The Inhibitory Role of Hydrogen Sulfide in UII-Induced Cardiovascular Effects and the Underlying Signaling Pathways

Urotensin II (UII) could increase blood pressure and heart rate via increased central reactive oxygen species (ROS) levels. We reported previously that hydrogen sulfide (H2S) exerts an antihypertensive effect by suppressing ROS production. The aim of the current study is to further examine the effects of endogenous and exogenous H2S on UII-induced cardiovascular effects by using an integrated physiology approach. We also use cell culture and molecular biological techniques to explore the inhibitory role of H2S on UII-induced cardiovascular effects. In this study, we found that cystathionine-β-synthase (CBS), the main H2S synthesizing enzyme in CNS, was expressed in neuronal cells of the rostral ventrolateral medulla (RVLM) area. Cellular distribution of CBS and urotensin II receptor (UT) in SH-SY5Y cells that are confirmed as glutamatergic were identified by immunofluorescent and Western blots assay. In Sprague–Dawley rats, administration of UII into the RVLM resulted in an increase in mean arterial pressure (MAP), heart rate (HR), ROS production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and phosphorylation of p47phox, extracellular signal-regulated protein kinase (ERK)1/2 and p38MAPK, but not stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK). These effects of UII were attenuated by application into the RVLM of endogenous (L-cysteine, SAM) or exogenous (NaHS) H2S. These results were confirmed in SH-SY5Y cells. UII-induced cardiovascular effects were also significantly abolished by pretreatment with microinjection of Tempol, Apocynin, SB203580, or PD98059 into the RVLM. Preincubated SH-SY5Y cells with Apocynin before administration of UII followed by Western blots assay showed that ROS is in the upstream of p38MAPK/ERK1/2. Gao activation assay in SH-SY5Y cells suggested that H2S may exert an inhibitory role on UII-induced cardiovascular effects by inhibiting the activity of Gαo. These results suggest that both endogenous and exogenous H2S attenuate UII-induced cardiovascular effects via Gαo-ROS-p38MAPK/ERK1/2 pathway.


Introduction
Urotensin II (UII), a cyclic neuropeptide, was originally found in teleost fish and later characterized in mammals [1]. It exerts a vasoactive effect through binding and activating an ancestral receptor, a G-protein coupled receptor, termed the UT receptor. UII and UT are widely expressed in the cardiovascular system, various peripheral organs, and the central nervous system (CNS). Our previous work [2] showed that microinjection of UII in the rostral ventrolateral medulla (RVLM) of spontaneously hypertensive rats (SHR) could increase blood pressure and heart rate through decreased central reactive oxygen species (ROS) and inhibition of NADPH oxidase.

Intracerebroventricular Infusion
After the rats were anesthetized, a hole (1.2-1.4 mm lateral to the midline and 0.8-1.0 mm posterior to bregma) was made in the skull. A stainless steel guide cannula (10 mm, 22 gauges) and a stainless steel injector, which was introduced through the guide cannula, were inserted into the hole. A total of 10 µL was delivered over 10 min into the ICV.

Microinjection into the RVLM
Animals were anesthetized with a mixture of chloralose (35 mg/kg) and urethane (700 mg/kg) and intubated to facilitate ventilation. Blood pressure and heart rate were recorded by inserting a cannula into the left femoral artery. Body temperature was maintained with a temperature-controlled table between 37 • C and 37.5 • C. Rats were then mounted in a stereotaxic frame, and microinjection was carried out with a micropipette tip (external tip diameter 0.4 mm). A total of 0.1 µL was delivered over 1 min into the RVLM (1.8 to 2.1 mm lateral to midline, 2.6 to 3.3 mm caudal to interaural line, and 0.3 to 0.9 mm from the ventral surface). The injection sites were confirmed by histological examination. The test agents, including L-cysteine (a CBS substrate), S-adenosyl-l-methionine (SAM, a CBS agonist), HA, Tempol, Apocynin, SB203580, PD98059, and NaHS, were purchased from Sigma Aldrich (St. Louis, MO, USA). UII was synthesized in GL biochemistry. BIM23127 was purchased from Tocris (Bristol, UK). Microinjection of artificial cerebral spinal fluid (aCSF) was used as vehicle control and volume.

