Effects of different intensities of continuous training on vascular inflammation and oxidative stress in spontaneously hypertensive rats

Abstract We aimed to study the effects and underlying mechanism of different intensities of continuous training (CT) on vascular inflammation and oxidative stress in spontaneously hypertensive rats (SHR). Rats were divided into five groups (n = 12): Wistar‐Kyoto rats sedentary group (WKY‐S), sedentary group (SHR‐S), low‐intensity CT group (SHR‐L), medium‐intensity CT group (SHR‐M) and high‐intensity CT group (SHR‐H). Changes in body mass, heart rate and blood pressure were recorded. The rats were euthanized after 14 weeks, and blood and vascular tissue samples were collected. Haematoxylin and Eosin staining was used to observe the aortic morphology, and Western blot was used to detect the expression of mesenteric artery proteins. After CT, the mean arterial pressures improved in SHR‐L and SHR‐M and increased in SHR‐H compared with those in SHR‐S. Vascular inflammation and oxidative stress levels significantly subsided in SHR‐L and SHR‐M (p < 0.05), whereas in SHR‐H, only vascular inflammation significantly subsided (p < 0.05), and oxidative stress remained unchanged (p > 0.05). AMPK and SIRT1/3 expressions in SHR‐L and SHR‐M were significantly up‐regulated than those in SHR‐S (p < 0.05). These results indicated that low‐ and medium‐intensity CT can effectively reduce the inflammatory response and oxidative stress of SHR vascular tissue, and high‐intensity CT can improve vascular tissue inflammation but not oxidative stress.

pyrin domain-containing protein 3 (NLRP3) [8][9][10] are directly involved in the increased inflammation and oxidative stress associated with the hypertensive cardiovascular system. Balancing vascular redox reactions in hypertension can protect cardiovascular homeostasis. 1 Toll-like receptors (TLRs) are key members of cell transmembrane receptors and pathogenic membrane recognition receptors in the innate immune system. TLR4 contributes to inflammation and oxidative stress and is associated with endothelial dysfunction and vascular remodelling in hypertension. 4 TLRs promote the release of inflammatory cytokines by recognizing aggressive immune response pathogens and interacting with NF-κB. Phosphorylation of IκB and p65 induces the degradation of IκB and subsequent translocation of p65 into the nucleus, thus activating the NF-κB pathway, which further synthesizes pro-inflammatory cytokines. 5 For example, tumour necrosis factorα (TNFα), interleukin-6 (IL-6) and interleukin-1β (IL-1β) cause chronic low-grade inflammation in hypertension.
Additionally, NLRP3 inflammasome can promote the conversion of IL-18 and IL-1β precursors into mature IL-18 and IL-1β, which plays a key role in atherosclerosis. 9 Increasing studies have confirmed that NLRP3 inflammasome is involved in the occurrence and development of hypertension. 10 In addition to the role of inflammation, free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are involved in the pathological progress of hypertension. 11 More importantly, inflammatory factors can activate ROS, 12 which in turn activate a variety of intracellular signal transduction pathways, including NF-κB and NLRP3, leading to a further increase in ROS production; thus, a positive feedback mechanism is formed, which ultimately leads to the progression of hypertension. 12 ROS derived from NADPH oxidase (NOX) is an important signalling molecule in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and it is involved in cell growth, migration, inflammation, fibrosis and contraction. 13 In hypertension, the increased activity of the three subtypes of NOX (NOX1, NOX2 and NOX4) in blood vessels is related to oxidative stress and abnormal redox signals, leading to ECs and VSMCs dysfunction, which further causes vascular damage. 14 Superoxide dismutase 2 (SOD2) is an antioxidant enzyme that can catalyse the disproportionation of superoxide anion radicals to oxygen and hydrogen peroxide and plays a crucial role in the balance of oxidation and antioxidants in vivo. 15 At present, continuous training (CT) is an effective strategy for the treatment of hypertension, and its induction of adaptive changes in the blood vessel wall (including ECs and VSMCs) has been supported by experimental and clinical studies. [16][17][18][19] In the early studies, CT was shown to affect arterial blood vessels mainly through improving risk factors, such as blood pressure, blood lipid levels, insulin resistance and obesity to indirectly affect the function and morphology of arterial blood vessels. 20,21 However, more studies later showed that CT directly provides a wide range of benefits to alleviate hypertension. For example, it can promote angiogenesis, 22 improve vascular structure, 19 improve inflammation 23 and balance oxidative stress. [24][25][26] Studies have shown that CT increases the production and bioavailability of nitric oxide, which is mediated by a variety of pathways, including the synthesis of molecular mediators, changes in neurohormone release and oxidant/antioxidant balance. Furthermore, CT can affect systemic molecular pathways related to angiogenesis and chronic anti-inflammatory effects, thereby affecting vascular function and structural changes. 27 In research involving CT and hypertension, the effects of different training intensities have increasingly been studied; different training intensities include physical activity, lowintensity training, medium-intensity training, high-intensity interval training. Changes in vascular structure and function may be related to the intensity of training load 22,23,28 ; therefore, investigating the effect of training intensity in hypertensive exercise rehabilitation is particularly important.
Therefore, we hypothesized that CT might improve blood pressure by altering oxidant and inflammatory profiles in the vascular tissue of spontaneously hypertensive rats (SHR). This study aims to explore the effects of different intensities of CT on vascular inflammation (TLR4/NF-κB/NLRP3) and oxidative stress (SOD2, NOX2/4) in SHR and investigate the associated underlying mechanism of action. This study reported the role of training in regulating NF-κB and NLRP3 pathways in SHR for the first time.

