The present study was conducted as a single-center, prospective, bilateral comparison study. Written informed consent was obtained from each patient prior to enrolment in the study. A total of 11 Chinese patients with plaque psoriasis (seven males and four females), aged 21.3–47.1 years (mean age 32.4 years), were recruited. For each individual, it was required that at least two target lesion pairs on each side of the body were of moderate to severe severity, and all comparative lesion pairs had to be in analogous anatomic locations and of approximately equal severity. Scalp, facial and genital psoriasis was neither treated nor assessed. Those patients who had received oral, topical, physical (i.e., ultraviolet or solarium treatment) and systemic antipsoriatic treatments within the previous six months were excluded. Other exclusion criteria included a current diagnosis of unstable psoriasis, pregnancy, benign or malignant tumor, topically serious skin trauma, uncontrollable systemic disease and a history of allergy.

Calcipotriol ointment alone was applied twice daily to the affected areas on one side of the body [monotherapy (M)group], randomly assigned for each patient. On the other side, calcipotriol ointment was applied only once in the evening and humectant containing P. oleracea extract once in the morning rotationally [combination (C) group]. Due to ethics restrictions, P. oleracea treatment alone could not be applied during the study. A fixed dosage was strictly required at a total of 0.5 g (a fingertip unit (22)) on each 10×10 cm² area for each application, which was calculated and strictly instructed by the investigators at the first visit of the patient. The therapeutic phase of the sides was equal at four weeks.

The severities of the paired psoriatic lesions were recorded on each visit (weeks 0, 2 and 4).Scales, plaque and erythema were usually considered as the main complaints of psoriasis. A nine-value rating scale with 0.5-point increments was used to evaluate the change in the degree of scales, plaque elevation and erythema, with the following classifications: 0, none; 1, mild; 2, moderate; 3, severe; and 4, very severe (23). Total scores were calculated as the sum of the points. Another assessment of overall efficacy was made by the patients using the following defined five-point grading scale on each visit: 1, excellent improvement (>75%); 2, marked improvement (51–75%); 3, moderate improvement (26–50%); 4, slight improvement (0–25%); and 5, no improvement or deterioration. Symptoms including itching, thermalgia, as well as any another abnormal sensations or adverse events, if any, were documented in detail on each visit.

To evaluate the condition of the skin barrier, patients were investigated instrumentally for TEWL, using a Tewameter^® TM 300 (Courage + Khazaka electronic GmbH, Cologne, Germany) under the manufacturer’s instructions. The condition of all subjects was first stabilized for 15–20 min, in a climate- and humidity-controlled room, with an ambient temperature range of 21–25°C and mean relative humidity range of 50–60%. Two topical comparative plaques of homologous anatomic positions and severity on each side were measured at each visit at weeks 0, 2 and 4. Additionally, five healthy volunteers were recruited as the control.

Skin tissues for downstream analysis were obtained from each patient at weeks 0 and 4 by a professional operator. Samples were approximately equally harvested on the bilateral body each time, and the regions at week 4 were close to those of week 0 while avoiding the scar. The tissues were fixed immediately according to the different downstream processes. Additionally, another five skin tissues without substantial lesions were obtained from the Department of Surgery in The First Affiliated Hospital of Chongqing Medical University as the normal control.

Routine H&E staining was conducted following paraffin sectioning of 5-μm-thick sections. The mean epidermal thickness was determined on H&E stained sections by measuring the distance between the outermost surface of the epidermis excluding the stratum corneum and the dermo-epidermal junction at five points through the entire length of three examined sections for each specimen.

To perform IHC, the slides were retrieved in a high-temperature antigen retrieval solution of citrate buffer and then incubated overnight at 4°C with primary antibodies (Abs) against K10, LOR, FLG (Abcam, Cambridge, MA, USA), TNF-α and IL-8 (Cell Signaling Technology, Inc., Danvers, MA, USA). Subsequently, the appropriate secondary Abs and detection kits were used. The tissues were visualized with 3,3′-diaminobenzidine substrate and counterstained with hematoxylin. Images were captured under closely comparable conditions.

Skin tissues from patients were homogenized and sonicated in lysis buffer (containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid, 1% Triton, 0.1% sodium dodecyl sulfate (SDS) and 1% protease inhibitors). Equal amounts of proteins were separated on 7–12% SDS-polyacrylamide gels in a minigel apparatus (Bio-Rad, Hercules, CA, USA) and then transferred electrophoretically to nitrocellulose membranes, followed by blocking with milk and incubation at 4°C overnight with anti-K10, LOR, FLG, TNF-α and IL-8 Abs, as well as anti-p65, phosphorylated-(p-)p65, inhibitor κBα (IκBα) and p-IκBα (phosphorylated at Ser32) Abs (Cell Signaling Technology, Inc.). Membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary Abs. Subsequent to immunoblotting, the films were scanned and the intensity of the immunoblotting bands was detected with a Bio-Rad GS-800 Calibrated Densitometer (Bio-Rad). GAPDH and α-tubulin were used as the loading controls.

Severity scores from the clinical assessments were compared at the baseline and across three time points using the Wilcoxon signed-rank test. The comparisons of the various severity scores between the M group and C group-treated lesion pairs were evaluated using the Mann-Whitney U test, and comparison of the adverse effects between the two groups was conducted using the Pearson’s χ² test. The results of the readings of TEWL, epidermis thickness and quantitative western blot images are all presented as the mean ± standard deviation, and were analyzed using the two-tailed unpaired Student’s t-test or paired-samples Student’s t-test. All analyses were performed using SPSS version 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Ten patients (78 target lesion pairs) that completed the phase of treatment were included in the statistical analysis for efficacy. The locations of the target lesions were on the legs (n=37), arms (n=24) and trunk (n=17). One patient dropped out due to rejection to the second skin biopsy at week 4, when the clinical manifestation had improved markedly.

