| Senescence, also known as aging, is performance of during the body function decline and the degradation of overall with physiological disorders, characterized by the functional structure dysfunction of progressive multi-organ system. Expressed as DNA, proteins, lipids and the like modified cell damage and harmful accumulation, the body’s aging process predetermined by gene regulation, on the other hand, it is caused by internal and external "wear and tear", which both occurred in the cellular and molecular level. It is common that the development of the body rests undifferentiated cells to differentiate, from differentiation to aging, and then by the process of aging to apoptosis. But with emergence of the concept about "autophagy", people began to re-understanding of the aging process and its mechanisms in this popular field of life sciences research.Cell homeostasis is achieved by precisely regulating the synthesis and degradation of cellular components. There are two degradation pathways of intracellular substances, one is the ubiquitin-proteasome system (UPS), specifically responsible for the degradation of the majority of cellular proteins, mainly short-lived protein, which is a highly efficient protein degradation pathway. Another one is autophagy, the major protein degradation and long life organelles. Autophagy is important for the degradation of eukaryotic cells, also known as type Ⅱ cell death and it is a process of cell autophagy-related gene in the regulation of the use of lysosomal degradation of damaged cells and macromolecules. There are three main types of autophagy in cell, namely giant autophagy (Macroautophagy), micro-autophagy (Microautophagy) and chaperone-mediated autophagy. Autophagy is the main way of the the cycle of giant cytoplasmic constituents, commonly referred to as autophagy. The common process of the autophagy is that protein degradation are all achieved within lysosomes. It uses its lysosomal degradation damaged cells and macromolecules to achieve the cell management and quality control of proteins and organelles. Cells "junk", including damage or aging organelles, long-lived proteins, errors or misfolded protein synthesis, etc., must be promptly removed by autophagy.Whether the cells are in a normal physiological or pathological status,autophagy is always an important body defense and protection mechanisms to maintain cellular homeostasis or self-assurance cell survival. The study found that autophagy aging process in a variety of eukaryotic organisms plays an important regulatory mechanism. Autophagy function will decline with aging,which may be due to the changes in cell structure and the age-related decrease of function. The study said that autophagy plays a key role in the aging process in organisms, and as part of an organic whole organism, aging autophagy of skin tissue may also be relevant with it. Foreign scholars found that older women’s skin own a higher amount of fibroblasts than young women during the dermis autophagy.The results showed that the degradation process autophagy was inhibited with older donor skin fibroblasts, leading to the accumulation of autophagic, which indicated that autophagy fibroblasts damaged skin in the elderly, leading to skin integrity and strength of the skin damage.The cell stress in vitro is a change or fluctuation of extracellular conditions, which causes the structure and function of macromolecules damage. Different stress factors trigger different cellular responses that called induce cell repair mechanism, which induce transient adaptation of a number of stress factors to cause the cell response, induce autophagy or trigger cell death. Irreparable damage or exposure to prolonged stress may lead to aging. In aging mammals, since the function of autophagy system disorders, autophagy as a holding cell homeostasis auto repair process that increase with age becomes less effective. The aging body suffers from trauma, infection and hypoxia blow may be cause the decline of autophagy that may not maintain cell homeostasis, and then toward worsening or delayed healing.Wound repair is a complex process, rely mainly on repair cells, inflammatory cells, growth factors and extracellular matrix synergy damage to soft tissue reconstruction. Aged-skin wound is the difficult healing, which the pathogenesis is quite complex and subject to multiple factors. The aging of cells and the whole body system, local recurrent ischemia-reperfusion injury, bacterial colonization may lead to delayed healing of wounds. As already mentioned,aged skin autophagic activity decreased with aging, and activity increased autophagy has anti-aging