Effects and Mechanisms of Lactococcus lactis Carrying Id2 Gene in the Treatment of Ulcerative Colitis
To address the issues of high production costs and poor stability in protein-based drug preparations, this study intends to introduce the Id2 gene into Lactococcus lactis to develop a new type of UC intervention drug that can simultaneously exert the immunomodulatory effect of the Id2 protein and the probiotic effect of LL in the gut. Result.1. The PCR verification of the bacterial suspension indicated that the size of the target fragment was 500 bp, suggesting that the engineered Lactococcus lactis LL-Id2 was successfully constructed. The WB results confirmed that the ID2 protein was mainly expressed intracellularly.2. LL, LL-Id2 and ID2 protein could all alleviate the colonic injury induced by DSS in UC mice: compared with the DSS group, the DAI scores of these three groups of mice decreased, the symptoms of colon shortening were relieved, the colonic pathological scores decreased, and the mRNA levels of pro-inflammatory cytokines decreased.3. The alleviation effect of LL-Id2 on UC was more significant than that of LL: the effect on alleviating weight loss was more obvious, the recovery effect of colonic mucosal barrier was better, the mRNA level of pro-inflammatory cytokine TNF- was lower, and the reduction in the number of neutrophils was more significant.4. LL-Id2 significantly enhanced the regulatory effect on the intestinal microbiota of UC mice: LL-Id2 significantly increased the abundance of beneficial bacteria such as Ligilactobacillus, norank Muribaculaceae and norank Prevotellaceae, while down-regulating the abundance of the commensal pathogen g Desulfovibrio. Compared with the DSS+LL group, the DSS+LL-Id2 group samples showed better intra-group repeatability, a greater separation distance from the DSS group samples, and fewer shared taxa with the DSS group. In addition, in terms of the community assembly process, the treatment of LL-Id2 reduced the random ecological processes in the DSS group and the DSS+LL group, and increased the proportion of deterministic ecological processes, promoting the reconstruction of the intestinal microbiota of UC mice towards a stable state.5. The results of transcriptome sequencing revealed that compared with LL, LL-Id2 down-regulated inflammatory-related pathways such as macrophage activation and inflammatory bowel disease, down-regulated genes such as Ifit3, Zbp1, and Xaf1, up-regulated repair processes such as innate immune response in mucosa and epithelial cell proliferation, and up-regulated the abundance of key genes of the Wnt signaling pathway such as Axin2, Nox1, and cell proliferation marker Tk1.6. The results of the correlation analysis between the microbiota and host gene expression suggest that the abundance of Axin2 and Nox1 genes is significantly positively correlated with norank Muribaculaceae and Lactococcus. The composition and changes of the microbiota indirectly regulate the transcriptional expression of host genes, and host gene expression also affects the community structure of intestinal microbiota. Both jointly play a regulatory role in the colon of UC mice.7.LL-Id2-EVs can more effectively inhibit the inflammatory response of Caco-2 cells: LL-Id2-EVs can significantly reduce the mRNA expression levels of pro-inflammatory factors IL-6, TNF-, and IL-1 in LPS-induced Caco-2 cells. In contrast, LL-EVs have no significant effect on the expression of the above inflammatory cytokines. Conclusion1. The engineered Lactococcus lactis carrying the Id2 gene was successfully constructed and the expression of ID2 protein was achieved.2. Compared with LL, the engineered strain LL-Id2 can significantly alleviate the pathological features of DSS-induced UC in mice. The mechanism is related to Lactococcus lactis regulating the intestinal microecological balance and the ID2 protein regulating the immune, inflammatory and barrier repair-related genes and pathways in the colon tissue transcriptome.3.