Evaluation Of The Effect Of Taurine Chelated Calcium On Skin Barrier Repair

Skin disease has become one of the most common diseases and frequently-occurring diseases recognized in the world today,which seriously affects human health.At present,the amino acid chelated calcium transdermal patch is clinically effective in the treatment of dermatitis and eczema diseases,but its types are few and urgently need to be developed.Taurine chelate calcium(TCCa)is a calcium preparation independently developed by our laboratory.Its transdermal absorption performance is better than commonly used calcium preparations.TCCa has previously completed preparation and characterization,and established a perfect penetration promotion system,but its mode of action and repair mechanism in vivo are not yet clear.Therefore,this paper studies the stability and dissociation mechanism of TCCa in body fluids,and elucidates the repair mechanism of TCCa to the skin barrier from the cellular level and the genetic level,which has important theoretical significance and practical value for the clinical application of TCCa.This article first studies the stability and dissociation mechanism of TCCa in body fluids.The stability constant of TCCa measured by spectrophotometer,was Kstability=4.08×1010.The experiment verified that TCCa is a two-linked six-membered ring structure formed by combining two amino and sulfonic acid groups of taurine with central calcium ion.D-Hanks balanced salt solution was used to simulate human calcium-deficient body fluids,and the effects of dissociation time,dissociation temperature and dissociation pH on the dissociation of TCCa were studied.Experimental data show that TCCa in calcium-deficient body fluids reached dissociation equilibrium in the first 2 h,and the dissociation rate of calcium reached 15.0±0.3%in human body fluid environment at 37℃ and pH=7.The dissociation rate and dissociation degree of TCCa are better than calcium L-aspartate,and the latter has a calcium dissociation rate of 13.2±0.6%.Secondly,this paper studied the repair mechanism of TCCa on the skin barrier from the cell level,and studied the effect of TCCa on the proliferation and differentiation of HaCaT cells in normal culture and calcium-deficient environment.In normal culture,TCCa appears to inhibit cell proliferation at a concentration of 5 mM,which may promote cell differentiation.TCCa has the best effect of promoting cell proliferation at a concentration of 1 mM.At this concentration,the attachment ratio is 77.50±4.41%,and the colony forming efficiency is 9.47±4.41%,and the proliferation rate is 116.67±8.52%.The effect of TCCa on cell proliferation after 24/48 h is basically the same.Compared with CaCl2,the concentration range of TCCa to promote cell proliferation is wider.In a calcium-deficient environment,TCCa has the best effect of promoting proliferation at 1 mM.At this concentration,the attachment ratio is 56.83±2.41%,the colony forming efficiency is 7.81±0.51%,and the proliferation rate is 149.83±4.95%.TCCa has a time-dependent effect on cell proliferation.Inorganic calcium such as CaCl2,organic acid calcium such as calcium gluconate(CG)and L-calcium lactate(L-CL),amino acid chelated calcium such as calcium L-aspartate(L-CA)are compared with TCCa.The results show that the proliferation rate of the TCCa group is 151.12±2.50%,which is higher than that of the CaC12 group of 106.4±4.4%.The calcium concentration of TCCa group to promote colony forming efficiency is 0.6-1.0 mM,which is slightly lower than the 1.0-2.0 mM of CG and L-CL groups.And the transdermal absorption effect of TCCa is better than CG and L-CL.TCCa is superior to L-CA in promoting cell proliferation and dissociation in body fluids.Therefore,compared with the above four calcium preparations,TCCa has obvious advantages in promoting cell proliferation,transdermal absorption,and dissociation in vivo.Finally,this article clarifies the repair mechanism of TCCa to the skin barrier from the genetic level.Fluorescence quantitative PCR is used to study the effect of calcium ion and TCCa on the mRNA expression of epidermal barrier-related genes in HaCaT cells.The results show that the mRNA expression of FLG,KRT 5,KRT 10,KRT 14,JAM 1,JAM 2,ZO 3 in cells treated with calcium-free culture is significantly reduced(P<0.05).It shows that calcium ions can regulate the proliferation and differentiation of cells and repair the damaged skin barrier by regulating the transcription expression of filaggrin,keratin and tight junction protein.Cells treated with 1 mM TCCa could significantly increase the mRNA expression levels of KRT 5 and KRT 14(P<0.05),indicating that 1 mM TCCa activated basal cell proliferation by regulating keratin expression.Cells treated with 5 mM TCCa could significantly up-regulate the mRNA expression levels of KRT 1,FLG 1,FLG 2,JAM 1,and ZO 2(P<0.05),it showed that 5 mM TCCa regulated the expression of keratin,filaggrin and tight junction protein,thereby promoting the early differentiation and late differentiation of cells and promoting the repair of skin barrier.Compared with CaCl2,TCCa has a higher ratio of up-regulating the mRNA expression of the above genes,indicating that TCCa is better than CaCl2 at promoting skin barrier repair.The study found that TCCa has strong thermal stability and can achieve good dissociation in body fluid environment.In addition,TCCa can promote the proliferation of HaCaT cells in normal culture and non-calcium culture,it is through regulating the expression of keratin,filaggrin and tight junctions to achieve the role of keratinocyte proliferation and differentiation and repair of the epidermal barrier.The research of this subject provides a theoretical basis and reference data for TCCa to treat diseases related to impaired skin barrier.