Ann Dermatol.  2019 Jun;31(3):279-285. 10.5021/ad.2019.31.3.279.

Inhibition of Poly(I:C)-Induced Inflammation by Salvianolic Acid A in Skin Keratinocytes

• Zhang QL
• Jiang RH
• Li XM
• Ko JW
• Kim CD
• Zhu MJ
• Lee JH

• KMID: 2444859
• DOI: http://doi.org/10.5021/ad.2019.31.3.279

Abstract

BACKGROUND
Skin keratinocytes participate actively in inducing immune responses when external pathogens are introduced, thereby contributing to elimination of pathogens. However, in condition where the excessive inflammation is occurred, chronic skin disease such as psoriasis can be provoked.
OBJECTIVE
We tried to screen the putative therapeutics for inflammatory skin disease, and found that salvianolic acid A (SAA) has an inhibitory effect on keratinocyte inflammatory reaction. The aim of this study is to demonstrate the effects of SAA in poly(I:C)-induced inflammatory reaction in skin keratinocytes.
METHODS
We pre-treated keratinocytes with SAA then stimulated with poly(I:C). Inflammatory reaction of keratinocytes was verified using real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot.
RESULTS
When skin keratinocytes were pre-treated with SAA, it significantly inhibited poly (I:C)-induced expression of inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α, and CCL20. SAA inhibited poly(I:C)-induced activation of nuclear factor-κB signaling. And SAA also inhibited inflammasome activation, evidenced by decrease of IL-1β secretion. Finally, SAA markedly inhibited poly(I:C)-induced NLRP3 expression.
CONCLUSION
These results demonstrate that SAA has an inhibitory effect on poly(I:C)-induced inflammatory reaction of keratinocytes, suggesting that SAA can be developed for the treatment of inflammatory skin diseases such as psoriasis.

Keyword

Inflammasomes; Keratinocytes; Poly(I-C); Salvianolic acid A

MeSH Terms

Blotting, Western
Cytokines
Enzyme-Linked Immunosorbent Assay
Inflammasomes
Inflammation*
Interleukin-6
Interleukin-8
Interleukins
Keratinocytes*
Necrosis
Psoriasis
Real-Time Polymerase Chain Reaction
Skin Diseases
Skin*
Cytokines
Inflammasomes
Interleukin-6
Interleukin-8
Interleukins

Figure

• 

  Fig. 1 (A) Structure of salvianolic acid A (SAA). (B) Cytotoxicity of SAA in keratinocytes. The immortalized SV40T-transformed human epidermal keratinocytes were treated with indicated concentrations of SAA for 24 hours. MTT assay was performed to measure cell viability. SAA did not show cytotoxicity up to 10 µM. The mean values±standard deviation are averages of triplicate measurements.

• 

  Fig. 2 Effect of salvianolic acid A (SAA) on poly(I:C)-induced inflammatory reaction in keratinocytes. (A) The immortalized SV40T-transformed human epidermal keratinocytes (SV-HEKs) were pre-treated with SAA at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml of poly(I:C) for 2 hours. The mRNA level was assessed by quantitative real-time polymerase chain reaction. SAA inhibited poly(I:C)-induced cytokine expressions. Data are expressed as fold induction. (B) SV-HEKs were pre-treated with SAA at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml of poly(I:C) for 24 hours. Secretion of cytokines was measured by enzyme-linked immunosorbent assay. SAA inhibited poly(I:C)-induced secretion of cytokines from SV-HEKs. The mean values±standard deviation are averages of triplicate measurements. IL: interleukin, TNF-α: tumor necrosis factor-α. *p<0.01.

• 

  Fig. 3 Effect of salvianolic acid A (SAA) on poly(I:C)-induced nuclear factor-κB (NF-κB) activation in keratinocytes. (A) The immortalized SV40T-transformed human epidermal keratinocytes were pre-treated with SAA at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml of poly(I:C) for 1 hour. The protein levels of phosphorylated-p65 (p-p65) and phosphorylated-IκBα (p-IκBα) were determined by Western blot. SAA inhibited poly(I:C)-induced activation of NF-κB. β-actin was used for internal control. (B) Effect of SAA on phosphorylation of mitogen-activated protein kinases (MAPKs). SAA inhibited poly(I:C)-induced phosphorylation of p38 MAPK, JNK, and ERK1/2.

• 

  Fig. 4 Effect of salvianolic acid A (SAA) on poly(I:C)-induced inflammasome activation in keratinocytes. (A) The immortalized SV40T-transformed human epidermal keratinocytes were pre-treated with SAA at the indicated concentrations for 1 hour, and then stimulated with 1 µg/ml poly(I:C) for 24 hours. Culture medium was collected and concentrated, then subjected to Western blot to assess the secreted protein levels for interleukin (IL)-1β and caspase-1. SAA inhibited poly(I:C)-induced secretion of IL-1β and caspase-1. Ponceau S staining was used for loading control. (B) After treatment with SAA, cellular extracts were prepared. The protein levels of NLRP3 and ASC were determined by Western blot. SAA inhibited poly(I:C)-induced NLRP3 expression. β-actin was used for internal control.

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