Chuangling Ye mitigates diabetic foot ulcer through suppressing keratinocyte ferroptosis via inhibiting ACSS2/ACSL4 axis

Background: Diabetic foot ulcers (DFU) is a serious complication of diabetes, and keratinocyte Ferroptosis has been shown to accelerate its pathological progression. Chuanglin Ye (CLY), widely used in clinical practice for the treatment of DFU, has been reported to exert anti-inflammation effects and promote skin regeneration. However, its pharmacological mechanisms on DFU remain unclear.

Methods: The DFU rats were treated with CLY (0.225 and 0.45 g/mL) for 21 days to evaluate its therapeutic efficacy. The chemical composition of CLY was analyzed using UPLC-MS/MS. Network pharmacology, western blot, and immunohistochemical staining were performed to assess the anti-ferroptosis effects of CLY. To validate the protective role of CLY against Ferroptosis, RSL3, a Ferroptosis inducer, was applied in this study. Subsequently, HaCaT cells were exposed to advanced glycation end products (AGEs) to establish an in vitro model of keratinocyte Ferroptosis, further confirming the inhibitory effects of CLY on Ferroptosis. RNA Sequencing was conducted to elucidate the molecular mechanisms underlying CLY-mediated protection against keratinocyte Ferroptosis. The functional involvement of the ACSS2/ACSL4 axis was verified through plasmid-mediated gene overexpression in vitro. Molecular docking analysis was employed to identify potential bioactive compounds in CLY that target ACSS2.

Results: CLY administration significantly accelerated wound healing in DFU rats. Network pharmacology analysis indicated the regulation of Ferroptosis underlies the therapeutic effect of CLY against DFU, which was further supported by its ability to ameliorate lipid peroxidation and iron overload. In contrast, the Ferroptosis inducer RSL3 attenuated the protective effects of CLY in DFU rats. In vitro, CLY markedly suppressed AGEs-induced lipid peroxidation and excessive ROS level. Mechanistically, RNA-seq analysis and subsequent validation experiments revealed the anti-ferroptosis effect of CLY depends on the inhibition of ACSL4 acetylation in keratinocytes. Furthermore, ACSS2 was identified as a key protein promoting ACSL4 acetylation, which was inhibiting by CLY. Finally, six major compounds in CLY were identified as active constituents targeting ACSS2.

Conclusion: Our findings demonstrate CLY promotes DFU wound healing by inhibiting keratinocyte Ferroptosis through suppressing ACSS2-mediated ACSL4 acetylation, highlighting its potential as a rational therapeutic approach for the management of DFU.