Korean Red Ginseng-Induced SIRT3 Promotes the Tom22–HIF-1α Circuit in Normoxic Astrocytes

Astrocytes play a key role in brain functioning by providing energy to neurons. Increased astrocytic mitochondrial functions by Korean red ginseng extract (KRGE) have been investigated in previous studies. KRGE administration induces hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in astrocytes in the adult mouse brain cortex. VEGF expression can be controlled by transcription factors, such as the HIF-1α and estrogen-related receptor α (ERRα). However, the expression of ERRα is unchanged by KRGE in astrocytes of the mouse brain cortex. Instead, sirtuin 3 (SIRT3) expression is induced by KRGE in astrocytes. SIRT3 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that resides in the mitochondria and maintains mitochondrial homeostasis. Mitochondrial maintenance requires oxygen, and active mitochondria enhance oxygen consumption, resulting in hypoxia. The effects of SIRT3 on HIF-1α-mediated mitochondria functions induced by KRGE are not well established. We aimed to investigate the relationship between SIRT3 and HIF-1α in KRGE-treated normoxic astrocyte cells. Without changing the expression of the ERRα, small interfering ribonucleic acid targeted for SIRT3 in astrocytes substantially lowers the amount of KRGE-induced HIF-1α proteins. Reduced proline hydroxylase 2 (PHD2) expression restores HIF-1α protein levels in SIRT3-depleted astrocytes in normoxic cells treated with KRGE. The translocation of outer mitochondrial membranes 22 (Tom22) and Tom20 is controlled by the SIRT3-HIF-1α axis, which is activated by KRGE. KRGE-induced Tom22 increased oxygen consumption and mitochondrial membrane potential, as well as HIF-1α stability through PHD2. Taken together, in normoxic astrocytes, KRGE-induced SIRT3 activated the Tom22–HIF-1α circuit by increasing oxygen consumption in an ERRα-independent manner.


Introduction
Astrocytic mitochondria functions appear to be intimately linked to the regulation of aging [1,2], and secretion of angiogenic/neurogenic factors [3][4][5][6][7] under physiological conditions. Since astrocytes boost communication between the vascular and neuronal systems [6,8,9], enhanced astrocytes may contribute to dynamic neurovascular communications. The Korean red ginseng extract (KRGE) enhances astrocyte proliferation in the subventricular zone, a neurogenic area of adult mice brains [4]. KRG injection triggers neurogenesis and the differentiation of neuroblasts in the hippocampus in mice [10]. KRGE can facilitate angiogenesis in both in vivo and in vitro models by activating the glucocorticoid receptor [11]. 1% triton X-100 in PBS at 4 • C overnight. After washing three times, the section slides were incubated in a mixture of fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG (1:300, Jackson ImmunoResearch) and/or tetramethylrhodamine (TRITC)-conjugated donkey anti-rabbit IgG (1:300, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The tissues were then washed with PBST (0.1% Tween-20 in PBS). The brain tissues were visualized using a mounting solution (Fluoro-Gel II containing 4 , 6-diamidino-2-phenylindole (DAPI), Electron Microscopy Sciences, Hatfield, PA, USA). The stained sections were subsequently examined using a fluorescence microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan). In each image, the average intensity of ten randomly chosen cells was described.

MitoTracker Staining
MitoTracker ® Deep Red FM, a mitochondrial membrane potential-sensitive reagent, was used to image intracellular active mitochondrial levels (Thermo Fisher Scientific). The astrocytes were plated in 12-well plates and cultured until they reached 70~75% confluence. After transfection with indicated si-RNA followed by treatment of 0.5 mg/mL KRGE for  23.5 h, cells were incubated with 2 µM MitoTracker-Red for 0.5 h. PBS was used for cell washing. The stained cells were imaged using a fluorescence microscope (Eclipse Ti2eU, Nikon), and the intensity of the red color was analyzed by ImageJ. In each image, the average intensity of five randomly chosen cells was described.

