Gynura procumbens Root Extract Ameliorates Ischemia-Induced Neuronal Damage in the Hippocampal CA1 Region by Reducing Neuroinflammation

Gynura procumbens has been used in Southeast Asia for the treatment of hypertension, hyperglycemia, and skin problems induced by ultraviolet irradiation. Although considerable studies have reported the biological properties of Gynura procumbens root extract (GPE-R), there are no studies on the effects of GPE-R in brain damages, for example following brain ischemia. In the present study, we screened the neuroprotective effects of GPE-R against ischemic damage and neuroinflammation in the hippocampus based on behavioral, morphological, and biological approaches. Gerbils received oral administration of GPE-R (30 and 300 mg/kg) every day for three weeks and 2 h after the last administration, ischemic surgery was done by occlusion of both common carotid arteries for 5 min. Administration of 300 mg/kg GPE-R significantly reduced ischemia-induced locomotor hyperactivity 1 day after ischemia. Significantly more NeuN-positive neurons were observed in the hippocampal CA1 regions of 300 mg/kg GPE-R-treated animals compared to those in the vehicle-treated group 4 days after ischemia. Administration of GPE-R significantly reduced levels of pro-inflammatory cytokines such as interleukin-1β, -6, and tumor necrosis factor-α 6 h after ischemia/reperfusion. In addition, activated microglia were significantly decreased in the 300 mg/kg GPE-R-treated group four days after ischemia/reperfusion compared to the vehicle-treated group. These results suggest that GPE-R may be one of the possible agents to protect neurons from ischemic damage by reducing inflammatory responses.


Introduction
Transient interruption of blood supply to the brain, referred to as transient forebrain ischemia, causes neuronal loss in the striatum, somatosensory cortex, and hippocampus [1,2]. Ischemic damage manifests as different phenotypes depending on the time post-injury and the affected regions. Mongolian gerbil (Meriones unguiculatus) is one of the most useful animals to induce transient forebrain ischemia because of its incomplete connections between basilar and internal carotid arteries at the base of the brain [3,4]. In this animal, damage to the hippocampus causes locomotor hyperactivity one day after ischemia/reperfusion

Experimental Animals
Sixty male gerbils (6 ± 0.5 months of age; 67 ± 4.3 g body weight) were used in the study. Animals were purchased from Japan SLC Inc. (Shizuoka, Japan) and mated each other. Only male gerbils were selected because they show larger infarct size, higher inflammatory responses, and increased activation of microglia after the ischemic insult [32]. Animals were housed in cages (four gerbils per cage) in a conventional area under standard conditions as described previously [33,34]. The experimental procedure was conducted according to international standards [35] and the protocol was approved by ethical committee (SNU-170807-11).

Preparation of GPE-R
For GPE-R, roots of Gynura procumbens (100 g) were purchased from a local oriental herb market (Kyung Dong Si Jang, Seoul, Korea). Roots of Gynura procumbens were ground, dried, and macerated using 80% ethanol (3 times) for 72 h at 20 • C, and washed with distilled water at 20 • C for 2 h. The extract was filtered using Whatman No.1 filter paper (Whatman, Maidstone, UK) and the insoluble materials were eliminated by centrifugation at 10,000× g for 30 min at 20 • C. The residual solvent was removed using rotary evaporator at 40 • C and the extract was freeze-dried. The final yield of was 9.49%.

Administration of GPE-R
The gerbils were divided into four groups: control group, vehicle-treated ischemic group, 30 mg/kg GPE-R-treated ischemic group, and 300 mg/kg GPE-R-treated ischemic group. Gerbils received oral administration of distilled water (vehicle-treated ischemic group) or GPE-R (30 mg/kg and 300 mg/kg GPE-R-treated ischemic groups) with a feeding needle once a day for 3 weeks before the surgery. Doses of GPE-R were chosen based on previous findings which showed that administration of 150-450 mg/kg of GP aqueous extract improved fertility [36] and administration of 25-50 mg/kg of GP stem extract (GPE-S) ameliorated ethanol-induced steatosis [37].

