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 DEFENCEVIETNAM MILITARY MEDICAL UNIVERSITY NGUYEN THI HOA A STUDY ON THE EFFECT OF NANO ALGINATE/CHITOSAN/LOVASTATIN ON BEHAVIORS AND LIP

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:

1 Pham Minh Dam, M.D., Ph.D.

2 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:

1 National Library of Vietnam

2 Library of Vietnam Military Medical University

1 Imperativeness

Obesity causes many serious consequences and complicationsaffecting the all of organs system in the body such as: lipid metabolismdisorders, diabetes, cardiovascular, skeletal, and respiratory diseases Inparticular, recently, there have been a number of studies showing thatobesity causes dysfunction of the central nervous system, which ismemory loss, cognitive decline, and reduced movement To study thepathogenesis as well as evaluate the effectiveness of therapeuticinterventions, many obesity models have been built on experimentalanimals The model of obesity that is considered to have the closestmechanism to the clinical reality of obesity is the high-energy diet Severalstudies have shown that this pattern also reflects the association ofdyslipidemia 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 thechanges of body shape, body weight, dyslipidemia but there are almost nostudies evaluating about the function of nervous centre systems on ananimal model of obesity The research team of the Institute of TropicalTechnology in Vietnam Academy of Sciences has successfully made thealginate/Chitosan nanoparticles from natural materials in Vietnam andload with Lovastatin, which is one classic drug of the statin group to testthe ability to increase the therapeutic effect of the drug Therefore, weperformed this study to, namely: ‘A study on the effect of Nano Alginate/Chitosan/Lovastatin on behaviors and lipidemia metabolism in rats ofobesity 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.

2 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 7 weeks via several criteriaincluding biometric parameters, blood lipids, and behavioral indices Thethesis has also proved the ameliorative effects on lipid metabolism of nanoAlginate/Chitosan/Lovastatin as well ameliorative effects on locomotion,exploration, and learning and memory in obese rats

3 Practical significance

The results of behaviors and changes of lipid metabolism contributesignificantly for the development of experimental animal models.Particularly, over 12 intervention weeks clearly shows the value ofapplying the model with results on the effects of nanoalginate/chitosan/lovastatin on behaviors and lipid metabolism in obesity

4 Structure of the dissertation

The thesis includes 126 pages: Introduction in 2 pages; Chapter 1(Literature review) in 42 pages; Chapter 2 (Study subjects and methods) in 13pages; Chapter 3 (Results) in 41 pages; Chapter 4 (Discussion) in 23 pages;Conclusion in 3 pages and Recommendation for Future researches in 1 pages.The thesis includes 20 tables, 26 figures, 184 references (5 inVietnamese and 179 in English, 39 papers are in recent five years)

CHAPTER 1 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 isabnormal or excessive fat accumulation that may impair health,increases the risk of with many diseases Body mass index (BMI) iscommonly 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 1.9billion 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 therise both in aldults and children, accounting for about 25% of thetotal adult population.

1.1.3 Etiology and consequences of obesity

The basic influence on the body weight status is the energy balanceand the level of physical activity of individuals In addition, that is theresult 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 humansand 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 hypothalamusand surgery in other organs including uterus and adipose tissue

1.2.2 Genetic models

There are over 50 different genetic models, these models are ofmonogenic 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 formssimilar to those of human obesity Some diets achieve their calorificvalue 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 andmetabolism 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 escapeplatform

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 theexoskeletons of crustaceans such as shrimp, crabs, squids, and in fungalcell walls; application in many fields as a biomaterial and medicine fortreating wounds, ulcers, tumor, anti-inflamation, and dyslipidemia

1.3.2 Alginate

Alginates are natural polysaccharide polymers isolated from brownseaweed (Phaeophyceae) For pharmaceutical applications, sodiumalginate 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 complexmaterial carriering variable drugs is a trend that has been attractingattention 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 arenaturally occurring in a low concentration in certain foods such as oystermushrooms and red yeast rice Lovastatin has effect on reducingcholesterolemia 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 andapplication in the world in treating many disorders, includingmetabolic disoders Alginate/Chitosan/Lovastatin complex has made

by the Institute of Tropical Technology in Vietnam Academy ofSciences Studying effect of this complex in experimental animals isnecessary

CHAPTER 2 SUBJECTS AND METHODS

2.1 Subjects

White rats provided by Animal Center of Vietnam Military MedicalUniversity (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 8 -9weeks 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 fedfor 7 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 6 groups