Oxygen Consumption in Live Cells
An oxygen consumption assay tool (Cayman, Ann Arbor, MI, USA) was utilized for detecting live cell oxygen consumption. Human brain astrocytes were transfected with the indicated 50 nM si-RNAs in a 60-mm dish (Falcon). After 5 h, the cells were subjected to trypsinization and seeded in a 96-well polystyrene microplate (black colored, Corning). The media were changed to DMEM without FBS and added with water or KRGE (0.5 mg/mL) for 24 h. The oxygen sensor probe was mixed with samples in each 96-well. The "No cell" well with oxygen sensor probe was set as the blank, approximately 100 µL mineral oil was laden to each well, and the 96-well plates were evaluated using a filter combination between the absorbance in the wells with water-treated cells and the absorbance in the wells with "no cells" was decided as "1", and the KRGE-administered groups were calculated according to the water-treated control group.
2.11. Intracellular NAD + /NADH Measurement Human brain astrocytes were grown in a 60-mm dish (Falcon). We used an intracellular NAD + /NADH quantification kit (BioVision, Waltham, MA, USA). Experiments were performed based on the manufacturer's instructions. Primary human brain astrocytes were lysed with NAD + /NADH extraction buffer (800 µL) followed by freeze/thaw cycles (two times repetition). To evaluate NADH, the sample lysis (400 µL) was put into a heating block at 60 • C for 30 min. The extracted sample (50 µL) with or without heating was added to a 96-well plate (Flacon), and NAD + cycling mixture (50 µL) was added. NADH developer (5 µL) was then added to the mixture, which was incubated for 1-4 h. A 450 nm wavelength was used for reading absorbance by the BioTek Epoch Microplate Spectrophotometer (Santa Clara, CA, USA).

ATP Levels in Astrocyte Cells
An BioVision ATP colorimetric assay kit was used to quantify ATP levels in human astrocytes. Cells were cultured on a 60-mm dish (Corning) and were transfected with the indicated si-RNAs followed by adding water or KRGE (0.5 mg/mL) for 24 h in DMEM without FBS. After trypsinization, the cells were moved to Eppendorf tubes. The cell pellet was lysed in ATP assay buffer (140 µL) and incubated for 5 min at room temperature. The Eppendorf tubes were centrifuged at 15,000 rpm for 5-10 min at 4 • C. Next, the supernatant was mixed with the reaction mixture reagents (1:1 ratio, total 100 µL) on 96-well plates (Corning). After incubation (0.5-2 h) without light at room temperature, absorbance (570 nm) was measured using Epoch Microplate Spectrophotometer (BioTek). Lysed cells were quantified using BCA reagent plus copper sulfate. ATP levels (nmol ATP/mg protein) in the water-treated control group were determined as "1", and the KRGE-treated groups were calculated to the control group.

Protein Intensity in GFAP + -Astrocytes
Intensity of immunoreactivity was measured by counting ten GFAP + -astrocytes' shape using ImageJ with random selection. The average of ten randomized immunoreactivity was evaluated. The average value of immunoreactivity intensities from water-treated group was decided to "1".

Data Evaluation
Quantification of protein expression by western blotting, MitoTracker staining, immunohistochemistry, and immunocytochemistry was achieved using ImageJ software (http://rsb.info.nih.gov/ij/, accessed on 15 August 2022). Comparisons were valued via one-way analysis of variance and Tukey's post-hoc test (mean ± standard deviation) in GraphPad Prism 6. Single comparisons were performed with paired t-test in Graph-Pad Prism 6.

KRGE Induces VEGF in Astrocytes Located near the Corpus Callosum
VEGF is an important growth factor related to regeneration (i.e., angiogenesis, neurogenesis, mitochondria biogenesis) [21,22,26,27]. We evaluated whether KRGE could induce VEGF expression in KRGE-administered mouse brain. Localization of VEGF in glial fibrillary acidic protein (GFAP)-positive astrocytes was detected near the corpus callosum approximately at bregma 1 mm, whose co-localization was clearly increased by KRGE (Figure 1a,b). Mice brains that were administered 0.25 mg/mL KRGE clearly showed VEGF expression in astrocytes compared with those administered 0.125 mg/mL KRGE ( Figure 1). Thus, KRGE upregulated VEGF expression in astrocytes near the corpus callosum.

