The effect of varied exercise intensity on antioxidant function, and aortic endothelial cell function and serum lipids in a non-alcoholic fatty liver disease rats

Exercise and diet may improve cardio-metabolic health in non-alcoholic fatty liver disease, but the optimal exercise prescription remains unclear. We aimed to compare the effects of diet and exercise at different intensities on antioxidant function, and aortic endothelial cell function and serum lipids in a non-alcoholic fatty liver disease rats. Fifty Sprague Dawley rats (180-220g) were randomly divided into two experimental groups and fed either standard rodent chow diet or a high-fat diet. After16 weeks, these animals that received the HFD were randomly separated into a high fat control group or three exercise training groups: HF and low intensity exercise, HF and moderate intensity exercise, HF and incremental intensity exercise, these experimental rats keep sedentary or training for the next 6 weeks. Markers of Aortic Oxidative stress were detected using assay kit. Immunohistochemical analysis was performed to determine the expression level of eNOS and ET-1. Lipid metabolism parameters were detected with an automatic analyzer. Exercise at different intensities improved lipid metabolism, enhanced anti-oxidation function, reduced MDA, increased NO, and improved the expression of eNOS and ET-1 protein levels. Decreased blood lipids were exhibited in all exercise groups. Notably, moderate intensity exercise demonstrated more effect on increasing GSH contents, and decreased the expression of ET-1 protein levels.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, 87 and it is the most prevalent liver disease worldwide(1). The prevalence of NAFLD is 88 approximately 30% in the United States and Europe, with a similar prevalence documented in 89 Asian countries (2). It encompasses a spectrum ranging from simple steatosis to fatty liver with 90 hepatocellular injury, termed nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis(3). 5 91 Moreover, the majority of deaths among NAFLD patients are not only associated with liver-92 related morbidity and mortality but also related to cardiovascular and other complications. A 93 large number of studies have shown that high-fat diets can cause lipid metabolism disturbances, 94 abnormal lipid accumulation, obesity, and NAFLD (4-6). Free fatty acids (FFA) can cause 95 oxidative stress which is a primary cause of intravascular dysfunction, and therefore long-term 96 high-fat diets can inhibit nitric oxide synthase expression in vascular endothelial cells, reduce 97 Nitric oxide (NO) production, resulting in abnormal blood vessels endothelial cell function and 98 vascular endothelial dysfunction. NO is produced via NO synthases, which are a family of 99 enzymes catalyzing the production of NO from L-Arginine. For the purposes of this work we 100 will consider total NO synthase (T-NOS), endothelial NO (e-NOS) and inducible NO (iNOS). 101 T-NOS as the name suggests is the aggregate NO synthase circulating at any particular time, 102 while e-NOS is the endothelial NOS generated in blood vessels and is involved with regulating 103 vascular function. i-NOS is inducible NOS which is usually raised in an oxidative environment. 104 As NO expression is altered with endothelial dysfunction, which in turn is associated with 105 NAFLD, finding an effective management solution is therefore a current research priority. vigorous and moderate exercise were equally effective in reducing intra-hepatic triglyceride 117 content, but body weight, body fat, waist circumference, and blood pressure with vigorous-118 moderate intensity exercise was lower than the moderate intensity group (13,14). Similarly, 119 Tsunoda K et al. showed vigorous-intensity was more effective than moderate-low intensity 120 exercise and moderate-high intensity protocols in preventing nonalcoholic fatty liver from 121 progressing to NASH (14). Two systematic reviews of published studies of NAFLD patients 122 participating in aerobic exercise programs showed that liver fat was significantly reduced, but 123 the optimal exercise intensity is undetermined(15, 16), although a growing number of 124 prospective data shows the effects on different types of exercise on NAFLD(17). Collectively, 125 these previous findings suggest that the intensity of exercise, rather than the volume or duration, 126 may play a critical role in magnifying the protective effects against NAFLD (18).

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The relationship between non-alcoholic fatty liver disease and aortic endothelial function is    The rats that failed to run regularly were excluded from the training protocol. The exercise  The secondary outcome measures were; presence of aortic enothelin-1 and eNOS, body mass 171 and liver mass and lipids. 172 We also confirmed the existence of NAFLD (liver histology) by hematoxylin and eosin (HE) 173 staining of embedded liver tissue samples.

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Markers of Aortic Oxidative stress 176 The aorta was separated from the ice plate and then put it in liquid nitrogen preserve the sample 177 for testing. Prepared fresh aorta samples were ground in saline solution to make 10% aorta    Table 1 shows that aortic T-NOS activity was higher in the CON versus HFC (P<0.01) and IE 233 (P<0.05) group, however only the low intensity (LE) group showed a significant elevation 234 compared to HFC (P<0.05). i-NOS activity was higher in the HFC and all exercise groups 235 versus CON group (P <0.01), however only the low LE and ME groups showed a significant 236 reduction compared to HFC (P<0.01), meanwhile the LE and ME groups showed a significant 237 reduction compared to the IE group (P <0.01).    Fig 1B). In addition, LE and ME groups exhibited significantly 261 increased GSH compared to the HFC group (P<0.05 and P<0.01 respectively; Fig 1D).  (Table 3 and Fig 2). The expression of ET-1 protein levels were significantly higher in the 13 275 HFC, LE, and IE groups compared to the CON group. The ET-1 levels were significantly 276 decreased by moderate intensity exercise training (P<0.01). (Table 4 and Fig 3).  ET-1, Endothelin-1; *, P < 0.05; **, p< 0.01 vs CON; ## , P < 0.01 vs HFC.  As shown in Fig 4, after 6 weeks treatment, body mass of the rats in each group were not 301 significantly different (Fig 4A). Liver mass were lower in the ME group than in the HFC group 302 (P<0.05), otherwise there was no significant difference among the exercise groups ( Fig 4B). lipid droplets were observed in the liver sections (Fig 5B). Lipid droplet volumes and quantities 315 were reduced with different exercise intensities (Figs 5C, 5D, and 5E).

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As shown in Table 5, the serum TC, TG, LDL-c and FFA were lowest in the CON group, but 317 TC, TG and LDL-c were significantly decreased in the LE, ME and IE groups compared with 15 318 the HFC group. No difference in serum HDL-c was observed between groups. Notably, TG, 319 TC, and LDL were not significantly different between the three exercise groups. to greater oxidative stress in the latter group, leading to iNOS consumption. ET-1 was reduced 384 in the moderate intensity group. Together these results suggest, exercise a low or moderate 385 intensity elicited greatest improvements in ET-1 and Nitric Oxide profile, implying that low to 386 moderate intensity exercise is optimal for protection of endothelial cell function.

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Other work has shown exercise training, at symptom-limited intensity, improves arterial 388 endothelial cell function in people with heart disease (30  409 We demonstrated that three different exercise training intensities were equally effective in 410 alleviating dyslipidemia as well as hepatic damage in a diet induced rat NAFLD model. These 411 results suggested that the therapeutic effect of exercise training in dyslipidemia and hepatic 412 damage is unrelated to exercise intensity. This notion is supported by meta-analytic work (41).

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Moreover, our study did not find any improvement in body mass or HDL-c in any group.

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However, it should be noted that liver mass was significantly decreased in the moderate 415 intensity group.

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Exercise plays an important role in improving lipid metabolism disorders, and is increasingly 417 seen as an adjunctive therapy for the prevention and treatment of NAFLD (16, 17, 42).