Qingre Huoxue Decoction, A Traditional Chinese Herbal Formulation, Impacts Angiogenesis in Psoriasis-Like Skin Through the HIF-1α/Flt-1/VEGF Pathway

Background: Qingre Huoxue Decoction (QHD), a traditional Chinese medicine (TCM) formulation, could alleviate psoriasis in our previous studies. The present work aimed to assess QHD’s effects on psoriasis and the underpinning mechanism in cultured cells and experimental animals. Methods: The CCK-8 assay was carried out for cell viability assessment. HUVEC migration was assessed by transwell and wound healing assays. QHD-induced suppression of capillary tube formation in HUVECs was detected by tube formation assay. In addition, the imiquimod (IMQ)-induced male BALB/c mouse model of psoriasis was established to examine the Psoriasis Area and Severity Index (PASI) after QHD administration. HIF-1α, Flt-1 and VEGF expression levels in vivo were assessed by immunoblot, qPCR and immunouorescence. Results: The results showed that QHD dose-dependently reduced viability in HUVECs. In addition, QHD suppressed tube formation in HUVECs at levels below those needed to inhibit HUVECs. Upon QHD administration, HUVEC migration was markedly decreased; QHD effectively prevented the migratory ability of HUVECs, as determined by wound areas at 0h, 12h and 24h, respectively. Finally, QHD starkly downregulated HIF-1α, Flt-1 and VEGF in the IMQ-induced mouse model, at the protein and mRNA levels. Conclusions: In summary, QHD inhibits angiogenesis in cultured cells and mice. HIF-1α/Flt-1/VEGF signaling is important in angiogenesis and psoriasis development. These ndings provide a rationale for developing QHD for clinical use against psoriasis.


Introduction
Psoriasis represents an incurable chronic in ammatory skin disease affecting about 3% of all humans 1 .
In Caucasians, an incidence of about 100,000 people per year has been reported. Histologically, psoriasis mostly features enhanced angiogenesis, unrestrained growth, abnormal keratinocyte differentiation and high amounts of in ltrated immune cells 2,3 . However, how psoriasis is initiated remains unknown. It was suggested that angiogenesis represents a hallmark of psoriasis and psoriatic arthritis 4 . Meanwhile, angiogenesis is accompanied by enhanced endothelial cell (EC) proliferation, adhesion and migration 5 .
Critical angiogenic factors, including vascular endothelial growth factor (VEGF) and its receptor Flt-1, show increased amounts in human psoriasis skin 6 . Mounting evidence indicates vascular endothelial growth factor (VEGF) upregulation constitutes an early and essential factor in psoriasis pathogenesis. It was recently demonstrated hypoxia-induced VEGF is a hallmark of psoriasis. In agreement, after psoriasis treatment with a TNF inhibitor, VEGF and Flt-1 are downregulated in P-DMSCs, and angiogenesis is reduced in the skin lesion. Hypoxia is characteristic of the skin tissue in patients with psoriasis, with HIF in keratinocytes and endothelial cells promoting angiogenesis by increasing the expression of VEGF and Flt-1, respectively. In psoriasis, abnormal rapid cell proliferation increases oxygen consumption, while epidermal thickening reduces oxygen supply. It was suggested local hypoxia in skin might be critical for adaptive upregulation of angiogenic proteins, including hypoxia inducible factor-1 alpha (HIF-1α).

Methods
The CCK-8 assay The CCK-8 assay was carried out for cell viability assessment. Cell seeding was performed in 96-well plates at 10 5 cells per well followed by a 24-h incubation at 37°C prior to the administration of QHD at 0, 25, 50, 100, 150 and 200µg/ml for 48h. Post-treatment, 0.5 mg/ml CCK-8 solution (100µl per well) was supplemented for a 2-h incubation. OD was read on a Varioskan Flash multiscan plate reader (Thermo, USA) at 450 nm. Triplicate assays were carried out.

Transwell migration assay
HUVECs migration was assessed in 24-well Transwell plates using 8-µm-pore polycarbonate membrane inserts (Corning, USA) with Matrigel (BD Biosciences) coating as directed by the manufacturer. Brie y, 3×10 5 cells in 100µl medium were placed in each upper compartment for a 24-h incubation at 37°C. Cells detected on the lower surface of the membrane by hematoxylin staining were numbered in ve randomly selected high-power elds. Triplicate assays were carried out.

Scratch assay
The scratch assay was performed to further examine HUVEC migration as reported previously 8 . In brief, cells were treated for 48h and a sterile 200-µl micropipette tip was used for scratching the well's center.
Upon washing with PBS, EBM with 1% FBS (Gibco Life Technologies, USA) was supplemented. The samples were incubated at 37°C, and gaps were examined at 0, 12 and 24h, respectively, using an inverted microscope equipped with an Olympus DP70 camera (Olympus, Japan). Image J was utilized for quantitation.

Tube formation assay
HUVECs were examined for tube formation with Geltrex-reduced growth factor basement membrane matrix (Invitrogen), as directed by the manufacturer. For quantifying tube formation, an inverted microscope (Olympus) was utilized to count meshes which were further analyzed with Image J.

