Nghiên cứu hiệu quả điều trị loét da mạn tính của cao TG

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 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, 14 th century) and “Compendium of Medical Material” (Shizhen Li, 1518-1593), is used to treat ulcers because of its functions: qing-re (clearing heat), antiinflammatory, pain relief, moisturizing and proliferation 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 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, 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 continuous and overlapping phases: coagulation, inflammation, proliferation and ECM remodeling 1.3 Pathophysiology of chronic skin ulcers CSUs 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), zhishi (remove dampness), hua-yu (resolve stasis), sheng-ji (stimulate the growth of new skin) to treat dampness-heat syndrome; 2)Tonify qi, huayu, 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, 14 th 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 rd of 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 grams of medicine per milliliter (Figure 2.1) 2.2 Subjects Determine the chemical composition of TG extract: 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 parallel therapies: Controlled ulcers: Group (10 rabbits): Change the dressing, wash and cover sores with gauze soaked in NaCl 0.9% solution Group (20 rabbits): Silver Sulfadiazine grease (SS) The study ulcers (30 rabbits): topical TG extract Change the dressing once every 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 st 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 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 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 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: 10 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; 0; [...]... loét da mạn tính trên động vật thực nghiệm được điều trị bằng cao TG, Tạp chí y học Việt Nam 437; 139-138 4 Lương Thị Kỳ Thủy, Lê Thị Cúc, Phạm Viết Dự, (2015), Đánh giá hiệu quả điều trị loét da mạn tính của cao TG trên lâm sàng, Tạp chí Y dược học cổ truyền Quân sự, 2(5), 34-42 5 Lương Thị Kỳ Thủy, Phạm Xuân Thắng, Đinh Văn Hân (2016), Nghiên cứu hiệu quả điều trị của cao TG đối với loét da mạn tính. .. tính an toàn của cao TG trên động vật thực nghiệm Tạp chí Y dược học cổ truyền Quân sự, 4(3); 48-56 2 Lương Thị Kỳ Thủy, Lê Đình Roanh, Phạm Viết Dự, Đinh Văn Hân, Phạm Lê Bách, (2014), Đánh giá tác dụng điều trị loét da mạn tính của cao TG trên mô hình thực nghiệm Tạp chí Y dược học cổ truyền Quân sự, 4(2), 15-22 3 Lương Thị Kỳ Thủy, Phạm Xuân Thắng, Lê Đình Roanh (2015), Nghiên cứu đặc điểm hình... 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... animals (LD50) of TG extract Thus, TG extract has no acute oral, subcutaneous and topical toxicity Determine the sub-chronic toxicity of TG extract Based on the guidance of the OECD (1981), we evaluated the subchronic toxicity of TG extract in 90 days on rabbit skin The study results showed that TG extract applied on skin continuously for 12 weeks at the highest dose (14,4g / kg / day) did not cause... nd day The TG extract group samples had strong positive with Vimentin on the 14 th 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 17 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 22nd day... of TG extract for chronic skin ulcers which are caused by diabetes, venous disease, irradiation and burn 2 Use more modern research techniques to further elucidate the mechanism of action of TG extract and the applicability in the treatment for chronic wounds CATEGORY OF PUBLISHED STUDIES LINKED TO THE THESIS 1 Lương Thị Kỳ Thủy, Phạm Viết Dự, Nguyễn Thị Tuyết Nga, (2014), Nghiên cứu tính an toàn của. .. conclusions: 1 TG extract contains polyunsaturated fatty acids: arachidonic acid, docosahexaenoic acid, oleic acid, linolenic acid and cholesterol TG extract did not irritate skin There was no acute toxicity by oral, subcutaneous and topical using Topical TG extract did not make chronic toxicity in the equivalent dose used in clinical practice (4,8g / kg / day) and triple dose (14,4g / kg / day) in 90 days under... 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. .. DESIGN total score between the groups Group DESIGN total score 0 day NaCl 0,9% (n=10) 21.01 SS (n=20) 21.3±0.72 TG extract (n=30) 21.2±0.63 p p1-2-3>0.05 th 14 day 16.8±2.9 16.5±3.2 6.3±0.9 p1-2,3