AAV-CBS increased CBS expression and H2S level in the PVN
As shown in Figure 1A, after injection of AAV into PVN, ZsGreen was strongly expressed in the PVN but not in the supraoptic nucleus (SON) or the subfornical organ (SFO). Additionally, SHR showed decreased CBS expression (Figures 1B and C) and H2S levels (Figure 1D) in their PVN compared to control rats. Administration of AAV-CBS to PVN could significantly increase PVN expression of CBS and H2S levels in SHR.
Endogenous H2S in the PVN lowered BP and HR of SHR
In our investigation, SBP was measured using a tail-cuff plethysmograph. As shown in Figure 2A, SHR had a significant increase in blood pressure compared with WKY rats (SHR + AAV- ZsGreen: 186±3 mmHg vs WKY+ AAV- ZsGreen: 125±4 mmHg, P<0.05, HR: SHR + AAV- ZsGreen: 186±3 mmHg vs WKY+ AAV- ZsGreen: 125±4 mmHg, P<0.05). From day 12, the blood pressure in the SHR + AAV- CBS rats gradually dropped and stayed lower than in the SHR + AAV- ZsGreen group by the end of our study (SHR + AAV- CBS: 155±5 mmHg vs SHR + AAV- ZsGreen: 186±3 mmHg, P<0.05). These SBP data were supported by MAP records (Figure 2B). Even though endogenous H2S in PVN also decreased SHR's heart rate (Figure 2C), the impact was insignificant. Additionally, we discovered that H2S had no discernible impact on WKY rats' BP and HR.
Endogenous H2S in the PVN decreased the expression of Fra-LI and plasma levels of NE of SHR
Plasma NE levels were an indirect predictor of sympathetic activity, and additionally, we examined Fra-LI expression in the PVN in order to evaluate the impact of endogenous H2S on neuronal activity. As shown in Figure 2, both Fra-LI expression in the PVN (Figure 2D) and plasma NE levels (Figure 2E) were much higher in SHR than those in WKY rats. Increased endogenous H2S in the PVN significantly reduced PVN Fra-LI expression and plasma NE concentration of SHR. Additionally, we discovered that in WKY rats, H2S had no discernible impact on Fra-LI expression or plasma NE levels.
Endogenous H2S in the PVN increased Nrf2 expression of SHR
According to immunofluorescence and western blotting analyses, compared to the WKY rats, Nrf2 expression in the PVN of SHR was lower in SHR, and Nrf2 expression was raised by an increase in endogenous H2S in SHR but not in WKY rats (Figures 3A and B).
Endogenous H2S in the PVN attenuated oxidative stress of SHR
We first detected ROS changes in the PVN using dihydroethidium (DHE) staining. Endogenous H2S in the PVN significantly decreased the increase of ROS in SHR but not in WKY rats as shown in Figure 3C.
The primary functional subunit of NADPH oxidase (NOX) is gp91phox. It and p22phox subunit are located on the plasma membrane. After being stimulated, the two combine to form a NOX complex. The p47phox subunit in the cytoplasm interacts with the p22phox subunit after phosphorylation, resulting in a conformational change. P47phox binds to other cytoplasmic subunits (p67phox and p40phox) to form the activated NOX. Their levels have been measured in numerous earlier investigations to represent NOX activity (Bai et al., 2017). Results revealed that compared to the control rats, the number of gp91phox (Figure 3D) and p47phox-positive neurons (Figures 3E), as well as their protein levels (Figure 3F) in the PVN of SHR rats, were significantly higher. After microinjection of AAV-CBS into the PVN bilaterally, the number of gp91phox and p47phox-positive neurons, as well as their protein levels, were decreased.
Superoxide dismutase (SOD), an antioxidant metalloenzyme, can catalyze the dismutation reaction of superoxide anion radicals to produce O2 and H2O2 in living things. SOD is essential for maintaining the proper balance of oxidation and antioxidants in the body (Su et al., 2021). SOD1 mainly exists in the cytoplasm of eukaryotic cells and is considered to be the most widely distributed one among the primitive biological groups. As shown in Figure 3F, compared to the WKY rats, SHR had SOD1 expression was lower in the PVN, but endogenous H2S in the PVN increased SOD1 expression in SHR.
