The Inhibition Of TRPV1 By Lysophosphatidic Acid In Cholestatic Pruritus

Pruritus is an unpleasant sensory perception which causes an intense desire to scratch.Besides dermatologic disorders,chronic pruritus is associated with systemic diseases,cancer,as well as psychological factors.Pruritus,which has a high impact on the quality of life,could cause insomnia,tristimania and suicidal tendency.Obstinate pruritus which is incurable could be a symptom of liver transplant operation.In C hina,pruritus is a common symptom of various liver disorders,whereas the pathogenesis of chronic cholestatic pruritus is complex and also remains enigmatic.How to relieve the intractable pruritus accompanied with cholestasic disorders is a conundrum to clinicians.Therefore,to find out the molecular mechanisms of the cholestatic pruritus will provide scientific proof for clinical treatments.Recent studies show that patients of cholestatic pruritus had a feature that the pain threshold was significantly increased.Morphine consumption was significantly decreased in surgery.Meanwhile,a number of media and receptors were involved in pruritus in cholestatic patients or cholestatic animal models.A new study suggested that the rise of lysophosphatidic acid(LPA)and autotoxin(ATX)in serum of patients with cholestasis might be a key factor to pruritus signal transduction system.Serum ATX was correlative with the degree of pruritus,which could be degraded by blocking serum ATX.ATX could hydrolyze lysophosphatidylcho line in serum into choline and LPA.LPA plays roles through LPA receptors.Studies have shown that LPA has at least 6 subtypes so far.According to our previous work,the expression of LPA1 receptor subtype was the most abundant both in sham mice and in cholestatic mice.In addition,compared with sham mice,the expression of LPA4 receptors in cholestatic mice increased most significantly among 6 subtypes.It has been well documented that the LPA1 receptors played an important role in pain signaling pathway.The LPA4 receptor expression increased significantly in the case of cholestasis,which suggested that LPA4 receptors might play an important role in the process of itch signal transduction.Therefore,we have focused on the roles of these two receptor subtypes in the cholestatic pruritus.Studies have shown that itch neurons are middle or small neurons which express gastrin releasing peptide(GRP)in the dorsal root ganglia of spinal cord.Their pivot protrusions which project to the layer I of the dorsal horn of spinal cord form synaptic connections with neurons that specificly express GRP receptors in layer I.Itch signals that come from the peripheral system are delivered to the central system.Therefore the neurons in which GRP expression are positive in DRG are regarded as itch neurons.TRPV1 is one of the important ion channels that mediate pain,and the peripheral pain signals are transmitted to the central nervous system through the pain signal pathways.Under normal circumstances,the pain signaling pathways are dominant and pain signaling pathways issue collateral to inhibit the itch signal transduction through inhibitory interneurons;and in cholestasis cases pain behavior decreased and itch behavior increased.Therefore,whether pain threshold in cholestatic patients elevated was associated with the reduction of TRPV1 expression and function was the work we will focus on.Therefore,we established a cholestatic mice model through common bile duct ligation,and applied the cheek model to observe itch like behavior such as wiping or scratching.We tested the function of LPA1 receptors and LPA4 receptors in cholestatic conditions via behavioral experiments methods.In addition,we used Western blot,whole cell patch clamp and immunofluorescence staining to observe the changes of the function of TRPV1 and the effect of LPA on TRPV1 in the case of cholestasis.So,to investigate the roles that LPA played in choleststic prutitus,we figured out the function and expression changes of TRPV1 channels,LPA1 receptors and LPA4 receptors in cholestatic mice.O ur study provides not only a new scientific proof for exploring the role and mechanisms of LPA in chronic cholestatic pruritus,but also a new method for the treatment of refractory cholestatic pruritus.The following are the main findings:1.LPA inhibited the function of TRPV1 in cholestatic disorders.(1)Western blot results showed that TRPV1 proteins were down-regulated in DRGs of BDL mice,compared with SHAM mice.(2)Whole cell patch clamp recordings in DRG itch neurons showed that inward current which evoked by capsaicin,TRPV1 agonist,was significantly decreased in cholestatic disorder.Behavioral study showed that the capsaicin-evoked pain behavior(wiping)was significantly decreased in BDL mice compared with SHAM mice,while scratching behavior had no significantly difference.These results suggested that TRPV1 function was reduced in cholestasis.(3)Whole cell patch clamp recordings in DRG neurons showed that inward current which evoked by capsaicin,TRPV1 agonist,was significantly inhibited by LPA in cholestatic disorder.Behavioral study showed that LPA could significantly reduce the numbers of wiping evoked by capsaicin in BDL mice.These results suggested that LPA inhibited the TRPV1 function in cholestasis.2.Scratching behavior could be enhanced by inhibiting LPA1 receptor.(1)Immunofluorescence staining showed that LPA1 receptor and GRP co-expressed in DRG neurons of mice.This result indicated that LPA1 receptor might be related to the occurrence of itch.(2)Behavioral study showed that inhibition of LPA1 receptors by using LPA1 receptor specific blocking agent K i16425 could reduce the pain reactio n induced by LPA,and increase the scratching response in SHAM mice.This result suggested that inhibition of LPA1 receptor could enhance the itch response.(3)Behavioral study showed that VPC31143(R),LPA1 receptor relative selective agonist,decreased wiping numbers in BDL mice,compared with SHAM mice.This result suggested that the function of LPA1 receptors decreased in the case of cholestasis.3.Scratching behavior could be enhanced by activation of LPA4 receptors.(1)Behavioral study showed that LPA evoked pain behavior in SHAM mice while VPC31144(S),LPA4 receptor selective agonist,evoked pruritus in SHAM mice.(2)Behavioral study showed tha t VPC31144(S)evoked significant pruritus behavior in both SHAM and BDL mice.These results suggested that LPA4 receptors might mediate itch response.(3)Behavioral study showed that VPC31144(S)produced scratch behavior in both SHAM and BDL mice in a dose-dependent manner.In summary,our study showed that under pathological conditions of cholestasis,the expression and function of TRPV1 decreased and LPA inhibited the function of TRPV1;LPA1 receptors function was inhibited in cholestasis;selective activation of LPA4 receptors induced scratching behavior and did not produce significantly pain behavior.These results suggested that LPA might increase pain threshold through inhibition of TRPV1 function and reduction of LPA1 receptors function,which may be one of the mechanisms of cholestatic pruritus.In addition,the enhancement of LPA4 receptor function might be one of the mechanisms of LPA-induced pruritus.