Serum lipidomics-based study of electroacupuncture for skin wound repair in rats

Lipid metabolism plays an important role in the repair of skin wounds. Studies have shown that acupuncture is very effective in skin wound repair. However, there is little knowledge about the mechanism of electroacupuncture. Thirty-six SD rats were divided into three groups: sham-operated group, model group and electroacupuncture group, with six rats in each group. After the intervention, orbital venous blood was collected for lipid metabolomics analysis, wound perfusion was detected and finally the effect of electroacupuncture on skin wound repair was comprehensively evaluated by combining wound healing rate and histology. Lipid metabolomics analysis revealed 11 differential metabolites in the model versus sham-operated group. There were 115 differential metabolites in the model versus electro-acupuncture group. 117 differential metabolites in the electro-acupuncture versus sham-operated group. There were two differential metabolites common to all three groups. Mainly cholesteryl esters and sphingolipids were elevated after electroacupuncture and triglycerides were largely decreased after electroacupuncture. The electroacupuncture group recovered faster than the model group in terms of blood perfusion and wound healing (p < 0.05). Electroacupuncture may promote rat skin wound repair by improving lipid metabolism and improving local perfusion.     

Clock gene Per2 modulates epidermal tissue repair in vivo

Wound healing can be influenced by genes that control the circadian cycle, including Per2 and BMAL1, which coordinate the functions of several organs, including the skin. The aim of the study was to evaluate the role of PER2 during experimental skin wound healing. Two groups (control and Per2-KO), consisting of 14 male mice each, were anesthetized by inhalation, and two 6 mm wounds were created on their dorsal skin using a punch biopsy. A silicone ring was sutured around the wound perimeter to restrict contraction. The wound healing process was clinically measured daily (closure index) until complete wound repair. On Day 6, histomorphometric analysis was performed using the length and thickness of the epithelial migration tongue, in addition to counting vessels underlying the lesion by immunofluorescence assay and maturation of collagen fibers through picrosirius staining. Bromodeoxyuridine (BrdU) incorporation and quantification were performed using the subcutaneous injection technique 2 h before euthanasia and through immunohistochemical analysis of the proliferative index. In addition, the qualitative analysis of myofibroblasts and periostin distribution in connective tissue was performed by immunofluorescence. Statistically significant differences were observed in the healing time between the experimental groups (means: 15.5 days for control mice and 13.5 days for Per2-KO; p = 0.001). The accelerated healing observed in the Per2-KO group (p < 0.05) was accompanied by statistical differences in wound diameter and length of the migrating epithelial tongue (p = 0.01) compared to the control group. Regarding BrdU immunoreactivity, higher expression was observed in the intact epithelium of Per2-KO animals (p = 0.01), and this difference compared to control was also present, to a lesser extent, at the wound site (p = 0.03). Immunofluorescence in the connective tissue underlying the wound showed a higher angiogenic potential in the Per2-KO group in the intact tissue area and the wound region (p < 0.01), where increased expression of myofibroblasts was also observed. Qualitative analysis revealed the distribution of periostin protein and collagen fibers in the connective tissue underlying the wound, with greater organization and maturation during the analyzed period. Our research showed that the absence of the Per2 gene positively impacts the healing time of the skin in vivo. This acceleration depends on the increase of epithelial proliferative and angiogenic capacity of cells carrying the Per2 deletion.     

