The results of the present study further indicated the molecular mechanism of Lp299v supplementation. Meanwhile, the hub genes and the results of enrichment analyses expanded the molecular mechanism of Lp299v supplementation in patients with stable CAD. Our experimental data were categorized into the DAU group and the non-DAU group. The former was composed of 8 samples, and the latter included 30 samples. The bioinformatics analysis was (Fig. 6) performed on both the DAU and the non-DAU groups. We found some differences between the results of two groups, which could reveal the weakening effect of alcohol on the protection of intestinal inflammation. This hypothesis will be further analyzed in the future experiments.

The composition of gut microbiota is very complex, including bacteroidetes, firmicutes, proteobacteria, fusobacteria, actinobacteria. In different populations with different diets, gut microbiota varies in terms of number and proportion (Yamashita et al. 2016). Previous studies have found that gut microbiota was associated with cardio-metabolic diseases (Zhou et al. 2020). Some scholars demonstrated that intestinal inflammation may be a risk factor for atherosclerosis, and gut microbiota and intestinal immunity could be used as therapeutic targets for the treatment of CAD (Yamashita et al. 2015). Lactobacillus plantarum is one of the important members of the genus Lactobacillus, and it has been identified as a probiotic, confirming its value for further research and application (Kleerebezem et al. 2010).

In both the DAU and non-DAU groups, the GO enrichment analysis indicated cell mitosis and reduced chemotactic movement, suggesting that Lp299v could promote immune regulation and attenuate immune response. Additionally, the GO enrichment analysis revealed that mitosis was up-regulated, apoptosis and cellular senescence were down-regulated, which may be secondary to the activation of the PI3K-Akt pathway. The KEGG pathway enrichment analysis showed apoptosis and senescence of cells in the DAU group, as well as the down-regulated expression of pathogen infection signaling pathways and cancer-promoting signaling pathways, as well as apoptosis and cellular senescence. While in the non-DAU group, pathogen infection signaling, oxidative stress signaling, and apoptosis signaling were down-regulated to varying degrees. In the DAU group, activation of these pathways inhibited cellular senescence and apoptosis, attenuated innate immune escape, and reduced inflammatory response. In the non-DAU group, activation of these pathways inhibited the proliferation and recruitment of macrophages, decreased the expression of interferon-β, and attenuated immune response and chemotactic cytokines. A previous study showed that alcohol intake could promote the growth of Gram-negative bacteria in the intestines and increase the permeability of the intestines, leading to systemic inflammations, which may explain the difference in the results of the KEGG analysis between the DAU and non-DAU groups (Parlesak et al. 2000). Another study showed that Lp299v reduced levels of IL-8, IL-12 and improved vascular endothelium, which supports our results (Malik et al. 2018). A number of scholars confirmed that Lp299v could attenuate the immune response in patients with coronary heart disease (Hofeld et al. 2021; Naruszewicz et al. 2002). A previous study reported that Lp299v significantly reduced cell apoptosis, which was consistent with the down-regulation of apoptosis pathway found in our study (Dykstra et al. 2011). Earlier studies showed that Lp299v-contained beverages could protect body cells against excessive production of reactive oxygen species, thereby protecting against oxidative damage (Gawlik-Dziki et al. 2021; Onning et al. 2003). These findings experimentally support our findings in the bioinformatics analysis.

We, in the present study, found genes that were associated with G protein-coupled receptors, immune responses, cell proliferation, and apoptosis regulation in the PPI networks. A previous study reported that Lp299v could be used in the treatment of some types of cancer, possibly because Lp299v promotes apoptosis, suppresses (Table 5) inflammation, and inhibits cell proliferation (Kazmierczak-Siedlecka et al. 2020). In our screening of hub genes, it was found that the majority of the hub genes were G protein-coupled receptor-related genes, followed by inflammation-activated chemotaxis-related genes.

Diverse types of chemokines act through G protein-coupled receptors, which are collectively known as chemokine receptors. Interleukin receptor and histamine receptor are involved in inflammation and allergic reactions. In the current study, it was hypothesized that one or more of the components of Lp299v could modulate the G protein-coupled receptor, thereby regulating the immune system, inhibiting inflammatory response, promoting cell mitosis and proliferation, and inhibiting cellular senescence and apoptosis.

We found the expressions of the genes encoding adenylate cyclase, and β- and γ-subunit of G protein-coupled receptor. The results of enriched MF of DEGs indicated that chemokine receptors, acting as a G protein-coupled receptor, could play important roles in the immune system cell signaling pathway. Using the constructed PPI networks, it was found that the expressions of CXCL11 and CXCR3 in the DEGs of this sample had a parallel relationship. Both CXCL11 and CXCR3 are pro-inflammatory factors. Hence, it was further hypothesized that Lp299v supplementation could modulate the expressions of chemokine receptors via a subsequent cellular effect.

In summary, Lp299v could treat patients with stable CAD by modulating inflammatory responses. Our study also found the role of G protein-coupled receptor, mitosis, apoptosis, and senescence of cell in the process of Lp299v supplementation. Cell mitosis, apoptosis, and senescence were associated with the PI3K-Akt pathway. The chemokine receptors could act as a G protein-coupled receptor, playing important roles in the immune system cell signaling pathway. It was supposed that chemokine receptors could have a cellular effect through the Gs-cAMP-PKA signaling pathway.

In this study, we found the following limitations. The small sample size might cause the bias in the experiment. We identified several hub genes and molecular pathways closely related to the effects of Lp299v supplementation, we didn’t explore the interactions between these hub genes yet. We used an unadjusted P-value < 0.05 as the threshold for significant difference and did not account for FDR, so the results of these analyses are purely exploratory.Thus, our findings remain to be further verified by additional in vitro experiments.