Unveiling the mechanism of high sugar diet induced advanced glycosylation end products damage skin structure via extracellular matrix–receptor interaction pathway

AGEs accumulate in the skin as a result of a high‐sugar diet and play an important role in the skin aging process.


| INTRODUC TI ON
The skin is the largest organ of the human body, and it not only protects the body from the external environment but also serves a cosmetic purpose.A youthful appearance positively affects people's social behavior.Skin aging has always been a hot issue of concern.Skin aging is affected by both intrinsic and extrinsic factors.Intrinsic factors of aging are unavoidable physiological processes that are visually characterized by dry lines and fine lines and gradual skin atrophy. 1 In contrast, the visual characteristics of aging caused by external factors are usually characterized by increased wrinkles, decreased elasticity, dullness, and sagging of the skin. 2,3chronic high-sugar diet leads to the accumulation of AGEs, inducing accelerated aging of the skin, making AGEs an extrinsic aging factor.Numerous studies have shown that volunteers with increased AGE levels in the skin have a higher incidence of skin problems such as yellowing and browning of the complexion, poorer elasticity, and deeper wrinkles.[4][5][6] The process of skin aging is closely related to structural and functional changes in ECM components such as collagens and their receptors.
High-sugar diets cause skin aging, but few studies have delved into the mechanisms by which high-sugar diets induce skin aging.
Currently, the mechanism is unclear, there is no chain of evidence, and there are no experimental imaging data.
In this study, we established an aging model of mice damaged by a high-sugar diet, compared the differences in body weight and skin physiological indexes of mice on a HSD group to those of mice in the control group, performed transcriptome sequencing and staining of pathological sections of skin tissues to observe the structural changes, and finally verified the differences in the expression of AGEs and ECMs by immunohistochemical staining.

| Animal grouping and modeling
Twenty-four female SPF-grade C57BL/6J mice, aged 3 weeks, were acclimatized to normal feed for 1 week and then randomly divided into two groups according to body weight: the control group and HSD group, with 12 mice per group.The HSD group was given daily high-sugar feed, and mice in the control group were given daily normal feed, without restriction of food and water for 28 days.

| Skin physiological indicator test
The 12 mice each in the control group and HSD group were weighed every 7 days.On the 28th day, the hair was removed from the skin using Veet hair removal cream in a rectangle that was 3 cm long and 2 cm wide above the root of the tail to allow for testing of skin physiological indexes.Skin water content, transcutaneous water loss, red pigment, melanin, oil, and gloss were measured using a skin bioquantification apparatus (CK, Germany), and each index was measured three times per animal.

| HE staining for skin histopathology
On the 28th day, three mice each from the control group and the HSD group (hair removed) were randomly selected, they were dissected and removed from the back with an area of approximately 2 cm 2 of skin and fixed in 4% formalin fixative for 72 h at room temperature, ensuring that the skin tissue was completely infiltrated in the fixative.After block fixation, the skin tissues were dehydrated and soaked in wax in a dehydrated box for 23 h and then removed and embedded in an embedding machine to make wax blocks for fixation.Wax blocks were sliced, and each block was cut into 4 μm sections.

| RNA extraction and detection
On the 28th day, a skin tissue block with an area of approximately 2 cm 2 on the back of 3 mice (randomly selected in 2.4) was dissected.
The subcutaneous tissue was removed, and the tissue was preserved in liquid nitrogen.A PEXBIO Total RNA Extraction Kit was used to extract total RNA from the skin tissues, followed by agarose gel electrophoresis to analyze the integrity of the RNA and the presence of DNA contamination of the samples, and the RNA samples were subjected to strict quality control.NEB library construction: The first strand of cDNA was synthesized with the M-MuLV reverse transcriptase system using fragmented mRNA as the template and random oligonucleotides as primers, followed by degradation of the RNA strand by RNaseH and synthesis of the second strand of cDNA by dNTPs in the DNA polymerase I system.The purified double-stranded cDNA was end-repaired, Atailed and ligated to the sequencing junction.cDNAs of approximately 200 bp were screened with AMPure XP beads, PCR amplification was performed, the PCR products were purified again using AMPure XP beads, and the library was finally obtained.
After library construction was completed, preliminary quantification was performed using a Qubit 2.0 Fluorometer, and the library was diluted to 1.5 ng/μl.Subsequently, the insert size of the library was detected using an Agilent 2100 bioanalyzer, and after the insert size was as expected, qRT-PCR was performed to accurately quantify the effective concentration of the library (the effective concentration of the library was higher than 2 nM) to ensure the quality of the library.