KRGE Induces Astrocytic HIF-1α Protein Levels near the Corpus Callosum
Regulation of VEGF can be mediated by HIF-1α and/or ERRα [19,20]. We analyzed the expression of HIF-1α and ERRα. In 0.25 mg/mL KRGE-treated mice brains, the upregulation of HIF-1α was markedly detected (Figure 2a) in astrocytes near the corpus callosum ( Figure 2b). HIF-1α immunoreactivity was almost co-localized with DAPI (nucleus marker) (Figure 2a,b). However, we were unable to find any significant difference in astrocytic ERRα expression between water and 0.25 mg/mL KRGEadministered mice (Figure 2c). ERRα immunoreactivity was almost co-localized with the nucleus (Figure 2d). These data demonstrated that astrocytic VEGF expression might stem from HIF-1α when mice were administered 0.25 mg/mL KRGE. Figure 1. KRGE induced VEGF expression in astrocytes located above the corpus callosum. (a) KRGE (0.125 mg/mL or 0.25 mg/mL for 3 days) was added into drinking water for mice. Expressive images of VEGF (green) and GFAP (red) are shown located above corpus callosum (rectangle) of the brain approximately at bregma 1 mm (n = 3 per group). Scale bar = 50 µm. (b) VEGF intensity in GFAP-positive (GFAP + ) cells of brains (a) was measured using Image J (n = 3 animals per group). Scale bar = 20 µm. ** p < 0.01. cc, corpus callosum.

KRGE Induces Astrocytic HIF-1α Protein Levels near the Corpus Callosum
Regulation of VEGF can be mediated by HIF-1α and/or ERRα [19,20]. We analyzed the expression of HIF-1α and ERRα. In 0.25 mg/mL KRGE-treated mice brains, the upregulation of HIF-1α was markedly detected (Figure 2a) in astrocytes near the corpus callosum ( Figure 2b). HIF-1α immunoreactivity was almost co-localized with DAPI (nucleus marker) (Figure 2a,b). However, we were unable to find any significant difference in astrocytic ERRα expression between water and 0.25 mg/mL KRGE-administered mice (Figure 2c).
ERRα immunoreactivity was almost co-localized with the nucleus (Figure 2d). These data demonstrated that astrocytic VEGF expression might stem from HIF-1α when mice were administered 0.25 mg/mL KRGE.

HIF-1α-Mediated Expression of Mitochondria, Tom20, and Tom22 in KRGE-Treated Astrocytes
Since crosstalk between HIF-1α and ERRα in astrocytes cells has been reported [17], we examined the interplay between HIF-1α and ERRα in KRGE-treated in vitro astrocytes. ERRα knockdown partly reduced KRGE-mediated HIF-1α protein levels and also showed a significant reduction in KRGE-induced mitochondria and Tom22 (Figure 5a). We then checked ERRα protein levels under HIF-1α-reduced conditions. KRGE-induced ERRα was not significantly altered by si-HIF-1α ( Figure 5a). Instead, the reduction of HIF-1α in the presence of KRGE resulted in a significant reduction of mitochondria, Tom20, and Tom22 protein levels ( Figure 5a). Synergistic effects of mitochondria, Tom20, and Tom22 by reductions of HIF-1α and ERRα were not observed (Figure 5a). These results demonstrated that HIF-1α could play a key role in the mitochondria mass and import systems without changing the ERRα in KRGE-treated astrocytes. Diminished ERRα or/and HIF-1α did not affect KRGE-induced SIRT3 expression (Figure 5a), indicating that SIRT3 acted as an upstream factor for HIF-1α. KRGE-mediated mitochondrial mass was partially reduced by si-Tom20 but significantly reduced by the si-Tom22 (Figure 5b). Treatment of cells with both si-Tom20 and si-Tom22 significantly reduced KRGEmediated mitochondrial mass (Figure 5b). Taken together, KRGE-induced SIRT3 regulated HIF-1α, possibly affecting proteins involved in the mitochondria import system (i.e., Tom20, Tom22) and mitochondrial mass.

HIF-1α-Mediated Expression of Mitochondria, Tom20, and Tom22 in KRGE-Treated Astrocytes
Since crosstalk between HIF-1α and ERRα in astrocytes cells has been reported [17], we examined the interplay between HIF-1α and ERRα in KRGE-treated in vitro astrocytes. ERRα knockdown partly reduced KRGE-mediated HIF-1α protein levels and also showed a significant reduction in KRGE-induced mitochondria and Tom22 (Figure 5a). We then checked ERRα protein levels under HIF-1α-reduced conditions. KRGE-induced ERRα was not significantly altered by si-HIF-1α ( Figure 5a). Instead, the reduction of HIF-1α in the presence of KRGE resulted in a significant reduction of mitochondria, Tom20, and Tom22 protein levels (Figure 5a). Synergistic effects of mitochondria, Tom20, and Tom22 by reductions of HIF-1α and ERRα were not observed (Figure 5a). These results demonstrated that HIF-1α could play a key role in the mitochondria mass and import systems without changing the ERRα in KRGE-treated astrocytes. Diminished ERRα or/and HIF-1α did not affect KRGE-induced SIRT3 expression (Figure 5a), indicating that SIRT3 acted as an upstream factor for HIF-1α. KRGE-mediated mitochondrial mass was partially reduced by si-Tom20 but significantly reduced by the si-Tom22 (Figure 5b). Treatment of cells with both si-Tom20 and si-Tom22 significantly reduced KRGE-mediated mitochondrial mass (Figure 5b). Taken together, KRGE-induced SIRT3 regulated HIF-1α, possibly affecting proteins involved in the mitochondria import system (i.e., Tom20, Tom22) and mitochondrial mass.

