The change of angiotensin Ⅱ production and its receptor expression during wound healing: possible role of angiotensin Ⅱ in wound healing

Objective This study was undertaken to observe the change in the local level of angiotensin Ⅱ (Ang Ⅱ) and the expression of its corresponding receptors AT1 and AT2 during wound healing, and explore the possible role of Ang Ⅱ in wound healing . Methods A model of full-thickness cutaneous wound was developed on the back of C57/BL6 mice. Specimens were taken from the wound of each mouse on the day 0, 1, 3, 5, 7, 9, 11, 13 and 15 after wounding. The change in the generation of Ang Ⅱ in wounded tissue during the healing process was detected with ELISA. The proliferation and the apoptosis of cells were detected by bromodeoxyuridine (Brdu) and terminal deoxyuncleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method in wounded skin during the healing process, respectively. The cellular localization and the mRNA level change of Ang Ⅱ receptors in wounded tissue during healing were detected with immunostaining and RT-PCR. Results Ang Ⅱ produced in wounded skin was increased in the first 7 days to reach the peak, and then gradually decreased during wound healing. BrdU labeling index was increased gradually in the first 7 days to reach the peak, and then gradually decreased during wound healing. The number of TUNEL-positive cells was increased slowly in the first 7 days after wounding. The increase in the number of TUNEL-positive cells was more markedly after epithelization of the wound. In normal mice, AT1 and AT2 receptor were found positively expressed in the whole epidermal layer, while positive expression was only found in the endothelial cells of the capillary vessels within the dermal layer, and positive expression was also found in appendages of the skin, i.e. hair follicle, sweat gland and sebaceous gland respectively. Positive staining signal of both AT1 and AT2 receptors were increased in the first 7 days to reach the peak, then gradually decreased. Expression of AT2R was increased again following the epithelization of wound. The result of RT-PCR showed that the expression of both AT1 and AT2 receptors was detectable, and AT1 receptor was increased in the first 7 days to the peak, and then gradually decreased during wound healing, while AT2 receptor expression reached its peak value on day 7, then gradually decreased, and increased again following the epithelization of wound. Conclusions These results indicate that Ang Ⅱ participate in wound repair and related to remolding in the late stage of wound healing through the change in production of angiotensin Ⅱ and expression of AT1 and AT2 receptors. AT1 receptor might be closely associated with cell proliferation,while AT2 receptor might play a role in cell apoptosis and remolding during wound healing.     

血管紧张素Ⅱ及其受体表达在创面愈合过程中的变化及作用

目的 观察创面愈合过程中血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)及其受体表达的动态变化,以及这种变化与创面愈合过程中细胞增殖和凋亡活动之间的关系,探讨AngⅡ在创面愈合过程中的可能作用.方法 建立小鼠背部全层皮肤缺损创面模型,于创面形成后第0、1、3、5、7、9、11、13、15天切取创面组织标本,用ELISA法检测创面局部组织AngⅡ产生的变化;采用BrdU及TUNEL染色检测创面细胞增殖和凋亡的变化;用免疫组织化学染色和RT-PCR检测创面局部组织AngⅡ受体AT1和AT2表达的组织细胞定位和mRNA水平的变化.结果 小鼠全层皮肤缺损创面局部组织AngⅡ、BrdU标记指数均在伤后逐渐增加,并于第7天达到峰值后即逐渐下降.TUNEL染色阳性细胞数在伤后即开始缓慢增加,并于创面完成上皮化后增加趋势更为明显.正常小鼠皮肤AT1和AT2受体在整个表皮层均有阳性表达,但在真皮层,AT1和AT2受体仅在微血管内皮细胞有阳性表达.AT1受体在角质形成细胞、成纤维细胞均有表达,阳性染色信号在伤后逐渐增加,在第7天最强,以后逐渐下降.AT2受体阳性染色信号也在伤后逐渐增加,7 d以后则逐渐下降.但当创面上皮化完成后,AT2受体阳性染色信号再次增加.RT-PCR结果 显示:AT1和AT2受体mRNA均有表达,AT1、AT2受体mRNA表达在伤后第7天均达到峰值,此后则逐渐下降,且AT2受体mRNA表达在创面上皮化完成后表达再次增加.结论 在创面愈合过程中,AngⅡ可能通过其产生及受体表达的变化调控创面的愈合及后期的塑形改建.AT1受体可能与细胞增殖活动密切相关;AT2受体可能与细胞凋亡及愈合过程中组织重建有关.