| Animals and CT protocol
All research procedures were approved by the Ethics Committee The treadmill inclination of each training group was 0. Chocolate (0.5 g) was given as a reward after each training session. After the experiment, the rats were euthanized. The aorta was excised to assess vascular function and morphometry, and the mesenteric artery was used for Western blot analysis.

| Weight, heart rate and blood pressure measurements
The rats were weighed using an animal scale (UX/UW, Shimadzu) from 8 to 12 a.m. every Monday. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were measured in conscious rats using a noninvasive tail-cuff system (Softron). Rats were habituated to the tail-cuff procedure prior to experimentation. Before the measurements, the rats were kept in an incubator (37℃) for 10 min.
The blood pressure of each rat was obtained by averaging three measurements.

| Vascular morphometry analysis
A 1.5-cm section of the ascending thoracic aorta was dissected from each rat. Paraffin sections (5 μm) were cut and stained with haematoxylin and eosin. ImageJ 1.52v software was used to calculate the thickness and cross-sectional area of the vascular intima-media layer.
Mean value of the vessel wall thickness from the endothelial surface to the boundary between medial and adventitia of blood vessel was determined using five different locations spanning the entire crosssection. All images were captured by a Leica DM4B upright metallurgical microscope (Leica Inc).

| Western blot analysis
Protein expression was measured by Western blot. 30,31 Briefly, tissues were lysed on ice for 1 h using a lysis buffer. The protein concentration was measured using the Bradford assay (Beyotime), and 40 μg of protein was used for the Western blot. Proteins were separated using 8%-10% SDS-PAGE and transferred onto PVDF membranes; the membranes were blocked with 5% non-fat milk for 1 h at 22℃ and then incubated with the primary antibodies overnight at 4℃. Membranes were reacted with secondary antibodies conjugated with horseradish peroxidase at 22℃ for 1 h.
Bands were detected by chemiluminescence detection reagent (Beyotime). The grey value of the bands was analysed using Image Lab 6.0, and the level of the target protein was represented by the ratio of the grey value of the target protein to the internal control protein (GAPDH). The level of phosphorylation was indicated by the ratio of the grey value of the phosphorylated protein to the total protein.

| Vascular reactivity experiment
The thoracic aorta was immediately dissected and put in physi-

| Analysis of biochemical parameters
The antioxidant and oxidative stress indicators in serum were measured using reagent kits provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). SOD was tested using the hydroxylamine method; glutathione peroxidase (GSH-Px) was tested using the colorimetric method; malondialdehyde (MDA) was tested using the thiobarbituric method (TBA method).

| Statistical analysis
All data were expressed as mean ± SEM. Statistical evaluation was performed using one-way or two-way ANOVA, followed by post hoc tests with Bonferroni adjustments. Statistical significance was set at p < 0.05. The GraphPad Prism 8.0 was used for this purpose.

