Qingxia Huang,Jing Li,Jinjin Chen,Zepeng Zhang,Peng Xu,Hongyu Qi,Zhaoqiang Chen,Jiaqi Liu,Jing Lu,Mengqi Shi,Yibin Zhang,Ying Ma,Daqing Zhao,Xiangyan Li
Ginsenoside compound K protects against cerebral ischemia/reperfusion injury via Mul1/Mfn2-mediated mitochondrial dynamics and bioenergy
Ginsenoside compound K,Cerebral ischemia/reperfusion injury,Mitochondrial dynamics,Bioenergy,Mul1/Mfn2 ubiquitination
Background Ginsenoside compound K (CK), the main active metabolite in Panax ginseng, has shown good safety and bioavailability in clinical trials and exerts neuroprotective effects in cerebral ischemic stroke. However, its potential role in the prevention of cerebral ischemia/reperfusion (I/R) injury remains unclear. Our study aimed to investigate the molecular mechanism of ginsenoside CK against cerebral I/R injury. Methods We used a combination of in vitro and in vivo models, including oxygen and glucose deprivation/reperfusion induced PC12 cell model and middle cerebral artery occlusion/reperfusion induced rat model, to mimic I/R injury. Intracellular oxygen consumption and extracellular acidification rate were analyzed by Seahorse multifunctional energy metabolism system; ATP production was detected by luciferase method. The number and size of mitochondria were analyzed by transmission electron microscopy and MitoTracker probe combined with confocal laser microscopy. The potential mechanisms of ginsenoside CK on mitochondrial dynamics and bioenergy were evaluated by RNA interference, pharmacological antagonism combined with co-immunoprecipitation analysis and phenotypic analysis. Results Ginsenoside CK pretreatment could attenuate mitochondrial translocation of DRP1, mitophagy, mitochondrial apoptosis, and neuronal bioenergy imbalance against cerebral I/R injury in both in vitro and in vivo models. Our data also confirmed that ginsenoside CK administration could reduce the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2 in cerebral I/R injury. Conclusion These data provide evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury via Mul1/Mfn2−mediated mitochondrial dynamics and bioenergy.
Ginsenoside compound K protects against cerebral ischemia/reperfusion injury via Mul1/Mfn2-mediated mitochondrial dynamics and bioenergy

Ginsenoside compound K (CK), the main active metabolite in Panax ginseng, has shown good safety and bioavailability in clinical trials and exerts neuroprotective effects in cerebral ischemic stroke. However, its potential role in the prevention of cerebral ischemia/reperfusion (I/R) injury remains unclear. Our study aimed to investigate the molecular mechanism of ginsenoside CK against cerebral I/R injury.

We used a combination of in vitro and in vivo models, including oxygen and glucose deprivation/reperfusion induced PC12 cell model and middle cerebral artery occlusion/reperfusion induced rat model, to mimic I/R injury. Intracellular oxygen consumption and extracellular acidification rate were analyzed by Seahorse multifunctional energy metabolism system; ATP production was detected by luciferase method. The number and size of mitochondria were analyzed by transmission electron microscopy and MitoTracker probe combined with confocal laser microscopy. The potential mechanisms of ginsenoside CK on mitochondrial dynamics and bioenergy were evaluated by RNA interference, pharmacological antagonism combined with co-immunoprecipitation analysis and phenotypic analysis.

Ginsenoside CK pretreatment could attenuate mitochondrial translocation of DRP1, mitophagy, mitochondrial apoptosis, and neuronal bioenergy imbalance against cerebral I/R injury in both in vitro and in vivo models. Our data also confirmed that ginsenoside CK administration could reduce the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2 in cerebral I/R injury.

These data provide evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury via Mul1/Mfn2−mediated mitochondrial dynamics and bioenergy.