Cellular and physiological upregulation of inducible nitric oxide synthase,arginase, and inducible cyclooxygenase in wound healing

Wound repair is regulated by overlapping cellular, physiological and biochemical events. Prostaglandins and nitric oxide have been a focus for inflammation research particularly since the discovery of their inducible isoforms nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Study of the cellular expression of iNOS and COX-2 and arginase which competes with iNOS for its substrate, in an in vivo model of wound healing could reveal important roles for these enzymes in the physiological progression of wound repair. Adult male rats received full thickness dermal wounds which were harvested at different times. Protein levels and activities of the enzymes were assessed by western blot and biochemical assays respectively. The cellular distribution and the colocalization were assessed by immunostaining. The protein levels and activities of iNOS, arginase, and COX-2 increased only during the inflammatory phase of wound. Immunocytochemistry showed that the three enzymes were coexpressed and the main cellular source was inflammatory cells mainly macrophages. iNOS was induced at the wound site and was the earliest to increase significantly (p < 0.05) for only up to 3 days postwounding. However, arginase and COX-2 significant ( p < 0.05) upregulation started at a later time points and continued for up to 14 days postwounding. Therefore iNOS, compared with arginase and COX-2, showed a temporal difference in expression during wound healing which could be explained by their products being required at different stages of the healing process. The coordinated expression of the three enzymes at different time points could account for the physiological progression of the healing process.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.     

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD⁺ precursor supplement, rescued the imbalanced NAD⁺/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.     

Antioxidant enzyme activities and lipid peroxidation induced by eicosapentaenoic acid (EPA) in PC12 cells

Eicosapentaenoic acid (EPA) is one of the major dietary polyunsaturated fatty acids and induces apoptosis in several cancer cells. In this study, the EPA induced lipid peroxidation and response of antioxidative enzymes have been investigated in rat pheochromocytoma PC12 cells to elucidate the mechanisms of apoptosis induced by the polyunsaturated fatty acid EPA. We have analyzed superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities and glutathione (GSH) contents in PC12 cells after exposure to different concentrations of EPA. Lipid peroxidation was shown to increase in the presence of EPA as an indication of the oxidative damage. Lipid peroxidation was enhanced by EPA in a dose-dependent manner, and the loss of cell viability was partially reversed by vitamin E. In the case of antioxidant enzyme activities, SOD and GPX activities and GSH contents increased significantly at 50 μmol/L EPA and were respectively 2.41-fold (p < 0.01), 3.49-fold (p < 0.05), and 1.43-fold (p < 0.05) higher than controls. The CAT activity at 10 μmol/L had the highest value and was increased by 25.83% (p < 0.05) compared to control. The results suggest that in PC12 cells the mechanism of apoptosis induced by EPA may be partly due to lipid peroxidation.     

Effects of High Zinc Levels on the Lipid Synthesis in Rat Hepatocytes

Zinc deficiency impairs the hepatic lipid metabolism. Previous studies were focused on the negative effects of zinc deficiency on the hepatic lipid metabolism. A few studies investigated the effects of high zinc levels on the lipid metabolism in hepatocytes. In this study, rat hepatocytes were cultured and treated with different and high concentrations of zinc to investigate the effects of high zinc levels on the lipid synthesis in hepatocytes in vitro. The levels of hepatocytes functional markers, including alkaline phosphatase, lactate dehydrogenase, and albumin, were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). The mRNA and protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) were significantly higher in the zinc treatment groups than in the control group (p?<?0.05, p?<?0.01). Furthermore, the mRNA expression levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) were significantly higher in the medium- and high-dose zinc treatment groups than in the control group (p?<?0.01). The mRNA levels of stearoyl-CoA desaturase-1 (SCD-1) were significantly higher in the high-dose group (p?<?0.01). These results indicate that high levels of zinc increase hepatocytes activity and SREBP-1c expression, which upregulate the expression of ACC1, FAS, and SCD-1, thereby improving the lipid metabolism in the hepatocytes.     

Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1

Maggot therapy is a simple and highly successful method for healing of infected and necrotic wounds. The increasing evidences indicate that Maggot excretions/secretions (ES) plays important roles in the wounds healing process. But the precise molecular mechanisms remain undefined. Herein, we investigated if ES induced cell migration during wound healing process using microvascular endothelial cells (HMEC-1) as model, and this effect was associated with the activation of AKT1 and ERK1/2. Wound healing and transwell migration assays were performed to study the effects of ES on HMEC-1 cell migration. Our data showed that ES significantly induced HMEC-1 cell migration in both wound healing and transwell assays, and time-dependently (P < 0.05) activated AKT1, but not ERK1/2. Moreover LY294002 (a PI3K inhibitor) partially attenuated (P < 0.05) ES-induced cell migration in wound healing assay while completely inhibited (P < 0.05) ES-induced AKT1 activation. These findings demonstrate that ES directly induces HMEC-1 cell migration and this event is partially mediated by the activation of AKT1.     

Stereological and gene expression examinations on the combined effects of photobiomodulation and curcumin on wound healing in type one diabetic rats

We examined the effects of photobiomodulation (PBM) independently and combined with curcumin on stereological parameters and basic fibroblast growth factor (bFGF), hypoxia-inducible factor-1α (HIF-1α), and stromal cell-derived factor-1α (SDF-1α) gene expressions in an excisional wound model of rats with type one diabetes mellitus (T1DM). T1DM was induced by an injection of streptozotocin (STZ) in each of the 90 male Wistar rats. One round excision was generated in the skin on the back of each of the 108 rats. The rats were divided into six groups (n = 18 per group): control (diabetic), untreated group; vehicle (diabetic) group, which received sesame oil; PBM (diabetic) group; curcumin (diabetic) group; PBM + curcumin (diabetic) group; and a healthy control group. On days 4, 7, and 15, we conducted both stereological and quantitative real-time PCR (qRT-PCR) analyses. The PBM and PBM + curcumin groups had significantly better inflammatory response modulation in terms of macrophages (P < .01), neutrophils (P < .001), and increased fibroblast values compared with the other groups at day 4 (P < .001), day 7 (P < .01), and day 15 (P < .001). PBM treatment resulted in increased bFGF gene expression on days 4 (P < .001) and 7 (P < .001), and SDF-1α gene expression on day 4 (P < .001). The curcumin group had increased bFGF (P < .001) expression on day 4. Both the PBM and PBM + curcumin groups significantly increased wound healing by modulation of the inflammatory response, and increased fibroblast values and angiogenesis. The PBM group increased bFGF and SDF-1α according to stereological and gene expression analyses compared with the other groups. The PBM and PBM + curcumin groups significantly increased the skin injury repair process to more rapidly reach the proliferation phase of the wound healing in T1DM rats.     

Rutin improves the antioxidant status in streptozotocin-induced diabetic rat tissues

Rutin, a polyphenolic flavonoid, was investigated for its antioxidant potential in streptozotocin (STZ)-induced diabetic rats. Rats were rendered diabetic by a single intraperitoneal injection of streptozotocin (50 mg/kg). The levels of fasting plasma glucose and insulin were estimated. Lipid peroxidative products and antioxidants were estimated in liver, kidney and brain. Histopathological studies were carried out in these tissues. A significant (p < 0.05) increase in the levels of fasting plasma glucose, lipid peroxidative products (thiobarbituric acid reactive substances [TBARS] and lipid hydroperoxides [HP]) and a significant (p < 0.05) decrease in plasma insulin, enzymic antioxidants (superoxide dismutase [SOD], catalase, glutathione peroxidase [GPx] and glutathione reductase [GRx]) and nonenzymic antioxidants (reduced glutathione [GSH], vitamin C and E) in diabetic liver, kidney and brain were observed. Oral administration of rutin (100 mg/kg) for a period of 45 days significantly (p < 0.05) decreased fasting plasma glucose, increased insulin levels and improved the antioxidant status of diabetic rats by decreasing lipid peroxidative products and increasing enzymic and nonenzymic antioxidants. Normal rats treated with rutin (100 mg/kg) showed no significant (p < 0.05) effect on any of the parameters studied. Histopathological studies of the liver, kidney and brain showed the protective role of rutin. Thus, our study clearly shows that rutin has antioxidant effect in STZ-induced experimental diabetes.     

Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split‐skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p‐value <0.05) in chronic wounds. Most were involved in biological processes including inflammation, angiogenesis, and cell mortality. Increased expression of the inflammatory response stimulating S100 proteins, predominantly S100A8 and S100A9 (almost tenfold), was identified. Matrix metalloproteinases (MMPs) MMP1, MMP2, and MMP8 were identified to be elevated in chronic wounds with significant impact on collagen degradation and tissue destruction. Further, proteins with antiangiogenic properties were found at higher expression levels in chronic wounds. Reduced angiogenesis leads to drastic shortage in nutrition supply and causes increased cell death, demonstrated by Annexin A5 exclusively found in chronic wound exudates. However, excessive nucleic and cytosolic material infers cell death occurring not only by apoptosis but also by necrosis. In conclusion, mass spectrometric investigation of exudates from chronic wounds demonstrated dramatic impairment in wound repair with excessive inflammation, antiangiogenic environment, and accelerated cell death.     

Rapid preparation of a noncultured skin cell suspension that promotes wound healing

Autologous skin cell suspensions have been used for wound healing in patients with burns and against normal pigmentation in vitiligo. To separate cells and the extracellular matrix from skin tissue, most researchers use enzymatic digestion. Therefore, this process is difficult to perform during a routine surgical procedure. We aimed to prepare a suspension of noncultured autologous skin cells (NCSCs) using a tissue homogenizer as a new method instead of harsh biochemical reagents. The potential clinical applicability of NCSCs was analyzed using a nude-rat model of burn healing. After optimization of the homogenizer settings, cell viability ranged from 52 to 89%. Scanning electron microscopy showed evidence of keratinocyte-like cell morphology, and several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the NCSCs. The rat model revealed that NCSCs accelerated skin regeneration. NCSCs could be generated using a tissue homogenizer for enhancement of wound healing in vivo. In the NCSC group of wounds, on day 7 of epithelialization, granulation was observed, whereas on day 14, there was a significant increase in skin adnexa regeneration as compared to the control group (PBS treatment; p < 0.05). This study suggests that the proposed process is rapid and does not require the use of biochemical agents. Thus, we recommend a combination of surgical treatment with the new therapy for a burn as an effective method.     

高乳清蛋白的肠内营养对大鼠伤口愈合的影响

探讨高乳清蛋白的肠内营养对大鼠创伤皮肤的促愈合作用.将36只SD大鼠随机分为3组,每组12只,即高乳清蛋白肠内营养组(A)、普通肠内营养组(B)、空白对照组(C),建立大鼠皮肤创伤模型后进行肠内营养支持,肠内营养用量为每天731.5kJ·kg-1,以灌胃的方式提供,分别于术后第7、14天测定血清蛋白、血红蛋白含量并计算创面愈合率.结果显示,术后第7、14天A组的血清蛋白、血红蛋白及伤口皮肤平均愈合率显著高于B、C组,差异具有统计学意义(p＜0.05).表明高乳清蛋白肠内营养具有较好的促进伤口愈合的作用.     

Studies of five microelement contents in human serum, hair, and fingernails correlated with aged hypertension and coronary heart disease

Using atomic absorption spectrometry (AAS), five microelements in human serum, hair, and fingernails of aged hypertension, coronary heart disease (diseased group) and aged health control (healthy group) were detected. Results of the t-test are as follows: The iron, zinc, and cadmium contents and Zn/Cu (mol/mol) ratio of the diseased group were significantly higher than that of the healthy group in serum (p<0.01, p<0.05, p<0.01, and p<0.05, respectively); the chromium contents in the serum, hair, and fingernails (p<0.05, p<0.01, and p<0.05, respectively); the iron and zinc contents in the hair and fingernails (p<0.01, p<0.001, p<0.05, and p<0.01 respectively) and Zn/Cu ratio in the hair (p<0.01) of the diseased group were significantly lower than that of the healthy group. Pathogenic factors of cardiovascular disease have three probabilities: (1) The iron contents of the diseased group were higher than that of the healthy group in serum and lower in hair and fingernails (i.e., abnormal iron metabolism); (2) the zinc contents and the Zn/Cu ratio of the diseased group were higher than that of the healthy group in the serum and the zinc contents in the hair and fingernails and the Zn/Cu ratio in the hair of the diseased group were significantly lower than that of the healthy group; (3) the chromium contents of the diseased group were significantly lower than that of the healthy group in the serum, hair, and fingernails.     

