Notoginseng Leaf Triterpenes Ameliorates OGD/R-Induced Neuronal Injury via SIRT1/2/3-Foxo3a-MnSOD/PGC-1α Signaling Pathways Mediated by the NAMPT-NAD Pathway

Background Cerebral ischemic stroke (CIS) is a common cerebrovascular disease whose main risks include necrosis, apoptosis, and cerebral infarction. But few therapeutic advances and prominent drugs seem to be of value for ischemic stroke in the clinic yet. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stem and leaf have been confirmed to have neuroprotective effects against mitochondrial damages caused by cerebral ischemia in vivo. However, the potential mechanisms of mitochondrial protection have not been fully elaborated yet. Methods The oxygen and glucose deprivation and reperfusion (OGD/R)-induced SH-SY5Y cells were adopted to explore the neuroprotective effects and the potential mechanisms of PNGL in vitro. Cellular cytotoxicity was measured by MTT, viable mitochondrial staining, and antioxidant marker detection in vitro.Mitochondrial functions were analyzed by ATP content measurement, MMP determination, ROS, NAD, and NADH kit in vitro. And the inhibitor FK866 was adopted to verify the regulation of PNGL on the target NAMPT and its key downstream. Results In OGD/R models, treatment with PNGL strikingly alleviated ischemia injury, obviously preserved redox balance and excessive oxidative stress, inhibited mitochondrial damage, markedly alleviated energy metabolism dysfunction, improved neuronal mitochondrial functions, obviously reduced neuronal loss and apoptosis in vitro, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL markedly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions and OGD/R-induced SH-SY5Y cells and regulated the downstream SIRT1/2-Foxo3a and SIRT1/3-MnSOD/PGC-1α pathways. And FK866 further verified that the protective effects of PNGL might be mediated by the NAMPT in vitro. Conclusions The mitochondrial protective effects of PNGL are, at least partly, mediated via the NAMPT-NAD+ and its downstream SIRT1/2/3-Foxo3a-MnSOD/PGC-1α signaling pathways. PNGL, as a new drug candidate, has a pivotal role in mitochondrial homeostasis and energy metabolism therapy via NAMPT against OGD-induced SH-SY5Y cell injury.


