Targeting Gut Microbiota and GPX4/xCT Signaling: A Mechanism by Tang Liping Formula Inhibits Pancreatic Ferroptosis in T2DM

Introduction: To explore the potential mechanism of the Chinese formula Tangliping (TLP) in the treatment of T2DM. Methods: This study employed bibliometric methods to conduct a thematic keyword cluster analysis of the drugs contained in TLP, and combined network pharmacology to explore the related mechanisms of TLP in T2DM. A total of 30 mice were included and divided into 5 groups, including the control group (n=6), model group (n=6), TLP-L group (n=6), TLP-H group (n=5; one mouse died during the experiment), and MET group (n=6). The T2DM mouse models were subsequently established, and the efficacy of TLP in treating T2DM was verified by measuring fasting blood glucose, conducting an oral glucose tolerance test, and assessing blood lipid and serum insulin levels. H&E staining and immunohistochemistry were used to observe damage in colonic and pancreatic tissues. The effect of TLP on intestinal flora was examined via fecal 16S rRNA sequencing. The impacts of TLP on ferroptosis in pancreatic cells of T2DM mice were evaluated by measuring serum levels of GSH and SOD, the immunohistochemical positivity rate of 4-hydroxynonenal (4-HNE) in pancreatic tissue，and by detecting the expression of GPX4, GSH, SLC7A11, FTH1, HO-1, HIF-1α, and NRF2 via RT-qPCR and Western blot. By analyzing data on gut microbiota and ferroptosis-related indicators, a heatmap was generated to reveal the potential association between TLP-mediated gut microbiota regulation and ferroptosis in T2DM. Results: The bibliometric and network pharmacology analyses suggested that TLP may exert therapeutic effects on T2DM by modulating the gut microbiota and ferroptosis. Our study demonstrated that TLP reduced fasting blood glucose, improved glucose tolerance and lipid profiles, ameliorated insulin resistance and islet dysfunction, alleviated pancreatic tissue damage, and modulated the abundance and composition of intestinal microbiota in T2DM mice. We found that TLP increased serum levels of SOD and GSH, decreased positivity rates of 4-HNE in mouse islets, upregulated the expression of GPX4, GSH, SLC7A11, FTH1, HO-1, HIF-1α, and NRF2, and downregulated the expression of TFRC. Furthermore, our study found that the Bacteroidota showed a negative correlation with 4-HNE expression, while Desulfobacterota exhibited a positive correlation with 4-HNE expression and a negative correlation with GPX4 expression. And the norank_f__ Desulfovibrionaceae, norank_f__Oscillospiraceae, and Lactobacillus also demonstrated significant correlations with key ferroptosis indicators, including GPX4, GSH, and 4-HNE. Discussion: The global prevalence of type 2 diabetes mellitus (T2DM) continues to rise, posing a serious threat to human quality of life. This study, by integrating pharmacodynamic results, gut microbiota analysis, and mechanism verification, confirmed that TLP can improve the gut microbiota structure in T2DM mice and inhibit pancreatic ferroptosis. The research findings provide a theoretical basis for the clinical application of traditional Chinese medicine in T2DM. The identified key gut microbiota and molecular targets represent a promising new avenue for clinical development. However, whether the inhibition of ferroptosis is a direct result of gut microbiota remodeling remains to be clarified. Verifying this mechanism association is a key objective for our future research. Conclusion: TLP exerts its therapeutic effect on T2DM by inhibiting pancreatic tissue ferroptosis through regulating the gut microbiota and GPX4/xCT signaling pathway.