| Background:A total of100million patients develop hypertrophic scars in the whole world each year. Excessive scar is a fibroproliferative disease (FPD) in the skin. Forming as a result of aberrations of physiologic wound healing and may develop following any insult to the deep dermis, including burn injury, lacerations, abrasions, surgery, piercings and vaccinations. Hypertrohpic scar usually cause pruritus, pain and contractures. Itch (73.3%) and pain (67.6%) are known to be extremely two common paresthesia in post-burn patients, with a reported incidence of100%in children and87%in adults. In the early stage of scarring, when the patients feel itch. They may scrach the scar frequently with a result of excessive scar. This process can dramatically affect a patient’s quality of life, both physically and psychologically.Pain and itch are transferred by small nerve fibers, which could impact opioid receptors and its ligands expressed on reparative cells in case of abnormal epidermis and dermis. Sensory neuropeptides contact nervous system and non-nervous system, and play a role in multiple aspects such as maintaining the integrity of the skin, tissue reconstruction etc. Previous studies have focused on substance P (SP), calcitonin gene-related protein (CGRP), neurokinin receptor (NKR), neurotrophic factors, and nerve growth factor (NGF) and their receptors, which affected wound healing. But the exact mechanism of paresthesia of pathological healing is unclear.In a recent study, opioid as a special neuropetide was produced by some cells such as keratinocytes in the skin, including three subtypes of β-endorphin, enkephalin and dynorphin. Opiate receptors are presented on keratinocytes surrounded with unmyelinated peripheral nerve fibers in the dermis and epidermis. Therefore, opioid system interact directly with skin and nerve tissue..Some studies indicate that μ-opioid receptor (MOR), δ-opioid receptor (DOR) and K-opioid receptor (KOR) have the capability of regulating non-neuronal cell, including immunocompetence and physiologic function. Human epidermal keratinocytes express MOR at both levels of the mRNA and protein, otherwise, DOR and KOR are localized on fibroblasts in dermis. This seems to fit with "layering hypothesis" about cutaneous itch and pain.A research using immunofluorescence and real-time PCR indicates that β-endorphin and MOR are expressed in the keratinocytes and fibroblasts. There is little P-endorphin in normal epidermis, and little opioid receptor in keratinocytes, they may transmit itch and pain. The phenotype of re-epithelialized epidermal cells and the activated fibroblasts changed after skin injuring, which leads to abnormal secretion and distribution of P-endorphin, and limited opioid receptors do not fully bind to their ligands. During the process of wound healing and scarring, a large number of free opioid peptides presets in epidermis and dermis of hypertrophic scars and keliods. MOR, KOR and DOR are significantly increased in the epidermis and dermis of hypertrophic scars comparing to normal skin. Upregulated opioid receptors enhance the perception and transmission of pruritus, leading to paresthesia in patients with hypertrophic scars. Cutaneous autocrine and paracrine opioid peptides influence biologicalproperty of keratinocytes, fibroblasts and immunocyts in epidermis and dermis. As expression of opioid and its corresponding receptor changes, various type of cytokines (such as interleukin) and histamine were released, thus results in paresthesia. Currently, researches of opioid and opioid receptors are focus on acute or chronic Dermatitis and allergic diseases while in hypertrophic scar has not been reported. In summary, to clarify the characteristics of distribution and of opioid peptides and opioid receptors and relationship between them in normal skin and hypertrophic scar, maybe the key point revealing paresthesia in patients with hypertrophic scar.Objective:1. To identify the relationship between opioid peptides and the outcome of pathological wound healing. By observing (3-endorphin, enkephalin,dynorphin and their corresponding receptors MOR DOR and KOR.2. To find a new therapeutic method of paresthesia in patients with hypertrophic scar, to utimately improve wound healing. By observing real-time regularity and diversity of endogenous opioid peptides and receptors in vivo, and effect caused by opioid antagonist in patients with hypertrophic scar who feel itch or pain.Methods:1. We detected dynamic changes of β-endorphin, enkephalin, dynorphin contects in skin of rats with itch. Adlut SD rats were used. Subcutaneous injection of dextran solution(60mg:1ml)0.2ml was performed on the rats, then itchy model was successfully established. After anaesthesia abdominal skin preparation, a microdialysis membrane was inserted horiaontally within the dermis through the guide cannula, physiological saline flowing through the probe. The10samples within3hours under three state:under normal physiological condition, after