| Information analysis
The DESseq R software was used to standardize the counts of mRNA of each sample (basemean), 7 and the negative binomial distribution test (NB) was used to analyze significant differences in the number of reads, from which specific genes associated with the condition were identified.The biological significance of specific differential genes was analyzed using algorithms with hypergeometric distributions, with functional differential analysis based on the GO database (GO enrichment analysis) (www.geneo ntolo gy.org) and the analysis of signaling pathways based on the KEGG database (pathway enrichment analysis) (www.genome.jp/ kegg/ pathw ay.html).

| IHC (Immunohistochemistry) staining of skin tissue
On the 28th day, five mice (randomly selected from the remaining mice) were dissected and removed from the back with an area of approximately 2 cm 2 of skin and fixed in 4% formalin fixative for 72 h at room temperature to ensure that the skin tissues were completely infiltrated in the fixative.After block fixation, the skin tissues were dehydrated and soaked in wax in a dehydrated box for 23 h and then removed and embedded in an embedding machine to make wax blocks for fixation.Skin tissues were subjected to immunohistochemistry (fluorescent labeling method).
Multiple IHC staining was performed on 4μm-thick paraffinembedded slides using the Opal Multiplex IHC System.After heatinduced epitope recovery, slides were blocked with PerkinElmer Antibody Dilution Blocking Buffer, and after closure, sections were incubated for 1 hour with the following primary antibodies: anti-AGE antibody, anti-Collagen I antibody, anti-Fibronectin antibody, anti-Laminin 5 antibody, and anti-Tenascin C antibody (Abcam, UK).
After washing in TBST, the slides were incubated with HRP Ms + Rb secondary antibody for 10 minutes at room temperature.The Opal fluorophore was pipetted onto each slide for 10 minutes at room temperature, and the slides were heated with a microwave to strip the primary and secondary antibodies.DAPI was pipetted onto each slide for 10 minutes at room temperature.Slides were covered with BDHM-BD-FM-1, and images were captured using the Vectra Polaris Automated Quantitative Pathology System and analyzed by inForm 2.8.0 software (PerkinElmer).

| Statistical analysis
The data of each group are presented as the mean ± S.D. and were analyzed using the unpaired t-test method in the GraphPad Prism software.Significant differences are indicated as *p < 0.05, **p < 0.01, and ****p < 0.0001.

| Effects of a high-sugar diet on the skin
As shown in Figure 1A, the body weights of the mice in both groups increased significantly after 7 days of feeding with control or special feed, and there was no significant difference in body weight between the HSD group and control group (p > 0.05).The mice in the control group continued to be fed with the control feed and showed a small change in body weight, with a nearly smooth straight-line change from the 7th to 28th days.The mice in the HSD group continued to be fed the high-sugar diet, and their body weights remained Microscopic histomorphometric observation (Figure 1D) showed that the epidermis and dermis of the back skin of mice in the control group were clearly demarcated.In the HSD group, the structure of the skin on the back of the mice was recognizable, and the dermal-epidermal boundary was still distinguishable (Line 1).The control group epidermis was regular, and the structure of the hair follicles, sweat glands, and sebaceous glands was intact.The HSD group epidermis was irregular with anomalous phenomena, the epidermis was thinned, the stratum corneum was discontinuous in the horizontal direction, the dermis lost its normal structure and showed vacuolated changes, and the number of pores was reduced (Line 2).
The control group cell layers of the stratum corneum, stratum pellucidum, and stratum basale were uniformly and densely arranged.
The HSD group boundary between the stratum corneum and the stratum pellucidum was blurred (Line 3).The HSD group adipocytes showed necrotic changes with scattered inflammatory cell infiltration (Line 4).