Ischemic Surgery
Ischemic surgery was conducted as described previously [32,33]. The animals were anesthetized with a mixture of 2.5% isoflurane (Hana Pharmaceutical Co., Ltd., Seoul, Korea) [38,39] in 33% oxygen and 67% nitrous oxide 1 h after the last vehicle or GPE-R treatment. The ventral neck region was shaved and a midline incision was made. Common carotid arteries were freed from the vagosympathetic trunk and isolated with Chromic Catgut #4. Occlusion of both arteries was done by non-traumatic aneurysm clips for 5 min and reperfusion was confirmed to observe the current flow of retinal central artery using an ophthalmoscope (HEINE K180 ® ; Heine Optotechnik, Herrsching, Germany). Bilateral common carotid occlusion was not performed in the control group (sham-operated). Animals after ischemic surgery were kept on the thermal incubator to maintain the body temperature without treatment with analgesics until they were euthanized because opioid agonists, anti-inflammatory drugs, and dexmedetomidine show neuroprotection against ischemic damage [40][41][42][43]. In addition, acetaminophen protects the brain tissue after middle cerebral artery occlusion, and thus should not be used to induce analgesia in rodents subjected to experimental stroke [43]. After ischemic surgery, animals were carefully monitored at least 4 times a day to identify animal welfare before reaching humane endpoint as described by Percie du Sert et al. [44]. Animals were sacrificed when the animals showed reduction of body weight (>20%), complete anorexia for 24 h, partial anorexia (<50% food intake) for 3 days, and inability and severe reluctance to stand for 24 h. In addition, animals were sacrificed when the animals show severe depression, lack of movement, and hypothermia.

Locomotor Activity
To observe the effects of GPE-R on locomotor hyperactivity caused by striatum damage, locomotor activity was traced for 60 min before and 1 day after ischemia, as described previously [33]. The apparatus was made of Plexiglas (25 × 20 × 12 cm) and locomotor activity was recorded with Photobeam Activity System-Home Cage (San Diego Instruments, San Diego, CA, USA).
The number of NeuN-positive nuclei in the dorsal hippocampus was measured in the hippocampal CA1 region using an image analysis system (software: Optimas 6.5, Cyber-Metrics, Scottsdale, AZ, USA). Analysis of the Iba-1 immunoreactivity in the hippocampal CA1 region was assessed by the optical density in 256-pixel gray levels. The hippocampal CA1 region was divided into 3 subregions such as stratum pyramidale, oriens, and radiatum. The optical density was measured in each subregion using ImageJ v. 1.80 software (National Institutes of Health) and results were transformed to a relative optical density (ROD) = log 10 (256/mean gray level) as described in previous studies [33,34].

ELISA for Pro-Inflammatory Cytokines
To measure changes in TNF-α, IL-1β, and IL-6 levels in the hippocampus, separate animals from the control, vehicle-treated ischemic, 30 mg/kg GPE-R-treated ischemic, and 300 mg/kg GPE-R-treated ischemic groups (n = 7 per group) were sacrificed with deep anesthesia with alfaxalone (100 mg/kg) and xylazine (15 mg/kg) and subsequent decapitation, and the extracted brains were used for enzyme-linked immunosorbent assay (ELISA) as described previously by our colleagues [47] with commercial kits (EMD Millipore, Billerica, MA, USA).

Statistical Analysis
Data were analyzed to check for significant differences between groups by one-way analyses of variance, followed by Bonferroni's post-hoc tests using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA). Level of significance was set at p < 0.05.

Body Weights
Gerbils showed similar body weight (65.93-67.04 g) before ischemia and body weight was slightly increased (68.19 g) in the control group, but ischemia-operated gerbils showed slightly decreases in body weight (62.48-64.95 g). However, there were no significant differences in body weights before and 4 days after ischemia in all groups ( Figure 1). with 3,3′-diaminobenzidine tetrachloride (Sigma). Primary antibodies were used as follows: mouse anti-neuronal nuclei (NeuN) antibody (1:1000; EMD Millipore, Temecula, CA, USA) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1; 1:500; Wako, Osaka, Japan). The number of NeuN-positive nuclei in the dorsal hippocampus was measured in the hippocampal CA1 region using an image analysis system (software: Optimas 6.5, CyberMetrics, Scottsdale, AZ, USA). Analysis of the Iba-1 immunoreactivity in the hippocampal CA1 region was assessed by the optical density in 256-pixel gray levels. The hippocampal CA1 region was divided into 3 subregions such as stratum pyramidale, oriens, and radiatum. The optical density was measured in each subregion using ImageJ v. 1.80 software (National Institutes of Health) and results were transformed to a relative optical density (ROD) = log10(256/mean gray level) as described in previous studies [33,34].

