Rat Nerve Cells Research Articles

Mechanism of Dabugan Decoction in treatment of generalized anxiety disorder based on network pharmacology and experimental verification

This study aims to explore the mechanism of Dabugan Decoction in the treatment of generalized anxiety disorder(GAD) based on network pharmacology, molecular docking, and animal experiments. Network pharmacology and molecular docking technology were used to obtain the possible targets and related signaling pathways of Dabugan Decoction in the treatment of GAD. The GAD rat model was established, and the corresponding drugs were given by gavage after randomization. After 28 days of continuous intervention, the anxiety state of rats was detected, and the pathological changes of the hippocampus were detected in each group. ELISA and Western blot were used to detect the protein expression levels of related molecules. A total of 65 drug compounds in Dabugan Decoction were obtained, involving 403 targets of action, 7 398 disease targets of GAD, and 279 common targets of "drug-disease". The key nodes in the protein-protein interaction(PPI) network were Akt1, TNF, IL-6, TP53, IL-1β, etc. Function analysis of Gene Ontology(GO) and enrichment analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG) showed that the PI3K-Akt signaling pathway was the most important pathway. The results of molecular docking showed that the core components of the drug had good binding activity with the corresponding key targets. Animal experiments showed that Dabugan Decoction could effectively improve the anxiety behavior of rats and increase the open arm end movement distance and total distance of rats in the elevated cross labyrinth, the number and stay time of entering the open box, and the time(%) and the number of entering the center of the open field. At the same time, HE staining and Nicil staining showed that the number of hippocampal nerve cells in rats increased, and they were closely arranged. The damage to the cell body was improved, and there was an increase in Nissl substances in the cells. The expression of TNF-α, IL-6, and IL-1β in rat hippocampus decreased, and the expression of TP53, p-Akt1, and p-PI3K increased. The mechanism may be related to the activation of the PI3K-Akt signaling pathway and the inhibition of inflammatory response. Dabugan Decoction can play a good therapeutic and regulatory role in GAD, reflecting the overall effect of traditional Chinese medicine(TCM) compound and the characteristics of multiple targets and multiple pathways. At the same time, it is preliminarily discussed that the state of GAD may be improved by Dabugan Decoction via-activating PI3K-Akt signaling pathway and inhibiting inflammatory response and anti-apoptosis, thus providing experimental data support for the clinical application of Dabugan Decoction.

Reducing the injury of hippocampal vascular endothelial cells after stroke via targeting SIRT1 by butylphthalide

Butylphthalide (NBP) can inhibit various pathological processes of ischemic stroke. This experiment explored the mechanism of NBP and SIRT1 on damage of hippocampal vascular endothelial cells after stroke. The neurons in the hippocampus of rats were stained with HE, and morphology and density of neurons were observed. Flow cytometry, commercial kits and Western blotting detected apoptosis of endothelial cells, levels of antioxidant enzymes and apoptotic proteins, intracellular calcium level and activity of Ca2+-ATPase. The damage to rat nerve cells was alleviated by butylphthalide to varying degrees, and the lost parts of rat nerve cells were recovered with decreased Bax and cleaved caspase-3 expression after butylphthalide treatment, and increased Bcl-2 (P <0.05), as well as decreased serum malondialdehyde (MDA) content and activity of catalase (CAT) decreased and elevated Superoxide dismutase (SOD) activity (P <0.05). The concentration of calcium ion also decreased but activity of Ca2+-ATPase increased (P <0.05) and mitochondria in the model group appeared with severe vacuolation and swelling. The vacuolation and swelling of mitochondria in the treatment group were improved. Additionally, mitochondrial membrane fluidity, potential and rat hippocampal ATPase activity in butylphthalide group were also increased. Compared to normal control group, model group, SIRT1 inhibitor group and butylphthalide+SIRT1 inhibitor group had lower levels of SIRT1 and higher p-NF-kB p65/p-IkBα levels. Butylphthalide has a protective effect on hippocampal neurons in stroke rats and can alleviate the damage degree of rat nerve cells.

Huperzine A ameliorates neurological deficits after spontaneous subarachnoid hemorrhage through endothelial cell pyroptosis inhibition.

