Tóm tắt Luận án Study on the therapeutic effectiveness of tg extract on chronic skin ulcers

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENSE MILITARY INSTITUTE OF TRADITIONAL MEDICINE LUONG THI KY THUY STUDY ON THE THERAPEUTIC EFFECTIVENESS OF TG EXTRACT ON CHRONIC SKIN ULCERS Speciality: Traditional medicine Code: 62720201 DOCTOR THESIS ABSTRACT HANOI – 2016 THIS STUDY WAS COMPLETED AT THE MILITARY INSTITUTE OF TRADITIONAL MEDICINE Science instructors: Associate Professor, Medicine Doctor LE DINH ROANH Associate Professor, Medicine Doctor PHAM VIET DU Reviewer 1: Associate Professor, Medicine Doctor LE LUONG DONG Reviewer2: Associate Professor, Medicine Doctor LE VAN DOAN Reviewer3: Medicine Doctor PHAM THI VAN ANH The thesis is protected by The National Council at The Military Institute of Traditional Medicine At (time) date month 2016 YOU CAN READ THIS THESIS IN: National Library Library of Central Medicine Information Military Institute of Traditional Medicine Library INTRODUCTION 1. Reason for choosing this subject A chronic skin ulcer (CSU) is an injury in the form of a sore on the skin or a mucous membrane. A CSU is defined as a wound lesion that lasts more than four weeks without remarkable healing tendency or as a frequently recurring wound. CSU seriously affects the quality of life of patients and causes immense costs to the healthcare system. Although the prevention and treatment of CSU has improved greatly, CSU still remains a great challenge for modern medicine. According to traditional medicine theories, CSU has various symptoms such as local sores, chronic pain and itching, it is also prone to relapse. Traditional medicine has been used for the prevention and treatment of CSU for many years and it has now expanded and is used worldwide. TG extract or chicken egg yolk oil which is mentioned in “Miraculous Effects of Southern Medicine” (Tue Tinh, 14th century) and “Compendium of Medical Material” (Shizhen Li, 1518-1593), is used to treat ulcers because of its functions: qing-re (clearing heat), anti-inflammatory, pain relief, moisturizing and proliferation. 2. Objectives of thesis 1) Determine the chemical composition and safety of TG extract. 2) Evaluate the effects of TG extract on CSUs in vitro rabbits. 3) Evaluate the clinical therapeutic efficacy of TG extract on CSUs stage II and III. 3. Scientific and practical significances - Determine the basic components of TG extract. - The results show that TG extract delivers effective treatment of CSUs: increases fibroblast migration, proliferation and activity, boosts the extracellular matrix (ECM) biosynthesis, increases angiogenesis and epithelial proliferation, and decreases MMP9, reduced destruction of the ECM, therefore CSUs heal faster. - Give a hypothesis of action mechanism of TG extract in the treatment of CSUs. - Provide a simple remedy which is easy to produce and can be used across-the-board in the treatment of CSUs. 4. The structure of the thesis Our thesis consists of 129 pages: Introduction (3 pages); Background (33 pages); Method (25 pages); Results (37 pages); Discussion (29 pages); Conclusion (1 page); Request (1 page). There are 158 references (Vietnamese: 21, English: 119, Chinese: 18); 29 tables, 1 chart, 68 figures, 14 appendixes in the thesis. Chapter 1: BACKGROUND 1.1. Skin histology The most common structural components within the dermis is the extracellular matrix (ECM). Understanding the structure and function of the ECM is the key to successful treatment of CSUs. 1.2. Pathophysiology of acute wound healing Acute wound repair process can be divided into 4 continuous and overlapping phases: coagulation, inflammation, proliferation and ECM remodeling. 1.3. Pathophysiology of chronic skin ulcers CSUs do not heal in normal phases and time scales. Despite intensive treatment, the wound is unable to close. The pathological disorders: (1) Proliferation reduction; (2) Imbalance between enzymes for protein hydrolysis and inhibitors; (3) The appearance of old and weak cells. 