Nghiên cứu tác dụng lên hành vi và chuyển hóa lipid máu của nano alginate chitosan lovastatin trên chuột cống gây mô hình béo phì TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE VIETNAM MILITARY MEDICAL UNIVERSITY NGUYEN THI HOA A STUDY ON THE EFFECT OF NANO ALGINATE/CHITOSAN/LOVASTATIN ON BEHAVIORS AND LIPIDEMIA METABOLISM IN RATS OF OBESITY MODEL Speciality: Biomedical sciences Code: 9720101 SUMMARY OF MEDICAL DOCTORAL THESIS HANOI - 2022 THIS WORK WAS CARRIED OUT AT VIETNAM MILITARY MEDICAL UNIVERSITY Supervisors: Pham Minh Dam, M.D., Ph.D Can Van Mao, MD., Ph.D., Assoc.Prof Reviewer: Nguyen Van Tuong, PhD., Assoc.Prof Reviewer: Pham Van Tran, PhD., Assoc.Prof Reviewer: Duong Thi Ly Huong, PhD., Assoc.Prof This thesis will be defended at University-level council at: h day month year 2022 This thesis will be available at: National Library of Vietnam Library of Vietnam Military Medical University INTRODUCTION Imperativeness Obesity causes many serious consequences and complications affecting the all of organs system in the body such as: lipid metabolism disorders, diabetes, cardiovascular, skeletal, and respiratory diseases In particular, recently, there have been a number of studies showing that obesity causes dysfunction of the central nervous system, which is memory loss, cognitive decline, and reduced movement To study the pathogenesis as well as evaluate the effectiveness of therapeutic interventions, many obesity models have been built on experimental animals The model of obesity that is considered to have the closest mechanism to the clinical reality of obesity is the high-energy diet Several studies have shown that this pattern also reflects the association of dyslipidemia and impaired brain function In Vietnam, the study of animal models of obesity has been performed by several scientists However, these studies only investigated about the changes of body shape, body weight, dyslipidemia but there are almost no studies evaluating about the function of nervous centre systems on an animal model of obesity The research team of the Institute of Tropical Technology in Vietnam Academy of Sciences has successfully made the alginate/Chitosan nanoparticles from natural materials in Vietnam and load with Lovastatin, which is one classic drug of the statin group to test the ability to increase the therapeutic effect of the drug Therefore, we performed this study to, namely: ‘A study on the effect of Nano Alginate/ Chitosan/Lovastatin on behaviors and lipidemia metabolism in rats of obesity model’ with the following objectives: - To evaluate behavioral changes and lipid metabolism disorder of experimentally induced obese rats - To evaluate the ameliorative effect on behaviors and lipid metabolism disorder of nano Alginate/Chitosan/Lovastatin complex in obese rats Scientific significance The thesis has showed a success in making an obesity model in rats by a high-fat diet with 38.9% of fat in foods over weeks via several criteria including biometric parameters, blood lipids, and behavioral indices The thesis has also proved the ameliorative effects on lipid metabolism of nano Alginate/Chitosan/Lovastatin as well ameliorative effects on locomotion, exploration, and learning and memory in obese rats Practical significance The results of behaviors and changes of lipid metabolism contribute significantly for the development of experimental animal models Particularly, over 12 intervention weeks clearly shows the value of applying the model with results on the effects of nano alginate/chitosan/lovastatin on behaviors and lipid metabolism in obesity Structure of the dissertation The thesis includes 126 pages: Introduction in pages; Chapter (Literature review) in 42 pages; Chapter (Study subjects and methods) in 13 pages; Chapter (Results) in 41 pages; Chapter (Discussion) in 23 pages; Conclusion in pages and Recommendation for Future