effects, but autophagy senile skin wound healing and what impact mechanism is not related to research, this study helps to understand the impact and mechanisms of autophagy plays a role in wound healing of the elderly, which can at the same time to promote wound healing by regulating autophagy.part1the expression of autophagy in different age groups of rats with acute skin wound healing processObjective To study the expression of autophagy-related genes LC3、 Beclin-1、 p62in cutaneous wound healing between the different age groups rats.Methods1. A full-thickness excisional lesion were performed on the rats back. We observed and compared wound tissue inflammatory conditions, dermal remodeling,collagen accumulation condition and adnexal development status by HE and masson’s stained methods.2. Observe the ultrastructure of autophagy in the skin of the old and young rats with electron microscopy.3. We established new monoclonal antibodies against the LC3、 Beclin-1、 p62used them for the specific western blot and envision immunohistochemistry detection at different time points (0,4,7,14,21d). Another, we used qRT-PCR detection the gene expression of LC3,Beclin-1,p62at different time points (0,4,7,14,21d).Results1. HE staining showed that the microstructures of skin were differente between the young and the old rats. The young rats normal epidermis and dermis fibrous connective tissue were more maturational. The subcutaneous tissue of the young rats were thicker, however, the boundaries were not very clear. The older rats normal epidermis were thinner, the connection of epidermis and dermis flattened, relaxed. The number of elastic fibers and collagen fibers were reducing, irregular and breaked into pieces. The small blood vessels were significant vasodilation, a large number of neutrophils, macrophages infiltration on4th day after wounding. There was a large number of granulation tissue formation on4th day after wounding in the younger group, in addition there were large number of fibroblasts. Granulation tissue had been substantially filled on7th day after wounding, fewer inflammatory cells. The wounds were completely healed on21th day after wounding on young rats. The wound were not completely healed on21th day after wounding on old rats.2. Masson trichrome staining showed:In the old rats21th days after injury, still shows a small amount of inflammatory infiltration, newborn dermal collagen accumulation loose, fewer, irregular, mostly filled with debris like, less collagen accumulation rule. All the wounds were healed in the young group21th day after injury, the number of collagen fibers increased and arranged regularly.3. Observed with electron microscopy, there were autophagy in keratinocytes and fibroblasts. The autophagy had bilayer or multilayer film. And there were residual organelle such as mitochondria, endoplasmic reticulum, ribosomes residue. There were more autophagy in older group.4. Immunohistochemistry:LC3,Beclin-1in the epidermis and dermis were expressed in normal skin group of young and old rats. The edge of the wound, the epidermis, dermis and hair follicles showed positive staining. In the newborn granulation tissue vascular endothelial cells, macrophages and fibroblasts are visible expression. Before wounding, two protein expression in the skin were more in the older group.LC3,Beclin-1protein expression were upward trend over time after wounding. LC3expression were more in the younger group, and Beclin-1expression is higher in older group on4,7,14,21th day afer wounding.5. qRT-PCR(1) LC3mRNA expressed in cutaneous wound healing between the different age groups rats.LC3mRNA expression were significant differences (P<0.05) on7d,14d,21d after wounding,4d<14d<7d<21d.The expression of LC3was significantly higher in the young rats (P<0.05).(2) Beclin-1mRNA expressed in cutaneous wound healing between the different age groups rats.Beclin-1mRNA expression were significant differences (P<0.05) on4d,7d,14d,21d after wounding,4d<14d<7d<21d. The expression of LC3was significantly less in the young rats.