Spontaneous subarachnoid hemorrhage (SAH) is a kind of hemorrhagic stroke which causes neurological deficits in survivors. Huperzine A has a neuroprotective effect, but its role in SAH is unclear. Therefore, we explore the effect of Huperzine A on neurological deficits induced by SAH and the related mechanism. In this study, Evans blue assay, TUNEL staining, immunofluorescence, western blot analysis, and ELISA are conducted. We find that Huperzine A can improve neurological deficits and inhibit the apoptosis of nerve cells in SAH rats. Huperzine A treatment can improve the upregulation of brain water content, damage of blood-brain barrier, fibrinogen and matrix metalloprotein 9 expressions and the downregulation of ZO-1 and occludin expressions induced by SAH. Huperzine A inhibit the expressions of proteins involved in pyroptosis in endothelial cells in SAH rats. The increase in MDA content and decrease in SOD activity in SAH rats can be partly reversed by Huperzine A. The ROS inducer H 2O 2 can induce pyroptosis and inhibit the expressions of ZO-1 and occludin in endothelial cells, which can be blocked by Huperzine A. In addition, the increase in the entry of p65 into the nucleus in endothelial cells can be partly reversed by Huperzine A. Huperzine A may delay the damage of blood-brain barrier in SAH rats by inhibiting oxidative stress-mediated pyroptosis and tight junction protein expression downregulation through the NF-κB pathway. Overall, Huperzine A may have clinical value for treating SAH.

MiR- 211 represses apoptosis of nerve cells in rats with cerebral infarction through PI3K/AKT signaling pathway.

To investigate the effect of micro ribonucleic acid (miR)-211 on the apoptosis of nerve cells in rats with cerebral infarction through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. A total of 36 Sprague-Dawley (SD) rats were randomly divided into sham operation group (n=12), model group (n=12) and miR-211 mimics group (n=12). Only the common carotid artery, external carotid artery, and internal carotid artery were exposed in sham operation group, and the models of cerebral infarction were constructed via suture method in the other two groups. After modeling, the rats in sham operation group and model group were intraperitoneally injected with normal saline, while those in miR-211 mimics group were given miR-211 mimics via intraperitoneal injection. At 2 weeks after intervention, samples were collected. Neurological deficit in rats was assessed using the Zea-longa score, and Nissl staining assay was performed to observe neuronal morphology. Western blotting (WB), quantitative polymerase chain reaction (qPCR) assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure the relative protein expressions of PI3K and phosphorylated AKT (p-AKT), mRNA expression of miR-211 and content of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), respectively. Additionally, the apoptosis was detected via terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. The neuronal morphology was normal in sham operation group, while it was disordered in model group, with damaged neurons. In miR-211 mimics group, the morphology of neurons was improved. The Zea-longa score was obviously higher in model group and miR-211 mimics group than that in sham operation group (P<0.05), while it was notably lower in miR-211 mimics group than that in model group (P<0.05). Compared with those in sham operation group, the relative protein expression levels of PI3K and p-AKT remarkably declined in model group and miR-211 mimics group (P<0.05), whereas they were clearly higher in miR-211 mimics group than those in model group (P<0.05). The relative expression level of miR-211 was lower in model group and miR-211 mimics group than that in sham operation group (P<0.05), while it was markedly higher in miR-211 mimics group than that in model group (P<0.05). In comparison with sham operation group, model group and miR-211 mimics group had remarkably increased content of Bax and evidently lowered content of Bcl-2 (P<0.05), whereas compared with model group, miR-211 mimics group displayed clearly reduced Bax content and notably raised Bcl-2 content (P<0.05). The apoptosis rate was distinctly higher in model group and miR-211 mimics group than that in sham operation group (P<0.05), while it was visibly lower in miR-211 mimics group than that in model group (P<0.05). MiR-211 represses the apoptosis of nerve cells in rats with cerebral infarction by up-regulating the PI3K/AKT signaling pathway, thereby protecting nerves.

Effects of combined electroacupuncture and medication therapy on the RhoA/ROCK-2 signaling pathway in the striatal region of rats afflicted by cerebral ischemia

ObjectiveTo investigate the effects of electroacupuncture(EA), gastrodin(Gas), and their combination on the signaling pathways involving Ras homologous gene family member A (RhoA) and Rho-associated frizzled helix protein kinase (ROCK-2) within the striatal region of rats subjected to cerebral ischemia. Additionally, we aim to elucidate the therapeutic effects and potential underlying mechanisms associated with the concurrent application of electroacupuncture and medication in the treatment of cerebral ischemia. MethodsRats were randomly assigned to one of five groups, namely, the sham operation (Sham) group, model group, EA group, Gas group, and the EA combined with Gas group (referred to as the "EA+Gas group"). Each group consisted of ten rats. Following the induction of cerebral ischemia, the EA group and EA+Gas group received EA stimulation at the Baihui(GV20) and Zusanli(ST36) acupoints for 30 min per session, administered once daily for 14 consecutive days. The Gas group and EA+Gas group were intraperitoneally injected with Gas at a dosage of 10 mg/kg, also administered once daily for 14 consecutive days. Nissl staining was employed to observe morphological alterations in the striatal nerve cells of rats in each group. Immunohistochemistry and western blot techniques were employed to evaluate the expression levels of striatal RhoA and ROCK-2 proteins. ResultsIn comparison to the Sham group, the model group exhibited a substantial reduction in the number of striatal nerve cells on the ischemic side, accompanied by notable changes in cell morphology, characterized by reduced cytoplasm, defective and atrophied cytosol, solidified nuclei, loosely arranged cells, and enlarged intercellular spaces. Additionally, there was a notable increase in the positive expression of RhoA and ROCK-2. In contrast, when compared to the model group, the EA, Gas, and EA+Gas groups demonstrated an elevated number of normal nerve cells within the ischemic striatal region, with a significant improvement in cell count and morphology. Furthermore, positive expression levels of RhoA and ROCK-2 were notably reduced in these groups. Compared with the EA group or the GAS group, the number of normal nerve cells in the striatum on the ischemic side of the EA+GAS group was further increased, and the positive expression level of RhoA and ROCK-2 were both further reduced. ConclusionThe protective mechanism underlying the therapeutic efficacy of EA combined with Gas against cerebral ischemic striatal injury in rats may be associated with the inhibition of the activation of the RhoA/ROCK-2 signaling pathway. Importantly, the therapeutic effects observed with the combination of electroacupuncture and medication were superior to those achieved with EA alone or the sole administration of Gas.

Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury

To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Electroacupuncture improves limb motor function by down-regulating cytosolic phospholi-pase A2 and reducing apoptosis of nerve cells in rats with spinal cord injury

To observe the effect of electroacupuncture（EA） on the expression of cytosolic phospholipase A2 （cPLA2） and apoptosis of nerve cells in rats with spinal cord injury （SCI）, so as to explore its mechanisms underlying improvement of SCI. Seventy-two female SD rats were randomly divided into model, EA, antagonist and EA+antagonist groups, with 18 rats in each group and other 18 rats were used as the sham operation （sham） group. The SCI model was established by referring to modified Allen's method with a weight impactor. The hindlimb motor function was assessed by using Basso-Beattie-Bresnahan （BBB） score. Rats of the EA group were subjected to EA stimulation at "Dazhui"（GV14）, "Yaoyangguan"（GV3）, bilateral "Ciliao"（BL32） and "Zusanli"（ST36） for 20 min, once a day for 14 days. Rats of the antagonist group received intravenous injection followed by intraperitoneal injection of arachidonyl trifluoromethyl ketone （AACOCF3, antagonist of cPLA2）, once every other day. Rats of the EA+antagonist group received EA treatment combined with antagonist injection. After the treatment, the rats were sacrificed and the spinal cord tissue was collected for detecting the protein expression of cPLA2, p-cPLA2, Bcl-2, Bax and Caspase-3 by Western blot, and the mRNA expression of cPLA2, Bcl-2, Bax and Caspase-3 using qRT-PCR. The morphological changes of the spinal cord were detected by Nissl staining. In comparison with the sham group, the BBB score, expression of Bcl-2 protein and mRNA were significantly down-regulated （P<0.01）, whereas the expression levels of Bax, Caspase-3 and p-cPLA2 proteins and mRNAs were considerably up-regulated in the model group （P<0.01）. Compared with the model group, the BBB score, expression levels of Bcl-2 protein and mRNA were significantly up-regulated （P<0.01, P<0.05）, while the expression levels of Bax, Caspase-3 and p-cPLA2 proteins in the EA, antagonist and EA+antagonist groups, Bax and cPLA2 mRNAs in both antagonist and EA+antagonist groups, and Caspase-3 mRNA in the EA+antagonist group were obviously down-regulated （P<0.01, P<0.05）. The effect of EA+antagonist was significantly superior to EA in increasing BBB score and in lowering expression of Bax and cPLA2 mRNAs （P<0.01, P<0.05）. Nissl staining showed reduced number of nerve cells and Nissl bodies, and striped dark blue cells in the model group, which was milder in the EA and antagonist groups, particularly in the EA+antagonist group. EA may improve the limb motor function of SCI rats, which may be related to its functions in down-regulating the expression of p-cPLA2, Bax and Caspase-3 and up-regulating Bcl-2 to reduce the apoptosis of nerve cells in the regional spinal cord.

Low androgen levels induce ferroptosis of rat penile cavernous endothelial cells.

