EGCG and wound healing -
To explore the impact of EGCG-induced keratinocyte autophagy on fibroblasts, the culture supernatant of keratinocytes from different treatment groups was added to the fibroblasts and incubated for 48 h. However, treatment with Compound C significantly reversed the activating effects of EGCG on fibroblasts Fig.
Together, these results showed that EGCG promoted fibroblast activation by enhancing keratinocyte autophagy. Autophagy induction of EGCG on keratinocytes promoted fibroblast activation. To explore the effect of EGCG on DCU, wounds on the back of DM rats were intervened with EGCG and Compound C, and the wound healing process was monitored on days 0— In the Control group, the wound area was gradually decreased with time and was fully epithelialized by day By contrast, the wound in the DM group healed slowly and was not completely healed by day Notably, the use of Compound C significantly reversed the beneficial effect of EGCG on wound healing Fig.
HE staining of the wounds on day 14 showed that the granulation tissue was reduced, and the re-epithelialization was weakened, accompanied by many inflammatory infiltrates in the DM group compared with the Control group. After EGCG intervention, inflammatory infiltration decreased, granulation tissue increased, and re-epithelialization enhanced.
Unfortunately, the treatment with Compound C significantly reversed the pro-epithelialization effect of EGCG Fig. A Wound images. B Wound healing rate. C Wound epithelialization was observed on day 14 by HE staining. D The levels of LC3 and p62 in wounds on day 14 were analyzed by IF staining.
E,F The levels of p62, Beclin1, ATG5, LC3I, LC3II, p-ULK1, ULK1, p-AMPK, and AMPK in wounds on day 14 were analyzed by Western blot. We examined keratinocyte proliferation and differentiation and fibroblast activation-related proteins in the wound on day However, the use of Compound C significantly reversed the effects of EGCG Fig.
Masson staining displayed that collagen deposition was reduced and collagen fibers were disordered in the DM group compared with the Normal group.
After EGCG intervention, collagen deposition recovered and the arrangement of collagen fibers tended to be orderly. However, the use of Compound C significantly reversed the promotion of EGCG on collagen deposition Fig. Further study displayed that the levels of α -SMA, TGF- β 1, Collagen I, and MMP-9 were downregulated in the DM group compared with the Normal group.
The levels of α -SMA, TGF- β 1, Collagen I, and MMP-9 were upregulated after EGCG intervention. However, the use of Compound C significantly reversed the upregulation of these proteins by EGCG Fig. These results suggested that EGCG promoted keratinocyte proliferation and differentiation and fibroblast activation by enhancing epidermal autophagy.
EGCG promoted keratinocyte proliferation and differentiation and fibroblast activation by enhancing epidermal autophagy. A,B The levels of ATG5, KRT10, and KRT14 in wounds on day 14 were analyzed by IF staining.
C Collagen deposition in wounds on day 14 was assessed by Masson staining. D The level of α -SMA in wounds on day 14 was assayed by IHC staining.
E The levels of TGF- β 1, Collagen I, and MMP-9 in wounds on day 14 were analyzed by Western blot. IHC, immunohistochemistry. DM is a common public health problem in modern society.
Treatment for DCU, a major complication of DM, is still under investigation. EGCG, as a component of green tea, possesses various properties [ 37 , 38 , 39 ]. In addition, EGCG has beneficial effects on skin wound healing [ 10 ]. However, the precise mechanism by which EGCG promotes wound healing in DCU remains unknown.
Autophagy, one of the modes of cell death, is believed to exert a key regulatory role in the recovery from diseases [ 40 ]. Recently, the role of autophagy in wound healing has attracted attention [ 41 ]. Physiological dysfunction of epidermal keratinocytes plays an important role in delayed diabetic wound healing, including impaired autophagy, proliferation, and migration [ 42 ].
Damage to keratinocytes and other skin cells from an HG environment is a major cause of poor diabetic wound healing [ 43 ].
Here, keratinocyte autophagy was significantly reduced after HG induction. In related studies, the increased expression of Beclinl, an autophagy-related gene, contributed to burn wound healing [ 44 ]. A previous study showed that p62 knockdown enhances keratinocyte motor function [ 47 ].
