In recent years, consumers pay more and more attention to the efficacy of cosmetics, and the efficacy evaluation of the traditional cosmetics industry is facing new challenges. With the introduction of a large number of new technologies such as molecular biology, cell biology and in vitro organ culture, in vitro detection has been gradually used in the industry. Because of it has some advantages such as short time-consuming, low cost and easy access to experimental materials. The traditional method of cell biology is limited by the characteristics of the cell experiment itself,so it is difficult to detect the formula of the cosmetics products,while the reconstructed skin model technology that has sprung up in recent years benefits from the omni-directional simulation of organ structure. It can well reproduce the role of the product on human skin, but its scientificity and stability still need to be compared with traditional cell biology experiments.
In this paper,three skin care formulas with anti-inflammatory effect were used as test products,The SDS-induced soothing effect evaluation model of reconstructed epidermis was established.The levels of inflammatory factors in epidermis equivalent and medium matrix were detected by molecular biological methods such as RT-QPCR,ELISA,and compared with the classical RAW264.7 macrophage inflammation model induced by LPS.The results show that there is a certain correlation between them in the efficacy evaluation of soothing products,which provides a scientific basis for the application of reconstructed epidermis model in the soothing efficacy evaluation of cosmetic products.
Keywords: inflammatory factors、RT-QPCR、ELISA、RAW 264.7 macrophages，reconstructed epidermis model
skin model
Methods:
In this paper,three basic skin care formulas with anti-inflammatory efficacy were selected as test samples to construct an in vitro soothing efficacy evaluation model of artificial epidermis model induced by SDS.The release of inflammatory mediators such as IL-1α,IL-8 and PGE2 in the supernatant of culture medium was detected by ELISA.The expression of inflammatory factors such as IL-4,IL-10 and IL-6 in skin model was detected by RT-qPCR technique.In addition,the evaluation model of LPS-induced relieving and anti-inflammatory of RAW264.7 cells was established.The content of NO in the supernatant of cell culture medium was detected by the Griess method,and the expression of inflammatory genes such as iNOS,IL-6,IL-1β,TNF-α and IL-10 in cells was detected by RT-qPCR.
Results and Discussion：
The detection of inflammatory mediators in human artificial epidermis model induced by SDS showed that compared with the SDS treatment group,the three cosmetic products could significantly reduce the expression of IL-1α,IL-8, IL-6 and other genes(p＜0.05).There is a certain correlation between the results of ELISA and RT-QPCR.In addition,in the results of RT-QPCR detection,the expression of iNOS gene was up-regulated in these three samples,which was different from that of IL-1α and IL-8.It is speculated that there are only keratinocytes in the skin model.
The expression of inflammatory factors in RAW264.7 cells induced by LPS in vitro showed that compared with the SDS treatment group,the three samples can significantly reduce the expression of iNOS,IL-6, IL-1β,TNF-α,IL-10 and other genes(P<0.05).However, there was no significant change in the content of NO detected in the supernatant of the culture medium.

Conclusion:

In this study, for three tested skin care formulas with anti-inflammatory properties,Elisa and RT-QPCR techniques were used to detect the expression of inflammatory factors in reconstructed epidermis model for three anti-inflammatory formulations.And compared with the classical methods of cell biology,the scientificity and availibility of reconstructed epidermis model in the evaluation of soothing effect were verified. It provides a new method and idea for the efficacy test of skin care formula in vitro.