Neuronal Apoptosis In Rats Research Articles

Oxidative stress mediates hippocampal neuronal apoptosis through ROS/JNK/P53 pathway in rats with PTSD triggered by high-voltage electrical burn.

The pathogenesis of post-traumatic stress disorder (PTSD) triggered by high-voltage electrical burn (HVEB) remains unclear and the oxidative stress plays a role in this process. The purpose of this study is to investigate the underlying mechanism of oxidative stress mediates hippocampal neuronal apoptosis in rats with PTSD triggered by HVEB. The PTSD rat model was developed by stimulating with high voltage electricity and screened using behavioral performance including Morris water maze (MWM), elevated plus-maze (EPM) and open-field test (OFT). The reactive oxygen species (ROS) generation was measured by DHE fluorescence staining or flow cytometry. Western blotting assay was used to detect the proteins of p-JNK, JNK, P53, PUMA, Bcl-2 and Bax in hippocampal tissue or HT22 cells treated with electrical stimulation. The serum MDA and 8-OHdG levels were increased (P < 0.001), while the activities of SOD and CAT were decreased (P < 0.001) significantly in patients with HVEB. Behavioral test results showed that high-voltage electric stimulation induced the PTSD-like symptoms and the ROS-JNK-P53 pathway was involved in the neuronal apoptosis in rats with PTSD induced by HVEB. In vitro experiments further confirmed the electrical stimulation induced neuronal apoptosis through ROS/JNK/P53 signaling pathway and the antioxidant NAC could rescued the ROS generation, activation of JNK/P53 proteins and improved the cell apoptosis rate in HT22 cells. Finally, the JNK inhibitor SP600125 could significantly inhibited the percentage of HT22 cell apoptosis induced by electrical stimulation (P < 0.001). These results indicated that oxidative stress mediates hippocampal neuronal apoptosis through ROS/JNK/P53 pathway in rats with PTSD triggered by HVEB.

Xenon postconditioning attenuates neuronal injury after spinal cord ischemia/reperfusion injury by targeting endoplasmic reticulum stress-associated apoptosis.

The purpose of the study is to explore the underlying mechanisms of xenon (Xe) which protects against spinal cord ischemia/reperfusion injury (SCIRI). A SCIRI rat model was induced by abdominal artery occlusion for 85 min and reperfusion. Xe postconditioning (50% Xe) was administered 1 h after 1 h of reperfusion. At reperfusion time points (2, 4, 6, and 24 h), rats were treated with spinal cord scans by MRI to assess the time of peak spinal cord injury after SCIRI. Subsequently, endoplasmic reticulum (ER) stress inhibitor sodium 4-phenylbutyrate (4-PBA) was administered by daily intraperitoneal injection (50 mg/kg) for 5 days before SCIRI. At 4 h after reperfusion, motor function, immunofluorescence staining, hematoxylin and eosin (HE) staining, Nissl staining, TUNEL staining, real-time reverse transcription polymerase chain (RT-PCR) reaction, and western blot analyses were performed to investigate the protective effects of Xe against SCIRI. In the rat I/R model, spinal cord edema peaked at reperfusion 4 h. SCIRI activated ER stress, which was located in neurons. Xe postconditioning remarkably alleviated hind limb motor function, reduced neuronal apoptosis rate, increased the number of normal neurons, and inhibited the expression of ER stress-related protein in spinal cord. Furthermore, the administration of the ER stress inhibitor 4-PBA strongly decreased ER stress-induced apoptosis following SCIRI. Xe postconditioning inhibits ER stress activation, which contributes to alleviate SCIRI by suppressing neuronal apoptosis.

Electroacupuncture attenuates chronic inflammatory pain and depression comorbidity by inhibiting hippocampal neuronal apoptosis via the PI3K/Akt signaling pathway