At the baseline, the severity of lesions between the two groups was similar in scaling, plaque elevation, erythema and overall lesional assessment. Subsequent to treatment, the two groups showed statistically significant amelioration in scaling, plaque elevation and erythema, as well as in the overall lesional severity, which was estimated by the patients themselves at weeks 2 and 4 (Fig. 1A–C). Comparing the two regimens, no statistically significant difference was identified at the two visits. However, according to the reports of the patients, the adverse effects in the M group were of significantly higher frequency than those in the C group during the first two weeks (P<0.05), including five individual complaints of skin dryness and four of transient burning sensation in the applied area, as well as three and two complaints of immediate temporary skin redness and/or itching following the application in the M group. However, all these complaints decreased gradually and were eventually eliminated in two weeks. By contrast, in the C group, the main complaints were the slightly aromatic odor of the humectant, an occasional slight burning sensation and itching (Fig. 1D).

At the beginning of the study, the TEWL of the patients was significantly higher than that of the normal controls, which indicated the impaired function of the skin barrier in the psoriatic lesions. In the M group, the TEWL was slightly lower following treatment than the initial TEWL at weeks 2 and 4; however, no statistically significant differences were identified, indicating that the calcipotriol ointment was not able to protect against water loss and improve the skin barrier function in the present study. By contrast, the value of TEWL in the C group was significantly decreased at the visits at weeks 2 and 4, accompanied by the normalization of the lesional condition. When compared with the M group, this clearly indicated that the improvement of skin barrier function with the combination regimen was more efficient than that with the monotherapy of calcipotriol (Table I).

To evaluate the keratinocyte proliferation status, epidermal thickness was investigated by routine H&E staining (Fig. 2). The results clearly showed that prior to treatment there was a pronounced increase in the epidermal thickness with the acanthotic appearance and rete ridges, accompanied by abnormal keratinocyte differentiation with the loss of the stratum granulosum. The stratum corneum was clearly thickened with parakeratosis. Subsequent to treatment, however, the layers of keratinocytes decreased along with the flattened rete ridges and the stratum granulosum cell layers increased. Comparison between the two regimens indicated that although the change of epidermal thickness was slightly greater in the M group than in the C group, no significant difference was statistically validated.

To further explore the keratinocyte differentiation status, the expression of protein markers reflecting the differentiation of the psoriatic keratinocytes was then investigated by IHC (Fig. 3A) and western blot analysis (Fig. 3B). These revealed that the K10 protein was mainly expressed in the stratum spinosum at moderate levels in normal tissue, while no expression was found in skin psoriatic lesions; however, K10 protein expression was strongly elevated following treatment in the two treatment groups. A notable quantity of LOR was detected in the stratum granulosum and lower corneum, which was significantly downregulated in the psoriasis lesions; however, evident elevation was detected following treatment. FLG expression of a moderate level was detected in normal controls and a lower expression was observed in untreated patients, with upregulation following treatment. Paralleled with the immunohistological outcomes, western blotting also revealed the same tendencies for the normal tissue, psoriatic skin and those following treatment. The protein expression levels of K10, LOR and FLG were significantly downregulated in the psoriatic lesions. However, following treatment, all these changes in protein expression levels were significantly reversed and accompanied by an improvement in skin condition. The combination therapy exerted markedly higher efficacies on inversing the abnormal expression levels of these differentiation markers for psoriatic keratinocytes.

TNF-α and IL-8, two pivotal inflammatory factors according to previous reports (3,24,25), are both involved in the pathogenesis of psoriasis, and have been proposed to be the crucial therapeutic targets for psoriatic patients. To explore if these factors were differentially regulated by the two regimens, their expression levels were investigated by IHC and western blot analysis. Fig. 4A shows that TNF-α and IL-8 expression was at a limited level and mainly distributed to the basal layers of the epidermis in normal tissue, but was widely upregulated within the whole epidermis in the psoriasis tissues, while being significantly decreased following treatment with the two regimens. Western blot analysis revealed a comparable tendency, as shown in Fig. 4B. However, following treatment, a significant difference between the effects of the combination therapy and monotherapy was statistically concluded for the TNF-α and IL-8 expression levels by quantitative analysis, thus revealing that the combination regimen exerted a more potent regulatory effect on these two inflammatory factors.

To further explore the potential mechanism by which P. oleracea aided calcipotriol in reversing the psoriatic condition, the status of the NF-κB pathway was investigated by western blot analysis. Since p65 is the main functional subunit of NF-κB, and its activation is mainly regulated by its inhibitory protein, IκBα (26,27), the expression levels of p65, p-p65, IκBα and p-IκBα were investigated. Fig. 5 shows that the expression levels of p65 were not significantly different between either the psoriatic lesions and normal controls, or prior and subsequent to treatment. However, the expression levels of p-p65 and p-IκBα were markedly increased, and IκBα levels were decreased in the psoriatic patients compared with those in normal tissue. Following treatment, the p-p65 and p-IκBα levels were decreased significantly, and subsequently the level of IκBα was reversed in the C group, but not in the calcipotriol M group. For the calcipotriol M group, no changes in the expression of p-p65, IκBα and p-IκBα were identified following treatment, which was consistent with a previous study (9), and therefore it was proposed that the combination regimen but not the calcipotriol directly suppressed the degradation of IκBα in the present study, which subsequently resulted in the accumulation of IκBα and inhibited the NF-κB activation and nuclear translocation.