researches in pages The thesis includes 20 tables, 26 figures, 184 references (5 in Vietnamese and 179 in English, 39 papers are in recent five years) CHAPTER LITERATURE REVIEW 1.1 Overview of obesity 1.1.1 The concept of obesity Obesity is an excess weight ratio over to heigh, in which there is abnormal or excessive fat accumulation that may impair health, increases the risk of with many diseases Body mass index (BMI) is commonly used to classify overweight and obesity adults 1.1.2 Prevalence of obesity in the world and in Vietnam According to a World Health Organization report in early 2015, the number of obese people has tripled since 1980, more than 1.9 billion adults, 18 years and older, were overweight, with 39%; over 600 million were obese The prevalence of overweight and obesity varies both by sexes and regions In Vietnam, obesity is also on the rise both in aldults and children, accounting for about 25% of the total adult population 1.1.3 Etiology and consequences of obesity The basic influence on the body weight status is the energy balance and the level of physical activity of individuals In addition, that is the result of factors that contribute to obesity, which are genetic, metabolic, environmental, behavioral and cultural factors 1.1.4 The effects of obesity on central nervous system Studies show that obesity reducing motor activity, exploration, recongnition, learning and memory, increasing anxiety in both humans and experimental animals They are premises to design behavioral tests to assess motor, learning and memory ability in experimental animals 1.1.5 Medications for treatment of obesity 1.1.5.1 Reducing food intake 1.1.5.2 Blocking nutrient absorption 1.1.5.3 Increasing energy expenditure 1.1.5.4 Modulating the central and peripheral controller regulating body weight 1.2 Experimental animal models of obesity 1.2.1 Surgical or chemical models of obesity Surgical induced obesity in animals mainly related the hypothalamus and surgery in other organs including uterus and adipose tissue 1.2.2 Genetic models There are over 50 different genetic models, these models are of monogenic or polygenic mutations which related to obese 1.2.3 Others animal model of obesity 1.2.4 High energy diet induced obese rats These are simple model of obesity exposing characteristics in forms similar to those of human obesity Some diets achieve their calorific value by adding carbohydrates and others by adding fat 1.2.5 Parameters used for assessement of obesity in the animal model The parameters used to assess including food intake, markers related to body weigh and adiposity, glycemia, insulin, lipid indexes and metabolism function of the liver 1.2.6 Methods for behavioural assessment in experimental animals 1.2.6.1 Morris water maze test Measured travelled distance and latency to finding the escape platform 1.2.6.2 Object recognition test Measured time and frequency exploring the novel and familiar object 1.2.6.3 Openfield test Measured travelled distance, mobile time, moving in/out zones 1.3 Overview of Nano Alginate/Chitosan/Lovastatin 1.3.1 Chitosan The deacetylated form of chitin as chitosan, abundant in the exoskeletons of crustaceans such as shrimp, crabs, squids, and in fungal cell walls; application in many fields as a biomaterial and medicine for treating wounds, ulcers, tumor, anti-inflamation, and dyslipidemia 1.3.2 Alginate Alginates are natural polysaccharide polymers isolated from brown seaweed (Phaeophyceae) For pharmaceutical applications, sodium alginate can be used as a stabilizer, emulsifier, or as a thickening agent in water-miscible gels 1.3.3 Alginate/Chitosan Study about production and application of AG/CS polymer complex material carriering variable drugs is a trend that has been attracting attention of many nationwide and worldwide scientists 1.3.4 Lovastatin and drugs carrier polymer complex 1.3.4.1 Introduction about