Endothelial dysfunction caused by low androgen levels in penile tissue can lead to erectile dysfunction. The exact mechanism of endothelial dysfunction has not been thoroughly studied. The study sought to verify whether low androgen levels induce ferroptosis of endothelial cells in rat penile tissue. Rat penile cavernous endothelial cells (CP-R133) were divided into a no-androgen group (Dihydrotestosterone (DHT): 0nmol/L), very low-androgen group (DHT: 0.1nmol/L), low-androgen group (DHT: 1nmol/L), DHT = 10nmol/L group, DHT (0nmol/L) + ferrostatin-1 (Fer-1) group, DHT (0.1nmol/L) + Fer-1 group, DHT (1nmol/L) + Fer-1 group, DHT (10nmol/L) + Fer-1 group. Cell viability, intracellular ferrous ion (Fe2+), malondialdehyde (MDA), GSH into oxidized glutathione (GSSG), reactive oxygen species (ROS), nitric oxide (NO), transferrin receptor 1 protein (TfR1), solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), endothelial nitric oxide synthase (eNOS), and phospho-eNOS (p-eNOS) were detected. Low androgen levels could induce ferroptosis of rat penile cavernous endothelial cells in vivo by upregulating the expressions of TfR1 and ACSL4 and downregulating the expressions of SLC7A11 and GPX4. Cell viability, the levels of glutathione (GSH), NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 0nmol/L group were lower than those in the other groups (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 0nmol/L group were higher than those in the other groups (P < .05). Cell viability and the levels of GSH, NO, SLC7A11, GPX4, and p-eNOS/eNOS in the DHT = 1nmol/L group were lower than those in the DHT (1nmol/L) + Fer-1 group, DHT = 10nmol/L group, and DHT (10nmol/L) + Fer-1 group (P < .05). The levels of Fe2+, ROS, MDA, GSSG, TfR1, and ACSL4 in the DHT = 1nmol/L group were higher than those in the DHT (1nmol/L) + Fer-1 group, DHT = 10nmol/L group, and DHT (10nmol/L) + Fer-1 group (P < .05). A ferroptosis inhibitor might be a novel drug for treating erectile dysfunction caused by low androgen level. The results of this study need to be further confirmed in in vitro and in human studies. Meanwhile, further investigation is needed to clarify whether low androgen levels affect ferroptosis of rat penile cavernous smooth muscle and nerve cells. Low androgen levels can induce ferroptosis of endothelial cells in rat penile tissue. Inhibition of ferroptosis can reverse endothelial dysfunction caused by low androgen levels.

Honokiol Reduces Mitochondrial Dysfunction and Inhibits Apoptosis of Nerve Cells in Rats with Traumatic Brain Injury by Activating the Mitochondrial Unfolded Protein Response.

This study was designed to determine the effects and underlying mechanism of honokiol (HNK) on traumatic brain injury (TBI). A rat TBI model was constructed using the modified Feeney free-fall percussion method and treatment with HNK via intraperitoneal injection. The brain tissues of the rats in each group were assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect the level of neuronal apoptosis. Western blots were used to detect the expression levels of apoptosis-related proteins (Bcl-2 and Bax), and ELISAs were used to measure the levels of pro-inflammatory cytokines (IL-18 and IL-1β) and the activity of caspase-1. In addition, the mitochondrial membrane potential, reactive oxygen species (ROS), and adenosine 5'-triphosphate (ATP) were also measured. Western blots and qRT-PCRs were used to determine the relative expression levels of the mitochondrial unfolded protein response (UPRmt)-related proteins and mRNAs. Based on the experimental results, treatment with HNK was associated with a decrease in the number of TUNEL-positive cells, downregulated Bax expression levels, elevated Bcl-2 expression levels, and inhibition of neuronal apoptosis in the brain tissue of TBI rats. HNK also suppressed neuroinflammation by decreasing IL-1β and IL-18 levels and caspase-1 activity. Additionally, HNK lowered the mitochondrial membrane potential and ROS levels, increased ATP levels, and improved mitochondrial dysfunction in neural cells. Furthermore, in the investigation of the mechanism of HNK on TBI, we observed that HNK could activate UPRmt by upregulating the mRNA and protein expression levels of HSPA9, CLPP, and HSP60 in the brain tissues of TBI rats. Collectively, HNK reduced mitochondrial dysfunction, inhibited the apoptosis of nerve cells, and attenuated inflammation in the brains of TBI rats. The protective effect of HNK may be achieved through the activation of UPRmt.

Huangqi Guizhi Wuwu Decoction can prevent and treat oxaliplatin-induced neuropathic pain by TNFα/IL-1β/IL-6/MAPK/NF-kB pathway

Objective: This study explored the effects and mechanisms of Huangqi Guizhi Wuwu Decoction on chemotherapy-induced neuropathic pain (CINP).Methods: Bodyweight and related behavioral testing of the rat model were utilized to investigate the effects of Huangqi Guizhi Wuwu Decoction on CINP. ELISA was used to measure the levels of TNF-α, IL-1β, and IL-6, in the serum of chronic CINP rats. Immunohistochemistry and Western blot analysis were performed to detect the expression of MAPK pathway related-proteins namely ERK1/2, p38, and JNK, and the expression of downstream essential proteins such as c-Fos, CREB, and NF-κB.Results: Body weight and related behavioral testing of the rat model suggests that Huangqi Guizhi Wuwu Decoction can improve the slow weight gain of oxaliplatin-induced chronic CINP model rats and effectively prevent and treat oxaliplatin-induced regular CIPN rat model of hyperalgesia. It can also oppress the mechanical pain threshold, cold pain threshold, and heat pain threshold decreased. Furthermore, by ELISA, immunohistochemistry, and western blot analysis, we found that Huangqi Guizhi Wuwu Decoction can down-regulate the levels of TNF-α, IL-1β, and IL-6 in the serum of chronic CINP rats induced by oxaliplatin. It also suppresses the expression of MAPK pathway related-proteins ERK1/2, p38, and JNK. This results in a decrease in the expression of downstream essential proteins, c-Fos, CREB, and Nf-κB.Conclusions: In conclusion, we found that Huangqi Guizhi Wuwu Decoction can combat nerve cell injury, reduce pain sensitization, and prevent and repair the damage of nerve cells in the oxaliplatin CINP model rats via TNFα/IL-1β/IL-6/MAPK/NF-kB pathway.