Abstract：

Objective This study was undertaken to observe the change in the local level of angiotensin Ⅱ (Ang Ⅱ) and the expression of its corresponding receptors AT1 and AT2 during wound healing, and explore the possible role of Ang Ⅱ in wound healing . Methods A model of full-thickness cutaneous wound was developed on the back of C57/BL6 mice. Specimens were taken from the wound of each mouse on the day 0, 1, 3, 5, 7, 9, 11, 13 and 15 after wounding. The change in the generation of Ang Ⅱ in wounded tissue during the healing process was detected with ELISA. The proliferation and the apoptosis of cells were detected by bromodeoxyuridine (Brdu) and terminal deoxyuncleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method in wounded skin during the healing process, respectively. The cellular localization and the mRNA level change of Ang Ⅱ receptors in wounded tissue during healing were detected with immunostaining and RT-PCR. Results Ang Ⅱ produced in wounded skin was increased in the first 7 days to reach the peak, and then gradually decreased during wound healing. BrdU labeling index was increased gradually in the first 7 days to reach the peak, and then gradually decreased during wound healing. The number of TUNEL-positive cells was increased slowly in the first 7 days after wounding. The increase in the number of TUNEL-positive cells was more markedly after epithelization of the wound. In normal mice, AT1 and AT2 receptor were found positively expressed in the whole epidermal layer, while positive expression was only found in the endothelial cells of the capillary vessels within the dermal layer, and positive expression was also found in appendages of the skin, i.e. hair follicle, sweat gland and sebaceous gland respectively. Positive staining signal of both AT1 and AT2 receptors were increased in the first 7 days to reach the peak, then gradually decreased. Expression of AT2R was increased again following the epithelization of wound. The result of RT-PCR showed that the expression of both AT1 and AT2 receptors was detectable, and AT1 receptor was increased in the first 7 days to the peak, and then gradually decreased during wound healing, while AT2 receptor expression reached its peak value on day 7, then gradually decreased, and increased again following the epithelization of wound. Conclusions These results indicate that Ang Ⅱ participate in wound repair and related to remolding in the late stage of wound healing through the change in production of angiotensin Ⅱ and expression of AT1 and AT2 receptors. AT1 receptor might be closely associated with cell proliferation,while AT2 receptor might play a role in cell apoptosis and remolding during wound healing.     

Effect of substance P released from peripheral nerve ending on endogenous expression of epidermal growth factor and its receptor in wound healing

Objective: To explore the relationship between substance P (SP) released from peripheral nerve endings and the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) during wound bealing. Methods: Fifty Wistar rats were randomly divided into 2 groups, injury group and capsaicin group. In the injury group, a full-thickness skin wound on the back of the rat was taken. The wound edge and granulation tissues were taken on the 1st, 3rd, 6th, 9th, 12th days after injury, respectively. In the capsaicin group, capsaicin was injected subcutaneously on the back of the rats to destroy the sensory nerve to prevent the secretion of SP, then a wound and sample was made in the same way.Immunohistochemistry and in situ hybridization were employed to detect the expression of SP, EGF/EGFR, and EGF mRNA/EGFR mRNA in the granulation tissues.Results: In the injury group,immunohistochemical stain of SP and EGF/EGFR was located on the hair follicles and sebaceous glands at the 1st day. And the stain of SP was obvious at the 3rd day in the granulation tissues, then decreased gradually. EGF/EGFR was at low level at the 3rd day, then increased gradually and reached the peak at the 9th day, then declined. In the capsaicin group, the stain of SP and EGF/EGFR was faint and without obvious change during the wound healing process. The tendency of the EGF mRNA/EGFR mRNA expression was similar to that of EGF/EGFR. Conclusions: During wound healing, SP may promote the healing process by affecting the expression of EGF/EGFR in the erunuation tissues.     

Animal study on expression of laminin and fibronectin in cornea during wound healing following alkali burn

Objective:To observe the expression of laminin and fibronectin in alkali-burned corneas in rats.Methods:A total of 18 normal Wistar rats were randomly divided into 6 groups(n=3 in each group).For each rat, one eye was injured by alkali burn,the other one was taken as the normal control.Then all the corneas were surgically removed and the expression of laminin and fibronectin was observed with immunohistochemistry respectively at 7 hours,1 day,3 days, 7 days,14 days and 28 days after alkali burn.Results:Compared with that of the normal controls, the expression of laminin and fibronectin of the burned eyes was dramatically higher at 7 hours, reached peak at 14 days and decreased to the normal level at 28 days after alkali burn.Conclusions:In the process of wound healing after alkali burn, the expression of laminin and fibronectin increases dramatically, which suggests that laminin and fibronectin may participate in the process of corneal wound healing.     

