Invest Clin 63(4): 327 - 343, 2022 https://doi.org/10.54817/IC.v63n4a01

Corresponding author: Ling Ruan. Department of Physical Education. Xi’an Shiyou University. Xi’an, Shaanxi,

710065, China. Phone 008618992591267. E-mail 191202@ xsyu.edu.cn

The effect of varied exercise intensity

on antioxidant function, aortic endothelial

function, and serum lipids in rats with

non-alcoholic fatty liver disease.

Ling Ruan1, Guanghua Wang1, Zhen Qing Lv1, Shoubang Li1, Qin Liu2, Yiling Ren1,

Quancheng Zhang1, Xianli Lv2, Rongping Wu1 and Zhan Jin1

1 Department of Physical Education, Xi’an Shiyou University, Xi’an, Shaanxi, China.

2 College of Physical Education, Ankang University, Ankang, Shaanxi, China.

Keywords: different exercise intensity; NAFLD; aortic endothelial cell function;

antioxidant; lipid metabolism.

Abstract. This study aimed to compare the effects of diet and exercise of

different intensities on antioxidant function, aortic endothelial cell function

and serum lipids in NAFLD (nonalcoholic fatty liver disease) rats. Fifty Sprague-

Dawley (SD) rats (180-220g) were randomly divided into two experimental

groups and fed either a standard rodent chow diet (CON; n=10) or a high-fat

diet (HFD; n=40). After 16 weeks, the animals that received the HFD were

randomly separated into a high-fat control group (HFC; n=10) or three ex-

ercise training groups: HFD and low-intensity exercise (LE; n=10), HFD and

moderate-intensity exercise (ME; n=10), and HFD and incremental intensity

exercise (IE; n=10). These experimental rats keep sedentary or trained for

the next six weeks. A detection kit was used to detect nitric oxide synthase

(NOs), nitric oxide (NO), malondialdehyde (MDA) and other markers of aor-

tic oxidative stress. The expression levels of endothelial nitric oxide synthase

(e-NOS) and endothelin-1 (ET-1) were detected by immunohistochemistry.

TC, TG, and other lipid metabolism parameters were detected by an auto-

matic analyzer. Exercise with different intensities could improve lipid me-

tabolism, enhance antioxidant function, reduce MDA (P<0.01), increase NO

(P<0.01), and improve the expression of e-NOS and ET-1 (P<0.01) protein

levels in NAFLD rats. Decreased blood lipids were exhibited in all exercise

groups. Notably, the moderate-intensity exercise demonstrated more effect

on increasing glutathione (GSH) contents (P<0.01) and decreased the ex-

pression of ET-1 protein levels (P<0.01). The results showed that exercise at

different intensities improved lipid metabolism and enhanced anti-oxidation

function. Moderate exercise could improve the function of aortic endothelial

cells.

328 Ruan et al.

Investigación Clínica 63(4): 2022

El efecto del ejercicio de intensidad variada sobre la función

antioxidante, la función endotelial aórtica y los lípidos séricos

en ratas con enfermedad del hígado graso no alcohólico.

Invest Clin 2022; 63 (4): 327 – 343

Palabras clave: diferente intensidad de ejercicio; NAFLD; función de las células

endoteliales aórticas; antioxidante; metabolismo de los lípidos.

Resumen. Este estudio tuvo como objetivo comparar los efectos de la

dieta y el ejercicio a diferentes intensidades sobre la función antioxidante, la

función de las células endoteliales aórticas y los lípidos séricos en ratas NAFLD

(con enfermedad del hígado graso no alcohólico) y alimentados con una

dieta estándar para roedores (CON; n = 10) o con una dieta alta en grasas

(HFD; n = 40). Después de 16 semanas, los animales que recibieron HFD se

separaron aleatoriamente en un grupo de control alto en grasas (HFC; n=10)

o tres grupos de entrenamiento físico: HFD y ejercicio de baja intensidad (LE;

n=10), HFD y ejercicio de intensidad moderada (ME; n=10), y HFD y ejercicio

de intensidad incremental (IE; n=10). Estas ratas experimentales se mantu-

vieron sedentarias o entrenadas durante las próximas seis semanas. Se utilizó

un kit de detección para determinar óxido nítrico sintetasa (NO), óxido nítrico

(NO), malondialdehído (MDA) y otros marcadores de estrés oxidativo aórtico.