Endogenous H2S in the PVN reduced PICs of SHR
Increased neuroinflammation in autonomic brain regions has been identified as strongly associated with hypertension. Therefore, we evaluated the changes of IL-1β, TNF-α, IL-6, and IL-10 in the PVN. Compared to the WKY + AAV- ZsGreen group, significantly increased numbers of IL-1β-positive neurons (Figure 4A) and decreased numbers of IL-10-positive neurons (Figure4B) were observed in the PVN of the SHR + AAV- ZsGreen group. Whereas, in SHR but not WKY rats, endogenous H2S in the PVN dramatically reduced the number of IL-1β-positive neurons and increased the number of IL-10-positive neurons. In addition, western blotting results revealed that the protein levels of TNF-α, IL-1β, and IL-6 in the PVN of SHR were significantly higher than those of WKY rats, while the protein level of IL-10 was significantly lower than that of WKY rats (Figure 4C). In SHR, endogenous H2S in the PVN dramatically boosted the level of IL-10 and lowered the level of TNF-α, IL-1β, and IL-6, but not in WKY rats. Additionally, we used an ELISA technique to measure IL-1β and IL-6 expression to ascertain the impact of endogenous H2S on plasma levels of pro-inflammation cytokines. Plasma levels of IL-1β (Figure S1A) and IL-6 (Figure S2B) were higher in the SHR+AAV-ZsGreen group than in the WKY+AAV-ZsGreen group, and these changes were significantly reduced in the group that received AAV-CBS PVN microinjections. Additionally, we discovered that in WKY rats, H2S had no discernible impact on the plasma level of pro-inflammatory cytokines.
In conclusion, the current results demonstrate that increased endogenous H2S in the PVN could restore the balance of anti- and pro-inflammatory cytokines and reduces the plasma level of inflammatory cytokines in SHR.
Endogenous H2S in the PVN restored neurotransmitter imbalance of SHR
In the PVN, GABA is a significant inhibitory neurotransmitter, and norepinephrine (NE) is a significant excitatory neurotransmitter. They are closely related to how sympathetic nerve activity is controlled in hypertension. A crucial enzyme in the manufacture of catecholamines (including NE, epinephrine, and dopamine) is tyrosine hydroxylase (TH). The enzyme glutamate decarboxylase (GAD) 67 is essential for the synthesis of GABA. The levels of NE and GABA may be indirectly reflected in the expression of TH and GAD67.
Immunofluorescence and immunohistochemistry results indicated that compared to the WKY group, SHR had significantly more TH-positive neurons (Figure 4E) and significantly fewer GAD67-positive neurons (Figure 4D) in the PVN. Endogenous H2S in the PVN markedly enhanced GAD67 expression while decreasing TH expression. The results of western blotting were consistent with immunofluorescence and immunohistochemistry studies (Figure 4F). Additionally, we discovered that in WKY rats, H2S had no discernible impact on TH or GAD67.
Microinjection of Nrf2 shRNA into PVN decreased the expression of Nrf2
Next, we observed the expression level of Nrf2 after injecting AAV-Nrf2 shRNA into PVN to assess the consequences of Nrf2 silencing. Western blotting results revealed that SHR treated with AAV-Nrf2 shRNA had considerably lower levels of Nrf2 protein in their PVN than SHR treated with AAV-EGFP NC (Figure 5A).
The antihypertensive impact of endogenous H2S in SHR is eliminated by PVN knockdown Nrf2
Next, we assessed whether endogenous H2S in the PVN ameliorates hypertension through the Nrf2 pathway. As demonstrated in Figure 5B, compared to the AAV-Nrf2 shRNA group, PVN endogenous H2S lowered blood pressure in the SHR+AAV-EGFP NC group. The findings showed that the hypotensive impact of endogenous H2S might be inhibited by decreasing Nrf2 expression in PVN.
The Nrf2-dependent antioxidant response of endogenous H2S in the PVN of SHR is eliminated by PVN knockdown Nrf2
The heme oxygenase-1 (HO-1) is a powerful antioxidant, antioxidant, and Nrf2 can directly regulate the activity of the HO-1 promoter. The results showed that endogenous H2S in the PVN increased the HO-1 positive neuron number (Figure 5C) and the expression level of HO-1 protein (Figure 5D) in SHR, but in the AAV-Nrf2 shRNA group, the effect of H2S was eliminated. These data suggest that the Nrf2/HO-1 pathway is the mechanism by which PVN endogenous H2S acts.
The effect of PVN endogenous H2S on oxidative stress of SHR is abrogated by PVN knockdown Nrf2
As shown in Figure 8, we found that endogenous H2S in the PVN reduced ROS formation (Figure 6A) and the expression of gp91phox (Figures 6B and C) and p47phox (Figure 6C), increased SOD1 (Figure 6C) protein expression level in SHR + AAV EGFP NC group. These changes were not present in the SHR + Nrf2 shRNA group. These findings imply that Nrf2 knockdown can completely reverse the effects of PVN endogenous H2S on oxidative stress and antioxidant capacity.
The effect of endogenous H2S on inflammation and neurotransmitter in the PVN of SHR is abrogated by PVN knockdown Nrf2
The result presented in Figures S2 and S3 demonstrated that PVN bilateral microinjection of AAV-CBS reduced the level of TNF-α (Figures S2A and B), IL-1β (Figures S2A and C), IL-6 (Figures S2A and D), and TH (Figures 3A, B and D), increased the level of GAD67 (Figures S3B and C) in the PVN in SHR + AAV EGFP NC group. These changes were not present in the SHR + Nrf2 shRNA group. These findings imply that Nrf2 knockdown can completely reverse the effects of PVN endogenous H2S on neuroinflammation and neurotransmitter.