Zhao Xiaomin, Zhao Yunshan, Qu Xin

Shanghai Technical Center, Ashland (China) Inc., China

Epigenetics is a derivative of genetics. Genetics is the study of the gene expression level changes which caused by DNA sequence change, for instance, gene mutation,loss of heterozygosity, etc.; whereas epigenetics refers to changes in a chromosome that affect gene activity and expression which can be stably heritable during biological development and cell proliferation. Examples of mechanisms that produce such changes are DNA methylation, genomic imprinting and non-coding RNA.In this article, microRNA (miRNA)—one kind of short non-coding RNA regulating skin biological function was studied and discussed.

miRNAs are a class of linear single stranded noncoding RNA containing about 22 nucleotides in length.miRNAs have been identified in nematode, mouse, plants and human beings and their related virus, that functions in RNA silencing and post-transcriptional regulation of gene expression. In 1993, Lee et al. discovered the first miRNA and named as lin-4 in nematode C. elegans.And in 2000, Reinhart et al. discovered the second miRNA—let-7 in nematode which regulates its biological development. It was a prelude to intensely researching miRNA. miRNA contains base sequences partially complementary to its targeted messenger RNA (mRNA).Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. miRNA participes in a series of important courses throughout the life cycle, such as hemopoiesis, organogenesis, cell apoptosis and proliferation. miRNA and small interfering RNA (siRNA) are the core members of RNA interference(RNAi). RNAi is intensely studied in gene therapy and new pharmacy development, but the study of skin care is rare. Isaacman et al. pointed out that RNAi represents a unique opportunity for the cosmetic scientist to develop revolutionary products that are capable of preventing or treating debilitating skin disorders.

In-vitro, ex-vivo and in-vivo experiments are performed to study the regulation effect on miRNA by hexapeptide-2,and then the influence of these miRNAs on collagen and melanin synthesis, and finally in-vivo effect after applying cream containing hexapeptide-2 versus placebo cream (not containing hexapeptide-2). In this way,hexapeptide-2 and its mechanism on epigenetics were studied progressively, skin whitening and anti-aging effects were performed to study its practical effects on skin of human being.

Experiments

Instruments and reagents

Spectrophotometer CM-2600D, Konica Minolta(Japan); Visia-CR, Canfield (USA); Photomax PRO,Derma medical systems (Austria); Visioline VL650,Courage+Khazaka (Germany); Image-Pro Analyzer 7.0, Media cybernetics (USA). DMEM culture medium,glucose, fetal bovine serum (FBS), L-glutamate, Lonza(Switzerland); Antibiotic Primocin, Invivogen (USA);Rabbit polyclonal antibodies, Antibodies to collagen I and III (Primary antibodies), Tebu-Rockland (France);Fluorescent marker second antibody, The donkey anti rabbit, Invitrogen(USA); The culture medium of melanocytes, Promocell (Germany); Acrylic Acid/VP Crosspolymer, Ashland(USA); Stearoxy Dimethicone/Dimethicone, Wacker (USA); PEG-100 Stearate and Glyceryl Stearate, Seppic (France); Butyrospermum Parkii(Shea)Butter, AAK (Switzerland); Cetyl Lactate, Isocetyl Stearoyl Stearate, Octyldodecyl Stearate, Ethylhexyl Palmitate, Hexapeptide-2 (trade name: Dermostatyl IS),Ashland (USA). The hexapeptide-2 mentioned below is a homogeneous solution of hexapeptide-2 and water and butanediol.

In-vitro and ex-vivo experimental method

The fibroblasts used for this study were extracted from normal human skin of 33-year-old (passage 4), 45-yearold (passage 8) and 66-year-old (passage 8) female donors.They were grown in DMEM medium containing 1 g/L glucose supplemented, 10% of FBS, 2 mM of L-glutamine and 100 ug/mL of Primocin. Cells were maintained at 37℃ in a humidified atmosphere containing 5% of CO2.Cells were seeded in 6-well plate, and each well was treated with 1% hexapeptide-2 aqua solution or kept untreated. Then real-time qPCR was used to assess the miR-29 expression level.

Norman human skin came from a plastic surgery intervention on the abdomen of a female donor (unknown age). Skin biopsies were obtained with a 6 mm diameter punch. The experiment is divided into 2 groups of 3 samples each. One group was treated with 1% hexapeptide-2 twice a day, adding 20 uL every time; the other one was not treated with hexapeptide-2. The two groups were cultured for 48 h. Then the immunofluorescence technique was used to analyze collagen I and III expression. This technique allowed to visualize specific protein in tissue sections by binding a specific primary antibody. A secondary antibody labeled with fluorochrome was used to recognized the primary antibody. Then immunofluorescence staining sample can be observed under the microscope, and the fluorescence intensity which represent the expression level of protein were analyzed with Image-pro Analyzer 7.0 (IPA 7.0).

