Exercise Training Attenuates Hypertension via Suppressing ROS/MAPK/NF-κB/AT-1R Pathway in the Hypothalamic Paraventricular Nucleus

Background: Aerobic exercise training (ExT) is beneficial for hypertension, however, its central mechanisms in improving hypertension remain unclear. Since the importance of the up-regulation of angiotensin II type 1 receptor (AT-1R) in the paraventricular nucleus (PVN) of the hypothalamic in sympathoexcitation and hypertension has been shown, we testified the hypothesis that aerobic ExT decreases blood pressure in hypertensive rats by down-regulating the AT-1R through reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK)/nuclear factors κB (NF-κB) pathway within the PVN. Methods: Forty-eight male Sprague-Dawley (SD) rats were assigned to the following groups: sham operation (SHAM) + kept sedentary (Sed), SHAM + exercise training (ExT), two kidney-one clamp (2K1C) + Sed, and 2K1C + ExT groups. Results: The 2K1C + Sed hypertensive rats showed higher systolic blood pressure (SBP), upregulated ROS, phosphorylated (p-) p44/42 MAPK, p-p38 MAPK, NF-κB p65 activity, and AT-1R expression in the PVN, and increased circulating norepinephrine (NE) than those of SHAM rats. After eight weeks of aerobic ExT, the 2K1C + ExT hypertensive rats showed attenuated NE and SBP levels, suppressed NF-κB p65 activity, and reduced expression of ROS, p-p44/42 MAPK, p-p38 MAPK, and AT-1R in the PVN, relatively to the 2K1C + Sed group. Conclusions: These data are suggestive of beneficial effects of aerobic ExT in decreasing SBP in hypertensive rats, via down-regulating the ROS/MAPK/NF-κB pathway that targets AT-1R in the PVN, and eventually ameliorating 2K1C-induced hypertension.


Introduction
Hypertension is a syndrome with elevated blood pressure as the primary clinical manifestation. A new study published in the Lancet journal reported that the number of hypertensive patients aged from 30 to 79 almost doubled in the world, from 650 million to 1.28 billion, during the 30-year period 1990-2019 [1]. According to the "2018 Chinese guidelines for the management of hypertension", the occurrence of hypertension keeps increasing and currently is 27.9% among residents aged 18 and over in China. These epidemiology studies demonstrate that hypertension has become a critical public health

Exercise Training Protocols
All rats were pre-adapted to the training program in the small animal treadmill (FT-200, Chengdu Techman Software Co., Ltd., Chengdu, China) for 5 days. Rats that could not adhere to the aerobic exercise training [28][29][30] (50 min/day, 5 days/week, 50-60% of maximal exercise capacity) were removed, and the rats that met the aerobic exercise training were retained and screened. The eligible rats were randomly divided into an aerobic exercise training group (ExT) or kept sedentary group (Sed). Each group was randomly split into two: sham operation (SHAM) group or two kidney-one clamp (2K1C) operation group, resulting in four groups namely SHAM + Sed, SHAM + ExT, 2K1C + Sed, and 2K1C + ExT. Briefly, the SD rats were anesthetized with isoflurane (500-700 mL/min). Through a right abdominal incision, the right renal artery was exposed for the acupuncture needle placement. The right renal artery and acupuncture needle were ligated with silk thread , and then the acupuncture needle was drawn out to induce hypertension [31]. A sham procedure was carried out as control. All rats recovered for one week after surgery. From the second week after the operation, the rats of the ExT group started running at 5.0 m/min for 50 min over 3-5 days. Then the speed gradually increased to 16.0 m/min from the three week till the eighth week ( Figure 1A).

