Neuroprotective effects of apigenin on retinal ganglion cells in ischemia/reperfusion: modulating mitochondrial dynamics in in vivo and in vitro models.

BACKGROUND: Retinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR). METHODS: An in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted. RESULTS: API treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission. CONCLUSION: API protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.

Polyphyllin I alleviates neuroinflammation after cerebral ischemia-reperfusion injury via facilitating autophagy-mediated M2 microglial polarization.

Microglial activation and polarization play a central role in poststroke inflammation and neuronal damage. Modulating microglial polarization from pro-inflammatory to anti-inflammatory phenotype is a promising therapeutic strategy for the treatment of cerebral ischemia. Polyphyllin I (PPI), a steroidal saponin, shows multiple bioactivities in various diseases, but the potential function of PPI in cerebral ischemia is not elucidated yet. In our study, the influence of PPI on cerebral ischemia-reperfusion injury was evaluated. Mouse middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation and reoxygenation (OGD/R) model were constructed to mimic cerebral ischemia-reperfusion injury in vivo and in vitro. TTC staining, TUNEL staining, RT-qPCR, ELISA, flow cytometry, western blot, immunofluorescence, hanging wire test, rotarod test and foot-fault test, open-field test and Morris water maze test were performed in our study. We found that PPI alleviated cerebral ischemia-reperfusion injury and neuroinflammation, and improved functional recovery of mice after MCAO. PPI modulated microglial polarization towards anti-inflammatory M2 phenotype in MCAO mice in vivo and post OGD/R in vitro. Besides, PPI promoted autophagy via suppressing Akt/mTOR signaling in microglia, while inhibition of autophagy abrogated the effect of PPI on M2 microglial polarization after OGD/R. Furthermore, PPI facilitated autophagy-mediated ROS clearance to inhibit NLRP3 inflammasome activation in microglia, and NLRP3 inflammasome reactivation by nigericin abolished the effect of PPI on M2 microglia polarization. In conclusion, PPI alleviated post-stroke neuroinflammation and tissue damage via increasing autophagy-mediated M2 microglial polarization. Our data suggested that PPI had potential for ischemic stroke treatment.

Annexin-A5 monomer as a membrane repair agent for the treatment of renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is one of the causes of acute kidney injury. Annexin A5 (AnxA5), a calcium-dependent cell membrane-binding protein, shows protective effects in various organ IRI models. This study explored the therapeutic effect of exogenous AnxA5 monomer protein on renal IRI and its potential mechanism of action. METHODS AND RESULTS: Different doses of AnxA5 were injected intravenously to treat bilateral renal IRI in SD rats. This model confirmed the protective effects of AnxA5 on kidney structure and function. In vitro, HK-2 cells were subjected to hypoxia for 12 h, followed by restoration of normal oxygen supply to simulate IRI. In vitro experiments demonstrated the mechanism of action of AnxA5 by measuring cellular activity and permeability. A comparison of the mutant AnxA5 protein M23 and the application of a calcium-free culture medium further validated the protective effect of AnxA5 by forming a network structure. CONCLUSIONS: Exogenous AnxA5 monomers prevented renal IRI by binding to the damaged renal tubular epithelial cell membrane, forming a two-dimensional network structure to maintain cell membrane integrity, and ultimately prevent cell death.

Utilizing network pharmacology, molecular docking, and animal models to explore the therapeutic potential of the WenYang FuYuan recipe for cerebral ischemia-reperfusion injury through AGE-RAGE and NF-κB/p38MAPK signaling pathway modulation.

BACKGROUND: Stroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment. OBJECTIVE: This study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe. MATERIALS AND METHODS: Active ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways. RESULTS: We identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, ß-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <-10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100ß and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated. CONCLUSION: This study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.

Xa inhibitor edoxaban ameliorates hepatic ischemia-reperfusion injury via PAR-2-ERK 1/2 pathway.