Introduction
Cerebral ischemic stroke (CIS) is one of the leading causes of death worldwide; it has the characteristics of high morbidity, lethality, disability, and recurrence rate [1][2][3]. The related researches and reports have revealed that cerebral ischemia and reperfusion (I/R) injury (CIRI) is involved in energy metabolism disorders [4], oxidative stress [5], Ca 2+ overload, excitatory neurotransmitters, apoptosis, and necrosis [5]. Currently, tissue plasminogen activator is the effective pharmacological therapy approved by the Food and Drug Administration for acute ischemic stroke since 1996. Still, its use remains limited due to the narrow therapeutic window [2,6]. Moreover, some neuroprotective drugs have been developed for treating I/R injury, such as butylphthalide and edaravone [6,7]. But it is still difficult to meet the needs for clinical treatment of ischemic stroke. Therefore, it is imperative to develop novel therapeutic strategies for stroke.
In the early stage of ischemia, a critical reduction of regional cerebral blood flow results in the severe oxygen and glucose deprivation [11,12], insufficient NAD+ synthesis, and a decrease in the ratio of NAD + /NADH. Then, it directly impairs H+ transmission in the oxidative respiratory chain, decreases intracellular ATP synthesis, and thus causes mitochondrial damages and energy metabolism disorders within minutes after ischemia [11]. Compared to other brain cells, neurons have higher energy demand, but their energy reserves are limited [11][12][13]. Depletion of ATP often triggers the ischemic cascades such as membrane ion pump failure, efflux of cellular potassium, an influx of sodium, chloride, and membrane depolarization [11][12][13]. These mitochondrial disorders may trigger mitochondrial quality control, recover mitochondrial morphology [14,15], and further aggravate the multiple pathological progresses of cerebral I/R injury, including excitotoxicity, mitochondrial response, free radical release, acidotoxity, protein misfolding, and inflammation [3][4][5]16]. Thus, mitochondrial metabolic disorder of energy is seen as one of the hallmarks of I/R-induced neuronal death. Currently, maintenance of mitochondrial homeostasis is being pursued as a new therapeutic target for stroke treatment and provides valuable insights for clinical strategies [11,13,17].
NAMPT is the rate-limiting enzyme for biosynthesizing NAD in mammals. Currently, much evidence supports NAMPT and the NAMPT-NAD+ biosynthesis pathway as therapeutic targets against ischemic stroke, including neuroprotection, vascular repair, and neurogenesis [8,10,18]. Base on the current reports, NAMPT could increase neuronal ischemic tolerance, inhibit neuronal apoptosis necrosis, and improve mitochondrial energy metabolism under ischemia [19][20][21]. On the one hand, NAMPT strongly reduces MMP depolarization, suppresses ischemia-induced neuronal death via inhibiting the activation of mitochondrial apoptotic signaling pathways, and promotes neuronal survival through inducing autophagy via regulating the mTOR-S6K1 signaling pathway [22,23]. NAMPT is critically involved in the regulation of such forms of cell death as apoptosis [20,24] and necroptosis [23,25] via connecting to sirtuin (SIRT) signaling [18,23], which constitutes a robust endogenous defense system against various stresses. On the other hand, NAMPT plays a dual role in redox metabolism and biological signaling via the NAMPT-NAD+ biosynthesis pathway [8,10], which is tightly associated with mitochondrial functions [19,20,24], AMPK signaling activation [26][27][28], and SIRT deacetylase activity alterations under ischemia stress [18,26]. All of these reports suggest that NAMPT is a crucial target for the prevention and treatment of CIS. Therefore, it is one of the hot tasks to find natural active substances and compounds around NAMPT targets for the prevention and treatment of ischemic stroke.
Ischemic stroke is a complex pathological process with multiple mechanisms [1][2][3]. Many pharmacological agents have been investigated for years, though with limited clinical success. Thus, it is appropriate to consider using pharmacological agents to improve mitochondrial quality and maintains mitochondrial functions under ischemia conditions or to develop multitarget drugs and multidrug therapies with mitochondria protection action via the NAMPT-NAD+ biosynthesis pathway [4,12,18]. Traditional herbal medicine and natural products commonly possess various pharmacological activities, such as antioxidation, anti-inflammation, antiapoptosis, mitochondrial function improvement, and neurofunctional regulation [29,30]. Therefore, it is a promising strategy to look for neuroprotective natural agents with mitochondria protection actions mediated by NAMPT-NAD+ pathway, which has great importance for the development of novel drugs for the treatment of ischemic stroke.
Currently, notoginseng leaf triterpenes (PNGL), as total saponins of Panax notoginseng stem and leaf, are isolated and purified from stems and leaves of Panax notoginseng (Burk) F. H. Chen, which is widely confirmed as the raw materials of medicinal resource, functional foods, and common foods. Our research team used PNGL to have developed a new fifth of Chinese medicines-"Qiye Tongmai Capsules." And we monitored eleven batches of PNGL samples by the chemical fingerprinting assay [31]. Our previous study has shown that PNGL exerts potent neuroprotective effects and antiapoptotic properties via attenuation of neuronal apoptosis caused by ischemia [31]. But the neuroprotective mechanisms of PNGL are not fully elaborated. In addition, our team previously has found that Panax notoginseng saponins (PNS) and its monomeric saponin components could reduce mitochondrial damages [32][33][34]. PNGL possessed pharmacological effects are similar to that of PNS. But it is essential to further verify whether PNGL exerts mitochondriaprotective effects against cerebral I/R injury. And the relevant mechanisms are unclear that how PNGL may alleviate mitochondrial dysfunction, improve energy metabolism and thus suppress cerebral ischemic damages, which needs to further explore in vitro.

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Oxidative Medicine and Cellular Longevity Therefore, this present research was to further explore the effects and mechanisms of PNGL against ischemic injury, mitochondrial injury, and metabolic disorder of energy in the OGD/R model in vitro, and FK866 was adopted to explore the regulation actions of PNGL on the NAMPT pathway.

