Effect of retinoic acid combined with narrow-band ultraviolet B on matrix metalloprotein 13 （ MMP13 ） in psoriasis

Matrix metalloproteinase 13 (MMP13) is a zinc-containing endopeptidase secreted by keratinocytes and skin fibroblasts and participates in many inflammatory diseases. Drugs for retinoic acid include tazarotene and acitretin. Tazarotene/acitretin and narrow-band ultraviolet B (NB-UVB) irradiation are used as a general treatment for psoriasis. However, their impact on MMP13 expression has yet to be determined. In this study, we measured the expression of MMP13 in patients with psoriasis, and investigated the effects of tazarotene and/or NB-UVB on MMP13 expression in a mouse model of psoriasis. After exposure to acitretin and/or NB-UVB, immortalized human HaCaT keratinocytes were analyzed for viability and MMP13 expression. Our results showed that MMP13 protein levels increased in skin lesions and serum samples in patients with psoriasis. Treatment with acitretin and NB-UVB irradiation alone or in combination suppressed cell viability and MMP13 expression in HaCaT cells. Consistently, tazarotene treatment and/or NB-UVB irradiation attenuated imiquimod-induced psoriasis-like dermatitis and inhibited MMP13 expression in a mouse model. Taken together, these results indicate that tazarotene/acitretin and NB-UVB irradiation can inhibit the expression of MMP13 in keratinocytes and psoriasis mouse models. Targeting MMP13 may represent a promising therapeutic strategy against psoriasis.


INTRODUCTION
Psoriasis is a chronic recurrent inflammatory disease caused by abnormal proliferation and differentiation of keratinocytes [1][2][3] . Matrix metalloproteinases (MMPs) are structurally related zinc-dependent endopeptidases capable of degradation of extracellular matrix (ECM) 4;5 . MMP13 has been reported to regulate multiple pathological processes such as arthritis, infection, and tumor progression [5][6][7] . Both topical and systemic vitamin A derivatives, are used in the treatment of psoriasis. Tazarotene is a topical agent and acitretin is a systemic retinoid 8 . Both are most effective in combination with other treatment modalities. Tazarotene and acitretin are selective retinoic acid receptor (RAR) agonists. They show the ability to regulate keratinocyte proliferation and differentiation and inhibit inflammation 8 . Narrow-band UVB (NB-UVB) that emits mostly 311/312 nm lights is commonly used in phototherapy 9 . NB-UVB has a more profound therapeutic efficacy for psoriasis than conventional UVB, with a shorter period of time and fewer adverse effects.
NB-UVB irradiation can result in lymphocyte apoptosis and decrease the number of Langerhans cells 10; 11 . Psoriasis resolves to a greater degree in patients treated with NB-UVB in combination with retinoic acid than in patients treated with either NB-UVB or retinoic acid alone 8; 12-14 . Despite these findings, the mechanism underlying the therapeutic benefits in psoriasis is still unclear.
In this study, we determined the expression of MMP13 in patients with psoriasis and investigated the influence of tazarotene/acitretin and NB-UVB irradiation on the expression of MMP13 in imiquimod (IMQ)-induced mice and keratinocytes.

Ethics statement
All studies involving human patients were approved by the General Hospital of Tianjin Medical University (Tianjin, China). Participants gave their informed consent in writing, and the study was conducted according to the declaration of Helsinki.

Tissue sample collection
Eighteen cases of skin lesions were collected from patients with psoriasis who were diagnosed in General Hospital of Tianjin Medical University between May 2019 and August 2019. Exclusion criteria were ongoing systemic anti-inflammatory treatment, other rheumatological diseases, other inflammatory dermatoses, pregnancy, intense UV exposure two weeks prior to study start, chronic infectious diseases, exposure to photosensitizing drugs, and age < 18 years. Ten cases of normal skin samples were obtained from patients undergoing cosmetic surgery in General Hospital of Tianjin Medical University. Blood samples were taken for preoperative HIV screening, and serum samples were stored at -80˚C. Skin biopsies were paraffin-embedded and subjected to immunohistochemistry.

Animal experiments
Twenty-five male BALB/c mice (6-8 weeks old) were purchased from Beijing Viton LiHua Co., LTD (Beijing, China). Mice were maintained at 24-25 • C under a 12 h light-dark cycle with free access to food and water. The study was performed in accordance with the European guideline for care in animal research and approved by the Institutional Review Board of General Hospital of Tianjin Medical University.
All the mice were randomly divided into 5 groups: control group, IMQ-induced group, IMQ+ tazarotene group, IMQ+NB-UVB irradiation group, and IMQ+ tazarotene+ NB-UVB irradiation group. Control mice were untreated. The IMQ-induced group received 62.5 mg IMQ cream on the shaved back daily for 7 days to induce psoriasis-like skin inflammation 15 . IMQ + tazarotene group, IMQ + NB-UVB irradiation group, and IMQ + tazarotene + NB-UVB irradiation groups received the IMQ treatment daily for 7 days together with tazarotene treatment, 300mJ/cm 2 NB-UVB irradiation, with 50mJ/cm 2 increase on the next day, and tazarotene plus NB-UVB irradiation, respectively. The 2 × 2 cm 2 skin on the back of mice was selected as the observation area. At the end of the experiment, all mice were sacrificed. Blood was taken from the eyeball of mice. The dorsal skin sample of mice was paraffin-embedded and analyzed for MMP13 expression. The epidermal thickness was quantified using a computer-assisted image analyzer.

Histology and immunohistochemistry
For histology, skin tissues were fixed in 4% buffered formalin, embedded in paraffin, and sectioned.
Consecutive sections were stained with hematoxylin and eosin (H&E) and Safranin-O. For immunohistochemistry, sections were deparaffinized and rehydrated. Endogenous peroxidase was blocked with 0.6% hydrogen peroxide in methanol. Sections were incubated with anti-MMP13 antibody overnight at 4˚C. An appropriate Bright Vision peroxidase system (Immunologic) was used. The staining was visualized with diaminobenzidine.

