Farnesyl Pyrophosphate Inhibits Epithelialization and Wound Healing through the Glucocorticoid Receptor

Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.     

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.     

Cortisol synthesis in epidermis is induced by IL-1 and tissue injury

Glucocorticoids (GCs) are known inhibitors of wound healing. In this study we report the novel finding that both keratinocytes in vitro and epidermis in vivo synthesize cortisol and how this synthesis regulates wound healing. We show that epidermis expresses enzymes essential for cortisol synthesis, including steroid 11 β-hydroxylase (CYP11B1), and an enzyme that controls negative feedback mechanism, 11β-hydroxysteroid dehydrogenase 2 (11βHSD2). We also found that cortisol synthesis in keratinocytes and skin can be stimulated by ACTH and inhibited by metyrapone (CYP11B1 enzyme inhibitor). Interestingly, IL-1β, the first epidermal signal of tissue injury, induces the expression of CYP11B1 and increases cortisol production by keratinocytes. Additionally, we found induction of CYP11B1 increased production of cortisol and activation of GR pathway during wound healing ex vivo and in vivo using human and porcine wound models, respectively. Conversely, inhibition of cortisol synthesis during wound healing increases IL-1β production, suggesting that cortisol synthesis in epidermis may serve as a local negative feedback to proinflammatory cytokines. Local GCs synthesis, therefore, may provide control of the initial proinflammatory response, preventing excessive inflammation upon tissue injury. Inhibition of GC synthesis accelerated wound closure in vivo, providing the evidence that modulation of cortisol synthesis in epidermis may be an important regulatory mechanism during wound healing.     

Targeting O-Glycosyltransferase (OGT) to Promote Healing of Diabetic Skin Wounds

Non-healing wounds are a significant source of morbidity. This is particularly true for diabetic patients, who tend to develop chronic skin wounds. O-GlcNAc modification of serine and threonine residues is a common regulatory post-translational modification analogous to protein phosphorylation; increased intracellular protein O-GlcNAc modification has been observed in diabetic and hyperglycemic states. Two intracellular enzymes, UDP-N-acetylglucosamine-polypeptide β-N-acetylglucosaminyl transferase (OGT) and O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (OGA), mediate addition and removal, respectively, of N-acetylglucosamine (GlcNAc) from intracellular protein substrates. Alterations in O-GlcNAc modification of intracellular proteins is linked to diabetes, and the increased levels of protein O-GlcNAc modification observed in diabetic tissues may in part explain some of the observed underlying pathophysiology that contributes to delayed wound healing. We have previously shown that increasing protein O-GlcNAc modification by overexpression of OGT in murine keratinocytes results in elevated protein O-GlcNAc modification and a hyperadhesive phenotype. This study was undertaken to explore the hypothesis that increased O-GlcNAc modification of cellular proteins in diabetic skin could contribute to the delayed wound healing observed in patients with diabetic skin ulcers. In the present study, we show that human keratinocytes cultured under hyperglycemic conditions display increased levels of O-GlcNAc modification as well as a delay in the rate of wound closure in vitro. We further show that specific knockdown of OGT by RNA interference (RNAi) reverses this effect, thereby opening up the opportunity for OGT-targeted therapies to promote wound healing in diabetic patients.     

组织工程复合皮肤应用于皮肤缺损创面的临床疗效观察

目的:临床观察组织工程复合皮肤对烧伤整形后需植皮患者的供皮区缺损创面的有效性及安全性。方法:试验选取不同临床中心烧伤整形后需植皮患者,在供皮区部分创面作为试验区应用组织工程复合皮肤覆盖,邻近创面采用盐水纱布替代作为对照区,应用后按常规方法包扎固定。临床试验时间为6个月,治疗期间观察统计患者的创面反应,愈合时间及愈合情况;对组织工程复合皮肤改善创面愈合质量及安全性进行临床评估。根据创面试验组和对照组的创面愈合时间,应用SPSS统计软件对数据进行方差齐性检验,根据检验结果分别进行独立样本t或t’检验。结果:试验共收集有效病例19例。临床观察显示应用后试验区创面无明显免疫排斥及炎性反应,患者自述疼痛明显减轻,试验区创面愈合时间与对照区相比缩短8d,统计学分析有显著性差异。愈后随访部分患者试验区愈合质量好于对照区,应用后患者疼痛、瘢痕形成等不良反应明显减少。结论:结果表明组织工程复合皮肤作为活型皮肤替代物用于医源性皮肤缺损的修复,这为促进供皮区的创面愈合提供了切实可行的方法。     

