This study aims to explore the effect of Xiaoxuming Decoction on synaptic plasticity in rats with acute cerebral ischemia-reperfusion. A rat model of cerebral ischemia-reperfusion injury was established by middle cerebral artery occlusion(MCAO). Rats were randomly assigned into a sham group, a MCAO group, and a Xiaoxuming Decoction(60 g·kg~(-1)·d~(-1)) group. The Longa score was rated to assess the neurological function of rats with cerebral ischemia for 1.5 h and reperfusion for 24 h. The 2,3,5-triphenyltetrazolium chloride(TTC) staining and hematoxylin-eosin(HE) staining were employed to observe the cerebral infarction and the pathological changes of brain tissue after cerebral ischemia, respectively. Transmission electron microscopy was employed to detect the structural changes of neurons and synapses in the ischemic penumbra, and immunofluorescence, Western blot to determine the expression of synaptophysin(SYN), neuronal nuclei(NEUN), and postsynaptic density 95(PSD95) in the ischemic penumbra. The experimental results showed that the modeling increased the Longa score and led to cerebral infarction after 24 h of ischemia-reperfusion. Compared with the model group, Xiaoxuming Decoction intervention significantly decreased the Longa score and reduced the formation of cerebral infarction area. The modeling led to the shrinking and vacuolar changes of nuclei in the brain tissue, disordered cell arrangement, and severe cortical ischemia-reperfusion injury, while the pathological damage in the Xiaoxuming Decoction group was mild. The modeling blurred the synaptic boundaries and broadened the synaptic gap, while such changes were recovered in the Xiaoxuming Decoction group. The modeling decreased the fluorescence intensity of NEUN and SYN, while the intensity in Xiaoxuming Decoction group was significantly higher than that in the model group. The expression of SYN and PSD95 in the ischemic penumbra was down-regulated in the model group, while such down-regulation can be alleviated by Xiaoxuming Decoction. In summary, Xiaoxuming Decoction may improve the synaptic plasticity of ischemic penumbra during acute cerebral ischemia-reperfusion by up-regulating the expression of SYN and PSD95.
Rats ; Animals ; Rats, Sprague-Dawley ; Brain Ischemia/drug therapy* ; Reperfusion Injury/metabolism* ; Infarction, Middle Cerebral Artery ; Neuronal Plasticity ; Reperfusion

BACKGROUND: Dysfunction of the gap junction channel protein connexin 43 (Cx43) contributes to myocardial ischemia/reperfusion (I/R)-induced ventricular arrhythmias. Cx43 can be regulated by small ubiquitin-like modifier (SUMO) modification. Protein inhibitor of activated STAT Y (PIASy) is an E3 SUMO ligase for its target proteins. However, whether Cx43 is a target protein of PIASy and whether Cx43 SUMOylation plays a role in I/R-induced arrhythmias are largely unknown. METHODS: Male Sprague-Dawley rats were infected with PIASy short hairpin ribonucleic acid (shRNA) using recombinant adeno-associated virus subtype 9 (rAAV9). Two weeks later, the rats were subjected to 45 min of left coronary artery occlusion followed by 2 h reperfusion. Electrocardiogram was recorded to assess arrhythmias. Rat ventricular tissues were collected for molecular biological measurements. RESULTS: Following 45 min of ischemia, QRS duration and QTc intervals statistically significantly increased, but these values decreased after transfecting PIASy shRNA. PIASy downregulation ameliorated ventricular arrhythmias induced by myocardial I/R, as evidenced by the decreased incidence of ventricular tachycardia and ventricular fibrillation, and reduced arrythmia score. In addition, myocardial I/R statistically significantly induced PIASy expression and Cx43 SUMOylation, accompanied by reduced Cx43 phosphorylation and plakophilin 2 (PKP2) expression. Moreover, PIASy downregulation remarkably reduced Cx43 SUMOylation, accompanied by increased Cx43 phosphorylation and PKP2 expression after I/R. CONCLUSION PIASy downregulation inhibited Cx43 SUMOylation and increased PKP2 expression, thereby improving ventricular arrhythmias in ischemic/reperfused rats heart.
Rats ; Male ; Animals ; Myocardial Reperfusion Injury/metabolism* ; Connexin 43/genetics* ; Sumoylation ; Down-Regulation ; Rats, Sprague-Dawley ; Arrhythmias, Cardiac/drug therapy* ; Myocardial Ischemia/metabolism* ; RNA, Small Interfering/metabolism*