KRGE-Induced SIRT3 Regulates Astrocytic Mitochondrial Functions
We found that 0.25 mg/mL KRGE increased SIRT3 expression mainly in astrocytes near the corpus callosum (Figure 6a,b). We evaluated the functions of in vitro astrocytic SIRT3. When SIRT3 is processed, it transforms from its 45 kilodalton (kDa) precursor form to its 28 kDa mature form. By means of mitochondrial matrix processing peptidase and mitochondrial intermediate peptidase, SIRT3 is present in the mitochondrial matrix [29]. Our results showed that the 28 kDa SIRT3 was restricted to the mitochondria, as assessed by the mitochondria-cytosol fraction assay (Figure 6c). The KRGE treatment increased the protein levels of SIRT3 and mitochondria (detection of mitochondrial mass) in the mitochondria-enriched fraction (Figure 6c). The KRGE treatment upregulated SIRT3 immunoreactivity, which was markedly reduced by si-SIRT3 in human astrocytes (Figure 6d). In KRGE-treated astrocytes, si-SIRT3 notably downregulated Tom20 and Tom22 expression in KRGE-treated astrocytes (Figure 6e). Moreover, diminished SIRT3 expression resulted in reduced mitochondrial mass (Figure 6e), OXPHOS complexes (i.e., Complex I and Complex IV) (Figure 6f), and ATP production (Figure 6g). However, SIRT3 did not affect the activation of AMPKα and vice versa in KRGE-treated astrocytes (Supplementary Figure S2a,b). These results demonstrated that KRGE-treated astrocytes played a key role in mitochondrial function through SIRT3 without affecting AMPKα activation.

KRGE-Induced SIRT3 Regulates Astrocytic Mitochondrial Functions
We found that 0.25 mg/mL KRGE increased SIRT3 expression mainly in astrocytes near the corpus callosum (Figure 6a,b). We evaluated the functions of in vitro astrocytic SIRT3. When SIRT3 is processed, it transforms from its 45 kilodalton (kDa) precursor form to its 28 kDa mature form. By means of mitochondrial matrix processing peptidase and mitochondrial intermediate peptidase, SIRT3 is present in the mitochondrial matrix [29]. Our results showed that the 28 kDa SIRT3 was restricted to the mitochondria, as assessed by the mitochondria-cytosol fraction assay (Figure 6c). The KRGE treatment increased the protein levels of SIRT3 and mitochondria (detection of mitochondrial mass) in the mitochondria-enriched fraction (Figure 6c). The KRGE treatment upregulated SIRT3 immunoreactivity, which was markedly reduced by si-SIRT3 in human astrocytes ( Figure  6d). In KRGE-treated astrocytes, si-SIRT3 notably downregulated Tom20 and Tom22 expression in KRGE-treated astrocytes (Figure 6e). Moreover, diminished SIRT3 expression resulted in reduced mitochondrial mass (Figure 6e), OXPHOS complexes (i.e., Complex I and Complex IV) (Figure 6f), and ATP production (Figure 6g). However, SIRT3 did not affect the activation of AMPKα and vice versa in KRGE-treated astrocytes (Supplementary Figure S2a,b). These results demonstrated that KRGE-treated astrocytes played a key role in mitochondrial function through SIRT3 without affecting AMPKα activation.

