Synergy of GHK‐Cu and hyaluronic acid on collagen IV upregulation via fibroblast and ex‐vivo skin tests

GHK‐Cu and HA are two commonly used skin care ingredients, both of which were reported to enhance collagen synthesis. This work aims to investigate their co‐effect on collagen regulation.


| Cell experiment
First, cell viabilities were assessed by GHK-Cu and HA solutions, where GHK-Cu concentration was from 0.0391 to 5 mg/mL, and HA concentration was from 0.0781 to 10 mg/mL. A series of HDF cell samples were cultured in KcGrowth medium with 10% FBS for 24 h. The culture condition was 37°C and 5% CO 2 , the same below.
Then, GHK-Cu and HA solutions were spiked into the mediums, respectively. The samples were cultured for another 24 h. After that, a mixture of KcGrowth medium and MTT (0.5 mg/mL in the medium) was dosed into the samples for another 4-h culture. Finally, the microplate reader (BioTek®) was used to measure optical density (OD) for cell viability calculations. with the identical total concentration were studied, as shown in Table 1. In Cell-5-13, the ratio of GHK-Cu and HA varies from 1:29 to 3:7 (wt:wt). In Cell-1-4, single-compound legs were tested. Blank, negative and positive controls were placed as the reference, that is, Cell-BC, -NC, and -PC.
In Cell-PC and Cell-1-13, the cells were cultured for 24 h after formula dose, and exposed to a 365-nm-wavelengthed UV light with the energy of 30 J/cm 2 for 26 mins. The negative control, Cell-NC, underwent the same UV exposure but without formula treatment.
The blank control, Cell-BC, was not treated by formulas or UV light.
After another 24-h culture, measurements were carried out. The levels of collagen-I, IV, and VII were detected by real-time reverse transcription PCR (qRT-PCR) assay with specific primers. Total RNA was reverse-transcribed to cDNA with PrimeScipt™ RT reagent kit.
PCR reaction was performed by using specific primers and SYBR Premix EX Taq™ II kit. Then, the fluorescence was quantified by the Bio-Rad detector (BioRad®).

| Ex-vivo skin model test
In the ex-vivo skin model test, two single-compound cases and one combination case were studied. Detailed information was shown in

| Statistical analysis
In each case, the average result and standard deviation were calculated from six replicates. A double-tailed t-test (α = 0.05) was used for data comparison, and the results were presented as letters, shown in Figures 2-4. If two cases contain identical letter(s), they are considered to be parity; while, if the letter(s) of two cases are totally different, they are considered to be significantly different.

| Cell test results
The results of cell viability were plotted in Figure 1. It can be seen that the curves of HA treatment tend to be higher than that of GHK-Cu treatment. For example, when HA concentration (three types of HA) is below 1.25 mg/mL, cell viability remains higher than While, a significant synergy between GHK-Cu and HA was discovered in collagen IV regulation, that is, Figure 3. The most outstanding data are Cell-5-7, that is, the mixture of GHK-Cu and LMW HA. As  Obviously, the concentration of collagen IV in Tissue-1, 2, 3 is much higher than that of negative control Tissue-NC, which is consistent with the learning of cell experiments. Among 3 test formulas, the combination case Tissue-3 shows the highest fluorescence intensity, which clearly verified the synergy of GHK-Cu and HA to upregulate collagen IV at skin level.