This study explored the protective effect of astragaloside Ⅳ(AS-Ⅳ) on oxygen-glucose deprivation(OGD)-induced autophagic injury in PC12 cells and its underlying mechanism. An OGD-induced autophagic injury model in vitro was established in PC12 cells. The cells were divided into a normal group, an OGD group, low-, medium-, and high-dose AS-Ⅳ groups, and a positive drug dexmedetomidine(DEX) group. Cell viability was measured using the MTT assay. Transmission electron microscopy was used to observe autophagosomes and autolysosomes, and the MDC staining method was used to assess the fluorescence intensity of autophagosomes. Western blot was conducted to determine the relative expression levels of functional proteins LC3-Ⅱ/LC3-Ⅰ, Beclin1, p-Akt/Akt, p-mTOR/mTOR, and HIF-1α. Compared with the normal group, the OGD group exhibited a significant decrease in cell viability(P<0.01), an increase in autophagosomes(P<0.01), enhanced fluorescence intensity of autophagosomes(P<0.01), up-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and down-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.05 or P<0.01). Compared with the OGD group, the low-and medium-dose AS-Ⅳ groups and the DEX group showed a significant increase in cell viability(P<0.01), decreased autophagosomes(P<0.01), weakened fluorescence intensity of autophagosomes(P<0.01), down-regulated Beclin1, LC3-Ⅱ/LC3-Ⅰ, and HIF-1α(P<0.05 or P<0.01), and up-regulated p-Akt/Akt and p-mTOR/mTOR(P<0.01). AS-Ⅳ at low and medium doses exerted a protective effect against OGD-induced autophagic injury in PC12 cells by activating the Akt/mTOR pathway, subsequently influencing HIF-1α. The high-dose AS-Ⅳ group did not show a statistically significant difference compared with the OGD group. This study provides a certain target reference for the prevention and treatment of OGD-induced cellular autophagic injury by AS-Ⅳ and accumulates laboratory data for the secondary development of Astragali Radix and AS-Ⅳ.
Rats ; Animals ; PC12 Cells ; Proto-Oncogene Proteins c-akt/genetics* ; Glucose/therapeutic use* ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Apoptosis ; Reperfusion Injury/drug therapy*

This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.
Rats ; Male ; Animals ; Myocardial Reperfusion Injury/drug therapy* ; bcl-2-Associated X Protein/metabolism* ; Rats, Sprague-Dawley ; Caspase 3/metabolism* ; Interleukin-18 ; Wolfiporia ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Creatine Kinase, MB Form ; Apoptosis ; Polysaccharides/pharmacology* ; Superoxide Dismutase/metabolism* ; 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives*

