Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis / 中国实验方剂学杂志

Objective::To observe the expressions of tight junction proteins (claduin-1, claudin-7 occludin)of psoriasis-like lesions in mice, and clarify the effect of Yangxue Jiedu decoction on the epidermal barrier of psoriasis, so as to provide scientific basis for the treatment of psoriasis with Yangxue Jiedu decoction. Method::C57BL/6J mice were randomly divided into control group, model group, methotrexate group and Yangxue Jiedu decoction.Methotrexate solution and water decoction of Yangxue Jiedu decoction were prepared, and the mice were given imiquimot to induce psoriasis-skin lesions after the hair was shaved.Daily photos were taken to record the forms of skin lesions and psoriasis area and severity index(PASI) scores.Water and oil test pens were used to detect skin moisture content. The pathological changes were observed by htoxylin eosin (HE) staining, and the epidermal thickness was measured.Ki67 was detected by immunofluorescence. Immunohistochemistry was used to detect the expressions of loricrin, CD3+ T lymphocyte infiltration and claduin-1, claudin-7, occludin.In addition, the expressions of claudin-7 and occludin in skin lesions were detected by Western blot.Meanwhile, interleukin-17(IL-17) (1 mg·L-1) was used to simulate the microenvironment of psoriasis skin lesions, and the intervention was conducted by making Yangxue component, Jiedu component and Yangxue Jiedu dry powder.The toxicity of the drug on Hacat cells was detected by cell counting kit-8(CCK-8)method.The effect of the drugs on the expressions of claudin-1, claudin-7, occludin in Hacat was detected by immunofluorescence assay. Result::Yangxue Jiedu decoction could significantly reduce the psoriasis skin lesions in mice, and reduce the PASI score and skin thickness (P<0.01). Compared with the model group, the skin moisture content in the lesion was increased (P<0.01). Abnormal expressions of ki67 and loricrin in epidermis and infiltration of CD3+ T cells were reduced (P<0.01). In addition, the expressions of claudin-1, claudin-7, occludin proteins (P<0.05), and the integrity of the tight junction structure were increased.In vitro studies, compared with the model group, the expressions of claudin-1, claudin-7, and occludin in the Yangxue Jiedu group and Yangxue group were increased (P<0.05). Compared with the model group, there was no statistically significant difference in protein expression in the Jiedu group. Conclusion::By regulating the expressions of tight junction proteins between keratinocytes, Yangxue Jiedu decoction can inhibit the abnormal proliferation and differentiation, and further restore the broken epidermal barrier.Yangxue Jiedu decoction plays a role in regulating tight junction mainly through Yangxue component.

Significance of Skin Barrier Dysfunction in Atopic Dermatitis

The epidermis contains epithelial cells, immune cells, and microbes which provides a physical and functional barrier to the protection of human skin. It plays critical roles in preventing environmental allergen penetration into the human body and responsing to microbial pathogens. Atopic dermatitis (AD) is the most common, complex chronic inflammatory skin disease. Skin barrier dysfunction is the initial step in the development of AD. Multiple factors, including immune dysregulation, filaggrin mutations, deficiency of antimicrobial peptides, and skin dysbiosis contribute to skin barrier defects. In the initial phase of AD, treatment with moisturizers improves skin barrier function and prevents the development of AD. With the progression of AD, effective topical and systemic therapies are needed to reduce immune pathway activation and general inflammation. Targeted microbiome therapy is also being developed to correct skin dysbiosis associated with AD. Improved identification and characterization of AD phenotypes and endotypes are required to optimize the precision medicine approach to AD.