Cerebral ischemia/reperfusion (I/R) injury is a pathological condition that may occur with the recovery of blood circulation in the treatment of ischemic stroke, which eventually leads to acute neurological deficit [1]. A growing body of preclinical and clinical evidence indicates that mitochondria, as dynamic organelles manage to retain the balance of fusion/fission dynamics in neuronal I/R situations [2]. Mitochondrial fusion accelerates the exchange of nutrition materials and assists in the repair of damaged mitochondria, while mitochondrial fission is an important step in the removal of defective mitochondria through mitophagy [3,4]. During I/R injury, the superoxide production by complex I reverse electron transport (RET) results in stunted mitochondrial fusion and fission, which in conjunction with traumatic mitophagy leads to dysfunctions of the Krebs cycle and oxidative phosphorylation (OXPHOS) [5]. Abnormal mitochondrial bioenergy leads to reduced mitochondrial respiration function and neuronal injury [6]. Hence, regulation of dynamics (elevated fusion and suppressed fission) and bioenergy promotion of mitochondria may play neuroprotective effects against cerebral I/R induced injury. Mitochondrial E3 ubiquitin ligase 1 (Mul1) is a multifunctional mitochondrial membrane protein and acts as the first-line surveillance of mitochondrial dynamics and energy supply in neuronal I/R injury [7,8]. In neurons, mitochondrial dynamic fusion is mainly controlled by optic atrophy protein 1 (OPA1) and mitofusins (Mfn1 and Mfn2), while fission depends on dynamin−related protein 1 (DRP1) and fission 1 protein (FIS1) [9,10]. In mammalian systems, the E3-active, C-terminal RING finger domain of Mul1 faces the cytosol, which can stabilize DRP1 and degrade mitofusins through ubiquitin-like modifiers and ubiquitination [11,12]. As expected from Mul1 protein with these proposed biochemical activities, I/R−induced Mul1 overexpression in neurons results in smaller and more fragmented mitochondria, enhanced mitophagy, inhibited OXPHOS, imbalance in energy homeostasis, and mitochondrial apoptosis [13]. Thus, the suppression of Mul1 activity and expression can regulate mitochondrial dynamics and mitophagy to enhance mitochondrial bioenergy against I/R damage in neurons. Ginsenoside compound K (CK), one of the main active metabolites from traditional Chinese medicine, Panax ginseng, has shown good safety and bioavailability in clinical trials, and exerts neuroprotective effects in neurodegenerative diseases and cerebral ischemic stroke [[14], [15], [16], [17]]. It has been shown that ginsenoside CK reduces the infarct volume of the cerebral I/R model induced by middle cerebral artery occlusion (MCAO) and suppresses microglial activation in the mice ischemic cortex [18]. Our previous studies have demonstrated that ginsenoside CK inhibits reactive oxygen species (ROS) burst and autophagy−mediated apoptosis, thereby acting against I/R injury [15]. Our literature review has found that ginsenoside CK can target mitochondrial ROS and function to inhibit metabolic stress [19]. Importantly, our latest in vivo and in vitro studies have shown that ginsenoside Rc, a key metabolic precursor of ginsenoside CK, can inhibit brain I/R injury by increasing mitochondrial biosynthesis and ATP production, and inhibiting mitochondrial apoptosis [20]. These studies have indicated that ginsenoside CK can significantly reduce cerebral I/R injury, and its effect may be related to mitochondrial function and energy metabolism. However, the underlying protective mechanism by which ginsenoside CK regulates mitochondrial function and bioenergy production against I/R damage remains to be elucidated. Hence, the present study was designed to investigate the pharmacological mechanism of ginsenoside CK against cerebral I/R induced injury based on Mul1/Mfn2−mediated mitochondrial dynamics and bioenergy in in vitro and in vivo studies.

