Glutamic-Acid Grafted Hyaluronic Acid Inhibits Inflammatory Factors via Fibroblast and Skin Model Tests

In the pursuit of more effective cosmetic ingredients for combating skin inflammation, the recent study, “Glutamic-Acid Grafted Hyaluronic Acid: A New Cosmetic Ingredient to Suppress Inflammatory Factor Expression in Cell and Skin Model Tests,” presents a novel approach. By conjugating Glutamic Acid (Glu) to low-molecular-weight Hyaluronic Acid (HA), the researchers aimed to leverage the anti-inflammatory properties of both compounds for improved skin health outcomes. 

The study describes how excessive inflammation can compromise the skin barrier and lead to issues like aging and various skin diseases [1-3]. Interleukins and tumor necrosis factors, specifically IL-1α, IL-6, and TNF-α, are reported to play pivotal roles in these processes [4-9]. HA, known for its regulatory role on inflammatory factors [10,11], was modified with Glu, an amino acid with reported influence on inflammation [12,13]. This modification aimed to combine the beneficial properties of both substances for enhanced skin care applications. 

The synthesis of HA-Glu involved a two-step process adapted from previous works [14,15], leading to the production of HA-Glu with varying degrees of grafting (Figure 1). The anti-inflammatory efficacy was assayed using human dermal fibroblasts and a 3D skin model, comparing the grafted compound against native HA. The results showed that HA-Glu significantly inhibits inflammatory markers IL-1α, IL-6, and TNF-α more effectively than HA alone (Figure 2 for molecular structures, Figure 3 for gene expression comparisons).

 Figure 1: Synthesis reaction of HA-Glu. 

Figure 2: 1H NMR spectra of native HA, L-Glu, and HA-Glus. 

Figure 3: The expression levels of IL-1α, IL-6 and TNF-α in cell tests were detected by qRT-PCR. 

Results of IL-1α (A), IL-6 (B), and TNF-α (C) expression in cell tests. (Mean± SD, n = 6). Cell-BC: blank control, Cell-NC: negative control, Cell-PC: 129 ng/mL Dexamethasone sodium phosphate, Cell-1: 150 μg/mL HA, Cell-2: 150 μg/mL Glu, Cell-3:150 μg/mL Graft-1, Cell-4: 150 μg/mL Graft-2, Cell-5: 150 μg/mL Graft-3. **P < 0.01 vs Cell-BC; ##P < 0.01 vs Cell-NC.

In addition to molecular and cellular assays, the 3D skin model demonstrated the functional benefits of HA-Glu on skin repair. HA-Glu was shown to ameliorate skin damage by enhancing the compactness of the stratum corneum and increasing the thickness of living cell layers (Figure 4 for ELISA results and Figure 5 for skin sample histology)[16-22].

 Figure 4: The expression levels of IL-1α, IL-6 and TNF-α in 3D model tests were detected by ELISA.

Results of IL-1α (A), IL-6 (B), and TNF-α (C) expression in 3D model tests. (Mean ± SD, n = 6). Model-BC: blank control, Model-NC: negative control, Model-PC1: 16.19 μg/mL Pirinixic acid, Model-PC2: 100 μg/mL Dexamethasone, Model-1: 500 μg/mL HA, Model-2: 250 μg/mL Graft-1, 250 μg/mL Graft-3. **P < 0.01 vs Model-BC; ##P < 0.01 vs Model-NC.

 Figure 5: H&E staining results of 3D skin model tests.

(Pink color: cytoplasm of cells; blue-purple color: nuclei) Model-BC: blank control, Model-NC: negative control, Model-PC₁: 16.19 μg/mL Pirinixic acid, Model-PC₂: 100 μg/mL Dexamethasone, Model-1: 500 μg/mL HA, Model-2: 250 μg/mL Graft-1, 250 μg/mL Graft-3.

The work suggests that the synergistic relationship between HA and Glu contributes to the improved outcomes. While HA facilitates Glu's interaction with cells and slows its own degradation, Glu provides additional antioxidant benefits and supports redox balance (Figure 6). The study concludes that HA-Glu holds significant promise for reducing inflammation-induced skin issues and signs of aging, positioning it as an effective ingredient in skin care formulations.

 Figure 6: Explanation of the inhibition mechanism of HA-Glu in HDFs.

Commentary: This innovative research offers a compelling advancement in cosmetic biochemistry by introducing a grafted molecule that could revolutionize skin care formulations. The meticulous approach to synthesizing and testing the HA-Glu compound underscores the potential of bio-conjugates in enhancing therapeutic benefits. Future studies may explore long-term clinical outcomes and potential side effects to further validate its efficacy and safety for widespread cosmetic use. Additionally, examining the interactions of HA-Glu with other skin components, such as the microbiome, could provide further insights into its holistic impact on skin health.