ROS
As described previously [2], superoxide production in the ventrolateral medulla was detected using a lucigenin-enhanced chemiluminescence assay. The ventrolateral medulla was administrated with different agents, then removed and homogenized in PBS containing 0.01 mM EDTA. After centrifuging, the supernatant was obtained and added to a buffer containing lucigenin (5 µmol/L) for O 2 − measurement. O 2 − production was expressed as the mean light unit per mg protein.
Superoxide production in SH-SY5Y cells was detected using ROS fluorescent probe, dihydroethidium (DHE, Sigma-Aldrich, St. Louis, MO, USA). Cells were treated with various agents, followed by incubation with 2 µmol/L DHE for 30 min. ROS production was analyzed by measuring the absorbance at 480 nm and 590 nm.

Measurement of NADPH Oxidase Activity
In the ventrolateral medulla, NADPH oxidase activity was analyzed by a luminescence assay. The assay was performed in PBS buffer containing EGTA (0.01 M/L) and lucigenin (5 µmol/L) as the electron acceptor and NADPH (100 mM/L) as the substrate. Background counts were recorded after dark adaptation, followed by the addition of a tissue homogenate (1 µL protein sample). The chemiluminescence value was recorded at 1 min − production in the incubation medium was measured.

Western Blotting
As described previously, tissue homogenates or cell lysates were subjected to Western blot analysis. BCA protein assay reagent (Pierce, Appleton, WI, USA) was used to assay protein content. Protein was resolved by SDS-PAGE in equal amounts and transferred to the PVDF membrane. Blots were incubated with primary antibody followed by horseradish peroxidase-conjugated secondary antibody. Anti-CBS antibodies were purchased from Abcam (Waltham, MA, USA). Anti-UT antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-total p47 phox antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-total or phosphorylated ERK1/2, p38MAPK, or JNK and phosphorylated p-p47 phox antibodies were purchased from Cell Signaling (Danvers, MA, USA). Protein bands were visualized by ECL (GE Healthcare, Chicago, IL, USA). The intensity (area × density) of the detected protein on Western blots was quantified by Image J software and was expressed as the ratio to the total protein amount.

Cell Viability Assay
Cell Counting Kit-8 (CCK-8, obtained from Dojindo, Munich, Germany) was used to determine cell viability. The SH-SY5Y cells were cultured in 96-well plates at 37 • C in a 5% CO 2 incubator. Cells were treated with different concentrations of UII and NaHS at different times. Then 10 µL CCK-8 solution was added to each well of plates then incubated for 2 h at 37 • C, 5% CO 2 . The cell viability was analyzed by measuring the absorbance at 450 nm.

Gα o Activation Assay
The activity of Ga o was determined using Gα o Activation Assay Kit (NewEast Biosciences, Wuhan, China) according to the manufacturer's instructions.

Statistical Analysis
Data are expressed as mean ± SEM. Differences between groups were compared using one-way ANOVA associated with Tukey-Kramer post hoc comparison or the unpaired Student's t-test for parametric data. Statistical significance was assumed when p-values below 0.05.

Expression of CBS and UT
To clarify the expression of H 2 S synthesizing enzymes and UT, we performed an immunofluorescent stain coupled with laser confocal microscopy on the RVLM area of SD rats and SH-SY5Y cells. The results confirmed that CBS, the main H 2 S synthesizing enzyme in CNS, was expressed in neuronal cells of the RVLM area ( Figure 1) and SH-SY5Y cells ( Figure 2). Cellular distribution of UT was identified in SH-SY5Y cells. The expression of CBS and UT in SH-SY5Y cells was further clarified by Western blots assay. We also found that SH-SY5Y cells are glutamatergic ( Figure 2).