Immunoblot
Prior to protein extraction, imiquimod (IMQ)-induced mice were treated with QHD for 24h. Total protein was extracted from IMQ-induced mouse samples using a protein extraction kit (Applygen Technologies,

QHD inhibits proliferation and capillary tube formation in HUVECs
We evaluated QHD's effects on HUVEC proliferation by the CCK-8 assay. It was found QHD dosedependently reduced proliferation in HUVECs (Figure.1A). Following a 24-h treatment, QHD's IC50 in HUVECs averaged 471.5µg/ml. Primary HUVECs incubated for 24 adhered to the plate. Following a 3 to 5day incubation, corroborating previously reported ndings, HUVECs showed cobblestone-like morphology. What's more, the angiogenic capability of HUVECs was dose-dependently altered by QHD. QHD at 20, 200 and 400µg/ml for 24h, respectively, markedly reduced tube formation in HUVECs in comparison with untreated cells (P<0.05) (Figure.1B). Jointly, the above data demonstrated QHD suppressed tube formation in HUVECs at levels starkly lower than needed for inhibiting HUVECs. Remarkably, QHD promoted capillary tube formation in HUVECs.

QHD suppresses HUVEC migration in vitro
Endothelial cell migration is important in neovascularization, and involves chemotactic, haptotactic and mechanical components. HUVEC migration was rst assessed by the transwell assay ( Figure.2A). In comparison with control cells, the QHD groups of HUVECs had starkly reduced migration. Next, HUVECs were examined for mechanical migration by the scratch assay. At 0, 12 and 24h upon wounding, respectively, shorter distances were travelled by HUVECs after QHD treatment (Figure.2B). These data also demonstrated that QHD effectively reduced the migratory ability of HUVECs.
QHD impacts angiogenesis through the HIF-1α/Flt-1/VEGF pathway in the imiquimod (IMQ)-induced mouse model In the process of angiogenesis, HIF-1α/Flt-1/VEGF signaling plays a critical role. To further assess QHD's effects, the IMQ-induced mouse model was established. At 3-5 days, mouse recombinant QHD was given by gavage. On the seventh day, psoriasis-like skin in ammation was observed as re ected by erythema and scaling (Figure.3). In this study, QHD-reduced angiogenesis was investigated using dorsal skin images of BALB/c mouse psoriasis models ( Figure.

Discussion
It was found that local hypoxia is an important feature of psoriasis. Hypoxia is a major factor in the early pathogenetic events of psoriasis, contributing to in ammatory cell recruitment; meanwhile, the cross-talk involving epidermal and immune cells likely control psoriasis development. Therefore, adjustment of the hypoxic microenvironment is an important direction in the treatment of psoriasis. Angiogenesis is one of the major mechanisms of tissue adaptation to hypoxia.
It is admitted local hypoxia in skin is essential in the adaptive induction of angiogenesis, upregulating multiple effectors such as hypoxia inducible factor-1 alpha (HIF-1α). Multiple reports have revealed HIF-1α is upregulated in psoriatic skin lesions and in blood.
HIF-1α is a heterodimeric transcriptional complex comprising the HIF-1α and HIF-1β subunits, with a function in controlling oxygen levels 9,10 . However, HIF-1 was recently shown to affect other stress response events beside hypoxia. Meanwhile, HIF-1α amounts are elevated in psoriatic lesions 11,12 , and in serum specimens from psoriasis cases. These elevated HIF-1α amounts alongside a high association of HIF-1α upregulation with disease severity suggest a major role for HIF-1α in psoriasis' pathogenetic mechanisms 13 . Therefore, angiogenesis, exaggerated proliferation and in ammatory response might constitute a vicious circle, with HIF-1α helping maintain and amplify this vicious circle. In agreement, drugs controlling HIF-1α expression might be e cient in disease management. HIF-1α could represent an important target to control in ammatory response and angiogenesis, since many effectors in various pathways seem to involve HIF-1α.
Traditional Chinese medicinal (TCM) preparations have been effectively utilized in treating psoriasis for years. Recently, angiogenesis in psoriasis was shown to be suppressed by TCM preparations. The present study showed QHD inhibits proliferation, migration and tube formation in cultured HUVECs. The present data also demonstrated QHD can signi cantly reduce the PASI in IMQ-induced mouse models. Multiple preliminary works showed angiogenesis is associated with the HIF-1α/Flt-1/VEGF pathway. The IMQinduced mouse model was established to further explore the mechanism underpinning the inhibitory effects of QHD on psoriasis. The present in vivo study showed that QHD effectively suppresses angiogenesis, which is associated with the HIF-1α/Flt-1/VEGF pathway.

Conclusions
In summary, this study rstly reported associations of HIF-1α, Flt-1 and VEGF with psoriasis via QHD intervention. These ndings indicate QHD inhibits angiogenesis both in cultured cells and in mice. HIF-1α/Flt-1/VEGF signaling is critical for angiogenesis, even in psoriasis. The TCM preparation QHD could inspire a rational development of new therapeutic options for clinical psoriasis. However, the molecular mechanism behind QHD's effect on the HIF-1α/Flt-1/VEGF pathway is unclear, which deserves further investigation.

Declarations Ethics approval
All animal studies were approved by the ethics committee of the Shuguang Hospital a liated to Shanghai University of Traditional Chinese Medicine and the principles of laboratory animal care were followed in all animal experiments. The study was carried out in compliance with the ARRIVE guidelines.  The Dorsal Skin Images of BALB/c Mouse Psoriasis Models Induced by Imiquimod. Psoriasis-like skin in ammation was induced to the back skin of the mice characterized by erythema and scaling.
QHD reduced angiogenesis was investigated through the dorsal skin images of BALB/c mouse psoriasis models induced by imiquimod. Statistical difference was analyzed by Student's t-test. *P<0.05 compared with that of control group and MTX group. The HIF-1α, Flt-1 and VEGF relative genes expression levels were calculated using the 2-ΔΔCT method. P < .05 was considered to be statistically signi cant. Graph shows mean ± SD. * p < .05, ** p < .01, ***p < .05.