In chronic inflammatory pain (CIP) and depression, neuroapoptosis has been identified as a contributing factor. Electroacupuncture (EA) shows promise as an alternative therapy for this comorbidity. However, the underlying mechanism remains unclear. This study aimed to investigate the effects of EA on hippocampal neuronal apoptosis in rats with CIP and depression. Rats received plantar injections of complete Freund’s adjuvant (CFA) on days 0 and 14. They were then divided into groups: sham operation, model, EA, and duloxetine. EA was administered at Hegu (LI4) and Taichong (LR3) from days 15 to 28, while the duloxetine group received duloxetine and distilled water daily (0.1 mg/ml). Pain behavior was assessed using the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) tests. Depression-like behavior was evaluated through the sucrose preference test (SPT), open-field test (OFT), and forced swim test (FST). Hematoxylin and eosin (HE) staining was employed to assess pathological changes in the hippocampus. Nerve cell apoptosis was determined using TUNEL fluorescence staining. Western blot analysis was conducted to measure the protein expression of Bcl-2, Bax, p-PI3K/PI3K, and p-Akt/Akt. EA demonstrated significant pain intensity reduction and alleviation of pain-related depressive symptoms. Our findings from the HE staining confirmed that CIP induced by CFA led to morphological changes in the hippocampus, while EA effectively reversed these pathological alterations. Moreover, EA intervention remarkably reduced neuronal apoptosis and exhibited an upregulation of Bcl-2 protein expression accompanied by a decrease in Bax expression. Additionally, EA activated the PI3K/Akt signaling pathway. Overall, our study suggests that EA holds the potential to improve pain and depressive behaviors in rats with CIP and depression comorbidity, potentially mediated through the activation of the PI3K/Akt pathway, leading to a reduction in hippocampal neuronal apoptosis.

Dendrobium nobile Lindl. alkaloids (DNLA) inhibits d-galactose-induced hippocampal neuronal senescence through the SIRT1-FoxO1-autophagy axis

Dendrobium nobile is a well-known traditional Chinese herbal medicine, and DNLA (Dendrobium nobile Lindl. alkaloids) is the active ingredient to have anti-brain aging effects in laboratory animals. The aim of the present study is to examine the anti-aging effects of DNLA in d-galactose induced primary hippocampal neuron senescence. Primary hippocampal neuron cultures were pretreated with DNLA (35 and 350 ng/mL) for 24 h, then exposed to d-galactose (50 mM) for 48 h. Cell viability was evaluated by the CCK-8 assay. Flow cytometry analysis was performed to evaluate the apoptosis rate of hippocampal neurons. β-Galactosidase staining was used to detect the senescence of neurons. Western blot analysis was used to detect the protein expression. The results show that DNLA markedly recovered the reduction in cell viability, inhibited the d-gal-induced hippocampal neuron apoptosis, decreased the senescence-associated β-galactosidase activity, and attenuated the protein expression of senescence marker p53 and p16 in d-galactose induced senescent hippocampal neurons. Furthermore, DNLA enhanced autophagy activity by increasing LC3-II/LC3-I ratio, Beclin1, and by decreasing p62 in d-galactose induced senescent hippocampal neurons. Moreover, DNLA upregulated the expression of autophagy-regulator SIRT1 and FoxO1. These novel findings demonstrate that DNLA is effective in protecting against d-galactose induced senescent hippocampal neurons in vitro. The anti-aging mechanisms of DNLA appear to be mediated, at least in part, through activated Sirt1-FoxO1-autophagy axis.

Effect of ligustrazine on hypoxic-ischemic encephalopathy in neonatal rats by regulating autophagy through the PINK1/Parkin pathway

To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.

Effects of electroacupuncture pretreatment on long-term postoperative cognitive dysfunction and neuron-inflammation in aged rats

