Keratinocytes (HaCaT, AW-CNH203, Abiowell, Changsha, China) and fibroblasts (HFF-1, AW-CCH222, Abiowell, Changsha, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, D5796, Sigma, Saint. Louis, MO, USA) and RPMI-1640 medium (2144322, Biological Industries, Kibbutz Beit-Haemek, Israel). These medium were supplemented with 10% of fetal bovine serum (FBS, 10099141, Gibco, Carlsbad, CA, USA) and 1% of Penicillin/Streptomycin (AWI0070a, Abiowell, Changsha, China). They were placed in an incubator (DH-160I, SANTN, Shanghai, China) containing 5% CO${}_2$ at 37 °C. In addition, cell slides were prepared by respective inoculation of appropriate densities of HaCaT and HFF-1 cells in culture dishes for Immunofluorescence (IF) staining and 5-Ethynyl-2${}^{\prime }$-deoxyuridine (EdU) staining analysis. The purchased Cell lines have been authenticated by short tandem repeat (STR) profiling. All the cells used in the experiment were tested and confirmed to be free of mycoplasma contamination.

For HaCaT cells, grouping 1 included 0 µM EGCG, 6.25 µM EGCG, 12.5 µM EGCG, 25 µM EGCG, 50 µM EGCG, and 100 µM EGCG. Grouping 2 included Control, HG, HG + 6.25 µM EGCG, HG + 12.5 µM EGCG, HG + 25 µM EGCG, and HG + 50 µM EGCG. The HaCaT cells in the Control group were cultured normally. The HaCaT cells in the other groups were treated with HG (50 mM glucose) for 24 h and EGCG (2503504, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) at different concentrations for 24 h [31]. Grouping 3 included Control, HG, HG + EGCG, and HG + EGCG + Compound C. Except for the Control group, HaCaT cells in other groups were treated with HG for 24 h. HaCaT cells in the HG + EGCG group were treated with 50 µM EGCG for 24 h after HG induction. HaCaT cells in the HG + EGCG + Compound C group were treated with 50 µM Compound C (D426448, Aladdin, Shanghai, China) and 50 µM EGCG for 24 h after HG induction [32]. The information about the processing of different groups was shown in Table 1. To induce the synthesis and release of CCL2, cells in each group were treated with 100 ng/mL TNF-$\alpha$ (MAB610-SP, R&D Systems, Minneapolis, MN, USA) for 24 h [21]. For HFF-1 cells, groups included Control, HG, HG + EGCG, and HG + EGCG + Compound C. TNF in the supernatant of HaCaT cells in grouping 3 was neutralized with 2 µg/mL anti-TNF-$\alpha$ antibody (AF-410-SP, R&D Systems, Minneapolis, MN, USA), and then the supernatant was added to HFF-1 cells for 48 h, respectively (the supernatant content was 20%).

Male adult Sprague-Dawley rats (SPF, 7–8 weeks, 180–200 g) were bought from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). After adaptive feeding, rats were randomly divided into Normal, DM, DM + EGCG, and DM + EGCG + Compound C groups with 10 rats in each group. The DM rat model was established according to a previously described method [33]. Rats were intraperitoneally injected with STZ (80 mg/kg) (AWH0492a, Abiowell, Changsha, China) dissolved in citrate buffer solution (0.1 M, pH 4.5) to induce type I DM [34], with the same dose of citrate buffer solution being the control in the Normal group. Follow-up experiments were performed when the blood glucose level reached 16.7 mM. After the rats were anesthetized with intraperitoneal injection of 1% pentobarbital (30 mg/kg), a 15 mm diameter wound was created on the back using the full-thickness skin defect method. Rats in Normal and DM groups were treated daily with 1% carboxymethyl cellulose (C501052, Aladdin, Shanghai, China). Rats in the DM + EGCG group were treated with 1 mg/mL EGCG daily [35]. Rats in the DM + EGCG + Compound C group were given 1 mg/kg Compound C near the wound at 30 min before EGCG intervention [36]. The information about the processing of different groups was shown in Table 2. On days 0, 3, 7, and 14, the wound area was measured and photographed, and the wound healing rate was calculated according to the methods described in the literature [35]. On day 14, rats were sacrificed by cervical dislocation, and neonatal epithelial tissues were taken for follow-up analysis.