Lovastatin Lovastatin is among commercial statin compounds, which are naturally occurring in a low concentration in certain foods such as oyster mushrooms and red yeast rice Lovastatin has effect on reducing cholesterolemia and preventing cardiovascular complications 1.3.4.2 Study on drug carrier polymer complex in the world and in Viet Nam Using nanoparticale system has been a new trend in research and application in the world in treating many disorders, including metabolic disoders Alginate/Chitosan/Lovastatin complex has made by the Institute of Tropical Technology in Vietnam Academy of Sciences Studying effect of this complex in experimental animals is necessary CHAPTER SUBJECTS AND METHODS 2.1 Subjects White rats provided by Animal Center of Vietnam Military Medical University (VMMU) had been used in two research aims below Research aim 1: Making experimental obese rat, assessing behavior and lipid metabolism in the modelled rats 72 whilte male rats aged -9 weeks old (weighted from 100 – 150 g) were randomly divided into two groups of normal diet (36 rats) and high-fat diet (36 rats), they were fed for weeks to prepare the treatment period Research aim 2: Evaluating effect on behavior and lipid metabolism disorder of nano alginate/chitosan/lovastatin complex in the obesity rats (the intervention period) for 12 weeks Rats were divided into groups (12 rats of a group) including: i) nomal diet-saline (C-NaCl) group, ii) nomal diet-lovastatin dosed mg/kg group (C-Lovastatin), iii) nomal diet- Alginate/Chitosan//Lovastatin nano combination of mg/kg (C-Nano/Lovastatin); iv) high-fat diet-saline (B-NaCl), v) high-fat diet- lovastatin dosed mg/kg (B-Lovastatin), and vi) high-fat diet-Alginate/Chitosan/Lovastatin nano combination dosed mg/kg (B- Nano/Lovastatin) 2.2 Methods 2.2.1 Research design An experimental, interventional, controlled, cross-sectional and longitudinal study, through weeks for modelling and 12 weeks of the intervention (orally administered of Lovastatin or physiological saline) 2.2.2 Materials Chemicals used in this study including natri clorid 0,9%, Lovastatin powder (Sigma Aldrich) Alginate/Chitosan/Lovastatin with ratio 8:2:10% Electronic scales to measure rats weight, internal organ weight and chemical; length scale to determine the length, chest and waist circumference of rats Blood component testing was performed at the Military Medical and Pharmaceutical Research Center, VMMU Behavioral studies in experimental chambers with instruments (open field, water maze ) Experimental rooms were kept quiet with temprature stable at 25 ± oC, equipped with the open-field, novel object recognition chamber, Moriss water maze, and behavioral recording and analyzing systems 2.2.3 Research procedures Research aim 1: Making experimental obese rat, assessing behavior and lipid metabolism in the modelled rats 72 male rats aged -9 weeks old were randomly divided into normal and high fat diet groups for weeks Evaluation parameters included anthropometric measurements, blood test, food intake, and behaviors 2.2.3.1 Anthropometric measurement, food intake and drinking Anthropometric parameters wrere measured at the start of the study and twice per week for weeks in the model period, and triple per week for 12 weeks of the follow intervention period Food intake and consumed drinking wrere measured twice per week during two the periods 2.2.3.2 Analysis lipidemia and glycemia Glycemina, triglyceridemia, and cholesterolemia were determined at the end of week 2, 4, and of the model period and at the end of week 3, 6, 9, 12 of the intervention period Research aim 2: Rats fed with normal or high-fat diet were given one of the substances, as Lovastatin, Nano/Lovastatin, or saline, divided into groups of rats during the intervention period Measure anthropometric indexes, food consumption, glycemia and lipidemia Behavioral assessment in the open field