1.3.5. Treatment for chronic skin ulcers Systemic therapy (according to etiology) Topical treatment: debridement, anti-inflammatory, anti-infection, maintaining moisture balance, biological agents, skin grafts and other forms of treatment. However, CSUs which are still a challenge for modern medicine, still don’t have a specific drug to treat them. 1.4. Traditional medicinal opinion on chronic skin ulcers Traditional medicine considers that CSU belongs to the “ulcer” branch of the Ulcer and Sores diseases. In traditional medicine, the pathogenesis of CSU is theoretically caused by “Re (heat)”. The pathogenic process of CSU was described first in “Lingshu: yongju” as follows: “harmful cold accumulates in the meridian and the results in a stiffness in blood flow and obstruction, which inhibits the circulation of defensive energy, leading to inflammation. Subsequently, harmful cold changes into harmful heat, which causes tissue damage and then pus formation.” The pathologic mechanisms underlying the refractory skin ulcers are that “long term disease results in a deficiency and stasis in both qi and blood, leading to a disorder of ying (nutrition) and wei (immunity) and skin dystrophy.” The “Xu (deficiency)” and “Yu (stasis)” are two major pathologic factors of the development of CSUs. Y. J. Wang and H. F. Que (2009) divided CSU into three syndrome types of blood stagnation with dampness-heat, qi deficiency with blood stasis and spleen deficiency with dampness encumbrance. Treatment of a CSU follow the principle of “Qing-Hua-Bu” method to treat for the syndrome differentiation: 1) Qing-re (clearing heat), zhi-shi (remove dampness), hua-yu (resolve stasis), sheng-ji (stimulate the growth of new skin) to treat dampness-heat syndrome; 2)Tonify qi, hua-yu, sheng-ji to treat qi deficiency with blood stasis syndrome; 3) Jian-pi (strengthen spleen), zhi-shi, sheng-jitotreatspleen deficiency with dampness encumbrance syndrome; and the last target is “qu-fu-sheng-ji, ji-ping-sheng-zhang” (removing necrotic tissues to stimulate the growth of new skin). 1.5. Experimental chronic skin ulcer models Rudolph R. (1979) introduced an animal model of Adriamycin ulceration. More than 50 days (rats) and 45-65 days (rabbits) of prolonged testing is suitable for experimental research. 1.6. TG extract TG extract or chicken egg yolk oil mentioned in “Miraculous effects of Vietnamese medicine – Ten therapies” (Tue Tinh, 14th century), can be used for treating ulcers, sores and burns. Chicken egg yolk oil is made by a traditional heating method and has a sweet, sour, warm, non-toxic characteristic. This remedy is useful for treating venereal diseases, ulcers and burns. Rastegar F. et al (2011) studied the effect of egg yolk oil in the healing of 3rd degree burn wounds in rats which showed that burn wounds healed faster and had abundant re-epithelialization without scarring. Chapter 2: SUBJECTS AND METHODS 2.1. Drugs and Chemistries TG extract which was made in the Pharmacy Department of The Military Institute of Traditional Medicine is a topical concentration of 6 grams of medicine per milliliter (Figure 2.1). 2.2. Subjects Determine the chemical composition of TG extract: 6 samples were taken at random from a batch of new products. Determine the safety of TG extract. Skin irritation: 03 New Zealand White rabbits. Acute toxicity of subcutaneous and oral administrations: Swiss white mice (National Institute of Hygiene and Epidemiology), at least 10 mice per batch. Dermal acute toxicity: 36 New Zealand White rabbits. Figure 2.1. TG extract packaged form Dermal subchronic toxicity: 30 New Zealand White rabbits. Experimental therapeutic effects: 30 New Zealand White rabbits. Evaluate clinical therapeutic effects: 64 chronic skin ulcers stage II and III of 59 inpatients and outpatients at Departments of Military Central Hospital 108 (A1, A2, A6, A7, A10, A15, B1A, B1B, B1C) from 10/2012 to 04/2015. Included criteria: stage II and III chronic skin ulcers. Excluded criteria: stage IV skin ulcers, syphilis sores, tuberculosis, leprosy; patients with systemic immunosuppression, systemic disease, mental illness, blood disease, life-threatening, patients not cooperating or dropped out of the treatment. 