Fig 2: SOCS3 or NRF2 knockdown compromised the protective effects of Rg1 in HAPI and PC-12 in vitro model. A and B HAPI cells were transfected with SOCS3 shRNA. Forty-eight hours after transfection, the cells were stimulated with LPS (1 μM) for 2 h, followed by co-incubation with Rg1 (5 μM, 10 μM, 20 μM) for a total 24 h LPS and Rg1 (A). The release of pro-inflammatory cytokines in supernatant of HAPI cells was detected by ELISA. B The protein level of COX-2, iNOS, and SOCS3 in HAPI cells was detected by western blots. C to E PC-12 cells were transfected with NRF2 shRNA. Forty-eight hours after transfection, the cells were stimulated with corticosterone (400 μM) for 2 h, followed by co-incubation with Rg1 (5 μM, 10 μM, 20 μM) for a total 24 h subjected to corticosterone stimulation and Rg1 treatment. C Mitochondrial ROS in PC-12 cells was detected by the mitoSOX kit. D Mitochondrial membrane potential in PC-12 cells was detected by the JC-1 assay. E The expression of NRF2 and HO-1 in PC-12 cells was detected by western blots. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 3: Rg1 abrogated microglial activation and attenuated mitochondrial dysfunction in CRS-induced rats. CRS rats were restricted in an adjustable cylindrical plastic bound pipe fitted to allow them to breathe for 4 h every day (from 09:00 to 13:00) with no food and water for 28 consecutive days. A group of rats (control) was not subjected to any treatment other than restraining food and water supply during 09:00–13:00. Rg1 (20 mg/kg/day) or escitalopram (1 mg/kg/day) was administered to the rats via the intragastric route 1 h after CRS every day. Hippocampus and serum were collected. A IBA-1 positive cells in the hippocampus were detected by immunofluorescence. B and C Pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the serum (B) and hippocampus (C) were detected by ELISA. D ATP level in hippocampus was detected. E The morphology of mitochondria in hippocampus was observed by TEM. F The copy number of mitochondrial DNA (mtDNA) in hippocampus. G The protein levels of SOCS3, NRF2, and EZH2 in hippocampus were detected by western blots. H The level of GAS5 was detected by qPCR. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: GAS5 knockdown suppressed microglial activation and attenuated mitochondrial dysfunction in CRS-induced rats. In vivo GAS5 knockdown was achieved by injecting lentivirus-packaged GAS5 shRNA (Lenti-shGAS5) into the lateral ventricle at the following coordinates: 0.3 mm behind the bregma and 1.0 mm lateral from the sagittal midline, at a depth of 2.2 mm from the skull surface on the last day of adaptation by using a Nanoject II (Drummond) system at a rate of 0.1 μl per min. Forty-eight hours after in vivo injection, the rats were subjected to CRS stimulation for 28 days. Hippocampus and serum were collected. A IBA-1 positive cells in the hippocampus were detected by immunofluorescence. B and C The levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the serum (B) and hippocampus (C) were detected by ELISA. D ATP level in hippocampus was detected. E The morphology of mitochondria in hippocampus was observed by TEM. F The copy number of mitochondrial DNA (mtDNA) in hippocampus. G The protein levels of SOCS3, NRF2, and EZH2 in hippocampus were detected by western blots. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 5: Nrf2 activation effect of K22 on H9c2 cells. (A) Cells were treated with K22 at various concentrations for 12 h. (B) Cells were exposed to K22 (10 μM) at various time points. qRT-PCR assay was applied to analyse the mRNA levels of Nrf2 and Nrf2-targeted gene. GAPDH was used to normalise the expression of these genes with DMSO-treatment group used as the blank control. (C) Cells were treated with K22 at various concentrations for 12 h. (D) Cells were exposed to K22 (10 μM) at various time points. Western blot assay was applied to determine the protein levels of Nrf2 and Nrf2-regulated proteins. (E&F) Statistics of the protein expression levels of Nrf2, HO-1 and NQO1. GAPDH was used as the internal control. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group.