Methods
2.1. Cell Culture. The human neuroblastoma cell line SH-SY5Y was obtained from the Institute of Basic Medical Sciences at the Chinese Academy of Medical Sciences and maintained in Dulbecco's modified eagle's medium (DMEM, NO. 1313831, Gibco, U.S.) with 10% fetal bovine serum (FBS, 1693361, Gibco, U.S.), 100 U/mL penicillin and 100 mg/mL streptomycin in a normal incubator, as a complete medium (CM) containing 4.5 g/L D-glucose. Cells incubated at 37°C in 95% humidity and 5% CO 2 . Confluent cells subcultured by diluting 1 : 3 in a 10 cm dish, and culture medium replaced once every two days.

OGD/R Induction.
To mimic ischemic-like conditions in vitro, SH-SY5Ycells were exposed to oxygen and glucose deprivation and reperfusion (OGD/R) as we described previously [35]. Briefly, cells washed with PBS, then replaced with glucose-free DMEM (11966025, Gibco, U.S.), and incubated in an anaerobic incubator (Type III, COY Laboratory, U.S.) under the conditions of 37°C, 95% N 2 , and 5% CO 2 for different time ( 2.3. Incubation and Treatment for Cells. The PNGL samples were obtained from by Jilin Academy of Chinese Medicine (LOT.2018-05-08). And we monitored eleven batches of PNGL samples by the chemical fingerprinting assay [31]. FK866, as a highly specific NAMPT inhibitor, could inhibit the NAMPT and NAD+ biosynthesis in mammals [23,36,37]. Our present experiment has suggested the potential mechanisms in vivo. To further explore the underlying mechanisms, FK866 (SECK-S2799, Selleck Chemicals, Beijing, China) was adopted.
The cell groups were showed as follows: the control group, the control group exposed to PNGL, the OGD/R group, the PNGL-pretreated groups exposed to OGD/R, the inhibitor Fk866-treated group exposed to OGD/R, and the coincubation group with the inhibitors and PNGL exposed to OGD/R. In the PNGL group, the cells were coincubated with different concentrations (1.56~100 μg per mL) for another 24 h, then followed an insult of OGD/R. The inhibitors were added 1 h before PNGL treatment until the end of the experiment.

Cell Apoptosis and Necrosis
Assay. SH-SY5Y cell apoptosis and necrosis were detected by using an ANNEXIN V/Dead Cell.
Apoptosis kit (194785, Thermo Fisher Scientific, USA) and a Hoechst 33342/PI kit (CA1120, Solarbio, Beijing, China) according to the operation protocol [38,39]. Briefly, after incubation and treatment, SH-SY5Y cells were harvested using 0.05% trypsin, centrifuged to remove the medium, washed twice with ice-cold PBS, and resuspended in 500 μL of 1X binding buffer. The 5 × 10 4 cells (30 μL of cell suspension) were mixed and incubated with 5 μL of Annexin V-FITc reagent at 37°C for 30 min under dark conditions, and followed by propidium iodide (PI, 5 μL) staining for 5 min. Stained cells were measured via a FACSCanto flow cytometry (BD, Biosciences, USA). The percentage of cells in quadrant 2 (Q2) and 4 (Q4) were quantitatively analyzed, generating the total percentage of apoptotic cells (apoptotic index) both early and late apoptotic stage. All experiments were performed in triplicate.
Meanwhile, after incubation and treatment, cells were washed three times with FBS-free DMEM and then incubated with the 10 μg/mL Hoechst 33342 staining solution for 20 min at room temperature in the dark, followed by PI staining for 5 min. Finally, after washed two times with DMEM, the cells were examined using a fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA).

Measurement of Intracellular ROS.
To measure intracellular ROS production, the 2 ′ , 7 ′ ,-dichlorofluorescein diacetate (DCFH-DA) assay was adopted in vitro according to the manufacturer instructions (20181219, Solarbio, Beijing, China). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF. After incubation and treatment, cells were washed three times with DMEM and then incubated with a DCFH-DA staining solution (10 μmol/L) 37°C for 30 min in the dark. A fluorescence microscope (EVOS M5000, Thermo Fisher Scientific, USA) was used to examine fluorescence values.

