Yin-Ku Lin

Clinical Assessment of Patients With Recalcitrant Psoriasis in a Randomized, Observer-Blind, Vehicle-Controlled Trial Using Indigo Naturalis

OBJECTIVE To evaluate the efficacy and safety of treatment with indigo naturalis in patients with recalcitrant plaque-type psoriasis. DESIGN Randomized, observer-blind, vehicle-controlled, intrapatient comparison study. SETTING Ambulatory department of a hospital. PARTICIPANTS Forty-two outpatients with chronic plaque psoriasis were enrolled in the study from May 1, 2004, to April 30, 2005. INTERVENTION The patients applied either indigo naturalis ointment or vehicle ointment topically to each of 2 bilaterally symmetrical psoriatic plaque lesions for 12 weeks (depending on the date of enrollment in the study). MAIN OUTCOME MEASURES The outcomes were assessed using the following criteria: the sum of erythema, scaling, and induration scores and the clearing percentage of the target plaque lesion assessed by 2 blinded observers. RESULTS Significant reductions in the sum of scaling, erythema, and induration scores (P < .001) (mean score, 6.3 after indigo naturalis treatment vs 12.8 in control subjects) and plaque area percentage (P < .001) (mean percentage, 38.5% after indigo naturalis treatment vs 90% in controls) were achieved with topical application of indigo naturalis ointment. Approximately 31 of 42 patients (74%) experienced clearance or near clearance of their psoriasis in the indigo ointment-treated lesion. CONCLUSION Topical indigo naturalis ointment was a novel, safe, and effective therapy for plaque-type psoriasis.

Anti-psoriatic effects of indigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component.

BACKGROUND Indigo naturalis has shown efficacy in treating psoriasis in our previous clinical studies. OBJECTIVES To investigate the potential effect of indigo naturalis on regulating keratinocyte proliferation and differentiation. METHODS Skin samples from six patients were analyzed for proliferating cell nuclear antigen (PCNA) and involucrin expression by immunohistochemical staining. In addition, indigo naturalis extracts from 10 to 500 microg/ml were added to cultured keratinocytes and cell viability determined. Real-time RT-PCR, Western blotting analysis and indirect immunofluorescent labeling were used to investigate the messenger (m)RNA and protein expressions of PCNA and involucrin. Finally, high-performance liquid chromatography (HPLC) was used to identify major components of indigo naturalis and their in vitro effects compared. RESULTS Immunohistochemical results demonstrated decreased PCNA and increased involucrin in psoriatic lesions after indigo naturalis treatment. Cultured keratinocytes decreased after indigo naturalis treatment, while G(0)/G(1) arrest was observed to dose-dependently increase. Staining revealed decreased PCNA-stained nuclei and increased cytosolic involucrin in treated keratinocytes. Decreased PCNA and increased involucrin at both the mRNA and protein levels were confirmed. Both major components, indirubin and indigo, could cause G(0)/G(1) phase arrest; however, only indirubin modulated the expressions of PCNA and involucrin similar to indigo naturalis. CONCLUSIONS Together, these findings indicate that the anti-psoriatic effects of indigo naturalis are mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes, with indirubin as the major active component.

The Efficacy and Safety of Topically Applied Indigo Naturalis Ointment in Patients with Plaque-Type Psoriasis

Background: It has been reported in the Chinese literature that indigo naturalis exhibits potential antipsoriatic effects in systemic therapy. Objective: To evaluate the efficacy and safety of topically applied indigo naturalis on treating plaque-type psoriasis and to analyze the histological change in skin tissues. Methods: Fourteen patients with chronic plaque psoriasis were enrolled. The patients were topically applied with either indigo naturalis ointment or vehicle ointment on contralateral skin lesions daily for 8 weeks. Efficacy was evaluated on the basis of the clinical scores, including induration, scaling, erythema and clearing percentage. At the end of treatment, skin punch biopsies were taken and prepared for the immunohistochemical analysis. Results: A significant reduction in clinical scores was achieved with topically applied indigo naturalis ointment. Analysis of biopsies showed a marked improvement of skin histology. The expressions of proliferating marker Ki-67 and inflammatory marker CD3 were decreased, but the differentiation marker such as filaggrin was increased in the epidermis after indigo naturalis ointment treatment. Conclusions: The results suggest that topical application of indigo naturalis ointment may be a novel, safe and effective therapy for psoriasis that is mediated, at least in part, by modulating the proliferation and differentiation of keratinocytes in epidermis, as well as by inhibiting the infiltration of T lymphocytes and therefore the subsequent inflammatory reactions in psoriatic lesions.

