Effect And Mechanism Of Salidroside On Palmitic Acid Transportation Across Vascular Endothelial Cells And Myocardial Insulin Resistance Induced By TNF-α

Objective:Salidroside is the main biological active ingredient of Rhodiola rosea.Studies have shown that salidroside can treat Type 2 Diabetes（T2DM）by improving insulin resistance（IR）.Tumor necrosis factor-α（TNF-α）can promote the transport of palmitic acid（PA）across vascular endothelial cells and aggravate PA-induced myocardial insulin resistance.The aim of this study is to investigate the effect and mechanism of salidroside on TNF-αpromoting PA transportation across microvascular endothelial cells and promoting myocardial insulin resistanceMethods:（1）The effect of salidroside and caspase-1 inhibitor（Ac-YVAD-cmk）on the TNF-α-promoting fatty acids across microvascular endothelial cells was detected by using fatty acid in-vitro transcytosis model.（2）The expression of NLRP3,pro-caspase-1,pro-interleukin-1β（pro-IL-1β）,caspase-1 and Interleukin-1β（IL-1β）were detected by Western blotting and the effect of salidroside on TNF-α-activated NLRP3 inflammasome was evaluated.（3）Using 2,7-dichlorodi-hydroflurescein diacetate（DCFH-DA）and MitoSOX^TMM Red fluorescent probes indicating cytoplasmic ROS and mitochondrial superoxide anion levels respectively,the effect of salidroside on TNF-α-promoted oxidative stress in CMECs was evaluated.（4）In the co-culture model of neonatal rat cardiomyocytes and CMECs,insulin-stimulated 2-NBDG uptake by cardiomyocytes was measured and the effect of salidroside on TNF-αaggravating PA-induced insulin resistance in cardiomyocytes was determined.（5）In the animal experiment,C57BL/6J mice were treated with TNF-α,PA,and or SAL,respectively.Using a small animals positron emission tomography（PET）,insulin-stimulated myocardial¹⁸F-FDG uptake was detected and the percent injection dose rate（%ID/cc）,were calculated.After PET scanning,the mice were sacrificed,the hearts were collected,and cardiac emission values were directly measured using a gamma counter.Results:（1）TNF-αincreased the transportation of fatty acids across CMECs using the in-vitro fatty acid transcytosis model,and salidroside could inhibit TNF-α-promoted fatty acids transcytosis.The caspase-1 inhibitor（Ac-YVAD-cmk）also inhibited TNF-α-promoted fatty acids transcytosis.Compared with Ac-YVAD-cmk alone,co-incubation with Ac-YVAD-cmk and salidroside didn’t show synergy.（2）Western Blotting results showed that TNF-αsignificantly increased the expression of NLRP3,pro-caspase-1,caspase-1 and IL-1β,while salidroside significantly inhibited the expression of NLRP3 and IL-1βinduced by TNF-α.（3）By using DCFH-DA fluorescent probe,TNF-α-caused a time-dependent increase in ROS levels in CMECs was observed;and salidroside could inhibit TNF-α-induced increase in ROS levels.Similar phenomenon were found in mitochondrial superoxide anion levels detected by using MitoSOX^TM Red.Under incubation of TNF-α,mitochondrial superoxide anion level in CMECs increased with increasing TNF-αincubation time and salidroside blocked this effect.（4）In in-vitro CMECs-cardiomyocyte co-culture experiment,TNF-αaggravated PA-induced insulin resistance in cardiomyocytes;while salidroside inhibited this effect.（5）Small animals PET results showed that under insulin stimulation,the mice in the group of TNF-α+PA had less uptake of ¹⁸F-FDG.Salidroside improved insulin-induced ¹⁸F-FDG uptake.The results of the cardiac gamma counter test were consistent with the PET scan results.Conclusion:TNF-αup-regulates ROS in cytoplasmic and superoxide anion in mitochondria and activates NLRP3 inflammasome in CMECs.Activation of NLRP3 inflammasome is involved in TNF-αpromoting PA transcytosis across CMECs,thereby aggravating PA-induced insulin resistance.Salidroside may play a role in inhibiting TNF-α-promoted fatty acid transcytosis and insulin resistance by reducing oxidative stress and NLRP3 activation induced by TNF-α.