To observe the effects of electroacupuncture pretreatment on postoperative cognitive dysfunction (POCD), neuronal apoptosis and neuron-inflammation in aged rats. Thirty-six male SD rats aged 20 months were randomly divided into sham operation group, model group and electroacupuncture (EA) group, with 12 rats in each group. The POCD rats model was prepared by internal fixation of left tibial fracture. Five days before modeling, EA stimulation (2 Hz/15 Hz, 1 mA, 30 min) was applied to "Zusanli" (ST36), "Hegu" (LI4) and "Neiguan" (PC6) on the unaffected side of rats in the EA group, once a day for consecutive 5 d. The learning and memory abilities of rats were evaluated by water maze test 31-35 days after operation. The apoptosis of hippocampal neurons was observed by Tunel/NeuN double staining. The expressions of high mobility group protein B1 (HMGB1) and phosphorylated (p)-nuclear factor (NF)-κB in microglia cells in hippocampal dentate gyrus were detected by immunofluorescence staining. The expression levels of interleukin (IL)-6 and IL-1β in the hippocampus were detected by Western blot. Compared with the sham operation group, the escape latency was prolonged (P<0.05); the frequency of crossing the original platform, ratio of the swimming distance and the time in the target quadrant of the Morris water maze were significantly decreased (P<0.05); the apoptosis rate of hippocampal neurons was significantly increased (P<0.05); the expressions of HMGB1 and p-NF-κB in microglia cells in the dentate gyrus and the expression levels of IL-6 and IL-1β in hippocampus were increased (P<0.05) in the model group. Compared with the model group, the results of the above indexes were all opposite (P<0.05) in the EA group. EA preconditioning can regulate hippocampal inflammatory response, alleviate neuronal apoptosis rate and long-term cognitive dysfunction in aged rats with POCD, the mechanisms may be related to the inhibition of microglia HMGB1/NF-κB pathway in hippocampal dentate gyrus.

TIMP2 facilitates CIRI through activating NLRP3-mediated pyroptosis.

This study aimed to investigate the underlying mechanisms of cerebral ischemia-reperfusion injury (CIRI) in mice using CIR and hypoxia/reoxygenation (H/R) cell models. The study evaluated brain tissue weight, pathological injury, and changes in the expression levels of TIMP2, p-ERK1/2 and NLRP3-mediated pyroptosis-related proteins in brain tissues and hippocampal neurons of CIR mice using established methods such as dry/wet weight measurement, HE staining, qPCR, TUNEL assay, and Western blotting. The results demonstrated a significant increase in brain water content and neuronal apoptosis rate in the experimental groups compared with those in the control group. In particular, the I/R+TIMP2 group showed the highest increase. Additionally, the control group exhibited a clear brain tissue structure, neatly and densely arranged cells with normal morphology, and evenly stained and clear hippocampal tissues. However, the I/R group showed hippocampal structure disorders, interstitial edema, deep nuclear staining, karyopyknosis, and karyorrhexis in brain tissues. The study results further revealed that TIMP2 could aggravate the pathological damage of brain tissues in the I/R+TIMP2 group compared with the I/R group and significantly reduced it in the TIMP2-KD group. Furthermore, the Western blotting results demonstrated that the protein expression levels of TIMP2, p-ERK1/2, t-ERK1/2, NLRP3, IL-1β, IL-18, GSDMD, Caspase-1, and ASC in brain tissues and hippocampal neurons were significantly higher in the experimental groups than those in the control group. The I/R+TIMP2 group displaying the highest increase and the TIMP2-KD group showing a significant decrease. In conclusion, TIMP2 can contribute to the occurrence and progression of CIRI by activating NLRP3-mediated pyroptosis.

The RNA Binding Protein HuR Promotes Neuronal Apoptosis in Rats with Spinal Cord Injury via the HDAC1/RAD21 Axis

The current research aims to study the regulation of the RNA binding protein HuR on neuronal apoptosis during spinal cord injury (SCI) and its underlying mechanism. SCI rat models were injected with HuR shRNA and/or pcDNA3.1-RAD21, followed by the evaluation of motor function, the degree of SCI, the expression of HuR and RAD21, and neuronal-like apoptosis. The co-localization of HuR-RAD21, RAD21-NeuN, and NeuN-cleaved caspase 3 was measured by immunofluorescence. Additionally, targeting relationships among HuR, HDAC1, and RAD21 were verified by chromatin immunoprecipitation and RNA immunoprecipitation. After transfection, apoptosis of PC12 cells was tested by flow cytometry. Results showed that silencing HuR or up-regulating RAD21 could alleviate SCI and reduce neuronal apoptosis. HuR could combine HDAC1 mRNA, and HDAC1 combined the promoter of RAD21. Further experiments revealed that HuR enhanced HDAC1 expression and reduced RAD21 promoter region acetylation. Overexpression of RAD21 reversed the enhancement in apoptosis of PC12 cells caused by overexpression of HuR. The injection of HuR shRNA in tail vein of SCI rats increased basso, beattie, and bresnahan score, relieved SCI, reduced HuR and HDAC1 expression, elevated RAD21 expression, and decreased neuronal-like apoptosis. However, this result was reversed by co-injection of pcDNA3.1-HDAC1. In conclusion, down-regulation of HuR alleviated SCI and neuronal apoptosis in rats by suppressing HDAC1 expression and promoting RAD21 expression.