Los niveles de expresión de la óxido nítrico sintetasa endotelial (e-NOS) y endo-

telina-1 (ET-1) se detectaron mediante inmunohistoquímica. El analizador au-

tomático detectó TC, TG y otros parámetros del metabolismo de los lípidos. El

ejercicio con diferente intensidad mejoró el metabolismo de los lípidos, mejoró

la función antioxidante, redujo la MDA (P <0,01), aumentó el NO (P <0,01) y

mejoró la expresión de los niveles de proteína e-NOS y ET-1 (P <0,01) en ratas

NAFLD. Se observó una disminución de los lípidos en sangre en todos los gru-

pos de ejercicio. En particular, el ejercicio de intensidad moderada demostró

un mayor efecto en el aumento del contenido de glutatión (GSH) (P<0,01) y

disminuyó la expresión de los niveles de proteína ET-1 (P<0,01). Los resultados

mostraron que el ejercicio a diferentes intensidades mejoró el metabolismo de

los lípidos y mejoró función antioxidante. El ejercicio moderado podría mejorar

la función de las células endoteliales aórticas.

Received: 09-05-2022 Accepted: 16-07-2022

INTRODUCTION

Nonalcoholic fatty liver disease

(NAFLD) is the hepatic manifestation of

metabolic syndrome, and it is the most

prevalent liver disease worldwide1. The

prevalence of NAFLD is approximately

30% in the United States and Europe,

with a similar prevalence has been doc-

umented in Asian countries2. It encom-

passes a spectrum ranging from simple

steatosis to fatty liver with hepatocellu-

lar injury, termed nonalcoholic steato-

hepatitis (NASH), fibrosis and cirrhosis3.

Effect of exercise intensity in rats with fatty liver disease 329

Vol. 63(4): 327 - 343, 2022

Moreover, the majority of deaths among

NAFLD patients are not only associated

with liver-related morbidity and mortal-

ity but also related to cardiovascular and

other complications. A large number of

studies have shown that high-fat diets

(HFD) can cause lipid metabolism dis-

turbances, abnormal lipid accumulation,

obesity, and NAFLD 4-6. Free fatty acids

(FFA) can cause oxidative stress, which is

a primary cause of intravascular dysfunc-

tion. Therefore, long-term HFD can in-

hibit nitric oxide synthase expression in

vascular endothelial cells; reduce nitric

oxide (NO) production, resulting in ab-

normal blood vessel endothelial cell func-

tion and vascular endothelial dysfunc-

tion. NO is produced via NO synthases,

which are a family of enzymes catalyzing

the production of NO from L-Arginine.

For this work, we will consider total ni-

tric oxide synthase (T-NOS), endothelial

nitric oxide synthase (e-NOS) and induc-

ible nitric oxide synthase (i-NOS). T-NOS,

as the name suggests, is the aggregate

nitric oxide synthase (NOS) circulating

at any time. At the same time, e-NOS is

the endothelial NOS generated in blood

vessels and is involved in the regulation of

vascular function. i-NOS is inducible NOS

which is usually raised in an oxidative envi-

ronment. As NO expression is altered with

endothelial dysfunction, which in turn is

associated with NAFLD, finding an effec-

tive management solution is, therefore,

a current research priority.

Previous experiments have shown

aerobic exercise can improve lipid me-

tabolism, oxidative stress 7, 8 and vascu-

lar endothelial function 9. Several phar-

macological and non-pharmacological

strategies have been proposed to relieve

NAFLD-associated deleterious altera-

tions 10. Among non-pharmacological

approaches, physical exercise-mediated

multi-systemic adaptations can promote

crosstalk between organs and orches-

trate pro-metabolic effects known to mit-

igate metabolism-related disorders such

as NAFLD 11. Keating et al. examined that

the efficacy of commonly prescribed ex-

ercise dose and intensity for reducing

liver fat and visceral adipose tissue in an

animal experimental model of NAFLD,

but no significant differences were found

between the dose or intensity of the ex-

ercise regimen and reductions in liver fat

or visceral adipose tissue 12. Paradoxical-

ly, it has been shown that vigorous and

moderate exercise were equally effec-

tive in reducing intrahepatic triglycer-

ide (TG) content, but body weight, body

fat, waist circumference, and blood pres-

sure with vigorous-moderate intensity

exercise were lower than the moderate-

intensity group 13, 14. Similarly, Tsunoda

et al. showed that vigorous intensity was

more effective than moderate-low inten-

sity exercise and moderate-high inten-

sity protocols in preventing NAFLD from

progressing to NASH 14. Two systematic

reviews of published studies of NAFLD

patients participating in aerobic exer-

cise programs showed that liver fat was

significantly reduced. Still, the optimal

exercise intensity is undetermined 15, 16,

although a growing number of prospec-

tive data shows the effects ofdifferent

types of exercise on NAFLD17. Collective-

ly, these previous findings suggest that

the intensity of exercise, rather than the

volume or duration, may play a critical

role in magnifying the protective effects

against NAFLD 18.

