Effect of Melatonin on Lipid Barrier in Rats’ Skin

In this work we studied the effect of intraperitoneal admin istration of melatonin in a dose of 1 mg per kg on lip ids and microstructure of rats’ skin. A single inject ion of melatonin induced a change in lipid profile most notably in phospholipids and triacylglycerol fractions which contents varied oscillatory in opposite each other with a maximum divergence at 3 hours. By 24 hours there was a significant increase in triglycerides, and after 48 hours the lipid profile approached the init ial values. After regular daily administration of melatonin for 6 days, the contents of almost all lipid fractions were significantly reduced (most in triacylg lycerols and phospholipids) with growing level of free fatty acids. By 21st day the contents of lip ids in the skin increased again, but did not reach the initial values. Reduction of lip id level was accompanied by prominent degenerative changes in the microstructure of the skin by the 6th day of experiment. In general, the administration of melatonin in any way caused the changes in skin’s lipid profile but after a single introduction parameters of lip id spectrum in two days were restored to their in itial values, and a regular admin istration for 6 days led to sustained changes in lip id profile and structure of the skin.


Introduction
One of the main functions of the skin is to form a barrier between the organism and the environment. The skin is a protector fro m mechanical damage, UV radiat ion, chemicals, pathogens, loss of water and electrolytes [1]. Proper function of the skin barrier is mostly provided by lip ids. These compounds are localized not only inside the cells, but also form co mp lex organized structures that fill the intercellular space of the epidermis [1,2]. Age-dependent changes in the skin and some diseases are directly related to a malfunction of the skin barrier [3,4,5]. The skin's physiological condit ion and metabolis m are regulated by endocrine and paracrine signaling. Skin cells contain receptors for neuropeptides, growth hormone, glucocorticoids, sex hormones, insulin-like gro wth factor, retinoids, eicosanoids, serotonin, melatonin and others [6]. Many of these compounds can be synthesized direct ly into skin cells.
Over the past few decades pineal hormone melatonin is increasingly used in the treat ment o f circad ian rhythms, card iovascu lar therapy , cancer, and n eurod egenerat ive disease [7,8,9]. It is one of the most evolutionarily conserved and pleiotropic hormone still act ive in hu mans and it has been implicated in v ital skin functions such as hair gro wth, fu r pig mentation as well as melano ma control [10]. Melatonin, as a multifunctional hormone, appears to regulate and modulate other functions in hu mans through the activation of its receptors and works as strong antioxidant that protects the DNA and prevents lip ids peroxidation [11,12]. Currently, there are some proposals that melatonin as a highly lipophilic co mpound penetrates easily through cellular membranes and therefore is able to efficiently protect read ily every intracellular structure including enzy mes, proteins, lip ids, mitochondria and the nucleus against oxidative damage [10,13].
Most of investigations regarding the different aspects of melatonin confirm that it is highly efficient anti-ag ing factor with immunoenhancing properties reducing skin o xidant damage and cancerogenesis [12,14,15,16]. Melatonin was successfully used as a protective substance for the skin exposed to UVR, as well as compound protecting whole-body irrad iated animals [17]. Recent reports showed that melatonin increases survival and decreases apoptosis of keratinocytes, fibroblasts and leukocytes subjected to UVR as a result of scavenging of free radicals, as well as inhibits apoptotic proteins and lipids pero xidation [18,19]. Melatonin is a scavenger of both oxygen-and nitrogen-based reactive molecu les, including pero xynitrite anion and its decomposition products, including hydro xyl radical, nitrogen dio xide, and carbonate radical [20]. Besides its ability to direct scavenge radicals and radical products, melatonin also augments the activities of antioxidative en zy mes, including glutathione pero xidase (GPx), supero xide dysmutase (SOD) and glutathione reductase [20,21].Enormous clinical interest was caused by the recentlypublished evidence which indicates the normalizat ion of lipid parameters in blood by exogenous melatonin in patients with obesity, diabetes, and hypercholesterolemia [22,23,24,25]. As it turns out, melatonin affects the intensity of lipid metabolism in different cell types such as liver, fat, bone, and so on [26,27,28,29,30].
Since the skin is also a typical target fo r the pineal hormones, we have assumed that melatonin along with other compounds may be involved in the maintain ing of skin lipid barrier. The purpose of this study was to evaluate the lip id content in the skin of rats under acute and prolonged administration of melatonin.