(3) p62mRNA expressed in cutaneous wound healing between the different age groups rats.p62mRNA expression were significant differences (P<0.05) on4d,7d, 14d,21d after wounding,4d>7d>14d>21d.On4d,7d, the expression of p62had no significant difference, the expression of p62was significantly less in the young rats.6. Western blottingLC3-II, Beclin-1protein expression was gradually increased4d,7d,14d,21d after wounding in the two groups, and the p62protein expression is gradually declining, that consistented with mRNA qRT-PCR results of the three genesConclusions1. Autophagic degradation decreased, resulting in the accumulation of autophagy and the destruction of the integrity and strength of the skin.2. In cutaneous wound healing, autophagy ability was enhanced. The skin of young rats can maintain steady self, and control autophagy in the normal range, thereby promoting wound healing. Regulatory function was disorder, resulting in the excessive autophagy itself occur, resulting in irreversible damage and delay the healing process in old rats.Part2Platelet-rich plasma (PRP) effect on the cell autophagy in the skin wound healing in aged rats with acute.Objective:platelet rich plasma (PRP) is extracted by centrifugation of blood out from the autologous platelet concentrate, after activated can form a gelatinous substance (platelet gel, PG). Due to the rich variety of growth factors, active peptide and other substances, mainly PDGF, TGF-β,VEGF, EGF, IGF.these cytokines can reduce wound inflammation and promote vascularization to accelerate healing, but also to promote the proliferation of epidermal and dermal cells to restore normal dermal matrix remodeling skin structure. Establish the rat model of acute back full-thickness skin defect to observe the efficacy of PRP to promote wound healing, and the detection process of autophagy genes and CD31expression, and investigate the effect of PRP on autophagy and also study the relationship between autophagy and wound healing.Method:1. Establish the SD rat model of acute back full-thickness skin defect. To use saline (control group), platelet-rich plasma group (PRP group), less PRP group (PPP group) to treated wounds respectively. And then take the wound skin specimens at0.4,7,14and21th day, wound tissue after section for HE and Masson staining was observed.2. Using transmission electron microscope to observe the skin autophagy morphology and structure in three groups at different times.3. Comparison the rate of wound healing of three groups.4.Using Envision immunohistochemical method, qRT-PCR and western-blotting assay the expression of LC3, Beclin-1, p62, CD31at different times in different age groups.Results1. The number of fibroblasts were increased at4d and then started to decrease at14d after wounding. There were a few collagen fibers in granulation tissue at7d. Epithelialization and collagen synthesis improved, the number of collagen fibers increased and arranged regularly in PRP group on14d. In control group, epithelial connection was still a gap, collagen fibers had not be mature, irregular arrangement. The number and state of collagen fibers of PPP group fall in between the other two groups.2. There were a small amount of vascular endothelial cells and fibroblasts in granulation tissue on4d. In control group, There were few vascular endothelial cells and fibroblasts. On7d, granulation tissue developed best in PRP group. Vascular endothelial cells, capillaries and fibroblasts increased. The expression of CD31increased. On14d,PRP group had new capillaries, lumen increased, showed that the initial formation of vascular smooth muscle.On21d, there were most capillaries and sebaceous glands,capillary network architecture was normal in PRP group. There were the lowest capillaries in control group.3. The number of autophagy in keratinocytes and fibroblasts increased on4d. The autophagy had bilayer or multilayer film. And there were residual organelle such as mitochondria, endoplasmic reticulum, ribosomes residue. There were fewer autophagy in PRP group.4. The wound healing rate of PRP group was significantly higher than the other groups(P＜0.05) on4d,7d,14d,21d.5. LC3mRNA expression were significant differences on14d,21d after wounding (P<0.05). On14d, the expression of LC3was highest. Beclin-1mRNA expression were significant differences (P<0.05) on4d,7d,14d,21d after wounding. The expression of Beclin-1was significantly less in the PRP group. The expression of p62was significantly less in the PRP group. Correlation analysis of data showed that the expression CD31of were correlated with p62and Beclin-1, on the4d and7d,negatively correlated with Beclin-1, was positively correlated with p62.6. LC3Ⅱ, Beclin-1and CD31protein expression was gradually increased after wounding, and the p62protein expression is gradually declining in the three groups. It was consistent with mRNA qRT-PCR results of the three genes.Conclusions1. PRP inhibited autophagy activity in wound healing in old rats and improve wound healing.2. Expression CD31of were correlated with autophagy related gene p62and Beclin-1,suggested that autophagy participated in the angiogenesis during wound healing. |
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