Study On The Underlying Mechanism For Tgase 1, Acpase And Aplpase Involved In Desquamation And Therapeutic Effects In Abnormal Desquamation Subjects

BackgroundThe normal epidermis consists of 95% keratinocytes (KC) and 5% melanocytes, Langerhans cells and Merkel' s cells. On the basement membrane some stem cells become hypertrophy and proliferate into the prickle cells. In the stratum spinosum there are differentiated products, including keratin filaments and lamellated bodies in the prickle cells. The content of the lamellated bodies, including the lysosomal enzymes, such as acid phosphotase, (ACPase), acid phospholipase A , (APLPase A2) and the lipid, secretes into the intercellular space. Through the transglutaminase 1, TGase 1, activity of cross-linking, both intercellular lipid envelope secreted from the lamellated bodies and the thickening cornified envelope, underlie the KC cytomembrane to form the epidermal barrier. The superficial KC of the stratum corneum becomes dissociation with each other, apoptosis and desquamation. The abnormal desquamation caused by un-dissociation of the superficial KC of the stratum corneum displays stacks of the patchy scales. The KC abnormal metabolism either in the lamellar ichythiosis (LI) subject accounted for congenital related gene, such as TGM1 gene defect, or in the psoriasis vulgaris subject accounted for immunogenetic inflammation also can result in parakeratosis or paraapoptosis to exhibit abnormal desquamation phenotype.In clinic, the drugs, such as urea, retinoic acid and steroids are commonly used for treatment of the subject with abnormal desquamation. These drugs are efficacy to certain extent with some side effects, such as epidermal atrophy, epidermal barrier function impairment and etc. Fartasch M applied the glycolic acid in low concentration to desquamation study and found that the desquamation was caused by degradation of the intercellular desmosome material in the superficial KC of the stratum corneum, which did not impair the epidermal barrier function. The information about the underlying mechanism for normal/abnormal desquamation and about its symptomatic therapeutic way in clinic has been limited.This study was designed to explore the way how to apply the glycolic acid in different concentrations to the subjects with different degrees in abnormal desquamation; and to explore the underlying mechanism for normal/abnormal desquamation through examining the activity of ACPase, APLPase A2nd TGase 1 associated with epidermal barrier function in normal/abnormal desquamation.Materials and Methods:1. Collection of samplesThe epidermal scales were collected from 30 subjects, including 10 cases of the lamellar ichythyosis (TGM1 gene was sequenced in individual case with heavy degree by Gene Company), 10 subjects of psoriasis vulgaris and 10 healthy volunteers.2. The way applied to the subjects with different degrees in abnormal desquamation with different concentrations of glycolic acid-glycerin.3-4% glycolic acid + 50% glycerin for heavy, 1-2% glycolic acid + 50% glycerin for the mild abnormality subjects, once topical treatment per day.3. Detection of the acid phosphotase activity The scale samples were fixed with 4% formaldehyde calcium for 30 min→0.585g sodium acetate in 5 ml DD.H2O→dip 2.5ml out of 5ml+1 ml 8.5% NaCl+0.1N HCl+4ml DD.H2O→substrate solution (dip 6ml out of it + 0.19ml 3.3% lead nitrate+16ml DD.H2O+2ml 3.2% beta-sodium glycerol + 0.5ml 0.6% MgSO4, pH adjusted to 5.6) filtrate before incubation on the slides. Incubate them at 37℃for 4h, washed with water, followed by 1% acetic acid for 30s, DH2O washing 3 times, 2% (NH₄)S₂ for 1-2 min, D H2O washing for 5min. Observe the slides under microscope and photo them. The negative controls performed were treated with no substrate.4. Detection of TGase 1 activity. Firstly, the scale spread slides and the HRP-monodansyl caverine conjugate (MDC) were prepared. The procedure of HRP-MDC preparation and detection of the TGase 1 was as follows:â‘ Activate /Oxidize HRP the HRP was activated and oxidized by sodium periodide (NaIO₄ and dialyzed at 4℃against 1 mmol/L acetate buffer overnight to remove recessive NaIO₄.â‘¡4mg mono-dansyl cadaverine (Sigma, US) dissolved in 1 ml PBS was mixed with the activated HRP, stirred at room temperature for 3h to form the conjugate.â‘¢Freshly, prepared 0.05ml 4mg/ml NaBH₄ (Sigma, US) was added and reacted to reduce the Schiff base at 4℃, for 2h.â‘£The TGase 1 activity detected by HRP-MDC: A solution including 10 mmol/L CaCl₂, 1% BSA mixed with B solution HRP-MD in 1:1 volume, and incubated the slides at 4℃overnight followed by DAB staining.⑤The negative controls were treated with no substrate.â‘¥The slides were observed under microscope and photo was taken.5. Detection of the APLPase A2 The incubation substrate (10ml 1% lecithine solution + 10 ml 2% Cal₂ in 0.1 mol/L glycine buffer, pH5.5) was used to incubate the slides at 37℃for 14-36h. After D.H2O washing, the slides were stained in the nile blue colloid at 60℃for 30min. Stop the reaction, observe the slides under microscope and photo them. The negative controls were treated with no substrate.6. Statistical analysis The experimental data were scanned with an Image Analyzer (Shan Fu, China) and the grey scanning mean (GSM) was analyzed with ANOV by the SPSS10.0software. The data showed as x±s, and a=0.05 was evaluated as the significant level.Results:1. The TGM1 gene was sequenced in one patient with sever LI ichythosis and thesequenced TGM1/TGase 1 gene mutation mainly showed a deleted T, 150th base, located in the exon 3, resulting in frameshift mutation.2. The abnormal desquamation phenotype in most of the LI subjects (80%), including the TGM1 gene sequenced in the individual patient with sever LI ichythosis was improved markedly to certain extent.3. In the control group the ACPase activity appears black-colored fine granules mainly located at the intercellular bridge, while the ACPase activity was almost not located in intercellular bridge with light grey color in the patient group. 4. In the control group the TGase 1 activity appears fine granules in brownish color located at the KC surface. However, the TGase 1 activity was poor or paucity in the patient group.5. In the control group the APLPase A2 activity appears fine granules in blue color located at the intercellular KC surface; while the APLPase A activity was poor or paucity in the patient group.Conclusions:1. The ACPase activity and APLPase A2 activity located in the intercellular space of KC show paucity in the patient group, which is involved in the normal/abnormal desquamation.2. The TGase 1 activity located at all over surface of KC involved in cross-linking of the cornified and lipid envelopes shows paucity in the patient group, and it is also involved in the normal/abnormal desquamation.3. The symptomatic therapy with glycolic acid in lower concentration plus glycerin can improve the abnormal desquamation profile in LI patients, even in one LI case (determined by TGM1 gene sequencing) with sever scaling.