Vitamin C Stimulates Epidermal Ceramide Production by Regulating Its Metabolic Enzymes

Ceramide is the most abundant lipid in the epidermis and plays a critical role in maintaining epidermal barrier function. Overall ceramide content in keratinocyte increases in parallel with differentiation, which is initiated by supplementation of calcium and/or vitamin C. However, the role of metabolic enzymes responsible for ceramide generation in response to vitamin C is still unclear. Here, we investigated whether vitamin C alters epidermal ceramide content by regulating the expression and/or activity of its metabolic enzymes. When human keratinocytes were grown in 1.2 mM calcium with vitamin C (50 mug/ml) for 11 days, bulk ceramide content significantly increased in conjunction with terminal differentiation of keratinocytes as compared to vehicle controls (1.2 mM calcium alone). Synthesis of the ceramide fractions was enhanced by increased de novo ceramide synthesis pathway via serine palmitoyltransferase and ceramide synthase activations. Moreover, sphingosine-1-phosphate (S1P) hydrolysis pathway by action of S1P phosphatase was also stimulated by vitamin C supplementation, contributing, in part, to enhanced ceramide production. However, activity of sphingomyelinase, a hydrolase enzyme that converts sphingomyelin to ceramide, remained unaltered. Taken together, we demonstrate that vitamin C stimulates ceramide production in keratinocytes by modulating ceramide metabolic-related enzymes, and as a result, could improve overall epidermal barrier function.

Overview of atopic dermatitis

Atopic dermatitis (AD) is a very common chronic disease that reportedly affects 10%-20% of the general population. The prevalence of AD appears to be steadily increasing, at least in developing countries. Two pathogenetic mechanisms have been mentioned. Traditionally immunological aberrations are thought to be a primary event in the initial development of AD ("inside-to-outside hypothesis"). Another hypothesis assumes that there is an intrinsic defect in epidermal barrier. Due to this barrier defect, allergens or irritants can easily penetrate the epidermal barrier, and induce immunologic reaction secondarily ("outside-to-inside hypothesis"). These days the epidermal barrier defect seems to gain more support as a primary event than immunological aberrations in the early changes of AD since the filaggrin mutation was reported in AD patients. Clinically AD initially affects face, and with age, flexural areas are typically involved. AD has many different clinical features. Diagnostic criteria for AD in each country may be a little different, although based on the criteria proposed by Hanifin and Rajka. AD can be controlled effectively with topical and/or systemic treatments and fortunately spontaneously disappears with age. However, in some cases very resistant to conventional therapies, additional treatments such as immunosuppressive agents are needed.

Epidermal Barrier in Atopic Dermatitis

Atopic dermatitis (AD) is a complex disease that affects up to 20% of children and impacts the quality of patients and families in a significant manner. New insights into the pathophysiology of AD point to an important role of structural abnormalities in the epidermis combined with immune dysregulation. Filaggrin (FLG) is synthesized as a large precursor, profilaggrin, and is expressed in the upper layers of the epidermis. FLG plays a critical role in the epidermal barrier, and FLG mutations cause abnormal epidermal function. FLG mutations are strongly associated with early-onset, and persistent severe AD. In addition, FLG deficiency in the epidermis is related to allergic sensitization and asthma. The basic skin care including repair and protection of the skin barrier with proper hydration and topical anti-inflammatory therapy is important to control the severity of skin disease in patients with AD.