Oligomycin B (ab143424), antimycin A (ab141904), and antibodies against total OXPHOS (ab110413), Mfn1 (ab104274), Mfn2 (ab56889), DRP1 (ab56788), ubiquitin (Abcam, ab140601), FIS1 (ab71498), and OPA1 (ab90857) were obtained from Abcam (Cambridge, MA, USA). Antibodies to TOM20 (#42406), LC3A/B (#4108), P-DRP1 (Ser637, #4867), and β-Actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, USA). Mul1 (16133-1-AP) and Parkin (14060-1-AP) antibodies were purchased from Proteintech (Wuhan, Hubei, China). Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, C2920), rotenone (R8875), and Mdivi-1 (M0199) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ginsenoside CK was purchased from Yuanye Bio-Technology (HPLC ≥ 98%, Shanghai, China).

Predesigned siRNAs for rat Mul1 (siG150514163245-1-5) and Mfn2 (siG141230171112-1-5) and negative control siRNA (siB160812022401-1-5) were provided by RiboBio (Guangzhou, China) and transfected into PC12 cells using Lipofectamine RNAiMAX (Invitrogen). Briefly, the diluted siRNA and transfection reagent in Opti-MEM (Invitrogen) were incubated for 5 min at room temperature to allow for the formation of siRNA-lipid complexes. After washing twice by Opti-MEM, the cells were incubated with the siRNA–lipid complexes for 48 h. We examined the efficiency of siRNA silencing by western blot and then subjected the cells to various treatments as planned [21].

Proteins from PC12 cells and brain tissues were extracted in NP-40 lysis buffer (Beyotime) containing a complete protease inhibitor cocktail (Sigma-Aldrich) [22]. The protein extracts were subjected to centrifugation at 12,000g for 12 min. After the specified antibodies were bound to protein lysate at 4°C overnight, the antigen–antibody complexes were immunoprecipitated with the A/G magnetic beads at room temperature for 1 h. After separating the beads with a magnetic rack, the pellets were eluted with sample buffer and boiled at 100 °C for 5 min. The magnetic beads were separated, and the supernatant was loaded onto an acrylamide gel.

To investigate the ATP production from OXPHOS and glycolysis, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using the Seahorse XFe24 analyzer (Seahorse Bioscience, Billerica, MA, USA) as reported previously [20]. Briefly, the cells were plated in a Seahorse XFe24 cell culture plate before pretreatment with ginsenoside CK and/or OGD/R incubation. The medium was then exchanged with Seahorse XF DMEM medium (Seahorse Bioscience) containing 2 mM glutaMAX (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 10 mM glucose, which were equilibrated for 30 min at 37°C before the experiment. Cellular ATP, OCR, or ECAR was monitored in basal conditions (before any addition) and after the addition of glucose (Glu), oligomycin (Olig), 2-D-glucose (2-DG), FCCP or rotenone (Rot) & antimycin A (AA) with three cycles of mixing (150 s), waiting (120 s) and measuring (210 s). The ATP, OCR, or ECAR values were normalized to cell numbers in the corresponding wells and expressed as pmol/min/104 cells or mpH/min/104 cells.

After pretreatment with ginsenoside CK and/or I/R injury, the brains were cut into 2-mm sections and fixed in ice-cold 2.5% glutaraldehyde solution for 15 min. Then, the tissue sections were postfixed in 0.7% potassium ferrocyanide, stained with 2.5% uranyl acetate in 0.1 M maleate, and embedded in Eponate (TedPella, CA, USA). The tissue sections were polymerized overnight and immersed in liquid nitrogen. Thin sections with 60–80 nm were cut with a diamond knife on a Leica EM UC7 ultramicrotome with ultra 45° (Daitome) and collected onto copper grids, which were stained with 4% uranyl acetate in 50% methanol and 5% citrate [23]. Images were captured with a transmission electron microscope (TECNAI G2 20 TWIN, FEI, Hillsboro, OR, USA).