Relationship between expression of Bax and Bcl-2 proteins and apoptosis in radiation compound wound healing of rats

Objective: To study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats. Methods : Apoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods. Results: (1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. ( 2 ) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bd-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level whenthe apoptosis decreased distinctively. Conclusions： Bax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.     

Vascular endothelial growth factor and its receptor expression during the process of fracture healing

Objective： To study the expression regularity of vascular endothelial growth factor （VEGF） during the process of fracture healing, and the type of VEGF receptor expressed in the vascular endothelial cells of the fracture site.
Methods： The fracture model was made in the middle part of left radius in 35 rabbits. The specimens from the fracture site were harvested at 8, 24, 72 hours and 1, 3, 5, 8 weeks, and then fixed, decalcified, and sectioned frozenly to detect the expression of VEGF and its receptor at the fracture site by in situ hybridization and immunochemical assays.
Results： VEGF mRNA and VEGF expression was detected in many kinds of cells at the fracture site during 8 hours to 8 weeks after fracture. Fltl receptor of VEGF was found in the vascular endothelial cells at the fracture site during 8 hours to 8 weeks after fracture, and strong expression of flkl receptor was detected from 3 days to 3 weeks after fracture.
Conclusions： The expression of VEGF and fltl receptor appears during the whole course of fracture healing, especially from 1 to 3 weeks. Flkl receptor is highly expressed in a definite period after fracture. VEGF is proved to be involved in the vascular reconstruction and fracture healing.     

荷负电气溶胶治疗对大鼠烫伤创面白细胞介素-8、10表达的影响及意义

Objective To discuss the influence of aerosol bioelectricity on the expression of interleukin (IL) -8 and IL-10 in wound healing of burned rats. Methods The deep Ⅱ degree scalding models were established in Sprague Dawley (SD) rats. Rats were randomly divided into experimental group (n1 =20) and control group (n2 =20). The rats in experimental group were treated with aerosol bioelectricity.Samples were collected at the first to eleventh day post-scalding. Immunohistochemistry and image analysis methods were conducted to examine the expression of IL-8 and IL-10 in both experimental and control groups. Results The average wound healing time in experimental group was 7. 00 ± 1. 15 days, and that in control group was 9. 00 ± 1. 34 days. IL-8 and IL-10 were observed mainly in polylmorphonuclear and mononuclear cells in both experimental and control groups on the 1 st day. On the third day, fibroblasts abounded, IL-8 expression was increased evidently and reached a peak. The peak value (6. 73 ± 1. 36) in experimental group was lower significantly than that in control group ( 2. 85 ± 0. 72, P ＜ 0. 01). From the 5th to 11th day, IL-8 expression was declined rapidly. IL-10 was expressed in keratode cells and had the peak value in experimental group (1. 24 ±0. 15) and control group (5. 69 ± 1. 32) on the 3rd day. IL-10 expression was declined gradually from the 5th to 11th days. The expression level of IL-10 in experimental group was significantly higher than in control group from the 3rd day to 11th days post-scalding (P＜0. 01). On the 3rd day, both IL-8 and IL-10 in experimental and control groups were expressed abundantly , and there was negative relationship between them (r = - 0. 862, P ＜ 0. 01). Conclusion Aerosol bioelectricity can indicate active cells proliferation through down-regulating the expression of IL-8 and up-regulating the expression of IL-10, accelerating burned wound healing.     