Epidermal Barrier in Human Fetus and Newborn / 대한피부과학회지

BACKGROUND: The lipids of the stratum corneum, which originate from polar lipid precursors provided by the cells of the stratum granulosum via the exocytosis of lamellar bodies, with cornified cell envelope form competent epidermal barrier structurally and functionally. The ontogeny of the epidermal barrier is not clearly defined because of difficulty of sampling and methodology which defines epidermal lipids. OBJECT: From ultrastructural observation of skin samples obtained from human fetuses and newborn on serial developmental timings, we tried to clarify the sequential development of epidermal barrier. METHODS: Skin samples were obtained from 13 human fetuses from EGA(estimated gestational age) 10 to 23wks and 2 newborns. Specimens were observed by fluorescent confocal microscopy with nile red to identify the distribution of epidermal lipids, by transmission electron microscope with lanthanum to investigate the functional permeability barrier, with RuO4 to observe the intercellular lipid bilayer and morphology of lamellar bodies, with ion capture cytochemistry to investigate the formation of epidermal calcium gradient. RESULT: In nile red stain, the amount of epidermal lipid increased during fetal period. At EGA 23wks, the lipid distribution revealed linear and continuous pattern. In lanthanum tracer study, the electron dense tracer permeated all the intercellular space of the epidermis up to periderm and subepidermal space until EGA 21wks. At EGA 23wks, the tracer permeated intercellular space of epidermis weakly. It might be predicted that incomplete epidermal barrier is present at this time. In RuO4 stain, precursor of lamellar body was observed at EGA 15wks, and intercellular lipid bilayer was observed at EGA 16wks. As gestation increases, there was a steady increase in epidermal lipid bilayers. In ion capture cytochemistry, epidermal calcium gradient was first observed in follicular epidermis at EGA 20wks, and in interfollicular epidermis at EGA 23wks. From these results, it is concluded that the basic structures of epidermal barrier are formed at EGA 23wks, but it is not complete, and epidermal barrier arises first from follicular epidermis.

Ultrastructural observation of the stratum corneum lipids in hairless mouse epidermis characterized by ruthenium tetroxied fixation / 医学研究生学报

Objectives: To reveal the morphological features of the stratum corneum lipids in hairless mouse epidermis. Methods: Ruthenium tetroxide and osmium tetroxide were compared as post fixative in the preparation of hairless epidermis for transmission electron microscopic examination. Results: Both reagents reveled characteristic membrane coating granules within the granular layer. Whereas, the transformation of the membrane coating granule contents into multiple lamellae at the interface between the granular and cornified layers and the persistence of these lamellae through all levels of the stratum corneum were demonstrated only by ruthenium tetroxide fixation. Conclusions: The distinctive patterning of the intercellular lamellae reflects the nonrandom organization of the stratum corneum lipids. In addition, the ruthenium tetroxide postfixation technique is a useful method in the investigation of the morphological features of the stratum corneum lipids.

Effects of Immersion on the Recovery of Epidermal Barrier after Acute Perturbation / 대한피부과학회지

BACKGROUND: If the skin is kept in continuous contact with water or other solutions after acute barrier perturbation, the pattern of barrier recovery will be different from that of the air exposure state at room temperature. Changes in the concentration of the solution also make a difference in the pattern of barrier recovery. However, there have been only a few studies on the difference of the barrier recovery or the changes in stratum corneum lipid between cases in the water immersion state and the air exposure state. OBJECTIVE: We studied the effect of immersion on the recovery of the epidermal barrier after acute perturbation so as to give aid to the basic research of the epidermal barrier and clinical application in prevention and treatment of contact dermatitis, hand eczema, and other occupational dermatoses. METHODS: After disruption of the epidermal barrier by tape stripping, hairless mice were immersed in temperature regulated hypotonic, isotonic, or hypertonic solutions, respectively. The pattern of barrier recovery with time was evaluated and compared with that of the air exposure group by means of TEWL measurement, and histochemical stain with Nile red. RESULTS: Immersion into water or solution without time to recovery after epidermal barrier disruption makes the recovery rate slower than that of the air exposure group except for the isotonic solution immersing group. CONCLUSION: For the normal recovery or prevention of the deterioration of the epidermal barrier, we should keep in mind to avoid the exposure to water or other solutions when acute or chronic barrier perturbation has developed. In addition, it would be better to use normal saline or other isotonic solutions, which do not disturb the normal barrier recovery process, to decrease damage to the epidermal barrier when it cannot help immersing into the solution.