All the in vivo and in vitro experimental groups were designed to establish equal size, blinding, and randomization [24]. All group sizes represent the numbers of experimental independent values; these independent values were used for statistical analyses, and statistical analyses were undertaken only for experiments where each group size (n) was ≥ 3. Values are expressed as mean ± SD in in vitro study and mean ± SEM in in vivo study. The normality test (D'Agostino–Pearson) and equal variance test (Brown–Forsythe) were applied. The two-tailed Student's t test was used for comparison between the two groups. Multigroup comparisons were performed using the one-way ANOVA followed by Tukey's post-hoc test. The post hoc tests were conducted only if the F value in ANOVA achieved the necessary statistical significance level and there was no significant variance inhomogeneity. The analyses were performed in GraphPad Prism 8.0. P < 0.05 was considered statistically significant [25].

Details of materials preparation and full experimental procedures, including cell culture and oxygen−glucose deprived/reperfusion (OGD/R) model, Animals and middle cerebral artery occlusion model, quantification of mitochondrial DNA content, mitochondria isolation, mitochondrial and mitophagy morphology, Western blot, immunofluorescence staining, ATP production assay, measurement of mitochondrial complex I–V activity, measurement of the mitochondrial membrane potential and reactive oxygen species, TTC staining, neurological deficit score, H&E staining, Nissl staining, and immunohistochemistry analysis are given in the Supplementary Information.

We analyzed whether ginsenoside CK regulates Mfn2 to restore mitochondrial function. First, the dose-dependent effect of ginsenoside CK on ATP content was evaluated by a luminescence assay. Multigroup studies comparisons were performed using the One-way ANOVA followed by a Tukey's post hoc test. P < 0.05 was considered statistically significant. Our results showed that ginsenoside CK at the concertation of 2, 4, and 8 μM significantly restored the decrease in ATP synthesis caused by OGD/R(Fig. S1A). We also found that ginsenoside CK increased the ATP content mainly from mitochondria, and had no obvious effect on ATP production from glycolysis (Fig. 1A). To further verify the origin of increased ATP mediated by ginsenoside CK, OCR was monitored in real-time to measure the key parameters of mitochondrial function by adding different modulators of respiration. As expected, we found that ginsenoside CK caused an enhancement in respiratory capacity, but had no significant effect on ECAR in the OGD/R-induced injury (Fig. 1B and C). To evaluate the effect of ginsenoside CK on the electron transport chain (ETC), we examined the expression levels and activities of the mitochondrial complex I–V, and the copy number of mitochondrial DNA. As shown in Fig. 1D-F and Fig. S1B, ginsenoside CK had no obvious effect on the expression of complex I–V and the ratio of mitochondrial DNA/nuclear DNA compared with the OGD/R group, but it had a significant promotion effect on the activities of mitochondrial complex I (NADH-CoQ reductase) and III (CoQ-Cytochrome C reductase). Importantly, we found that the effect of ginsenoside CK pretreatment in promoting mitochondrial oxygen consumption in OGD/R-injured PC12 cells was completely abolished by Mfn2 knockdown (Fig. 1G and Fig. S1C), compared with the si-Ctrl group. These results suggest that ginsenoside CK rescues OGD/R-induced mitochondrial dysfunction, which might be dependent on Mfn2 signaling.

