Experimental Study On Anti-aging Effects Of Dimethylamin-oethanol And Compound Amino Acid In Aging Rat Skin

Keeping youth forever is an eternal topic and dream of human being. In the recent years, mesotherapy has been arousing everyone’s interest as an anti-aging strategy. Pioneered by the French physician Dr. Michel Pistor in1952, Mesotherapy is a minimally invasive procedure that is widely used in Europe and elsewhere to treat various injuries and medical conditions. It was recognized by the French National Academy of Medicine in1986as an integral part of traditional medicine, and has been becoming highly popular within the public. In China, Mesotherapy was approved by Ministry of Health and CFDA. Mesotherapy, a safe, simple, less painful procedure which is one of the so-called "lunchtime cosmetic procedures", requires no recovery time and is perfect for professionals and successful people in the fast-paced modern life. Although there are a wide range of products of Mesotherapy, less foundimental reasearchs have been proceeded. Moreover, there is some controversy over its efficacy and safety.DMAE is an analog of vitamin B choline that has been used to treat a number of conditions affecting the brain and central nervous system. There is some evidence that DMAE may be helpful for attention deficit hyperactivity disorder (ADHD). More widely marketed today as a memory and mood enhancer, DMAE is said to improve intellectual functioning. The basis for such claims probably stems from its purported ability to increase levels of a neurotransmitter called acetylcholine, although this has not been proven. Studies suggest that the skin is an active site of acetylcholine synthesis, storage, secretion, metabolism, and receptivity. The role of DMAE as a modulator of acetylcholine-mediated functions in the skin remains to be elucidated. DMAE is receiving more attention as a new skincare regimen today for its acute effects of anti-aging, anti-wrinkle and skin firmness. From three recent clinical reports,3%DMAE facial gel formula has been shown to be efficacious in the mitigation of forehead lines and periorbital fine wrinkles, and in improving lip fullness and shape and the overall appearance of facial skin. The safety concern of the long-term application of DMAE gel for up to1year has also been further documented.Age-related skin changes are inevitable and include thinning, sagging, wrinkling, loss of elasticity and areas of dryness. Histological changes include dermal thinning, collagen thickening with degeneration and reduced amount of fibers, and elastin breaking down. In infant and young adult skin, type Ⅰ collagen is the most abundant protein in the dermis accounting for approximately80%, while type Ⅲ collagen is much less with about10%of dermal mass. When skin becomes aged, the collagen synthesis is reduced for type Ⅰ but up-regulated for type Ⅲ collagen, resulting in an inversed turnover of type Ⅰ/Ⅲ ratio in the skin. Though it is rarely seen in normal adult skin, increased expression of matrix Metalloproteinase-1(MMP-1) is a prominent feature of some pathological situations, such as wound healing, tissue repairing and remodeling. MMP activity is a key driver in wound healing to facilitate the motility of inflammatory cells and to enhance the availability of inflammatory mediators to set the stage for healing. However the tissue inhibitor of metalloproteinase (TIMP), a natural inhibitor of MMP, can restrain the activation of MMP and its activity in the wound healing process by forming a ternary complex with MMP.Proliferating cell nuclear antigen (PCNA) was first found in serum of SLE patients by Miyachi et al, which only express in nomal proliferating cell or tumor cell. Subsequent studies showed that PCNA is close to DNA synthesis and play an important role in trigger of cell proliferation. It is a ideal indicator of state of cell proliferation.In order to evaluate potential anti-aging effects of DMAE and compound Amino Acid in Mesotherapy, tissue structure and collagen metabolism of D-galactose (gal) induced aging skin were measured in this study. Their considered mechanism of action in the skin was also described.Methods:Aging was artificially induced by subcutaneous injection with D-gal at the dose of125mg/kg.d for6weeks (42days).80Wistar rats were randomly divided into aging groups receiving D-gal treatment, including aging control group (D-gal for6weeks); NS (D-gal for6weeks+normal saline for4weeks); AA (D-gal for6weeks+compound amino acid for4weeks);0.1%DMAE (D-gal for6weeks+0.1%DMAE for4weeks);0.2%DMAE (D-gal for6weeks+0.2%DMAE for4weeks);0.1%DMAE+AA (D-gal for6weeks+0.1%DMAE/AA for4weeks);0.2%DMAE+AA (D-gal for6weeks+0.2%DMAE/AA for4weeks) and sham control group which was not given D-gal. At the end of each treatment, histologic changes of aging skin were observed; tissue water content and hydroxyproline were determined according to assay kits; PCNA stains of each group were observed by immunochemical methods; RT-PCR was used to detect mRNA expression of type I/III procollagen, MMP-1and TIMP-1. Simple PCI image analysis software were quantified the thickness of epidermis, dermis, collagen fibre density and PCNA positive stains. Data were expressed as means±standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) using SPSS13.0software. The results were taken to be statistically significant at a probability level of p<0.05.Results:1.Animal:There were some aging phenomenon, such as lean and small, low weight, yellow and no luster fair, thinned skin, decreased activities, neurological impairment in aging group rats after rats were subcutaneous injected with D-gal at the dose of125mg/kg.d for6weeks.2. Histological changes:When compared to sham control group, aging skin showed decreased thickness for both epidermis and dermis (P<0.05). With aging, the epidermis reduced its numbers of cell layers, and the dermal collagen fibers appeared sparse, slender or broken. Although the histologic changes of0.1%DMAE+AA group