Effcacy-oriented compatibility for Tianma (), Yanlingcao () and Bingpian () on improving cerebral ischemia stroke by network pharmacology and serum pharmacological methods.

To evaluate the compatibility of Tianma (, TM), Yanlingcao (, YLC) and Bingpian (, BP), and their efficacy in the treatment of cerebral ischemic stroke. Network pharmacology was used to determine the compatibility of TM, YLC, and BP, and their potential mechanism. The middle cerebral artery occlusion (MCAO) rat model was used to evaluate the curative effect of the six combinations of TM, YLC, and BP (TZB1-TZB6) on cerebral ischemia, by using the weight matching method to form. The potential component changes of TM and YLC in the blood and brains of rats were analyzed using ultra performance liquid chromatography-mass spectrometry. Finally, molecular docking linked the results of animal experiments and network pharmacology, determining the potential component contributors of TM and YLC to treating ischemic stroke. TZB reduced the cerebral infarct volume and protected the nerve cells in MCAO rats. The components of TM and YLC were also identified in the blood and brain homogenate, and BP can facilitate the entry of the components of TM and YLC into the blood and brain. Diosgenin, pennogenin, and gastrodin induced effective binding activities with adenosine receptor a1. We investigate an approach that improves the means of folk prescription combined with multi technology that maybe promote the transformation of Chinese medicinal prescription into component-based Chinese medicine.

Tauroursodeoxycholic acid protects rat spinal cord neurons after mechanical injury through regulating neuronal autophagy

BackgroundTo study the protective effect of tauroursodeoxycholic acid (TUDCA) on the spinal cord nerve cells (SCN) of SD rats and to explore the protective mechanism of TUDCA against mechanical injury of the SCN. Material and methodsThe SCN of SD rats were cultured in vitro, and a mechanical injury models of 1 mm, 3 mm, and 5 mm SCN were established. The cell survival rate was determined using the MTT assay to determine the optimal degree and time of injury. Different concentrations (0.5, to 20 mmol/L) of TUDCA were used to detect SCN cell survival rate after mechanical injury. MTT assay was used to determine the optimal TUDCA intervention dose. SCN autophagy in different experimental groups were observed by electron microscopy after the best degree of mechanical injury, time of injury, and TUDCA concentration. Beclin-1 and LC3 II/I expressions were detected by western blotting and immunohistochemistry. ResultsSurvival rate of SCN was close to 50% when the injury interval was 3 mm and the injury time was 24 h, significantly different from those of each group (P < 0.05). At 3 mm injury interval and 24 h injury time, SCN survival rate was approximately 80% when TUDCA concentration was 4 mmol/L, which was significantly different from those of each group (P < 0.05). Cell morphology of the normal control group was complete, with few autophagy lysosomes. Compared with the normal control group, the number of autophagic lysosomes in the mechanical injury group increased, and cell damage was more severe. Compared with the mechanical injury group, the number of autophagy lysosomes in the mechanical injury + TUDCA intervention group increased significantly, and cell damage was less severe. Further, compared with the normal control group, beclin-1 and lc3ii / I expressions in the mechanical injury group were significantly higher (P < 0.05); compared with the mechanical injury group, beclin-1 and lc3ii / I expressions in the mechanical injury + TUDCA intervention group were significantly higher (P < 0.05). ConclusionTUDCA can protect SCN from mechanical injury in vitro, which may be related to the enhancement of the expression of autophagy-related protein beclin-1 and LC3 II/I.

Mechanism of Electroacupuncture at Baihui and Shenting Alleviating Cerebral Ischemia-reperfusion Injury via Regulating BNIP3L-Mediated Mitophagy