Exercise Training Protocols
All rats were pre-adapted to the training program in the small ani (FT-200, Chengdu Techman Software Co., Ltd) for 5 days. Rats that could the aerobic exercise training [28][29][30] (50 min/day, 5 days/week, 50-60% of cise capacity) were removed, and the rats that met the aerobic exercise tra tained and screened. The eligible rats were randomly divided into an ae training group (ExT) or kept sedentary group (Sed). Each group was rando two: sham operation (SHAM) group or two kidney-one clamp (2K1C) op resulting in four groups namely SHAM + Sed, SHAM + ExT, 2K1C + Sed ExT. Briefly, the SD rats were anesthetized with isoflurane (500-700 mL/m right abdominal incision, the right renal artery was exposed for the acupu placement. The right renal artery and acupuncture needle were ligated w (2-0), and then the acupuncture needle was drawn out to induce hypert sham procedure was carried out as control. All rats recovered for one week From the second week after the operation, the rats of the ExT group started m/min for 50 min over 3-5 days. Then the speed gradually increased to 16 the three week till the eighth week ( Figure 1A). (n = 5 rats, * p < 0.05 or & p < 0.01 compared with the SHAM + Sed group or t group; # p < 0.01 relatively to 2K1C + Sed group).

Recording of Blood Pressure
SBP of all rats were recorded using the tail-cuff method daily before th previously described [32]. During the experiment, the SBP was measured o (n = 5 rats, * p < 0.05 or & p < 0.01 compared with the SHAM + Sed group or the SHAM + ExT group; # p < 0.01 relatively to 2K1C + Sed group).

Recording of Blood Pressure
SBP of all rats were recorded using the tail-cuff method daily before the operation as previously described [32]. During the experiment, the SBP was measured once per week.

Renal Sympathetic Nerve Recordings
RSNA parameter measurements were conducted as described earlier [33]. Briefly, under general anesthesia with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) Nutrients 2022, 14, 3968 4 of 12 (intraperitoneal), rats underwent a retroperitoneal laparotomy and the left renal nerves were identified for RSNA recording.

Collection of Brain Tissue Samples and Blood
Animals were anesthetized with isoflurane at the end of 8th week, plasma specimens and brain tissue were gathered and stored at −80 • C as previously described [34].

Immunofluorescence Staining
The intact brain was removed from the skull and fixed in 4% paraformaldehyde for 12 h and then transferred to 30% sucrose solution prepared with 0.01 M phosphate buffer for 3 days. The fixed brain tissue was embedded by OCT, then quickly frozen, and then frozen sectioned with a thickness of 18 µm. As mentioned previously, immunofluorescence studies were performed in floating sections [35]. The primary antibodies for p-p44/42 MAPK (Thr202/Tyr204, 1:200 dilution) and p-p38 MAPK (Thr180/Tyr182, 1:200 dilution) were bought from CST, AT-1R (ab124505, 1:20 dilution) was bought from Abcam. Dihydroethidium (DHE, Molecular Probes) was to check ROS generation.

ELISA Studies
Circulating norepinephrine (NE) and NF-κB p65 activity in the PVN were measured using ELISA kits, as previously described [22].

Quantitative Real-Time PCR (RT-qPCR)
AT-1R mRNA in PVN was quantified through RT-qPCR and the list of primer sequences is presented in Table 1 [37]. RT-qPCR was performed as noted earlier [34]. GAPDH level is standardized by the gene expression level of AT-1R mRNA.

Statistical Analysis
Results were presented as mean ± SEM and statistical significance was considered for p < 0.05. Data were analyzed by two-way ANOVA followed by a post-hoc Tukey test, and blood pressure data were analyzed by repeated measures ANOVA.

Blood Pressure
The blood pressure of the tail arteries of the SD rats in the four groups was measured at a fixed time point every week. The basal SBP in each group was similar. Figure 1B shows significant increases in SBP of the 2K1C rats starting from the third week to the end of the study (compared to the sedentary rats in SHAM group, * p < 0.05 & p < 0.01). Aerobic ExT significantly attenuated SBP starting from the 6th week in 2K1C + ExT (contrasted to 2K1C + Sed group, # p < 0.01). Our results showed that aerobic ExT attenuated blood pressure in hypertensive rats.

Renal Sympathetic Nerve Activity
Recent studies have found that the kidneys are only innervated by the sympathetic nerves, but not by vagus nerves [38]. The renal sympathetic nerve plays a vital role in the regulation of systemic autonomic balance. Excessive renal sympathetic nerve activation can promote the progression of angiocardiopathy like hypertension, therefore we measured the level of RSNA. Figure 2A,B show that sedentary rats in the 2K1C renovascular hypertension group exhibited higher RSNA than the sedentary rats in SHAM group (p < 0.001). With eight-week aerobic exercise training treatment, RSNA was significantly reduced comparatively to sedentary 2K1C animal group (p < 0.05). These results revealed that RSNA was negatively regulated by aerobic ExT in 2K1C rats.
shows significant increases in SBP of the 2K1C rats starting from the third week to the end of the study (compared to the sedentary rats in SHAM group, * p < 0.05 & p < 0.01). Aerobic ExT significantly attenuated SBP starting from the 6th week in 2K1C + ExT (contrasted to 2K1C + Sed group, # p < 0.01). Our results showed that aerobic ExT attenuated blood pressure in hypertensive rats.