We further evaluated the effect of ginsenoside CK on mitochondrial dynamics and mitophagy in OGD/R injury and whether it is Mfn2-dependent. As shown in Fig. S2A–Fig. S2B, ginsenoside CK incubation decreased mitochondrial fragmentation in a dose-dependent manner, compared with the OGD/R group. Consistent with the expectations, knockdown of Mfn2 blocked the inhibitory effect of ginsenoside CK on mitochondrial fission in OGD/R-injured PC12 cells (Fig. 2A). The p62 protein interacts with LC3II, which is degraded in the autolysosomes. Immunoblot analysis showed that ginsenoside CK pretreatment increased the level of p62 and decreased the ratio of LC3II/LC3I in OGD/R injury (Fig. 2B). In addition, we observed that the autolysosomes (mCherry-positive/GFP-positive; yellow dots) decreased significantly after ginsenoside CK administration prior to OGD/R incubation (Fig. 2C and Fig. S2C), confirming the effect of ginsenoside CK in reducing autophagy. To determine whether Mfn2-regulated mitophagy contributes to neuroprotection of ginsenoside CK against OGD/R injury, Parkin translocation to mitochondria, and co-localization of mitochondria and lysosomes were evaluated in si-Ctrl or si-Mfn2 transfected cells. The translocation of Parkin to mitochondria significantly induced by OGD/R was reversed by ginsenoside CK, while Mfn2 knockdown markedly abrogated the mitigation effect of ginsenoside CK in terms of mitophagy (Fig. 2D and Fig. S2D). Similarly, Mfn2 knockdown counteracted the effect of ginsenoside CK in reducing the co-localization of mitochondria and lysosomes (Fig. 2E and Fig. S2E). These results indicate that the effects of ginsenoside CK on mitochondrial dynamics and mitophagy are mediated by the Mfn2 signaling in the OGD/R-induced PC12 cell model.

To further clarify the regulatory effect of ginsenoside CK on Mul1 and mitochondrial dynamics–related signaling pathways during OGD/R injury, we separated cytoplasmic and mitochondrial proteins of PC12 cells untreated or treated with ginsenoside CK for western blot analysis. OGD/R incubation caused the decreased DRP1 phosphorylation at S637 and the slightly increased DRP1 in the cytoplasmic and mitochondrial proteins (Fig. 3A and Fig. S3A). Meanwhile, we observed that OGD/R incubation also resulted in the significant decreases in mitochondrial fusion–related proteins and the obvious increases in Mul1 and mitochondrial fission–related proteins (Fig. 3A and Fig. S3A). Importantly, our results showed that ginsenoside CK pretreatment prior to OGD/R incubation significantly reduced the expression of Mul1 and the translocation of DRP1 to mitochondria, and increased Mfn2 expression in mitochondria (Fig. 3A and Fig. S3A). Since Mul1 mainly regulates mitochondrial dynamics through ubiquitination of Mfn2, we then conducted a co-IP experiment to verify the regulatory effect of ginsenoside CK on the Mul1/Mfn2 signaling pathway. As expected, when Mul1 was immunoprecipitated, the protein binding of Mfn2 with Mul1 was significantly reduced after ginsenoside CK treatment, which means that ginsenoside CK reduced the degradation of Mfn2 by Mul1 after OGD/R incubation (Fig. 3B). In contrast, after Mfn2 was immunoprecipitated, OGD/R-induced Mul1 expression and the level of ubiquitination were obviously reduced by ginsenoside CK, which suggests that ginsenoside CK pretreatment did reduce Mul1-mediated Mfn2 ubiquitination and degradation during the OGD/R injury (Fig. 3B). To identify whether the contribution of ginsenoside CK firstly regulates the binding of Mul1 and Mfn2, and then influences mitochondrial fission, mitophagy, and mitochondrial apoptosis, Mdivi-1, a widely used inhibitor of mitochondrial fission, was introduced to investigate the mechanism of ginsenoside CK in OGD/R-induced injury. In combination with Mdivi-1 treatment at 10 μM for 24 h, the effect of ginsenoside CK on reducing Mul1 expression and increasing Mfn2 expression was preserved, which means that mitochondrial fission occurred after the Mul1/Mfn2 signaling pathway (Fig. 3C). In addition, Mfn2 knockdown reversed ginsenoside CK–related inhibition of the production of mitochondrial ROS to prevent OGD/R injury (mito-ROS, Fig. 3D and Fig. S3B). These results suggest that ginsenoside CK mainly regulates Mul1 to reduce the ubiquitination and degradation of Mfn2, resulting in increased Mfn2 expression to inhibit mitochondrial fission, mitophagy, and mitochondrial apoptosis in OGD/R neuronal injury.