<sec><title>Objective</title> The purpose of this study is to explore the mechanism of electroacupuncture at Baihui and Shenting acupoints to alleviate cerebral ischemia-reperfusion injury by regulating BNIP3L-mediated mitophagy. </sec><sec><title>Methods</title> A total of 60 SD rats were randomly divided into sham operation group (<italic>n</italic>=15) and operation group (<italic>n</italic>=45) by using the random number table method. Only the blood vessels were separated and exposed in the sham operation group, without ligation and no nylon thread inserted. The rats in the operation group were treated with the middle cerebral artery occlusion (MCAO) method to prepare the focal cerebral ischemia-reperfusion (I/R) model, and the Zea Longa method was used to score the neurological function 2h after the operation. Finally, 30 rats were included in the operation group. The included operation group rats were further randomly divided into the model group, electroacupuncture group, and electroacupuncture+3-MA group, with 10 rats in each group. After grouping, each group was treated with different methods such as electroacupuncture for 7 days, and the Zea Longa method was used to evaluate the neurological function again on the 1, 3, 5, and 7 days. After 7 days of intervention, the left cerebral cortex tissue of each group was obtained, hematoxylin-eosin (HE) staining was used to observe the pathological changes of the brain tissue, the expression level of LC3-Ⅱ/LC3-Ⅰ protein was detected by Western blot, and the tissue immunofluorescence technology was applied to detect the expression and colocalization of mitophagy-related protein BNIP3L and SQSTM1 labeled. The TUNEL method detected the apoptosis rate of rat nerve cells in each group. </sec><sec><title>Results</title> 1) The neurological scores of the rats in the operation group were significantly increased 2 hours after the MCAO, and the rats in the sham-operation group did not show symptoms of neurological deficit. On the 7th day after the intervention, the neurological scores of the rats in the model group and the electroacupuncture+3-MA group still increased significantly, which were significantly different from those in the sham operation group (<italic>P</italic><0.05). However, the neurological scores of rats in the electroacupuncture group reduced after electroacupuncture intervention, which was significantly different from the model group and electroacupuncture+3-MA group (<italic>P</italic><0.05). 2) The results of HE staining showed that the cell body of neurons in the sham operation group was large, polygonal (multiple protrusions), the nucleus was lightly colored, and the characteristic structure of Nissl body could be seen in the cytoplasm. In the model group, neurons shrank, the cytoplasmic structure was unclear, cytoplasmic eosinophilic staining was enhanced, Nissl bodies gathered or disappeared, nuclear pyknosis, and some cell bodies deformed and shrunk to triangles and the spaces around the cells widened. The electroacupuncture group could reduce the pathological damage caused by ischemia-reperfusion to a certain extent. 3) Western blot results showed that the autophagy level (LC3-Ⅱ/LC3-Ⅰ) of the model group decreased on the 7th day after MCAO, there wass a significant difference compared with the sham operation group (<italic>P</italic><0.05), while the electroacupuncture group could activate the level of autophagy, which was significantly different from the model group (<italic>P</italic><0.05). 4) In the model group, the level of mitophagy in the cortical infarction area was absent, as evidenced by the weakened red fluorescence and the intensity of green fluorescence. However, the intervention of electroacupuncture could activate the mitophagy of nerve cells in the infarct area, which was manifested by increased expression of red fluorescence and green fluorescence and enhanced colocalization (orange). 5) TUNEL test results indicated the apoptotic rate of rats in the model group rising, and there was a significant difference compared with the sham operation group (<italic>P</italic><0.05). However, the apoptotic rate of the electroacupuncture group decreased, which was significantly different from that of the model group (<italic>P</italic><0.05). The results of the electroacupuncture+3-MA group showed that the autophagy inhibitor 3-MA could reverse the neuroprotective effect of electroacupuncture in MCAO model rats. </sec><sec><title>Conclusion</title> Electroacupuncture at Baihui and Shenting acupoints can alleviate neurological damage in rats with cerebral ischemia-reperfusion. The specific mechanism is related to regulating BNIP3L-mediated mitophagy to reduce cerebral ischemia-reperfusion injury and cell apoptosis. </sec>

Xylazole inhibits NO-cGMP pathway in fetal rat nerve cells.

BackgroundXylazole (Xyl) is a veterinary anesthetic that is structurally and functionally similar to xylazine. However, the effects of Xyl in vitro remain unknown.ObjectivesThis study aimed to investigate the anesthetic mechanism of Xyl using fetal rat nerve cells treated with Xyl.MethodsFetal rat nerve cells cultured for seven days were treated with 10, 20, 30, and 40 μg/ mL Xyl for 0, 5, 10, 15, 20, 25, 30, 45, 60, 90, and 120 min. Variations of amino acid neurotransmitters (AANTs), Nitric oxide-Cyclic GMP (NO-cGMP) signaling pathway, and ATPase were evaluated.ResultsXyl decreased the levels of cGMP and NO in nerve cells. Furthermore, Xyl affected the AANT content and Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity in nerve cells. These findings suggested that Xyl inhibited the NO-cGMP signaling pathway in nerve cells in vitro.ConclusionsThis study provided new evidence that the anesthetic and analgesic effects of Xyl are related to the inhibition of the NO-cGMP signaling pathway.

Poria cocos Regulates Cell Migration and Actin Filament Aggregation in B35 and C6 Cells by Modulating the RhoA, CDC42, and Rho Signaling Pathways.