1. Quaresma JAS (2019) Organization of the Skin Immune System and Compartmentalized Immune Responses in Infectious Diseases. Clin Microbiol Rev 32: 18-34.
2. Balato A, Schiattarella M, Lembo S, Mattii M, Prevete N, et al. (2013) Interleukin-1 family members are enhanced in psoriasis and suppressed by vitamin D and retinoic acid. Arch Dermatol Res 305: 255-262.
3. Millrine D, Jenkins RH, Hughes STO, Jones SA (2022) Making sense of IL-6 signalling cues in pathophysiology. FEBS Lett 596: 567-588.
4. Bou-Dargham MJ, Khamis ZI, Cognetta AB, Sang Q-XA (2017) The Role of Interleukin-1 in Inflammatory and Malignant Human Skin Diseases and the Rationale for Targeting Interleukin-1 Alpha. Med Res Rev 37: 180-216.
5. Li Y, Zhao J, Yin Y, Li K, Zhang C, et al. (2022) The Role of IL-6 in Fibrotic Diseases: Molecular and Cellular Mechanisms. Int J Biol Sci 18: 5405-5414.
6. Khan K, Xu S, Nihtyanova S, Derrett-Smith E, Abraham D, et al. (2012) Clinical and pathological significance of interleukin 6 overexpression in systemic sclerosis. Ann Rheum Dis 71: 1235-1242.
7. Firlej E, Kowalska W, Szymaszek K, Rolinski J, Bartosinska J (2022) The Role of Skin Immune System in Acne. J Clin Med 11: 1579.
8. Borg M, Brincat S, Camilleri G, Schembri-Wismayer P, Brincat M (2013) The role of cytokines in skin aging. Climacteric 16: 514-521.
9. Brotas AM, Cunha JMT, Lago EHG, Machado CCN, Carneiro SCdaS (2012) Tumor necrosis factor-alpha and the cytokine network in psoriasis. An Bras Dermatol 87: 673-681.
10. Rayahin JE, Buhrman JS, Zhang Y, Koh TJ, Gemeinhart RA (2015) High and low molecular weight hyaluronic acid differentially influence macrophage activation. ACS Biomater Sci Eng 1: 481-493.
11. Zheng BW, Wang BY, Xiao WL, Sun YJ, Yang C, et al. (2022) Different molecular weight hyaluronic acid alleviates inflammation response in DNFB-induced mice atopic dermatitis and LPS-induced RAW 264.7 cells. Life Sci 301: 120591.
12. Hinoi E, Yoneda Y (2011) Possible involvement of glutamatergic signaling machineries in pathophysiology of rheumatoid arthritis. J Pharmacol Sci 116: 248-256.
13. Pacheco R, Oliva H, Martinez-Navío JM, Climent N, Ciruela F, et al. (2006) Glutamate released by dendritic cells as a novel modulator of T cell activation. J Immunol 177: 6695-6704.
14. Schanté CE, Zuber G, Herlin C, Vandamme TF (2012) Improvement of hyaluronic acid enzymatic stability by the grafting of amino-acids. Carbohydrate Polymers 87: 2211-2216.
15. Magnani A, Rappuoli R, Lamponi S, Barbucci R (2000) Novel polysaccharide hydrogels: Characterization and properties. Polymers for Advanced Technologies11: 488-495.
16. Neves-Petersen MT, Klitgaard S, Skovsen E, Petersen SB, Tømmeraas K, et al. (2010) Biophysical properties of phenyl succinic acid derivatised hyaluronic acid. J Fluoresc 20: 483-492.
17. Wen X, Huang A, Hu J, Zhong Z, Liu Y, et al. (2015) Neuroprotective effect of astaxanthin against glutamate-induced cytotoxicity in HT22 cells: Involvement of the Akt/GSK-3beta pathway. Neuroscience 303: 558-568.
18. Ohta S, Harigai M, Tanaka M, Kawaguchi Y, Sugiura T, et al., (2001) Tumor necrosis factor-alpha (TNF-alpha) converting enzyme contributes to production of TNF-alpha in synovial tissues from patients with rheumatoid arthritis. J Rheumatol 28: 1756-1763.
19. Loi C, Cynober L (2022) Glutamate: A Safe Nutrient, Not Just a Simple Additive. Ann Nutr Metab 78: 133-146.
20. Hensley CT, Wasti AT, DeBerardinis RJ (2013) Glutamine and cancer: cell biology, physiology, and clinical opportunities. J Clin Invest 123: 3678-3684.
21. Schanté C, Zuber G, Herlin C, Vandamme TF (2011) Synthesis of N-alanyl-hyaluronamide with high degree of substitution for enhanced resistance to hyaluronidase-mediated digestion. Carbohydr. Polym 86: 747-752.
22. Luan X, Cong Z, Anastassiades TP, Gao Y (2022) N-Butyrylated Hyaluronic Acid Achieves Anti-Inflammatory Effects In Vitro and in Adjuvant-Induced Immune Activation in Rats. Molecules 27.