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils

ETHNOPHARMACOLOGICAL RELEVANCE Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. AIM OF THE STUDY Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. MATERIALS AND METHODS Superoxide anion (O2(.-)) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. RESULTS QD significantly inhibited O2(.-) generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. CONCLUSIONS Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O2(.-) generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Development and Evaluation of Perfluorocarbon Nanobubbles for Apomorphine Delivery

Apomorphine is a dopamine receptor agonist for treating Parkinsons disease. However, its clinical application is limited by its instability and the need for frequent injections. The aim of the present work was to develop acoustically active perfluorocarbon nanobubbles (PNs) for encapsulation of both apomorphine HCl and base forms to circumvent these delivery problems. The PNs were prepared using coconut oil and perfluoropentane as the inner phase, which was emulsified by phospholipids and cholesterol. The morphology, size, zeta potential, and drug release of the PNs were characterized. The particle size ranged from 150 to 380 nm, with differences in the oil or perfluorocarbon ratio in the formulations. Atomic force microscopy confirmed oval- or raisin-shaped particles and a narrow size distribution of these systems (polydispersity index = 0.25-0.28). The stability experimental results indicated that PNs could protect apomorphine from degradation. Evaporation of the PNs at 37 degrees C was also limited. Apomorphine HCl and base in PNs showed retarded and sustained release profiles. Ultrasound imaging confirmed the echogenic activity of PNs developed in this study. The apomorphine HCl release by insonation at 1 MHz showed enhancements of two- to fourfold compared to the non-ultrasound group, illustrating a possible drug-targeting effect. On the contrary, apomorphine base showed a decreased release profile with ultrasound application. Apomorphine-loaded PNs showed promising stability and safety. They were successful in sustaining apomorphine delivery.

Comparison of Refined and Crude Indigo Naturalis Ointment in Treating Psoriasis: Randomized, Observer-Blind, Controlled, Intrapatient Trial

individual cycles in secondary analyses. Cox proportional hazards regression models were used to estimate relative risks (RRs) and 95% CIs. Analyses were updated because the main exposure, outcome, and covariates were all time varying. We had multivariate models with or without smoking. Analyses were conducted using SAS software, version 9.2 (SAS Institute Inc). The study was approved by the institutional review board of Brigham and Women’s Hospital. Our receipt of each completed questionnaire implied participant’s informed consent of the present study.

Inhibitory effect of indigo naturalis on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells.

The use of indigo naturalis to treat psoriasis has proved effective in our previous clinical studies. The present study was designed to examine the anti-inflammatory effect of indigo naturalis in primary cultured human umbilical vein endothelial cells (HUVECs). Pretreatment of cells with indigo naturalis extract attenuated TNF-α-induced increase in Jurkat T cell adhesion to HUVECs as well as decreased the protein and messenger (m)RNA expression levels of vascular cell adhesion molecule-1 (VCAM-1) on HUVECs. Indigo naturalis extract also inhibited the protein expression of activator protein-1 (AP-1)/c-Jun, a critical transcription factor for the activation of VCAM-1 gene expression. Since the reduction of lymphocyte adhesion to vascular cells by indigo naturalis extract could subsequently reduce the inflammatory reactions caused by lymphocyte infiltration in the epidermal layer and help to improve psoriasis, this study provides a potential mechanism for the anti-inflammatory therapeutic effect of indigo naturalis extract in psoriasis.

Indirubin, an acting component of indigo naturalis, inhibits EGFR activation and EGF-induced CDC25B gene expression in epidermal keratinocytes