ELISA for Pro-Inflammatory Cytokines
To measure changes in TNF-α, IL-1β, and IL-6 levels in the hippocampus, separate animals from the control, vehicle-treated ischemic, 30 mg/kg GPE-R-treated ischemic, and 300 mg/kg GPE-R-treated ischemic groups (n = 7 per group) were sacrificed with deep anesthesia with alfaxalone (100 mg/kg) and xylazine (15 mg/kg) and subsequent decapitation, and the extracted brains were used for enzyme-linked immunosorbent assay (ELISA) as described previously by our colleagues [47] with commercial kits (EMD Millipore, Billerica, MA, USA).

Statistical Analysis
Data were analyzed to check for significant differences between groups by one-way analyses of variance, followed by Bonferroni's post-hoc tests using GraphPad Prism 5.01 software (GraphPad Software, Inc., La Jolla, CA, USA). Level of significance was set at p < 0.05.

Body Weights
Gerbils showed similar body weight (65.93-67.04 g) before ischemia and body weight was slightly increased (68.19 g) in the control group, but ischemia-operated gerbils showed slightly decreases in body weight (62.48-64.95 g). However, there were no significant differences in body weights before and 4 days after ischemia in all groups ( Figure 1). There are no significant changes in body weight before and after ischemia or GPE-R treatment. Error bars represent standard errors of the means.

Effects of GPE-R on Locomotor Activity before and 1 Day after Ischemia
The distance traveled was measured to compare the locomotor hyperactivity among the groups, both before and one day after ischemia/reperfusion. In the control group, spontaneous motor activity showed no significant difference before and after sham surgery. In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, gerbils showed significantly higher locomotor hyperactivity 1 day after ischemia compared to one day before. In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, the distance traveled was 2.97-fold and 2.82-fold one day after vs. before ischemia, respectively. In the 300 mg/kg GPE-R-treated ischemic group, locomotor activity was increased one day after ischemia; however, it showed significant lower levels compared to that in the vehicle-treated ischemic group (1.94-fold one day after ischemia vs. one day before ischemia, p < 0.001) (Figure 2). groups. There are no significant changes in body weight before and after ischemia or GPE-R treatment. Error bars represent standard errors of the means.

Effects of GPE-R on Locomotor Activity before and 1 Day after Ischemia
The distance traveled was measured to compare the locomotor hyperactivity among the groups, both before and one day after ischemia/reperfusion. In the control group, spontaneous motor activity showed no significant difference before and after sham surgery. In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, gerbils showed significantly higher locomotor hyperactivity 1 day after ischemia compared to one day before. In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, the distance traveled was 2.97-fold and 2.82-fold one day after vs. before ischemia, respectively. In the 300 mg/kg GPE-R-treated ischemic group, locomotor activity was increased one day after ischemia; however, it showed significant lower levels compared to that in the vehicle-treated ischemic group (1.94-fold one day after ischemia vs. one day before ischemia, p < 0.001) (Figure 2).

Effects of GPE-R on Ischemia-Induced Neuronal Death 4 Days after Ischemia
In the control group, many NeuN-positive neurons were detected in the CA1 region four days after sham surgery ( Figure 3A). In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, only a few NeuN-positive neurons were observed in the CA1 region four days after ischemia/reperfusion and most surviving neurons were identified as non-pyramidal cells, based on morphology and location ( Figure 3B,C). In these groups, the percentage of NeuN-immunoreactive neurons was 5.56% and 5.50% of the control group, respectively ( Figure 3E). In the 300 mg/kg GPE-R-treated ischemic group, some NeuN-positive neurons were observed in the stratum pyramidale of CA1 region ( Figure 3D) and the number of NeuN-immunoreactive neurons was significantly increased to 42.47% of the control group compared to that in the vehicle-treated group (p < 0.001) ( Figure 3E).

