Bile Acids Elicited Endothelium-Dependent Vasoconstrictor Hypo-activity Through TRPV4 Channels In Obstructive Jaundice

ObjectiveObstructive jaundice(OJ)is a commonly seen symptom caused by intra-or extra-hepatic bile duct obstruction,resulting in various pathophysiology changes to the body,including cardiovascular dysfunction.During anesthesia,the blood pressure of OJ patient's is lower,and the vascular reactivity to vasoactive drug is weakened.Previous studies of vascular reactivity suggest that changes of vascular endothelial cell activity and permeability changes in aorta is participated in this process.However,the etiology of vascular hypo-reactivity in OJ patients remains uncertain.The transient receptor potential cation channel V4(TRPV4)is widely expressed on endothelial cells,and participates in regulation of vascular reactivity.The aim of this study was to explore the regulatory role of TRPV4 channel in the vascular hypo-reactivity in OJ patients.Method1.The bile duct ligation(BDL)model is established to mimic the clinical obstructive jaundice.The thoracic aortic segments of the model rats are isolated and trimmed to 3-4 mm vascular rings,and the vascular tension is measured under vascular tone meter.The expression of TRPV4 protein and its mRNA in aortic vessels are detected at different time points,and the correlation between its expression and time after BDL,as well as the vascular tone is analyzed.2.Serum concentration of bile acids and bilirubin are tested.Rat thoracic aortic endothelial cells are cultured in vitro,with BAs and bilirubin are added to the ECG medium as experiment group,an equal amount of medium is added to the control group.We then observe the TRPV4 protein expression by measuring the expression of TRPV4 protein and mRNA in endothelial cells stimulated by BAs or bilirubin,and we use immunofluorescence to explore the co-expression of TRPV4 with endothelial cell Dil protein.3.The vascular-constriction and diastolic state of thoracic aorta in BDL rats are observed under different concentrations of vasoconstrictor norepinephrine and vasodilator acetylcholine,and the recovery of vasoconstriction and diastolic are observed with application of TRPV4 inhibitor HC-067047.We further explore the downstream pathway of TRPV4 protein,using L-NAME(a nitric oxide synthase inhibitor),indomethacin(a non-selective cyclooxygenase inhibitor)and celecoxib(a selective COX2 inhibitor).1.The contractile ability of rat thoracic aorta rings are timely decreased after BDL in KCL solution,meanwhile,the expression of TRPV4 protein and mRNA in thoracic aorta rings showed a time-dependent increasing trend.We also discover that relaxation of thoracic aortic rings is concentration-dependently and time-dependently increased after using the TRPV4 protein agonist GSK1016790 A.2.Serum concentration of bile acids and bilirubin are abnormally higher in BDL rats.Compared with the control group,the expression of endothelial cell TRPV4 in BAs medium was significantly increased,while no elevation of the endothelial cell TRPV4 in bilirubin medium can be found.Immunofluorescence showed that TRPV4 protein is co-labeled with Dil protein in endothelial cell membrane.Our results also show that intracellular calcium concentration in the BAs medium is more prominent.3.After using HC-067047,vasodilation effects caused by acetylcholine is slightly changed,while the vasoconstriction effects caused by norepinephrine is significantly decreased.The use of different inhibitors against the downstream molecules of TRPV4 show that there is a significant the reversal effect of COX inhibitors on the vasoconstriction.Further experiments show that the use of HC-067047 can significantly reduce the expression of COX2 expression.ConclusionFollowing conclusions can be drawn from our results:1.Obstructive jaundice induces an increased expression of TRPV4 protein and decreased vascular reactivity.Our results suggest that the effect of vasoconstriction is decreased after BDL,and this trend is consistent with the increase of TRPV4 protein in the aorta ring.Further,pharmacological inhibition of TRPV4 can cause a concentration-dependent relaxation response of aortic ring in BDL rats.2.Bile acid mediates TRPV4 protein expression in aortic vascular endothelial cell.Bile acid increased the expression of TRPV4,while this phenomenon is not significant in bilirubin,suggesting that bile acid is the key molecule that causes vascular tone changes in OJ.The results of intracellular calcium imaging confirmed this conclusion and verified the presence of bile acid and calcium influx.3.TRPV4 protein participates in vascular hypo-responsiveness through COX pathway in OJ.The vascular hypo-reactivity of OJ is manifested by a decreased response to Result vasoconstrictors and vasodilators.TRPV4 protein is involved in the regulation of vascular hypo-responsiveness and is manifested as insensitive to vasoconstrictors.Downstream molecular screening reveals that the COX2 pathway may play a role in this process.