The incomplete degradation of tumour cells by macrophages (Mφ) is a contributing factor to tumour progression and metastasis, and the degradation function of Mφ is mediated through phagosomes and lysosomes. In our preliminary experiments, we found that overactivation of NADPH oxidase 2 (NOX2) reduced the ability of Mφ to degrade engulfed tumour cells. Above this, we screened out liquiritin from Glycyrrhiza uralensis Fisch , which can significantly inhibit NOX2 activity and inhibit tumours, to elucidate that suppressing NOX2 can enhance the ability of Mφ to degrade tumour cells. We found that the tumour environment could activate the NOX2 activity in Mφ phagosomes, causing Mφ to produce excessive reactive oxygen species (ROS), thus prohibiting the formation of phagolysosomes before degradation. Conversely, inhibiting NOX2 in Mφ by liquiritin can reduce ROS and promote phagosome-lysosome fusion, therefore improving the enzymatic degradation of tumour cells after phagocytosis, and subsequently promote T cell activity by presenting antigens. We further confirmed that liquiritin down-regulated the expression of the NOX2 specific membrane component protein gp91 phox, blocking its binding to the NOX2 cytoplasmic component proteins p67 phox and p47 phox, thereby inhibiting the activity of NOX2. This study elucidates the specific mechanism by which Mφ cannot degrade tumour cells after phagocytosis, and indicates that liquiritin can promote the ability of Mφ to degrade tumour cells by suppressing NOX2.
The primary pathogenic mechanism of inflammatory bowel disease (IBD) involves elevated levels of reactive oxide species (ROS) in the gut, leading to oxidative stress and damage to the intestinal barrier function. We engineered a non-pathogenic bacterial strain, Escherichia coli Nissle 1917 (EcN), for oral CRISPR-Cas9 delivery to edit NOX2 (encoding the gp91 phox subunit of NADPH oxidase 2), thereby alleviating IBD symptoms by reducing ROS. EcN expressing the Cas9/sgRNA ribonucleoprotein (RNP) was encapsulated in a hydrogel (composed of hyaluronic acid, chitosan, and MgCl 2 ), which protected EcN-RNP from degradation and increased survival from 0.07% to 12%. EcN-RNP hydrogel reduced NOX2 expression by 47% and ROS by 88% in lipopolysaccharide-induced RAW264.7 cells. Moreover, oral delivery of EcN-RNP hydrogel mitigated inflammation in dextran sodium sulfate-induced colitis mouse models. Mechanistically, the hydrogel could activate the NRF2-HO1/GPX4 pathway by inhibiting NOX2 expression, enhancing oxidative defense, and promoting glutathione accumulation. Thus, the EcN-RNP hydrogel offers a promising therapeutic strategy for IBD.
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