Effects of H 2 S on UII-Induced MAP and HR
We first performed microinjection of UII (0.5 nmol/0.1 µL) into the RVLM of SD rats. The results showed that the MAP and HR of SD rats were increased within 5 min, while coadministration of NaHS (400 pmol/0.1 µL), an exogenous H 2 S donor, discernibly attenuated the effect induced by UII ( Figure 3). Furthermore, increase in the production of endogenous H 2 S through pretreatment with microinjection of L-cysteine (a CBS substrate, 0.034 nmol/0.1 µL, Figure 4B) and SAM (a CBS agonist, 0.01 nmol/0.1 µL, Figure 4C), but not HA (a CBS antagonist, 3 nmol/0.1 µL, Figure 4D), into the RVLM decreased the induced upregulation of MAP and HR by UII (0.5 nmol/0.1 µL).

Effects of H 2 S on UII-Induced O 2 − Production and NADPH Oxidase Activity
We reported previously [2] that microinjection of H 2 S into the RVLM of SHR rats decreased the level of superoxide anion (O 2 − ) and NADPH oxidase activity. To further identify the underlying mechanism of the inhibitory effect of H 2 S on UII-induced cardiovascular effect, ROS production and NADPH oxidase activity were detected.
In vivo, the ROS production was detected after RVLM was administrated with UII (0.5 nmol/0.1 µL). The results revealed that the superoxide anions (O 2 − ) production in the RVLM was significantly enhanced upon UII administration, and this effect of UII was blocked by pretreatment with NaHS (400 pmol/0.1 µL) into the RVLM ( Figure 5A,B).
NADPH oxidase activity in the RVLM was dramatically increased by UII, and this effect was diminished by pretreatment with microinjection of NaHS (400 pmol/0.1 µL) into the RVLM ( Figure 5C). Phosphorylation of the p47 phox subunit is an important step for the activation of NADPH oxidase. Pretreated the RVLM area ( Figure 5D) or SH-SY5Y cells ( Figure 5E) with UII activated the p47 phox subunit of NADPH oxidase. The UIIinduced increase in phosphorylation of the p47 phox subunit was significantly decreased by coadministration of NaHS, indicating that NaHS decreases the production of superoxide via suppression of the activity of NADPH oxidase.
To further confirm the effects of ROS on UII-induced hypertension, we performed the microinjection into the RVLM area of SD rats. Microinjection of UII (0.5 nmol/0.1 µL) into the RVLM evoked an appreciable increase in MAP and HR that lasted for 10 min. This pressor response was significantly inhibited by pretreatment with microinjection of Tempol (a superoxide dismutase mimic, 50 nmol/0.1 µL, Figure 6A) or Apocynin (NADPH oxidase inhibitor, 5 nmol/0.1 µL, Figure 6B) into the RVLM. However, UII or NaHS had no effect on SOD (data not shown).
In vitro, we firstly incubated the SH-SY5Y cells with several concentrations of UII (10 −10 , 10 −9 , 10 −8 , 10 −7 , and 10 −6 M, Figure 7A) and different exposure times (5, 10, 15, and 20 min, Figure 7B), respectively. Dihydroethidium (DHE) fluorescence intensity results showed that UII significantly increased ROS levels in SH-SY5Y cells at the dose of 10 −8 mol/L for 10 min. The effect of UII is both concentration-and time-dependent. Preincubating SH-SY5Y cells with UII (10 −8 mol/L) with various doses of NaHS (10, 50, 100, and 200 µM, Figure 7D) for 5 min before treatment, we found that NaHS exerts its inhibitory effect on UII at the dose of 50 µmol/L. L-cysteine (a substrate for H 2 S, 1 mmol/L) and SAM (a CBS agonist, 200 µmol/L) pretreatment can also significantly inhibit the increase in ROS level caused by UII, while pretreatment with HA (a CBS inhibitor, 10 mmol/L) had no effect on ROS production ( Figure 7F). Cell Counting Kit-8 (CCK-8) assay showed that UII ( Figure 7C) and NaHS ( Figure 7E) treatment had no significant effect on the SH-SY5Y cells viability. These results suggested that both exogenous and endogenous H 2 S could inhibit the increase in ROS levels caused by UII.