Poria is used as a traditional Chinese herbal medicine with anti-inflammatory, anticancer, and mood-stabilizing properties. Poria contains triterpenoids and polysaccharides, which are reported to regulate the cytoplasmic free calcium associated with the N-methyl-D-aspartate receptor and affect the cell function of neonatal rat nerve cells and hippocampal neurons. Although the modulatory effects of Poria on neuronal function have been widely reported, the molecular mechanism of these effects is unclear. Cell migration ability and the reorganization of actin filaments are important biological functions during neuronal development, and they can be regulated mainly by the Rho signaling pathway. We found that the cell migration ability and actin condensation in B35 cells enhanced by P. cocos (a water solution of P. cocos cum Radix Pini (PRP) or White Poria (WP)) might be caused by increased RhoA and CDC42 activity and increased expression of downstream ROCK1, p-MLC2, N-WASP, and ARP2/3 in B35 cells. Similar modulations of cell migration ability, actin condensation, and Rho signaling pathway were also observed in the C6 glial cell line, except for the PRP-induced regulation of RhoA and CDC42 activities. Ketamine-induced inhibition of cell migration and actin condensation can be restored by P. cocos. In addition, we observed that the increased expression of RhoA and ROCK1 or the decreased expression of CDC42 and N-WASP caused by ketamine in B35 cells could also be restored by P. cocos. The results of this study suggest that the regulatory effects of P. cocos on cell migration and actin filament aggregation are closely related to the regulation of RhoA, CDC42, and Rho signaling pathways in both B35 and C6 cells. PRP and WP have the potential to restore neuronal cell Rho signaling abnormalities involved in some mental diseases.

Gambogic Acid Relieves Neuropathic Pain in Rats by Regulating CXCR4-TXNIP/NLRP3 Pathway

Neuropathic pain is caused by abnormal sensory processing in the central nervous system (CNS). The immune response of the CNS is related to the function of glial cells and is critical in neuropathic pain. Agents based on cytokines and glial cells in the CNS have potential for the treatment of neuropathic pain. Gambogic acid (GA) is one of the main components of Garcinia cambogia, which has anti-inflammatory, antitumor, and analgesic effects. However, the effect of GA on neuropathic pain and related mechanisms are still unclear. Previous studies indicated that GA could reduce CXCR4 expression in neuropathic pain. In this study, we found that GA could alleviate nerve pain behavior in rats by measuring the incubation period of heat shrinkage and mechanical pain threshold. Also, GA reduced inflammation in chronic constriction injury rats. We further found GA reduced the apoptosis of spinal cord nerve cells in chronic constriction injury rats. Mechanically, we noticed GA relieved neuropathic pain in rats via regulating CXCR4-TXNIP/NLRP3 pathway. Our data confirmed that GA could serve as a promising drug for the treatment of neuropathic pain.

The Cerebroprotein Hydrolysate-I Plays a Neuroprotective Effect on Cerebral Ischemic Stroke by Inhibiting MEK/ERK1/2 Signaling Pathway in Rats.

ObjectiveTo investigate the neuroprotective effect and mechanism of cerebroprotein hydrolysate-I (CH-I) on cerebral ischemia/reperfusion injury in rats.MethodsA total of 100 adult healthy male SD rats were randomly divided into a sham group, model group, CH-I treated group, and cerebrolysin (CBL) positive group, consisting of 20 rats in each group. The middle cerebral artery occlusion/reperfusion (MCAO/R) model of rats was built by inserting a suture into the left external carotid artery (ECA) through the internal carotid artery (ICA). Treatment was performed by intraperitoneal injection of CH-I (20 mg/kg). The neurobehavioral function of rats was evaluated by modified neurological severity scores (mNSS). TTC staining was used to detect the cerebral infarction volume (CIV) of rats. The morphological and structural changes of nerve cells were observed by HE staining and the neuronal apoptosis was counted by TUNEL assay. Immunohistochemical (IHC) analysis was used to detect BDNF and pMEK1/2 expressions. The expressions of BDNF, pMEK1/2, pERK1/2, and pCREB were determined with Western blotting.ResultsAfter treatment with CH-I, the mNSS and CIV of rats were improved (P<0.05). And the CH-I can reduce the degeneration and apoptosis of nerve cells in rats (P<0.01). Western blotting showed that the expressions of pMEK1/2, pERK1/2, and pCREB in rats were increased, while the expression of BDNF was decreased after modeling (P<0.05). After treatment, the expressions of pMEK1/2, pERK1/2, and pCREB in the CH-I group were decreased (P<0.05), while the expression of BDNF was significantly increased (P<0.05) compared with the model group. IHC showed that the expression of BDNF and pMEK1/2 was consistent with Western blotting.ConclusionIt is suggested that the CH-I might play a neuroprotective role by inhibiting the expression of MEK-ERK-CREB and enhancing the expression of BDNF after cerebral ischemia/reperfusion injury, thus improving the neurobehavioral function of MCAO/R rats.

