Effect Of TRPV1 On Induceing Release Of Inflammatory Mediators In Human Keratinocytes And Intervention Of Drugs

Skin is the largest organ of human body, covering the entire body surface. Keratinocytes are its major component, by making keratin protein, function as a protective barrier against exogenous stimuli. As Keratinocytes have been demonstrated to produce various kinds of cytokines, skin plays an important role in immunologic and inflammatory responses of the body. Evidence is accumulating to show the significant contribution of cytokines to the pathogenesis or severity of certain diseases. Keratinocytes are activated by environmental stimuli to produce a variety of cytokines that can affect the immune response as well as the cell growth and differentiation of Keratinocytes themselves and other cell components in the skin in an autocrine or paracrine fashion.TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of TRP family. The receptor is a nonselective cationic channel and both rat and human TRPV1 were recently cloned. TRPV1 is activated by capsaicin, heat and protons, and can be hypersensitized by injury. These findings indicate that TRPV1 acts as a pivotal molecular integrator of noxious chemical and thermal stimuli in the peripheral terminals of primary afferents involved in nociception and inflammation. Therefore, the presence of functional TRPV1 receptors in keratinocytes has generated interest in skin–nerve crosstalk under physiological and pathological conditions. It has become a promising target for the development of a new generation of anti-inflammatory and analgesic agents.TRPV1 expression has been identified in non-neuronal cells as well as neuronal cells, for example in bronchial epithelial cells, cardiomyocytes, urinary bladder epithelial cells, gastric epithelial cells, oral epithelium, keratinocytes. Activation of TRPV1 in human keratinocytes by capsaicin results in a dose-dependent expression of cyclooxygenase-2. Capsaicinoids cause the calcium-dependent production of IL-6 and calcium- independent cell death via TRPV1 in cultured human lung cells and primary cultured human airway epithelial cells. These findings suggest that TRPV1 plays a key role in inflammation by producing various cytokines in non-neuronal cells. The biological role of the receptors in each of these cells remains elusive. Understanding the regulatory pathways of TRPV1 activation in keratinocyte will offer a great potential for treating skin diseases such as genetic abnormalities, infections, and skin cancer. The mechanism of TRPV1 in inflammation should be to study. The drugs target at TRPV1 will bring a bright future to producing therapeutics for the inflammation diseases of skin targeting human TRPV1.In the study, in order to clarify the physiological role of TRPV1 in non-neuronal tissues of the human skin, we examined inflammatory mediators'expression and relationship with the expression of TRPV1 in normal human skin keratinocytes and HaCaT cells stimulated with CAP. The intercellular Ca2+ concentration changes and NFκB activation were examined by FCM and Dual Luciferase Assay System. The effect of drugs on the production of pro-inflammation mediators, Ca2+ concentration changes and NFκB activation from primary cultured human keratinocytes and HaCat cells were also invesgated.The results are shown as followed:1. The effect of capsaicin on TRPV1 mRNA induction in primary skin keratinocytes and HaCaT cells was assessed by Reverse Transcript PCR. The TRPV1 mRNA level increased by CAP. The concentration of IL-8 and PGE2 protein in the keratinocytes culture medium evaluated by ELISA, was increased 62.75% and 54.72% treated by CAP. The capsaicin-mediated accumulation of IL-8 and PGE2 production was concentration-dependent. Capsazepine significantly inhibited the 8μM capsaicin-mediated increase in IL-8 and PGE2 production.2. Intracellular Calcium Levels were detected by FCM. 2-16μmol/L CAP increased the level of intracellular calcium by concentration-dependent manner. However, 3μmol/L CPZ,10μmol/L CPZ and 20μmol/L CPZ decreased the CAP induced related fluence ratio by 32.11%,38.33% and 34.66%.3. The stably transfected HEK293T cells expression TRPV1 were constructed using Lipofectamine reagent. A stably transfected clone was also isolated by selection in G418, identified by FCM and Western blotting. The intracellular Calcium Levels were induced by CAP via TRPV1 activation, and extracllular calcium result in the elevation.4. The pNFκB and pRL-TK plsamids were transiently transfected to HEK293T cells, HaCaT cells and HEK 293T stably expression of TRPV1, respectively. The related NFκB activation ratio was invesgated by Dual Luciferase Assay System. The result showed that CAP can significantly increase the related NFκB activation ratio in HaCaT cells and HEK 293T-TRPV1 cells. The HEK293T cells have not changed. CPZ, a TRPV1 antagonist, can decrease the related NFκB activation ratio.5. Incubation the cells with PD98059 and Wortmannin before treated with CAP, the inhibitors of PI3K pathway and MAPK pathway can suppress the level of NFκB activation 52.42% and 24.52%,respectively. That indicated that CAP induced NFκB activation via PI3K pathway and MAPK pathway.6. Ginsenoside Rb1 significantly decreased the expression level of TRPV1 mRNA and the activation of NFκB in HaCaT cells induced by CAP, as well as inhibited the secretion of IL-8,IL-6 and PGE2 by 26.72%,38.23% and 57.01%.7. Paeoniflorin significantly decreased secretion of IL-8,IL-6 and PGE2 induced by CAP in HaCaT cells 11.39%,51.96%。75.98%. The effects of PF are different on the calcium influx in between HaCaT cells and HEK293T-TRPV1 cells, which suggested that PF was not directly effect calcium influx through the TRPV1 channel.8. CAP has no significant influence on the production of TNFα. Notoginsenoside R1 and Ginsenoside Rg1 has no significant influence on the TRPV1 activation, calcium influx and signal transfer pass way.9. The result of TRPV1 stimulated by CAP was shown by proteomic technology to find the unreported proteins related with the function of TRPV1. As a result of MOLDI-TOF MS/MS, Triosephosphate isomerase, enolase 1 variant and KERATIN 14 in HaCaT cells treated with CAP have significant changed. The function and role of these proteins on the inflammation in skin should be investigated further.