In this study, the human epidermal melanocytes were derived from a 45-year-old female donor (passage 3).They were grown in melanocyte growth medium supplemented with 1 ng/mL human basic fibroblast growth factor, 0.052 g /L medium bovine pituitary extract,0.5 mg/L hydrocortisone, 5 mg/L human recombinant insulin, 10 ng/L phorbol 12-myristate 13-acetate (PMA)and 0.1 g/L Primocin. Cells were maintained at 37℃ in a humidified atmosphere containing 5% of CO2. Then realtime qPCR was used to measure the miR-218 expression level.

In-vivo experimental method

35 healthy Chinese residents (age 36 to 65, 17 males and 18 females) were measured by double-blind splitface test. The code number for subjects was from 001 to 035. All the subjects had healthy skin, no allergy history,and no obvious scar on testing zone. All the subjects had uneven skin tone, at least 2 freckles or age spots on each side of face with diameter at least 2 mm, and had fine lines or deep wrinkles in crows-feet area. The study lasted for 56 days. During this study, the subjects were requested to apply samples with 1% hexapeptide-2 was or not on each side of face randomly. The side of face applied sample with hexapeptide-2 marked as biofunctional treatment group, the other side as placebo group, twice per day, and the application dosage was 2 mg/cm2. The subjects were tested on day 0 (D0), D7, D14, D28, D42 and D56, respectively. Anti-wrinkle test ended on D42.On the day of test, the subjects were asked to wash their face with warm water following a standard procedure,then stayed relaxed in the controlled lab under constant temperature [(22 ± 1)℃] and humidity (ranging from 55% to 60%) conditions. Visia-CR was used to take full face pictures and then analyzed with Image-Pro Analyzer 7.0 (IPA 7.0). Photomax Pro was used to take the pictures of freckles or age spots and then analyzed with IPA 7.0.Spectrophotometer to determine normal skin tone and skin spots color by L*ab value (L* represents normal skin whitening or spots lightening) and a ITA° value which is based on the following formula. As final step, Visioline VL650 was used to analyze wrinkles.

L* represents normal skin whitening, the bigger value,means whitener skin. b* represents skin yellowness, the bigger value means yellower skin.

Statistical method

The statistical analysis was performed with JMP 10.0 software. Student’s T test (one-tailed) or Wilcoxon Signed Rank test (one-tailed) were used depending on whether the data were normal distribution. For threshold of significance, if α ＞ 10%, marked as “NS”, it is not significant and showed as P ＞ 0.10; if 5%＜α≤10%,marked as “Δ”, it is directional significant and showed as 0.05 ＜ P ≤ 0.10; if α ≤ 5%, marked as“*”, it’s significant and showed as P ≤ 0.05; if α ≤ 1%, marked as “**”, it’s very significant and showed as P ≤ 0.01; if α ≤ 0.5%,marked as “***”, it’s highly significant and showed as P ≤ 0.005; if α ≤ 0.1%, marked as “****”; it’s extremely significant and showed as P ≤ 0.001.

For each parameter, if the change value is bigger on bio-functional side than on placebo side, the subjects remarked as effective population, the effective population proportion compared to total population remarked as effective population percentage. At each test time point,the increase or decrease of percentage for each parameter is calculated as follows (L*is used as an example):

ΔLArepreasents the L* value change on the biofunctional side, ΔLPrepreasents the L* value change on the placebo side.

The same priciple was used on the calculation of the change percentage of other parameters.

Results and discussion

In-vitro and ex-vivo results

miR-29 expression regulated by hexapeptide-2 and its influence on collagen synthesis

miR-29 is partly complementary to the mRNA of collagen I, then the collagen I translation can be silenced,it means less collagen I can be synthesized. With aging,miR-29 expression will increase[6,7]which will interfere collagen I synthesis more and more intensely, and cause skin aging. As shown in Figure 1, the untreated control group had an increase in miR-29 expression compared with treatment group. And accordingly, for the ex-vivo skin (Figure 2), the collagen I and III expression of the treatment group increased significantly by 57% and 55%respectively compared with the untreated group. These above results showed that hexapeptide-2 may inhibit miR-29 expression level which then increased collagen I and III synthesis.