Retraction Note: microRNA-195 attenuates neuronal apoptosis in rats with ischemic stroke through inhibiting KLF5-mediated activation of the JNK signaling pathway

Retraction Note: microRNA-195 attenuates neuronal apoptosis in rats with ischemic stroke through inhibiting KLF5-mediated activation of the JNK signaling pathway

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation.

Microglia, which are the resident macrophages of the central nervous system, are an important part of the inflammatory response that occurs after cerebral ischemia. Vav guanine nucleotide exchange factor 1 (Vav1) is a guanine nucleotide exchange factor that is related to microglial activation. However, how Vav1 participates in the inflammatory response after cerebral ischemia/reperfusion injury remains unclear. In this study, we subjected rats to occlusion and reperfusion of the middle cerebral artery and subjected the BV-2 microglia cell line to oxygen-glucose deprivation/reoxygenation to mimic cerebral ischemia/reperfusion in vivo and in vitro, respectively. We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral artery and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation. Silencing Vav1 reduced the cerebral infarct volume and brain water content, inhibited neuronal loss and apoptosis in the ischemic penumbra, and improved neurological function in rats subjected to occlusion and reperfusion of the middle cerebral artery. Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3 (NLRP3) inflammasome in the ischemic penumbra, as well as the expression of inflammatory factors. In addition, Vav1 knockdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxygenation. Taken together, these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/reperfusion through inhibiting the activation of microglia and NLRP3 inflammasome.

Melatonin restores endoplasmic reticulum homeostasis to protect injured neurons in a rat model of chronic cervical cord compression.

Cervical spondylotic myelopathy (CSM) refers to a chronic injury of the cervical cord caused by cervical intervertebral disc degeneration. Endoplasmic reticulum (ER) homeostasis is essential to counteract neuronal apoptosis. ER stress, an integral part of ER homeostasis, was observed in a rat model of chronic cervical cord compression in our previous study. However, the correlation between ER homeostasis and CSM remains unknown. The antioxidant melatonin is known to exert therapeutic effects in acute spinal cord injury, but the specific effects and their potential mechanisms in the pathological processes of CSM require further exploration. The present study hypothesized that ER homeostasis is essential for neuronal apoptosis in the CSM and that melatonin maintains this homeostasis. The results showed that ER stress led to neuronal apoptosis in rats with chronic cervical cord compression. Conversely, melatonin attenuates protein kinase R-like ER kinase-eukaryotic initiation factor 2α-C/EBP-homologous protein, inositol-requiring enzyme 1, and transcription factor 6 signaling pathways to release ER stress and prevents Bax translocation to the mitochondrion, thereby promoting motor recovery and protecting neurons in vivo. It also rescued primary rat cortical neurons from ER stress-induced glutamate toxicity in vitro. Moreover, melatonin remodels the ER morphology and restores homeostasis via ER-phagy in injured neurons. FAM134B, CCPG1, RTN3, and Sec. 62 are four known ER-phagy receptors. In this study, Sec. 62 was identified as a key melatonin factor in promoting ER-phagy and restoring ER homeostasis in damaged neurons in vivo and in vitro. In conclusion, melatonin suppresses neuronal apoptosis by reducing ER stress and promoting ER-phagy to restore ER morphology and homeostasis. The current results suggested that melatonin is a promising treatment for CSM owing to its restorative effect on ER homeostasis; however, well-designed randomized controlled trials must be carried out to further investigate its clinical effects.

Lncfos/miR-212-5p/CASP7 Axis-Regulated miR-212-5p Protects the Brain Against Ischemic Damage.