Determination of MMP.
Mitochondrial membrane potential (MMP) was assessed as described previously [38,39] using a JC-1 MMP assay kit (C2006, Beyotime, Shanghai, China) according to the operating manual. Briefly, cells were washed twice with FBS-free DMEM and then incubated with JC-1 (200 μM) for 20 min at 37°C, followed by washing with DMEM to remove excess JC-1. MMP was calculated by the ratio of red to green fluorescence. The fluorescence images of JC-1 in the SH-SY5Y cells were acquired by a fluorescence microscopy (EVOS M5000, Thermo Fisher Scientific, USA).

Assessment of Mitochondrial Viability.
After incubation and treatments, the mitochondrial viability was evaluated as described previously [40]. The MitoTracker Red CMXRos probe solution was prepared according to the manufacturer instructions. Cells were washed twice with DMEM and then incubated with a MitoTracker Red CMXRos probe (50 nM) 3 Oxidative Medicine and Cellular Longevity for 45 min at 37°C, followed by washing with DMEM. The fluorescence images of mitochondrial viability in the SH-SY5Y cells were acquired by a fluorescence microscopy (EVOS M5000, Thermo Fisher Scientific, USA).
2.9. ELISA Assay. After incubation and treatment, SH-SY5Y cells were collected and sonicated by ultrasound homogenization in ice-cold lysis buffer. The supernatants from the ischemic brain tissues and the pretreatment cells were    2.12. Statistical Analysis. Data are presented as the mean values ± standard error of the mean (SEM). All analyses were performed by using the GraphPad Prism 8.0 statistical software (GraphPad Software, Inc., La Jolla, San Diego, CA, USA). Two-way analysis of variance (ANOVA) was used with drug and treatment as independent factors. Group differences after significant ANOVAs were measured by post hoc Bonferroni test, and P < 0:05 was considered statistically significant.

OGD/R Treatment Induces an Ischemia and Hypoxia Cell
Model In Vitro. Our previous research proved the neuroprotective effects of PNGL against cerebral I/R injury [42]. And it was preliminarily found that PNGL-induced mitochondrial protection in vivo. To further investigate the neuronal actions and mechanisms of PNGL against mitochondrial oxidative injury, an ischemia and hypoxia neuronal cell model was built to mimic the pathological changes of stroke in vitro. SH-SY5Y cells were exposed to OGD/R induction. MTT assay was employed to assess the cell viability.
The results showed that as cells were exposed to the OGD for 3-7 h periods then followed by 24 h of reperfusion, and cell viability has a time-dependent reduction, which reached 55% following 5 h of OGD (Figure 1(a), P < 0:01 versus control group); after exposure of OGD for 4 h, the cell viability was partially improved along with the reperfusion time (Figure 1(b) and 1(c), P < 0:01). However, with OGD for 6 h, the cell viability partially decreased and aggravated (Figure 1(b)-1(d), P < 0:01), indicating that the 5 h of OGD treatment may be the critical point for human neurons to resist ischemic injury [28,43]. Hence, the 4 h OGD treatment was used as the conditions of subsequent experiments.
Moreover, FK866, as a highly specific NAMPT inhibitor, was used to block the NAMPT enzymatic function and inhibit the NAD synthesis neurons, which is to verify the protective effects and mechanisms of PNGL (Figure 1(e) and 1(f)). The results revealed that treatment with PNGL did not significantly inhibit cell viability at concentrations of 1.56 to 100.0 μg/mL in SH-SY5Y cells (Figure 1(f)); FK866 (0.1~1 nm) showed no significant inhibition on neurons, but at the 10-100 nm, the cell viability obviously decreased (Figure 1(f), P < 0:01), which was in line with the reported concentration of 1.83 nM in SY5Y cells [20,42,44,45]. Therefore, SH-SY5Y cells were pretreated with FK866 at concentrations of up to 1.0 nm for 1 h, and then followed by coincubation with PNGL for 24 h in the subsequent experiments.