300 mT恒磁场对于糖尿病大鼠创伤愈合的影响

目的:研究300mT恒磁作用对于糖尿病大鼠皮肤创伤愈合的修复效果,旨在为其系统的临床应用提供理论依据.方法:8周龄雌性SD大鼠,体重220±20g,行腹腔注射50mg/kg链脲做菌素,3天后空腹血糖高于16.7 mmol/L的大鼠用于实验.32只糖尿大鼠病和16只正常大鼠的背部构建直径2cm的圆形创口,将动物随机分为对照组、糖尿病组、糖尿病恒磁场作用组,每组16只,术后一天采用无菌纱布将创口进行包裹,磁场作用组的纱布表面粘附有300 mT中心场强的钕铁硼恒磁片.分别于第6、13、20天每组处死4只大鼠,对其进行创伤愈合率和创伤表皮组织生物力学实验评估.结果:相比于糖尿病组,糖尿病恒磁场治疗大鼠的伤口愈合速度在第6、13、20天显著提高(P＜0.05),伤口组织的抗张强度亦显著提升(P＜0.05),其愈合速度接近于正常状态下的大鼠.结论:300mT恒磁场作用对于促进糖尿病大鼠皮肤创伤的愈合速率具有积极的治疗作用,恒磁场治疗凭借经济、简单、方便等特点,具有广阔的临床应用前景.     

全蝎软膏对大鼠糖尿病皮肤溃疡创面愈合的效果评价

目的:探讨全蝎软膏治疗糖尿病皮肤创面愈合的作用及其机制。方法:采用SPF级SD大鼠构建糖尿病皮肤溃疡模型,将其随机平均分为对照组、模型组、全蝎软膏组和湿润烧伤膏组。造模后,分别在创面处涂抹全蝎软膏或湿润烧伤膏处理,空白组和模型组用PBS处理,观察和比较各组创面的愈合情况,并收集用药后3 d、7 d、10 d和14 d的创面肉芽组织进行HE染色,通过ELISA及荧光定量PCR检测肉芽组织内碱性成纤维细胞生长因子(bFGF)、血管性假血友病因子(v WF)、肿瘤坏死因子α(TNF-α)与Smad 4基因的表达。结果:成功构建大鼠糖尿病皮肤溃疡模型,成模率为96.15%。创伤后第3 d、7 d、10 d和14 d,各治疗组创面愈合率均显著高于模型组(P0.05),给药14 d时,全蝎软膏组创面愈合率已接近对照组水平。HE染色结果显示与模型组相比,全蝎软膏组中肉芽组织生长较快、毛细血管数量增多且组织纤维化程度较低。ELISA和荧光定量PCR检测结果显示与模型组和湿润烧伤膏组相比,全蝎软膏组肉芽组织内bFGF、v WF含量上调更显著(P0.05),而TNF-α含量与Smad 4 m RNA显著降低(P0.05)。结论:全蝎软膏可促进糖尿病皮肤溃疡创面肉芽组织的生长,可能与其抑制炎症反应并改善血管功能有关。     

