NLRP3 Inflammasome Is Involved in Calcium-Sensing Receptor-Induced Aortic Remodeling in SHRs

Figure 1. Calhex 231 attenuated NLRP3 inflammasome activation in the aortas of SHRs. (a) Immunohistochemistry was used to detect CaSR expression in the aortas of rats. Quantification of the relative CaSR protein levels in these arteries demonstrated that CaSR was increased in SHRs and decreased by antagonist of CaSR, bar = 50 μm, n = 5. (b) Western blot analysis of CaSR, NLRP3, ASC, and caspase 1. (c–g) Bar graphs showed the quantitative analyses of the proteins from B. (h) Representative western blot images of pro-IL-1β, IL-1β, and IL-18 in the aortas from experimental rats. (i–k) Quantitative analyses of proteins from in H. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. WKY; # P < 0.05, ## P < 0.01, and ## P < 0.001 vs. SHRs.

Figure 2. Suppression of CaSR prevented aortic fibrosis in SHRs. (a) Masson staining was used to detect aortic fibrosis, bar = 50 μm, n = 5. (b) Quantification of the fibrosis area to vascular area of these arteries. (c) Western blots of collagen I, collagen III, MMP2, MMP9, and TIMP2 expressions. (d–h) Quantitative analyses of the proteins from C. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. WKY; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. SHRs.

Figure 3. Calhex 231 prevented the activation of NLRP3 inflammasome in VSMCs induced by Ang II. (a) Western blot images of CaSR and NLRP3 expression induced by Ang II in a time-dependent manner. (b) Quantification of the proteins from A. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. 0 h, n = 4. (c) CaSR regulated NLRP3 inflammasome activation induced by Ang II. Western blot images of CaSR, NLRP3, ASC, and caspase 1. (d–h) Quantitative analyses of the proteins from C. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control; # P < 0.05 and ## P < 0.01 vs. Ang II.

Figure 4. Calhex 231 inhibited the release of IL-1β and IL-18 in VSMCs induced by Ang II. (a, b) IL-1β and IL-18 levels measured with enzyme-linked immunosorbent assay (ELISA). (c) Western blot images of pro-IL-1β, IL-1β, and IL-18. (d–f) Quantitative analyses of the proteins from C. ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs. control; # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. Ang II.

Figure 5. Calhex 231 suppressed collagen synthesis in VSMCs cultured with Ang II. (a) Western blots of collagen I, collagen III, MMP2, MMP9, and TIMP2 expressions. (b–f) Quantitative analysis of the proteins from A. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. control; # P < 0.05 and ## P < 0.01 vs. Ang II.

Figure 6. CRID3 inhibited the formation of NLRP3 inflammasomes and production of IL-1β and IL-18 in VSMCs stimulated with Ang II. (a) CRID3 (25 μM) pretreatment for 30 min prevented increase in ASC and caspase 1 induced by Ang II. The analysis tables are shown in (b–d). (e, f) IL-1β and IL-18 in supernatants were analyzed by ELISA. Protein expressions of pro-IL-1β and IL-1β in VSMCs detected by western blot are shown in (g). (h, i) Bar graphs showed increased expression of pro-IL-1β and IL-1β induced by Ang II. CRID3 prevented the release of mature IL-1β, but failed to decrease pro-IL-1β. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. control; # P < 0.05, ## P < 0.01, ### P < 0.001, and ns P > 0.05 vs. Ang II.

Figure 7. CRID3 inhibited collagen synthesis in VSMCs stimulated with Ang II. (a) Protein expression of collagen I, collagen III, MMP2, MMP9, and TIMP2 was detected by western blot. VSMCs were treated with CRID3 (25 μM) for 30 min before adding Ang II. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. control; # P < 0.05 and ## P < 0.01 vs. Ang II.

Figure 8. Calhex 231 pretreatment inhibits Ang II-induced increase in cytosolic [Ca2+]i in VSMCs. (a) The cytosolic [Ca2+]i was stained with fluo3/AM and the levels of cytosolic [Ca2+]i were represented by intensity of fluorescence. Ang II induced an increase in cytosolic [Ca2+]i, which was prevented when pretreatment of Calhex 231 and BAPTA/AM, n ≥ 10. (b) The schematic diagram of the mechanism of CaSR and NLRP3 inflammasome in vascular remodeling during hypertension. In VSMCs, cytosolic [Ca2+]i increase induced by CaSR triggers NLRP3 inflammasome activation; NLRP3 inflammasome induces cytokines IL-1β and IL-18 release, which leads to aortic fibrosis.