2026 年 49 巻 2 号 p. 249-253
Hepatocyte growth factor (HGF) exhibits mitogenic, motogenic, and morphogenic activities. Enhancing HGF production could serve as a therapeutic approach for organ regeneration, wound healing, and embryogenesis. Notably, HGF demonstrates therapeutic potential in the treatment of neurodegenerative diseases. In this study, we found that quercetin promotes HGF production in normal human dermal fibroblasts (NHDF) at low concentrations. cAMP response element binding protein (CREB), a transcription factor, is phosphorylated. Additionally, this activity may result from quercetin’s interaction with the β2 adrenaline receptor (β2AR). Further pharmacological analysis suggested that HGF production is promoted via PKA pathway. In conclusion, quercetin shows potential as a drug for treating organ-related diseases, including neurodegenerative disorders, by enhancing HGF production.
Hepatocyte growth factor (HGF) plays a pivotal role in liver regeneration.1) HGF is a heterodimer consisting of a heavy chain and a light chain.2) By binding with c-Met, HGF exhibits multiple activities, including mitogenic, motogenic, morphogenic, and anti-apoptotic effects.3,4) These effects can serve as therapeutic approaches for organ regeneration, wound healing, and embryogenesis.5–7) Recently, studies have suggested that HGF has potential as a therapeutic drug for neurodegenerative diseases such as amyotrophic lateral sclerosis8,9) and Alzheimer’s disease.10)
Conversely, inhibition of HGF may be a potent strategy for cancer treatment. Because HGF is involved in cell growth, proliferation, and survival, its inhibition is being explored as an anticancer therapy.11) For instance, an endogenous inhibitor of HGF protease has shown a potential to interfere with colorectal cancer progression.12)
Therefore, molecules capable of modulating HGF production could serve as valuable therapeutic tools. In our previous studies, we demonstrated that caffeoylquinic acid, acteoside, and caffeic acid promote HGF production.13) Additionally, we found that cyanidin-3-glucoside (C3G) enhances HGF production, while cyanidin alone does not.14) In this research, we concluded that sugar moiety plays a key role in HGF promotion. Additionally, the mechanism of HGF production was elucidated, showing that C3G binds to the β2 adrenaline receptor (β2AR) and induces subsequent events such as an increase in cAMP levels.
In this study, we identified a novel small chemical compound capable of inducing HGF production: quercetin. Quercetin is a plant-based natural product that possesses various biological activities, including anti-inflammation, anticancer, and neurodegenerative effects.15–17) However, its ability to promote HGF production has not been reported previously. Our investigation suggests that quercetin can enhance HGF production by binding with β2AR.
The test compounds used in this study were obtained from the following manufacturers: C3G (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), cyanidin chloride (FUJIFILM Wako Pure Chemical Corporation), quercetin (Funakoshi, Tokyo, Japan), piceatannol (Funakoshi), catechin (Tokyo Chemical Industry, Tokyo, Japan), isoorientin (Tokyo Chemical Industry), epigallocatechin gallate (EGCG) (Nacalai Tesque, Inc., Kyoto, Japan), platelet-derived growth factor-BB (PDGF-BB) (FUJIFILM Wako Pure Chemical Corporation), forskolin (Nacalai Tesque, Inc.), isoproterenol (Nacalai Tesque, Inc.), ICI 118551 (ICI, Cayman Chemical, Ann Arbor, MI, U.S.A.), and KT5720 (KT, Cayman Chemical).
Cell CultureNormal human dermal fibroblasts (NHDF) cells, purchased from Kurabo Co., Ltd. (Osaka, Japan), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Co. LLC., St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, U.S.A.) at 37°C in humidified 5% CO2 atmosphere. Human Embryonic Kidney 293 T cells (HEK293T), provided by RIKEN Bioresource Research Center (Ibaraki, Japan), were cultured in DMEM containing 10% fetal bovine serum and 1% of 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, Co., LLC.) at 37°C in humidified 5% CO2 atmosphere.
Enzyme-Linked Immunosorbent Assay (ELISA)/BCA AssayNHDF cells were seeded in a 96-well plate (Nunc, Roskilde, Denmark) at a density of 1 × 104 cells/well and incubated overnight. The cells were treated with test samples dissolved in 100 μL of DMEM supplemented with 0.5% FBS for 120 h. After treatment, the conditioned medium was collected for the quantification of HGF. Cell layers were washed with phosphate-buffered saline (PBS) and lysed with 0.5% Triton X-100 in PBS. Subsequently, the amount of cellular protein was quantified using a BCA assay (TaKaRa Bio, Inc., Shiga, Japan).