荷负电气溶胶治疗对大鼠烫伤创面白细胞介素-8、10表达的影响及意义

Objective To discuss the influence of aerosol bioelectricity on the expression of interleukin (IL) -8 and IL-10 in wound healing of burned rats. Methods The deep Ⅱ degree scalding models were established in Sprague Dawley (SD) rats. Rats were randomly divided into experimental group (n1 =20) and control group (n2 =20). The rats in experimental group were treated with aerosol bioelectricity.Samples were collected at the first to eleventh day post-scalding. Immunohistochemistry and image analysis methods were conducted to examine the expression of IL-8 and IL-10 in both experimental and control groups. Results The average wound healing time in experimental group was 7. 00 ± 1. 15 days, and that in control group was 9. 00 ± 1. 34 days. IL-8 and IL-10 were observed mainly in polylmorphonuclear and mononuclear cells in both experimental and control groups on the 1 st day. On the third day, fibroblasts abounded, IL-8 expression was increased evidently and reached a peak. The peak value (6. 73 ± 1. 36) in experimental group was lower significantly than that in control group ( 2. 85 ± 0. 72, P ＜ 0. 01). From the 5th to 11th day, IL-8 expression was declined rapidly. IL-10 was expressed in keratode cells and had the peak value in experimental group (1. 24 ±0. 15) and control group (5. 69 ± 1. 32) on the 3rd day. IL-10 expression was declined gradually from the 5th to 11th days. The expression level of IL-10 in experimental group was significantly higher than in control group from the 3rd day to 11th days post-scalding (P＜0. 01). On the 3rd day, both IL-8 and IL-10 in experimental and control groups were expressed abundantly , and there was negative relationship between them (r = - 0. 862, P ＜ 0. 01). Conclusion Aerosol bioelectricity can indicate active cells proliferation through down-regulating the expression of IL-8 and up-regulating the expression of IL-10, accelerating burned wound healing.     

离体冻伤模型创面愈合过程中基质细胞衍生因子1促表皮干细胞迁移的研究

Objective To study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo. Methods A three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohisto-chemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 μL per wound) , SDF-1 group (treated with 100 ng/mL SDF-1, 50 μL per wound) , and AMD3100 group [treated with 100 ng/mL AMD3100 (50 μL per wound) for 30 minutes, and then SDF-1 50 μL was added per wound]. The redistribution of ESC around wound was observed. Results The expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin β1-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis. Conclusions SDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.     

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.     

mRNA and protein expression of transcription factor c—fos in burned rats and their effects on wound healing

Objective:To explore the expression of mRNA and its protein in burned rats and their effects of burn wound healing.Methods:A partial-thickness burn of 30% total body surface area was created on the back of 40 Wistar rats.In situ hybridization and immunohistochemical methods were used to exaluate the location and the amount of the c-fos mRNA and its protein in normal skin and the burned skin,respectively,at 3h,6h,1d,3d,7d and 14d after burn.Results:Under a light microscope,both the expression of c-for mRNA and its protein could be found in the normal skin,but their induction levels were much higher in the burned skin.The level of for protein expression reached peak at 3h after burn while that of c-for mRNA reached peak at 6h after burn.Conclusions:The expression of c-fos can be induced by burns.And the peak level expression of c-for mRNA comes later than that of c-fos protein.It indicates that the action of fos protein is induced by post-translational modification of pre-existing fos molecules.     

离体冻伤模型创面愈合过程中基质细胞衍生因子1促表皮干细胞迁移的研究

Objective To study the promotion effect of stromal cell-derived factor 1 (SDF-1) on the migration of epidermal stem cells (ESC) in the healing process of frostbite-wound model ex vivo. Methods A three-dimensional model of full-thickness frostbite of skin was constructed (with slot-like wound) out of skin equivalent. The expression of SDF-1 in wound stroma was observed with immunohisto-chemistry staining on post injury days (PID) 3 and 7. The model frostbite wounds were divided into control group (treated with PBS 50 μL per wound) , SDF-1 group (treated with 100 ng/mL SDF-1, 50 μL per wound) , and AMD3100 group [treated with 100 ng/mL AMD3100 (50 μL per wound) for 30 minutes, and then SDF-1 50 μL was added per wound]. The redistribution of ESC around wound was observed. Results The expression of SDF-1 in wound stroma increased gradually on PID 3 and 7. Compared with those in control and AMD3100 groups, there were more ESC and epithelial cell layers, and more integrin β1-positive cells appeared at the basal layer of wound in SDF-1 group, and some of the positive cells migrated upward to epidermis. Conclusions SDF-1 contributes to wound repair through promoting ESC to migrate toward and gather around wound edge. This may be one of the mechanisms of ESC participating in wound repair.     

