Effects Of Hypoxic Hepatocytes With Keap1 Knock-down On The Expression And Activity Of MMP-2 In Hepatic Stellate Cells

Hepatocytes are the main cells of the liver,accounting for approximately 70% of the cells in the liver,and are susceptible to damage by various pathogenic factors.Among these various pathogenic factors,hypoxia is not only the source of hepatocytes damage,but also the accomplice of other pathogenic factors to damage liver cells.Oxidative stress is a key mechanism of the hepatocyte injury.Damaged hepatocytes release a large number of pro-inflammatory and pro-fibrotic factors,activate hepatic stellate cells(HSCs),result in abnormal deposition of the extracellular matrix,while matrix degradation is reduced.Previous studies have found that double knock-down of intracellular hepatic oxygen and oxidative stress receptors can inhibit the activation of HSCs,reduce collagen production,and delay the development of liver fibrosis.However,it is unclear whether matrix metalloproteinases associated with matrix degradation change simultaneously.This experiment explored under hypoxic conditions the effects of knockdown oxidative stress receptors in hepatocytes on matrix metalloproteinases of hepatic stellate cells,and the regulatory molecules involved in the regulation of matrix metalloproteinases activity.Objectives An interventional model of knockdown Kelch-like ECH-associated protein 1(Keap1)in hepatocytes was established to investigate the influence of hypoxic hepatocytes with Keap1 knock-down on the matrix metalloproteinase-2(MMP-2),and the corresponding regulatory molecules such as matrix metalloproteinase inhibitor(TIMP-2),membrane-type 1 matrix metalloproteinase(MT1-MMP or MMP14),caveolin(caveolin-1,Cav-1)in HSCs.Methods The Keap1 shRNA plasmid vector was constructed and the transfection efficiency in hepatocytes was observed by the fluorescence microscope.The hepatocytes with Keap1 knock-down were cultured in hypoxia,and the expression levels of Keap1 and its downstream regulator Nrf2 in the hepatocytes were verified by Western Blot and Q-PCR.The hepatocyte stellate cell line(HSC-T6)was intervened by the above-mentioned hepatocyte culture supernatant,and the protein expression of MMP-2,caveolin-1,MT1-MMP,TNF-α,TIMP-2 of mouse HSC-T6 was detected by Western Blot.Gelatin zymography was used to detect MMP-2 activity in the supernatant.Finally,TNF-α expression inhibitor was used to treat hypoxic hepatocytes,the related indexes were detected,and the possible mechanism was preliminarily discussed.Results The stable hepatocytes with Keap1 knock-down was successfully established,and the expression of Nrf2,which has protective effect on hepatocytes,was increased after knocking down the oxidative stress receptor Keap1 in hepatocytes,while the expression of TNF-α was decreased.The supernatant of knocking-down Keap1 liver cell significantly influenced the expression of MMP-2,TIMP-2,MT1-MMP and caveolin-1 proteins in HSCs.Hepatocytes with Keap1 knock-down were cultured in hypoxia conditions,and the supernatant(CM)was collected to culture HSC-T6 cells.Compared with the DMEM group,CM of the hepatocytes made the expression of MMP-2,caveolin-1,TIMP-2,MT1-MMP protein decreased,while the activity of MMP-2 was increased in HSC-T6 cells.Not only the TNF-α expression decreased,but also the activity of MMP-2 increased in the supernatant of the HSC-T6,after the cells were cultured in the CM derived from hypoxic hepatocytes treated by TNF-α inhibitor.The above differences were statistically significant.Conclusion When knocking-down the hepatocyte oxidative stress receptor Keap1,the level of anti-oxidative stress molecule Nrf2 is increased,the degree of hepatocyte injury is reduced,and the synthesis and release of TNF-α is decreased,which may be related to the expression of caveolin-1 in hepatic stellate cells.When the stimulating effect is reduced,the interference of MMP-2 activation on the membrane surface is alleviated,and the MMP-2 activity is increased.