BACKGROUND Topical indigo naturalis ointment is clinically proved to be an effective therapy for plaque-type psoriasis. Indirubin, as the active component of indigo naturalis, inhibits cell proliferation of epidermal keratinocytes. However, the detailed underlying mechanism is not fully understood. OBJECTIVE To further investigate the anti-proliferating effects of indigo naturalis and indirubin on epidermal keratinocytes. METHODS The decreased expression of CDC25B in indigo naturalis- or indirubin-treated epidermal keratinocytes, as revealed by cDNA microarray analysis, was studied. The CDC25B expression was examined under different serum concentrations and compared between primary and immortalized keratinocytes. The activation of EGFR and the effect of EGF on the cell proliferation and CDC25B expression were also investigated in epidermal keratinocytes. RT/real-time PCR and western blot method were used to analyze the CDC25B expression at the mRNA and protein levels, respectively. RESULTS Indigo naturalis and indirubin were confirmed to down-regulate CDC25B expression significantly at both the mRNA and protein levels. The growth-dependent expression of CDC25B was demonstrated by the increased expression in serum-stimulated and immortalized keratinocytes. The activation of EGF receptor, known to be highly expressed in psoriatic lesions, was inhibited by indigo naturalis or indirubin. The cell proliferation and CDC25B expression of epidermal keratinocytes were induced by EGF alone and confirmed to be inhibited by indigo naturalis or indirubin. CONCLUSION Except being a common therapeutic target in various cancers, CDC25B also plays an important role in the hyper-proliferation of epidermal keratinocytes which can be suppressed by anti-psoriatic drug indigo naturalis and its component, indirubin.

Using Imiquimod-Induced Psoriasis-Like Skin as a Model to Measure the Skin Penetration of Anti-Psoriatic Drugs

Objective Psoriasis is a chronic inflammatory skin disease and topical therapy remains a key role for treatment. The aim of this study is to evaluate the influence of psoriasis-like lesions on the cutaneous permeation of anti-psoriatic drugs. Methods We first set up imiquimod-induced dermatitis in mice that closely resembles human psoriasis lesions. The development of the lesions is based on the IL-23/IL17A axis for phenotypical and histological characteristics. Four drugs, 5-aminolevulinic acid (ALA), tacrolimus, calcipotriol, and retinoic acid, were used to evaluate percutaneous absorption. Results The most hydrophilic molecule, ALA, revealed the greatest enhancement on skin absorption after imiquimod treatment. Imiquimod increased the skin deposition and flux of ALA by 5.6 to 14.4-fold, respectively, compared to normal skin. The follicular accumulation of ALA was also increased 3.8-fold. The extremely lipophilic drug retinoic acid showed a 1.7- and 3.8-fold increase in skin deposition and flux, respectively. Tacrolimus flux was enhanced from 2 to 21 μg/cm2/h by imiquimod intervention. However, imiquimod did not promote skin deposition of this macrolide. The lipophilicity, but not the molecular size, dominated drug permeation enhancement by psoriatic lesions. The in vivo percutaneous absorption of ALA and rhodamine B examined by confocal microscopy confirmed the deficient resistance of epidermal barrier for facilitating cutaneous delivery of drugs via psoriasis-like skin. Conclusion We established the topical delivery profiles of anti-psoriatic drugs via imiquimod-treated psoriasis-like skin.

An In Vitro Study of the Antimicrobial Effects of Indigo Naturalis Prepared from Strobilanthes formosanus Moore

Indigo naturalis is effective in treating nail psoriasis coexisting with microorganism infections. This study examines the antimicrobial effects of indigo naturalis prepared from Strobilanthes formosanus Moore. Eight bacterial and seven fungal strains were assayed using the agar diffusion method to examine the effects of indigo naturalis and its bioactive compounds. The bioactive compounds of indigo naturalis were purified sequentially using GFC, TLC, and HPLC. Their structures were identified using mass spectrometry and NMR spectroscopy. UPLC-MS/MS was applied to compare the metabolome profiles of indigo naturalis ethyl-acetate (EA) extract and its source plant, Strobilanthes formosanus Moore. The results of in vitro antimicrobial assays showed that indigo naturalis EA-extract significantly (≥1 mg/disc) inhibits Gram-positive bacteria (Staphylococcus aureus, S. epidermis and methicillin-resistant S. aureus (MRSA)) and mildly inhibits non-dermatophytic onychomycosis pathogens (Aspergillus fumigates and Candida albicans), but has little effect on dermatophyes. Isatin and tryptanthrin were identified as the bioactive compounds of indigo naturalis using S. aureus and S. epidermis as the bioassay model. Both bioactive ingredients had no effect on all tested fungi. In summary, indigo naturalis prepared from Strobilanthes formosanus Moore exhibits antimicrobial effects on Staphylococcus and non-dermatophytic onychomycosis pathogens. Tryptanthrin and isatin may be its major bioactive ingredients against Staphylococcus and the inhibitory effect on MRSA may be due to other unidentified ingredients.