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. 2022 Jan 18:12:695039.
doi: 10.3389/fphar.2021.695039. eCollection 2021.

The Japanese Herbal Medicine Hangeshashinto Induces Oral Keratinocyte Migration by Mediating the Expression of CXCL12 Through the Activation of Extracellular Signal-Regulated Kinase

Kanako Miyano  1   2 , Seiya Hasegawa  3 , Noriho Asai  2   3 , Miaki Uzu  4 , Wakako Yatsuoka  5 , Takao Ueno  5 , Miki Nonaka  2 , Hideaki Fujii  3 , Yasuhito Uezono  2   6
Affiliations

The Japanese Herbal Medicine Hangeshashinto Induces Oral Keratinocyte Migration by Mediating the Expression of CXCL12 Through the Activation of Extracellular Signal-Regulated Kinase

Kanako Miyano et al. Front Pharmacol. .

Abstract

Several clinical studies have reported that Japanese herbal medicine Hangeshashinto (HST) has beneficial effects on chemotherapy-induced oral ulcerative mucositis (OUM). Our previous research demonstrated that HST improves chemotherapy-induced OUM through human oral keratinocyte (HOK) migration, which was suppressed by mitogen-activated protein kinase (MAPK) and C-X-C chemokine receptor 4 (CXCR4) inhibitors. However, the association between these molecules and HOK migration was unclear. Here, we examined the effects of HST on the expression of CXCR4/CXCR7 and C-X-C motif chemokine ligands 11 and 12 (CXCL11/CXCL12) in HOKs. Our results indicated that HST upregulated CXCL12, but not CXCR4, CXCR7, nor CXCL11 in HOKs. HST-induced expression of CXCL12 was significantly suppressed by an inhibitor of extracellular signal-regulated kinase (ERK), but not of p38 and c-Jun N-terminal kinase (JNK). In addition, HST induced phosphorylation of ERK in HOKs. These findings suggest that HST enhances HOK migration by upregulating CXCL12 via ERK.

Keywords: CXCL12; extracellular signal-regulated kinase; hangeshashinto; oral keratinocytes; oral ulcerative mucositis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of MAPK inhibitors on Hangeshashinto (HST)-induced migration of human oral keratinocyte (HOKs). HOKs were scratched and co-treated with HST, CXCR4 inhibitor BDPA-Zn, ERK inhibitor U0126, JNK inhibitor II, or p38 inhibitor SB202190 for 72 h. Data are expressed as the mean ± SEM (bars, n = 12–50). *** indicates p < 0.001, compared with vehicle; #, ##, #### indicates p < 0.05, p < 0.01, p < 0.001 compared with HST alone. Bonferroni’s comparison test following ANOVA.
FIGURE 2
FIGURE 2
Effect of Hangeshashinto (HST) on the mRNA expression of CXCR4, CXCR7, CXCL11, or CXCL12 in human oral keratinocytes (HOKs). HOKs were scratched and treated with vehicle or HST for 48 h mRNA expression was determined by RT-qPCR. (A) CXCR4 (n = 5), (B) CXCR7 (n = 6), (C) CXCL11 (n = 5), or (D) CXCL12 (n = 6). Data are presented as the mean ± SEM (bars). * indicates p < 0.05, compared with vehicle; unpaired t-test.
FIGURE 3
FIGURE 3
Involvement of MAPKs in Hangeshashinto (HST)-induced expression of CXCL12 mRNA in human oral keratinocytes (HOKs). HOKs were scratched and co-treated with HST and inhibitors of (A) ERK (U0126; n = 3–5), (B) JNK (JNK inhibitor II; n = 3–5) or (C) p38 (SB202190; n = 3–8) for 48 h. mRNA expression was determined by RT-qPCR. Data are expressed as the mean ± SEM (bars). *, ** indicates p < 0.05, p < 0.01 compared with vehicle, respectively; # indicates p < 0.05 compared with HST alone. Bonferroni’s comparison test following ANOVA (A–C). n.s. indicates not significant (B,C).
FIGURE 4
FIGURE 4
Effect of Hangeshashinto (HST) on ERK phosphorylation in human oral keratinocytes (HOKs). HOKs were scratched and treated with vehicle or HST for 48 h. ERK expression was calculated using the ratio of the pERK-specific band density/ERK-specific band density determined by western blotting (n = 3). Data are expressed as the mean ± SEM. (bars). * indicates p < 0.05 compared with vehicle. Unpaired t-test.
FIGURE 5
FIGURE 5
Effect of components of Hangeshashinto (HST) on the expression of CXCL12 mRNA in human oral keratinocytes (HOKs). HOKs were scratched and treated with vehicle, 6-shogaol (1 µM), 10-gingerol (10 µM), or glycyrrhetinic acid (10 µM) for 48 h. mRNA expression was determined by RT-qPCR. Data are expressed as the mean ± SEM. (bars). * indicates p < 0.05 compared with vehicle. Bonferroni’s comparison test following ANOVA.
FIGURE 6
FIGURE 6
Schematic diagram showing the mechanism of the Japanese herbal medicine HST inducing oral keratinocyte migration by mediating the expression of CXCL12 through the activation of ERK. HST induces ERK phosphorylation and upregulates CXCL12, which activates its receptor CXCR4, and induces cell migration.
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