The relationship between NAFLD

and aortic endothelial function is poorly

understood. It is unclear whether exer-

cise could affect aortic endothelial func-

330 Ruan et al.

Investigación Clínica 63(4): 2022

tion in a dietary-induced rat model of

NAFLD. Moreover, different exercise in-

tensities may produce varying effects on

endothelial function in rat NAFLD mod-

els. Therefore, this study compared the

impact of different exercise intensities

on markers of aortic endothelial func-

tion in a HFD-induced NAFLD rat model.

MATERIALS AND METHODS

Animals

Male Sprague–Dawley (SD) rats (180-

220g) were purchased from the Guangdong

Medical Laboratory Animal Centre (GDM-

LAC) (Guangzhou, China). According to

the principles of the Helsinki declaration,

this experiment was approved by the animal

experiment ethics checklist of South China

Normal University (iacuu-2008-0020). Rats

were raised in a specific pathogen free (SPF)

facilities environment (23 ± 1°C, humidity

60-70%, 12h light/dark cycle), in the Labo-

ratory Animal Center. SD rats (n=50) were

randomly divided into two experimental

groups, a control group fed standard rodent

chow diet (CON; n=10), and a high-fat diet

group (HFD; n=40). After 16 weeks, ani-

mals that received the HFD were randomly

separated into a sedentary control high fat

group (HFC; n=10) or three exercise train-

ing groups: HFD and low-intensity exercise

(LE; n = 10), HFD and moderate-intensity

exercise (ME; n = 10), HFD and incremen-

tal intensity exercise (IE; n =10). For the

next six weeks, the CON group received ad

libitum feeding with standard rodent chow

diet and remained sedentary, the HFD group

received ad libitum feeding with a high-fat

diet and remained sedentary. The LE, ME

and IE groups, were fed with high a fat diet

and were trained with different exercise

training intensities. At the end of six weeks

of treatment, the rats were sacrificed af-

ter overnight fasting. Blood samples, aorta

samples, and liver samples were harvested

for analysis. All procedures were performed

following the “Guidelines for the Care and

Use of Laboratory Animals” published by the

National Institutes of Health (NIH Publica-

tion No. 85-23, revised in 1996).

Composition of the HFD

The HFD contained 5% sucrose, 18%

lard, 15% egg yolk powder, 0.5% sodium cho-

late, and 1% cholesterol, added to the 60.5%

basic standard rodent chow diet.

Exercise experimental protocol

All animals were familiarized with

treadmill running (DSPT202, Qianjiang

Technology Company, Hangzhou, China)

at 0-15 m/min, 10-20 min per day, for

six consecutive days. An electrified grid

(0.6–mA intensity) was placed behind

the belt of the treadmill to induce run-

ning. The rats that failed to run regularly

were excluded from the training proto-

col. The exercise program involved 60

min/day, five days per week, for a total

of six weeks. The rats performed exercises

based on a protocol described previously19-21,

with some modications. The daily train-

ing intensity program was for each group

respectively: LE group: 15 m/min, ME

group: 20 m/min, and IE group consist-

ed of running 10 minutes at 15 m/min,

followed by a gradual increase in intensi-

ty at 20 m/min for 30 min, and increase

in intensity to 27 m/min for 20 min on a

motor–driven treadmill.

Outcome Measures

The primary outcome measures

were the markers of oxidative stress in the

aorta; T-NOS and i-NOS. superoxide dis-

mutase (SOD), malondialdehyde (MDA),

catalase (CAT), glutathione (GSH) and

Total antioxidant capacity (T–AOC).

The secondary outcome measures

were; the presence of aortic endothelin-

Effect of exercise intensity in rats with fatty liver disease 331

Vol. 63(4): 327 - 343, 2022

1(ET-1) and e-NOS, body mass, liver mass

and lipids.

We also confirmed the existence of

NAFLD (liver histology) by hematoxylin

and eosin (H&E) staining of embedded

liver tissue samples.

Lipids

Blood samples were collected from the

abdominal aorta and centrifuged at 3000

rpm for 15 min, and then serum was col-

lected. The serum TG levels (mmol/l), total

cholesterol (TC) levels (mmol/l), low–den-

sity lipoprotein cholesterol (LDL–c) levels

(mmol/l), high-density lipoprotein choles-

terol (HDL–c) levels (mmol/l), were detect-

ed with an automatic analyser (Toshiba Ac-

cuteTBA-40FR, Toshiba Corporation, Tokyo,

Japan).