HaCaT cells in the logarithmic growth phase were digested with trypsin (AWC0232, Abiowell, Changsha, China) to prepare a cell suspension. Cells were inoculated into 96-well plates at a density of 1 $\times$ 10${}^4$ cells/well. After adherent growth, cells were treated with EGCG for 24 h. After the removal of the EGCG-containing medium, 100 µL of medium containing 10% CCK-8 reagent (NU679, DOJINDO, Kumamoto, Japan) was added. After being incubated for 4 h, the cells were analyzed with a multifunctional microplate reader (MB-530, HEALES, Shenzhen, China) to obtain the optical density (OD) at 450 nm.

Total proteins were extracted from HaCaT cells and neonatal epithelial tissues using RIPA lysate (AWB0136, Abiowell, Changsha, China). The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (NC) membranes. NC membranes were blocked with 5% skimmed milk (AWB0004, Abiowell, Changsha, China) and incubated with the following primary antibodies respectively, including p62 (1:10,000, 66184-1-Ig, Proteintech, Chicago, IL, USA), Beclin1 (1:2000, 11306-1-AP, Proteintech, Chicago, IL, USA), ATG5 (1:5000, ab108327, Abcam, Cambridge, UK), LC3 (2 µg/mL, ab48394, Abcam, Cambridge, UK), phosphorylated-AMPK (p-AMPK, 1:5000, ab92701, Abcam, Cambridge, UK), AMPK (1:5000, 10929-2-AP, Proteintech, Chicago, IL, USA), p-ULK1 (1:5000, 29006-1-AP, Proteintech, Chicago, IL, USA), ULK1 (1:20,000, ab133747, Abcam, Cambridge, UK), CCL2 (1:2000, 66272-1-Ig, Proteintech, Chicago, IL, USA), transforming growth factor-$\beta$1 (TGF-$\beta$1, 1:1000, ab235578, Abcam, Cambridge, UK), matrix metallopeptidase-9 (MMP-9, 1:1000, ab38898, Abcam, Cambridge, UK), Collagen Ⅰ (1:1000, ab270993, Abcam, Cambridge, UK), and $\beta$-actin (1:5000, 66009-1-Ig, Proteintech, Chicago, IL, USA) Then, NC membranes were incubated with the following secondary antibodies respectively, including horseradish peroxidase (HRP)-goat anti-mouse IgG (1:5000, SA00001-1, Proteintech, Chicago, IL, USA) and HRP-goat anti-rabbit IgG (1:6000, SA00001-2, Proteintech, Chicago, IL, USA). NC membranes were incubated with Super ECL Plus detection reagent (AWB0005, Abiowell, Changsha, China) for chemiluminescence imaging. Quantity One 4.6.6 (Bio-Rad Inc., Hercules, CA, USA) was employed to obtain the gray values of protein bands, and the expression level of each protein was calculated with $\beta$-actin being an internal reference.

Cell slides were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton, and blocked with 5% bovine serum albumin (BSA). For tissue slices, antigen repair was performed after dewaxing to water. The tissue slices were washed with PBS, and sequentially placed in sodium borohydride solution, 75% ethanol solution, and Sudan black dye solution. After washing with PBS, the tissue slices were blocked with 5% BSA. The cell slides were incubated respectively with primary antibodies of LC3 (1:100, 14600-1-AP, Proteintech, Chicago, IL, USA), p62 (1:50, 18420-1-AP, Proteintech, Chicago, IL, USA), alpha-smooth muscle actin ($\alpha$-SMA, 1:50, BM0002, Boster, Pleasanton, CA, USA), and Collagen I (1:100, 14695-1-AP, Proteintech, Chicago, IL, USA). The tissue slices were incubated respectively with primary antibodies of LC3, p62, ATG5 (1:50, 66744-1-Ig, Proteintech, Chicago, IL, USA), keratin 10 (KRT10, 1:50, 18343-1-AP, Proteintech, Chicago, IL, USA), and keratin 14 (KRT14, 1:50, 10143-1-AP, Proteintech, Chicago, IL, USA). The cell slides and tissue slices were incubated respectively with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:200, SA00013-2, Proteintech, Chicago, IL, USA) or CoraLite594-conjugated Goat Anti-Mouse IgG (H + L) (1:200, SA00013-3, Proteintech, Chicago, IL, USA). Then, nuclei were stained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) staining solution (AWI0331a, Abiowell, Changsha, China) and washed with PBS. The cell slides and tissue slices were sealed with buffered glycerin and the images were collected by a microscope (BA410T, Motic, Wetzlar, Germany).