To investigate the therapeutic effect of ginsenoside CK on cerebral I/R injury, we evaluated the infarct volume, neurological deficit score, and brain water content in the rat model. TTC staining showed that the infarct volume of the ginsenoside CK pretreatment group was significantly smaller than that of the I/R group (Fig. 4A and B). After ginsenoside CK pretreatment, the neurological function score decreased significantly after 24 h, compared with the I/R group (Fig. 4C). Moreover, ginsenoside CK pretreatment relieved cerebral edema caused by I/R injury (Fig. 4D). In addition, H&E and Nissl staining showed that the neurons in the cortex, CA1, CA3, striatum, and gyrus in rats with cerebral I/R injury were arranged loosely, and exhibited cell body shrinkage, partial nuclear fragmentation, nuclear pyknosis, nucleolar blurring, and even degeneration (Fig. 4E and Fig. S4A-S4B). Compared with the I/R injury group, ginsenoside CK administration significantly restored the damage to neurons in the cerebral cortex and CA1 regions (Fig. 4E). Detection of a neuron marker MAP2 by immunofluorescence staining also verified the neuroprotective effect of ginsenoside CK pretreatment (Fig. 4F and G). To further confirm the inhibitory effect of ginsenoside CK on I/R-induced mitochondrial dysfunction, we extracted mitochondria from fresh brain tissues to determine oxygen consumption and mitochondrial complex enzyme activity. As shown in Fig. 4H and I, ginsenoside CK pretreatment significantly restored the reductions in maximal respiration capacity (no effect on basal respiration) and the activities of complex I and III induced by I/R. Similar to in vitro experiments, ginsenoside CK had no effect on the protein expression of the mitochondrial complex in the I/R-injured rat model (Fig. S4C–Fig. S4D). Taken together, these results verified the effect of ginsenoside CK on the inhibition of neuronal damage and bioenergy imbalance in the in vivo I/R model.

We further verified the regulatory role of ginsenoside CK in mitochondrial dynamics and mitophagy in the rat I/R model. First, we observed the ultrastructure of mitochondria through TEM and analyzed the mitochondrial aspect ratio, the number of mitochondria and autophagosomes. As shown in Fig. 5A, ginsenoside CK pretreatment significantly reduced mitochondrial division and autophagy, and slightly increased the mitochondrial account, compared with the I/R model group. Moreover, we observed the expressions of LC3, Parkin, and TOM20 in the different areas of brain tissue using a laser confocal microscope, and performed the co-localization analysis. In the CA1 region, the increase in LC3 expression after I/R incubation was significantly offset by ginsenoside CK pretreatment, while this phenomenon was not observed in the CA3 region (Fig. 5B and Fig. S5A–Fig. S5C). Interestingly, we found that ginsenoside CK pretreatment significantly inhibited the increase of Parkin–TOM20 co-localization induced by I/R in all regions of rat brain tissues, including cortex, gyrus, CA1, and CA3 regions (Fig. 5C and D). Taken together, these in vivo results demonstrate that ginsenoside CK administration significantly reduces I/R-induced mitochondrial fission and mitophagy in brain tissue.

By immunoblot analyses, we found that ginsenoside CK pretreatment significantly abolished I/R-induced increased Mul1 expression, DRP1 translocation, and reduced Mfn2 expression, especially in mitochondria, which was similar with the results of our in vitro experiments (Fig. 6A and Fig. S6A). Confocal imaging further revealed that the reduction in Mfn2 expression induced by I/R was inhibited by ginsenoside CK pretreatment in the cortex and CA1 regions (Fig. 6B and Fig. S6B–S6C). Importantly, ginsenoside CK pretreatment augmented Mfn2 expression, accompanied by a significant reduction in ubiquitinated protein expression after I/R processing (Fig. 6C and Fig. S6D). To verify whether ginsenoside CK pretreatment directly reduces the effect of Mul1-mediated ubiquitination of Mfn2, we analyzed the interaction of Mul1 and Mfn2 by a co-IP experiment. Indeed, after Mul1 was immunoprecipitated, ginsenoside CK significantly blunted the binding of Mul1 to Mfn2 in cortex tissues during I/R (Fig. 6D). Similarly, the binding of Mfn2 to Mul1 was also inhibited by ginsenoside CK pretreatment, when Mfn2 was immunoprecipitated. Importantly, the ubiquitination of Mfn2 was significantly inhibited in the CK group, compared with the I/R group (Fig. 6D). Collectively, these data confirm that ginsenoside CK pretreatment reduces the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2 and attenuating mitochondrial translocation of DRP1 against I/R injury.