Markers of aortic oxidative stress

The aorta was removed to an ice

plate, then placed in liquid nitrogen and

the sample was saved for testing. Pre-

pared fresh aorta samples were ground

in saline solution to make 10% aorta ho-

mogenates, followed by centrifugation

for 20 min at 4°C. The resulting super-

natant was collected using specific kits

according to the manufacturer’s instruc-

tions. NOS (T-NOS and i-NOS) were de-

tected using an assay kit (Colorimetric

method), (Nanjing Jiancheng Corp.,

Nanjing, China), NO assay kit (Nitrate

reductase method).

MDA contents in the aorta were quan-

tified using a lipid peroxidation MDA assay

kit (TBA method) (Beyotime Institute of

Biotechnology, Jiangsu, China) according

to the manufacturer’s protocol. CAT activ-

ity assay kit (Visible light method), SOD,

T–AOC activity and GSH contents were de-

termined using a reagent kit (Colorimetric

method), (Nanjing Jiancheng Corp., Nan-

jing, China).

Endothelin-1 and Nitric oxide synthase

ET-1 and e-NOS were measured by im-

mune-histochemical analysis. After the ab-

dominal cavity was opened, the aorta was

wholly and quickly separated; the fixed aorta

was embedded in paraffin, sliced into 5-μm-

thick sections, and mounted on glass slides.

The immunohistochemistry was performed

with a PowerVision two-step immunohisto-

chemistry detection kit. ET-1 and e-NOS an-

tibodies were obtained from (Bioss Biotech-

nology Co., Ltd. Beijing, China). Samples

were observed through JVC3-CCD camera

(Nikon Corp., Tokyo, Japan), and Image-Pro

Plus image (Media Cybernetics Corp., USA)

processing software system was used for

image acquisition and analysis. The brown

granules visible in the cytoplasm or nucle-

us were considered the positive expression

of aortic endothelial cells. The number of

positive cells per section were counted in 10

random elds (400x magnification), and the

percentage of positive cells (positive cells/

total cells × 100%) was calculated. Three

non-consecutive sections were selected from

each specimen, and those indices were aver-

aged.

Characterization of non-alcoholic fatty

liver disease

To characterize NAFLD, rat livers

were fixed with 10% formalin, and the

paraffin-embedded liver tissue samples

were cut into 10μm sections for H&E

staining. At least three randomly se-

lected liver section images were then

captured digitally (400x magnification),

and each set of images was examined and

photographed using nikon Eclipse Ci

light microscope (Nikon Tokyo, Japan).

Statistical analysis

The statistical package for the social sci-

ences (SPSS version 20.0, IBM Corp., USA)

software was used for one-way ANOVA

and Tukey’s significant difference post

332 Ruan et al.

Investigación Clínica 63(4): 2022

analysis. The Graph Pad Prism (version

5.0; Graph Pad Prism Software, La Jolla,

CA, USA) software was used to draw the

chart. The statistical results were ex-

pressed as means ± standard deviations

(M ± SD). P value of ≤ 0.05 denoted a

statistically significant difference.

RESULTS

Effect of different exercise training

intensities on lipid metabolism disorders

and liver histology

As shown in Fig. 1, liver histology

was evaluated by H&E staining. CON

group rats tissue exhibited well-arranged

hepatic cords, cells with round and cen-

tral nuclei, a lobular structure and an

array of wheel–shaped cells along the

centrilobular vein. However, in the HFC

group, lipid droplets were observed in

the liver sections (Fig. 1B). Lipid drop-

let volumes and quantities were reduced

with different exercise intensities (Figs.

1C, 1D, and 1E).

As shown in Table 1, the serum TC, TG,

LDL-c and FFA were lowest in the CON group,

but TC, TG and LDL-c were significantly de-

creased in the LE, ME and IE groups com-

pared with the HFC group. No difference in

serum HDL-c was observed between groups.

Notably, TG, TC, and LDL were not signifi-

cantly different between the three exercise

groups.

Fig. 1. The optical microscope image of H&E staining in Rat Liver Tissue (400×).

CON= control group (A); HFC= high fat control group (B); LE= HF and low intensity exercise (C);

ME= HF and middle intensity exercise (D); IE= HF and incremental intensity exercise (E).

Effect of exercise intensity in rats with fatty liver disease 333

Vol. 63(4): 327 - 343, 2022

Effect of different exercise training

intensities on body mass and liver mass

As shown in Fig. 2, after six weeks

of treatment, body mass of the rats in

each group were not significantly dif-

ferent (Fig. 2A). Liver mass were lower

in the ME group than in the HFC group

(P<0.05), otherwise there was no sig-

nificant difference among the exercise

groups (Fig. 2B).

Effect of different exercise training

intensities on aortic endothelial cell

oxidative stress

As shown in Fig. 3, compared with

the CON group, CAT, GSH, and T–AOC

showed a significant reduction in the

HFC group (P<0.01; Fig 3C, 3D and 3E).

