigate the preventive and therapeutic effects of iridoid glycosides from Boschniakia rossica （IGBR） on precancerous lesions of liver cancer in rats， and to clarify its possible mechanism. Methods Thirty Wistar rats were selected and the precancerous liver lesion model was established using the modified Solt-Faber method. The rats were randomly divided into sham operation group， model group， and IGBR group， and there were 10 rats in each group. The morphology of liver tissue of the rats in various groups were observed； the liver weights， liver indexes and liver regeneration degrees of the rats in various groups were measured； HE staining was used to observe the pathomorphology of liver tissue of the rats in various groups； immunohistochemistry method was used to detect the expression of glutathione-S-transferase（GST）-Pi protein in liver tissue of the rats in various groups； colorimetric method was used to detect the activities of γ-glutamyl transpeptidase （γ-GT）， catalase （CAT）， superoxide dismutase （SOD）， glutathione peroxidase （GSH-Px） and glutathione S-transferase （GST）， and the level of malondialdehyde （MDA） in liver tissue and mitochondria of the rats in various groups； Western blotting method was used to detect the expression levels of α-smooth muscle actin （α-SMA）， collagen type Ⅰ alpha 1 chain （ColⅠα1）， matrix metalloproteinase （MMP） 13， MMP2， tissue inhibitor of metalloproteinase （TIMP） 1， TIMP2， transforming growth factor β1 （TGF-β1）， TGF-β1 receptor （TβR）， mothers against decapentaplegic homolog （Smad） 2/3， Smad4， and Smad7 proteins in liver tissue of the rats in various groups. Results Compared with sham operation group， the liver weight， liver index and degree of liver regeneration of the rats in model group were increased （P<0.05）； compared with model group， the liver weight and liver index of the rats in IGBR group showed a decreasing trend， but the difference was not statistically significant （P>0.05）. The HE staining results showed that the liver lobule structure in sham operation group was intact and clear， with large hepatocytes， abundant eosinophilic cytoplasm， and hepatocytes arranged in single cords radiating from the central vein； there were irregular hepatic sinusoids between plates， with only a small amount of collagen fibers and inflammatory cell infiltration in the portal area， and no degeneration or necrosis of hepatocytes. In model group， the normal arrangement of hepatocytes disappeared， the liver lobule structure was disrupted， small cell hyperplasia （mainly oval cells） was observed in the portal area， with massive collagen deposition and significant fibrous tissue hyperplasia in the fibrous septum； hepatocytes showed extensive degenerative edema， hydropic degeneration or even ballooning degeneration and focal necrosis； basophilic hepatocytes formed proliferative areas with clear cytoplasm， centrally located nuclei and 1-2 prominent nucleoli； glassy hepatocytes with enlarged nuclei and pale transparent cytoplasm were also observed. In IGBR group， the liver lobule structure was basically preserved， with reduced inflammatory lesions， mild edema， and scattered spotty or focal necrosis； nuclear atypia and pathological mitotic figures or binucleation were observed. The immunohistochemistry results showed GST-Pi protein positive foci with brown-yellow cytoplasmic staining in round or oval nodules. The GST-Pi protein positive foci were observed in liver tissue of the rats in model group， indicating successful establishment of precancerous liver lesion model. The scattered GST-Pi protein positive foci were observed in IGBR group， which were significantly reduced compared with model group. Compared with sham operation group， the activity of γ-GT in liver tissue of the rats in model group was increased （P<0.05）； compared with model group， the activity of γ-GT in liver tissue of the rats in IGBR group was decreased （P<0.05）. Compared with sham operation group， the GST activity and MDA level in liver tissue and liver mitochondria of the rats in model group were increased （P<0.05）， while the activities of SOD， CAT， and GSH-Px were decreased （P<0.05）； compared with model group， the GST activity and MDA level in liver tissue and liver mitochondria of the rats in IGBR group were decreased （P<0.05）， while the activities of SOD， CAT， and GSH-PX were increased （P<0.05）. The Western blotting results showed that compared with sham operation group， the expression levels of α-SMA， ColⅠα1， TIMP1， and TIMP2 proteins in liver tissue of the rats in model group were increased （P<0.05）， while the expression levels of MMP13 and MMP2 proteins were decreased （P<0.05）， and the ratios of TIMP1/MMP13 and TIMP2/MMP2 were increased （P<0.05）； compared with model group， the expression levels of α-SMA， ColⅠα1， and TIMP2 proteins in liver tissue of the rats in IGBR group were decreased （P<0.05）， while the expression levels of MMP13 and MMP2 proteins were increased （P<0.05）， and the ratios of TIMP1/MMP13 and TIMP2/MMP2 were decreased （P<0.05）. Compared with sham operation group， the expression levels of TGF-β1 and Smad2/3 proteins in liver tissue of the rats in model group were increased （P<0.05）； compared with model group， the expression levels of TGF-β1， Smad2/3 and Smad 4 proteins in liver tissue of the rats in IGBR group were decreased （P<0.05）. Conclusion IGBR can inhibit precancerous liver lesions and liver fibrosis in rats， and its mechanism may be related to enhancing the antioxidant capacity of liver tissue， inhibiting TGF-β/Smad signaling pathway and regulating TIMP/MMP balance.

Key words: Iridoid glycosides from Boschniakia rossica, Precancerous liver lesions, Liver fibrosis, Transforming growth factor-β1, Superoxide dismutase