The sandwich human HGF ELISA was performed at room temperature. 96-well plates were coated with antihuman HGF monoclonal antibody (0.2 μg/mL, diluted in PBS) (R&D Systems, Inc., Minneapolis, MN, U.S.A.) and incubated overnight at 4°C. The wells were washed with 0.05% Tween 20 in PBS and incubated with PBS containing 1% BSA, 5% Tween 20, and 5% sucrose for 1 h. After washing, the conditioned medium was added to the wells. Simultaneously, human HGF (R&D Systems, Inc.) for the standard curve was also added within the range of 0–50 ng/mL. After a 2-h incubation, the wells were washed and subsequently incubated with biotinylated goat antihuman HGF antibody (250 ng/mL, diluted in PBS) (R&D Systems, Inc.) for 90 min. After further washing, streptavidin-HRP conjugate (200 ng/mL, diluted in PBS) (Sigma-Aldrich, Co., LLC.) was added and incubated for 30 min. The wells were washed again, and a substrate solution containing 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 0.3 mg/mL) (Sigma-Aldrich, Co., LLC.) was mixed with H2O2 at a ratio of 1 : 1000, added to the wells, and incubated for additional 30 min. Stop solution (1 M H2SO4) was added, and the optical density of each well was measured at 450 nm using a microplate reader (Varioskan Lux, Thermo Fisher Scientific). The HGF levels were expressed as pg/μg cellular protein.
Western BlotNHDF cells were treated with 15 μM test samples and 1 μM folskolin, and then lysed in RIPA buffer (Nacalai Tesque, Inc.) mixed with a 1% phosphatase inhibitor cocktail (Nacalai Tesque, Inc.). Total cellular protein samples were boiled in 5× SDS sample buffer for 5 min, separated by 10% SDS-PAGE, and transferred to Immobilon-P PVDF membrane (Merck Millipore, Burlington, MA, U.S.A.). The blot was incubated with a blocking solution (2% BSA in TBS/T solution) and then probed with anti-pCREB (1 : 1000, Cell Signaling Technology, Danvers, MA, U.S.A.) or anti-GAPDH (1 : 3000, Thermo Fisher Scientific). Horseradish peroxidase (HRP)-conjugated anti-rabbit (Cell Signaling Technology) or anti-mouse secondary antibodies (Cell Signaling Technology) were used at a 1 : 3000 dilution. Immunoreactive bands were visualized using immobilon Western Chemiluminescent HRP substrate (Merck Millipore) using LuminoGraphI (WSE-6100, ATTO Corporation, Tokyo, Japan).
cAMP Response Element (CRE) Reporter AssaypcDNA3.1-β2AR was constructed by SLiCE as mentioned in a previous study.14) HEK293T cells were transfected with pGL4.29 (Promega Corporation, Madison, WI, U.S.A.) and pcDNA3.1-β2AR using Hilymax (Dojindo, Kumamoto, Japan). Transfected cells were treated with test samples, 10 μM isoprotenol, and 10 μM ICI for 24 h. CRE promoter activity was measured using a luciferase assay system (Promega Corporation). Luminescence values (RLU) corresponding to CRE promoter activity were quantified using a microplate reader (Synergy H1, WakenBtech, Kyoto, Japan).
Cell Viability AssayConfluent NHDF cells were seeded into a 96-well plate at a density of 1 × 104 cells/well. The cells were treated with quercetin for 120 h. WST-1 reagent (Dojindo) solution was added to the cell culture. After 2 h, the absorbance was measured at 570 nm using a microplate reader (Synergy H1, WakenBtech).
Measurement of mRNA Abundance of HGF GeneNHDF cells were treated with test samples and inhibitors, 20 μM KT or 10 μM ICI, for 48 h, and total RNA was extracted using the FastGeneTM RNA premium kit (Nippon Gene Co., Ltd., Tokyo, Japan). Reverse transcription was performed with the Superscript ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Co., Ltd., Osaka, Japan). Real-time PCR analysis was carried out using the THUNDERBIRD Next SYBR qPCR Mix (Toyobo, Co., Ltd.) and analyzed on a Quant Studio 5 system (Applied Biosystems, Waltham, MA, U.S.A.). Gene expression levels were normalized to β-actin. The Primer sequences used were as follows: HGF, forward: 5′-ACGAACACAGCTTTTTGCCTT-3′, HGF, reverse 5′-AACTCTCCCCATTGCAGGTC-3′, β-actin, forward 5′-CTGTGGCATCCACGAAACTACC-3′, β-actin, reverse 5′-GCAGTGATCTCCTTCTGCATCC-3′.
Statistical AnalysisAll data are shown as the mean ± standard deviation (S.D.). Statistical analysis was performed by coding in Python on Google colaboratory and Dunnett’s test was adopted.