miR-212-5p has been reported to be involved in many biological processes. However, the role of miR-212-5p in ischemic stroke remains unclear. This study explored the biological role and potential mechanism of miR-212-5p in ischemic stroke by investigating the lncfos/miR-212-5p/CASP7 axis. A total of 32 patients with ischemic stroke and 32 age- and sex-matched healthy controls (HCs) were enrolled in this study. In addition, 336 rats were used in this study. The rats were subjected to middle cerebral artery occlusion (MCAO) and intracerebroventricular injection of a microRNA (miRNA) agomir, a miRNA antagomir, a short hairpin RNA (shRNA) lentiviral vector, or a negative control. The neurological deficit score was calculated; the infarct volume was measured; histopathological assays were performed; the neuronal apoptosis rate was determined; and the lncfos, miR-212-5p, and CASP7 expression levels in the peri-infarct area were assessed. In this study, we found that the expression level of miR-212-5p was significantly downregulated in the peri-infarct area and blood of the MCAO model rats and the blood of patients with ischemic stroke. A double-luciferase experiment showed that CASP7 was a direct target gene of miR-212-5p and that miR-212-5p was a target miRNA of lncfos. Lateral ventricular injection of the miR-212-5p agomir effectively inhibited the apoptosis induced by ischemic brain damage, reduced the infarct volume, attenuated the neurological deficit symptoms, and downregulated the expression of CASP7 in the peri-infarct area of the MCAO model rats. Suppressing lncfos with sh-fos led to the upregulated expression of miR-212-5p and played a neuroprotective role in the rat MCAO models. We concluded that miR-212-5p plays a neuroprotective role in ischemic stroke and that its function is regulated by the lncfos/miR-212-5p/CASP7 axis. Moreover, miR-212-5p may be a potential biomarker and therapeutic target for ischemic stroke.

Retracted: Pramipexole Inhibits Neuronal Apoptosis in Rats with Parkinson's Disease.

[This retracts the article DOI: 10.1155/2022/7002630.].

Effect of electroacupuncture at Baihui ameliorated neurologic deficit and hemodynamic stability in rat model of post-cardiac arrest syndrome

To observe the results of electroacupuncture (EA) on the resuscitation of a rat model of asphyxia cardiac arrest (CA). And to explore its effect on the neurologic deficits and hemodynamic instability of post-cardiac arrest syndrome (PCAS). A total of 107 male SD rats were randomly divided into sham, CA, and EA groups. Each group received arterial catheterization and tracheal intubation. The sham group was not induced asphyxia. Asphyxial cardiac arrest was established by endotracheal tube clamping. Rats in the CA group received basic respiratory support and fluid resuscitation in return of spontaneous circulation (ROSC) and rats in the EA group received EA at Baihui based on the treatment of CA group after ROSC, with a dense-dispersed wave at frequencies of 4-20 Hz, while the current intensity was adjusted minimum to induce a twitch of the scalp, the course of treatment was 30 minutes. The baseline data, hemodynamics after ROSC, neurological deficit score (NDS), pathological changes of brain tissue, and levels of serum biomarker were recorded and compared among the three groups. The 72-hour survival of rats was analyzed by Kaplan-Meier survival curve. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of necrotic neurons in the hippocampal CA1 region of rat brain. Meanwhile, Nissl staining and TdT-mediated dUTP nick-end labeling (TUNEL) were used to detect cell apoptosis and injury. Compared with the CA group, the mean arterial pressure (MAP) in the EA group increased significantly at 15 minutes after ROSC [mmHg (1 mmHg ≈ 0.133 kPa): 125.00 (94.00, 136.25) vs. 92.00 (72.00, 122.50), P < 0.05]. There was no significant difference in the NDS score between the EA group and the sham group. Still, the NDS score of the rats in the CA group at 6 hours after ROSC were significantly lower than that in the sham group (46.00±10.61 vs. 80.00±0.00, P < 0.05). Kaplan-Meier survival curve analysis showed that EA did not improve the 72-hour survival rate of rats (100% in the sham group, 25% in the CA group, and 30% in the EA group, P > 0.05). The analysis by TUNEL showed that the apoptosis rate of neurons in CA1 region of the hippocampus in EA group at 6 hours after ROSC was significantly lower than that in CA group [(62.84±2.67)% vs. (71.29±3.70)%, P < 0.05]. Compared with the CA group, the level of serum S100 calcium binding protein B (S100B) in the EA group at 6 hours after ROSC was significantly lower (ng/L: 19.30±13.87 vs. 132.28±31.67, P < 0.05), but there were no significant differences in the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) between these two groups. In the present study, EA at Baihui can stabilize the hemodynamic, moreover, it has a particular neuroprotective effect on PCAS rats. Still, EA at Baihui does not reduce the systemic inflammatory response and improve the survival rate of rats, and its mechanism remains to be verified in further research.