Effects of H 2 S on UII-Induced MAPK Activation
According to the study of Chan et al. [7], MAPK signaling pathway is involved in the Ang-II-induced pressor effect. Thus, we hypothesized that H 2 S might exert its inhibitory role through the MAPK signaling pathway. The results suggest that microinjection of UII (0.5 nmol/0.1 µL) into the RVLM resulted in the activation of p38MAPK or ERK1/2 but not SAPK/JNK. The UII-induced p38MAPK or ERK1/2 phosphorylation was significantly antagonized by coadministration of NaHS (400 pmol/0.1 µL, Figure 8A). Microinjection of SB203580 (0.5 µg/0.1 µL, Figure 8B) or PD98059 (0.1 µg/0.1 µL, Figure 8C), inhibitors of p38MAPK and ERK1/2, respectively, into the RVLM significantly blunted the increase in MAP and HR induced by UII.

ROS Is in the Upstream of p38MAPK/ERK1/2
In exploring whether ROS is in the upstream of p38MAPK/ERK1/2, we detected the change in the phosphorylation of ERK1/2 and p38MAPK in SH-SY5Y cells after preincubation with Apocynin. The results revealed that SH-SY5Y cells pretreated with Apocynin could significantly inhibit the increase in phosphorylation of ERK1/2 and p38MAPK caused by UII (Figure 9). These results suggested that ROS is upstream of p38MAPK/ERK1/2.

Effects of H 2 S on UII-Induced Gα o Activation
UII specifically binds the UII receptor, which displayed coupling to Gα o and induced ERK1/2 and p38MAPK phosphorylation. As we mentioned above, UT can activate the MAPK signaling pathway coupled to Gα o , and ROS is upstream of the MAPK pathway, so we hypothesized that H 2 S might decrease ROS levels through the inhibition of Gα o activity. Firstly, we performed the microinjection in the RVLM area of SD rats. The results showed that pretreatment with microinjection of BIM23127 (BIM, a UII receptor antagonist, 0.2 µg/µL) into the RVLM can significantly attenuate the increase in MAP and HR induced by UII ( Figure 10A).

ROS Is in the Upstream of p38MAPK/ERK1/2
In exploring whether ROS is in the upstream of p38MAPK/ERK1/2, we detected the change in the phosphorylation of ERK1/2 and p38MAPK in SH-SY5Y cells after preincubation with Apocynin. The results revealed that SH-SY5Y cells pretreated with Apocynin could significantly inhibit the increase in phosphorylation of ERK1/2 and p38MAPK caused by UII (Figure 9). These results suggested that ROS is upstream of p38MAPK/ERK1/2. Then Gα o protein expression and its activity were measured in SH-SY5Y cells 10 min after the cells were preincubated with BIM or NaHS and before the treatment of UII. The results showed that coadministration with BIM or NaHS could significantly attenuate the UII-induced Gα o activity. However, the expression of the Gα o protein remained unchanged ( Figure 10B). Antioxidants 2022, 11, x FOR PEER REVIEW 13 of 19

Effects of H2S on UII-Induced Gαo Activation
UII specifically binds the UII receptor, which displayed coupling to Gαo and induced ERK1/2 and p38MAPK phosphorylation. As we mentioned above, UT can activate the MAPK signaling pathway coupled to Gαo, and ROS is upstream of the MAPK pathway, so we hypothesized that H2S might decrease ROS levels through the inhibition of Gαo activity. Firstly, we performed the microinjection in the RVLM area of SD rats. The results showed that pretreatment with microinjection of BIM23127 (BIM, a UII receptor antagonist, 0.2 μg/μL) into the RVLM can significantly attenuate the increase in MAP and HR induced by UII ( Figure 10A).
Then Gαo protein expression and its activity were measured in SH-SY5Y cells 10 min after the cells were preincubated with BIM or NaHS and before the treatment of UII. The results showed that coadministration with BIM or NaHS could significantly attenuate the UII-induced Gαo activity. However, the expression of the Gαo protein remained unchanged ( Figure 10B).