Effects of GPE-R on Ischemia-Induced Neuronal Death 4 Days after Ischemia
In the control group, many NeuN-positive neurons were detected in the CA1 region four days after sham surgery ( Figure 3A). In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, only a few NeuN-positive neurons were observed in the CA1 region four days after ischemia/reperfusion and most surviving neurons were identified as non-pyramidal cells, based on morphology and location ( Figure 3B,C). In these groups, the percentage of NeuN-immunoreactive neurons was 5.56% and 5.50% of the control group, respectively ( Figure 3E). In the 300 mg/kg GPE-R-treated ischemic group, some NeuN-positive neurons were observed in the stratum pyramidale of CA1 region ( Figure 3D) and the number of NeuN-immunoreactive neurons was significantly increased to 42.47% of the control group compared to that in the vehicle-treated group (p < 0.001) ( Figure 3E). Nutrients 2021, 13, x FOR PEER REVIEW 6 of 12

Effects of GPE-R on Ischemia-Induced Activation of Microglia 4 Days after Ischemia
In the control group, Iba-1 immunoreactive microglia were diffusely found in the CA1 region and had a morphology of a round soma with thin processes ( Figure 4A). In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, Iba-1 immunoreactivity was showed in microglia, which had hypertrophied somas with very short or no processes (phagocytic form) in the stratum pyramidale, while in the stratum oriens and radiatum, they showed hypertrophied somas with bushy processes (reactive form) ( Figure 4B,C). In these groups, Iba-1 immunoreactivity was significantly increased in the stratum pyramidale of CA1 region to 1334.1 and 1094.5% of the control group, respectively ( Figure 4E). In the stratum radiatum of CA1 region, Iba-1 immunoreactivity was significantly increased only in the vehicle-treated group compared to that in the control group, while in the stratum oriens, there were no significant differences on Iba-1 immunoreactivity among groups ( Figure 4E). In the 300 mg/kg GPE-R-treated ischemic group, there were few Iba-1-immunoreactive microglia in the phagocytic form in the stratum pyramidale, although many reactive microglia were present in the stratum oriens and radiatum ( Figure 4D). In this group, significantly lower levels of Iba-1 immunoreactivity were

Effects of GPE-R on Ischemia-Induced Activation of Microglia 4 Days after Ischemia
In the control group, Iba-1 immunoreactive microglia were diffusely found in the CA1 region and had a morphology of a round soma with thin processes ( Figure 4A). In the vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups, Iba-1 immunoreactivity was showed in microglia, which had hypertrophied somas with very short or no processes (phagocytic form) in the stratum pyramidale, while in the stratum oriens and radiatum, they showed hypertrophied somas with bushy processes (reactive form) ( Figure 4B,C). In these groups, Iba-1 immunoreactivity was significantly increased in the stratum pyramidale of CA1 region to 1334.1 and 1094.5% of the control group, respectively ( Figure 4E). In the stratum radiatum of CA1 region, Iba-1 immunoreactivity was significantly increased only in the vehicle-treated group compared to that in the control group, while in the stratum oriens, there were no significant differences on Iba-1 immunoreactivity among groups ( Figure 4E). In the 300 mg/kg GPE-R-treated ischemic group, there were few Iba-1-immunoreactive microglia in the phagocytic form in the stratum pyramidale, although many reactive microglia were present in the stratum oriens and radiatum ( Figure 4D). In this group, significantly lower levels of Iba-1 immunoreactivity were de-tected in the stratum pyramidale of CA1 region compared to the vehicle-treated ischemic group (621.48% of the control group) ( Figure 4E).
Nutrients 2021, 13, x FOR PEER REVIEW 7 of 12 detected in the stratum pyramidale of CA1 region compared to the vehicle-treated ischemic group (621.48% of the control group) ( Figure 4E).

Effects of GPE-R on ISCHEMIA-Induced Release of Pro-Inflammatory Cytokines 6 h after Ischemia
Six hours after ischemia/reperfusion, IL-6, IL-1β, and TNF-α levels in the vehicletreated ischemic group were significantly increased in hippocampal homogenates compared to the control group. IL-6, IL-1β, and TNF-α levels in the vehicle-treated ischemic group were 3.30-, 5.17-, and 24.72-fold of those in the control group, respectively. In the 30 mg/kg GPE-R-treated ischemic group, IL-6 (p = 0.2477), IL-1β (p = 0.2009), and TNF-α (p = 0.5792) levels in the hippocampus were similar to those of the vehicle-treated ischemic group. In the 300 mg/kg GPE-R-treated ischemic group, IL-6 (p = 0.0019), IL-1β (p = 0.0165), and TNF-α (p < 0.0001) levels were significantly decreased to about half of those of the vehicle-treated group. IL-6 (p = 0.0206) and TNF-α (p = 0.0003) levels were significantly decreased in 300 mg/kg GPE-R-treated ischemic group compared to that in the 30 mg/kg