Rat model
The experiments were conducted on white male Wistar rats (200-250 g) in the summer period. The animals were kept on a standard diet under natural light exposure. All experimental procedures were conducted in accordance with the Rules of laboratory practice in the Russian Federation (2003) and with Princip les on Good Laboratory Practice (OECD № ENV/MC/ CHEM (98) 17,1997).
Rats were decapitated under ether anesthesia. Skin samples (100-150 mg) o f interscapular region cleared fro m hair and subcutaneous adipose tissue were used for preparation of lip id extracts, and their further qualitative and quantitative analysis.

Melatonin Admi nistrati on
Rats received intraperitoneal injections of melatonin in 1 ml of a sterile physiological solution with a dose of 1 mg/ kg of body weight at 10 am. It is believed that in th is dosage melatonin induces the most expressed changes of lipid and carbohydrate metabolism [31].
Animals were decapitated 1, 2, 3, 4, 5, 6, 24 or 48 hours after inject ion of a single dose of melatonin. The control group consisted of intact animals which d id not receive melatonin.
In case of prolonged admin istration of melatonin, animals were decapitated after 6 or 21 days of daily regular injections of melatonin. In this case, apart from intact animals, groups of comparison received injections of physiological solution for 6 and 21 days.

Li pi d Extraction
Lipid extracts fro m the skin samp les were obtained by Folch Method [32]. The extract was used for further qualitative and quantitative analysis of lipids.
Fractionation of lipids was performed using the method of thin layer chro matography [33] on silica gel L 5/ 40 Chemapol.
Separation of total lip ids on the fractions was carried out in a solvent system of hexane -d iethyl ether -methanolacetic acid at a rat io of 9:2:0.2:0.3 by volu me. Thereby the following classes of lipids were reliab ly determined: phospholipids (PL), diacylg lycerols (DA G), cholesterol (C), free fatty acids (FFA), triacylglycerols (TA G), cholesteryl esters (CE).
Detection was carried out by the exposure of dried chromatogram in iodine vapor. Elution of lip id fractions was performed by a mixture of chloro form and ethanol (3:2 by volume) for DA G, C, FFA, TA G, CE, and chloroform: ethanol:14N NН 4 ОН (5.6:4.2:0.2 by volume) for PL. Obtained extracts were dried and used for the quantitative determination of individual lipid fractions.

Quantitati ve Determination of Li pi ds
Determination of total lipids (TL) and their fract ions were carried out by heating o f the dried extracts with concentrated H 2 SO 4 for 15-20 minutes at 190-200 0 С [33]. After cooling down, the samples were diluted with water 1:1, and absorbance was measured at a wavelength of 490 nm (for total lip ids) or 400 n m (for individual fractions). The contents of total lip ids and individual lipid fractions in the samples were determined by means of ca libration standards purchased from Sig ma-Aldrich.
Quantitative determination of total phospholipids (TPL) and their fractions was carried out on the content of lip id phosphorus using malachite green method in modification [34]. Dried ext racts were mineralized with perchloric acid for 25 min at 225 °C. M ineralizates were dissolved in 1.5 ml o f water, then 2 ml of dye reagent were added (1 volu me of 4.2% solution of ammoniu m mo lybdate in 5 N HCl + 3 volu mes of 0.2% solution of malachite green) and 0.1 ml of 1.5% solution of Tween-20. After 1 hour, absorbance was measured at wavelength of 670 n m. Phosphate was determined by previously plotted calibration graph.