and0.2%DMAE+AA group were much better improved than that of aging control group, their indicators were all significantly lower than that of sham control group (P<0.01). In addition, when compared to aging control,0.2%DMAE alone also significantly increased epidermal thickness and density of collagen fiber (P<0.01), and0.1%DMAE increased epidermal thickness (P<0.01).3. Water contents in aging skin:There was no statistically significant difference in water contents among all groups (P>0.05).4. Hydroxyproline contents in aging skin:Hydroxyproline contents were apparently increased in aging skin treated with0.1%DMAE+AA or0.2%DMAE+AA complex (P<0.01), which showed a significant difference when compared to all other aging groups. Interestingly, hydroxyproline contents in0.2%DMAE+AA group reached an equivalent level to that of sham control group (P>0.05) even though both groups had much higher hydroxyproline contents than any of other6aging groups (P<0.05). There was no significant difference between0.1%DMAE+AA and0.2%DMAE+AA groups (P>0.05).5.Changes of collagen synthesis:(1)Collagen type I expression was greatly increased in response to the treatment of both0.1%DMAE+AA and0.2%DMAE+AA complex (P<0.01). Although there was no statistically significant difference between these two groups and sham control (P>0.05), all other aging groups showed much less expression of collagen type I (P <0.01).(2) When treated with0.1%DMAE+AA,0.2%DMAE+AA or0.2%DMAE alone, the mRNA expression for collagen type III was also considerably elevated over the aging control group (P<0.01), but still significantly less than sham control (P<0.01).6.Chagens of collagen catabolism:(1) MMP-1, a potential key regulator in aging skin, was also observed in this study, and results showed that its mRNA expression significantly increased in rats skin tissue treated with either0.1%DMAE+AA or0.2%DMAE+AA. These up-regulations of MMP-1activity showed a statistically significant difference to that of aging control group (P<0.01), but not to sham control(P>0.05).(2) In contrast, the transcripts for TIMP-1of all aging groups was down regulated and reached significantly lower levels to that of sham control (P<0.01).7. Expression of PCNA:PCNA expression of sham control is higher than all aging groups (P<0.01), and there were no difference among every aging group(P>0.05).Conclusion:1. Mesotherapy with combined application of DMAE and compound AA could improve the thickness of aging skin, and play an anti-aging role. 2. Both DMAE and compound AA may have limited effects on skin moisturizing which suggests that DMAE and compound AA had limited effects on skin moisturizing. This might be due to a lower level of concentration and shorter action period of local available DMAE in epidermal layer when direct subcutaneous injection was used for DMAE delivery, which differs from tropical application of DMAE.3. Mesotherapy with combined application of DMAE and compound AA could increase collagen contents of aging skin, especially, effect of0.2%DMAE+AA could be better which increased hydroxyproline contents is equal to sham control.4. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen type I synthesis.5. Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting catabolism to remodel skin texture.6. Intradermal injection of DMAE or AA alone showed no effects on hydroxyprolin contents or messages for collagen type I and MMP-1in the aging skin. These data indicate that combined application of DMAE and AA is the only way to play their anti-aging action in this D-gal induced aging skin model by modulating collagen type I metabolism and remolding the structure of aging skin.7. Intradermal injection of0.1%DMAE and0.2%DMAE did increase the dermal collagen fiber density over the aging control group. This result suggest that0.1%DMAE and0.2%DMAE treatment may potentially trigger aging skin to thicken the collagen fibre, but not apparently affect dermal collagen metabolism. Previous studies have shown that cultured rabbit dermal fibroblasts responded to DMAE by massive vacuolization in a concentration-dependent manner. The epidermis of rabbit external ear was also significantly thickened and exhibited clear perinuclear swelling indicative of vacuolization in response to tropical application of3%DMAE. It was suggested that vacuolar cytopathology may not be dissociable from the improvement of skin appearance that is rapidly produced by topically administered DMAE, and could be the cellular basis of the anti-wrinkle effect of DMAE. From our results, the vacuolization of the dermal fibroblast might occur in both0.1%and0.2%DMAE treated aging skin, resulting in cellular swelling and increased collagen fiber density. Epithelial cells of aging skin could also respond to the higher concentration of0.2%DMAE by vacuolization, which presented as increased epidermal thickness. However, these may need further studies to clarify this matter.8. Mesotherapy with0.2%DMAE+AA,0.1%DMAE+AA,0.2%DMAE,0.1%DMAE, AA, NS could not effect expression of PCNA.Mesotherapy caused minimum invasion and discomfort, without any posttreatment pain. All patients can return to their everyday activities immediately after treatment. In present study mesotherapy with DMAE+AA compound solution were administered directly and accurately into the region to be treated, and effect of medicines are more immediate and effective at minimum dose with the advantage of avoiding side-effects accompany with systemic administration at high dose.In conclusion, Mesotherapy with combined application of DMAE and compound AA could ameliorate skin collagen metabolism by promoting collagen synthesis and catabolism to remodel skin texture, and improve the thickness of aging skin. At the same time, complex of DAMA and AA could not induce fibroblast proliferation. This new strategy shows a promising potential in slowing skin aging in D-gal induced aging model, while further investigations should be conducted to demonstrate the biological bases of its anti-aging effects.