Hepatic ischemia-reperfusion injury causes liver damage during surgery. In hepatic ischemia-reperfusion injury, the blood coagulation cascade is activated, causing microcirculatory incompetence and cellular injury. Coagulation factor Xa (FXa)- protease-activated receptor (PAR)-2 signaling activates inflammatory reactions and the cytoprotective effect of FXa inhibitor in several organs. However, no studies have elucidated the significance of FXa inhibition on hepatic ischemia-reperfusion injury. The present study elucidated the treatment effect of an FXa inhibitor, edoxaban, on hepatic ischemia-reperfusion injury, focusing on FXa-PAR-2 signaling. A 60 min hepatic partial-warm ischemia-reperfusion injury mouse model and a hypoxia-reoxygenation model of hepatic sinusoidal endothelial cells were used. Ischemia-reperfusion injury mice and hepatic sinusoidal endothelial cells were treated and pretreated, respectively with or without edoxaban. They were incubated during hypoxia/reoxygenation in vitro. Cell signaling was evaluated using the PAR-2 knockdown model. In ischemia-reperfusion injury mice, edoxaban treatment significantly attenuated fibrin deposition in the sinusoids and liver histological damage and resulted in both anti-inflammatory and antiapoptotic effects. Hepatic ischemia-reperfusion injury upregulated PAR-2 generation and enhanced extracellular signal-regulated kinase 1/2 (ERK 1/2) activation; however, edoxaban treatment reduced PAR-2 generation and suppressed ERK 1/2 activation in vivo. In the hypoxia/reoxygenation model of sinusoidal endothelial cells, hypoxia/reoxygenation stress increased FXa generation and induced cytotoxic effects. Edoxaban protected sinusoidal endothelial cells from hypoxia/reoxygenation stress and reduced ERK 1/2 activation. PAR-2 knockdown in the sinusoidal endothelial cells ameliorated hypoxia/reoxygenation stress-induced cytotoxicity and suppressed ERK 1/2 phosphorylation. Thus, edoxaban ameliorated hepatic ischemia-reperfusion injury in mice by protecting against micro-thrombosis in sinusoids and suppressing FXa-PAR-2-induced inflammation in the sinusoidal endothelial cells.

The Promising Effects of Erdosteine and Vitamin B in the Liver Ischemia/Reperfusion Model in Anesthetized Rats.

Background and Objectives: Erdosteine (Erd) is an antioxidant and anti-inflammatory drug. Vitamin B has been reported to exert anti-inflammatory and antioxidant effects. In this study, we investigated the effect of erdosteine and vitamin B complex on a liver ischemia/reperfusion (I/R) model. Materials and Methods: Thirty-two Wistar Albino male rats weighing 350-400 g were used. The animals were randomly selected and divided into four groups. The groups are as follows: first group (Sham), second group (I/R), third group (I/R + vit B), and fourth group (I/R + vit B + Erd). Rats were subjected to 45 min of hepatic ischemia, followed by a 45 min reperfusion period in the I/R and Vitamin B + Erd groups. An amount of 150 mg/kg/day of erdosteine was given orally for 2 days, and 0.05 mL/kg of i.p. vitamin B complex was given 30 min before the reperfusion. Serum biochemical parameters were measured. Serum Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) were measured, and the Oxidative Stress Index (OSI) was calculated. Hepatic tissue samples were taken for the evaluation of histopathological features. Results: In terms of all histopathological parameters, there were significant differences in the I/R + vit B group and I/R + vit B + Erd group compared with the I/R group (p < 0.01). In terms of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), TNF-alpha, and IL-6 levels, there were significant differences between the I/R group and treatment groups (p < 0.01). The lowest TOS and OSI levels were obtained in the treatment groups, and these groups had statistically significantly higher TAS levels compared with the sham and I/R groups (p < 0.01). Conclusions: As a preliminary experimental study, our study suggests that these agents may have potential diagnostic and therapeutic implications for both ischemic conditions and liver-related diseases. These results suggest that the combination of vit B + Erd may be used to protect against the devastating effects of I/R injury. Our study needs to be confirmed by clinical studies with large participation.

