Effects of tocotrienol-rich fraction (TRF) on farnesoid- x-receptor (FXR) gene and its signalling pathways in the heart of high fat diet-fed mice

Cardiovascular Disease (CVD) is the number one cause of mortality globally of non- communicable disease. High fat diet (HFD) as a risk factor for CVD development. FXR and its target genes (shp and stat3) are expressed in the heart. The activation of FXR and its target genes in the heart demonstrated anti-inflammatory and anti-atherosclerotic effects, improved cardiac remodelling, and reduced blood pressure. Similarly, tocotrienol-rich fraction (TRF) is known to possess cardioprotective effects such as anti- atherosclerosis, anti-inflammatory, anti-oxidant activities, anti-thromboembolic and anti-adipogenic effects. However, whether TRF activates FXR and its target genes in the HFD animal model is not known. A total of 28 male Jax mice leptin-knockout B6.Cg-LepOb/J strain were used. All mice were given HFD consisting 60% energy of from fat, 20% from carbohydrate and 20% of protein. One group was given HFD without intervention, another three groups were fed with HFD mixed with palm kernel olein (PKO), tocotrienol-rich fraction with long-chain triglyceride (TRF-LCT) and tocotrienol-rich fraction with medium-`chain triglyceride (TRF-MCT) at 200mg/kg/day via oral route. After 6 weeks of treatment, anthropometric indices and random blood glucose were assessed and the heart was collected for measurement of fxr, shp, stat3, sod1, sod2 and gpx1 gene expression by real-time quantitative polymerase chain reaction (RT-qPCR), STAT3 Total protein expression and STAT3 pY705 by enzyme linked immunosorbent assay (ELISA). Serum samples were collected for untargeted metabolomic profiling from all mice. The result showed a significant increment of 3.18- fold in fxr expression for the HFD+TRF-MCT group (3.18±0.61) compared to HFD. Meanwhile, a significant 10-fold increment in fxr expression was seen in the HFD+PKO group (10.0±2.05) compared to HFD. No significant difference was seen in the shp, sod1 and sod2 expression between HFD+TRF-MCT and the HFD group. A significant decrease of stat3 expression was seen in both HFD+PKO (0.49±0.048) and HFD+TRF- MCT (0.53±0.053) groups. A significant increment of 1.79-fold higher gpx1 expression was observed in HFD+TRF-MCT group (1.789±0.164). No significant differences for STAT3 Total and STAT3 pY705 protein expression were seen in all groups. In metabolomic profiling, 31248 peaks were generated in positive mode, and 15204 peaks were detected in negative mode analysis when comparing HFD+TRF-LCT group against HFD group. Metabolites involving bile acids secretion, biotin metabolism and cholesterol metabolism were found to be significantly involved (p<0.05).TRF supplementation in high fat diet-fed mice confers its protective effects via the regulation of cardiac fxr and its target genes, protection against inflammation and oxidative stress through multiple biochemical signalling pathways