RETRACTED: MicroRNA-29a-3p strengthens the effect of dexmedetomidine on improving neurologic damage in newborn rats with hypoxic-ischemic brain damage by inhibiting HDAC4

Hypoxic-ischemic brain damage (HIBD) is a common brain injury caused by hypoxia or ischemia of the brain. This study aims to investigate the effect of dexmedetomidine (Dex) post-treatment on neurological impairment of newborn rats with HIBD via modulating microRNA-29a-3p (miR-29a-3p) and histone deacetylase 4 (HDAC4). HIBD model of newborn rats was established. Newborn modeled rats were injected with Dex, miR-29a-3p mimic or HDAC4 siRNA to figure their roles in learning and memory abilities, left hemisphere atrophy, brain tissue injury, inflammatory response and apoptosis rate of nerve cells of rats. The expression of miR-29a-3p and HDAC4 in hippocampal tissues of rats were detected, and the potential relationship between miR-29a-3p and HDAC4 was analyzed. Decreased miR-29a-3p and elevated HDAC4 were found in hippocampal tissues of rats with HIBD. In addition, Dex, elevated miR-29a-3p or declined HDAC4 enhanced spatial learning and memory abilities in rats with HIBD. Moreover, Dex, up-regulated miR-29a-3p or declined HDAC4 alleviated brain atrophy, repressed brain tissue injury, retrained the inflammation, repressed the apoptosis of neurons in the hippocampal region of rats with HIBD. HDAC4 was targeted and negatively regulated by miR-29a-3p. The study concludes that miR-29a-3p strengthened the effect of Dex on improving neurologic damage in newborn rats with HIBD by inhibiting HDAC4.

Role of MLC901 in increasing neurogenesis in rats with traumatic brain injury

BackgroundTraumatic brain injury is a dangerous life threatening condition. This study examines the role of MLC901 in increasing neurogenesis. The aim of this study was to demonstrate the role of MLC901 in increasing neuron cell (neurogenesis) in rat with traumatic brain injury using the synaptophysin marker. MethodsThe synaptophysin levels were measured as a marker for neuron cell (neurogenesis) of brain nerve cells in Sprague-Dawley rats aged 10–12 weeks, weighing 200–300 g. All rats (n = 10) were performed as traumatic brain injury using The Modified Marmourou Model, then they were divided into 2 group, one group was given MLC901 (n = 5) and the other group was not given MLC901 (n = 5). The synaptophysin levels in both groups were assessed after 6 weeks and also carried out an examination of immnuhistochemical from the brain tissue of both groups. ResultsThere was an increase in the number of neuron cells as evidenced by synaptophysin ihc staining in the rats given MLC 901 (Neuroaid II) compared to those without MLC 901. Synaptophysin levels were lower in the control group than in the MLC 901 group (81.6, SD: 13.52 vs 118.4, SD: 12.198, p = 0.062). ConclusionThese research suggest that MLC901 can increase neurogenesis in traumatic brain injury and also appeared as synaptophysin antibody that binding to cytoplasm of neuronal cells in the rat brain.

Coriolus versicolor polysaccharides (CVP) regulates neuronal apoptosis in cerebral ischemia-reperfusion injury via the p38MAPK signaling pathway.

BackgroundThis study aims to investigate the regulation of herbal polysaccharide, Coriolus versicolor polysaccharides (CVP), on neuronal apoptosis in a rat cerebral ischemia-reperfusion injury (CIRI) model. We also intend to explore the mechanisms and effectiveness of CVP in the treatment of neuronal apoptosis in CIRI rats, including neurological function, cerebral infarction volume, inflammatory factors, and the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway as well as its downstream protein cleaved-Caspase-3.MethodsA CIRI model was established in rats using the Longa method of middle cerebral artery occlusion. Neurological function scores and cerebral infarction volumes were measured in CIRI rats. Annexin V-fluorescein isothiocyanate (FITC) were used to measure neuronal apoptosis in CIRI rats. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in CIRI rats were determined using enzyme-linked immunosorbent assay (ELISA). A western blot assay was used to measure the protein expression levels of p38MAPK, phospho-p38MAPK (p-p38MAPK), Bcl-2, Bax and cleaved-Caspase-3 in brain tissue of CIRI rats.ResultsCVP can effectively improve the neurological function of rats after 6, 12, 24, and 48 h of CIRI. It can also improve the behavioral test, reduce the cerebral infarction volume and inhibit the apoptosis of nerve cells in CIRI rats. The protein expression levels of p-p38MAPK and cleaved-Caspase-3 exhibited a decreasing trend following CVP administration.ConclusionsCVP can significantly reduce the pathological characteristics of CIRI in rats and inhibit the apoptosis of nerve cells around the lesions. The mechanism of its effectiveness is related to inhibiting the activation of the p38MAPK signaling pathway.