PNGL Improves Cell Viability and Inhibits
Apoptosis/Necrosis, Partly Reversed by FK866 In Vitro. SH-SY5Y cells were simultaneously stained with AV−FITC/PI and Hoechst33324/PI, followed by the flow cytometry and fluorescence microscope analysis. It demonstrated that OGD-induced SH-SY5Y cell viability significantly decreased compared with the control group (Figure 2(a), P < 0:01); however, at concentrations of 1.56 to 12.50 μg/mL, PNGL dose-dependently increased SH-SY5Y cell viability following OGD injury (Figure 2(a), P < 0:01), and, thus the concentration of 6.25 μg/mL was selected to conduct the investigation in vitro.
Under normal conditions, a low level of neuronal apoptosis and necrosis levels were noted (Figures 2(c) and 2(d)). After the OGD/R inductions, the percentage of apoptotic and necrosis cells increased (Figures 2(c) and 2(d), P < 0:01), accompanied by the cell viability decreases (Figure 2(b), P < 0:01). In contrast, pretreatment with PNGL suppressed the apoptosis rates (Figure 2(d), P < 0:01) and improved cell viability (Figure 2(b), P < 0:01), respectively, but these effects of PNGL against neuronal injury were partly abolished by the further FK866 incubation (Figures 2(b)-2(d), P < 0:01). Besides, there were no significant differences in cell viability and apoptosis rates between control cells and PNGL-treated cells, which indicated that these concentrations of PNGL were nontoxic under normal conditions.
Hence, these results indicate that PNGL decreases the OGD-induced ischemia injury on SH-SY5Y cells in vitro, and the neuroprotective effects may be associated with the NAMPT.

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Oxidative Medicine and Cellular Longevity

PNGL Raises MMP and Reduced ROS Levels, Partly
Reversed by FK866 In Vitro. Intracellular ROS production, stimulation, and reduction of MMP following OGD/R were thought to be important markers for the OGD/R-induced neurotoxicity and apoptosis. To evaluate the mitochondrial injury, we quantified the mitochondria membrane potential and intracellular ROS levels via JC-1 and DCFH-DA kits.
Our data showed that the OGD/R group, compared to the control group, markedly increased intracellular ROS pro-duction (Figure 3(a), P < 0:01) and decreased the MMP levels ( Figure 3(b), P < 0:01). Conversely, cotreatment with PNGL reduced intracellular ROS production (Figure 3(a), P < 0:01) and improved the MMP and the red value (Figure 3(b), P < 0:01). Treatment with PNGL alone shows no significant effect on ROS and MMP levels. Furthermore, the coincubation of FK866 partly reversed the ROS decreases and the MMP improvement (Figures 3(a)  and 3(b), P < 0:01, P < 0:01).  These results suggest that PNGL has a vital role in the inhibition of mitochondrial injury, preservation of redox balance, and improvement of neuronal survival, which may be mediated by the target NAMPT.

PNGL Upregulates the NAMPT-SIRT1/2/3 Pathway, Partly
Reversed by FK866 In Vitro. NAMPT, as a ratelimiting enzyme of NAD biosynthesis in the salvage pathway, positively regulates the activity of sirtuins and exerts protective effects in many cell types [46,47]. To further explore the relationship between the PNGL and the NAMPT-NADmediated SIRT1/2/3 pathways in vitro, we also used FK866 to investigate in vitro, which are involved in neuronal energy metabolism and mitochondrial functions following neuronal oxidative stress.

PNGL Improved Mitochondria and Energy Metabolism,
Partly Reversed by FK866 In Vitro. Mitochondria and mitochondrial metabolism play a vital role in the ischemia and hypoxia process; inhibition of mitochondrial viability leads to energy metabolism dysfunction and a cascade of mitochondria-dependent apoptosis [4,48]. Hence, we assessed the mitochondrial structure and energetic metabolism in vitro via the FK866 and Mito-Tracker Red CMXRos assay (Figure 4).
Next, we assessed the NAD+ and NADH levels mediated by the NAMPT in the OGD/R-induced SH-SY5Y cells. OGD/R induction significantly decreased intracellular NAD+ and NADH levels, and yet PNGL alleviated the energy metabolic disorder caused by OGD (Figures 4(f Taken together, all of these results suggest that PNGL effectively inhibits oxidative stress injury, alleviates energy metabolism dysfunction, improves mitochondrial function, and thus reduces the neuronal loss and apoptosis in vitro, which may be closely associated with the NAMPT and the SIRT1/2.
3.6. PNGL Improves the Downstream Foxo3a-MnSOD/PGC-1α Pathway, Partly Reversed by FK866 In Vitro. At last, we further determined the PGC-1α, MnSOD, and phosphorylated FOXO expression levels, as the critical downstreams of the NAMPT-NAD-SIRT1/2/3, which regulates transcriptional activity of the targeted antioxidant and cell cycle genes, including the UCP2, PECPK, PGC-1α, MnSOD, and catalase genes. The downstreams exert crucial homeostatic effects via inhibition of mitochondrial oxidative injury and improvement of mitochondrial energy recovery.
As revealed in Figure 5, the OGD/R treatment decreased the phosphorylated Foxo3a expression levels (Figures 5(a)-