on locomotion, object recongnition and learning activities in the water maze Weighted organs after intervention 2.2.3.3 The procedure to evaluate rat behavior causes obesity model Using locomotion, exploration test in the open-field, object recognition test with three sessions and the Moris water maze test 2.2.3.4 The procedure to weight organs and make pathohistological analysis 2.3 Data analysis Data has been anlalyzed by the software SPSS Statistics ver 20.0 (IBM Inc., USA) and significant value was set at p < 0.05 2.4 Research ethics Experimental procedures and care of experimental animals were performed according to the guideline of Animal Center, VMMU CHAPTER RESULTS 3.1.1 Results of the model of obese rats 3.1.1 Anthropometric indexes, food intake and consumed drinking 3.1.1.1 Anthropometric indexes Figure 3.1 Bodyweigh (A) and Length (B) in the two groups of rats over weeks *: p < 0.05; **: p < 0.01; ***: p < 0.001 The body weight of the high-fat diet group was higher than that of the normal diet rats from the end of week (p < 0.05) The body length of the high-fat diet group was longer than that of the normal diet rats over weeks and marked at the end of week (p < 0.05) 3.1.1.2 Food intake and consumed drinking 3.1.2 Results of several lipidemic components and glycemia 3.1.2.1 Glycemia concentration 3.1.2.2 Several lipidemic components Table 3.5 Triglyceridemia (mmol/L) of the two groups of rats over weeks Group Normal diet High-fat diet p Time point (̅x ± SD) (̅x ± SD) Start 1.20 ± 1.08 1,17 ± 0.89 0.909 End of week 0.105 End of week 1.63 ± 1.06 1.97 ± 1.36 4.24 x 10-4 End of week 3.63 x 10-5 End of week 1.43 ± 0.65 2.77 ± 2.10 3.18 x 10-4 F(1, 69), p 1.37 ± 1.00 3.29 ± 2.38 1.06 ± 0.64 4.23 ± 4.50 42.730, 9.58 x 10-9 Figure 3.7 The discrimination time ratio for each object in the sample phase (A) and the test phase (B) of the two groups of rats at the end of the modelling period **: p < 0.01 In the sample phase, no difference in discrimination ratios between two objects of the two groups In the test phase, the discrimination ratios between the familiar object and the novel object were different in normal diet group but indiferent in high-fat diet group 3.1.3.3 Learning, memory activity in Moriss water maze Figure 3.9 Travelled distance (A) and time to find the submerged platform (B) of the two groups of rats over training days in the water maze at week 3.1.4 Histological results of modelled animals 3.2 Results of effect on behaviors and blood lipid metabolism disorder of nano Alginate/Chitosan/Lovastatin in obesity rat model 3.2.1 Anthropometric indexes, consumed food and drinking in the intervention period 3.2.1.1 Anthropometric indexes Figure 3.11 Body of the weight (gram) investigated groups of rats in normal diet (A), high-fat diet (B), and their body length (cm) in normal diet (C), high-fat diet (D) over 12 weeks of intervention 3.2.1.2 Consumed food and drinking 3.2.2 Effects on lipidemic disorder and glycemia in the intervention period 3.2.2.1 Glycemia concentration 3.2.2.2 Several lipidemic components Table 3.14 Triglyceridemia (mmol/L) of the investigated groups of rats over 12 weeks of intervention Time point Before End of week End of week End of week End of week 12 Diet intervention intervention intervention intervention intervention C-NaCl 1.17 ± 0.70 1.17 ± 0.97 1.43 ± 0.63 1.40 ± 1.15 1.19 ± 1.18 C-Lovastatin 1.15 ± 0.67 0.90 ± 0.54 0.97 ± 0.37 0.88 ± 0.50 0.88 ± 1.28 Normal C-Nano/ 1.21 ± 0.65 0.82 ± 0.40 1.27 ± 0.56 0.84 ± 0.63 1.22 ± 0.86 Lovastatin F-normal diet (2, 33) = 1.190, p = 0.317 B-NaCl 4.27 ± 4.90 1.51 ± 1.48 3.45 ± 1.71a 4.83 ± 2.96bc 2.54 ± 2.88 B-Lovastatin 5.37 ± 5.94 1.15 ± 0.71 2.18 ± 1.55 2.07 ± 1.61c 1.40 ± 1.37 High-fat B-Nano/ 2.92 ± 2.21 1.30 ± 0.87 1.98 ± 1.19a 1.13 ± 0.80b 1.35 ± 0.97 Lovastatin F-high-fat diet (2, 32) = 4.486, p = 0.019, pa < 0.05, pb < 0.01, pc < 0.05 F(2, 65) = 