KRGE Induces Astrocytic HIF-1α by Enhancing Tom22-Mediated Oxygen Consumption
The relationship between the Tom20/Tom22 complex and HIF-1α was investigated. HIF-1α protein was elevated by KRGE while being considerably decreased by si-Tom22 but not si-Tom20 (Figure 7a). Increased mitochondrial functions could stabilize HIF-1α because of upregulated oxygen consumption. Therefore, we observed the oxygen consumption, and our results showed that KRGE-induced oxygen consumption was blocked by si-Tom22, but not by si-Tom20 (Figure 7b). In the presence of KRGE, si-HIF-1α reduced the immunoreactivity of Tom22 while si-Tom22 reduced the immunoreactivity of HIF-1α (Figure 7c,d), indicating a possible interaction between the two proteins. The mitochondrial membrane potential was detected using the MitoTracker assay when cells were treated with si-Tom22 or si-HIF-1α. KRGE enhanced mitochondrial membrane potential, which was significantly reduced by si-Tom22 or si-HIF-1α (Figure 7e). Our findings demonstrated the crucial activities of the Tom22-HIF-1α circuit in the mitochondrial functions of KRGEtreated astrocytes (i.e., oxygen consumption and mitochondrial membrane potential).

KRGE-Mediated SIRT3 and Tom22 Stabilize HIF-1α Protein through PHD2
In the presence of KRGE, si-Tom22-mediated reduction of HIF-1α protein was recovered by co-knockdown of PHD2 (Figure 8a). During normoxia, PHD2 is the key oxygen sensor in the degradation of HIF-1α using oxygen [30,31]. Similar to Figure 8a, the si-SIRT3-mediated reduction of HIF-1α protein was stabilized by the co-knockdown of PHD2 (Figure 8b). These results suggested that KRGE upregulated mitochondrial SIRT3 and Tom22, leading to mitochondrial activation (i.e., oxygen consumption) and consequent inhibition of HIF-1α degradation.  immunoreactivity of HIF-1α (Figure 7c,d), indicating a possible interaction between the two proteins. The mitochondrial membrane potential was detected using the MitoTracker assay when cells were treated with si-Tom22 or si-HIF-1α. KRGE enhanced mitochondrial membrane potential, which was significantly reduced by si-Tom22 or si-HIF-1α ( Figure  7e). Our findings demonstrated the crucial activities of the Tom22-HIF-1α circuit in the mitochondrial functions of KRGE-treated astrocytes (i.e., oxygen consumption and mitochondrial membrane potential).

KRGE-Mediated SIRT3 and Tom22 Stabilize HIF-1α Protein through PHD2
In the presence of KRGE, si-Tom22-mediated reduction of HIF-1α protein was recovered by co-knockdown of PHD2 (Figure 8a). During normoxia, PHD2 is the key oxygen sensor in the degradation of HIF-1α using oxygen [30,31]. Similar to Figure 8a, the si-SIRT3-mediated reduction of HIF-1α protein was stabilized by the co-knockdown of

Discussion
KRGE has been known to activate mitochondria functions which can be required for regeneration and anti-aging effects [1,4,11,[32][33][34]; however, it is still unclear what exactly caused the active mitochondrial states it generated. Our previous data showed that an important transcription factor for cell survival and metabolism, HIF-1α, plays an essential