Discussion
It is well established that UII participates in central cardiovascular control through the activation of UT. H 2 S elicits an antihypertensive effect through reduced ROS production. In the present study, we provide the first evidence demonstrating that H 2 S may exert an inhibitory role in UII-induced cardiovascular effects via Gα o -ROS-p38MAPK/ERK1/2 pathway. Our statement is supported by the following four findings: (1) The metabolic system of H 2 S was present in the RVLM of SD rat and SH-SY5Y cells. Pretreatment with microinjection of H 2 S into the RVLM significantly attenuated UII-induced MAP and HR rise. (2) H 2 S decreased the UII-induced MAP and HR by inhibiting the activity of NADPH oxidase, phosphorylation of its subunit, p47 phox , and ROS production. (3) Decreased phosphorylation of p38MAPK and ERK1/2 plays an active role in the inhibitory role of H 2 S on UII-induced cardiovascular effects, and p38MAPK/ERK1/2 pathway is in the downstream of ROS. (4) The activity of Gα o was also involved in the inhibitory role of H 2 S on UII-induced cardiovascular effects ( Figure 11).  UII is the most potent vasoactive peptide identified in mammals, which is involved in the pathophysiology of systemic and pulmonary hypertension. Increased UII levels have been reported in patients with hypertension [4] or cardiovascular diseases [15]. Although the peripheral effects of UII are reported to be different, its central effects are invariable in different species [16]. Intracerebroventricular administration of UII in vertebrates provokes hypertension [17]. We previously reported that intracerebroventricular infusion UII significantly increased MAP in SHR rats [6]. However, in different brain regions, UII exerts distinct roles in cardiovascular regulation were reported [18]. In the present study, we found that in the RVLM, a key area for the regulation of arterial blood pressure, MAP was significantly increased by the administration of UII in SD rats (Figure 3). This result is supported by many other studies [16,17]. However, a recent prospective study demonstrated that UII is unlikely to play a role in the development of hypertension [19]. Since it was an observational study, the authors may need to provide more evidence to confirm their conclusions.
As a novel gasotransmitter, evidence highlights a crucial role of H2S in numerous UII is the most potent vasoactive peptide identified in mammals, which is involved in the pathophysiology of systemic and pulmonary hypertension. Increased UII levels have been reported in patients with hypertension [4] or cardiovascular diseases [15]. Although the peripheral effects of UII are reported to be different, its central effects are invariable in different species [16]. Intracerebroventricular administration of UII in vertebrates provokes hypertension [17]. We previously reported that intracerebroventricular infusion UII significantly increased MAP in SHR rats [6]. However, in different brain regions, UII exerts distinct roles in cardiovascular regulation were reported [18]. In the present study, we found that in the RVLM, a key area for the regulation of arterial blood pressure, MAP was significantly increased by the administration of UII in SD rats (Figure 3). This result is supported by many other studies [16,17]. However, a recent prospective study demonstrated that UII is unlikely to play a role in the development of hypertension [19]. Since it was an observational study, the authors may need to provide more evidence to confirm their conclusions.