3.009, p = 0,056 Table 3.15 showed that the blood triglyceride concentration of rats in both the normal and high-fat diet groups were difference when using the drug, but not at a significant level Table 3.16 Cholesterolemia (mmol/L) of the investigated groups of rats over 12 weeks of intervention Time point Before End of week End of week End of week End of week 12 Diet intervention intervention intervention intervention intervention C-NaCl 1.29 ± 0.18 1.15 ± 0.35 1.29 ± 0.25 1.50 ± 0.30 0.95 ± 0.46 C-Lovastatin 1.21 ± 0.45 1.39 ± 0.31 1.11 ± 0.33 1.47 ± 0.27 1.08 ± 0.49 Normal C-Nano/ 1.28 ± 0.16 1.21 ± 0.22 1.21 ± 0.28 1.38 ± 0.53 1.27 ± 0.41 Lovastatin F-normal diet (2, 33) = 0.116, p = 0.891 B-NaCl 2.07 ± 0.65 1.45 ± 0.63 2.23 ± 0.59a 4.20 ± 3.21bc 1.86 ± 0.97d B-Lovastatin 1.82 ± 0.80 1.43 ± 0.23 1.89 ± 0.58 1.69 ± 0.27c 1.33 ± 0.36 High-fat B-Nano/ 1.83 ± 0.44 1.45 ± 0.13 1.45 ± 0.65a 1.50 ± 0.39b 1.14 ± 0.25d Lovastatin F-high-fat diet (2, 32) = 6.831, p = 0.004; pa, pb, pc , pd < 0.05 F(2, 65) = 7.640, p = 0.001 Table 3.16 showed that the blood cholesterol concentration of rats in the normal and high-fat diet groups were significant decreased difference when using the drug Table 3.17 Blood HDL-cholesterol concentration (mmol/L) of the investigated groups of rats over 12 weeks of intervention Drug NaCl Lovastatin Nano/ Diet Lovastatin Before 0.92 ± 0.18 1.02 ± 0.30 0.91 ± 0.24 Normal After 12 week of 0.74 ± 0.19 0.71 ± 0.27 0.78 ± 0.22 intervention F-normal diet (2, 33) = 0.188 p = 0.829 Before 0.86 ± 0.41 0.57 ± 0.37 0.87 ± 0.36 High-fat After 12 week of 0.79 ± 0.31 0.65 ± 0.31 0.85 ± 0.17 intervention F–high-fat diet (2, 32) = 2.878, p = 0.071 F(2, 65) = 2.908, p = 0.062 Table 3.17 showed that the concentration of HDL-cholesterol in the blood of rats in the normal and high-fat diet groups when using the drug were different but not at a significant level Table 3.18 Blood LDL-cholesterol concentration (mmol/L) of the investigated groups of rats over 12 weeks of intervention Drug NaCl Lovastatin Nano/ Diet Lovastatin Before 0.57 ± 0.11 0.60 ± 0.16 0.57 ± 0.10 Normal After 12 weeks 0.53 ± 0.09 0.51 ± 0.12 0.53 ± 0.21 of intervention F-normal diet (2, 33) = 0.018, p = 0.982 Before 1.04 ± 0.45 1.02 ± 0.60 0.84 ± 0.27 High-fat After 12 weeks 0.70 ± 0.31 0.60 ± 0.13 0.60 ± 0.21 of intervention F–high-fat diet (2, 32) = 0.743, p = 0.484 F(2, 65) = 0.708, p = 0.496 Table 3.18 showed that there were no difference in the concentration of LDL-cholesterol in the blood of rats in both the normal and high-fat diet groups when using the drug 3.2.3 Effects on behaviors of animals after drug intervention 3.2.3.1 Spontaneous locomotor activity in the open-field Figu re 3.13 Travelled distance (m) in the open field of the investigated groups of rats in normal diet (A) and high-fat diet (B) after drug intervention Figure 3.13 about the locomotion length after 12 weeks of intervention, in the high-fat diet the group using Nano/Lovastatin tended to be more mobile than the group using Lovastatin and the group using NaCl Figure 3.15 Mobile time in the open field of the investigated groups of rats in normal diet (A) and high-fat diet (B) after drug intervention Figure 3.15 of locomotion time showed no difference in diet as well as drug factor 3.2.3.2 Object recognition activity after the intervention period The results on the entries and the discrimination ratios to explore objects of rats in the study groups of the two diets in the test phase after intervention are presented in Figures 3.23 and 3.24 Figu re 3.23 Object approaches in the test phase in the open-field of the investigated rat groups in normal diet (A) and high-fat diet (B) after drug intervention In the test phase (Figure 3.23), there was no difference in the entries of explore between the novel and familiar objects as well as the entries of explore between the two diets and the entries of explore