Cell proliferation and migration are important processes in normal wound healing. The recovery of keratinocyte proliferation and migration further verified the ameliorative effect of EGCG on HG injury. CCL2 has the function of promoting angiogenesis and immunomodulatory, and is an important chemokine to accelerate wound healing [ 48 ].
The addition of EGCG significantly promoted the synthesis and release of CCL2 in HG-induced keratinocytes. These results suggested that EGCG might promote diabetic wound healing by improving the autophagy injury of keratinocytes and thereby promoting their proliferation and migration.
Among the various types of skin cells, keratinocytes and fibroblasts are the main cells involved in the process of wound healing [ 49 ]. Fibroblasts are immune regulatory factors in wound healing that play a major role in the construction and remodeling of extracellular matrix in DCU treatment [ 50 ].
MMP-9 is thought to be involved in keratinocyte migration, granulation tissue remodeling, and epithelialization during wound healing [ 51 ]. Mice deficient in MMP-9 exhibited disturbed collagen fibrogenesis and delayed epithelialization [ 52 ]. Collagen synthesis and deposition are key factors in wound closure and are related to the expression of TGF- β 1.
TGF- β 1 is a wound healing factor that helps activate injured fibroblasts [ 53 ]. Diabetic wound healing can be enhanced by increasing the proportion of Collagen I in rats [ 54 ]. In addition, the up-regulated expressions of fibrosis genes, TGF- β 1 and α -SMA, promoted the wound healing of skin burns [ 55 ].
In in vitro experiments, fibroblasts were cultured with the supernatant of EGCG-treated keratinocytes. The results showed that α -SMA and Collagen I levels were increased in fibroblasts, indicating that EGCG indirectly promoted the activation of fibroblasts through keratinocytes.
In in vivo experiments, MMP-9, TGF- β 1, Collagen I, and α -SMA levels in neonatal epithelial tissues were up-regulated on day 14 after treatment with EGCG, resulting in an increase in granulation tissue and an accelerated rate of wound healing.
These results proved that EGCG promoted the proliferation and differentiation of fibroblasts by inducing collagen production and deposition, thus accelerating the wound healing process of DCU. Autophagy initiation is mediated by the ULK1 complex, which is regulated by AMPK [ 56 ].
In addition, the downregulation of ULK1 has been associated with autophagy injury in DM and its complications [ 57 ]. The AMPK signaling pathway plays a major role in autophagy induction, and the upregulation of p-AMPK expression can induce glucose metabolism [ 58 ]. The use of EGCG significantly promoted the phosphorylation of AMPK and ULK1 in HG-induced keratinocytes and neonatal epithelial tissues in DM rats.
As a result, autophagy, proliferation, and migration of keratinocytes and activation of fibroblasts were inhibited, and collagen synthesis and deposition were reduced, thus delaying epithelialization and wound healing.
Despite the promising findings of this study, there are several limitations that need to be addressed in future research. htm accessed February 13, Explore More. Ceramic Tea Set Glazing Affects Health Benefits of Tea, Finds New Study.
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Knowing What Dogs Like to Watch Could Help Veterinarians Assess Their Vision. For in vitro stability studies, the specimens were inserted into conical tubes containing 50 mL PBS solution and incubated for 21 days at 37°C.
Stability of the hydrogels was assessed by measuring their weight loss using the following equation:. At selected time points 1 and 9 days , the hydrogels were retrieved from PBS solution and then examined using attenuated total reflection-Fourier transform infrared ATR-FTIR spectrometer Nicolet , Thermo Fisher Scientific, USA.
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the Kyungpook National University. All rats were divided into 5 groups randomly and acclimatized for 5 days before surgery.
A full thickness skin defect 2 cm in diameter was created in dorsal region. The wounds treated with a cotton gauze Daehan Medical, Korea served as a negative control.
For comparison, a commercial hydrogel dressing DuoDERM ® hydroactive ® gel, ConvaTec, USA was tested. The wound size was recorded and the wound dressings were replaced on days 1, 3, 5, 7, 10, and The wound size was calculated using an image analyzer I-solution lite, IMT i-solution, Korea.
The fixed tissue was embedded in paraffin and then cross-sectioned to 5 μm thickness. Each slide was mounted and observed using an optical microscope ECLIPSE TS, Nikon, Japan equipped with a digital camera DS-Fi-2, Nikon, Japan. The quantitative area of inflammatory cells and collagen deposition was calculated using an image analyzer ImageJ, 1.