2.3. Method 2.3.1. The chemical composition analysis and the safety of TG extract 2.3.1.1. Chemical composition analysis Determine fatty acids in Gas Chromatography Mass Spectrometry (GC/MS). Quantitate some metals in Atomic Absorption Spectroscopy (ASS). Determine pH in Seven Easy device. 2.3.1.2. Determine the safety of TG extract * Evaluate acute toxicity: oral, subcutaneous (WHO guidelines) and topical administration (the guidance of the Organization for Economic Co-operation and Development (OECD). * Evaluate chronic toxicity: topical administration on rabbit for 90 days (the guidance of the OECD number 411, 1981). 2.3.2. The therapeutic efficacy of TG extract on experimental chronic skin ulcers Method: Controlled experimental study on animals. Use the model of Rudolph R et al: Experimental skin ulcers produced by Adriamycin on rabbits. Experimental treatment model of TG extract Each rabbit was treated with 2 parallel therapies: Controlled ulcers: Group 1 (10 rabbits): Change the dressing, wash and cover sores with gauze soaked in NaCl 0.9% solution. Group 2 (20 rabbits): Silver Sulfadiazine grease (SS). The study ulcers (30 rabbits): topical TG extract. Change the dressing once every 2 days and photograph the ulcers. Location: Laboratory - National Institute of Burns. Figure 2.5. Measure the size of CSUs using the Image Pro Plus software Evaluate the therapeutic efficacy in general anatomy Measuring size: perform on ulcer photographs (with standard ruler (cm): Askina, B. Braun) on the 1st day, 14th day and 22nd day of treatment using polygon mode of the Image Pro Plus 4.5 software (America) (Figure 2.6). Evaluate the effectiveness of TG extract by rate of ulcer size reduction: Percentage change in wound area of a CSU (%) = (initial size – inspection time size)/initial size x 100 Evaluate the effectiveness of experimental treatment on scoring system by Sanada DESIGN H et al (2004) (Table 2.2.). Histopathology Biopsy the ulcers 3 times: 1st day, 14th day, 22nd day of the treatment. Histopathological test: H&E (hematoxylin eosin) staining, scanned by light microscope. Immunohistochemistry: Randomly selected biopsy samples of 5 animals, immunohistochemical staining (ABC method) to represent CD34, Vimentin and MMP9. Ultrastructure: Template biopsies were scanned on electron microscope (JSM 5410LV, JEOL, Japan), and transmission electron microscope (JEM 1400, JEOL, Japan). 2.3.3. Evaluate clinical therapeutic effectiveness of TG extract Method: clinical trial, longitudinal and cross-sectional study. Systemic therapy: follow to protocol. Site treatment: prepare ulcers bed: clean the surface, excision. All lesions had their dressing changed, cleansed and ointment was applied once per day. Medication: Apply TG extract on ulcer surface once per day. Dosage: 0.5 to 1 ml of TG extract per 1000 mm2 of skin. Cover and hold a thin piece of gauze in place. Method of evaluating the clinical therapeutic effectiveness Measuring ulcer size: Measure the size of ulcers on photographs taken at the moments: the start, the middle and the end of treatment using polygon mode of Image Pro Plus 4.5 software (US). Evaluate the ulcer size changing: Percent of ulcer size reduction (%) = (initial size – inspection time size)/initial size x 100 Table 2.2. Ulcers scoring based on the DESIGN criteria DESIGN criteria Values Score Depth I, II, III, IV stage Exudate - No; - Less to medium; - Much 0; 3; 6 Size (mm2) 0; <400; 400 to<1600; 1600 to < 3600; 3600 