In this study, we, at first, treated quercetin with NHDF cells. As a result, quercetin upregulates HGF production at the mRNA level (Fig. 1A). Previous research indicates that cyanidin, a flavonoid, did not enhance HGF production, suggesting the necessity of sugar moieties in this process.14) However, certain flavonoids, such as quercetin, were capable of promoting HGF production, emphasizing the critical role of the flavonoid C-ring. In this context, the sugar moiety in C3G may act as an alternative functional element to the C-ring of cyanidin. Quercetin is also known for their therapeutic roles in neurodegenerative diseases. To investigate this at the protein level, we performed an ELISA assay to measure HGF production. The results showed that quercetin increased HGF protein production. While quercetin effectively promoted HGF production at lower concentrations, its promotive effect diminished at higher concentrations (Fig. 1B). Importantly, quercetin demonstrated no toxicity even at 50 μM (Fig. 1C). These findings suggest that quercetin may function as a partial agonist.
(A) HGF increases in mRNA level (n = 3). Statistical analyses were performed using the Dunnett’s test (***p < 0.01). Comparisons without any markings are not significant. (B) HGF production measured by ELISA. PDGF is used as a positive control. The values were represented as mean ± standard deviation (n = 4). Statistical analyses were performed using Dunnett’s test (**p < 0.05). Comparisons without any markings are not significant. (C) Cytotoxicity measured by WST-1 assay (n = 4).
Previous research has suggested that the CRE-binding protein (CREB) may regulate HGF expression. To determine the signaling pathway involved, we measured the phosphorylation of CREB by 15 μM quercetin (Fig. 2). Forskolin was used as the positive control. The results showed that quercetin induced the phosphorylation of CREB. These findings indicate that CREB could act as a promoter of HGF production, similar to the C3G.
Quercetin was treated for 15 or 60 min. Forskolin is used as a positive control. GAPDH is used as endogenous control.
To identify the receptor for quercetin, we conducted a luciferase assay using HEK293T cells (Fig. 3A). When quercetin was applied to HEK293T cells transfected with p.GL 4.29, luminescence increased partially in a concentration-dependent manner. However, owing to the limited number of receptors in HEK293T cells, we co-transfected it with β2AR. Following β2AR transfection, similar trends were observed, with β2AR activation occurring at approximately 10 μM. ICI, an inhibitor of adrenaline receptors, suppressed HGF production even in the presence of β2AR transfection.
(A) Activation of β2AR measured by luciferase assay. Quercetin was treated for 24 h. Subsequently, luminescence was measured. Co-transfection of β2AR and pGL 4.29 is described as +β2AR. ISO is isoproterenol (positive control). The values were represented as the mean ± standard deviation (n = 3). Statistical analyses were performed using the Dunnett’s t-test (***p < 0.01; **p < 0.05; *p < 0.1). Each statistical test was calculated separately for transfected and non-transfected samples. Comparisons without any markings are not significant. (B) HGF production is inhibited by PKA inhibitor (KT) and β2AR inhibitor (ICI) in qPCR in NHDF cells (n = 3). Statistical analyses were performed using the Dunnett’s test (***p < 0.01; **p < 0.05). Comparisons without any markings are not significant.
In order to examine if quercetin’s bind with β2AR, we performed RT-qPCR again with inhibitors: ICI and KT5720 (KT). As a result, both inhibitors interfere with HGF production. ICI is a β2AR’s inhibitor. KT is a PKA inhibitor. Therefore, β2AR and CREB are surely involved with promotion of HGF production (Fig. 3B). Additionally, what KT interferes with HGF production means that PKA pathway is involved with quercetin’s induction of HGF production.
These results indicate that β2AR serves as a receptor of quercetin. Interestingly, at higher concentrations, quercetin inhibited the activation of β2AR, suggesting its role as a partial agonist. This behavior corresponds to ELISA results. In another study, caffeic acid promotes HGF production. Caffeic acid and quercetin are similar to adrenalin13) (Fig. 4). Especially, both quercetin and caffeic acid have the same catechol moiety in common with adrenalin. Therefore, these structures could be important to bind with adrenalin receptor.
Quercetin might induce other cytoprotective factors like BDNF as well. A recent study suggests that β2AR selective agonist, salmeterol, induces BDNF production.18) Quercetin also activates β2AR, so this compound can induce BNDF via the same pathway as well. We should investigate if quercetin has such ability in future research.
In summary, quercetin at low concentrations promotes HGF production by binding to β2AR, which activates the PKA pathway and phosphorylates CREB. Thus, quercetin holds potential as a therapeutic agent for organ repair, neurodegenerative disease inhibition, and related conditions. However, at higher concentrations, quercetin does not contribute to HGF production.
This work was partially supported by JSPS KAKENHI Grant Number: 23K23547.
The authors declare no conflict of interest.
