基于体外试验评价透明质酸眼刺激保护作用

In vitro study on the protective effects of hyaluronic acid on eye irritation

通过荧光素渗漏试验和重组人角膜上皮-眼刺激(ET_(50))试验,研究了高分子、低分子、酶切寡聚三种不同分子量的透明质酸对眼刺激的保护作用。参照OCED TG460选择七种物质在本实验室验证荧光素渗漏试验;并进一步采用荧光素渗漏试验和重组人角膜上皮-眼刺激(ET_(50))试验评价了三种透明质酸对荧光素钠渗漏率、细胞存活率等的影响,得到了透明质酸对眼刺激的保护作用体外评价结果。荧光素渗漏试验证实,10 g/L透明质酸能显著抑制0.25 g/L苯扎溴铵造成的MDCK细胞荧光素渗漏率增加;重组人角膜上皮-眼刺激试验证实,20 g/L酶切寡聚透明质酸能显著抑制乙酸甲酯造成的细胞存活率降低;重组人角膜上皮-眼刺激(ET_(50))试验证实,10 g/L透明质酸能显著抑制0.5 g/L苯扎溴铵造成的组织模型细胞存活率降低,增加ET_(50)值。结果表明,三种透明质酸均具有眼刺激性保护作用,揭示了透明质酸在化妆品应用的新前景,同时也为透明质酸眼刺激舒缓功效的评价开发了新方法。

Fluorescein leakage(FL)test and recombinant human corneal epithelial cell model eye stimulation(ET_(50))test were both took to investigate the protective effects of three different molecular weight hyaluronic acids(HA)including high,low and oligomeric on eye irritation.Firstly,seven verification substances in different stimulation levels including benzalkonium chloride(5%),4-carboxy-benzaldehyde,promethazine hydrochloride,ethyl-2-methylaceto-acetate,glycerol,sodium lauryl sulfate(15%,w/%)and sodium hydroxide(10%,w/%)were used to verify the luciferin leakage test in our own laboratory.Then,fluorescein leakage test and in vitro artificial cornea model-eye stimulation ET_(50)test were both conducted to evaluate the inhibitory effect of three hyaluronic acids on eye irritation by the index of fluorescence leakage rate as well as cell survival rate.Fluorescein leakage test method is established successfully and is confirmed that 10 g/L mass concentration of hyaluronic acid can significantly reduce the increase of fluorescence leakage rate caused by 0.25 g/L mass concentration of benzalkonium bromide.What’s more,incubating for another 24 h after exposure to 10 g/L mass concentration of hyaluronic acid and 0.25 g/L mass concentration of benzalkonium bromide for 30 min,the cell survival rate does not change,but the morphological change of the cells can be observed tending to be normal under the microscope,compared to the cells treated by 0.25 g/L mass concentration of benzalkonium bromide only.Meanwhile,in vitro artificial corneal model-eye stimulation test confirms that oligo hyaluronic acid can significantly inhibit the decrease of cell survival rate which causes by methyl acetate at mass concentration of20 g/L rather than 10 g/L.Accordingly,in vitro artificial corneal model-eye stimulation test(ET_(50))confirms that 10 g/L mass concentration of hyaluronic acid can significantly inhibit the decrease of cell survival rate and increase the ET_(50)value which is induced by 0.5 g/L mass concentration of benzalkonium bromide,but it falls flat on serious irritation induced by10%(w/%)content of Brij35 solution.In conclusion,these results show that high,low and oligomeric molecular weight hyaluronic acid can all significantly reduce the irritation of ocular stimulation induced by mild positive substances,which provides a basis for the protective effect of hyaluronic acid,reveals a new prospect of the application of hyaluronic acid in cosmetics and presents a new method of efficacy evaluation on cosmetic raw materials.