to < 6400; 6400 to< 10000; ≥ 10000 0; 3; 6; 8; 9; 12; 15 Infection No; obvious symptoms or localized infection 0; 3 Granulation tissue Unknown (closed wounds) ≥50% area of injury < 50% area of injury 0 3 6 Necrotic tissue No; Yes 0; 3 Pocketing No; Yes 0; 6 Evaluate the clinical effectiveness of treatment based on DESIGN scoring system of Sanada H et al (2004) (Table 2.2). Use the following formula to determine the change of detail scores: Changing in detail scores according to DESIGN: Changing in detail score of ulcers = (inspection time score) - (initial score) Proportion of effective treatment for ulcers Efficiency ratio (%) = (Number of effective ulcers) / (number of effective ulcers + number of inefficient ulcers) x100 Effective: reducing ≥50% of total score compared to the initial score. Ineffective: reducing<50% of the total score compared to the initial score. Percentage of complete healing (%) = (number of < 1 cm2ulcers at the end of the treatment) * 100 / total number of ulcers. 2.4. Statistical Analysis. Use Epi Info 2008 and SPSS 13.0 software and statistical analysis methods in medical research and evaluate the results. Chapter 3: RESULT 3.1. The chemical composition and the safety of TG extract 3.1.1. The chemical composition of TG extract: shown in Table 3.1. Table 3.1. Fatty acid composition of TG extract No. Composition n Content (mg/ml) (± SD) 1 Palmitic acid (C16:0) 6 21,01±0,63 2 Palmitoleate (C16:1) 6 1,72±0,16 3 Stearic acid (C18:0) 6 15,27±1,55 4 Oleic acid (C18:1) 6 38,13±3,20 5 Linolenic acid (18:2) 6 6,79±0,64 6 Arachidonic acid (C20:4) 6 1,14±0,09 7 Docosahexaenoic acid (C22:6) 6 1,77±0,30 8 Cholesterol 6 21,01±0,63 Essential components of TG extract are the long-chain fatty acids: oleic acid (omega-9), linolenic acid (omega-3, omega-6), docosahexaenoic acid (DHA), arachidonic acid (a type of omega-6) and cholesterol. Magnesium (Mg) (3,28±0,63), zinc (Zn) (3,95±1,93) are presented in TG extract. pH of TG extract was 5.2 ± 0.2, acidic. 3.1.2. The safety of TG extract Determine the acute toxicity of TG extract Identify the acute toxicity of subcutainous administration: With volume, the highest concentration of TG extract 60g medicine per kg in rats, there were no signs of acute toxicity. LD50 of subcutainous TG extract was unidentified. Determine acute toxicity of oral administration: maximum dose of 450 mg per kg in rats did not show signs of acute toxicity, after a week of tracking. LD50 of oral TG extract was unidentified based on the Litchfield – Wilcoxon method. Identify the acute toxicity of topical administration: The lethal dose 50% of the animals (LD50) of TG extract with the highest dose (14,4g per kg) was not able to be determined. The sub-chronic toxicity of TG extract After 6 weeks and 12 weeks of topical TG extract, no special expression was figured out in the control group and two study groups. The hematopoietic function, liver and kidney functions tests in both 2 study groups have no significant difference compared to the control group and before treatment (p > 0.05). Liver, kidney and skin histopathology did not show any pathological lesions. TG extract had no experimental sub-chronic toxicity. 3.2. Therapeutic efficacy of TG extract on experimental chronic skin ulcers 3.2.1. Experimental chronic skin ulcers in rabbits 2 skin ulcers were produced successfully on each of 30 rabbits with similar sizes and characteristics. Average time to produce ulcer was 21.3 ± 2.6 days, average size was 11.1 ± 3.5 cm2. 