Fig 1: Rgs1 knockdown decreases levels of inflammation and apoptosis markers in hippocampal neurons in the context of OGD/R injury, whereas miR-27a-3p downregulation reverses the protective effect of Rgs1 silencing. (A) Rgs1 expression level as determined by RT-qPCR assay. (B) Rgs1 expression as determined by Western blotting assay. (C) Measurement of TNFα, IL-1β, and IL-6 levels by RT-qPCR assay. (D) Determination of TNFα, IL-1β and IL-6 levels in the culture supernatant by ELISA assay. (E) NF-κB (p65) expression levels as determined by RT-qPCR assay. (F) Measurement of NF-κB (p-p65) protein expression by Western blotting. (G) Evaluation of viability of hippocampal neurons by CCK-8 assay. (H) Measurement of apoptosis by flow cytometry. (I) Determination of caspase-3, Bax, and Bcl-2 expression by RT-qPCR assay. (J) C-caspase-3, Bax, and Bcl-2 protein levels. Results are shown as Mean ± SD. (n=3). *P<0.05, ***P<0.001, compared to normal; ##P<0.01, ###P<0.001, compared to shRgs1-NC; &P<0.05, &&P<0.01, &&&P<0.001, compared to shRgs1.
Fig 2: Biliverdin upregulates miR-27a-3p and downregulates Rgs1 expression in OGD/R-injured hippocampal neurons and MCAO/R-injured rat brains. The miR-27a-3p expression level as determined by RT-qPCR. (A) Hippocampal neurons. (B) Cortex. (C) Hippocampus. RT-qPCR analysis of Rgs1 expression. (D) Hippocampal neurons. (E) Cortex. (F) Hippocampus. Western blot assessment of Rgs1 protein expression. (G) Hippocampal neurons. (H) Cortex. (I) Hippocampus. Results are presented as Mean ± SD. (n=3 for cell, n=5 for brain). **P<0.01, ***P<0.001, compared to normal or sham; #P<0.05, ##P<0.01, ###P<0.001, compared to OGD/R or MCAO/R.
Fig 3: Rgs1 is a miR-27a-3p target and anti-Rgs1 shRNA screening. (A) The binding region of miR-27a-3p on the 3 ʹ-UTR of Rgs1 was predicted using TargetScan version 7.2. (B) Dual luciferase reporter assay was conducted to evaluate the interaction between miR-27a-3p and its binding site on the 3ʹ-UTR of the Rgs1 gene. (C) RT-qPCR assessment of Rgs1 expression after transfection of hippocampal neurons. (D) Rgs1 protein level was detected by RT-qPCR after transfection of hippocampal neurons. (E) Expression levels of Rgs1 mRNA and protein upon Rgs1 silencing in hippocampal neurons with different anti-Rgs1 shRNA. Results are presented as Mean ± SD. (n=3). *P<0.05, compared to shRgs1-NC; **P<0.01, compared to normal; ***P<0.001, compared to normal or miR-mimic-NC; #P<0.05, ###P<0.001, compared to miR-mimic-NC; &&&P<0.001, compared to miR-inhibitor-NC.
Fig 4: Summary of the protective effects and mechanisms of biliverdin in cerebral I/R injury. Biliverdin upregulates miR-27a-3p to target Rgs1, thereby inhibiting inflammation by suppressing NF-κB signaling, and reducing apoptosis in hippocampal neurons in vitro. It also confers neuroprotective effects against cerebral I/R injury by reducing brain infarct volume and apoptosis of neurocytes in vivo.
Fig 5: Localization of Rgs1 and identification of hippocampal neurons, expression of miR-27a-3p and Rgs1 at 3, 6, 9, and 12 h after OGD/R, and cell viability analysis at various timepoints and upon treatment with different concentrations of biliverdin. (A) Immunofluorescence results showing Rgs1 localization in normal rat cortex and hippocampus. (B) Immunohistochemical staining for NeuN in hippocampal neurons. (C) Results of CCK-8 assay showing the viability of hippocampal neurons. (D) miR-27a-3p expression level as determined by RT-qPCR assay. (E) Rgs1 expression level as determined by RT-qPCR assay. (F) Cell viability analysis after treatment with various biliverdin concentrations. Results are presented as Mean ± SD. (n=3). *P<0.05, **P<0.01, ***P<0.001, compared to normal; #P<0.05, compared to OGD/R.
Supplier Page from Abcam for Anti-RGS1 antibody