| Basic data (effects of different training intensities on the body mass, blood pressure and heart rate of rats)
We measured the weight of the rats once a week and observed different degrees of increase in the weight of each group ( Figure 1A).
There was a significant difference in weight between WKY-S and

| Vascular structure
To explore the potential impact of different intensities of training on the vascular morphology of SHR, the vascular morphology of the thoracic aortas of the rats involved in this study was examined

| Change of vasodilation function of aorta
Decreased eNOS activity in vascular endothelial cells is a hallmark of endothelial dysfunction, characterized by impaired endotheliumdependent relaxation (ACh-induced), which is an early marker for hypertension. We explored the changes in vasodilation function of different groups. Results were analysed by using two-way ANOVA.
Compared with WKY-S, ACh-induced dilation function was impaired in SHR-S (p < 0.05) ( Figure 3F). LICT and MICT significantly improved ACh-induced vasodilation function of aorta, compared with SHR-S (p < 0.05). However, HICT impaired the ACh-induced vasodilation function in SHR (p < 0.05). These results indicate that LICT and MICT have a protective effect on the endothelium-dependent relaxation function in SHR, whereas HCT has a potentially harmful effect on the endothelium-dependent relaxation function.

| Changes in NLRP3, ASC, Caspase-1 and IL-1β expression in the mesenteric artery
The role of NLRP3 inflammasome in hypertensive vascular dysfunction has been confirmed; inhibition of the NLRP3 pathway improves hypertensive vascular dysfunction. 8

| Changes in SOD2, NOX2, NOX4 expression in mesenteric artery
Oxidative stress has been proven to damage blood vessels in the

| Changes in activity of SOD, GSH-Px and MDA level in serum
To further examine the effects of chronic CT with different intensities on oxidative stress in SHR, the SOD and GSH-Px

