The Role Of TRPV1 In The Itch And Antipruritic Effect Of Osthole

Background and purpose:Itch is an unpleasant feeling that different from the pain,which causes the desire to scratch.It is not only the clinical symptom of various skin diseases,but also is the accompany symptoms of systemic diseases.In daily life,mosquito bites,exposure to allergens,taking drugs can lead to the occurrence of scratch.Chronic itch,which appear frequently,also accompany with depression,insomnia,anxiety.It can be said that itch is a common disease,which gravely affect people’s daily lives and quality of life.Nowadays,the mechanism of itch is still unknown,effective drugs are need for clinic.TRPV1 is transient opened and nonselective cation channel,which can be activated by heat(>43 ℃),weak acid,capsaicin and some endogenous ligand.TRPV1 plays an important role in the initiation and conduction of pain.However,more and more studies suggested that TRPV1 plays an important role in itch,such as in histamine-dependent itch.First,TRPV1 is co-expressed with histamine on dorsal root ganglion(DRG)neurons,and many histamine-sensitive neurons are also capsaicin-sensitive.Secondly,histamine-evoked itch behaviors are attenuated in both TRPV1 antagonist treated and TRPV1-deficient mice.Finally,histamine H1 receptor coupled G protein-coupled receptors signals via PhospholipaseCβ3 to open TRPV1 ion channel and trigger itch by activation of somatosensory neurons.However,besides histamine H1 receptor,there is another key receptor-H4 receptor in histamine evoked itch.Currently,the role of TRPV1 in histamine H4 receptor-mediated acute itch is unclear,and whether TRPV1 is participate in chronic itch is not clear.Studies show that the boundaries between chronic pain and chronic itch are ambiguous under pathological conditions.Under chronic itch conditions,noxious nociceptive irritation lead to more obvious scratching behavior which lead to pain behavior in normal conditions.how this phenomenon forms is unknown.TRPV1,the important mediator in pain and itch,which role was played in the process of painful stimulation switch to itchy sensation is necessary to study.Cnidium monnieri(L.)Cuss is a dry and ripe fruit of an annual umbrella plant,which has an effect on warming kidney yang,relieving rheumatism and insecticidal antipruritic.By screening of ancient and modern Chinese medicine agents which is itch related,we found that:Chinese medicine Cnidium has long been used to treat various of itch diseases.The ethanol extract of Cnidium can significantly attenuate the scratching behaviors induced by substance P and dermatitis model.Osthole is the main coumarin component of Cnidium,which had an inhibit effect on compound 48/80 or DNCB model induced scratching behaviors.This suggests that osthole may be effective for histamine-dependent itch.However,the effect of osthole on histamine-dependent itch remains to be clear and whether the antipruritus effect of osthole target to the TRPV1 is need to study.Therefore,the main purpose of our study are as following:1.To study the function of TRPV1 in histamine H4 receptor-mediated acute itch.2.To investigate whether TRPV1 ion channel is involved in chronic itch and the process of chronic itching,and to explore the mechanism of pain-to-itch switch under chronic itchy conditions.3.To study the antipruritic mechanism of osthole by target to TRPV1,looking for effective clinical antipruritic drugs.Experimental method:In the first chapter,cultured DRG neurons of WT mice were used to study the effect of immepip,which is the histamine H4 receptor agonist.Calcium imaging were also used to study the effect of G protein coupled receptor,PLC pathway,TRPV1 ion channel,TRPA1 ion channel blockers on neurons activated by immepip.The behavioral experiments were used to investigate whether the blockers of PLC pathway and TRPV1 ion channel had an inhibit affect on immepip evoked scratching behaviors.In the second chapter,the AEW(acetone/ether and water)model was used to imitate dry skin symptom,which present in many chronic itchy conditions.Behavioral assays were applied to study the behavioral changes induced by subcutaneous injection of capsaicin in the WT and TRPV1-/-AEW-treated mice.The