Effect Of Arotinoid-trometamol On Proliferation And Differentiation Of Cultured Hacat Cells

Psoriasis is a common, chronic, and recurrent inflammatory diseaseof the skin characterized by circumscribed erythematous, dry, scalingplaques of various sizes. It is characterized by three main pathologicfeatures: keratinocyte hyperproliferation, abnormal differentiation,and T cell infiltration in dermis. The pathogenesis of psoriasis isunclear until now. It is recognized that psoriasis is closely associatedwith an autoimmune disease induced by multiple gene and mediated by Tlymphocyte, involving in the effect of the factors such as inherit,infection, abnormal metabolism, trauma, mind stress on immunologic systemof body.Retinoic acids can inhibit the proliferation of epidermal cell,promote differentiation of keratinocyte and decrease inflammation. It hasattained good clinical effect in the treatment of psoriasis, either usedalone or combined with other methods. Its mechanism has not beenelucidated clearly, but perhaps relates with the three factors mentionedabove.Acording to their chemical structures, retinoic acids can be dividedinto three generations. The third generation of retinoic acids alwayshas a character of receptor selection. It can specially activate signalpathways of retinoic acids so that the third generation of retinoic acidscan minimize the side effects and maximize the therapeutic action. Therefore, more and more attentions have been paid on the research anddevelopment of the third generation of retinoic acids.Arotinoid-trometamol is a third generation of retinoic acidsdeveloped by Chongqing Huabang pharmaceutical limited company. It is anew retinoic acids receptor agonist with chemical structures similar withTTNPB. It is not clear whether Arotinoid-trometamol can effectivelyinhibit the epithelial proliferation and promote differentiation. In thisresearch, human keratinocyte HaCat cells are selected as the cell modelwhose proliferation and differentiation are similar to that of psoriasis,and Keratin 6 and TGase 1 are chosen as our observation targets forepidermal hyperproliferation and differentiation respectively. TheRT-PCR method is adopted to detect the expression of KRT6 and TGase1mRNAafter Arotinoid-trometamol acts on HaCat cells. The result is thencompared with that of Acitretin to evaluate the effect ofArotinoid-trometamol on epidermal proliferation and differentiation,which will provide rationale for Arotinoid-trometamol's furtherapplication for clinical treatment of psoriasis and other diseases.PARTâ… Detection of the Dose-Time Effect of Arotinoid-trometamol onProliferation in Cultured HaCat CellsObjective: To investigate the effect of Arotinoid-trometamol on theproliferation of Hacat cells cultured in serum free medium, and discussthe dose-time effect.Methods: Various doses (10^-6-10^-9mol/l) of Arotinoid-trometamol wereadded into confluent Hacat cells cultured in serum free media. The totalRNA was abstracted in 6 hours, 12 hours and 24 hours respectively. TheRT-PCR method was adopted to detect the expression of mRNA of acharacteristic marker, Keratin 6 of the proliferation of Hacat cells. Weobserved moroghologic change of Hacat cell after adding Arotinoid-trometamol by high power microscope. The proliferativeeffect was assessed by means of incorporation of [³H]thymidine.Results: In 12 hours after Arotinoid-trometamol acts on HaCat cells, aconsiderable drop can be seen on the expression of Keratin 6 at theconcentration of 10^-6-10^-8mol/l. There is a significant difference for eachexperimental group compared with the negative control group, P＜0.05. In24 hours after Arotinoid-trometamol acts on HaCat cells, there is asignificant difference between each experimental group and the negativecontrol group at the concentration of 10^-6-10^-9 mol/l, P＜0.05. This suggeststhat at the point of 24 hours after Arotinoid-trometamol acts on HaCatcells, Arotinoid-trometamol at the concentration of 10^-6-10^-9 mol/l caneffectively inhibit the proliferation of Hacat cells and has a dosedependent manner.At