Effect of copper sulfate on experimental atherosclerosis

Serum copper concentration increases significantly (p<0.01) in rats with experimental atherosclerosis compared to a control group. The serum zinc, the zinc, and copper concentration in abdominal aorta and in liver decreases significantly (p<0.05) compared to the control group. Administration of copper sulfate for 100 d in these animals induces a significant increase of serum copper (p<0.01), decrease of serum cholesterol (p<0.05) and increase of liver copper concentration as compared with the group fed only a high cholesterol diet. In the aorta of these animals the copper concentration increases and edema and lipid infiltration are considerably less than in the group of animals fed only a high lipid diet.     

Effects of diode laser therapy on the acellular dermal matrix

Acellular dermal matrix (ADM) was subcutaneously implanted into calvarian skin of male Wistar rats (n = 40). Low-level laser (λ 685 nm, 4 J/cm²) was locally applied in experimental group (n = 20) above the skin flap. Grafts were harvested at 1, 3, 7 and 14 days after surgery and underwent histological analyses. In treated animals, the extent of edema and the number of inflammatory cells were reduced (P < 0.05). The amount of collagen in graft treated with low-level laser were significantly higher than those of controls (P < 0.05) and were statistically more prominent on the 14th day after surgery. The mean count of fibroblasts was significantly higher in the low-laser therapy group within the 3rd day, showing a marked influx of fibroblasts into area. In conclusion, wound healing of the ADM appear to be positively affected by laser therapy.     

局部应用PTD-SOD、SOD对小鼠皮肤创伤的抗氧化应激损伤保护效果及其比较

目的探讨局部应用PTD-SOD、SOD对小鼠皮肤创伤的抗氧化应激损伤保护效果及其差异。方法制备机械性创伤小鼠模型和不同浓度的PTD-SOD(1000、3000、6000 U)及SOD(1000、3000、6000 U)溶液,分别用上述溶液进行治疗,同时设立模型对照组和生理盐水对照组,各组均连续治疗13 d。观察各组创伤愈合情况,记录创伤愈合率和愈合天数;于创伤后第14天取各组小鼠创伤愈合部位皮肤,一部分制成10%组织匀浆液用于检测抗氧化酶(SOD、CAT、GSH-Px)活性及丙二醛(MDA)、羟脯氨酸(Hyp)含量,一部分制成病理组织切片用于皮肤组织学观察。结果①与模型对照组相比,PTD-SOD各组或SOD各组的抗氧化酶活性和Hyp含量显著(P0.05)或极显著(P0.01)升高,MDA含量显著(P0.05)或极显著(P0.01)降低,能显著(P0.05)或极显著(P0.01)提高创伤愈合率、缩短创伤愈合时间;与生理盐水对照组相比,结果类似。②在同等剂量下,从促创伤愈合时间、抗氧化酶活性、MDA含量、Hyp含量等方面比较,PTD-SOD组明显(P0.05)或极明显(P0.01)优于SOD组。③适当剂量的PTD-SOD促创伤愈合效果优于高剂量PTD-SOD的促创伤愈合效果。结论 PTD-SOD或SOD在皮肤创伤治疗中具有良好的抗氧化应激损伤效果,这种保护效果在创伤愈合的早期最显著;同等剂量下,PTD-SOD在促创伤愈合的效果上明显优于SOD。