Hydrogen Bonds Of Bilirubin And 5-HT_3A Receptor Contribute To The Increased Pain Thresholdtin Obstructive Jaundice

ObjectiveObstructive jaundice is characterized by cholestasis caused by the obstruction of intrahepatic or extrahepatic bile duct with stone,cancer,or pancreas diseases.Liver damage and complex pathophysiological changes in the body occurred gradually.In obstructive jaundice patients,pain threshold is increased and they usually show insensitive to nociceptive stimulation.In clinical experience,the increased pain threshold easily leads to delayed treatment,relative excess use of analgesic drugs,respiratory depression and delayed recovery in perioperative period.This study is to investigate the mechanism that increased level of bilirubin in obstructive jaundice increases the release of inhibitory neurotransmitter?-aminobutyric acid?GABA?,enhances the activity of GABAergic neurons by activating 5-hydroxytryptamine 3A(5-HT_3A)receptor in the spinal dorsal horn,and then mediates the increased pain threshold in obstructive jaundice.Methods1 Obstructive jaundice model was established by bile duct ligation?BDL?,and liver function indexes in serum and cerebrospinal fluid?CSF?were analyzed by automatic biochemical analyzer.Pain threshold of Sprague-Dawley?SD?rats after BDL and bilirubin intrathecal injection was measured.Subunits of 5-HT₃ receptor expression were analyzed by western blot in BDL,bilirubin intrathecal injection rats and cultured spinal dorsal horn neurons.Activation of spinal dorsal horn neurons and 5-HT_3A receptor expression were further observed by immunofluorescence.2 Pain threshold was measured by injection of 5-HT_3A receptor antagonist,GABA_A receptor antagonist and GABA_B receptor antagonist.Cultured spinal dorsal horn neurons were exerted to identify 5-HT_3AA receptor expression on GABAergic neurons,further subunits of GABA receptor expression were analyzed after BDL,bilirubin intrathecal injection and cultured spinal dorsal horn neuron.GABA concentrations in CSF were determined by enzyme-linked immuno sorbent assay?ELISA?.Electrophysiological recording was used to test the effect of 5-HT_3AA receptor agonist and bilirubin on GABAergic spontaneous inhibitory postsynaptic current?sIPSC?.3 The effects of bilirubin and 5-HT_3A receptor agonists on human embryonic kidney293?HEK293?cells over-expressing human 5-HT_3A receptor(HEK293-5-HT_3A)were recorded by electrophysiological test.The affinity of bilirubin and 5-HT_3A receptor was determined by radioligand binding assay.Further,the spatial structure,binding mode and binding site of bilirubin and 5-HT_3A receptor were predicted by homologous modeling and molecular docking.Results1 Total bilirubin?TB?,direct bilirubin?DB?,indirect bilirubin?IB?,total bile acid?TBA?,alanine aminotransferase?ALT?and aspartate aminotransferase?AST?increased after BDL,while only DB and IB were significantly higher in CSF than sham group.Mechanical and thermal pain thresholds were significantly higher in rats after BDL or bilirubin intrathecally,and intrathecal ondansetron in BDL3d rats reversed the increased pain threshold in a concentration-dependent manner.5-HT_3A receptor expression was increased in BDL,bilirubin intrathecal injection rats and cultured spinal dorsal horn neuron with bilirubin.Immunofluorescence showed that the number of activated neurons and 5-HT_3AA receptor expression in spinal dorsal horn increased.2 Intrathecal ondansetron and bicuculline group significantly reduced pain threshold in BDL rats,while the effect of CGP55845 was weak.Ondansetron and bicuculline also reversed the increased pain threshold induced by intrathecal 5-HT_3A receptor agonist mCPBG and bilirubin.Immunofluorescence in cultured spinal dorsal horn neurons showed the co-expression of 5-HT_3A receptor and GABAergic neurons,and the expression of GABA_A receptor was increased in BDL,bilirubin intrathecal injection rats and cultured spinal dorsal horn neuron with bilirubin.ELISA showed that the concentrations of GABA in CSF were significantly increased after BDL and bilirubin intrathecally,and were decreased after ondansetron intervention.Electrophysiological recording suggested that mCPBG and bilirubin enhanced the sIPSC of GABAergic neurons,and ondansetron intervention weakened this effect.3 Bilirubin induced inward current of HEK293-5-HT_3A cells.Radioligand binding assay confirmed the weak affinity between bilirubin and 5-HT_3AA receptor.Homologous modeling and molecular docking showed bilirubin entered the hydrophobic gap formed by5-HT_3A receptor with benign complementarity,and hydrogen bonds formed.ConclusionThis study draws the following conclusions:1 Bilirubin increased pain threshold in obstructive jaundice through 5-HT_3A receptor.The mechanical and thermal pain thresholds were significantly increased in BDL rats,and the level of bilirubin in CSF increased.Intrathecal bilirubin also increases pain threshold in rats which can be inhibited by 5-HT_3A receptor antagonist.Meanwhile 5-HT_3A receptor expression increased significantly,and spinal dorsal horn neuron activity increased after bilirubin intervention2 Bilirubin regulates the function of GABA pathway through 5-HT_3A receptor.5-HT_3A receptor was expressed extensively in GABAergic neurons of spinal dorsal horn.Bilirubin can increase GABA concentrations in CSF,GABA_A receptor expression and GABAergic sIPSC,which could be blocked by 5-HT_3A receptor antagonist ondensetron.3 Bilirubin and 5-HT_3A receptor were combined by hydrogen bonds.Bilirubin can directly induce the inward currents of HEK293-5-HT_3A cells with low affinity.Bilirubin can enter the hydrophobic gap of 5-HT_3A receptor with benign complementarity in spatial structure and can form hydrogen bonds.In conclusion,the elevated bilirubin in central nervous system in obstructive jaundice can enhance the activity of GABAergic pathway by hydrogen bonds with 5-HT_3A receptor in spinal dorsal horn,which significantly contributes to the increased pain threshold in obstructive jaundice.