The Iontophoresis Effect on Recovery After Acute Epidemal Barrier Disruption / 대한피부과학회지

BACKGROUND: The stratum corneum(SC) has a permeability barrier function which regulates percutaneous absorption by the inhibition of transepidermal water loss(TEWL). Acute mechanical or chemical disruption of the SC induces the impairment of the permeability barrier and so increases the TEWL. The calciumtion has been recognized as an important ion in the recovery of the skin barrier. Recently the main delivery pathway of iontophoretic drugs have been suggested by SC interstices. However the morphologic changes in the SC interstices and calcium after iontophoresis have not been reported. OBJECTIVE: The aim of our study is to confirm that iontophoresis may induce changes in the SC interstices and delay the recovery of the barrier after disruption. MATERIALS AND METHODS: After tape stripping the hairless mouse flank skin, the iontophoresis power supply (6V, 0.6mA) was connected to the patch atiached for 2.5 hours to the stripped site. We checked the THWL 0, 2.5, 6, 12, 18, 24 hours after the tape stripping and obtained specimens and performed osmium tetroxide, ruthenium tetroxide postfixation and calcium ion-capture cytochemical stains for electron microscopic study. RESULTS: The recovery rate of the TEWL in iontophoresis was lower than in the control. This was especially so in the anouse which had received anode iontophoresis for 6 hours. It showed statistically lower TEWL than in the control(p<0.05). Anode iontophoresis induced low calcium in stratum granulosum (SG), but cathode iontophoresis induced high calcium in SC. After iontophoresis there were changes in the SC interstices structures. CONCLUSION: Iontophoresis can induce changes in the SC interstices and calcium distribution and so may help the topical drug delivery system.

Epidermal Lipid Homeostasis

Stratum corneum lipids, which are enriched in sphingolipids, free fatty acids, and cholesterol, are required for epidermal barrier function. When the epidermal permeability barrier is perturbed, the transepidermal water loss returns to normal by 24-48 hours in parallel with the reappearance of stratum corneum lipids, derived from secreted lamellar bodis and accelerated lipid synthesis. Recent evidence shows that topical application of individual lipids interferes with barrier recovery while complete mixtures of cholesterol, fatty acids, and ceramides facilitate recovery after barrier disrupton. Metabolic imbalances and perturbed barrier function can be either the cause or the consequences of the pathobiology of scaling disease. Many skin diseases relating cornification and dryness are indeed related to abnormality of one or several combinations of lipids. Recently the cytokines which have changed during barrier recovery seem to be important in understanding of epidermal lipid homeostasis as well as barrier recovery.

Differences in the Recovery Rate after Perturbation of Epidermal Barrier by Means of Acetone Treatment and Tape-Stripping Technique

BACKGROUND: The epidermal permeability barrier necessary for terrestrial life resides in the intercellular spaces of the stratum corneum and is composed of lipids. OBJECTIVE: Since strrtum corneum lipid may be important for the permeability barrier, we studied the differences and effects of experimentally altered barrier function using acetone and tape-stripping technique. METHODS: The permeability barrier of hairless mouse was disrupted by tape-stripping and acetone treatment and the recovery rate was assessed by histochemical staining, electron microscopic examination and lipid analysis. RESULTS: Although the transepidermal water loss recovered completely by 48 hours in both of the acute models, acetone treated samples seem to have on over-all better recovery rate than tape-stripped samples. The return of barrier function to normal in both tape-stripped and acetone-treated skin was accompanied by a comparable return of normal nile red and ruthenium tetroxide staining. The amount of lipid in stratum corneum paralleled both the return of barrier function towards normal and the extent of prior damage to the barrier in acetone treated skin, yet, the lipid synthesis in tape-stripped skin showed a slower return of lipid content. CONCLUSION: The difference in the recovery rate of the two acute models may be due to the fact that acetone mainly extracts intercellular lipids, whereas, tape-stripping has a prolonged effect by removal of comeocyte in addition to the intercellular lipids. This shows the importance of comeocytes as well as the intercellular lipid bilayer in the recovery of normal barrier function.