Early period of fracture healing in ovariectomized rats

Objective. To evaluate the effect of osteoporosis on fracture healing through observing the hlstomorphological changes, bone mineral density of callus and expression and distribution of transforming growth factor beta 1 (TGF-β1 ), basic fibroblast growth factor (bFGF)and bone morphogenetic protein.2 (BMP-2) in ovariectomized rats. Methods. Sixty female Sprague-Dawley rats ( aged 12 weeks and weighing 235 g on average ) were randomly divided into an ovariectomized (OVX) group (n =30) anda sham-operated (SO) group ( n = 30). Ovariectomy was performed in the OVX rats and same incision was made in the SO rats. Three months later, fracture of femoral shaft was made on all the rats. Then they were killed at different time points. Callus formation was observed with histological and imethods. Results: A reduction in callus and bone mineral density in the healing femur and a decrease of osteoblasts expressing TGF-β1 near the bone trabecula were observed in the OVX rats 3-4 weeks after fracture.Histomorphological analysis revealed a higher content of soft callus in the OVX rats than that in the SO rats.Immunohistochemistry results showed that no remarkable difference in expression and distribution of BMP-2 and bFGF between the OVX and SO groups was found. Conclusions: Osteoporosis influences the quantity and quality of callus during the early period of fracture healing. The effect of osteoporosis on fracture healing has no relationship with the expression of BMP-2 or bFGF. The decreased expression of TGF-I31 in osteoblasts may cause a decrease in quality of facture healing after osteoporosis.     

Chitosan green tea polyphenol complex as a released control compound for wound healing

Objective： In recent years, oxidative stress has been implicated in a variety of degenerative process and diseases, including acute and chronic inflammatory conditions such as wound healing. Green tea polyphenols have shown anti-oxidant property. The present study discussed the application of chitosan green tea polyphenol complex on the wound healing. Methods： The wound healing effect ofchitosan green tea polyphenol complex was studied in tenweek-old healthy male Sherman rats weighing 150-180 g by two wound models. The rats were randomly chosen and divided into four groups （n=5）, administered with distilled water in Group A as control group, epigallocatechin-3-gallate （EGCG） in Group B, chitosan-EGCG complex in Group C and chitosan-green tea polyphenols complex in Group D, respectively. In rats＇incision wound model, two straight paravertebral incisions were made and skin tensile strength was measured using continuous water flow technology on the 10th day. In rats＇excision wound model, wound contraction and period of epithelization were measured. The polyphenols release from the complex was continuously monitored by an elution technique in aqueous solution at different pH values （pH=4, 5, 6, 7）. Results： The treatment groups showed significantly enhanced the breaking strength in incision wound （328±14.5） g and 021±18.5） g compared with control （264±16.7） g. In the excision wound model, the wound contraction percentage in treatment groups was relatively increased during the recovery period. Respectively, the percentage of wound contraction ranged from 47.60%±2.15% on day 4 to 107.98% ± 1.26% on day 16 compared with control group （8.46%±5.42% to 59.80%±4.47%）. The complex demonstrated a gradual increase in the release rate from the initial stage and slow increase at different pH values. The release rate approximated 0.6-0.7 in the complex and remained stable 6 hours after injury, which may be the end of the release process. Conclusions： In our study, chitosan polyphenol complex has enhanced the healing of incision wounds by increasing the breaking strength of the wounds. In excision wound model, the complex hastens the period of epithelialization. The study on the optimal release of complex among various pH values could be applied in the wound test, which can lead to a gradually active substance （polyphenols） release and efficient coverage of epithelial layers found in the healing of incision and excision wound.     

Impact of mechanical stress and tension-stress on angiogenesis in wound healing

Angiogenesis plays a fundamental role in the development of the embryonic vascular tree as well as in several normal and pathologic conditions during postnatal life. Blood supply, established by neovascularization. is imperative for histogenesis during wound healing as well as the limb lengthening applied extensively in the treatment of skeletal trauma sequalae. But little attention has been paid to this area. This review aims to summarize angiogenesis regulation, the process of angiogenesis in wound healing and angiogenesis under mechanical stress, particularly in association with the tension-stress principle.     

Effect of recombinant human basic fibroblast growth factor on angiogenesis during mandible fracture healing in rabbits

Objective: To investigate the effect of recombinant human basic fibroblast growth factor ( rhbFGF) on angiogenesis during mandible fracture healing in rabbit.Methods: Fifty adult white rabbits were used for animal model and randomly divided into a control group (25 rabbits) and an experimental group (25 rabbits). The membranous complex of rhbFGF and bovine type I collagen was prepared and implanted into the rabbit mandible fracture site under periosteum. The animals were sacrificed on 7, 14, 28, 56 and 84 days respectively after operation and the whole mandibles were harvested. The expression of factor Vm related antigen (F8-RA) in callus was examined with immimohistochemical staining.Results: The amounts of microvascular formation in calluses in the rhbFGF-treating group on days 7, 14, 28 and 56 were more than those of the control group (P < 0.01).Conclusions: The results indicated that rhbFGF could stimulate microvascular formation during mandible fracture healing in rabbits.