Renal Sympathetic Nerve Activity
Recent studies have found that the kidneys are only innervated by the sympathetic nerves, but not by vagus nerves [38]. The renal sympathetic nerve plays a vital role in the regulation of systemic autonomic balance. Excessive renal sympathetic nerve activation can promote the progression of angiocardiopathy like hypertension, therefore we measured the level of RSNA. Figure 2A,B show that sedentary rats in the 2K1C renovascular hypertension group exhibited higher RSNA than the sedentary rats in SHAM group (p < 0.001). With eight-week aerobic exercise training treatment, RSNA was significantly reduced comparatively to sedentary 2K1C animal group (p < 0.05). These results revealed that RSNA was negatively regulated by aerobic ExT in 2K1C rats.

Plasma NE
The plasma NE level indicates the degree of the SNA. Figure 3A shows that compared to the sedentary rats in SHAM group, sedentary rats in 2K1C renovascular hypertension group had higher plasma NE (p < 0.001). Eight weeks of aerobic exercise training decreased the level of circulating NE in 2K1C + ExT group as compared to the 2K1C + Sed rats (p < 0.05). These results demonstrated that aerobic ExT decreased plasma NE level in 2K1C rats.

Plasma NE
The plasma NE level indicates the degree of the SNA. Figure 3A shows that compared to the sedentary rats in SHAM group, sedentary rats in 2K1C renovascular hypertension group had higher plasma NE (p < 0.001). Eight weeks of aerobic exercise training decreased the level of circulating NE in 2K1C + ExT group as compared to the 2K1C + Sed rats (p < 0.05). These results demonstrated that aerobic ExT decreased plasma NE level in 2K1C rats.
shows significant increases in SBP of the 2K1C rats starting from the third week to the end of the study (compared to the sedentary rats in SHAM group, * p < 0.05 & p < 0.01). Aerobic ExT significantly attenuated SBP starting from the 6th week in 2K1C + ExT (contrasted to 2K1C + Sed group, # p < 0.01). Our results showed that aerobic ExT attenuated blood pressure in hypertensive rats.

Renal Sympathetic Nerve Activity
Recent studies have found that the kidneys are only innervated by the sympathetic nerves, but not by vagus nerves [38]. The renal sympathetic nerve plays a vital role in the regulation of systemic autonomic balance. Excessive renal sympathetic nerve activation can promote the progression of angiocardiopathy like hypertension, therefore we measured the level of RSNA. Figure 2A,B show that sedentary rats in the 2K1C renovascular hypertension group exhibited higher RSNA than the sedentary rats in SHAM group (p < 0.001). With eight-week aerobic exercise training treatment, RSNA was significantly reduced comparatively to sedentary 2K1C animal group (p < 0.05). These results revealed that RSNA was negatively regulated by aerobic ExT in 2K1C rats.

Plasma NE
The plasma NE level indicates the degree of the SNA. Figure 3A shows that compared to the sedentary rats in SHAM group, sedentary rats in 2K1C renovascular hypertension group had higher plasma NE (p < 0.001). Eight weeks of aerobic exercise training decreased the level of circulating NE in 2K1C + ExT group as compared to the 2K1C + Sed rats (p < 0.05). These results demonstrated that aerobic ExT decreased plasma NE level in 2K1C rats.

DHE Expression in the PVN
To examine the ROS production in PVN, DHE staining was performed. The fluorescent intensity of DHE was quantified by NIH Image J2x software (Washington, DC, USA). Figure 4A,B show that compared with SHAM rats, 2K1C + Sed rats exhibited an increased ROS level (p < 0.001). Eight weeks of aerobic exercise training attenuated the ROS compared with the sedentary rats in 2K1C group rats (p < 0.05). These data therefore revealed aerobic ExT reduced ROS production in 2K1C hypertensive rats.