低强度激光照射对糖尿病大鼠创伤愈合的治疗效果研究

目的:低强度激光的生物效应已经得到广大科研工作者和临床医生的广泛关注,诸多研究证明低强度激光能够促进创伤组织的修复,但是其对于糖尿病创伤的组织愈合效果目前仍鲜有报道.本文旨在系统探索650 nm波长的低强度激光照射对糖尿病大鼠皮肤创伤愈合的作用效果.方法:3月龄雄性SD大鼠,腹腔注射50 mg/kg的链脲做菌素,3天后检测空腹血糖值,高于16.7mmol/L的大鼠用于实验.于48只大鼠(32只糖尿病+16只正常)的背部构建直径2 cm的圆形创伤切口,将大鼠随机分为对照组、糖尿病组、糖尿病激光治疗组,每组16只,激光组大鼠伤口每天暴露于650 nm激光下2h.每组分别于第5、12、19天处死4只大鼠,对其进行创伤愈合率和创伤表皮组织生物力学实验评估.结果:相比于对照组,糖尿病大鼠的伤口愈合速度在第5、12、19天均显著降低(P＜0.05),伤口完全愈合时间显著增加(P＜0.05),伤口抗张强度显著下降(P＜0.05).低强度激光刺激显著提高了第5、12、19天大鼠伤口的愈合速度(P＜0.05),显著降低了伤口的愈合时间(P＜0.05),显著提高了伤口抗张强度(P＜0.05).结论:650nm低强度激光能够显著加速糖尿病大鼠创伤的延迟愈合,使其愈合速度接近于正常状态的组织,该研究揭示了650nm波长下的低强度激光治疗具有良好的用于促进糖尿病患者伤口愈合临床应用前景.     

Mathematical Modelling of Aerosolised Skin Grafts Incorporating Keratinocyte Clonal Subtypes

Severe burns can be very traumatic for the patient, and while burns caused by industrial or domestic accidents are common, there are also increasing numbers of burns associated with terrorism. A novel technique to assist in the healing process is to spray skin cells, keratinocytes, that are cultured from the patient’s own tissue, directly onto the burn site. This process involves taking some undamaged skin from the patient, allowing the skin cells to proliferate rapidly in the laboratory over a period of 5–10 days, harvesting and separating the cells and then spraying them onto the burn. This paper deals with keratinocytes that have been cultured in vitro for a short period of time (early passage cultured cells). The spraying process has yet to be optimised with respect to the seeding density required for fastest re-epithelisation and thus there is a need for this process to be modelled. In this paper, we review some of the skin biology and develop a mathematical model of the growth patterns of cell colonies after they have been applied using a aerosolised technique. The model allows us to predict coverage over time and can be used as a decision support tool for clinicians. PACS: 92B05     

恒古骨伤愈合剂联合塞来昔布与针灸对寒湿型腰痛的治疗效果及运动功能的影响

摘要 目的：探讨恒古骨伤愈合剂联合塞来昔布与针灸对寒湿型腰痛的治疗效果及运动功能的影响。方法：选取牡丹江医学院附属第二医院2022年7月到2023年7月收治的80例寒湿型腰痛患者,分为对照组（n=30）、针灸组（n=25）和联合组（n=25）。对照组常规口服塞来昔布,针灸组采取塞来昔布+针灸治疗,联合组应用塞来昔布+针灸+恒古骨伤愈合剂治疗,对比三组患者临床疗效,治疗前后的寒湿症候积分与症状体征积分,疼痛程度,并应用直腿抬高实验、Osweatry功能障碍指数（ODI）、日本骨科协会评估（JOA）来评价两组患者治疗前后的运动功能。结果：治疗后,联合组总有效率为96%,针灸组为72%,对照组为53.33%。联合组高于针灸组和对照组（P<0.05）;治疗后三组患者症候积分与症状体征积分均降低,且联合组低于针灸组和对照组（P＜0.05）;治疗后三组患者疼痛情绪评分（PRI-S)、疼痛感觉评分（PRI-A）、疼痛总分（PRI-T)、现实疼痛状况（PPI）以及视觉模拟量表（VAS）评分均降低,且联合组低于针灸组和对照组（P<0.05）;治疗后三组运动功能指标有差异,与针灸组和对照组相比,联合组直腿抬高角度、JOA评分升高ODI指数降低（P＜0.05）。结论：对寒湿型腰痛患者采用塞来昔布+针灸+恒古骨伤愈合剂治疗能够减轻患者临床症状,疗效显著,且能够改善患者腰腿疼痛情况,提升腰腿功能与运动功能。     

In vitro reconstruction of full thickness human skin on a composite collagen material

Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support.