As postmitotic and high-energy-demanding cells, neurons are inevitably more prone to mitochondrial pathology [26]. Mitochondria undertake fusion and fission processes all the time for maintaining the balance of bioenergetic efficiency and energy expenditure, which is involved in the pathogenesis of cerebral I/R injury [27,28]. Mitochondrial fusion is a multistep and conserved process that begins with the mitofusins proteins-mediated juxtaposition and tethering of adjacent mitochondria, followed by conformational changes of mitofusins oligomers driven by GTP hydrolysis [29]. Mechanically, tethering and fusion for the outer mitochondrial membrane are mainly regulated by Mfn1, Mfn2, and homologous proteins that contain a GTPase domain (DRP1) [30]. Indeed, we observed that the phenotype of I/R-induced changes in mitochondrial morphology was accompanied by a significant reduction in the expression of Mfn1, Mfn2, and OPA1, and an elevation in mitochondrial Mul1, DRP1 and FIS1 expression. Importantly, we only observed significant changes in Mul1 and Mfn2 levels and DRP1 translocation upon manipulation of ginsenoside CK administration, which led us to consider that the Mul1-mediated Mfn2 and DRP1 expression could drive the effect of ginsenoside CK against I/R injury. Multiple studies have reported that Mul1 retards mitochondrial fusion through the ubiquitination and degradation of Mfn2 in the C-terminal RING finger domain, which is involved in the imbalance of mitochondrial dynamics, mitophagy, and bioenergy during metabolism stress [13]. Using a co-IP experiment, we found that ginsenoside CK pretreatment significantly inhibited the binding of Mul1 to Mfn2 to mediate Mfn2 degradation, thereby promoting the expansion and fusion of healthy mitochondria and further preventing mitochondrial dysfunction in response to I/R and OGD/R damage. Our findings provided new insights into the potential targets during I/R injury and the new molecular mechanism of ginsenoside CK, which are associated with mitochondrial dynamics and bioenergy. Mitochondrial fragmentation–induced mitophagy and apoptosis are almost always observed during cerebral I/R injury, and DRP1 has therefore been mechanistically implicated in programmed cell apoptosis [31,32]. Emerging evidence suggests that DRP1 stimulates Bax oligomerization and Cyto C release by promoting mitochondrial fission and mitophagy, which are widely recognized as a culprit of neuronal injury, even programmed cell death [33,34]. However, in some studies, genetic or chemical overexpression of DRP1, accompanied by strengthened mitophagy and decreased Cyto C release, has shown a critical neuroprotective response against neuronal I/R injury [35]. Thus, there is an ongoing debate about the pros and cons of inhibiting fragmentation-induced mitophagy during cerebral I/R injury [33]. In this respect, we observed that I/R- or OGD/R incubation caused the changes in mitochondrial morphology, bioenergy imbalance, and neuronal injury, which are all accompanied by the translocation of DRP1 to mitochondria. Importantly, these phenotypes above were significantly reversed by ginsenoside CK pretreatment in the cell and animal models. In addition, the targeted inhibition of DRP1 by Mdivi-1 completely abolished the restraining effect of ginsenoside CK on I/R-induced mitophagy and mito-ROS burst but did not abrogate the interaction of Mul1 with Mfn2, which indicated that ginsenoside CK pretreatment inhibited the Mul1-regulated ubiquitination of Mfn2 to abolish DRP1-mediated fission, mitophagy, and apoptosis. Ginsenoside CK is a secondary ginsenoside biotransformed from major ginsenosides, such as Rb1, Rb2, or Rc. Previous reports have shown that ginsenoside CK significantly inhibits I/R-induced rat neurological damage