Mechanism of DT-13 regulating macrophages in diabetic wound healing

Abstract

Background

Diabetes is a complex metabolic system disease, and one of the main reasons why it is difficult to heal is that macrophages cannot realize the transition from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype. Liriope spicata Lour. is a traditional Chinese medicine. According to the theory of traditional Chinese medicine, Liriope spicata Lour. has nourishing Yin Sheng Jin, moistening lung clear heart, used for lung dryness dry cough, Yin deficiency cough, throat arthralgia throat pain, thirst, internal heat thirst, upset insomnia, intestinal dryness constipation, is the classification of Yin tonifying drugs. Liriope muscari baily saponins C (DT-13) is one of the main active ingredients of Liriope spicata Lour., has significant anti-inflammatory, anti-tumor, and immunomodulatory effects.

This thesis aims to explore the role of DT-13 in angiogenesis by regulating the polarization of macrophages, and ultimately play an anti-inflammatory role in regeneration by regulating immune cells.

Methods

We conducted in vivo experiments using a diabetic mouse ulcer model to verify the effect of DT-13 in promoting wound healing. Spatial transcriptome sequencing technology was utilized to perform RNA transcript analysis on wound tissues from type II diabetic mice and non-diabetic mice. Subsequently, we used the CCK-8 assay to evaluate the impact of DT-13 on the viability of THP-1 cells (human monocytes). ELISA, immunofluorescence, and Western blot techniques were employed to study the mechanisms by which DT-13 inhibits the sustained inflammation and polarization process of M1 macrophages induced by LPS. The Transwell assay was used to assess the influence of DT-13 treatment on the co-culture of M1 macrophages induced by LPS under high glucose conditions with HUVEC cells. Finally, the chick embryo chorioallantoic membrane (CAM) assay was applied to explore the effects of DT-13 on angiogenesis stimulated by M1 macrophages under high glucose conditions with LPS stimulation.

Results

We found that DT-13 can promote wound healing in a diabetic ulcer model in mice. Through spatial transcriptome sequencing results, we discovered that type II diabetic mice had higher levels of inflammation at the wound site and abnormal expression of macrophage characteristic proteins. The CCK-8 assay detected that DT-13 at 20 μmol/L had an effect on THP-1 cells. Through Q-PCR, ELISA, immunofluorescence, and Western blot results, we found that the mechanism by which DT-13 exerts anti-inflammatory effects on M1 macrophages with sustained inflammation induced by LPS under high glucose conditions may be through the TLR4-NFKB signaling pathway, and the mechanism for inducing the polarization of M1 macrophages to M2 type may be through the ERK-STAT3 signaling pathway. Interestingly, through the Transwell assay, we found that M1 macrophages induced by LPS under high glucose conditions, after treatment with DT-13 and co-cultured with HUVECs, could increase the migratory ability of HUVEC cells. This indicates that M1 macrophages induced by LPS under high glucose conditions, after treatment with DT-13, can promote angiogenesis.

Conclusion

DT-13 can significantly promote healing wounds in diabetic mice, and its mechanism may be to participate in promoting the formation of blood vessels by changing the polarization of macrophages.