As a novel gasotransmitter, evidence highlights a crucial role of H 2 S in numerous diseases, including hypertension, cardiovascular disease, and neurodegenerative diseases. CBS is the major H 2 S-producing enzyme in the CNS. CBS produces H 2 S by using L-cysteine as the main substrate, and its activity is regulated by SAM and HA, agonist and inhibitor of CBS, respectively. In the present study, the expression of CBS was confirmed in the RVLM ( Figure 1) and in SH-SY5Y cells (Figure 2), a neuroblastoma cell line. SH-SY5Y cells were identified as glutamatergic neurons (Figure 2). H 2 S has been reported to protect SHR against hypertension [20]. Treatment with NaHS for 3 months can significantly reduce blood pressure of SHR [21]. Dillon GA et al. firstly found in adults with hypertension that H 2 S-dependent microvascular vasodilation is improved following 16 weeks of sulfhydryl-donating antihypertensive treatment [22]. Furthermore, IP administration of NaHS in a rat model lowered angiotensin II-induced blood pressure rise and prevented the development of hypertension [23]. In agreement with these studies, our results show that pretreatment with microinjection of NaHS (Figure 3), SAM, or L-cysteine ( Figure 4) into the RVLM reduced the UII-induced MAP and HR in SD rats. These studies indicate that increase in the expression of both endogenous and exogenous H 2 S may significantly inhibit UII-induced cardiovascular effects. However, the mechanisms that underlie this antihypertensive effect remain largely unknown.
It is worth noting that higher oxidative stress was reported in hypertensive patients [4], and oxidative damage is crucial in the pathogenesis associated with cardiovascular injury in hypertension [24]. In CNS, an imbalance between ROS production and degradation caused by oxidative stress was reported to contribute to the pathophysiology of hypertension [25], elevated O 2 − , H 2 O 2 , and baseline ROS in the RVLM, leading to hypertension in SHR [26]. Our current study shows that ROS production and NADPH oxidase activity were also increased by UII ( Figure 5). Coadministration with Tempol or Apocynin remarkably inhibited the vasoactive effect caused by UII in the RVLM of SD rats ( Figure 6). We also found in SH-SY5Y cells that ROS production significantly increased after the cells were treated with UII (10 −8 mol/L) for 10 min (Figure 7). These results indicate that ROS play a vital role in UII-induced hypertension.
H 2 S has been proven to be an antihypertensive substrate and reduced ROS production may contribute to its antihypertensive effects [27]. H 2 S also modulates blood pressure via KATP and Kv channels [28]. We reported previously that H 2 S exerts an antihypertensive effect by suppressing ROS production. Microinjection of NaHS, SAM, Apocynin, or Tempol decreased the level of O 2 − in the RVLM of SHR. NADPH oxidase is a major enzyme for superoxide production in the brain. We assessed the activity of NADPH oxidase and found that microinjection of NaHS, SAM, and Apocynin decreased NADPH oxidase activity significantly [2]. In this regard, we pretreated the RVLM or SH-SY5Y cells with NaHS, L-cysteine, or SAM, and the results showed that the increased ROS production induced by UII could be significantly decreased by either exogenous or endogenous H 2 S, while preincubated SH-SY5Y cells with HA had no effect on UII-induced ROS production ( Figures 5 and 7). These observations suggest that ROS may be involved in the inhibitory role of H 2 S on UII-induced oxidative stress.
The main source of ROS in CNS is NADPH oxidase [29,30]. Increased ROS generation in the RVLM elevated the MAP and sympathoexcitation of SHR, probably by upregulating NADPH oxidase [31]. NADPH oxidase consists of two membrane-bound gp91 phox and p22 phox and three cytosolic subunits, such as p47 phox and p67 phox, as well as small GTPase Rac. Phosphorylation of p47 phox is key to the assembly process of the activated NADPH oxidase [32]. Increased enzyme activity of NADPH oxidase was observed in the RVLM of SHR. The enhanced central sympathetic outflow in SHR was blunted when the gene transcription of p47 phox , gp91 phox , and p22 phox subunits were knocked down in the RVLM [5,33]. The mRNA and protein expressions of the p47 phox , gp91 phox , p67 phox , and p40 phox are upregulated in the RVLM of the UII-induced neurogenic hypertension model [7,34,35]. The present study proved that the NADPH oxidase activity was increased in the RVLM of the UII-induced hypertension rat. Pretreatment with microinjection of H 2 S into the RVLM exerted an antihypertensive role in the UII-induced hypertension rat model through decreased phosphorylation of p47 phox . We observed the same mechanism in SH-SY5Y cells ( Figure 5). These results demonstrated that H 2 S in the RVLM is attributable to attenuating UII-induced hypertension by inhibiting the phosphorylation of p47 phox .