OBJECTIVE: To investigate protective effect of Cordyceps sinensis (CS) through autophagy-associated adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway in acute kidney injury (AKI)-induced acute lung injury (ALI). METHODS: Forty-eight male Sprague-Dawley rats were divided into 4 groups according to a random number table, including the normal saline (NS)-treated sham group (sham group), NS-treated ischemia reperfusion injury (IRI) group (IRI group), and low- (5 g/kg·d) and high-dose (10 g/kg·d) CS-treated IRI groups (CS1 and CS2 groups), 12 rats in each group. Nephrectomy of the right kidney was performed on the IRI rat model that was subjected to 60 min of left renal pedicle occlusion followed by 12, 24, 48, and 72 h of reperfusion. The wet-to-dry (W/D) ratio of lung, levels of serum creatinine (Scr), blood urea nitrogen (BUN), inflammatory cytokines such as interleukin- β and tumor necrosis factor- α, and biomarkers of oxidative stress such as superoxide dismutase, malonaldehyde (MDA) and myeloperoxidase (MPO), were assayed. Histological examinations were conducted to determine damage of tissues in the kidney and lung. The protein expressions of light chain 3 II/light chain 3 I (LC3-II/LC3-I), uncoordinated-51-like kinase 1 (ULK1), P62, AMPK and mTOR were measured by Western blot and immunohistochemistry, respectively. RESULTS: The renal IRI induced pulmonary injury following AKI, resulting in significant increases in W/D ratio of lung, and the levels of Scr, BUN, inflammatory cytokines, MDA and MPO (P<0.01); all of these were reduced in the CS groups (P<0.05 or P<0.01). Compared with the IRI groups, the expression levels of P62 and mTOR were significantly lower (P<0.05 or P<0.01), while those of LC3-II/LC3-I, ULK1, and AMPK were significantly higher in the CS2 group (P<0.05 or P<0.01). CONCLUSION CS had a potential in treating lung injury following renal IRI through activation of the autophagy-related AMPK/mTOR signaling pathway in AKI-induced ALI.
Rats ; Male ; Animals ; AMP-Activated Protein Kinases/metabolism* ; Cordyceps/metabolism* ; Rats, Sprague-Dawley ; Kidney/pathology* ; Acute Kidney Injury/metabolism* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Cytokines/metabolism* ; Acute Lung Injury/drug therapy* ; Mammals/metabolism*

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*

Renal ischemia-reperfusion injury (IRI) is histologically characterized by tubular cell death. Diverse pathways of regulated cell death (RCD) have been reported to contribute to renal IRI in recent studies. In this review, we discuss the signaling pathways, regulators and crosstalk of RCD, including necroptosis, ferroptosis and pyroptosis, and their role in renal IRI in order to pave the way for new therapeutic opportunities.
Apoptosis ; Ferroptosis ; Humans ; Kidney/metabolism* ; Necroptosis ; Regulated Cell Death ; Reperfusion Injury/drug therapy*

Objective: To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury (CI/ RI), and to explore its neural protection mechanisms of the decoction. Methods: Rat middle cerebral artery ischemia/reperfusion injury model (ischemia for 2 h, reperfusion for 24 h) was prepared by the improved line plug method. Sixty male SD rats were randomly divided into sham operation group, model group, butylphthalide group(0.054 g/kg), Zhongfeng capsule high-dose groups (1.08 g/kg), Zhongfeng capsule middle-dose groups (0.54 g/kg), Zhongfeng capsule low-dose groups (0.27 g/kg), with 10 rats in each group. Rats were treated with Zhongfeng capsule by gavage once a day for 10 days. The rats were sacrificed and the brain tissue was obtained after the experiment in each group. Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group. The pathological changes of brain tissue were observed by HE staining. The serum levels of estradiol (E2) and follicle stimulating hormone (FSH) were determined by ELISA. The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively. Results: Compared with the sham operation group, the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group, while the brain index, neurological deficit score, gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P＜0.05 or P＜0.01). In the model group, nerve cells of brain tissue were loosely packed, interstitial edema, triangular in shape, nuclear pyknosis and dark-blue staining were observed. Compared with the model group, the body weight of rats was increased obviously, the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group (P＜0.05). The brain index, the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01), while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01). Conclusion: Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats, the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.
Animals ; Autophagy-Related Proteins/pharmacology* ; Beclin-1/metabolism* ; Body Weight ; Brain ; Brain Ischemia/metabolism* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/drug therapy*

