Abstract
Purpose
Oxidative stress plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). TRPM2 ion channel functions as a molecular sensor for oxidative stress. The aim of this study was to examine the protective effects of Salidroside, a powerful antioxidative plant, on TRPM2 in an established in vitro model of NAFLD.
Methods
NAFLD model was established by palmitic acid (PA) in hepatic L02 cell lines and was added to the media at a final concentration of 400 μM. Cells were used as normal group, PA group and PA receiving varied concentrations of Salidroside (75μg/mL, 150μg/mL, 300μg/mL). After treating 24 hrs, MTT assay was used to detect cell viability, and ALT level was measured using an appropriate kit assay. Intracellular lipid accumulation was observed by Oil red O staining. Cytosolic Ca2+ concentrations were evaluated by flow cytometer with Fluo-3/AM. Quantitative RT-PCR was used to measure the mRNA expression of TRPM2, IL-1β and IL-6, and the protein expressions of TRPM2, p-CaMKII and autophagy (LC3B, p62) were determined using Western blot.
Results
Treatment with Salidroside effectively restored liver injury and alleviated lipid droplet deposition in a dose-dependent manner, which was associated with inhibition of TRPM2/Ca2+/CaMKII pathway. Additionally, autophagic clearance was enhanced by intervention with Salidroside in a dose-dependent manner. Further investigation indicated that Salidroside down-regulated the mRNA expression of IL-1β and IL-6-pro-inflammatory cytokines.
Conclusion
These results suggest that Salidroside could alleviate inflammatory injury and steatosis via autophagy activation mediated by downregulation of the TRPM2/Ca2+/CaMKII pathway. Targeting the TRPM2 ion channel is a novel treatment strategy for oxidative stress-induced liver in NAFLD.
Acknowledgment
This research was funded by Liaoning Nature Science Fund (20180550081), China.
Abbreviations
NAFLD, non-alcoholic fatty liver disease; NASH, Non-alcoholic steatohepatitis; TRPM, transient receptor potential melastatin; Sal, Salidroside; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; CaMKII, calmodulin-stimulated protein kinaseII; PA, palmitic acid; ALT, alanine aminotransferase; qRT-PCR, quantitative real-time PCR; LC3B, Light chain 3B; IL, interleukin; ROS, reactive oxygen species.
Author Contributions
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Disclosure
The authors report no conflicts of interest in this work.