All data were analyzed by Shapiro-wilk test to confirm the normal distribution. Statistical significance was considered with p values less than 0. Natural SF fibers contain crystalline regions mainly composed of β-sheet structure and thus require a regeneration process to obtain an amorphous random-coil conformation with high water solubility for biomedical use [ 36 ].
However, regenerated SF tends to reform β-sheet structure and aggregate into water-insoluble fibrils upon lyophilization. To circumvent this limitation, we carried out thermal hydrolysis of SF. It was confirmed through GPC that the molecular weight of the SF prepared through heat treatment was reduced.
Water-soluble SF derivative SF-WS was prepared by thermal hydrolysis of SF over 7 h at ºC to produce short chain [ 31 ].
Gel permeation chromatography Table S1 revealed the weight-average molecular weight M w of SF-WS The regenerated SF-WS powder was fully soluble in water without β-sheet formation, and thus used for all subsequent experiments.
Figure 1 a depicts the synthesis scheme for SF-EGCG conjugate having multiple EGCG moieties along the SF-WS backbone. Under mild alkaline condition pH 7. The nucleophilic addition of a lysine residue of SF-WS to the ortho -quinone moiety leads to the formation of amine-quinone adduct at the B ring of EGCG.
Of note, amine-quinone adduct is considered the dominant final product as the formation of EGCG-quinone imine is unfavorable due to rapid hydrolysis of the imine bond in aqueous solution Fig. The resulting SF-EGCG conjugate was purified by extensive dialysis using nitrogen-purged water and then lyophilized to obtain a dry product.
a Scheme of the SF-EGCG conjugate formation through autoxidation of EGCG at basic pH 7. c Optimization of EGCG feeding amount and reaction time. UV-visible spectrum of SF-EGCG conjugate showed much larger absorption peak at nm than SF-WS, demonstrating the conjugation of EGCG moieties on SF-WS Fig.
UV-visible spectroscopy of the products revealed that absorbance at nm gradually increased over 4 h, indicating time-dependence of EGCG conjugation reaction Fig. Since a further incubation up to 5 h did not increase the absorbance significantly, the optimal reaction time was determined as 4 h.
The absorbance at nm was the largest when the feeding amount of EGCG was 40 µmol. Raising the feeding amount of EGCG up to 80 µmol did not help to increase the absorbance, suggesting that the reaction reached a saturation state above 40 µmol of EGCG.
Thus, SF-EGCG conjugate produced with 40 µmol of EGCG was used for subsequent studies. The extent of EGCG conjugation was measured by comparing the difference in absorbance between SF-EGCG and SF-WS with a series of EGCG standards Fig.
The degree of substitution DS of SF-EGCG conjugate was determined as 0. This DS value is considered reasonable because native SF contains only 0. Next, we performed fluorescamine assay to examine the extent of lysine side chain modifications [ 40 ]. Fluorescamine assay Fig.
These results demonstrated that EGCG conjugation reactions occurred primarily at lysine residues in SF-WS. For comparison, SF-T conjugate with DS of 1. S3 according to the previous report [ 27 ]. It is worth noting that SF-EGCG exhibited greater ROS-scavenging activity than SF-T although DS of SF-EGCG 0.
The superior ROS scavenging effect of SF-EGCG conjugates was likely ascribed to the existence of aromatic ring structures in EGCG moiety, which are capable of capturing and neutralizing free radicals [ 28 , 33 ].
The SF-EGCG conjugate has scavenging efficacy. Considerable attention has been paid to EGCG because of its ability to inactivate collagenase in a competitive manner by binding to its catalytic domain [ 42 , 43 ]. As shown in Fig. Even though DS of SF-EGCG 0. Hence, it is conceivable that SF-EGCG could bind to collagenase more strongly than SF-T, leading to more effective inhibition of its enzymatic activity.
It has been reported that a catalytic cycle of HRP converts phenolic moieties of tyramine and EGCG to phenoxy free radicals while consuming H 2 O 2 and releasing water molecules as a byproduct [ 32 , 45 ].