After six weeks of exercise training, SOD,

CAT, and T–AOC in LE, ME, and IE group

were significantly higher compared to

Table 1

The change of blood lipids in each group.

Groups TG

(mmol/L)

LDL–c

(mmol/L)

HDL–c

(mmol/L)

FFA

(umol/L)

CON 0.45±0.13 1.36±0.33 0.26 ± 0.06 0.41 ± 0.01 313.1 ± 23.1

HFC 1.55±0.21** 5.09±0.24** 1.88 ± 0.13** 0.32 ± 0.07 627.2 ± 97.8**

LE 0.87±0.30##** 4.17±0.20##** 1.13± 0.11##** 0.35 ± 0.11 569.2 ± 39.4**

ME 0.80±0.20##** 4.06±0.18##** 1.06± 0.17##** 0.37 ± 0.08 558.2 ± 68.1**

IE 0.79±0.18##** 4.12± 0.16##** 1.05± 0.16##** 0.37 ± 0.09 567.6 ± 49.7**

**, P < 0.01 compared with CON group; ##, P < 0.01 compared with HFC group.

All data are expressed as mean ± SD; 8–10 animals per group were used. CON= control group; HFC= high fat

control group; LE= HFD and low intensity exercise; ME=HFD and middle intensity exercise; IE= HFD and incre-

mental intensity exercise.

Fig. 2. Effects of exercise training on body mass and liver mass.

Body mass (A), liver mass (B) of each group. *, P< 0.05 compared with CON group; #, P < 0.05

compared with HFC group;

CON= control group; HFC= high fat control group; LE= HF and low intensity exercise; ME= HF and middle

intensity exercise; IE= HF and incremental intensity exercise.

334 Ruan et al.

Investigación Clínica 63(4): 2022

Fig. 3. Effects of exercise training on markers of

oxidative status in the aortic endothelial

cell.

SOD (A), MDA (B), CAT (C), GST (D),

and T–AOC (E). **, P < 0.01 compared

with CON group; #, P < 0.05; ##, P < 0.01

compared with HFC group; CON= control

group; HFC= high fat control group; LE=

HF and low intensity exercise; ME= HF

and middle intensity exercise; IE= HF and

incremental intensity exercise.

Effect of exercise intensity in rats with fatty liver disease 335

Vol. 63(4): 327 - 343, 2022

the HFC group (Fig. 3A, 3C and 3E),

whereas the MDA levels were decreased

compared to the HFC group (P<0.01;

Fig. 3B). In addition, LE and ME groups

exhibited significantly increased GSH

compared to the HFC group (P<0.05

and P<0.01 respectively; Fig. 3D).

Effect of exercise training intensity

on aortic NOS and NO activities

Table 2 shows that aortic T-NOS ac-

tivity was higher in the CON versus HFC

(P<0.01) and IE (P<0.05) groups; how-

ever, only the low intensity (LE) group

showed a significant elevation compared

to HFC (P<0.05). i-NOS activity was

higher in the HFC versus CON group (P

<0.01); the LE and ME groups showed a

significant reduction compared to HFC

(P<0.01), while the LE and ME groups

showed a significant reduction compared

to the IE group (P<0.01).

Table 3 shows that the NO content

in the aorta was higher in the CON versus

HFC (P<0.01) and all exercise (P<0.01)

groups, all exercise groups showed a

significant elevation compared to HFC

(P<0.01).

Effect of different exercise training

intensities on e-NOS and ET-1 expression

in the aorta

The expression of e-NOS protein lev-

els in the aorta was significantly lower

in the HFC group than in CON group

(P<0.05), whereas there was no differ-

ence between the three exercise groups

(Table 4 and Fig. 4). The expression of

ET-1 protein levels was significantly high-

er in the HFC, LE, and IE groups com-

pared to the CON group. The ET-1 levels

were significantly decreased by moder-

ate-intensity exercise training (P<0.01)

(Table 5 and Fig. 5).

DISCUSSION

The aim of our study was to inves-

tigate the effects of varying exercise in-

Table 2

The change of aortic nitric oxide synthase (NOS) activity in each group (U/mg prot).

CON HFC LE ME IE

T-NOS 6.04 ± 0.32 3.09 ± 0.31** 4.79 ± 0.25#4.19 ± 0.31 4.17 ± 0.19*

iNOS 1.29 ± 0.14 2.85 ± 0.27** 2.03 ± 0.17**##&& 2.18 ± 0.23**##&& 2.63 ± 0.16**

T-NOS, Total Nitric Oxide Synthase; iNOS, inducible nitric oxide synthase; *, P<0.05; **, P<0.01 vs CON; #, P

<0.05; ##, P < 0.01 vs HFC; &&, P < 0.01 vs IE.

