Shexiang Tongxin Dropping Pills targeting Piezo1/Ca²⁺/NLRP3 axis attenuates vascular endothelial inflammation.

Figures

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  Fig. 1. STDP ameliorates LCWE-induced vasculitis. A The weight changes of mice in each group. B Spleen coefficients of mice in each group. C Percentage of peripheral blood monocytes of mice in each group on days 0, 3, and 7. D Representative image about the abdominal aorta of mice in each group. E Statistical analysis about the abdominal aortic area of mice in each group. F Statistical analysis about the maximum diameter of the abdominal aorta of mice in each group. G Frozen sections of mouse hearts were taken for dual fluorescence co-localisation of vascular endothelial cells labelled with vWF (red) with F4/80 (green), scale bar = 10 μm. Statistical analysis of the Mandel co-localization coefficient between the two was performed. H Dual fluorescence co-localization detection was performed for vascular endothelial cells (labeled with vWF, red) and ICAM-1 (green), and statistical analysis was conducted using the Mandel co-localization coefficient, scale bar = 10 μm. I Dual fluorescence co-localization detection was performed for vascular endothelial cells (labeled with vWF, red) and VCAM-1 (green), and statistical analysis was conducted using the Mandel co-localization coefficient, scale…

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  Fig. 2. STDP attenuates the inflammation in LCWE-induced MVECs. A Cytotoxicity was assessed by LDH release assay. B Cell viability was measured by CCK-8 assay. C, D The expression level of ICAM-1 was analyzed by western blotting and the relative protein expression level were determined by densitometric analysis. E, F Representative fluorescent confocal images of VCAM-1 (green) and DAPI (blue) in MVECs and the summarized data of the immunofluorescence intensity, scale bar = 10 μm. N = 4–6, **P < 0.01, ***P < 0.001 compared to control group; ns P > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001 compared to LCWE group

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  Fig. 3. STDP attenuates vascular endothelial dysfunction by NLRP3 inhibition. A–D The expression levels of NLRP3, pro-IL-1β and cle-IL-1β were analyzed by western blotting and the relative protein expression level were determined by densitometric analysis. E, F The expression levels of ICMA-1 were analyzed by western blotting and the relative protein expression level were determined by densitometric analysis. G, H Representative fluorescent confocal images of VCAM-1 (green) and DAPI (blue) in MVECs and the summarized data of the immunofluorescence intensity, scale bar = 10 μm. N = 4–6, *P < 0.05, ***P < 0.001 compared to control group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared to LCWE group

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  Fig. 4. STDP decreases Piezo1 expression in LCWE-induced MVECs. A, B Among the key pathways identified through enrichment analysis of these targets, the fluid shear stress and atherosclerosis pathway showed the highest enrichment score. C LCWE stimulated MVECs and administered STDP as well as the positive drug ASP (2 mmol/L) Protein levels of Piezo1 were detected by WB. D Piezo1/GAPDH gray values were calculated for each group. E-K The expression levels of TRPC1, TRPC4, TRPC5, TRPV4, TRPV3 and Piezo2 were determined by Western blotting and the relative protein expression level were determined by densitometric analysis. N = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; ns P > 0.05, #P < 0.05, ##P < 0.01 compared to LCWE group

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  Fig. 5. STDP attenuates inflammasome activation via regulation of Ca2+ signaling. A In the differential analysis of gene expression matrices in vasculitis diseases, the Ca2+ pathway was significantly enriched. B, C MVECs in each group were observed under the Ca2+ imaging equipment and 5 mmol/L of Yoda1 (Piezo1 channel agonist) was added to cells after the baseline was smooth for about 1500 s, and the average of peak and baseline Ca2+ concentration was calculated in eight cells selected from each group. Representative images and statistical charts of intracellular Ca2+ changes. D–J The protein expression levels of NLRP3, cleaved caspase-1 (cle-caspase1), and IL-1β were analyzed by Western blotting and the relative protein expression level were determined by densitometric analysis. N = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared to LCWE group

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  Fig. 6. Piezo1 knockdown abrogates STDP-mediated endothelial protection. A, B Knockdown Piezo1 representative images as well as statistical graphs. C, D The protein expression levels of NLRP3 was analyzed by Western blotting and the relative protein expression level were determined by densitometric analysis. E, F Immunofluorescence double staining to co-localize NLRP3 with Caspase1. G, H The levels of IL-18 and IL-1β in cell culture supernatants were measured by ELISA. I, J The expression levels of pro-Caspase-1, cle-Caspase-1 were analyzed by Western blotting and the relative protein expression level were determined by densitometric analysis. K, L The protein expression levels of ICAM-1 was analyzed by Western blotting and the relative protein expression level were determined by densitometric analysis. M, N Representative fluorescent confocal images of VCAM-1 (green) and DAPI (blue) in MVECs and the summarized data of the immunofluorescence intensity, scale bar = 10 μm. N = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group, #P < 0.05, ##P < 0.01, ###P < 0.001 compared to LCWE group

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  Fig. 7. STDP attenuates vascular endothelial inflammation by modulating the Piezo1/Ca2+/NLRP3 signaling axis