Effect of GluN2C knockdown and overexpression using magnetic nano-gene carriers on cerebral ischemia-reperfusion injury

To investigate the effects of the GluN2C subunit on cerebral ischemia-reperfusion injury, we fabricated magnetic nano-gene vectors to inhibit and overexpress GluN2C in rat hippocampal neurons. The present study comprised six experimental groups: normal control (Control); oxygen and glucose deprivation withreoxygenation (OGD/R); Glu2NC si-RNA knockdown (si-RNA); GluN2C si-RNA knockdown and oxygen-glucose deprivation with reoxygenation (si-RNA+OGD/R); GluN2C overexpression (GluN2C); and GluN2C overexpression and oxygen-glucose deprivation with reoxygenation (GluN2C+OGD/R). Cells were then cultured for 6 h, 12 h, and 24 h under standard conditions. GluN2C expression was detected by Western blotting. Cell activity was determined using MTT assays. Apoptosis was measured by flow cytometry. GluN2C protein levels in the GluN2C and GluN2C+OGD/R groups increased with reoxygenation time. GluN2C protein levels were significantly higher in the GluN2C group compared to the GluN2C+OGD/R group at all measured timepoints. No differences in GluN2C protein levels were observed between the other four experimental groups at any timepoint. After 6 h of reoxygenation, neuronal activity in the si-RNA+OGD/R group was significantly higher than in the GluN2C+OGD/R group (0.451±0.006 vs. 0.419±0.005; P &lt;0.05). Compared to the OGD/R group, neuronal activity was higher in the si-RNA+OGD/R group and lower in the GluN2C+OGD/R group after 12 h and 24 h of reoxygenation. Neuronal activity was significantly higher in the si-RNA+OGD/R group compared to the GluN2C+OGD/R group. Compared to the OGD/R group, the rate of neuronal apoptosis was lower in the si-RNA+OGD/R group and higher in the GluN2C+OGD/R group increased. The rate of neuronal apoptosis rate was significantly lower in the si-RNA+OGD/R group compared to the GluN2C+OGD/R group at 6 h, 12 h, and 24 h after reoxygenation. The findings of the present study demonstrate the role of the GluN2C subunit in nerve injury during cerebral ischemia-reperfusion injury.

Effects of repeated sevoflurane exposure on hippocampal cell apoptosis and long-term learning and memory ability in neonatal rats

Objective: To investigate the effects of repeated sevoflurane exposure on hippocampal cell apoptosis and long-term learning and memory ability in neonatal rats and its regulation on PI3K/AKT pathway. Methods: Ninety SD rats were randomly divided into the control group (25% oxygen), the single exposure group (inhalation of 3% sevoflurane and 25% oxygen on the 6th day after birth), the 3-times exposure group (inhalation of 3% sevoflurane and 25% oxygen on the 6th, 7th, and 8th day after birth), the 5-times exposure group (inhalation of 3% sevoflurane and 25% oxygen on the 6th, 7th, 8th, 9th and 10th day after birth), and the 5-times exposure + 740Y-P (PI3K activator) group (intraperitoneal injection of 0.02 mg/kg 740Y-P after inhalation of sevoflurane for 5 times) according to the random number table method. Morris water maze was used to measure the learning and memory ability; HE staining and transmission electron microscopy were used to observe the morphological and structural changes of neurons in the hippocampus; TUNEL was used to detect the apoptosis of hippocampal nerve cells; Western blot was used to detect the expressions of apoptosis-related proteins (Caspase-3, Bax, Bcl-2) and PI3K/AKT pathway related proteins in the hippocampus of rats. Results: Compared with the control group and the single exposure group, the learning and memory abilities of rats in the 3-times exposure group and the 5-times exposure group were severely reduced, the morphology and structure of hippocampal neurons were severely damaged, and the apoptosis rate of hippocampal nerve cells was increased (P＜0.05), the expressions of Capase-3 and Bax proteins were significantly increased (P＜0.05), and the expressions of Bcl-2 protein and PI3K/AKT pathway protein were significantly decreased (P＜0.05). With the increase in the number of exposures to sevoflurane, the learning and memory abilities of rats were significantly reduced, the hippocampal neuron cells were severely damaged, the hippocampal neuronal apoptosis rate was significantly increased (P＜0.05), and the expressions of PI3K/AKT pathway proteins were significantly reduced (P＜0.05). Compared with the 5-times exposure group, the learning and memory abilities and hippocampal neuron structure of rats in the 5-times exposure +740Y-P group were restored to a certain extent, and the hippocampal neuronal apoptosis rate, the levels of capase-3 and Bax protein were significantly reduced (P＜0.05), while the expressions of Bcl-2 protein and PI3K/AKT pathway protein were increased significantly (P＜0.05). Conclusion: Repeated exposure to sevoflurane can significantly reduce the learning and memory abilities of neonatal rats and exacerbate hippocampal neuronal apoptosis, which may be mediated by inhibiting the PI3K/AKT pathway.