TRPV1-PLAP+/-transgenic mice were used for alkaline phosphatase staining to study the expression of TRPV1 in the skin and trigeminal ganglion(TG)of AEW-treated mice.Real-time PCR and western blotting(WB)were used to detect the mRNA level and protein level of TRPV1 in TGs.In the calcium imaging experiment,Pirt-Gcamp3+/-mice were used to study the sensitivity of cultured TG neurons or whole TGs to capsaicin stimulation in control and model lateral of AEW-treated mice.In the third chapter,the antipruritic effect of osthole was studied by behavioral assays.The effect of osthole on the itch sensory signal conduction induced by histamine and histamine receptors agonist HTMT and VUF8430 were studied by calcium imaging.And whole cell patch clamp recording and calcium imaging were used to study the effect of osthole on the activity of TRPV1 ion channel.Experimental results:1.In the first chapter,through the calcium imaging experiments we found that in the peripheral sensory neurons,histamine H4 receptor agonist immepip can active DRG neurons,and can activation of the same neuron with histamine or capsaicin.The antagonists of the PLC pathway and the TRPV1 ion channel can inhibit the immepip-induced response in DRG neurons.Although ruthenium red can block immepip-induced extracellular calcium influx,TRPA1 blocker HC-030031 does not inhibit immepip-induced extracellular calcium influx,suggesting that TRPV1 is the downstream ion channel of histamine H1 receptor.Behavioral experiments have demonstrated this:immepip-induced scratching behaviors were significantly inhibited by the PLC pathway blocker U73122 and TRPV1 ion channel blocker AMG9810.2.In the second chapter,using the acetone-ether-water model,we imitated dry skin symptom of the chronic itch model.In behavior experiments,capsaicin usually evokes pain-related wiping behaviors in WT mice.But in AEW treated mice,capsaicin elicited robust itch-related scratching behaviors and wiping behaviors were unchanged.TRPV1-PLAP+/-transgenic mice were used for AEW model,and stained to study the expression of TRPV1.The results showed that the density of TRPV1 positive fibers increased in the skin of AEW-treated mice,and the expression of TRPV1 positive neurons increased in TGs.Real-time PCR and Western blot results confirmed that both the mRNA level and protein level of TRPV1 were significantly up-regulated in TGs after AEW treatment.The calcium imaging experiments were further found that AEW treatment sensitized the TRPV1 ion channel,which lead the neurons more sensitive to capsaicin stimulation.In other words,the central sensitization of itch-signaling pathway could magnify capsaicin induced sensory inputs from peripheral itch-sensing neurons,which overpowers the pain pathway and mediates pain-to-itch switching in chronic itchy conditions.3.In the third chapter,through behavioral experiments we found that osthole could suppress histamine,HTMT,VUF8430-induced scratching behaviors,but did not inhibits 8mM CQ-induced nonhistamine-dependent itch.It indicating that the antipruritic effect of osthole is mainly acting on histamine-dependent itch.In the calcium imaging experiments,osthole had an inhibitory effect on histamine,HTMT and VUF8430-induced extracellular calcium influx,suggesting that osthole may not be a selective agent of H1 or H4 receptor directly,but plays a partial role in the downstream signaling transduction.Calcium imaging experiments and whole-cell patch clamps recordings had shown that osthole can inhibits calcium influx and inward current induced by capsaicin in the DRG neurons.In one words,osthole can regulate the activity of TRPV1 ion channels.Experimental conclusion:In summary,we can get the following conclusions by behavioral assays,calcium imaging,electrophysiological experimentsand related molecular biology experiments.TRPV1 plays an important role in itch.1.TRPV1 and PLC participate in histamine H4 receptor-mediated itch.2.In AEW chronic itchy model,TRPV1 expression was up-regulated and neuronal hypersensitivity to capsaicin,which underlies the pain-to-itch switch under chronic itchy conditions.3.Osthole has antipruritic effect on the histamine-dependent itch by modulating of TRPV1 ion channel activity in peripheral sensory neurons.