HaCaT cells (1 $\times$ 10${}^5$) in the logarithmic growth stage were inoculated into 6-well plates, and then treated with corresponding drugs. According to the instructions of the EdU detection kit (C10310, RIBOBIO, Guangzhou, China), cells in each group were subjected to EdU labeling, immobilization, Apollo staining, and DNA staining, and images were collected by a fluorescence microscope.

The scratch assay was applied to detect the migration ability of HaCaT cells in each group. HaCaT cells from different groups were digested to make a cell suspension. The 5 $\times$ 10${}^5$ cells were uniformly inoculated in 6-well plates. When the cells had filled the plates, lines were drawn and viewed under a microscope to record the initial scratch width. After 24 h and 48 h, the images were photographed with a microscope and scratch widths were measured using ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA).

The HaCaT cell cultures in different groups were centrifuged at 1000 g at 4 °C for 15 min, and the supernatant was used for detection. The content of CCL2 released to supernatant was assessed according to the instruction of the CCL2 ELISA kit (CSB-E04655h, CUSABIO, Wuhan, China).

The epithelial tissue slices were dewaxed in xylene and dehydrated with gradient ethanol (75–100%). They were stained with hematoxylin (AWI0001a, Abiowell, Changsha, China), and then returned to blue in PBS. Next, the slices were stained with eosin (AWI0029a, Abiowell, Changsha, China), and dehydrated with gradient alcohol (95–100%). The slices were cleared in xylene and then sealed with neutral gum (AWI0238a, Abiowell, Changsha, China) for observation with a microscope.

After being dewaxed to water, the epithelial tissue slices were immersed in citrate buffer (0.01 M, pH 6.0) (AWI0206a, Abiowell, Changsha, China), and boiled for antigen retrieval. Subsequently, the endogenous enzymes were inactivated with 1% periodic acid. The slices were incubated with the primary antibody of $\alpha$-SMA (1:500, BM0002, Boster, Pleasanton, CA, USA) overnight followed by poly-HRP-anti-mouse-IgG. Next, diaminobenzidine (DAB) was added to slices for catalytic color development. The slices were re-stained with hematoxylin, rinsed with distilled water, returned to blue in PBS, and dehydrated with gradient alcohol (60–100%). After being cleared in xylene and sealed with neutral gum, the slices were observed by a microscope.

The epithelial tissue slices were dewaxed to water and stained with hematoxylin. They were washed with tap water and distilled water in turn. Next, the slices were soaked in PBS to make the nucleus return blue and stained with an acid fuchsin stain solution. Then, the slices were reacted with a phosphomolybdic acid differentiation solution, stained with aniline blue counterstain, and rinsed with absolute ethanol. The slices were blow-dried, cleared in xylene, and then sealed with neutral gum before observation with a microscope.

Data were analyzed by GraphPad Prism 8.0.1 (GraphPad Software Inc., San Diego, CA, USA) and expressed as mean $\pm$ standard deviation. Kolmogorov-Smirnov test and exploratory descriptive statistics test were used to analyze whether the data conformed to a normal distribution and homogeneity of variance. One-way analysis of variance (ANOVA) and Tukey’s post-hoc test were employed in comparison between groups. Comparisons between groups at different time points were analyzed by two-way ANOVA with Bonferroni as a post hoc test. The difference was statistically significant when p 0.05. All the experiments followed randomization and blind analysis to avoid experimental bias.