Microscopy of Skin Samples
For microscopic examination semi-thin sections (1 µm) were used. Samp les were fixed in 10% formalin solution, washed in cacodylate buffer (p H 7,2-7,4), and postfixed by 1% solution of OsO 4 . After that the samp les were dehydrated in ascending alcohol concentrations and embedded in epo xy resin Epon 812. Sect ions were stained with a three-dye (methylene blue, azure II, basic fuchsine), embedded in Canada balsam and examined under light microscope [35].

Statistical Analysis
To demonstrate statistically significant differences between groups of control and experimental animals, Student' s t-test was used with p-values less than 0.05.

Single Administrati on of Melatoni n
After in jection of melatonin, significant increase of total lip ids in the skin was observed only after 24 hours ( Table 1). The elevation was occurred mainly due to the contribution of TA G, whereas the contents of other lipid fract ions were relatively constant. The subsequent reduction of TAG returned the quantity of total lipids to almost the init ial level after 48 hours.
Nevertheless, during the first 6 hours after ad ministration of melatonin, the changes in the ratio of individual lip id fractions were not as distinct. In this period, the most significant oscillatory changes were observed in TAG and PL fractions, the amounts of which varied in opposite phase, accompanied with a slight increase in FFA ( fig. 1) The character of changes in TL level was fully consistent with the changes in PL fract ion.
Thus, we can note that in the first hours after the administration of melatonin, most labile lip id co mponents of skin are PL, and only after 24 hours the level of TL begins to be determined by change of TAG content. Exogenous melatonin induced significant changes in the phospholipid spectrum of the skin ( Table 2). In the early period (3-5 hrs after injection), the contents of PI, PS, PE were reduced. By 24 hrs, the proportion of PC decreased in favor of other memb rane glycerophospholipids (PE and PS). On the second day, the distribution of phospholipid fractions reached the values of intact animals.

Prolonged Admi nistration of Melatonin
At regular injections of melatonin, by the 6th day a significant decrease in the contents of almost all lip id fractions in the skin was coupled with growing levels of FFA (Table 3). However, in this case, the greatest decreases were also in the levels of TG and PL, against wh ich the other fractions, except FFA, changed insignificantly.
By the 21st days the contents of almost all lipid fract ions were elevated again, but did not reach the initial values. Nevertheless, it can be explaned as a partial adaptation to the systematic introduction of the hormone.
Among the phospholipids' fractions the biggest changes were noted in the contents of PC, PE, PS (Table 4). In addition, lysophospholipids, the products of PL hydrolysis, were accu mulated.

Microscopy of Skin
After prolonged ad min istration of melatonin the structure of the epidermis and dermis undergoes some changes. Figure 2 represents the microscopy image of the intact skin (A) and skin after 6 days of regular ad min istration of melatonin (B). As the figure shows, the collagen bundles of the dermis under the influence of melatonin acquire a homogenous structure, their boundaries are poorly distinguished. Epidermis in some areas is damaged and flaky, wh ile its fragments are visible on the surface of the dermis. Dyes stained the tissue poorly, wh ich can be probably related to changes of its tinctorial properties.

