Fig 2: Successful adenoviral transduction and knockdown efficiency of UCP2. (A) Knockdown efficiency of shRNA-UCP2 in the astrocytes. The mRNA levels of UCP2 were determined by RT-qPCR with normalization to β-actin. *P<0.05 vs. the control group. (B and C) Immunoblotting showing the expression of UCP2 at different concentrations (MOI) of sh-UCP2. *P<0.05 vs. the shRNA-NC group. (D) Compared to the shRNA-UCP2 group, sepsis caused a 2.08-fold increase in UCP2 mRNA as noted in the LPS+IFN-γ+shRNA-UCP2 group. *P<0.05 vs. shRNA-NC group; **P<0.05 vs. shRNA-UCP2 group; #P<0.01 vs. shRNA-NC group. (E and F) Compared to the shRNA-UCP2 group, sepsis caused a 2.22-fold increase in UCP2 protein expression as noted in the LPS+IFN-γ+shRNA-UCP2 group. *P<0.05 vs. shRNA-NC group; **P<0.05 vs. shRNA-UCP2 group; #P<0.01 vs. shRNA-NC group. UCP2, uncoupling protein 2; LPS, lipopolysaccharide; IFN-γ, interferon-γ; MOI, multiplicity of infection.

Fig 3: Effect of UCP2 knockdown on mitochondrial function and morphology after LPS+IFN-γ treatment. Astrocytes were transduced with Ad-shRNA-NC or Ad-shRNA-UCP2 adenovirus followed by treatment with or without LPS+IFN-γ. (A-C) Astrocytes were stained with JC-1 to determine the mitochondrial membrane potential (ΔΨm) by confocal microscopy (original magnification, ×400) (A) and flow cytometry (B and C) *P<0.05 vs. shRNA-NC group; **P<0.05 vs. LPS+IFN-γ+shRNA-NC group. (D) Intracellular ATP levels were determined with an ATP assay kit with a luminometer. *P<0.05 vs. shRNA-NC group; **P<0.05 vs. LPS+IFN-γ+shRNA-NC group; ns, not significant. Data are presented as the means ± SD (n=3). (E) The mitochondrial ultrastructure was imaged using a transmission electron microscope (original magnification ×20,000). UCP2, uncoupling protein 2; LPS, lipopolysaccharide; IFN-γ, interferon-γ; ATP, adenosine triphosphate.

Fig 4: Effects of UCP-2 knock down on neurological function at 24 h after subarachnoid hemorrhage (SAH). (A) Expression of UCP-2 after administration of UCP-2 siRNA by intracerebroventricular injection in naive and SAH rats. (B-C) The neurological functions of SAH groups after administration of UCP-2 siRNA. Data represent with mean ± SD (n = 6 per group). @@P < 0.01 vs. naive+scr siRNA group; &P < 0.05, &&P < 0.01 vs. SAH+scr siRNA group; * *P < 0.01 vs. Sham group; ##P < 0.01 vs. SAH+vehicle group; vehicle, 10% DMSO dissolved in corn oil; AVE, AVE 0991; siRNA, small interfering ribonucleic acid; scr siRNA, scrambled siRNA.

Fig 5: UCP2 gene silencing leads to ROS-induced inhibition of autophagy.A ROS levels were assessed in microvascular endothelial cells (RBMEC) silenced for 24 h for UCP2 (siUCP2) or transfected with a plasmid overexpressing UCP2 (ovUCP2), either in the presence or in the absence of high-salt treatment (NaCl 20 mM) for 72 h (N = 8) B ROS levels were assessed in RBMEC after treatment with different concentrations of H2O2 for 12 h (N = 3). C, D Evaluation of autophagy in RBMEC after treatment with different concentrations of H2O2 for 12 h. Representative western blot of LC3-I/II (C) and corresponding quantification (D) are shown (N = 6). E, F Autophagic flux evaluation in RBMEC after treatment with different concentrations of H2O2 for 12 h either in the presence or in the absence of 100 nM bafilomycin for the last 3 h. Representative western blot of LC3-I/II (E) and corresponding quantification (F) are shown (N = 6). CTR indicates not transfected and not treated cells. *p < 0.05, ***p < 0.001 obtained using the one-way ANOVA followed by Bonferroni post hoc analysis. Data are reported as mean ± SEM.