between the treatment groups Figure 3.24 The discrimination time ratio in the test phase in the open-field of the investigated rat groups in normal diet (A) and high- fat diet (B) after drug intervention *: p < 0.05; **: p < 0.01 The ratio of time to explore objects in the test phase of the normal group and the high-fat group showed no interaction between the three factors There was a difference in the ratios of time to explore between the old and new objects, but no difference in the time rate to explore between the two diets and the ratios of time to explore between the therapeutic drug groups 3.2.3.3 Learning - memory in Moriss water maze after drugs intervention Figue 3.25 Travelled distance to find the platform of rat groups over training days in the wate maze after 12 weeks of drug intervention Two factors (diets and drugs) (analysis ANOVA with two non - repeated factors) However, the travel distance of rats to find the submerged platform of all three drug groups (using NaCl, Lovastatin and using Nano/Lovastain) of the two diets (normal and high-fat) were reduced compared to day1 of training Figure 3.26 Travelled time to find the platform of rat groups over training days in the wate maze after 12 weeks of drugs intervention Figure 3.26 showed that the time that find the submerged platform in the drug groups of the two diets gradually decreased from day to day Regarding the last days of training, rats in the B-Nano/Lovastatin group of high-fat diet tended taking less time to find platform than the Lovastatin group of high-fat diet and the B-NaCl group Table 3.20 Travelled time (s) and distance (m) of the investigated groups of rats at the platform removed quardant of the water maze on the last training day after 12 weeks of intervention Diet Normal High-fat Index C-NaCl C-Lovastatin C-Nano/ B-NaCl B-Lovastatin B-Nano/ Lovastatin Lovastatin Time (s) 27.87 ± 7.10 28.55 ± 6.90 27.61 ± 8.24 28.31 ± 5.78 28.99 ± 10.79 26.41 ± 6.56 F–normal det (2, 27) = 0.043, p = 0.958 F–high-fat diet (2, 28) = 0.284, p = 0.755 F(2, 55) = 0.076, p = 0.927 Distance 8.32 ± 2.36 8.03 ± 2.14 7.76 ± 2.13 8.48 ± 1.02 7.98 ± 2.66 7.89 ± 2.11 (m) F–normal diet (2, 27) = 0.156, p = 0.857 F-high-fat diet (2, 28) = 0.268, p = 0.767 F(2, 55) = 0.015, p = 0.985 Table 3.20 showed that there was no difference in the travelled time and the distance to swim in the quadrant in the previous training days of the treatment groups in the two diets and in each diet CHAPTER DISCUSSIONS 4.1 Assessing behavioral alterations and lipidemic metabolism in the modelled obese rats 4.1.1 Alterations in anthropometric measurements In this study, according to the diet formula for obese rats of Seo et al (2012), we conducted a mouse model of obesity with the intention of increasing blood lipids in 8-week-old Wistar male rats with a high-fat diet The combined results have shown the success of the model, which is performed in many research indicators, including bodyweight and other biometric measurements (body length, thoracic circumference, abdominal circumference and body weight/ body length ratio) increased energy consumption of food and concentration of blood lipid components and blood glucose concentration 4.1.2 Alterations in lipidemic metabolism and glycemia ... diet-saline (B-NaCl), v) high-fat diet- lovastatin dosed mg/kg (B -Lovastatin) , and vi) high-fat diet -Alginate/ Chitosan/ Lovastatin nano combination dosed mg/kg (B- Nano/ Lovastatin) 2.2 Methods 2.2.1 Research... weeks via several criteria including biometric parameters, blood lipids, and behavioral indices The thesis has also proved the ameliorative effects on lipid metabolism of nano Alginate/ Chitosan/ Lovastatin. .. Measured travelled distance, mobile time, moving in/out zones 1.3 Overview of Nano Alginate/ Chitosan/ Lovastatin 1.3.1 Chitosan The deacetylated form of chitin as chitosan, abundant in the exoskeletons