DHE Expression in the PVN
To examine the ROS production in PVN, DHE staining was performed. The fluorescent intensity of DHE was quantified by NIH Image J2x software (Washington, USA). Figure 4A,B show that compared with SHAM rats, 2K1C + Sed rats exhibited an increased ROS level (p < 0.001). Eight weeks of aerobic exercise training attenuated the ROS compared with the sedentary rats in 2K1C group rats (p < 0.05). These data therefore revealed aerobic ExT reduced ROS production in 2K1C hypertensive rats.

P-p44/42 MAPK Expression in the PVN
We then sought to understand the underlying signaling pathway mediating effects of ExT on hypertension in PVN. Figures 5A,B, 6B and S1 show that sedentary rats in the 2K1C renovascular hypertension group had enhanced p-p44/42 MAPK protein expression compared to that in sedentary rats in SHAM group (p < 0.001). With 8-week ExT, the increased p-p44/42 MAPK activation in 2K1C + Sed rats was attenuated ( Figures 5A,B, 6B and S1, p < 0.05). No statistical difference in p44/42 MAPK expression among the four groups ( Figure 6C, p > 0.05) was detected. We observed that aerobic ExT decreased the p-p44/42 MAPK level in hypertensive rats.

P-p44/42 MAPK Expression in the PVN
We then sought to understand the underlying signaling pathway mediating effects of ExT on hypertension in PVN. Figure 5A,B, Figure 6B and Figure S1 show that sedentary rats in the 2K1C renovascular hypertension group had enhanced p-p44/42 MAPK protein expression compared to that in sedentary rats in SHAM group (p < 0.001). With 8-week ExT, the increased p-p44/42 MAPK activation in 2K1C + Sed rats was attenuated ( Figure 5A,B, Figure 6B and Figure S1, p < 0.05). No statistical difference in p44/42 MAPK expression among the four groups ( Figure 6C, p > 0.05) was detected. We observed that aerobic ExT decreased the p-p44/42 MAPK level in hypertensive rats.     Figures 6D, 7A,B and S2 show that sedentary rats in the 2K1C renovascular hypertension group had enhanced p-p38 MAPK protein expression in comparison with the sedentary rats in SHAM group (p < 0.01). With 8-week ExT, the increased p-p38 MAPK activation in 2K1C + Sed rats was attenuated ( Figures 6D, 7A,B and S2, p < 0.05). No statistical difference in p38 MAPK expression among the four groups ( Figure 6E, p > 0.05)  Figure 6D, Figure 7A,B and Figure S2 show that sedentary rats in the 2K1C renovascular hypertension group had enhanced p-p38 MAPK protein expression in comparison with the sedentary rats in SHAM group (p < 0.01). With 8-week ExT, the increased p-p38 MAPK activation in 2K1C + Sed rats was attenuated ( Figure 6D, Figure 7A,B and Figure S2, p < 0.05). No statistical difference in p38 MAPK expression among the four groups ( Figure 6E, p > 0.05) was detected. These results indicated decreased p-p38 MAPK protein expression in hypertensive rats.   Figure 3B shows that NF-κB p65 expression in sedentary rats of the 2K1C renovascular hypertension group was higher than that in SHAM group (p < 0.001). Eight weeks of aerobic exercise training attenuated the increase in 2K1C + ExT group as contrasted   Figure 3B shows that NF-κB p65 expression in sedentary rats of the 2K1C renovascular hypertension group was higher than that in SHAM group (p < 0.001). Eight weeks of aerobic exercise training attenuated the increase in 2K1C + ExT group as contrasted with the 2K1C + Sed group (p < 0.05). We observed that ExT inhibited the activity of NF-κB in hypertensive rats. Figure 8A-C, Figure 6F and Figure S3 show that sedentary rats in the 2K1C renovascular hypertension group had enhanced AT-1R positive cells ( Figure 8A,B, p < 0.001) and protein expression ( Figures 6F and S3, p < 0.001), and mRNA expression ( Figure 8C, p < 0.01) in comparison with the sedentary SHAM rats. With 8-week ExT, the increased AT-1R positive cells ( Figure 8A,B, p < 0.05), protein expression ( Figure 6F and Figure S3, p < 0.05) and mRNA expression ( Figure 8C, p < 0.05) in 2K1C + Sed rats were attenuated. These data together supported that aerobic ExT inhibited AT-1R expression in 2K1C rats.