| Mechanism of high-sugar diet on skin
As shown in Figure 2A, each mouse skin sample sent for testing was free of macromolecular contamination and was intact and nondegraded.All of them passed the quality control evaluation and were ready for transcriptome sequencing.
As shown in Figure 2B,C, gene expression in mouse skin tissues changed significantly when given a high-sugar diet versus a normal diet for 28 days.In particular, 1555 genes were upregulated genes, and 1699 were downregulated in the HSD group compared with the control group.
Gene function annotation was performed based on the GO database, and the significance level of each function was calculated using Fisher's test to screen out the significant functions embodied by the genes.As shown in Figure 2D, the upregulated gene functions in the high-sugar group were reflected in the domains of muscle contraction, fatty acid metabolism, redox reactions, lipid metabolism process, ion transport, response to toxic substances, and glycogen biosynthesis process.As shown in Figure 2E, the downregulated gene functions in the high-sugar group were reflected in the domains of immune system response, inflammatory response, cell adhesion, defense response, neutrophil chemotaxis, mitosis, and collagen catabolism, among others.
Signaling pathway annotation was performed based on the KEGG database, and Fisher's test and multiple comparisons test were used to screen the significant signaling pathways represented by the genes.As seen in Figure 3A, the upregulated pathways in the highsugar group were enriched mainly in the transporter-related pathway, PPAR signaling pathway, fatty acid degradation, ion channel pathway, drug metabolism-cytochrome P450 pathway, cytoskeletal proteins, lipid synthesis proteins, and so on.As shown in Figure 3B, the downregulated pathways in the high-sugar group were enriched mainly in the CD molecular immunodifferentiation pathway, pattern recognition receptor, glycosaminoglycan-binding protein, ECM-receptor interaction pathway, platelet activation, osteoclast differentiation, and NF-κB signaling pathway.
Based on the results of transcriptomics sequencing, it was found that among the differentially expressed genes in the skin of mice in the high-sugar and control groups, the GO downregulated genes and KEGG downregulated pathways were more significant than the upregulated genes and upregulated pathways.Among them, the extracellular matrix (ECM)-receptor interaction signaling pathway showed significant downregulation (Figure 3C), which is most closely and directly related to the altered state of the skin aging process.
IHC staining of skin tissues verified that a high-sugar diet induced upregulation of the expression of AGEs in mouse skin (Figure 4A) and downregulation of the expression of the protein components of the ECM, such as type I collagen (Figure 4B-E).The expression of AGEs was significantly increased in the epidermal dermis of mouse skin tissues in the high-sugar-diet group compared to the control group (5.20% ± 0.02% vs. 40.67%± 0.04%) (p < 0.0001).Collagen I expression was significantly decreased (23.85% ± 0.07% vs. 1.40% ± 0.01%) (p < 0.0001), fibronectin 1 expression was significantly decreased (22.39% ± 0.02 vs. 6.38% ± 0.04) (p < 0.01), laminin 5 expression was significantly decreased (7.22% ± 0.03% vs. 0.48% ± 0.00%) (p < 0.01) and tenascin C expression was significantly decreased TA B L E 1 Effects of changes in the level of downregulation of the extracellular matrix-receptor interaction signaling pathway on the skin.

Categories Downregulated genes Name
Effects on the skin Ref.

ECM proteins Col1a1 Col1a2
Collagen I 1. Promotes skin cell migration and adhesion 2. Closely related to the tensile strength and stability of dermal tissue and blood vessels 14-16