Enzyme-linked immunosorbent assay (ELISA) for MMP13
MMP13 concentrations in the serum samples and supernatants of cells were quantified using a commercially available MMP13 ELISA kit according to the protocols provided by the manufacturers. All analyses were performed in triplicate. Optical densities were measured at 450 nm using a microplate reader.

CCK-8 analysis
100µl of cell suspension (1×10 3 cells/well) was dispensed in a 96-well microtiter plate, and then pre-incubated in an incubator with humidified atmosphere and 5% CO 2 at 37 °C for 24h. HaCaT cells were exposed to 1µM acitretin and/or to 50-100mJ/cm 2 NB-UVB, cell cultures were incubated for 24h in the incubator.10µl of CCK-8 solution (Solarbio Co., Ltd., Beijing, China) was added to each well and then incubated for 4h in the incubator. A microplate reader was used to measure the absorbance at 450nm.

Quantitative real-time PCR analysis
Total RNA from cells were extracted using Trizol RNA isolation reagent (Thermos Fisher Scientific, USA) according to the manufacturer's protocol. One microgram of total RNA was used for the synthesis of first-strand cDNA. Real-time PCR assays were carried out using AceQ Universal SYBR qPCR Master Mix. The PCR primers were used as follows: forward 5′-AACGCCAGACAAATGTGACC-3′ and reverse 5′-AAAACAGCTCCGCATCAACC-3′ for MMP13; forward 5′-GTCCACCGCAAATGCTTCTA-3′ and reverse 5′-TGCTGTCACCTTCACCGTTC-3′ for β-actin. The cycling conditions used were as follows: 95 °C for 5 min, followed by 40 cycles of denaturation at 95°C for 10s, annealing at 60°C for 30s, and extension at 72°C for 15s. The relative amount of MMP13 transcript was normalized by the amount of β-actin transcript. The results were analyzed using the formula 2 −ΔΔCT .

Western blot analysis
HaCaT cells were lysed in lysis buffer. The protein concentrations were measured by Bicinchoninic Acid (BCA) Protein Assay. Protein samples (20 µg) were separated by SDS-PAGE (10%) and transferred onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% fat-free milk and incubated overnight at 4°C with anti-MMP13 antibody (Abcam, USA). After washing, the membrane was then exposed to secondary antibodies coupled to horseradish peroxidase. Immunoreactivities were detected using ECL reagents (Biyuntian Biotechnology Co., LTD., shanghai, China). Protein bands were quantified using Image J software.

Statistical analysis
All quantitative data represent the results of three independent experiments conducted in triplicate.
Each value is expressed as the mean ± standard deviation (SD). The data were analyzed with the unpaired, two-tailed Student's t test or one-way analysis of variance (ANOVA), using SPSS 23.0 and GraphPad Prism 8.0 software. Differences with P values of less than 0.05 were considered statistically significant.

Upregulation of MMP13 in patients with psoriasis
MMP13 staining was strongly positive in each layer of skin tissues in patients with psoriasis. In contrast, MMP13 staining was weak in normal skin tissues and mainly confined to the spinous and basal layers (Figure 1A). Consistent with the upregulation of MMP13 in skin tissues, we showed that the serum MMP13 level in patients with psoriasis was significantly higher than that in the control group ( Figure 1B).

Effects of pharmacological treatment and NB-UVB irradiation on pathological changes in a mouse model of psoriasis
we investigated the effect of different treatments with mice. As shown in Figure 2A, the skin changes in mice after treatment with tazarotene and/or NB-UVB irradiation. Compared to the control group, IMQ-induced mice showed increased skin thickness, hypertrophic spinous layer, and prolonged epidermal ridge (Figure2C). IMQ-induced epidermal thickness was significantly attenuated by treatment with tazarotene and/or NB-UVB irradiation. The combination of tazarotene and NB-UVB irradiation led to a more profound improvement in skin thickness than each treatment alone ( Figure 2B).

Assessment of MMP13 expression in psoriatic mice
Compared to control mice, skin lesions from IMQ-induced psoriatic mice had increased MMP13 expression. Treatment with tazarotene or NB-UVB irradiation alone or in combination markedly prevented the IMQ-induced elevation of MMP13 in skin tissues (Figure 2D). Similarly, IMQ-induced mice had significantly greater levels of serum MMP13 than control mice. Serum MMP13 levels were lowered by treatment with tazarotene and/or NB-UVB irradiation (Figure 2B).

Acitretin and NB-UVB irradiation inhibit cell viability and MMP13 expression in HaCaT cells
Next, we investigated the effect of different treatments on HaCaT cells. As shown in Figure 3A, the HaCaT cell viability was reduced after treatment with acitretin and/or NB-UVB irradiation.

DISCUSSION
In this study, we showed that the levels of MMP13 in skin lesions and serum samples of patients with psoriasis were higher than those in the normal group.

CONCLUSION
In summary, we demonstrate that MMP13 expression is increased in skin lesions and serum samples of patients with psoriasis. Tazarotene treatment plus NB-UVB effectively inhibits MMP13 expression in a mouse model of psoriasis. Moreover, Acitretin and NB-UVB treatment suppress HaCaT cell proliferation and MMP13 expression. Our findings suggest that tazarotene/acitretin and NB-UVB exert their synergistic beneficial effects on psoriasis likely through regulation of MMP13 expression and secretion. Further study is necessary to reveal the detailed molecular mechanism involved in the improvement of psoriasis by tazarotene/acitretin and NB-UVB.