Tetrahydropalmatine Attenuates Liver Fibrosis by Suppressing Endoplasmic Reticulum Stress in Hepatic Stellate Cells
Liver fibrosis is a pathological condition characterized by the activation of hepatic stellate cells (HSCs) and the excessive deposition of extracellular matrix. This process is driven by various pathogenic mechanisms, among which endoplasmic reticulum stress (ERS) in HSCs has been identified as a critical contributor. ERS can lead to the activation of HSCs, exacerbating liver fibrosis. Despite extensive research on liver fibrosis, effective anti-fibrotic drugs remain scarce. Tetrahydropalmatine (THP), an active ingredient derived from the Chinese herb Corydalis yanhusuo, has shown hepatoprotective effects in previous studies. This study aimed to investigate the effects of THP on liver fibrosis and elucidate the underlying mechanisms.
The study utilized C57BL/6J male mice weighing 20–25 grams, which were randomly divided into five groups: an Oil group, a carbon tetrachloride (CCl4) group, and three THP treatment groups with varying dosages (THP-Low [THP-L], 20 mg/kg; THP-Middle [THP-M], 40 mg/kg; and THP-High [THP-H], 80 mg/kg). The THP powder was suspended in a 0.5% sodium carboxymethylcellulose (CMC-Na) solution. All mice received intraperitoneal injections of either Oil or a 20% CCl4 solution in oil (5 mL/kg, twice per week for six weeks). Concurrently, CCl4-induced mice were administered THP intragastrically on a daily basis.
Histopathological examinations, including hematoxylin-eosin (H&E) staining, revealed marked necrosis, inflammatory cell infiltration, and disruption of tissue architecture in the livers of mice treated with CCl4. These pathological changes were significantly ameliorated by THP treatment. Sirius red staining demonstrated that THP treatment significantly reduced collagen deposition. Additionally, immunohistochemical analysis showed that the expressions of α-smooth muscle actin (α-SMA) and collagen I were markedly downregulated in CCl4-induced mice treated with THP.
Serum alanine aminotransferase (ALT) levels, which were significantly elevated in the CCl4 group, were markedly reduced by THP treatment. Quantitative real-time PCR (qPCR) assays indicated that hepatic mRNA levels of profibrotic genes, such as α-SMA, collagen I, platelet-derived growth factor, connective tissue growth factor, and tissue inhibitor of metalloproteinase-1, were significantly reduced by THP treatment in CCl4-induced mice. Western blotting analysis further confirmed that THP downregulated α-SMA in the livers of CCl4-induced mice. These findings collectively suggest that THP attenuates chronic liver injury, inflammation, and fibrosis in CCl4-induced mice.
To explore the molecular mechanisms underlying the anti-fibrotic effects of THP, RNA sequencing (RNA-seq) was performed to identify significantly enriched signaling pathways and differentially expressed genes (DEGs). The DEGs were selected based on a P value of less than 0.05 and a log2 (Fold Change) greater than 1. Gene ontology enrichment analysis revealed that the main signaling pathways involved in liver fibrosis included MAPK pathways and 26 pathways related to ERS, inflammation, and fibrosis. The heatmap analysis demonstrated that genes associated with ERS, inflammation, and fibrosis pathways were significantly downregulated by THP treatment.
ERS has been shown to play a pivotal role in HSC activation. When the ER is overwhelmed with unfolded or misfolded proteins, protein kinase RNA-like ER kinase (PERK) undergoes autotransphosphorylation. The major substrate of PERK phosphorylation is eukaryotic translation initiation factor 2α (eIF2α). Phosphorylation of eIF2α leads to global translation attenuation while permitting the selective translation of activating transcription factor 4 (ATF4). ATF4 primarily acts through the unconventional splicing of transcription factor X-box binding protein 1 (XBP1). Spliced XBP1 (sXBP1) contributes to HSC activation by facilitating cargo secretion through the expansion of ER capacity and upregulation of protein secretion. C/EBP homologous protein (CHOP), a downstream target of ATF4, is implicated in the progression of liver disease.
To verify the effect of THP on inflammation and ERS, qPCR assays were performed. The results showed that hepatic mRNA levels of pro-inflammatory genes, such as C-C motif chemokine ligand 2 and C-X-C chemokine receptor 2, and ERS-related genes, including Ddit3 (Chop), Hspa5 (glucose-regulated protein 78, Grp78), Atf4, and sXbp1, were significantly downregulated by THP treatment in fibrotic mice. Immunofluorescence assays for co-staining of CHOP/α-SMA indicated a significant reduction in the positive area in the THP treatment groups, further confirming the inhibitory effect of THP on ERS in HSCs.
The study also examined the effects of THP on ERS in LX-2 cells, an activated human HSC line. qPCR and Western blotting analyses revealed significant downregulation of ATF4, CHOP, and α-SMA after THP treatment for 24 hours. Extracellular signal-regulated kinase 1/2 (ERK1/2), which is implicated downstream of inositol-requiring enzyme 1α (IRE1α) kinase activity, plays important roles in HSC activation. Previous studies have reported that L-THP pretreatment alleviates hepatocyte injury caused by ischemia/reperfusion by inhibiting p-ERK1/2 and NF-κB. In line with these findings, co-staining of p-ERK1/2/α-SMA on liver sections showed obvious downregulation in the THP treatment groups.
Recent studies have demonstrated that levo-tetrahydropalmatine prevents liver fibrosis through the PPAR gamma/NF-kappaB and TGF-beta1/Smad pathways. However, whether the inhibitory effects of THP on ERS are direct or mediated through other signaling pathways, such as the TGF-beta1/Smad pathway, requires further investigation. Collectively, the data suggest that THP inhibits HSC activation and liver fibrosis by suppressing ERS in HSCs.
In summary, this study elucidates a novel mechanism by which THP attenuates liver fibrosis. The inhibitory effect of THP on HSC activation is partially dependent on the suppression of ERS. These findings provide a foundation for the potential therapeutic application of THP in the treatment of liver fibrosis.
doi.org/10.1097/CM9.0000000000001883
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