Col4a1 Col4a2
Collagen IV 1.It is mainly found in the dermal-epidermal junction of the skin, and plays an important role in the stabilization of the basement membrane 2. Impaired expression leads to abnormal thinning of the basal lamina and, in severe cases, to epidermolysis bullosa  for 90% of the dry weight of the skin and is the main component that makes up the skin. 10Fibroblasts are the main cells that synthesize collagen.The integrity of the mechanical characteristics and function of the skin depends on the structural integrity of the dermal constituents. 11The result gave us the first intuitive evidence that a high-sugar diet truly damages the skin, so we used the transcriptomics results to make the bold hypothesis of AGES-ECM-skin damage.Then, immunohistochemistry was used to corroborate this finding.
Aging is often thought to be associated with alterations in the skin's ECM, a complex collection of macromolecular protein components, including collagen type I, collagen type IV, and fibronectin 1, that provide positional support for cells in tissues.
A variety of stimuli can cause the ECM components to be out of balance, but there are no related studies that analyze which protein components are involved in the high-sugar dietary factors that induce damage to the skin's ECM components.In this study, significant downregulation of the ECM-receptor interaction pathway was analyzed by skin transcriptome sequencing, and the protein components and receptor types that were downregulated and expressed by this pathway are shown in Table 1.Thirteen ECM protein components were downregulated and expressed, and six ECM protein receptor components were downregulated.Changes in ECM composition affecting receptor activation may lead to receptor insufficiency, and receptor insufficiency may affect ECM action.Normal expression and anchoring of ECM proteins, such as collagen, fibronectin, laminin, and tenascin, and ECM receptors, such as integrins, contribute to the normalization of cell behavioral functions, such as cell proliferation, migration, and adhesion (Table 1).Integrin is a heterodimeric receptor on the cell membrane that attaches the cytoskeleton to the ECM microenvironment, acts as a biochemical/mechanical signal receptor for the cell, and is one of the determinants affecting cellular tone, 12 playing a role in regulating cell proliferation, migration, and adhesion.
The TGFβ signaling pathway plays a central role in the biosynthesis of ECMs, and elevated MMP expression is the main reason for the disruption of protein components in ECMs. 13 summarize, the hypothesized mechanism of skin aging induced by a high-sugar diet is shown in Figure 5.A high-sugar diet causes senescence damage to the skin.In relation to aging, the damaging effect of a high-sugar diet on skin is manifested by promoting the accumulation of AGEs, which in turn disrupts the balance of protein components of ECMs, inhibits their receptor expression, and affects the normal function of ECM receptors, leading to impaired cell behavioral functions such as cell proliferation, migration, and adhesion.
This result provides a chain of evidence that a high-sugar diet leads to aging-related damage in the skin, and these findings can be used to develop efficacious ingredients that protect the skin from aging stimulated by a high-sugar diet.
All experimental procedures were approved by the Institutional Animal Care and Use Committee of Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences in 2021(Ethics Number: 2021D057).
HE staining: Sections were deparaffinized with xylene (xylene I 10 min; xylene II 10 min) and then dipped in water after graded ethanol deparaffinization (anhydrous ethanol I 5 min: anhydrous ethanol II 5 min; 90% ethanol 5 min; 70% ethanol 5 min).Excess staining solution was washed away with running water for 10-15 min, and hydrochloric acid alcohol differentiation was observed under the microscope for 1-2 s.The nuclei were washed again with water for 10 min, and the nuclei were returned to blue by ammonia for 30 s.The nuclei were rinsed with running water for 10 min, and 1% eosin staining solution (Solarbio, China) was washed with water for 1 min and washed with water rapidly for 1-2 min.Step-by-step ethanol dehydration and xylene dehydrogenation were performed.Neutral gum was used to seal the slices, and the slices were air-dried by an air-cooling process and then observed and photographed under a light microscope.

2. 5 . 2 |
Library construction and quality control Acquisition of mRNA: A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations, and index codes were added to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.Ribosomal RNA (rRNA) is highly abundant in mouse total RNA, and its removal is desirable in order to reveal the biological significance of less abundant transcripts.The NEBNext rRNA Depletion Kit (Mouse) employs the NEBNext RNase H-based RNA Depletion Workflow.The mRNA obtained was subsequently exposed to divalent cations in NEB Fragmentation Buffer, and library construction was performed according to the NEB method.
After passing the library inspection, different libraries were pooled according to the effective concentration and 10G target data volume for Illumina sequencing.Four types of fluorescently labeled dNTPs, DNA polymerase, and junction primers were added to the sequencing flow cell for amplification.When extending the complementary strand of each sequencing cluster, each fluorescently labeled dNTP will release the corresponding fluorescence, and the sequencer will capture the fluorescence signals and convert the light signals into sequencing peaks through computer software, thus obtaining the sequence information of the fragment to be tested.
Figure 1C, the TWEL, ERYTH, L, b* and ITA° value of the high-sugar group were highly significant (p < 0.01) compared with those of the control group.Additionally, the TWEL, L and ITA° value were lower in the high-sugar group than in the control group, the ERYTH value and b* value were higher in the high-sugar group than in the control group, and the GLOSS_DSC and a* values were significantly higher in the high-sugar group than in the control group (p < 0.05).Furthermore, the CM and MEXA values were lower in the high-sugar group than in the control group, but the difference was not significant.Compared to the control group (a* 8.19, b* 1.65, L 51.68, ITA° 40.37), the skin color of mice in the HSD model group (a* 10.44, b* 3.32, L 46.14, ITA° -46.70) tended to be redder, yellower, darker, and deeper.The glossiness of the mice in the HSD group was significantly higher, which might be caused by the elevated oil secretion in mice in the HSD model group.