The Role of the GRP78/PERK/ATF4 Pathway in the Ability of Gua Lou Gui Zhi Decoction to Attenuate Apoptosis by Inhibiting Endoplasmic Reticulum Stress after Ischemia-Reperfusion Injury.

Endoplasmic reticulum stress (ERS) is a key part of the apoptotic cascade that is initiated after cerebral ischemia-reperfusion injury and is very important for research on poststroke rehabilitation. In addition, the unfolded protein response (UPR) plays an important role in ERS because it activates downstream apoptotic signal transduction and induces apoptosis through the glucose-regulated protein 78 (GRP78)/protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) pathway. The Gua Lou Gui Zhi Decoction (GLGZD) ameliorated neuronal apoptosis of ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO) had been proved in our previous study. The present study aims to underly the regulatory ability of GLGZD in ERS-induced apoptosis mediated by the GRP78/PERK/ATF4 pathway. GLGZD was analyzed by HPLC. The effects of GLGZD were obversed on MCAO-induced ischemic rats. The cerebral infarct volume was detected by 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) Staining. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) were used to detect apoptosis. Transmission Electron Microscopy (TEM), Ca2+ levels and reactive oxygen species (ROS) detection were used to determine the function of endoplasmic reticulum. The GRP78/PERK/ATF4 signaling pathway was assessed by western blotting and immunohistochemistry. Our results showed that GLGZD exerted its effects on ischemia-reperfusion injury by significantly promoting the restoration of the quantity and morphology of the rough ER and reducing the neuronal apoptosis rate in the ischemic cortex. Moreover, both of the intracellular ROS and Ca2+ levels in ischemic cortical cells were found significantly reduced by GLGZD. The GLGZD-treated group showed increased levels of phosphorylation in both of PERK and eukaryotic translation initiation factor 2α (eIF2α), activation of cysteinyl aspartate-specific proteinase-3 (Caspase-3), upregulation of the total protein levels of sarcoplasmic/endoplasmic Ca2+ ATPase 2α (SERCA 2α) and B-cell leukemia/lymphoma gene 2 (Bcl-2). These findings suggest that GLGZD reduces oxidative stress-induced injury and promotes a dynamic calcium balance, thereby inhibiting ERS and exerting an antiapoptotic effect on neuronal ischemic injury, which are closely related to the activation of GRP78/PERK/ATF4 signaling pathway.

Pinus massoniana needle extracts attenuate oxidative stress injury in cerebral ischemia reperfusion rats by regulating JNK3/caspase-3 signal transduction.