Discussion
The new findings are as follows: (i) The ROS/MAPK/NF-κB/AT-1R pathway was activated within the PVN during hypertension progression, (ii) long-term aerobic ExT significantly suppressed the ROS/MAPK/NF-κB/AT-1R pathway in the PVN, (iii) long-term aerobic ExT weakened RSNA and SBP in 2K1C hypertension. These findings revealed that aerobic ExT attenuated blood pressure likely via the ROS/MAPK/NF-κB/AT-1R pathway in PVN during hypertension.
In this study, in comparison with the control group, rats in 2K1C + ExT group showed significant decreases of SBP during the sixth to eighth weeks of the experimental period. The present study demonstrated that long-term aerobic ExT could attenuate 2K1C-induced hypertension and delay the progression of high blood pressure. Similar effects from other research groups have also been reported [39,40]. Aerobic ExT has been recognized as the basis treatment of hypertension [41].
PVN is considered as a pivotal nucleus in the brain that functions in balancing SNA and blood pressure [42]. Lots of findings have reported that the increased ROS generation, and NF-κB activation in the PVN contribute to elevated SNA in ANG II-and high salt-induced hypertensive rats [22,[43][44][45]. Similarly, the outcomes of this study suggest that compared with SHAM rats, 2K1C induced hypertensive rats had enhanced ROS generation, NF-κB p65 activation and AT-1R expression within the PVN. In our experimental setting, eight weeks of aerobic ExT significantly decreased the expressions of ROS and AT-1R, as well as weakened NF-κB activation in the PVN.
It is also well established that enhanced ROS generation is associated with NF-κB activation in PVN [46,47]. Recently, it was shown that ROS is sufficient to activate NF-κB