Effects of GPE-R on ISCHEMIA-Induced Release of Pro-Inflammatory Cytokines 6 h after Ischemia
Six hours after ischemia/reperfusion, IL-6, IL-1β, and TNF-α levels in the vehicletreated ischemic group were significantly increased in hippocampal homogenates compared to the control group. IL-6, IL-1β, and TNF-α levels in the vehicle-treated ischemic group were 3.30-, 5.17-, and 24.72-fold of those in the control group, respectively. In the 30 mg/kg GPE-R-treated ischemic group, IL-6 (p = 0.2477), IL-1β (p = 0.2009), and TNF-α (p = 0.5792) levels in the hippocampus were similar to those of the vehicle-treated ischemic group. In the 300 mg/kg GPE-R-treated ischemic group, IL-6 (p = 0.0019), IL-1β (p = 0.0165), and TNF-α (p < 0.0001) levels were significantly decreased to about half of those of the vehicle-treated group. IL-6 (p = 0.0206) and TNF-α (p = 0.0003) levels were significantly Nutrients 2021, 13, 181 8 of 12 decreased in 300 mg/kg GPE-R-treated ischemic group compared to that in the 30 mg/kg GPE-R-treated ischemic group, while IL-1β (p = 0.2706) levels did not show any significant changes between 30 and 300 mg/kg GPE-R-treated ischemic group ( Figure 5).

Discussion
Brain ischemia is the most severe type of stroke, and many studies have investigated its pathophysiology, molecular mechanisms, and potential therapeutic agents [48,49]. However, most studies have failed due to toxicity and side effects [17]. Medicinal plants have been studied as alternative preventive or therapeutic agents in various diseases including brain ischemia because of their minimal toxic effects [31,[50][51][52].
In the present study, we selected GPE-R as a possible candidate to prevent the neuronal damage induced by ischemia, because GPE has various functional components including chlorogenic acid, quercetin-3-0-rhamnosyl, kaempferol-3-0-glucoside, and kaempferol-3-0-rutinoside [37,53]. We assessed the neuroprotective effects of GPE-R in gerbils one day after ischemia based on their total distance traveled, which had been proved to be increased after ischemic hippocampal cell damage in previous studies [54,55]. However, the locomotor activity is decreased to basal levels seven days after ischemia [56,57]. Hence, the locomotor activity test is a useful predictable model to assess hippocampal damage early [57]. Administration of 300 mg/kg GPE-R significantly reduced the distance traveled one day after ischemia/reperfusion compared to both vehicletreated ischemic and 30 mg/kg GPE-R-treated ischemic groups. We also confirmed the neuroprotective effects of GPE-R against ischemic damage in the hippocampal CA1 region by NeuN immunohistochemistry. Most pyramidal cells showed neuronal death and surviving neurons were relatively few (about 4-10% in total). In this regard, we conducted NeuN staining to detect surviving neurons in the hippocampal CA1 region. Administration of 300 mg/kg, but not 30 mg/kg, GPE-R significantly reduced CA1 neuronal death four days after ischemia. GPE has high phenolic content and is a potential source of treatment for various diseases [19]. Furthermore, extracts from GPE-S mitigated ethanol-induced lipid accumulation in the livers of mice [37] and treatment with GPE-L significantly ameliorated ultraviolet B-induced matrix metalloproteinase-1 (MMP-1) expression in the culture medium of human dermal fibroblasts [27]. However, this study provides new insight that GPE-R has in vivo neuroprotective effects against ischemic damage in gerbils, although one poster presentation demonstrated the neuroprotective effects of GPE against glutamate-induced neurotoxicity in HT22 cells by modulation of intracellular calcium, reactive oxygen species levels, and mitochondrial membrane potential level [58].
Ischemia-induced reactive microglia produce and release neurotoxic molecules and pro-inflammatory cytokines [59,60]. In the present study, administration of 300 mg/kg