Discussion
Ischemic stroke remains one of the leading causes of death worldwide, which is mainly caused by cerebral ischemia and reperfusion injury [6,49]. Currently, scholars have done lots of researches on how to prevent and treat cerebral ischemia and reperfusion injury [6,7]. Compared with the actual clinical application need, it is still seriously insufficient. Therefore, efficient drug treatments for ischemic stroke are urgently needed. In our previous works, PNGL exerted the neuroprotective effects against cerebral I/R injury via apoptosis suppression and mitochondrial protection [29,31]. But the neuroprotective mechanisms of PNGL are not completely elaborated. In the present paper, the protection mechanisms of PNGL against I/R injury were further investigated. The study demonstrates that PNGL significantly improves cell viability (Figures 1 and 2), decreases the ROS levels, raised MMP (Figure 3), and inhibits apoptosis and necrosis ( Figure 2). The in vitro results further suggest that PNGL is a promising agent for preventing and treating ischemic stroke. Knowingly, a critical reduction of regional cerebral blood flow or severe oxygen and glucose deprivation leads to mitochondrial dysfunction within minutes after ischemia [11,12], which could trigger mitochondrial quality control [9], including ROS scavenging, mitochondrial dynamics, and mitophagy [11,12,17]. Meanwhile, the related study demonstrates that depletion of ATP production is one of the major 9 Oxidative Medicine and Cellular Longevity initiators, which triggers ischemic cascades, such as membrane ion pump failure, efflux of cellular potassium, influx of sodium, and membrane depolarization. Maintaining the mitochondrial function is critical in promoting neuron survival and neurological improvement [11,12,17]. Our research results showed that treatment with PNGL remarkably alleviated mitochondrial structure injury caused by OGD/R (Figure 4), increased ATP and ATPase levels (Figures 4(a)-4(e)), increased the mitochondrial viability (Figures 4(a)-4(e)), indicating that the neuroprotection of PNGL might be tightly associated with inhibiting mitochondrial injury and improving metabolism of energy. NAD+, as a coenzyme, plays a vital role in energy balance and cellular redox reactions in ischemic stroke [50][51][52]. In the cytosol, NAD is translated to NADH during glycolysis, and the tricarboxylic acid cycle (TAC) enzymes reduce the NAD+ molecules [9,52]. Under the hypoxia-ischemia conditions, NADH gets oxidized in the cytoplasm via the reduction of pyruvate to lactate (Figure 6), which leads to mitochondria dysfunction and NAD decrease. And then ATP synthesis was inhibited [11,18,23]. Thus, the absence of the mitochondrial NAD+ pool causes oxidative damages and excessive ROS production [28,50,52], which aggravates mitochondria impairment, including the function of mitochondria structure, depletion of ATP production, and depolarization of MMP [12,13]. In our study, the results showed after OGD/R induction, the NAD+ and the ratio of NAD +/NADH level significantly decreased (Figure 4), the oxidative injuries increased ( Figure 3) in vitro, and MMP depolarized, which was consistent with the previous reports. While PNGL pretreated OGD/R-induced cells, it reversed these alterations of NAD+, ROS, MMP, and ATP caused by I/R. All of these indicated that PNGL might exert mitochondria protective effects via the maintenance of the mitochondrial NAD+ pool and inhibition of oxidative injury in vitro.
NAMPT is the rate-limiting enzyme in the NAD biosynthetic pathway [18,53,54]. As shown in Figure 6, intracellular NAMPT converts nicotinamide into NAD as the ratelimiting enzyme for mammalian NAD+biosynthesis [55][56][57][58]. The in vitro experiments demonstrated that the intracellular NAMPT level was induced by ischemia and OGD along with the NAD+ decrease (Figure 3(c)), which was in accordance with the related previous researches. In contrast, PNGL treatment markedly improved the intracellular level