injection of dextran, and after the injection of naltrexone and dextran at the same time. Then detect the concentrations of P-endorphin, enkephalin and dynorphin in all samples using ELISA2. We detected the expression of β-endorphin, enkephalin, dynorphin and MOR, KOR, DOR in hypertrophic scar tissue. Hypertrophic scar samples from42patients with hypertrophic scar (who were divided into3groups:group A:without itch and pain (n=20); group B:with itch (n=14), group C:with itch and pain (n=8)), and normal skin samples from5healthy individuals from April2010to September2011. We used immunofluorescence method to observe the expression of β-endorphin in scar and normal skin tissues. And the tissue homogenate concentrations of β-endorphin, enkephalin and dynorphin were detected using enzyme-linked immunosorbent essay (ELISA) method. Other nine patients were divided into four groups (group with itch and pain, group with itch, gourp with pain, group without itch and pain). The real-time quantitative polymerase chain reaction (RT-PCR) was used to evaluate MOR, KOR and DOR mRNA in all groups.3.We detected real-time regularity and diversity of β-endorphin, enkephalin, dynorphin in vivo, using microdialysis and administration of opioid antagonist.Clinic trail. Four hypertrophic scar patients with itch or pain were collected, the scars are red, hard and prominent leather. After routine disinfection, a microdialysis membrane was inserted horizontally4-7mm under the scar using a guide cannula with length of40mm. The guide was then with drawn, leaving the40mm membrane horizontal within the scar. A microdialysisi probe was inserted to the microdialysis pumb and sample collection tube. We collected3samples within10hours. Chose each patient’s own normal skin as a control, and repeated the above operation. Then we detected concentrations of β-endorphin, enkephalin and dynorphin in all samples using ELISA. The patients were asked to use0.02%naltrexone TAT gel on site of paresthesia, twice a day, for one week. Before and after treatment, each patient was evaluated with visual analogue scale (VAS) score of each patient, and the scar were observed.4.Statistical methods:we evaluated data using spss13.0software. We used one-way ANOVA for multi-comparison, LSD for pairwise comparison, and p<0.05was considered statistically significant.Results:1. After injection of dextran, the itch model of rat was successfully established, the β-endorphin content of rat skin significantly increased in comparison with that was under normal physiological condition. After injection of dextran and naltrexone at the same time, the β-endorphin content of rat skin decreased.2. The β-endorphin immunofluorescence rection positioning expression arised in all samples, and it expressed around peripheral nerve fibers in the dermis, fibroblasts and monocytoid cells, and expressed on the keratinocyte membrane principally. β-endorphin expressed significantly stronger in group B and group C than in group A.3. The β-endorphin content of human hypertrophic scar significantly increased in comparison with that of normal scar. In addition, the content of β-endorphin in group B was significantly higher than that in group A (P=0.001) and group C (P=0.008), and there were no significant differences between group A and C (P=0.881). The enkephalin content of hypertrophic scar significantly increased compared with that of normal scar in group A (P=0.011) and group B (P=0.008), there was no significant differences among other groups (P>0.05). The dynorphin content of hypertrophic scar significantly increased in comparison with that of normal scar, and there was no significant differences among other groups (P>0.05).4. MOR, KOR and DOR mRNA were detected in all samples. The expressions of MOR mRNA in group with itch and pain was significantly stronger than those in the other groups. The expression of KOR and DOR mRNA in group with pain and goup with itch and pain were significantly stronger than those in the other groups.5. In vivo, the β-endorphin and enkephalin Enk content of hypertrophic scar significantly increased in comparison with that of normal scar (P<0.01), while the Dyn content of normal sikn had no significant differences (P>0.05).After administration of opioid antagonist the VAS scores of each patient was lower than before.Conclusions:1. Development of curtaneous itch is associated with opioid petides and their respective receptors.2. β-endorphin, enkephalin, dynorphin and their corresponding receptor subtype (MOR, DOR, and KOR) are important factors in hypertrophic scar patients with paresthesia and β-endorphin and MOR are closely linked with the sense of "itching" after administration of MOR antagonist, the sense of "itching" significantly reduces.3. As the three types of opioid peptides regulating each other, thus affecting the match with their respective receptors, so they play important role in paresthesia, and further study is needed. |
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