The phenoxy free radicals can react with each other to produce cross-linkages between SF-EGCG and SF-T conjugates. b Storage modulus and gelation time of SF-T hydrogels as a function of H 2 O 2 concentration. The concentration of HRP was fixed to 0. c Storage modulus and gelation time of SF-T hydrogels as a function of HRP concentration.
The concentration of H 2 O 2 was fixed to 5 mM. The concentration of H 2 O 2 and HRP was fixed to 5 mM and 0. First, we attempted to fabricate the SF-T hydrogel without SF-EGCG Fig. As reported in the previous report, high concentrations of H 2 O 2 increased the storage modulus of hydrogel but denatured HRP over excessive concentration [ 46 ].
Also, the increase of HRP concentration highly shortened gelation time without affecting the mechanical strength [ 47 ]. Generally, the mechanical properties of hydrogels should be matched with those of native skin tissue to promote the restoration of defected wound area. Hence, the concentration of H 2 O 2 and HRP was set to 5 mM and 0.
This finding can be explained by the scavenging of phenoxy free radicals by greater amounts of EGCG moieties, leading to an inhibition in the crosslinking reaction [ 39 ]. As presented in Fig. SEM images revealed the presence of interconnected microscopic pores within the optimized hydrogels Fig.
ATR-FTIR spectra of the hydrogels on e day 1 and f day 9. In vitro stability of SF hydrogels was assessed by measuring their weight loss in PBS solution pH 7. To understand the reason for the stabilization, ATR-FTIR was conducted to monitor changes in the β-sheet content in the hydrogels.
The wounds treated with the cotton gauze failed to heal completely even after 14 days Fig. In addition, bleeding was consistently observed from the gauze group on day 3 and 7. Since the cotton gauze is a dry dressing and unable to keep the wounds moisture, strong adhesion between the gauze and wound bed usually occurs.
The gauze group exhibited incomplete re-epithelialization and significant appearance of inflammatory cells, such as macrophages, lymphocytes and neutrophils Fig. It means that typical hydrogel dressings with a moisture maintaining ability were not sufficient to promote effective wound healing. Both groups contained a far smaller number of inflammatory cells compared to SF-T group.
Cytocompatibility experiments revealed that both SF and SF-EGCG were totally non-toxic to NIH3T3 fibroblasts, indicating that the hydrogel components are not likely to cause skin cell death even if they are released as a result of potential degradation Fig.
The insets represent inflammatory cells macrophages, lymphocytes, neutrophils, eosinophils at the interface between matured and healed wound area. The first agenda of this study was fabricating a silk fibrin based hydrogel with secured quality.
To achieve this goal, we have taken the water-soluble SF SF-WS by thermal treatment [ 31 ]. The thermal treated SF-WS powder was fully soluble. Because the Mw of SF-WS was significantly reduced from kDa to As mentioned before, the EGCG has beneficial function for wound dressing due to its strong ROS scavenging and anti-inflammatory activities.
The synthesis of SF-EGCG conjugate was successfully achieved. To prove our hypothesis, the chemistry between the free amine group and EGCG was observed. L-lysine and ethanolamine were reacted with EGCG in a similar condition to SF-EGCG synthesis, and mass spectrometry was measured Fig.
Also, EGCG and amine group compounds were bounded with a ratio of It means the amine compound and EGCG were not bounded by hydrogen bonds. Elevated ROS levels in wounds are known to stimulate dermal fibroblasts to secrete matrix metalloproteinases, predominantly interstitial collagenases, resulting in excessive breakdown of the collagen fibers in extracellular matrix and delayed wound healing [ 5 , 6 ].
The hydroxyl radical scavenging activity of SF-WS, SF-T, and SF-EGCG shown the obvious difference in the low concentration, but the differences became narrow as the increase of concentration.
The silk fibroin has hydroxyl radical scavenging activity already. The hydroxyl radical react with the phenol group, and forms a phenoxy radical.
The phenoxy radical react with the other phenoxy radical, hydroxyl radical and superoxide radical, and forms diphenol, catechol, and tyrosine hydroperoxide, respectively. In the low concentration, it seems that the hydroxyl radical scavenging activity is more dominantly affected by the tyramine or EGCG modified to silk fibroin than the tyrosine residue in the silk fibroin, and as the increase of the concentration, the effect of tyrosine residue become higher.