All data are expressed as mean ± SD; 8–10 animals per group were used. CON= control group; HFC= high fat

control group; LE= HFD and low intensity exercise; ME=HFD and middle intensity exercise; IE= HFD and incre-

mental intensity exercise.

Table 3

The change of aortic NO in each group (U/mg prot).

CON HFC LE ME IE

NO 202.34±11.46 150.21±7.72** 176.61±9.07**## 183.18±8.10**## 175.43±7.02**##

NO, Nitric Oxide ;*, P < 0.05; **, P < 0.01 vs CON; #, P < 0.05 vs HFC.

All data are expressed as mean ± SD; 8–10 animals per group were used. CON= control group; HFC= high fat

control group; LE= HFD and low intensity exercise; ME=HFD and middle intensity exercise; IE= HFD and

incremental intensity exercise.

336 Ruan et al.

Investigación Clínica 63(4): 2022

tensities on aortic endothelial function

in high fat diet-induced NAFLD rats. The

specific process is shown in Fig 6. We can

confirm that HFD induced vascular en-

dothelial dysfunction in NAFLD rats. We

found that exercise enhanced anti-oxida-

tion function and improved some mark-

ers of aortic endothelial cell function.

T-NOS activity appeared to respond best

to low intensity exercise. i-NOS activity

was lower only in the LE and ME groups.

Moderate intensity exercise demonstrat-

Table 4

The change of e-NOS expression in each group.

CON HFC LE ME IE

eNOS 0.52±0.13 0.35±0.08* 0.43±0.02 0.41±0.07 0.38±0.21

eNOS, Endothelial nitric oxide synthase; *, P < 0.05; vs CON.

All data are expressed as mean ± SD; 8–10 animals per group were used. CON= control group; HFC= high fat

control group; LE= HFD and low intensity exercise; ME=HFD and middle intensity exercise; IE= HFD and

incremental intensity exercise.

Fig. 4. Effects of exercise training on aortic e-NOS expression.

CON= control group (A); HFC= high fat control group (B); LE= HF and low intensity exercise (C);

ME= HF and middle intensity exercise (D); IE= HF and incremental intensity exercise (E).

Table 5

The change of ET-1 expression in each group.

CON HFC LE ME IE

ET-1 0.25 ± 0.03 0.39 ± 0.04** 0.33 ± 0.05* 0.29 ± 0.02## 0.35 ± 0.09**

ET-1, Endothelin-1; *, P < 0.05; **, P< 0.01 vs CON; ##, P < 0.01 vs HFC.

All data are expressed as mean ± SD; 8–10 animals per group were used. CON= control group; HFC= high fat

control group; LE= HFD and low intensity exercise; ME=HFD and middle intensity exercise; IE= HFD and

incremental intensity exercise.

Fig. 5. Effects of exercise training on aortic ET-1 expression.

CON= control group (A); HFC= high fat control group (B); LE= HF and low intensity exercise (C);

ME= HF and middle intensity exercise (D); IE= HF and incremental intensity exercise (E).

Effect of exercise intensity in rats with fatty liver disease 337

Vol. 63(4): 327 - 343, 2022

ed the greatest effect on decreasing the

expression of the potent vasoconstric-

tor ET-1 levels and the expression of NO,

whereas GSH was raised in this group.

Decreased blood lipids were exhibited in

all exercise groups.

The impact of different exercise training

intensities on lipid metabolism disorders

We demonstrated that three different

exercise training intensities were equally ef-

fective in alleviating dyslipidaemia as well as

hepatic damage in a diet-induced rat NAFLD

model. These results suggested that the

therapeutic effect of exercise training in dys-

lipidaemia and hepatic injury is unrelated to

exercise intensity. This notion is supported

by meta-analytic work 22. Moreover, our study

did not find any improvement in body mass

or HDL-c in any group. However, it should

be noted that liver mass was significantly de-

creased in the moderate-intensity group.

Exercise plays a vital role in improving

lipid metabolism disorders and is increas-

ingly seen as adjunctive therapy for the

prevention and treatment of NAFLD16,

17, 23. Machado et al. showed that exercise

intensity would be more effective in improv-

ing metabolic parameters than frequency or

duration 24. A retrospective study indicat-

ed that moderate and vigorous-intensity

physical activity yielded similar health

benefits to low, in terms of the measured

body adiposity and serum TG 25. Two stud-

Fig. 6．Effects of different exercise intensities on antioxidant function aortic endothelial cell function and

blood lipid in rats with non-alcoholic fatty liver disease.