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Oxidative Medicine and Cellular Longevity in OGD-induced cells (Figure 3(c)). All of these results indicate that PNGL may regulate the NAMPT pathway against the mitochondria dysfunction and cerebral I/R injury. FK866, a potent inhibitor of NAMPT, significantly inhibits NAD biosynthesis [36,59], which verifies that the NAMPT-mediated systemic NAD biosynthesis plays a critical role in the regulation of mitochondrial functionality [8,23]. Hence, FK866 was adopted to demonstrate whether PNGL might regulate the NAMPT-NAD+ pathway in vitro. Our results showed that FK866 incubation partly reversed the improvement of NAD induced by PNGL (Figures 4(f)-4(h)), aggravated the mitochondrial injury, impaired the mitochondrial homeostasis (Figures 4(a)-4(c)), and further blocked the metabolic energy (Figures 4(d) and 4(e)). All these indicate that PNGL exerts mitochondrial protective effects via the NAMPT-NAD+ pathway. Moreover, accompanied by the NAD pool impairment and the deteriorated dysfunction of mitochondria (Figures 4(a)-4(c)), FK866 partly inhibited the antioxidant capability (Figure 3), the MMP improvement, and the neural cell viability (Figure 2), which indicated that the mechanisms might be closely associated with the NAMPT-mediated systemic NAD biosynthesis.
In addition, SIRT1 is not the only mediator of NAMPT to maintain mitochondrial NAD+ pool [75]. Sirt3 is the primary mitochondria-targeted deacetylase, predominantly expressed in highly metabolic tissues, and binds to and deacetylate several metabolic and respiratory enzymes that regulate mROS generation and mitochondrial functions [60, 62,   [68,72,76] through upregulation of PGC-1α [62,65,75] and SOD2 [66,75], which is similar to the regulatory role of SIRT1. PGC-1α and SOD2 stimulate mitochondrial biogenesis and electron transport activity [14,62,65]. And they suppress ROS production and protect cells from mROS-induced oxidative damages. Thus, the SIRT1/3-mediated PGC-1α and SOD2 may be the critical proteins. Our data indicated that treatment with PNGL increased the PGC-1α and SOD2 levels in the OGD/R-induced cells ( Figure 5), resulting in the inhibition of mitochondrial oxidizing injury, upregulation of the mitochondria TAC enzymes, improvement of energy metabolism, and maintenance of mitochondrial homeostasis. All of these results were further confirmed by FK866.
Overall, our results ( Figure 6) suggest that PNGL can possess neuroprotective effects against cerebral I/R injury, notedly exert antioxidative and mitochondria-protective effects, improve the energy metabolism, and thus inhibit neuronal apoptosis and necrosis. The underlying mechanisms may be tightly associated with the NAMPT-NAD+ biosynthesis pathway and its downstream SIRT1/2/3-Foxo3a-MnSOD/PGC-1α signaling pathways. However, the molecular mechanisms of the mitochondrial biogenesis have not been completely elaborated. Therefore, further investigations will be required to more deeply elucidate.

Conclusions
In the present study, we explored and verified the protection effects and mechanisms of PNGL in OGD/R-induced SH-SY5Y cells. As elaborated in Figure 6, PNGL strikingly improves cell viability, significantly preserves redox balance, inhibits excessive ROS levels, alleviates mitochondrial injury, improves energy metabolism function (MMP, NAD, ATP, and ATPase levels), raises neuronal mitochondrial viability, reduces the neuronal necrosis and apoptosis, and thus notedly improves neuronal survival under ischemia and hypoxia conditions. In general, this study finds that the protective effects of PNGL are, at least partly, mediated through the NAMPT-NAD+ pathway and its key downstream SIRT1/2/3-Foxo3a-MnSOD/PGC-1α signaling pathways. PNGL, as a new drug candidate, has excellent application prospects for CIS.