Figure 1. Quantitative real-time PCR evaluation of miRNA-29a level in fibroblast from donors with different age

Figure 2. Collagen I and III immunofluorescent staining in skin biopsies after 48 hours of treatment

miR-218 expression level regulated by hexapeptide-2 then influencing melanin synthesis

miR-218 is partly complementary to the mRNA of key regulating genes of melanin synthesis. This will lead to the related mRNA degradation, then silencing the key regulating genes expression and melanin synthesis is inhibited. The in-vitro experiment results on human melanocytes showed that the treatment group by 1%hexapeptide-2 can highly significantly increased the expression of miR-218 by 60% (P ＜ 0.005) compared with the control group. In this way, it’s deduced that hexapeptide-2 can inhibit melanin synthesis and then had whitening effect.

In-vivo results

Based on the above in-vitro and ex-vivo results, it indicated that hexapeptide-2 might regulate anti-wrinkle pathway and skin whitening pathway by regulating related miRNA. In the following clinical experiment, a series of conducted experiments had been used to prove hexapeptide-2 has skin whitening and anti-wrinkle effects.

Skin whitening efficacy

As shown in Figure 3, compared to placebo side,the L* value on the side applying cream containing 1%hexapeptide-2 increased significantly from D7. L* value kept increasing except at D14 on the bio-functional group.It showed that 1% hexapeptide-2 had good skin whitening effect. On the other side, L* value also showed some increase at D42 and D56 on the placebo group. This was mainly because the experiment was performed in winter,there is less UV radiation, the melanin content in skin tends to decrease in this season naturally.

Figure 3. Whitening efficacy of clinical test

Spots lightening efficacy

Figure 4 showed comparing with placebo group,on D7 and D14, the L* value of the bio-functional group was bigger than that of the placebo group,but there was no significant difference between the two groups. From D28, the L* value of spots on biofunctional group was significant than that of the placebo.What’s more, the image analysis results based on the images taken by Photomax Pro showed the spots color lightened significantly on the bio-function group than on the placebo group (P ＜ 0.01 on D42 and P ＜ 0.05 on D56). Figure 5 demonstrated the spots treated by 1%hexapeptide-2 turned lightener obviously, while the spots on the placebo group was largely unchanged or slightly deepened. All the results manifested that 1%hexapeptide-2 had excellent spots-lightening efficacy.

Figure 4. Spots lightening effect of clinical test

Skin erythema improvement efficacy

Figure 6 showed that a* value of the bio-functional group treated by 1% hexapeptie-2 was more significant throughout the study (P ＜ 0.05) than that of placebo group. And the a* value increased in the placebo group.It may because the experiment was conducted in winter,cold and dry weather can irritate the skin. But 1%hexapeptide-2 can improve erythema significantly and obviously in this condition.

Figure 6. Erythema improvement effect of clinical test

Remove darkness efficacy

As shown in Figure 7, at all the time points, increase of ITA° value in the bio-functional group was significant than that in the placebo group, the increase percentage was 750%, 723%, 158%, 40% and 32% on D7, D14, D28,D42 and D56 with P ＜ 0.05. All these results showed that 1% hexapeptide-2 can help lighten the skin tone.

Figure 5. The demonstration of spots lightening effect

Figure 7. Skin dullness improvement effect on clinical test

Anti-wrinkle efficacy

The anti-wrinkle test lasted 42 days. On D42,compared to placebo group, the treatment group containing 1% hexapeptide-2 showed a significant wrinkle decrease in wrinkle area by 431% (P ＜ 0.01), in wrinkle number decrease by 579% (P ＜ 0.05), in wrinkle length decrease effect by 333% (P ＜ 0.05) and total wrinkle depth decrease by 731% (P ＜ 0.01). Figure 8 demonstrated that the No.008 subject showed obvious wrinkles improvement effect in the bio-functional group, but in the placebo group, it didn’t show any change. Based on all these results, it showed 1% hexapeptide-2 has good anti-wrinkle efficacy.

Figure 8. Anti-wrinkle effect demonstration by silicon replica printing

Conclusion

In summary, by regulating miR-29 and miR-18 which is based on epigenetics technology, hexapepetide-2 can decrease the silencing of collagen synthesis related genes,and increase the silencing of melanin synthesis related genes. It was found that hexapeptide-2 is an effective ingredient for use in anti-aging cosmetics formulation.It can whiten skin, lighten spots, improve skin erythema,remove darkness and anti-wrinkle.

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