F I G U R E 1
Effects of high-sugar diet on body weight and skin physiopathologic indexes in mice.(A) Changes in body weight of mice in the HSD and control diet groups during the 28-day test cycle.(B) Schematic diagram of the location of hair removal on Day 28 of the skin index test in mice.(C) Results of the skin physiological index test in mice.(D) HE staining results of mouse skin histopathology (from top to bottom: whole skin, skin structure, epidermis, subcutaneous adipose tissue).*p < 0.05, **p < 0.01.F I G U R E 2 Results of transcriptome sequencing and analysis of mouse skin-1.(A) Total RNA agarose gel electrophoresis plot of each sample.(B) Differential gene volcano plots for the high-sugar and control groups.(C) Differential gene clustering diagram.(D) Comparison of top 40 upregulated differential gene GO function enrichment in the high-sugar group and control group.(E) Comparison of GO functional enrichment of the top 40 downregulated DEGs between the high-sugar group and the control group.F I G U R E 3 Results of transcriptome sequencing and analysis of mouse skin-2.(A) Comparison of KEGG pathway enrichment of the top 40 upregulated differentially expressed genes between the high-sugar group and the control group.(B) Comparison of KEGG pathway enrichment of the top 40 downregulated differentially expressed genes between the high-sugar group and the control group.(C) Downregulation of ECM-receptor interaction signaling pathway protein expression.F I G U R E 4 Effect of a high-sugar diet on the protein expression of AGEs and ECMs in mouse skin.(A) The high-sugar diet induced high expression of AGEs in skin tissues.(B) The high-sugar diet decreased collagen I expression in skin tissues.(C) The high-sugar diet reduced fibronectin 1 expression in skin tissues.(D) The high-sugar diet reduced laminin 5 expression in skin tissues.(E) The high-sugar diet decreased tenascin C expression in skin tissues.*p < 0.05, ***p < 0.001, ****p < 0.0001.

F I G U R E 5 1 . 33 Itga4 Integrin alpha 4 1 . 35 Itga9 Integrin alpha 9 1 .migration 36 Itgb8 Integrin beta 8 1 .barrier 37 Cd44 CD44 antigen 1 . 1 .
Diagram of the mechanism of high-sugar-diet-induced skin damage (by FigDraw, ID: WIAWYbb407).The high-sugar diet induces a large accumulation of AGEs in the skin, causing downregulation of TGFβ expression related to ECM production and upregulation of MMP expression related to ECM protein degradation, and disruption of the stable state of the ECM manifests as a decrease in cell behavioral functions, such as migration, adhesion, and proliferation at the cellular level in tissues, and aging-related skin damage at the skin level.Composition of the αIIbβ3 integrin receptor 2. Expressed primarily at the platelet site, impaired function leads to bleeding and thrombocytopenia 32,Composition of α4β1 and α4β7 integrin receptors 2. α4β1 integrins play a role in skin immunity and neutrophil migration and regulate fibroblast differentiation status 3. α4β7 integrins are expressed mostly on leukocytes and regulate leukocyte migration and skin inflammation 34,Composition of the α9β1 integrin receptor 2. Expressed in the basal lamina and regulates the process of cell Composition of αvβ8 integrin receptors 2. It is preferentially expressed on skin regulatory T cells and regulates skin inflammation following disruption of the skin Combines with hyaluronic acid to have an anti-allergic effect and regulate cell growth and movement 38Acts as a HA receptor in nonadherent cells and regulates CD44 receptor expression 40 TA B L E 1 (Continued) (10.20% ± 0.05% vs. 3.41% ± 0.03%) (p < 0.05) in the epidermal dermis of skin tissues of the mice in the high-sugar-diet group when compared with the control group.4| DISCUSS IONWith the prolongation of the modeling time, the weight of the mice in the HSD group gradually decreased compared with that in the control group, which is consistent with the typical clinical symptoms of diabetes mellitus.It has been shown that AGEs are causative factors for chronic metabolic diseases and skin complications, such as diabetic skin ulcers, susceptibility to infections, and slow or even nonhealing wounds,8 and are responsible for the frequency of skin problems caused by a high-sugar diet.Cell behavioral functions such as migration, adhesion, and proliferation decrease with deeper aging,9 and these changes are shown in Figure2E.The incidence of skin problems, such as yellowing and browning of skin, poorer elasticity, and deeper wrinkles, increases with the increase in AGEs in the skin.[4][5][6]This is consistent with the results of the skin physiological index test: The skin color of mice in the HSD group tended to become redder, yellower, darker, and deeper.Skin histopathological and morphological observations showed that stimulation with a high-sugar diet led to skin thinning, structural damage, irregular deformation of the epidermis, and loss of the normal structure of the dermis.Meanwhile, transcriptomics results showed significant downregulation of the ECM-receptor interaction pathway.The ECM components can provide support for skin cells and maintain the normal structure of skin tissue, which is directly related to skin aging changes.The dermis is rich in fibroblast and ECM components.Collagen in the dermis accounts It is found mainly in the papillary layer of the dermis, around blood vessels, hair follicles, and glandular structures 2. Helps regulate skin elasticity and the ability of skin cells to differentiate, proliferate, and migrate