Discussion
Brain ischemia is the most severe type of stroke, and many studies have investigated its pathophysiology, molecular mechanisms, and potential therapeutic agents [48,49]. However, most studies have failed due to toxicity and side effects [17]. Medicinal plants have been studied as alternative preventive or therapeutic agents in various diseases including brain ischemia because of their minimal toxic effects [31,[50][51][52].
In the present study, we selected GPE-R as a possible candidate to prevent the neuronal damage induced by ischemia, because GPE has various functional components including chlorogenic acid, quercetin-3-0-rhamnosyl, kaempferol-3-0-glucoside, and kaempferol-3-0-rutinoside [37,53]. We assessed the neuroprotective effects of GPE-R in gerbils one day after ischemia based on their total distance traveled, which had been proved to be increased after ischemic hippocampal cell damage in previous studies [54,55]. However, the locomotor activity is decreased to basal levels seven days after ischemia [56,57]. Hence, the locomotor activity test is a useful predictable model to assess hippocampal damage early [57]. Administration of 300 mg/kg GPE-R significantly reduced the distance traveled one day after ischemia/reperfusion compared to both vehicle-treated ischemic and 30 mg/kg GPE-R-treated ischemic groups. We also confirmed the neuroprotective effects of GPE-R against ischemic damage in the hippocampal CA1 region by NeuN immunohistochemistry. Most pyramidal cells showed neuronal death and surviving neurons were relatively few (about 4-10% in total). In this regard, we conducted NeuN staining to detect surviving neurons in the hippocampal CA1 region. Administration of 300 mg/kg, but not 30 mg/kg, GPE-R significantly reduced CA1 neuronal death four days after ischemia. GPE has high phenolic content and is a potential source of treatment for various diseases [19]. Furthermore, extracts from GPE-S mitigated ethanol-induced lipid accumulation in the livers of mice [37] and treatment with GPE-L significantly ameliorated ultraviolet B-induced matrix metalloproteinase-1 (MMP-1) expression in the culture medium of human dermal fibroblasts [27]. However, this study provides new insight that GPE-R has in vivo neuroprotective effects against ischemic damage in gerbils, although one poster presentation demonstrated the neuroprotective effects of GPE against glutamate-induced neurotoxicity in HT22 cells by modulation of intracellular calcium, reactive oxygen species levels, and mitochondrial membrane potential level [58].
Ischemia-induced reactive microglia produce and release neurotoxic molecules and pro-inflammatory cytokines [59,60]. In the present study, administration of 300 mg/kg GPE-R significantly reduced the phagocytic form of microglia in the stratum pyramidale of CA1 as well as the reactive form of microglia in the stratum oriens and radiatum. In addition, we observed the significant reduction of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α in hippocampal homogenates 6 h after ischemia. We observed the levels of IL-6, IL-1β, and TNF-α in the hippocampus 6 h after ischemia because these pro-inflammatory cytokines are increased biphasically, with a prominent increase at this early period after ischemia [61,62]. In addition, nuclear factor κB (NF-κB) is a transcription factor involved in the regulation of the induction of pro-inflammatory genes such as IL-1, IL-2, IL-6, IL-8, and TNF-α, and functions in both innate and adaptive immune cells [63]. GPE has been shown to inhibit the nuclear transfer of NF-κB [64] as well as the release of pro-inflammatory cytokines such as IL-6 and IL-8 in human HaCat keratinocytes [27]. GPE also decreases inflammatory response stimulated by lipopolysaccharide (LPS) by inhibiting nitric oxide production and inducible nitric oxide synthase expression in RAW 264.7 cells [65]. Essential oils from Gynura procumbens have shown anti-inflammatory effects by inhibiting overexpression of COX-2 and reducing migration of RAW 264.7 macrophages in cell culture [66]. In addition, GPE also has vasodilatory effects via the inhibition of angiotensin converting enzyme [22], bradykinin [67], and calcium channel [68], which play an important role in neuronal death induced by ischemia in gerbils [69,70].
In summary, the pretreatment with GPE-R significantly promotes the survival of neurons in the hippocampal CA1 region after ischemia/reperfusion by reducing reactive microglia and pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α. GPE-R can be a candidate functional food to protect neurons from ischemic damage.