338 Ruan et al.

Investigación Clínica 63(4): 2022

ies showed that vigorous-intensity inter-

val training and continuous moderate-in-

tensity exercise have the same effect on

lowering the serum FFAs, TG of NAFLD

in animals 17, 26. Suk et al. also indicated

that high-intensity exercise improved

lipid metabolism in the liver of rats27.

Fisher et al. also did not find any improve-

ments in body weights and HDL-c between

groups of differing exercise intensities28.

However, Khammassi et al. showed that

high-intensity interval training may be

particularly useful in overweight/obese

youth to improve body composition and

lipid profile 29.

The impact of different exercise training

intensities on the antioxidant function

of aortic endothelial cells

Our study showed that CAT, GSH,

and T–AOC was significantly reduced in

the HFC group, compared with the CON

group, which confirmed the expected ef-

fect of HFD induced NAFLD rat model.

After six weeks of exercise training SOD,

CAT, and T–AOC were significantly in-

creased, conversely, MDA was reduced

considerably in all exercise groups. Fur-

thermore, low intensity and moderate-

intensity exercise increased GSH.

Previous work has shown that HFD

increase lipid peroxidation and destroy

the balance of the oxidative and anti-

oxidative systems30. Moreover, oxidative

stress and increased ROS production are

the primary cause of dysfunction in aor-

tic endothelial cells 19, 20. The relationship

between exercise and oxidative stress is ex-

tremely complex, depending on the mode,

intensity, and duration of the exercise. Pingi-

tore et al. noted that regular moderate train-

ing in humans appears beneficial for oxidative

stress and health. Conversely, acute exercise

leads to increased oxidative stress21, presum-

ably as their period of adaptation that is miss-

ing from acute exercise training. Pereira et

al. also showed high-intensity exercise might

induce oxidative stress31. Li et al. reported

that SOD activity and GSH were signifi-

cantly raised after rats were exercised at

medium intensity 32. Radak et al. also indi-

cated that moderate exercise significantly

increased the activity of antioxidant en-

zymes33. However, Lu et al. reported that

high-intensity exercise was superior to the

moderate-intensity in attenuating oxidative

stress and improving glucolipid metabolism

in post-MI rat myocardium34. Jamurtas et al.

also found high intensity to be superior

to moderate intensity for reducing oxida-

tive stress in healthy male humans35. It,

therefore, remains unclear which exercise

intensity is optimal for improving antioxi-

dant function.

In our study, we elucidated that all

exercise training intensities, especially

for low intensity and moderate intensity,

enhanced antioxidant enzyme activity and

suppressed NAFLD rats’ oxidative stress.

We speculated, and our data support the

notion that incremental exercise may

increase reactive oxygen species (ROS)

production during incremental exercise

leading to the oxidation of protein, lip-

ids or nucleic acid 36. The production of

ROS during exercise is also accompanied

by a reduction of antioxidant capacity 37.

However, our data lack measures of ROS

production and other related oxidative

stress markers. So further studies of the

molecular mechanisms involved in anti-

oxidation may be indicated.

The impact of different exercise training

intensities on endocrine function of aortic

endothelial cells

Studies have shown that a high-fat

diet can lead to lipid abnormalities, vascu-

lar endothelial damage, reduced NO con-

Effect of exercise intensity in rats with fatty liver disease 339

Vol. 63(4): 327 - 343, 2022

tent produced by endothelial cells, result-

ing in impaired endothelial function 38.

The role of NO in the liver largely depends

on the type of NOS that catalyzes its pro-

duction. e-NOS plays a beneficial role in

alcoholic liver disease, while i-NOS plays

an important role in alcohol-induced liver

damage. In the process of oxidative stress,

the production of i-NOS can be induced,

and a large amount of NO from i-NOS

can aggravate the liver damage caused by

oxidative stress, while NO produced from

e-NOS can resist the effect of superoxide

39. The results of this experiment showed

that compared with the CON group, the

expression of ET-1 (P＜0.01) in the aorta

of the HFC group increased significant-

ly, while the expression of no and e-NOS

(P＜0.05) decreased, and the expression

of i-NOS (P＜0.01) increased, suggesting

that long-term high-fat diet will cause oxi-

dative stress in rats and then lead to vas-

cular endothelial dysfunction.

Exercise can effectively improve the

function of aortic endothelial cells, but

the optimal exercise intensity is still

unclear. The study of Hambrecht et al.

showed that exercise training, at symp-

tom-limited intensity, improves arterial

endothelial cell function in people with

heart disease 40. Several previous studies

have shown low and moderate-intensity

exercise to have a positive effect on aor-

tic endothelial cell function of rats 41.

Shaodong et al. indicated that aerobic

exercise, of unknown intensity, decreas-

es the production of lipid oxidation prod-

ucts, and thus prevents damage to endo-

thelial cells in rats with dyslipidemia42.