3.3.2. The therapeutic effectiveness of TG extract Treatment duration: Table 3.13. Compare treatment duration between groups (day(s)) Group n ± SD p NaCl 0,9%(1) 10 29.9±2.4 p1-2<0,01 SS (2) 20 26.7±2.0 p2-3<0,001 TG extract (3) 30 22.3±2.9 p1-3<0,001 Healing time of the treatment group was significantly shorter than the 2 control groups (p <0.001), (t-test). Changes in ulcer size: Table 3.14. Changes in ulcer size after 14 and 22 days of treatment Time Group n ± SD p After 14 days of treatment TG extract (1) 30 2.99±1.31 p1-2<0.05 NaCl (2) 10 5.48±2.11 p1-3<0.05 SS (3) 20 4.95±1.57 p2-3>0.05 After 22 days of treatment TG extract (1) 30 0.52±0.55 p1-2<0.01 NaCl (2) 10 3.44±1.31 p1-3<0.01 SS (3) 20 2.15±0.67 p2-3<0.05 The ulcer size of TG extract group decreased faster than the 2 control groups did (p <0.01). The proportion of complete healing is calculated by the ratio of ulcer which completely closed or size <1cm2 on the 22nd day. Table 3.15. Compare effectiveness of completely closed ulcer on the 22nd day Group Effectiveness Ineffectiveness Total p TG extract (1) 29 (96.7%) 1 (3.3%) 30 p1-2<0.001; NaCl 0,9%(2) 1 (10%) 9 (90%) 10 p1-3<0.001; SS (3) 11 (55%) 9 (45%) 20 p2-3<0.05 Total 41 19 60 TG extract group had healing rates significantly higher than control groups (p <0.001) (Fisher test). Evaluate ulcers according to DESIGN system Table 3.16. Compare DESIGN total score between the groups Group DESIGN total score p 0 day 14th day 22nd day NaCl 0,9% (n=10) 21.01 16.8±2.9 11.1±3.51 pn0-n22<0.05 SS (n=20) 21.3±0.72 16.5±3.2 12.5±3.12 pn0-n22<0.05 TG extract (n=30) 21.2±0.63 6.3±0.9 0.9±1.63 pn0-n14,n22<0.01 p p1-2-3>0.05 p1-2,3<0.01 p1-2,3<0.01 After 14 and 22 days of treatment, total score of TG extract group decreased significantly faster compared to two control groups (p <0.01) (t-test). The therapeutic efficacy according to the rate >50% of reduction on the DESIGN total score Table 3.17. Compare therapeutic efficacy between the groups according to DESIGN Group Effectiveness Ineffectiveness Total p TG extract (1) 29 (96.7%) 1 (3.3%) 30 p1-3<0.05 NaCl 0,9%(2) 4 (40%) 6 (60%) 10 p1-2<0.001 SS (3) 15 (75%) 5 (25%) 20 p2-3>0.05 Total 45 15 60 The effective treatment proportion of TG extract group was significantly higher compared to control groups (p <0.05, p <0.001) (Fisher test). Histopathology The similar ulcers were made in rabbits: frozen necrotic tissue, inflammatory cell infiltration (Figure 3.6). ab Figure 3.6 and 3.10. Histopathology of skin ulcers which were treated with TG extract. a - 3.6. Initial treatment, frozen necrotic tissue (**), inflammatory cells (®). b – 3.10. TG extract group on the 14th day of treatment: Fibroblasts proliferation (®), angiogenesis (¬- -), epithelial cells (**) . The control groups still showed remarkable inflammatory process after 14 days of treatment. There was moderate proliferation of fibroblast and blood vessel after 22 days. In the TG extract group, there were dominant fibroblast proliferation, angiogenesis, some samples showed epithelial cells on the 14th day (Figure 3.10). Extracellular matrix structure similar to normal skin, a thin epidermis covered after 22 days of treatment: Immunohistochemistry staining CD34 staining Figure 3.14.CD34 staining of skin samples which was treated with TG extract at 14th day. Positive blood vessels with dark brown CD34 (Rabbit 22). X400 (cited). The positive level with CD34 of the groups was different over time. It was slightly positive with CD34 of control groups on the 14th day, strongly positive on the 22nd day. The TG extract group had a strong positive with CD34 on the 14th day and decreased after 22 days of treatment. Vimentin antibody staining Figure 3.15. Vimentin antibody staining, TG extract group, 14th day, X400 At the beginning, all skin ulcer samples were negative with Vimentin. On the 14th day, the control groups samples had light positive with Vimentin, increased on the 22nd day. The TG extract group samples had strong positive with Vimentin on the 14th day and had decreased on the 22nd day. MMP9 antibody staining Figure 3.16. 0.9% NaCl control group on the 14th day, strong positive with antibodies MMP9. X400. At the beginning, all ulcers were strongly positive with MMP9. After 14 days, the control groups ulcer had strong positive with MMP9 and reduced on the 22