To screen suitable EGCG concentration, keratinocytes were treated with a series of EGCG concentrations (0, 6.25, 12.5, 25, 50, and 100 µM) for 24 h, and the cell viability was measured with the CCK-8 assay. No significant effect on cell viability was observed with EGCG at 0, 6.25, 12.5, 25, and 50 µM. However, However, keratinocyte viability was significantly decreased with 100 µM EGCG. Thus, 50 µM EGCG was selected for subsequent experiments (Fig. 1A). To explore the effect of EGCG on autophagy impairment, keratinocytes were first induced with HG (50 mM glucose) for 24 h, and then treated with EGCG for another 24 h, after which the levels of autophagy-related proteins were analyzed. After HG induction, autophagy was impaired in keratinocytes, showing decreased LC3II/LC3I, Beclin1, and ATG5 levels, and increased p62 level. However, after EGCG treatment, LC3II/LC3I, Beclin1, and ATG5 levels raised whereas p62 level declined in HG-treated keratinocytes, and the extent of these changes was EGCG concentration-dependent (Fig. 1B). Collectively, the above results demonstrated that EGCG improved HG-induced autophagy impairment in keratinocytes.

Fig. 1.

EGCG induced autophagy in HG-treated keratinocytes. (A) The viability of keratinocytes treated with different concentrations of EGCG was evaluated by the CCK-8 assay. (B) The levels of p62, Beclin1, ATG5, LC3I, and LC3II in keratinocytes were analyzed by Western blot. &p 0.05 vs. 0 µM EGCG, *p 0.05 vs. Control, #p 0.05 vs. HG.

To clarify the regulation of the AMPK/ULK1 pathway in autophagy induction by EGCG in HG-treated keratinocytes, an AMPK inhibitor (Compound C) was utilized to intervene, and proteins related to autophagy and the pathway were analyzed. HG induction led to a reduction in LC3, Beclin1, and ATG5 levels while a rise in p62 level in keratinocytes. Compared with the HG group, LC3, Beclin1, and ATG5 levels were raised and p62 level was declined in keratinocytes in the HG + EGCG group. However, the use of Compound C significantly reversed the autophagy induction of EGCG (Fig. 2A,B). Further analysis demonstrated that HG induction decreased the phosphorylation of AMPK and ULK1 in keratinocytes. Compared with the HG group, phosphorylation of AMPK and ULK1 in keratinocytes increased in the HG + EGCG group. However, the use of Compound C significantly reversed the effect of EGCG on AMPK/ULK1 pathway activation (Fig. 2C). These results suggested that EGCG enhanced autophagy in HG-treated keratinocytes by activating the AMPK/ULK1 pathway.

Fig. 2.

Activation of the AMPK/ULK1 pathway could promote autophagy induction of EGCG to HG-treated keratinocytes. (A) The levels of LC3 and p62 in keratinocytes were detected by IF staining. (B,C) The levels of p62, Beclin1, ATG5, LC3I, LC3II, p-ULK1, ULK1, p-AMPK, and AMPK in keratinocytes were determined by Western blot. *p 0.05 vs. Control, #p 0.05 vs. HG, &p 0.05 vs. HG + EGCG.

Based on the above results, we further conducted EdU staining, scratch assay, and Western blot analyses on the proliferation and migration of keratinocytes. The proliferation and migration of keratinocytes were significantly inhibited following HG induction. Compared with the HG group, the proliferation, and migration of keratinocytes were significantly raised in the HG + EGCG group. However, the use of Compound C significantly reversed the promoting effects EGCG on the proliferation and migration of keratinocytes (Fig. 3A,B). Through the detection of chemokine CCL2, we observed a decrease in the synthesis of CCL2 in keratinocytes, as well as a decline in the content of CCL2 released into the culture supernatant after HG induction. Compared with the HG group, the synthesis and release of CCL2 in keratinocytes were raised in the HG + EGCG group. Notably, the use of Compound C significantly reversed the promoting effects of EGCG on the synthesis and release of CCL2 (Fig. 3C,D). These results displayed that EGCG promoted the proliferation and migration of HG-treated keratinocytes by enhancing autophagy.