Discussion
The new findings are as follows: (i) The ROS/MAPK/NF-κB/AT-1R pathway was activated within the PVN during hypertension progression, (ii) long-term aerobic ExT significantly suppressed the ROS/MAPK/NF-κB/AT-1R pathway in the PVN, (iii) longterm aerobic ExT weakened RSNA and SBP in 2K1C hypertension. These findings revealed that aerobic ExT attenuated blood pressure likely via the ROS/MAPK/NF-κB/AT-1R pathway in PVN during hypertension.
In this study, in comparison with the control group, rats in 2K1C + ExT group showed significant decreases of SBP during the sixth to eighth weeks of the experimental period. The present study demonstrated that long-term aerobic ExT could attenuate 2K1C-induced hypertension and delay the progression of high blood pressure. Similar effects from other research groups have also been reported [39,40]. Aerobic ExT has been recognized as the basis treatment of hypertension [41].
PVN is considered as a pivotal nucleus in the brain that functions in balancing SNA and blood pressure [42]. Lots of findings have reported that the increased ROS generation, and NF-κB activation in the PVN contribute to elevated SNA in ANG II-and high saltinduced hypertensive rats [22,[43][44][45]. Similarly, the outcomes of this study suggest that compared with SHAM rats, 2K1C induced hypertensive rats had enhanced ROS generation, NF-κB p65 activation and AT-1R expression within the PVN. In our experimental setting, eight weeks of aerobic ExT significantly decreased the expressions of ROS and AT-1R, as well as weakened NF-κB activation in the PVN.
It is also well established that enhanced ROS generation is associated with NF-κB activation in PVN [46,47]. Recently, it was shown that ROS is sufficient to activate NF-κB in various modes of fibroblast senescence [48]. Previous studies have already revealed that ROS promote NF-κB activation in the PVN, further increasing ROS generation in hypertensive rats [49,50]. In addition, chronic NF-κB blockade in PVN significantly attenuates RSNA in heart failure rats [51], inhibits ROS expression, and reduces blood pressure in hypertensive rats [46]. There may be a mutually promoting relationship between ROS and NF-κB in the PVN in hypertension. In addition, activated NF-κB in PVN is a master regulator of AT-1R up-regulation, and contributes to increased sympathetic activity in hypertensive [49] as well as heart failure rats [52].
MAPK, including p44/42 MAPK, JNK, and p38 MAPK as the major family members, plays pivotal roles in heart failure [53] and hypertension [54]. Recently, it was reported that phosphorylation of the MAPK within the PVN contributes to excessive RSNA, and the p44/42 MAPK inhibitor, PD-98059, micro-injected in the PVN significantly reduces RSNA in both heart failure [26] and ANG II-induced hypertensive rats [54]. This suggests that p44/42 MAPK signaling in PVN plays a key role in sympatho-excitation in the pathophysiology of heart failure and hypertension. Researchers also found p-p44/42 MAPK activation and AT1-R expression in the PVN were substantially elevated in hypertensive model in rats. Injection of PD-98059 into PVN reduced the expression of p-p44/42 MAPK and AT1-R in the PVN and consequently decreased blood pressure [55]. Therefore, the up-regulation of AT1-R in PVN may be mediated by MAPK signaling stimulation in PVN during hypertension. In this study, we found that PVN activities of p-p44/42 MAPK and p-p38 MAPK in 2K1C groups were enhanced more than those of SHAM groups. Eight weeks of aerobic ExT suppressed the PVN activities of p44/42 MAPK and p38 MAPK in 2K1C rats. Moreover, the existing research from our laboratory and other groups have reported that MAPK affects NF-κB activity as the upstream regulator in the PVN [26,47].
In summary, our data showed that the 2K1C surgery activated the PVN ROS/MAPK/NF-κB signaling overloading the AT-1R overexpression. This study revealed that aerobic ExT attenuated hypertension in 2K1C rats, and suppressed the ROS/MAPK/NF-κB/AT-1R pathway in the PVN of 2K1C hypertensive animals. Figure 9 illustrates the central mechanism of aerobic ExT attenuating hypertension. Aerobic ExT might be a potential treatment for hypertension through modulating the ROS/MAPK/NF-κB/AT-1R pathway in 2K1C hypertension.
Nutrients 2022, 14, x FOR PEER REVIEW the p44/42 MAPK inhibitor, PD-98059, micro-injected in the PVN significantly RSNA in both heart failure [26] and ANG II-induced hypertensive rats [54]. This s that p44/42 MAPK signaling in PVN plays a key role in sympatho-excitation pathophysiology of heart failure and hypertension. Researchers also found p MAPK activation and AT1-R expression in the PVN were substantially elevate pertensive model in rats. Injection of PD-98059 into PVN reduced the expre p-p44/42 MAPK and AT1-R in the PVN and consequently decreased blood press Therefore, the up-regulation of AT1-R in PVN may be mediated by MAPK s stimulation in PVN during hypertension. In this study, we found that PVN acti p-p44/42 MAPK and p-p38 MAPK in 2K1C groups were enhanced more than SHAM groups. Eight weeks of aerobic ExT suppressed the PVN activities of MAPK and p38 MAPK in 2K1C rats. Moreover, the existing research from our lab and other groups have reported that MAPK affects NF-κB activity as the upstre ulator in the PVN [26,47].
In summary, our data showed that the 2K1C surgery activated th ROS/MAPK/NF-κB signaling overloading the AT-1R overexpression. This study r that aerobic ExT attenuated hypertension in 2K1C rats, and suppress ROS/MAPK/NF-κB/AT-1R pathway in the PVN of 2K1C hypertensive animals. illustrates the central mechanism of aerobic ExT attenuating hypertension. Aero might be a potential treatment for hypertension through modulati ROS/MAPK/NF-κB/AT-1R pathway in 2K1C hypertension.

Conclusions
Data from the current study supported that long-term aerobic ExT significa hibited the ROS/MAPK/NF-κB signaling in PVN and subsequently down-re AT-1R expressions, attenuated blood pressure of 2K1C rats, and consequently

Conclusions
Data from the current study supported that long-term aerobic ExT significantly inhibited the ROS/MAPK/NF-κB signaling in PVN and subsequently down-regulated AT-1R expressions, attenuated blood pressure of 2K1C rats, and consequently ameliorated the development of 2K1C-induced hypertension.

Informed Consent Statement: Not applicable.
Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files.