Fig 2: The ratio of M1/M2 polarization and release of pro-inflammatory/immune-regulatory factors in the RVLM of SIH rats. A Immunofluorescence detection of M1 and/or M2 phenotype microglia in the RVLM showed robust switching to M1 phenotype in SIH rats. B Relative ratio of M2 versus M1 phenotype microglia in the RVLM expressed as a percentage. C, D qRT-PCR showing the release of pro-inflammatory/immune-regulatory factors was increased in the RVLM of SIH rats. E, F The PRR antagonist, PRO20, decreased the M1 polarization in SIH rats. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; #P < 0.05 vs SIH; scale bars, 200 µm in (A) and (E).

Fig 3: Expression of NLRP3 is downregulated by PRO20, a PRR antagonist. A Effect of PRO20 on the mRNA expression of NLRP3/ASC/caspase-1 using qRT-PCR analyses. B, C Representative immunoblots and quantitative analysis showing that NLRP3, ASC, caspase-1, and pro-IL-1ß expression in the RVLM of SIH rats was upregulated, while PRO20 treatment downregulated their expression. D Representative images showing that NLRP3 (red) co-localized with the microglial marker (OX42). E, F Relative fluorescence intensity calculation of NLRP3-immnoreactivity with or without OX42-immnoreactivitiy in different groups. G–I Representative images and quantitative analysis showing co-localization of PGP 9.5 (neuronal marker, green) and NLRP3 (red) in the RVLM. J, K Representative images of double immunofluorescent staining for GFAP (astrocyte marker, green) and NLRP3 (red) and densitometric quantification of immunoreactivity in the RVLM. Notably, immunofluorescence staining analysis showed that NLRP3 mostly localized with microglia and neurons (arrows). Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; #P < 0.05 vs SIH; scale bars, 200 µm in (D), (G), and (J).

Fig 4: Expression of prorenin/PRR in the RVLM of rats. A qRT-PCR showing both prorenin and PRR are increased in the RVLM of SIH rats. B Representative western blots of prorenin and PRR in the RVLM of SIH rats. C, D Densitometric quantification showing prorenin and PRR are higher in the SIH group than in controls. Data are presented as the mean ± SEM. *P < 0.05 vs Ctrl, n = 6/group.