Discussion
Our data suggest that melatonin may alter lipid metabolism in the skin o f rats, and thus interferes with the functioning of the skin barrier. Effect of the drug depends on the duration of a course of injections. The response of the skin lipids to a single dose of melatonin is extended in time, but early alterations of parameters are detected even a few hours after injection, and full return to initial values does not occur even after two days. The dynamics of change is complex; oppositely directed changes in concentrations of major lip id fractions are observed. Prolonged uptake of melatonin results in a reduction of lip id level in the skin that may cause persistent disruption of its barrier function. This is indirectly confirmed by the results of microscopy of histological preparations of the skin.
It is known that the lip id skin barrier is formed due to the functioning of the sebaceous glands and differentiating keratinocytes. Sebocytes and keratinocytes have lipoprotein lipase on their surface, LDL receptors, and various transporters of fatty acids [36,37]. Part of the skin lipids are of nutritional origin. However, skin cells are capable of independent synthesis and transformat ion of fatty acids. One can find a full set of enzy mes necessary for the formation o f t rig lycerides, phospholipids, sphingolipids, and cholesterol [36,37].
Hormonal regulat ion of tissue lipid metabolis m is carried out at different stages. Expected targets for endogenous and exogenous regulators are in a system of lipid transport in the blood, lipoprotein receptors, membrane transporters of lip ids, quantity and activity of intra-and ext racellular enzy mes responsible for metabolism of these compounds [1,2,36]. It is not clear wh ich of the potential targets can be used by melatonin to suppress production of lip ids in the skin. Ho wever, data obtained by other researchers for different cell types, allow us to propose the following explanations.
First, melatonin may impair the tissue supply of alimentary lipids by reducing the number of circulating lipoproteins, TA G, and C. Similar results were obtained in humans and various animals [6,38,39]. Second, melatonin is able to modify the transport of lipids through the plasma memb rane. Thus, in mononuclear leukocytes of humans the hormone reduces the number of receptors for LDL, which leads to lower levels of intracellular cholesterol [40]. In adipocytes melatonin inhibits fatty acid transporters [41]. In postmenopausal wo men with normolipidemia p rolonged administration of the hormone inhibits the activity of LP-lipase [8]. Finally, intracellular en zy mes may serve as a target for melatonin. Thus, in adipocytes isolated from the inguinal fat pads, melatonin inhibits lipolysis induced by izoproterenol [42].
Despite the progress made in deciphering the intracellu lar signaling of melatonin, the questions remain open regarding the mechanisms of lipid metabolis m regulat ion by this hormone. Possibilit ies widely discussed in the literature include both direct action o f melatonin [28,29] through specific receptors (membrane МТ1, МТ2 and nuclear RZR/ RORα, RZR/ ROR) [43] and effects mediated by other hormones (insulin, glucocorticoids, growth hormone, leptin, etc.) [41,44].
Specialized melatonin receptors are present anywhere in the skin. Membrane and nuclear receptors have been found in fibroblasts, keratinocytes, melanocytes, the cells of hair follicles, eccrine glands, and the endothelium of blood vessels [27]. Thus, there is the potential of direct action of melatonin on lip id metabolis m. Indirect evidence favouring this mechanism is reported in the present work as rapid changes in the skin content of TAG and PL in the first 6 hours after admin istration of melatonin.
The possibility of indirect action of melatonin is indicated in the study by B. Bo jková et al [45], where it has been found that prolonged administration of melatonin led to increased levels of cort icosterone in the blood of male rats. Glucocorticoids negatively affect the skin barrier function, because they reduce lip id synthesis in the epidermis [46]. It has been shown [47] that melatonin, with the participation of receptors МТ1, suppresses insulin secretion by isolated pancreatic islets. Hypoinsulinemia also results in suppression of lipogenesis and stimulat ion of lipolysis.
The action of melatonin, mediated by the endocrine glands and, thus, combined with the more or less long biosynthetic processes must be developed with a certain delay. This assumption is not excluded as a possible explanation fo r the significant growth of TA G level by 24 hours and its subsequent reduction to 48 hours after administration of melatonin reported here.

Conclusions
Our study demonstrates a distinct effect of melatonin on the skin: the administration of melatonin, in any case, causes changes in the skin's lip id profile but after a single injection the parameters of lip id spectrum are restored to their orig inal values after two days, and a regular administration for 6 days leads to sustainable changes in lip id spectrum and the structure of the skin.
Uptake of exogenous melatonin is one of the reasons for significant changes in tissue structure and, consequently, the barrier function of skin, causing in both a decrease total lip ids of the skin, and a change its lipid profile. This fact may be considered as one of the side effects of melatonin preparations.