Furthermore, a previous experiment

demonstrated that moderate-intensity

exercise could reduce the expression of

ET-1 levels, induced by aortic injury in

mice 43. Wang et al. and Archana et al.

found that moderate-intensity exercise

is optimal for raising serum NO 7,44.

However, Morishima et al. indicated en-

dothelial function was maintained by con-

ducting high-intensity resistance exercise45.

Meta-analysis showed that high-intensity

training seems to have a superior effect

on the improvement of endothelial func-

tion compared with moderate exercise in

cardiac patients 46.

Our results show that although the re-

duction of e-NOS expression level in the ME

group is not significant compared with the

HFC group, it still shows a downward trend

and is stronger than the IE group, with the

fastest decreasing trend in the LE group. In

the results of NO index, compared with the

HFC group, the expression levels of NO in

different exercise intensity groups were sig-

nificantly increased (P<0.01), but the ME

group had the greatest increase. In the ex-

perimental results of ET-1, we found that the

expression levels of ET-1 in different exercise

intensity groups were significantly reduced,

and the ME group and IE group showed a very

significant difference. At the same time, we

found that compared with the IE group, the

i-NOS activity of the LE group and the ME

group was significantly decreased (P<0.01),

which may be due to the intensity of the lat-

ter can better improve oxidative stress, lead-

ing to the reduction of i-NOS. To sum up,

these results show that moderate-intensity

exercise can make the most significant im-

provement in endothelin-1 and nitric oxide

levels, which means that moderate-intensity

exercise is the most ideal for protecting en-

dothelial cell function.

Our experiments show that exer-

cise can reduce the expression of aortic

e-NOS and ET-1 protein levels, improved

lipid metabolism. However, the role and

underlying mechanism of exercise train-

ing in NAFLD related aortic endothelial

cell function remain poorly understood.

Notably, moderate intensity exercise

340 Ruan et al.

Investigación Clínica 63(4): 2022

demonstrated more effect on decreasing

the expression of ET-1 protein levels, and

GSH. Therefore, we believe that moder-

ate exercise demonstrated improved aor-

tic endothelial cell function, is underlined

by the following: (i) Moderate-intensity

exercise improves lipid metabolism, pro-

moting fat mobilization and lipid energy

catabolism25. (ii) Moderate-intensity ex-

ercise has a beneficial anti-oxidative ef-

fect. (iii) The reduced ET-1 and increased

NO expression were significant in the ME

group, ultimately improving aortic endo-

thelial cell function47,48. We do, however,

concede that results of other works are

conflicting.

ACKNOWLEDGEMENTS

First and foremost, I would like to show

my deepest gratitude to Dr. Neil Smart, a

respectable, responsible and resourceful

scholar, who has provided me with valuable

guidance. His keen and vigorous academic

observation enlightens me. I shall extend my

thanks to Dr. Li for all his kindness and help.

Funding

This project was supported by Shaanxi

Science and Technology Association Project

(2021JQ586, 2021JQ834), a research grant

from the Special Research Project of the

Education Department of Shaanxi Province

(19JK0017), Projects of Shaanxi Provincial

Bureau of sports(2021037), and Shaanxi So-

cial Science Foundation Project (2020Q017).

We would also like to thank Shou-bang Li

who assisted in designing the project and

performing daily training over the course of

the study and also thank Guang-hua Wang,

Rong-ping Wu, Zhen-Qing Lv, and Zhan Jin

who assisted in improving the quality of the

figures. We would also like to thank Yi-ling

Ren, Quan-cheng Zhang, Qin Liu and Xian-li

Lv for their work in collecting and querying

the information.

Conflict of interest

No conflicting financial interests exist.

Author’s ORCID numbers

• Ling Ruan (LR):

0000-0002-6338-8052

• Guanghua Wang (GW):

0000-0002-0729-3147

• Zhen Qing Lv (Z-QL):

0000-0003-2303-2104

• Shoubang Li (SL):

0000-0003-2413-9632

• Qin Liu (QL): 0000-0003-0116-3840

• Yiling Ren (YR):

0000-0002-4434-8688

• Quancheng Zhang (QZ):

0000-0002-1740-4699

• Xianli Lv (XL):

0000-0001-7237-0893

• Rongping Wu (RW):

0000-0001-6080-1094

• Zhan Jin (ZJ):

0000-0002-7343-3654

Contributions of authors

LR and GW drafted and revised the ma-

nuscript; SL assisted in designing the pro-

ject and performing daily training over the

course of the study; GW, RW, Z-QL, and ZJ

assisted in improving the quality of the figu-

res; YR, QZ, QL and XL work in collecting

and querying the information.

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