and OGD/R-induced neuronal autophagy and apoptosis [15,18]. Furthermore, ginsenoside CK administration can regulate multiple signaling pathways related to energy metabolisms, such as PI3K and AMPK in OGD/R-induced injury models [15,36]. Ginsenoside Rc, a metabolic precursor of ginsenoside CK, can significantly restore the imbalance of neuronal energy metabolism and mitochondrial dysfunction induced by OGD/R or I/R injury [20]. Inspired by this interaction, we speculated that ginsenoside CK could regulate mitochondrial dynamics and bioenergy, thereby protecting neurons from cerebral I/R injury. Therefore, when we studied the mechanism of I/R damage based on mitochondrial dynamics and bioenergetics, we carried out the screening for different ginsenoside monomers. We expectedly found a typical dose-dependent effect of ginsenoside CK on mitochondrial aspect ratio, ATP level, and OCR in the OGD/R-induced neuronal injury model. More importantly, ginsenoside CK pretreatment inhibited the expression of Mul1 to regulate Mfn2 ubiquitination, mitochondrial dynamics and bioenergy, which contributed to the maintenance of mitochondrial integrity and neuroprotective properties in in vivo I/R and in vitro OGD/R models. These results provide a new mechanism for the beneficial effects of ginsenoside CK as natural preventive agent against cerebral I/R injury. Of note, this study still had some limitations. First, mito-ROS was produced from complex I and complex III in cerebral I/R injury. In this study, total mito-ROS production was determined using MitoSOX™ dye, which was not able to distinguish the ROS from complex I or complex III. Particularly, accumulating evidence has suggested that ROS produced by complex I causes the neuronal deleterious effects, whereas ROS produced by complex III had the neuroprotective effects in mice with cerebral I/R injury [37]. Future studies are needed to determine how ginsenoside CK regulates ROS production by complex I or complex III to explain the potential function of ginsenoside CK in the reduction of mito-ROS production and mitochondrial depolarization. Second, previous studies have shown that patients with ischemic stroke or transient ischemic attack were characterized by significantly increased concentration of platelets and circulating platelet-leukocyte aggregate in blood [38,39]. Noteworthily, Jiahong Han. et al demonstrated ginsenoside CK (5mg/kg and 10 mg/kg) treatment significantly increased the levels of platelet and red blood cells, while inhibited the percentage of leukocyte in mice induced by cyclophosphamide [40]. An increase in platelets may increase the risk of stroke, so further studies are needed to clarify the effect of different doses and times of ginsenoside CK on blood platelets. Finally, more robust data should be provided to substantiate these findings for a protective effect of ginsenoside CK against cerebral injury using Mul1 loss-of-function animal models.

In conclusion, ginsenoside CK pretreatment could attenuate mitochondrial translocation of DRP1, mitophagy, mitochondrial apoptosis, and neuronal bioenergy imbalance against cerebral I/R injury in both in vitro and in vivo models. Our data also confirmed that ginsenoside CK administration could reduce the binding affinity of Mul1 and Mfn2 to inhibit the ubiquitination and degradation of Mfn2, thereby elevating the protein level of Mfn2. Our findings provided new insights into the potential targets during I/R injury and the new molecular mechanism of ginsenoside CK, which are associated with mitochondrial dynamics and bioenergy. These data provide evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury via Mul1/Mfn2−mediated mitochondrial dynamics and bioenergy.

The authors have no conflicts of interest to declare.