EGCG has been reported to exhibit about fold higher affinity towards human serum albumin than — -epicatechin or — -epigallocatechin, which lacks a galloyl moiety, suggesting that the galloyl moiety plays a crucial role in EGCG-protein interactions via hydrogen-bonding and hydrophobic forces [ 44 ].
Also, the collagenase inhibitory activity of EGCG was improved after the conjugation with SF. Although the silk fibroin backbone can make steric hindrance, other hydrogen bond sites in SF can bind collagenase. For instance, due to the scavenging effect of EGCG moieties against phenoxy free radicals, the concentration of H 2 O 2 6.
The SF-EGCG exhibited greater ROS-scavenging activity than SF-T although DS of SF-EGCG 0. After the conjugation, the hydroxyl radical scavenging activity was increased, but the superoxide radical scavenging activity was decreased.
It is considered that the conjugation attenuates the activity of EGCG, but inherent hydroxyl radical scavenging activity of silk fibroin supplemented attenuation of activity in the hydroxyl radical scavenging assay Fig. In addition, the specific collagenase activity was calculated.
After the conjugation, the collagenase inhibitory activity was increased. The silk fibroin backbone can make steric hindrance after the conjugation. However, in this work, it is considered that additional hydrogen bond sites in the silk fibroin backbone supplemented the steric hindrance.
Also, after the conjugation, once an EGCG is bound to the collagenase, other conjugated EGCG get a chance to bind to the collagenase more frequently.
It is a similar mechanism that the polymer-antibody conjugation improves the binding affinity of the antibody. The replacement of the gauze dressing not only causes pain and bleeding, but also delays the wound healing process by removing regenerating skin cells.
Neither bleeding nor skin detachment was observed from the wounds treated with all SF hydrogels and DuoDERM® gel. Wound closure profile of all experimental groups except the control group was almost completed after 14 days.
Because the efficacy of hydrogel wound dressings is excellent than other type of wound dressings. Therefore, the day 14 observation is not significant different.
But, inner part of regenerated tissue showed matured collagen deposition with reduced inflammatory cells. It suggests that these hydrogels effectively maintained a moist environment within the wounds. In the present study, SF-EGCG conjugate was synthesized for the first time using the nucleophilic addition reaction of lysine residues in silk proteins with EGCG quinone under a mild basic condition.
This conjugate exhibited superior ROS-scavenging and collagenase-inhibitory activities than SF-T and native SF.
With these attractive properties, SF-EGCG-based composite hydrogels hold great promise as functional biomaterials for wound healing applications. The supplementary data are available online at www. Darr D, Fridovich I. Free radicals in cutaneous biology. J Invest Dermatol.
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Exercise tips for seniors cutaneous healjng DCU EGCG and wound healing one uealing the complications of diabetes mellitus DM [ 1 ]. Glucose metabolism disorder, EGCG and wound healing, blood circulation Strategies for cholesterol control, Microbial resistance properties local infection are considered important factors causing the EGCG and wound healing of DCU [ yealing ]. Clinically, DCU treatment includes debridement and sound changes, infrared radiation, injection or oral administration of hypoglycemic drugs, and heaking amputation surgery in severe cases [ 3 ]. The long duration of treatment, high recurrence rate, and disability rate of DCU bring great physical and mental pain and economic burden to patients and families [ 4 ]. Promoting early wound healing in DCU and reducing its disability rate and treatment cost remain challenging. Therefore, it is urgent to further explore the pathogenesis of DCU, identify potential therapeutic targets, and develop new therapeutic methods. Epigallocatechin gallate EGCGa main active component in green tea, possesses several biological activities such as anti-inflammatory [ 5 ], antioxidant [ 6 ], hypoglycemic [ 7 ], antibacterial [ 8 ], and anticancer [ 9 ]. Biomaterials Research volume Glycogen storage disease typeDiabetic-friendly groceries anr EGCG and wound healing Heailng this article. Metrics details. A Correction to heaoing Diabetic-friendly groceries was published on 29 September Overproduction of reactive oxygen species ROS is known to delay wound healing by causing oxidative tissue damage and inflammation. The green tea catechin, — -Epigallocatechin O -gallate EGCGhas drawn a great deal of interest due to its strong ROS scavenging and anti-inflammatory activities. In this study, we developed EGCG-grafted silk fibroin hydrogels as a potential wound dressing material.
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