Fig. 3.

EGCG promoted the proliferation and migration of HG-treated keratinocytes by enhancing autophagy. (A) EdU staining of keratinocyte proliferation. (B) Scratch assay of keratinocyte migration. (C) The synthesis of CCL2 in keratinocytes was analyzed by Western blot. (D) The content of CCL2 in the keratinocyte culture supernatant was determined by ELISA. *p 0.05 vs. Control, #p 0.05 vs. HG, &p 0.05 vs. HG + EGCG.

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. Fibroblast markers, including $\alpha$-SMA and Collagen Ⅰ, were measured by IF staining. The levels of $\alpha$-SMA and Collagen Ⅰ in fibroblasts of the HG group were lower than those in the Control group. The levels of $\alpha$-SMA and Collagen Ⅰ in fibroblasts of HG + EGCG group were raised compared with those in the HG group. However, treatment with Compound C significantly reversed the activating effects of EGCG on fibroblasts (Fig. 4A,B). Together, these results showed that EGCG promoted fibroblast activation by enhancing keratinocyte autophagy.

Fig. 4.

Autophagy induction of EGCG on keratinocytes promoted fibroblast activation. (A,B) The levels of $\alpha$-SMA and Collagen Ⅰ in fibroblasts were analyzed by IF staining. *p 0.05 vs. Control, #p 0.05 vs. HG, &p 0.05 vs. HG + EGCG.

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 0–14. In the Control group, the wound area was gradually decreased with time and was fully epithelialized by day 14. By contrast, the wound in the DM group healed slowly and was not completely healed by day 14. However, wound healing in the DM + EGCG group was significantly improved, with the wound appearing nearly fully healed by day 14. Notably, the use of Compound C significantly reversed the beneficial effect of EGCG on wound healing (Fig. 5A,B). 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. 5C). Further analysis showed that compared with the Control group, the levels of LC3II/LC3I, Beclin1, ATG5, and the phosphorylation of AMPK and ULK1 declined in the DM group, whereas the level of p62 was increased. After EGCG intervention, the levels of LC3II/LC3I, Beclin1, ATG5, and phosphorylation of AMPK and ULK1 were increased, whereas the level of p62 was decreased. Again, the use of Compound C significantly reversed the promoting effects of EGCG on epidermal autophagy and AMPK/ULK1 pathway activation (Fig. 5D–F). These results illustrated that EGCG enhanced epidermal autophagy through the AMPK/ULK1 pathway to promote diabetic wound healing.

Fig. 5.

EGCG enhanced epidermal autophagy through the AMPK/ULK1 pathway to promote diabetic wound healing. (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. *p 0.05 vs. Control, #p 0.05 vs. HG, &p 0.05 vs. HG + EGCG.

We examined keratinocyte proliferation and differentiation and fibroblast activation-related proteins in the wound on day 14. IF staining showed a decrease in the number of ATG5/KRT14- and ATG5/KRT10-positive cells in the DM group compared with the Normal group. After EGCG intervention, the number of ATG5/KRT14- and ATG5/KRT10-positive cells was increased. However, the use of Compound C significantly reversed the effects of EGCG (Fig. 6A,B). 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. 6C). Further study displayed that the levels of $\alpha$-SMA, TGF-$\beta$1, Collagen I, and MMP-9 were downregulated in the DM group compared with the Normal group. The levels of $\alpha$-SMA, TGF-$\beta$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. 6D,E). These results suggested that EGCG promoted keratinocyte proliferation and differentiation and fibroblast activation by enhancing epidermal autophagy.

Fig. 6.

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 $\alpha$-SMA in wounds on day 14 was assayed by IHC staining. (E) The levels of TGF-$\beta$1, Collagen I, and MMP-9 in wounds on day 14 were analyzed by Western blot. *p 0.05 vs. Control, #p 0.05 vs. HG, &p 0.05 vs. HG + EGCG. IHC, immunohistochemistry.