OBJECTIVE: To reveal the neuroprotective effect and the underlying mechanisms of a mixture of the main components of Panax notoginseng saponins (TSPN) on cerebral ischemia-reperfusion injury and oxygen-glucose deprivation/reoxygenation (OGD/R) of cultured cortical neurons. METHODS: The neuroprotective effect of TSPN was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and live/dead cell assays. The morphology of dendrites was detected by immunofluorescence. Middle cerebral artery occlusion (MCAO) was developed in rats as a model of cerebral ischemia-reperfusion. The neuroprotective effect of TSPN was evaluated by neurological scoring, tail suspension test, 2,3,5-triphenyltetrazolium chloride (TTC) and Nissl stainings. Western blot analysis, immunohistochemistry and immunofluorescence were used to measure the changes in the Akt/mammalian target of rapamycin (mTOR) signaling pathway. RESULTS: MTT showed that TSPN (50, 25 and 12.5 µ g/mL) protected cortical neurons after OGD/R treatment (P<0.01 or P<0.05). Flow cytometry and live/dead cell assays indicated that 25 µ g/mL TSPN decreased neuronal apoptosis (P<0.05), and immunofluorescence showed that 25 µ g/mL TSPN restored the dendritic morphology of damaged neurons (P<0.05). Moreover, 12.5 µ g/mL TSPN downregulated the expression of Beclin-1, Cleaved-caspase 3 and LC3B-II/LC3B-I, and upregulated the levels of phosphorylated (p)-Akt and p-mTOR (P<0.01 or P<0.05). In the MCAO model, 50 µ g/mL TSPN improved defective neurological behavior and reduced infarct volume (P<0.05). Moreover, the expression of Beclin-1 and LC3B in cerebral ischemic penumbra was downregulated after 50 µ g/mL TSPN treatment, whereas the p-mTOR level was upregulated (P<0.05 or P<0.01). CONCLUSION TSPN promoted neuronal survival and protected dendrite integrity after OGD/R and had a potential therapeutic effect by alleviating neurological deficits and reversing neuronal loss. TSPN promoted p-mTOR and inhibited Beclin-1 to alleviate ischemic damage, which may be the mechanism that underlies the neuroprotective activity of TSPN.
Animals ; Beclin-1 ; Brain Ischemia/metabolism* ; Glucose ; Infarction, Middle Cerebral Artery/drug therapy* ; Mammals/metabolism* ; Neuroprotection ; Neuroprotective Agents/therapeutic use* ; Oxygen ; Panax notoginseng ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Reperfusion Injury/metabolism* ; Saponins/therapeutic use* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVES: To evaluate the effect of echinacoside (ECH) on cognitive dysfunction in post cerebral stroke model rats. METHODS: The post stroke cognitive impairment rat model was created by occlusion of the transient middle cerebral artery (MCAO). The rats were randomly divided into 3 groups by a random number table: the sham group (sham operation), the MCAO group (received operation for focal cerebral ischemia), and the ECH group (received operation for focal cerebral ischemia and ECH 50 mg/kg per day), with 6 rats in each group. The infarct volume and spatial learning were evaluated by triphenyl tetrazolium chloride staining and Morris water maze. The expression of α7nAChR in the hippocampus was detected by immunohistochemistry. The contents of acetylcholine (ACh), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), activities of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and catalase (CAT) were evaluated by enzyme linked immunosorbent assay. The neural apoptosis and autophagy were determined by TUNEL staining and LC3 staining, respectively. RESULTS: ECH significantly lessened the brain infarct volume and ameliorated neurological deficit in infarct volume and water content (both P<0.01). Compared with MCAO rats, administration of ECH revealed shorter escape latency and long retention time at 7, 14 and 28 days (all P<0.01), increased the α7nAChR protein expression, ACh content, and ChAT activity, and decreased AChE activity in MCAO rats (all P<0.01). ECH significantly decreased MDA content and increased the GSH content, SOD, and CAT activities compared with MCAO rats (all P<0.05). ECH suppressed neuronal apoptosis by reducing TUNEL-positive cells and also enhanced autophagy in MCAO rats (all P<0.01). CONCLUSION ECH treatment helped improve cognitive impairment by attenuating neurological damage and enhancing autophagy in MCAO rats.
Acetylcholinesterase ; Animals ; Autophagy ; Brain Ischemia/metabolism* ; Cerebral Infarction ; Cognitive Dysfunction/drug therapy* ; Glutathione/metabolism* ; Glycosides ; Infarction, Middle Cerebral Artery/drug therapy* ; Neuroprotective Agents/therapeutic use* ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury/drug therapy* ; Stroke/drug therapy* ; Superoxide Dismutase/metabolism* ; alpha7 Nicotinic Acetylcholine Receptor