Role and Mechanism of Matrine Alone and Combined with Acitretin for HaCaT Cells and Psoriasis-Like Murine Models
Psoriasis is a chronic inflammatory skin disease characterized by well-demarcated, scaly plaques, which reflect the underlying processes of inflammation and epidermal hyperproliferation. The disease has a high incidence and is prone to relapse, with a complex pathogenesis involving factors such as genetics, abnormal keratinocyte proliferation, differentiation, and immune regulation disorders. Traditional treatments for psoriasis, including topical medications, phototherapy, and systemic therapies like methotrexate and acitretin, have limited efficacy. Although biological agents targeting cellular immunity mechanisms have shown promise, long-term safety data are still lacking. Acitretin, a systemic retinoid, is commonly used for moderate to severe psoriasis, but its efficacy as a monotherapy is inconsistent, and higher doses increase the risk of adverse effects. Therefore, combination therapies are often employed to enhance efficacy and reduce side effects.
Matrine, an active ingredient derived from the traditional Chinese medicine Sophora, has demonstrated various pharmacological activities, including anti-inflammatory, anti-allergic, anti-viral, and anti-cancer effects. It has been used to treat chronic hepatitis, tumors, and skin diseases like atopic dermatitis and eczema. Matrine’s anti-cancer mechanisms include cell cycle arrest, apoptosis induction, and inhibition of angiogenesis. Given its potential, this study aimed to investigate the role and mechanisms of matrine alone and in combination with acitretin in treating psoriasis using in vitro and in vivo models.
Methods
Cell Culture and Treatment
HaCaT cells, a spontaneously immortalized human keratinocyte cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum. Cells were treated with matrine at concentrations of 0, 0.2, 0.4, 0.8, and 1.6 mg/mL for 24, 48, and 72 hours. Acitretin was dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 10 mmol/L. For combination therapy, HaCaT cells were treated with matrine (0.4 mg/mL) and acitretin (10 mmol/L).
Cell Proliferation Assay
Cell viability was assessed using the MTS assay. HaCaT cells were seeded in 96-well plates and treated with matrine, acitretin, or their combination. Absorbance was measured at 490 nm to determine cell viability.
Cell Cycle and Apoptosis Analysis
Cell cycle analysis was performed using 7-aminoactinomycin D (7-AAD) staining, and apoptosis was assessed using annexin-V-FITC/7-AAD staining. Flow cytometry was used to quantify cell cycle phases and apoptosis rates.
Autophagy Observation
Autophagy was observed using transmission electron microscopy (TEM). HaCaT cells were treated with matrine, acitretin, or their combination, and autophagic vacuoles were detected.
Western Blotting
Protein expression was analyzed using Western blotting. Total proteins were extracted from treated HaCaT cells, and specific antibodies were used to detect autophagy-related proteins (LC3, Beclin1, p62), cell cycle-related proteins (p21, cyclin D1), and components of the PI3K/Akt/mTOR pathway.
Animal Experiments
Psoriasis-like mouse models were established using imiquimod cream. Mice were treated with matrine (50 mg/kg), acitretin (4.5 mg/kg), or their combination for seven consecutive days. The severity of skin inflammation was scored based on erythema, scaling, and thickening. Skin lesions were collected for histological and immunohistochemical analysis.
Results
Effects of Matrine on HaCaT Cells
Matrine inhibited HaCaT cell proliferation in a dose- and time-dependent manner. At 24 hours, the proliferation percentages for matrine concentrations of 0.2, 0.4, 0.8, and 1.6 mg/mL were 94.36%, 89.00%, 77.80%, and 64.42%, respectively. By 72 hours, these percentages decreased to 81.05%, 54.15%, 36.45%, and 30.36%, respectively. Matrine induced G0/G1 phase cell cycle arrest in a dose-dependent manner, with the percentage of cells in the G0/G1 phase increasing from 38.80% in the control group to 82.31% at 1.6 mg/mL. Matrine also increased the expression of autophagy-related proteins LC3II/I and Beclin1 and the cell cycle arrest-related protein p21, while decreasing cyclin D1 expression. TEM revealed more autophagosomes in matrine-treated cells compared to the control group.
Effects of Matrine Combined with Acitretin
The combination of matrine and acitretin significantly enhanced the inhibition of HaCaT cell proliferation compared to either treatment alone. At 48 hours, the proliferation percentage in the combination group was 58.67%, compared to 79.95% and 74.40% in the acitretin and matrine groups, respectively. The combination treatment also induced a higher percentage of G0/G1 phase arrest (74.96%) compared to matrine (64.77%) or acitretin (43.66%) alone. Western blotting showed that the combination treatment increased the expression of p21 and LC3II/I and decreased cyclin D1 and p62 expression more significantly than either treatment alone. TEM confirmed that the combination treatment induced more autophagosomes than matrine or acitretin alone.
Effects on the PI3K/Akt/mTOR Pathway
Matrine and acitretin, both alone and in combination, downregulated the PI3K/Akt/mTOR pathway in HaCaT cells. The combination treatment significantly reduced the expression of phosphorylated PI3K, Akt, mTOR, and p70S6K compared to matrine or acitretin alone.
Animal Model Results
In the psoriasis-like mouse model, the combination of matrine and acitretin significantly reduced the cumulative inflammation score (1.480) compared to matrine (2.370) or acitretin (2.888) alone. Histological analysis showed improved skin lesions in the combination group, with reduced epidermal thickening and inflammatory cell infiltration. Immunohistochemistry revealed higher LC3 expression in the combination group compared to the matrine, acitretin, or control groups.
Discussion
Matrine demonstrated therapeutic potential for psoriasis by inhibiting keratinocyte proliferation, inducing autophagy, and arresting the cell cycle at the G0/G1 phase. These effects were mediated through the downregulation of the PI3K/Akt/mTOR pathway. The combination of matrine and acitretin showed synergistic effects, enhancing autophagy induction and cell cycle arrest more significantly than either treatment alone. This synergy was also observed in the psoriasis-like mouse model, where the combination treatment improved skin lesions and reduced inflammation more effectively than monotherapy.
The study provides evidence that matrine, particularly in combination with acitretin, could be a promising therapeutic strategy for psoriasis. By modulating autophagy and the cell cycle, matrine may enhance the efficacy of acitretin while reducing its side effects. Further research is needed to validate these findings and explore the clinical applications of matrine-based combination therapies for psoriasis.
doi.org/10.1097/CM9.0000000000000412
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