| DISCUSS ION
Our study revealed that different intensities of CT had different effects on oxidative stress and vascular inflammation in SHR. All three training paradigms could effectively reduce the activation of the There is evidence that different training intensities have different effects on the vascular function of hypertensive animal models. [32][33][34][35] Appropriate training intensity is increasingly becoming the key to training therapy. Inflammatory response and oxidative stress have been extensively studied in SHR. This research aimed to explore the effects of different intensities of training (35% VO 2 max, 50% VO 2 max, 65% VO 2 max) on inflammation and oxidative stress in blood vessels of SHR.
Seven key findings were observed in this research ( Our results suggested that LICT and MICT had significant effects on blood pressure, including the SBP and DBP. Moderate training had a more significant effect in reducing blood pressure, whereas HICT further increased the DBP in SHR. DBP is closely related to peripheral vascular resistance, suggesting that HICT may cause damage to peripheral resistance blood vessels. In this study, we examined proteins associated with inflammation and oxidative stress in mesenteric artery rather than aorta, considering that the vascular function of small blood vessels or resistance vessels can better reflect the real peripheral resistance blood vessels in various organs of the body. However, considering that vascular morphometry analysis and vascular reactivity experiment about the secondary branches of mesenteric artery are difficult, we only conducted these tests in the aorta.
Early studies reported that the mechanism by which training improves blood pressure is related to the eNOS-NO pathway. 38 NO in vascular endothelial cells plays an important role in angiogenesis, regulating vascular tension, inhibiting smooth muscle cell proliferation and migration, and inhibiting platelet aggregation and adhesion. 38 Decreased level of NO produced by eNOS in vascular endothelial cells is a hallmark of endothelial dysfunction, characterized by impaired endothelium-dependent relaxation, which is an early marker for hypertension. The results of this study found that eNOS expression was up-regulated in training groups with different intensities, but the blood pressure and vascular morphology in the SHR-H showed no positive changes. We speculated that HICT caused eNOS uncoupling and caused inhibition of the eNOS function. 39 In the past few years, iNOS has been reported to be mainly induced by cytokines, which is closely related to the vascular dysfunction of hypertension. 40 We observed that the vascular iNOS expression in SHR-S rats was significantly higher than that in WKY-S rats and chronic CT could reduce the expression of iNOS in vascular tissues.
Acute high-intensity muscle training promotes the production of ROS, which is related to oxidative damage and the activation of F I G U R E 8 Graphic abstract various biochemical signalling pathways. 41 HICT causes an increase in the level of vascular oxidative stress, leading to an increase in the ROS-related uncoupling of eNOS, and the balance between NO and ROS is lost, which in turn causes a vicious cycle of reduced NO bioavailability and increased ROS. 42 In human studies, it has been confirmed that high-intensity training increases oxidative stress. 41 Our results revealed that high-intensity training had no effect on the activity of SOD and GSH-Px, MDA level in serum, and SOD2, NOX2 and NOX4 expression in the mesenteric arteries of SHR, whereas low-and medium-intensity training could significantly improve oxidative stress. Previous studies have investigated the relationship between CT and oxidative stress level in vivo by measuring the SOD and GSH-Px activities and the MDA level. [45][46][47] Although many studies have shown that CT can reduce oxidative stress, there is a paradox in the relationship between training and the production of ROS. Training can also induce oxidative stress under certain conditions, such as acute high-intensity training and ultramarathon training. Additionally, appropriate training may cause the body to be exposed to mild oxidative stress repeatedly, which may initiate an adaptive process. in mitochondrial biosynthesis and homeostasis. 58 Sirtuins act as cell sensors to detect energy supply and regulate metabolic processes.
Mammalian sirtuin 1 and sirtuin 3 are the two cores that control metabolic processes and are located in the nucleus and mitochondria, respectively. 59 Changes in the function of SIRT1 and SIRT3 significantly affect the vascular function associated with hypertension. 60 There are enough studies to prove that training increases the oxygen consumption of mitochondria, leading to an increase in the production of ROS, 63,64 and proper exercise can effectively reduce inflammation and oxidative stress in hypertension. 65 Excessive exercise promotes oxidative stress. 63,64 Reactive nitrogen and oxygen compounds' (RONS) levels can influence the phosphorylation of Ser485-AMPKα1/Ser491-AMPKα2, thereby inhibiting the phosphorylation of Thr172-AMPKα, 66 and excessive RONS production reduces Thr172-AMPKα phosphorylation during hypoxia. 67 Another important mechanism may be that the accumulation of lactic acid produced by long-term high-intensity exercise is accompanied by a decrease in pH, and the relative abundance of AMPK decreases in a long-term low-pH environment. 68,69 Thr172-AMPKα phosphorylation is reduced, consequently reducing mitochondrial biogenesis, which is not conducive to the increase of functional protein PGC1α.
We speculated this change might be one of the important reasons why AMPK function was inhibited in SHR rats with high-intensity training in our study; we plan to conduct further research on this topic.
Among the three intensities we set: LICT (35% VO 2 max), MICT (50% VO 2 max) and HICT (65% VO 2 max), we believe that there existed a certain beneficial training intensity between 50%-65% VO 2 max in aerobic training, and training intensity higher than this value is not suitable to treat hypertension.
In conclusion, this study showed that CT with different intensities had different effects on oxidative stress and vascular inflammation in SHR. All three training paradigms could effectively reduce the activation of the TLR4/NF-κB/NLRP3 inflammatory pathway in SHR, but only low-intensity and medium-intensity training could improve the blood pressure and the oxidative stress in SHR, whereas high-intensity training did not show any positive effects. Therefore, we suggest choosing medium-or low-intensity (≤50% VO 2 max) continuous exercise training as a therapeutic strategy for hypertension patients. Future research on the relationship between training intensity and hypertension should be conducted to further explore the critical point between medium-intensity and high-intensity training and to explain the differences in the changes to oxidative stress caused by different intensities of exercise.

ACK N OWLED G EM ENTS
We thank Francesca Cornero from the University of Cambridge for editing and reviewing this manuscript for the English language.
Thanks to He Linlong as a volunteer for supervising the rat treadmill experiment. This work was supported by the scientific research project of Chongqing Sports Bureau (No. B202014) and The key scientific research project of Chongqing Medical University (No. 201729).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data utilized in this study are included in this article, and all data supporting the findings of this study are available on reasonable request from the corresponding author.