the same level of the concentration, the expression of KRT6 will beweakened as the time that Arotinoid-trometamol acts on HaCat cellsextends. The effect that Arotinoid-trometamol inhibits the proliferationof Hacat cells has a time dependent manner.Morphology: Hacat cell became small, diaperal distribution and cellmembrane shrinkaged after adding Artinoid-trmetamol.Arotinoid-trometamol at the concentration of 10^-6-10^-9 mol/l caneffectively inhibit the proliferation of Hacat cells and has a dosedependent manner by means of [³H]thymidine.Conclusion: After its action for 12 hours, Arotinoid-trometamol at theconcentration of 10^-6-10^-8mol/l can significantly inhibit theproliferation of Hacat cells and thus it is dose dependent. After itsaction for 24 hours, Arotinoid-trometamol at the concentration of10^-6-10^-9mol/l can effectively inhibit the proliferation of Hacat cells andthus it is dose dependent. Meanwhile, we can find that it has a timedependent manner as well. PARTâ…¡Detection of the Dose-Time Effect of Arotinoid-trometamol onDifferentiation in Cultured HaCat CellsObjective: To investigate the effect of Arotinoid-trometamol ondifferentiation of Hacat cells cultured in serum free medium and discussthe dose-time effect.Methods: Various doses (10^-6-10^-9mol/l) of Arotinoid-trometamol were addedinto confluent Hacat cells cultured in serum free media. The total RNAwas abstracted in 6 hours, 12 hours and 24 hours. The RT-PCR method wasadopted to detect the expression of mRNA of a characteristic marker,TGasel of the differentiation of Hacat cells.Results: After its action on Hacat cells for 6, 12 and 24 hours,Arotinoid-trometamol at the concentration of 10^-6-10^-9mol/l cansignificantly increase the expression of TGasel. There is a significantdifference between each experimental group and the negative control group,P＜0.05.At the same level of the concentration, the expression of TGasel will bebuilt up as the time that Arotinoid-trometamol acts on HaCat cellsextends. The effect that Arotinoid-trometamol inhibits thedifferentiation of Hacat cells has a time dependent manner.Conclusion: After its action for 6, 12 and 24 hours, Arotinoid-trometamolat the concentration of 10^-6-10^-9mol/l can significantly promote thedifferentiation of Hacat cells and thus it is dose dependent. Meanwhile,we can find that it has a time dependent manner as well.PARTâ…¢Comparison Study between TGasel and KRT6 of the Effect ofArotinoid-trometamol on Hacat CellsObjective: To compare the effect of Arotinoid-trometamol and acitretinon proliferation and differentiation of Hacat cells cultured in serum free medium and discuss the clinical application perspective ofArotinoid-trometamol.Methods: Various doses (10^-6-10^-9mol/l) of Arotinoid-trometamol andacitretin were added into confluent Hacat cells cultured in serum freemedia respectively. Their total RNAs were abstracted in 24 hours. TheRT-PCR method was adopted to detect the expression of mRNA ofcharacteristic markers, TGasel and KRT6 of the proliferation anddifferentiation of Hacat cellsResults: Arotinoid-trometamol can inhibit the proliferation of Hacatcells down to the similar level compared with acitretin. Literaturely,there is no significant difference between them (P＞0.05). On the otherhand, Arotinoid-trometamol can promote the differentiation of Hacatcells up to the similar level compared with acitretin. Likewise, thereis no significant difference between them (P＞0.05).Conclusion: Arotinoid-trometamol has a similar effect as acitretin onthe epidermal proliferation and differentiation, which may be a new wayto treat posriasis. Since Arotinoid-trometamol can activate the signalpathways of retinoic acids in a specific way, it will avoid the badreactions due to the activation of other retinoic acids and thusfacilitate it to minimize the side effects of drugs. In terms of its usagein the treatment of psoriasis, the side effects of Arotinoid-trometamolis obviously less than that of acitretin. From this point of view, it hasa relative broader clinical application perspective.