Ursolic Acid Alleviates the Injurious Effects of Busulfan Plus Cyclophosphamide on Hepatic Sinusoidal Endothelial Cells By Inhibiting the TNFα/NF-Κb Signaling Pathway

Background The combination of busulfan and cyclophosphamide (BuCy) is a commonly employed myeloablative regimen for allogeneic hematopoietic stem cell transplantation (HSCT). However, its detrimental effects on hepatic sinusoidal endothelial cells (HSCs) can elevate the risk of hepatic sinusoidal obstruction syndrome (SOS), a severe complication that threatens patient survival post-HSCT. Despite this, the underlying molecular mechanisms of hepatic SOS remain elusive, hindering the development of specific therapeutic targets. Ursolic acid (UA), a compound known to mitigate organ damage induced by agents such as BU and CY, has yet to be explored for its protective role in hepatic SOS.

Objective This study aimed to investigate the therapeutic potential of UA in hepatic SOS, elucidating the molecular mechanisms underlying its protective effects.

Methodology Primary rat-derived HSCs were cultured, and the half-inhibitory concentrations of UA and BuCy were determined using the Cell Counting Kit-8. Experimental groups comprised a blank control, DMSO control, and BuCy treatment groups. Reactive oxygen species (ROS) levels and apoptosis were assessed via flow cytometry. Furthermore, mRNA high-throughput sequencing was conducted to analyze the molecular mechanisms mediating BuCy-induced HSC damage. To validate the findings in vivo, experiments were performed in SD rats, grouped into blank control, DMSO control, and BuCy treatment cohorts. Liver tissues underwent hematoxylin and eosin (HE) staining, immunohistochemistry, and ROS activity analysis. Additionally, Enzyme-Linked Immunosorbent Assay (ELISA) and Western blot (WB) were employed to quantify relevant molecular changes. Finally, in vitro cellular experiments were conducted to explore the effects of BuCy and UA on HSCs, including OE-NF-κB overexpression, BuCy combined with UA, and BuCy combined with an NF-κB inhibitor.

Results In vitro experiments demonstrated that HSC apoptosis was significantly elevated in the BuCy group compared to the control (p<0.001), whereas UA combined with BuCy reduced apoptosis (p<0.05). Similarly, BuCy treatment increased ROS levels compared to the control (p<0.001), and UA mitigated ROS production (p<0.05). mRNA sequencing identified 536 downregulated and 4330 upregulated genes, enriched in pathways related to apoptosis, TNFα, MAPK, among others. Notably, TNFα, MMP9, TAK1, IKKα, and NF-κB mRNA expression levels were significantly upregulated in the BuCy group (p<0.05), corroborated by ELISA results for TNFα protein (p=0.003). These findings suggest that BuCy may damage HSCs via the TNFα/NF-κB signaling pathway. Further validation using OE-NF-κB and NF-κB inhibitor experiments confirmed the role of NF-κB in BuCy-induced apoptosis and ROS generation. Additionally, UA inhibited the expression of TNFα/NF-κB pathway factors, including TNFα, IKKα/β, IκBα, and p65, both in vitro and in vivo.

Conclusion Our findings emphasize the pivotal role of the TNFα/NF-κB signaling pathway in BuCy-induced HSC damage, enhancing our understanding of the molecular mechanisms underlying hepatic SOS and suggesting potential therapeutic avenues.

No relevant conflicts of interest to declare.