Artesunate Induces Ferroptosis in Hepatic Stellate Cells and Alleviates Liver Fibrosis

Artesunate (Art) has emerged as a promising therapeutic agent for liver fibrosis due to its ability to induce ferroptosis in hepatic stellate cells (HSCs), which play a central role in the progression of liver fibrosis. Here is a detailed explanation of how Artesunate works and its potential therapeutic benefits:

### 1. **Hepatic Stellate Cells and Liver Fibrosis**

• Liver fibrosis is a condition characterized by excessive deposition of extracellular matrix (ECM) proteins, primarily driven by the activation of HSCs.
• Activated HSCs are responsible for producing fibrogenic proteins like α-SMA, Collagen I, and Fibronectin, which contribute to the scarring and progression of fibrosis.
• Targeting HSCs to inhibit their activation or induce their death is a key strategy for combating liver fibrosis.

### 2. **Ferroptosis as an Anti-Fibrosis Strategy**

• Ferroptosis is a regulated form of cell death characterized by iron overload, reactive oxygen species (ROS) accumulation, lipid peroxidation, and depletion of glutathione (GSH).
• Inducing ferroptosis in activated HSCs offers a novel approach to reducing fibrogenic activity and alleviating liver fibrosis.

### 3. **Artesunate Induces Ferroptosis in HSCs**

• Artesunate (Art), an anti-malarial drug, has been found to selectively induce ferroptosis in activated HSCs (e.g., LX2 cells) while sparing normal hepatocytes (LO2 cells) at therapeutic concentrations.
• Mechanisms of ferroptosis induction by Art include:
• Reduction of intracellular GSH levels.
• Increase in free iron levels and lipid peroxidation (measured by malondialdehyde, MDA).
• Elevation of ROS levels, as confirmed by DCFH-DA fluorescence assays.
• These changes lead to ferroptotic death in HSCs, characterized by mitochondrial shrinkage, loss of cristae, and a shift in JC-1 fluorescence signals.

### 4. **Artesunate Suppresses HSC Activation**

• Art downregulates key markers of HSC activation, including α-SMA, Collagen I, and Fibronectin, thereby reducing the fibrogenic potential of these cells.
• This suppression of HSC activation further contributes to the alleviation of liver fibrosis.

### 5. **The ROCK1/ATF3 Axis: Key Regulatory Pathway**

• Artesunate exerts its effects on HSCs through the ROCK1/ATF3 signaling axis:
• Art promotes the degradation of ROCK1 (Rho-associated protein kinase 1) via the ubiquitin–proteasome pathway, while ROCK2 remains unaffected.
• The reduction in ROCK1 levels decreases ATF3 phosphorylation, allowing ATF3 to accumulate in the nucleus.
• Nuclear ATF3 suppresses the transcription of SLC7A11, a key component of the cystine/glutathione (GSH) pathway, leading to reduced cystine uptake and GSH depletion.
• This cascade ultimately drives ferroptosis in HSCs.

### 6. **Key Experimental Findings**

• **ATF3's Role:** ATF3 is essential for the induction of ferroptosis. Knockdown of ATF3 using siRNA reverses Art-induced ferroptosis, restores GSH levels, and reactivates HSCs.
• **ROCK1's Role:** Overexpression of ROCK1 blocks the effects of Art, preventing ferroptosis, restoring mitochondrial function, and reversing the suppression of fibrogenic markers like α-SMA and Collagen I.
• **Ferroptosis Inhibitors:** Agents like NAC (N-acetylcysteine, which increases intracellular GSH) and Fer-1 (a ferroptosis inhibitor) rescue HSCs from Art-induced ferroptosis, confirming the role of oxidative stress and lipid peroxidation in this process.

### 7. **In Vivo Evidence**

• Artesunate has shown significant anti-fibrotic effects in animal models of liver fibrosis (e.g., CCl₄-induced mouse models):
• Art reduces collagen deposition, improves hepatocyte architecture, and alleviates liver fibrosis.
• Serum biomarkers of liver fibrosis, such as hyaluronic acid (HA), laminin (LN), procollagen III (PC-III), type IV collagen (IV-C), and liver enzymes (AST, ALT, ALP), improve significantly after Art treatment.
• Targeting HSC-specific ATF3 or ROCK1 alters the outcomes of Art treatment in vivo. For example, ATF3 interference or ROCK1 overexpression diminishes Art's anti-fibrotic effects, further validating the importance of the ROCK1/ATF3 axis.

### 8. **Therapeutic Potential of Artesunate**

• Artesunate's ability to selectively induce ferroptosis in activated HSCs while sparing normal hepatocytes makes it a highly promising candidate for anti-fibrosis therapy.
• By targeting the ROCK1/ATF3 axis, Art effectively suppresses HSC activation, reduces fibrogenic protein production, and alleviates liver fibrosis in preclinical models.

### Conclusion:

Artesunate represents a novel and effective therapeutic approach for liver fibrosis by inducing ferroptosis in activated HSCs through the ROCK1/ATF3 axis. Its ability to selectively target HSCs, coupled with strong preclinical evidence of efficacy, positions Artesunate as a potential anti-fibrotic agent for future clinical applications.