Central blockade of NLRP3 reduces blood pressure via regulating inflammation microenvironment and neurohormonal excitation in salt-induced prehypertensive rats

Figures

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  Fig. 1. Effects of NLRP3 on MAP in salt-induced prehypertension rats. a The blood pressure of the high-salt group increased gradually after 2 months of high-salt diet in comparison with control group. b Compared with HS + vehicle rats, chronic PVN infusion of MCC950 a specificity NLRP3 antagonist for 4 weeks attenuated hypertension. Blood pressure continued to arose after 4 weeks of discontinuation treatment. Values are expressed as means ± SEM, n = 6–8. *P < 0.05, NS vs HS, or NS + vehicle vs HS + vehicle, †P < 0.05, HS + MCC950 vs HS + vehicle, #P < 0.05, HS + MCC950’ vs NS + MCC950’

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  Fig. 2. The expression of NLRP3 pathway protein in PVN. a A representative immunohistochemistry image of NLRP3, scale bar 100 μm, immunofluorescence image of IL-1β, scale bar 50 μm, showing the changes of NLRP3, IL-1β in the high-salt diet rats from 2 to 3 months. b A representative immunoblot of NLRP3/ASC/IL-1β. c Analysis of NLRP3 pathway expression in various groups; n = 3. Values are expressed as means ± SEM. *P < 0.05, **P < 0.001, vs control (NS), 3 V, third ventricle

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  Fig. 3. a A representative immunohistochemistry image of NLRP3 in the heart and aorta, scale bar 50 and 20 μm, showing the changes of NLRP3 in the high-salt diet rats from 2 to 3 months. b Analysis integrated OD of NLRP3 in various heart tissue groups, n = 6–8. c Analysis integrated OD of NLRP3 in various aorta tissue groups, n = 6–8. d The tissue sections of the left ventricles were stained by HE staining, scale bar 50 μm. e ELISA detection plasma TNF-α expression in the various groups, n = 6–8. f ELISA detection plasma IL-6 expression in the various groups, n = 6–8.Values are expressed as means ± SEM. **P < 0.001, ***P < 0.0001 vs control (NS)

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  Fig. 4. The expression of Iba-1 in the microglia was increased. a A representative immunohistochemistry image of Iba-1 in cortex and hippocampus, scale bar 10 and 50 μm. b A representative immunoblot of Iba-1. c Analysis of Iba-1 expression in various groups, n = 3. Values are expressed as means ± SEM. *P < 0.05, **P < 0.001, vs control (NS)

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  Fig. 5. Flow cytometric analysis T cells in peripheral circulation. a CD4+ T cell (CD3+CD4+). b CD8+ T cell (CD3+CD8+). Data is represented as the percentage of CD3+CD4+, CD3+CD8+

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  Fig. 6. Increased appearing of CD8+ microglias/macrophages in the brain of high-salt diet rats. A representative immunohistochemistry image of CD8 molecule microglias/macrophages and neurons in cortex (ctx), corpus callosum (cc), hippocampus (hipp), choroid plexus (CP), and hypothalamic paraventricular nucleus (PVN), scale bar 50 μm, showing the changes of CD8+ cells in the high-salt diet rats for 3 months, n = 6–8, 3 V, third ventricle

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  Fig. 7. Central blockade NLRP3 decreases ASC/Pro-caps-1/IL-1β expression of prehypertensive rats. a A representative western blot of NLRP3 pathway. b Densitometric analysis expression of NLRP3 pathway in the PVN of different groups, n = 3. c A representative immunofluorescence image of ASC, scale bar 100 μm, and immunohistochemistry image of IL-1β, scale bar 50 μm, showing the changes of ASC and IL-1β expression when central blockade NLRP3 was stopped. d Densitometric analysis of immunofluorescent intensity of ASC in various groups. e Densitometric analysis of integrated OD of IL-1β in various groups, n = 6–8. Values are expressed as means ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001, 3 V, third ventricle

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  Fig. 8. Central blockade NLRP3 suppresses of VCAM-1, CXCR3, and CCL2 expression in the PVN of prehypertensive rats. a A representative immunoblot of VCAM-1, CXCR3, and CCL2. b Densitometric expression analysis of VCAM-1, CXCR3, and CCL2 in various groups, n = 3. c Terminated treatment of anti-NLRP3, a representative immunoblot of VCAM-1, CXCR3, and CCL2. d Densitometric protein analysis of VCAM-1, CXCR3, and CCL2 in various groups; n = 3. e ELISA detection IL-6 expression in the PVN of the various groups, n = 6–8. f ELISA detection TNF-α expression in the PVN, n = 6–8. Values are expressed as means ± SEM. *P < 0.05, **P < 0.001. ***P < 0.0001

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  Fig. 9. Central blockade NLRP3 regulates PVN excitatory and inhibitory neurotransmitters. a A representative immunohistochemistry image of TH and GAD67, scale bar 10 and 20 μm, showing the changes of TH and GAD67 in the high-salt diet rats after central blockade NLRP3. b Expression of TH in various groups; n = 6–8. c Expression of GAD67 in various groups, n = 6–8. d ELISA detection plasma NE expression in various groups, n = 6–8. e ELISA detection GABA expression in various groups, n = 6–8.Values are expressed as means ± SEM. *P < 0.05, ***P < 0.0001, 3 V, third ventricle