Hydrogen Sulfide Attenuates Hypertensive Inflammation via Regulating Connexin Expression in Spontaneously Hypertensive Rats

Abstract

BACKGROUND Hydrogen sulfide (H2S) has anti-inflammatory and anti-hypertensive effects, and connexins (Cxs) are involved in regulation of immune homeostasis. In this study, we explored whether exogenous H2S prevents hypertensive inflammation by regulating Cxs expression of T lymphocytes in spontaneously hypertensive rats (SHR). MATERIAL AND METHODS We treated SHR with sodium hydrosulfide (NaHS) for 9 weeks. Vehicle-treated Wistar-Kyoto rats (WKYs) were used as a control. The arterial pressure was monitored by the tail-cuff method, and vascular function in basilar arteries was examined by pressure myography. Hematoxylin and eosin staining was used to show vascular remodeling and renal injury. The percentage of T cell subtypes in peripheral blood, surface expressions of Cx40/Cx43 on T cell subtypes, and serum cytokines level were determined by flow cytometry or ELISA. Expression of Cx40/Cx43 proteins in peripheral blood lymphocytes was analyzed by Western blot. RESULTS Chronic NaHS treatment significantly attenuated blood pressure elevation, and inhibited inflammation of target organs, vascular remodeling, and renal injury in SHR. Exogenous NaHS also improved vascular function by attenuating KCl-stimulated vasoconstrictor response in basilar arteries of SHR. In addition, chronic NaHS administration significantly suppressed inflammation of peripheral blood in SHR, as evidenced by the decreased serum levels of IL-2, IL-6, and CD4/CD8 ratio and the increased IL-10 level and percentage of regulatory T cells. NaHS treatment decreased hypertension-induced Cx40/Cx43 expressions in T lymphocytes from SHR. CONCLUSIONS Our data demonstrate that H2S reduces hypertensive inflammation, at least partly due to regulation of T cell subsets balance by Cx40/Cx43 expressions inhibition.

Figure 1

Effect of NaHS on spontaneous hypertension induced increase in blood pressure (BP) in SHR. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, systolic blood pressure in WKY, SHR, and NaHS+SHR was measured. SHR had significantly increased blood pressure compared to WKY (** P<0.01). Long-term administration of NaHS significantly reduced BP in the SHR+NaHS group (^## P<0.01 vs. SHR). Data analyzed by comparing area under the curve values using one-way ANOVA and the t test, n=15 animals in each group.

Figure 2

Long-term NaHS treatment alleviates vascular remodeling and infiltration of inflammatory cells in BA and kidney tissues of SHR. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, kidneys and basal cerebral arteries were harvested and were stained by hematoxylin-eosin. A, cross-sections of BA stained with hematoxylin-eosin staining (magnification×200. scalar bar=4.5 μm). B, longitudinal sections of kidney tissues stained with hematoxylin-eosin staining (magnification×100, scalar bar=4.5 μm) (n=15).

Figure 3

The inhibitory effect of NaHS on the contractile response in basilar arteries (BA) induced by 40 mM KCl. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, BA were isolated and their contractile response were determined as described in “Materials and methods”. Contraction of BA in response to KCl was greater in SHR than in WKY rats (* P<0.05). Long-term NaHS treatment suppressed vascular contraction of cerebral arteries from SHR in response to KCl (^# P<0.05 vs. SHR). All data-points are from 7 BA. Results are means ±SEM of 4–6 experiments.

Figure 4

Long-term NaHS treatment reversed the changes of different T lymphocyte subtypes in SHR. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, PBMCs were harvested and subjected to flow cytometry analysis. Reported dot plots are generated gating on living PBMCs in the scatter (FSC vs. SSC) dot plot (not shown). A, Representative flow cytometry analysis showing percentages of circulating T lymphocytes subtypes in the peripheral blood of 15 SHR and 15 age-matched WKY rats. B, Bar graph shown are proportion of CD3⁺, CD4⁺, CD8⁺ and CD25⁺ T cells expressing CD4⁺ as well as the ratio of CD4⁺/CD8⁺ in the peripheral blood of SHR and WKY rats. The vertical axis represents the frequency of various T lymphocyte subtypes. Quantitative analysis of the mean percentage of cells ±SEM. ** P<0.01, compared with the WKY rats; ^# P<0.05, compared with SHR (n=15 animals in each group).

Figure 5

Effect of long-term NaHS treatment on the production of pro-inflammatory and anti-inflammatory cytokines in serum of SHR. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, serum level of IL-2 (A), IL-6 (B) and IL-10 (C) were determined as described in “Materials and methods”. Data represented as total amount of cytokine produced in pg/ml in peripheral blood; the results shown are the mean ±SEM; * P<0.05 and ** P<0.01, compared with the WKY rats; ^## P<0.01, compared with SHR (n=15 animals in each group).

Figure 6

Effect of long-term NaHS treatment on surface expressions of Cx40 and Cx43 in different T lymphocyte subtypes of SHR. A, Representative flow cytometry plots are presented for Cx40 and Cx43 expression levels on gated single-positive CD4⁺ T lymphocytes or CD8⁺ T lymphocyte populations in the peripheral blood from 15 SHR and 15 WKY rats. Fresh, resting PBMCs from SHR and WKY rats underwent surface staining with antibodies against CD3, CD4, and CD8 molecules. After surface staining, the cells were fixed, permeabilized, and stained with unlabeled anti-Cx40 or anti-Cx43 plus FITC-labeled secondary antibodies. Based on the CD4⁺ or CD8⁺ gate, the cells were further gated based on Cx40 and Cx43 expression levels, and the frequency of CD4⁺ or CD8⁺ T cells expressing Cx40 and Cx43 was determined. B, Bar graph shown are the percentage of CD4⁺ or CD8⁺ T cell population expressing Cx40 and Cx43. Both Cx40 and Cx43 expression levels are significantly increased in CD4⁺ or CD8⁺ T cells of SHR compared with those of WKY rats. Long-term NaHS treatment inhibited the expressions of Cx40 and Cx43 in CD4⁺ and CD8⁺ T cells from the peripheral blood of SHR, and their expressions in SHR returned to the levels seen in WKY rats. Values are mean ± SEM. * P<0.05 and ** P<0.01, compared with WKY rats; ^# P<0.01, compared with SHR rats (n=15 animals in each group).

Figure 7

Effect of treatment with NaHS on protein levels of Cx40 and Cx43 in peripheral blood lymphocytes of SHR. We treated 9-week-old male SHR and WKY rats with NaHS (56 μmol/kg⁻¹·day⁻¹, i.p.) or the same volume of normal saline and continued every day. At 9 weeks after NaHS injection, peripheral blood lymphocytes were harvested and examined by Western blot for the expression levels of Cx40 (A) and Cx43 (B). After densitometric analysis, the data were expressed as ratios of Cxs to β-actin. The data represent the mean ±SEM of 3 experiments (n=15 animals in each group). ** P<0.01 vs. WKY rats; ^## P<0.01 vs. SHR.