(12 rats of a group) including: i) nomal diet-saline (C-NaCl) group, ii) nomal diet-lovastatin dosed 4 mg/kg group (C-Lovastatin), iii) nomal diet-

Alginate/Chitosan//Lovastatin nano combination of 4 mg/kg

(C-Nano/Lovastatin); iv) high-fat saline (B-NaCl), v) high-fat lovastatin dosed 4 mg/kg (B-Lovastatin), and vi) high-fat

diet-diet-Alginate/Chitosan/Lovastatin nano combination dosed 4 mg/kg Nano/Lovastatin)

(B-2.2 Methods

2.2.1 Research design

An experimental, interventional, controlled, cross-sectional andlongitudinal study, through 7 weeks for modelling and 12 weeks of theintervention (orally administered of Lovastatin or physiological saline)

2.2.2 Materials

Chemicals used in this study including natri clorid 0,9%, Lovastatinpowder (Sigma Aldrich) Alginate/Chitosan/Lovastatin with ratio8:2:10%

Electronic scales to measure rats weight, internal organ weightand chemical; length scale to determine the length, chest and waistcircumference of rats Blood component testing was performed at theMilitary Medical and Pharmaceutical Research Center, VMMU.Behavioral studies in experimental chambers with instruments (openfield, water maze )

Experimental rooms were kept quiet with temprature stable at 25 ± 1

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 8 -9 weeks old were randomly divided into normaland high fat diet groups for 7 weeks Evaluation parameters includedanthropometric 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 andtwice per week for 7 weeks in the model period, and triple per week for 12weeks of the follow intervention period Food intake and consumed drinkingwrere measured twice per week during two the periods

2.2.3.2 Analysis lipidemia and glycemia

Glycemina, triglyceridemia, and cholesterolemia were determined at theend of week 2, 4, 6 and 7 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 6groups of rats during the intervention period Measure anthropometricindexes, food consumption, glycemia and lipidemia Behavioralassessment in the open field on locomotion, object recongnition andlearning 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 recognitiontest with three sessions and the Moris water maze test

2.2.3.4 The procedure to weight organs and make pathohistological analysis

3.1.1.1 Anthropometric indexes

Figure 3.1 Bodyweigh (A) and Length (B) in the two groups of rats

over 7 weeks *: p < 0.05; **: p < 0.01; ***: p < 0.001.

The body weight of the high-fat diet group was higher than that ofthe normal diet rats from the end of week 5 (p < 0.05) The bodylength of the high-fat diet group was longer than that of the normaldiet rats over 7 weeks and marked at the end of week 2 (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 7

weeks

Group

Time point

Normal diet (x̅ ± SD)

Since the end of week 4, triglyceridemia of high-fat diet groupwas significantly increased compared with the normal diet group

Table 3.6 Cholesterolemia (mmol/L) of the two groups of rats over

7 weeks

Group

Time point

Normal diet (x̅ ± SD)

Table 3.7 Blood HDL-cholesterol concentration (mmol/L) of the

two groups of rats over 7 weeks

Group

Time point

Normal diet (x̅ ± SD)

Table 3.8 Blood LDL-cholesterol concentration (mmol/L) of the two

groups of rats over 7 weeks

Group

Time point

Normal diet (x̅ ± SD)

High-fat diet

End of week 7 0.58 ± 0.12 0.96 ± 0.46 2.33 x 10

Therewere

difference in blood LDL-cholesterol content between two groups ofthe end of week 7 (p < 0.05) in table 3.8 (p < 0.001)

3.1.3 Behavior results of model animals

3.1.3.1 Spontaneous locomotor activity in the open-field

Fi

gure 3 3 Travelled distance (A) and average speed (B) in the open

field of the two groups of rats ***: p < 0.001

Total travelled distance and average speed in the open field of thenormal diet group were significantly higher than those in the high-fatdiet (p < 0.001) (Fig 3.3)

3.1.3.2 Object Recognition activity

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 betweentwo objects of the two groups In the test phase, the discrimination ratiosbetween the familiar object and the novel object were different in normaldiet 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 7.

3.1.4 Histological results of modelled animals

3.2 Results of effect on behaviors and blood lipid

Alginate/Chitosan/Lovastatin in obesity rat model

3.2.1 Anthropometric indexes, consumed food and drinking in the intervention period