Discussion
KRGE has been known to activate mitochondria functions which can be required for regeneration and anti-aging effects [1,4,11,[32][33][34]; however, it is still unclear what exactly caused the active mitochondrial states it generated. Our previous data showed that an important transcription factor for cell survival and metabolism, HIF-1α, plays an essential role in KRGE-mediated astrocytic mitochondrial biogenesis [4]. Our novel findings suggested that mitochondrial astrocytic SIRT3 is an upstream factor for HIF-1α and that the SIRT3-HIF-1α axis regulates KRGE-mediated Tom22 expression. Notably, Tom22 is a crucial factor in mitochondrial oxygen consumption in KRGE-treated astrocytes, leading to the stabilization of the HIF-1α protein. Therefore, KRGE boosted the astrocytic mitochondrial function via the SIRT3-mediated Tom22-HIF-1α circuit, leading to the enhancement of the regenerative factor, VEGF (Figure 8c).
In our observation, HIF-1α may be detected in neurons as well as astrocytes; in addition, we assume that KRGE may increase the HIF-1α expression in neurons. Neuronspecific inactivation of the HIF-1α increases brain injury in a mouse model of transient focal cerebral ischemia [35]. In embryonic neural stem cells, deletion of the HIF-1α gene leads to a decrease in self-renewal of neural stem cells, paralleled by an enhancement in neuronal differentiation [36]. Therefore, KRGE may function in neuronal cells by regulating HIF-1α. In this study, the relative prevalence of SIRT3 and VEGF expression in KRGE-administered mice brains can be higher in astrocytes than in neurons localized above the corpus callosum. Thus, we focus on astrocytic VEGF expression through SIRT3-HIF-1α axis.
Nampt-mediated NAD + biosynthesis is essential for SIRT3 activation [28]. KRGEinduced ERRα expression was downregulated by Nampt knockdown. However, diminished Nampt expression did not downregulate HIF-1α protein levels in KRGE-treated astrocytes. In normoxic conditions, astrocytes treated with KRGE did not result in an increase in the NAD + /NADH ratio. An increase in SIRT3 activation may cause a reduction in NAD + levels in the mitochondria because the deacetylation activity performed by SIRT3 converts NAD + into nicotinamide and acetyl ADP-ribose [37]. We hypothesize that KRGEinduced SIRT3 activation may lead to NAD + reduction, even though KRGE produces NAD + by Nampt. Our findings suggested that Nampt can regulate ERRα in a SIRT3-independent manner and that SIRT3 can regulate HIF-1α in a Nampt-independent manner.
How does KRGE increase the expression of SIRT3? Ginsenoside Rg1 may increase SIRT3 expression at both the mRNA and protein levels in HSCs/HPCs cells of an aging rat model [38]. In rat retinal capillary endothelial cells, Rb1 recovers the protein expression of SIRT1 and SIRT3 during high glucose-induced endothelial damage [39]. Rb1 also increases SIRT activity, which is decreased by high glucose [39]. In this study, KRGE contains 0.83 mg/g ginsenoside Rg1 and 5.52 mg/g ginsenoside Rb1. Therefore, we assume that KRGE increases the expression of SIRT3 possibly through specific ginsenoside(s)-mediated SIRT3 gene expression.
Energy production is important for brain homeostasis and function. Our previous study demonstrated that KRGE-induced ATP production could be downregulated by si-HIF-1α [4] or si-Tom22 [34]. In this study, KRGE-induced ATP production was completely inhibited by si-SIRT3. KRGE-induced OXPHOS complexes I and IV were reduced with lower SIRT3 expression, indicating that SIRT3 may be implicated in KRGE-mediated mitochondrial OXPHOS and ATP synthesis.
The relationship between HIF-1α and Tom22 in normoxic astrocytes has not been well established. Here, Tom22 may act as an oxygen sensor by stimulating oxygen consumption and HIF-1α stabilization. In contrast, Tom20 did not affect the protein stability of HIF-1α, even though Tom20 knockdown reduced ATP production in KRGE-treated astrocytes [34]. Since our data demonstrate that HIF-1α is mainly expressed in the nucleus of brain tissues, we assume that Tom22 regulates HIF-1α via an indirect pathway. The SIRT3-Tom22 axis regulated the mitochondrial oxygen consumption, leading to enhanced HIF-1α stabilization through PHD2 inactivation due to oxygen depletion in KRGE-treated astrocytes.
The association between Tom22 and VDAC (known as Por1 in yeast) is important to regulate mitochondrial protein gate assembly [40]. We speculate that the Tom22-VDAC complex-related import system, including Tom40, may be involved in mitochondrial functions. In recent reports, HIF-1α binds with mitochondrial protein VDAC1 [41]. Under normoxia, transcriptionally inactive forms of unmodified HIF-1α or its C-terminal domain alone can be targeted to mitochondria, stimulating production of a C-terminally truncated active form of VDAC1 [41]. Thus, we cannot exclude the possible interaction between HIF-1α and Tom22 in the mitochondrial outer membrane.
Interestingly, the Tom22-HIF-1α circuit influenced the Tom20 expression. As mitochondrial mass can be clearly regulated by the combination of Tom20 and Tom22, the HIF-1α-mediated increase in mitochondrial mass can be modulated by both Tom22 and Tom20. The SIRT3-HIF-1α axis is a critical regulator of Tom20 and Tom22 without affecting ERRα expression under KRGE-treated normoxic conditions.

Conclusions
In conclusion, KRGE enhanced astrocytic VEGF, HIF-1α, and SIRT3 expression near the corpus callosum; however, the precise mechanism by which this occurs is not yet completely understood. According to our novel research, KRGE increased the expression of SIRT3 in astrocytic mitochondria, and SIRT3 promoted mitochondrial biogenesis and ATP synthesis, increasing oxygen consumption. These results triggered the Tom22-HIF-1α circuit through mitochondria-nucleus crosstalk, possibly leading to VEGF upregulation.