According to the above observed phenomenon, H 2 S could attenuate the enhanced MAP and HR induced by UII via decreased ROS production, while the underlying signaling pathways remain unclear. It has been reported that multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway, are associated with ROS actions in the RVLM. The activities of MAPK (p38MAPK, ERK1/2, and JNK) are strengthened by angiotensin II through an AT1R-dependent ROS production in CNS [7,36,37]. In H 2 O 2 -induced apoptotic cardiomyocytes, UII promotes the phosphorylation of ERK1/2 and increases H 2 S production via upregulating CSE levels [38]. Thus, we speculate that the MAPK signaling pathway may participate in the antihypertensive effect of H 2 S. In the present study, the detection of the phosphorylation of the MAPK family in the RVLM revealed that UII potentiated the activation of p38MAPK and ERK1/2 but not JNK. Pretreated with H 2 S significantly suppressed UII-induced enhanced phosphorylation of p38MAPK and ERK1/2. This result was further confirmed by microinjection of selective p38MAPK or ERK1/2 inhibitor into RVLM. Similarly, we also found in SH-SY5Y cells that H 2 S inhibits UII-induced activation of p38MAPK and ERK1/2 ( Figure 8). These results indicatethat ROS and p38MAPK/ERK1/2 pathway are both involved in the inhibitory role of H 2 S on UII-induced cardiovascular effects. However, the upstream-downstream relationship between ROS and p38MAPK/ERK1/2 pathway remains to be explored.
It is generally accepted that ROS production induces the activation of p38MAPK and ERK1/2 [7,39]. However, there are reports that support the opposite opinion [40]. To confirm the upstream-downstream relationship between ROS and p38MAPK/ERK1/2, we used Apocynin and UII in SH-SY5Y cells. We found that NADPH oxidase activity was significantly inhibited by the administration of Apocynin, followed by suppression of UII-induced p38MAPK and ERK1/2 phosphorylation (Figure 9). Our observations suggest that in the underlying signaling pathways of H 2 S in UII-induced cardiovascular effects, ROS may be in upstream of the p38MAPK/ERK1/2 pathway.
As we all know, H 2 S has many cellar and molecular targets. The antihypertensive role of H 2 S in UII-induced cardiovascular effects is highly complex. UII exerts a vasoactive effect through binding to UT, which is primarily coupled to several G proteins, such as Gα i/o and Gα q/11 . UT coupled to Gα i/o , leads to activation of the MAPK signaling pathway. Since ROS is upstream of the MAPK signaling pathway, we hypothesized that H 2 S might decrease ROS levels through the inhibition of Gα o activity. RVLM administration of BIM (UT antagonist, 0.2 µg/0.1 µL) significantly prevented the UII-induced cardiovascular effects. Our results further indicated that UII significantly increased the activity of Gα o , while pretreated with H 2 S attenuated the activity of Gα o . The protein expression of Gα o remains unchanged in each group ( Figure 10). These observations indicate that H 2 S may exert an inhibitory role in UII-induced cardiovascular effects via Gα o -ROS-p38MAPK/ERK1/2 pathway, and Gα o might be the potential target of H 2 S.

Conclusions
In summary, the present study indicated that both endogenous and exogenous H 2 S attenuate UII-induced cardiovascular effects through Gα o -ROS-p38MAPK/ERK1/2 pathway. This may provide a novel approach to counteract sympathoexcitation in cardiovascular disorders that are associated with enhanced brain UII. Our study revealed the important value of H 2 S as a new potential therapeutic pathway against hypertension. Institutional Review Board Statement: The animal study protocol was approved by the animal experimental Ethics Committee of Fudan University (20210302-038).