Mechanism of Si Junzitang in Inhibiting Hepatocellular Carcinoma Progression Through Regulating O-GlcNAcylation of NF-κB p65 in Paracancerous Tissues / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo investigate the molecular mechanism by which Si Junzitang in intervening in the development of hepatocellular carcinoma （HCC） by regulating the O-linked β-N-acetylglucosamine modification （O-GlcNAcylation） of nuclear factor kappa-B （NF-κB） p65 in the paracancerous tissues. MethodThe orthotopic liver cancer mouse model was established. Twenty-four C57BL/6 mice were randomly divided into four groups： Normal group， model group， Si Junzitang low-dose group （10 g·kg-1）， and Si Junzitang high-dose group （25 g·kg-1）， with 6 mice in each group. The O-GlcNAcylation level and phosphorylation modification level of p65 in the paracancerous tissues were detected using Western blot. The O-GlcNAcylation of p65 was assessed using immunoprecipitation （IP）. The mRNA expression of interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， transforming growth factor-β1 （TGF-β1）， vascular endothelial growth factor A （VEGFA）， matrix metalloproteinase-2 （MMP-2）， matrix metalloproteinase-9 （MMP-9） in the paracancerous tissues was detected using real-time quantitative polymerase chain reaction （Real-time PCR）. The tumor number， liver weight， locomotor activity， grip strength， and Qi status of the mice were observed and analyzed. ResultCompared with the normal group， the model group showed a significant decrease in O-GlcNAcylation in the paracancerous tissues （P<0.01）， a significant decrease in p65 O-GlcNAcylation （P<0.01）， a significant increase in p65 phosphorylation （P<0.01）， significantly elevated mRNA levels of cytokines IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9 （P<0.01）， significantly increased liver weight （P<0.01）， significantly declined grip strength， number of grid crossings， and number of vertical stand-ups （P<0.01）， and significantly dwindled Qi status （P<0.01）. Compared with model group， the Si Junzitang low-dose and high-dose groups showed significantly increased levels of O-GlcNAcylation in the paracancerous tissues （P<0.05， P<0.01）， significantly upregulated p65 O-GlcNAcylation levels （P<0.05， P<0.01）， and significantly decreased p65 phosphorylation levels （P<0.01）. In the Si Junzitang low-dose group， the mRNA levels of IL-6， TGF-β1， and VEGFA significantly decreased （P<0.05， P<0.01）. In the Si Junzitang high-dose group， the mRNA levels of IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9 significantly decreased （P<0.01）， the number of tumors larger than 3 mm in diameter significantly decreased （P<0.01）， and liver weight significantly decreased （P<0.05）. Additionally， grip strength， number of grid crossings， and number of vertical stand-ups significantly increased （P<0.05， P<0.01）， along with a significant increase in qi status （P<0.01）. ConclusionSi Junzitang can inhibit the progression of orthotopic HCC in mice， which may be achieved by increasing the O-GlcNAcylation level in the paracancerous tissues， enhancing the O-GlcNAcylation of p65， inhibiting the phosphorylation modification of p65， and ultimately suppressing the expression of downstream IL-6， TNF-α， TGF-β1， VEGFA， MMP-2， and MMP-9.