To investigate the effect and mechanism of Pinus massoniana needle extracts (PNE) on oxidative stress injury in cerebral ischemia reperfusion rats. The SD male rats were randomly divided into sham group, model control group, Edaravone (3 mg/kg) group, PNE low-dose (200 mg/kg), medium-dose (400 mg/kg) and high-dose (800 mg/kg) groups. PNE was administered by gavage for 7 d before modeling and 6 h after modeling in PNE treatment groups; Edaravone was given by intraperitoneal injection 7 d before modeling and 6 h after reperfusion. The rat model of cerebral ischemia reperfusion injury was established by middle cerebral artery occlusion method. After 24 h of reperfusion, the neurological deficit score, brain water content and cerebral infarction volume of rats were measured. The pathological changes of cerebral cortex and hippocampus were observed by HE staining, and the number of normal nerve cells was counted. The apoptosis rate of neurons in cerebral cortex was detected by TUNEL method. The content of nitric oxide (NO), malondialdehyde (MDA) and superoxide dismutase (SOD) activity in ischemic brain tissue were detected. The protein expression of c-Jun N-terminal kinase (JNK) 3, phosphorylated JNK3 (p-JNK3), B-cell lymphoma protein(Bcl) -2, Bcl-2 associated X (Bax), cytochrome C and caspase-3 in cerebral cortex were detected by Western blotting method. Compared with the model control group, the behavioral score, brain water content and cerebral infarction volume in PNE groups were significantly reduced (all P<0.05), the pathological damage of cerebral cortex and hippocampal CA1 area was significantly alleviated, and the number of normal nerve cells in ischemic cortex and hippocampal CA1 area was increased (all P<0.05). The medium-dose PNE group had the best effect. Compared with the model control group, the apoptosis rate of cortical neurons, the content of NO and MDA in cerebral cortex, the ratio of p-JNK3/JNK3, the expression level of cytochrome C and caspase-3 protein in PNE medium-dose group were significantly reduced , and the activity of SOD, the Bcl-2/Bax ratio were significantly improved (all P<0.05). PNE ameliorates brain injury after cerebral ischemia reperfusion in rats, which may be related to scavenging NO and MDA, inhibiting oxidative stress-mediated JNK3/caspase-3 signsal transduction to inhibit neuronal apoptosis.

Eucommia ulmoides extract alleviated spinal cord injury in rats by inhibiting endoplasmic reticulum stress and oxidative stress

Spinal cord injury (SCI) is the major cause of severe disability worldwide, which leads to neuron death, neuronal degeneration, and functional changes in the spinal cord. Eucommia ulmoides extract (EUE) is composed of multiple iridoids and phenols and possesses anti-oxidative and anti-inflammatory effects in various pathologies. This study aims to evaluate the effects of EUE on SCI and the underlying mechanisms. Male adult Sprague-Dawley rats (250–280 g) were applied to mimic SCI in vivo. Western blot and ELISA kits assessed the expressions of apoptosis, oxidative stress, and endoplasmic reticulum stress (ER stress)-relevant proteins. The apoptosis rate of neurons in spinal cord specimens was measured by TUNEL assay. Finally, our data indicated that EUE inhibited SCI-induced apoptosis, oxidative stress, and ER stress through the suppression of mitogen-activated protein kinase (MAPK) pathway. Our data manifest EUE as a potential therapeutic target in SCI.

Investigation into Antiepileptic Effect of Ganoderic Acid A and Its Mechanism in Seizure Rats Induced by Pentylenetetrazole.

Ganoderic acid A (GAA) exhibited neuron protection in in vitro epilepsy study, but no study has been done in vivo. Rats were administered (i.p.) pentylenetetrazole daily for 28 days to induce seizure. Rats with grade II or above of epileptic score were divided into three groups and given placebo, sodium valproate, or GAA treatment, respectively, for 7 days. The electrical signals of brain were monitored with electroencephalography (EGG); epileptic behavior was assessed using the Racine scale; morphological changes and apoptosis rate of cortical neurons were assessed with H&E staining and TUNEL staining, respectively. Protein expression of calcium-sensing receptor, p-ERK, p-JNK, and p-p38 in hippocampal tissue and Bcl-2, cleaved caspase-3, and Bax in cortical tissues was observed by Western blot and immunohistochemistry assay, respectively. After GAA treatment, apparent seizure-like EEG with significant arrhythmic disorder and spike waves was reduced or disappeared, and wave amplitude of EEG was reduced significantly. GAA showed similar effect with sodium valproate treatments on epilepsy. There were an apparent improvement of the epileptic behavior and a significant increase in the epileptic latency and shortening of the epileptic duration in the treatment group compared to control. GAA treatment ameliorated the nuclear pyknosis of neurons which appeared seriously in the epilepsy group. GAA treatment significantly reduced the cortical neuron apoptosis of epilepsy and the expression of calcium-sensing receptor, p-P38, p-JNK, cleaved caspase-3, and Bax but increased the expression of both p-ERK and Bcl-2. In conclusion, GAA treatment showed strong antiepileptic effect by decreasing apoptosis in cortical neuron and the expression of calcium-sensing receptor and stimulating the MAPK pathway.