Effects of butyrate on intestinal ischemia-reperfusion injury via the HMGB1-TLR4-MyD88 signaling pathway.

BACKGROUND: This study combined bioinformatics and experimental verification in a mouse model of intestinal ischemia-reperfusion injury (IRI) to explore the protection mechanism exerted by butyrate against IRI. METHODS: GeneCards, Bioinformatics Analysis Tool for Molecular Mechanisms of Traditional Chinese Medicine and GSE190581 were used to explore the relationship between butyrate and IRI and aging. Protein-protein interaction networks involving butyrate and IRI were constructed via the STRING database, with hub gene analysis performed through Cytoscape. Functional enrichment analysis was conducted on intersection genes. A mouse model of IRI was established, followed by direct arterial injection of butyrate. The experiment comprised five groups: normal, sham, model, vehicle, low-dose butyrate, and high-dose butyrate. Intestinal tissue observation was done via transmission electron microscopy (TEM), histological examination via hematoxylin and eosin (H&E) staining, tight junction proteins detection via immunohistochemistry, and Western blot analysis of hub genes. Drug-target interactions were evaluated through molecular docking. RESULTS: Butyrate protected against IRI by targeting 458 genes, including HMGB1 and TLR4. Toll-like receptor pathway was implicated. Butyrate improved intestinal IRI by reducing mucosal damage, increasing tight junction proteins, and lowering levels of HMGB1, TLR4, and MyD88. Molecular docking showed strong binding energies between butyrate and HMGB1 (-3.7 kcal/mol) and TLR4 (-3.8 kcal/mol). CONCLUSIONS: According to bioinformatics predictions, butyrate mitigates IRI via multiple-target and multiple-channel mechanisms. The extent of IRI can be reduced by butyrate through the inhibition of the HMGB1-TLR4-MyD88 signaling pathway, which is related to senescence.

A study on the mechanism of Beclin-1 m6A modification mediated by catalpol in protection against neuronal injury and autophagy following cerebral ischemia.

OBJECTIVE: Catalpol (CAT) has various pharmacological activities and plays a protective role in cerebral ischemia. It has been reported that CAT played a protective role in cerebral ischemia by upregulaing NRF1 expression. Bioinformatics analysis reveals that NRF1 can be used as a transcription factor to bind to the histone acetyltransferase KAT2A. However, the role of KAT2A in cerebral ischemia remains to be studied. Therefore, we aimed to investigate the role of CAT in cerebral ischemia and its related mechanism. METHODS: In vitro, a cell model of oxygen and glucose deprivation/reperfusion (OGD/R) was constructed, followed by evaluation of neuronal injury and the expression of METTL3, Beclin-1, NRF1, and KAT2A. In vivo, a MCAO rat model was prepared by means of focal cerebral ischemia, followed by assessment of neurological deficit and brain injury in MCAO rats. Neuronal autophagy was evaluated by observation of autophagosomes in neurons or brain tissues by TEM and detection of the expression of LC3 and p62. RESULTS: In vivo, CAT reduced the neurological function deficit and infarct volume, inhibited neuronal apoptosis in the cerebral cortex, and significantly improved neuronal injury and excessive autophagy in MCAO rats. In vitro, CAT restored OGD/R-inhibited cell viability, inhibited cell apoptosis, LDH release, and neuronal autophagy. Mechanistically, CAT upregulated NRF1, NRF1 activated METTL3 via KAT2A transcription, and METTL3 inhibited Beclin-1 via m6A modification. CONCLUSION: CAT activated the NRF1/KAT2A/METTL3 axis and downregulated Beclin-1 expression, thus relieving neuronal injury and excessive autophagy after cerebral ischemia.

All-trans retinoic acid pretreatment of mesenchymal stem cells enhances the therapeutic effect on acute kidney injury.

A promising new therapy option for acute kidney injury (AKI) is mesenchymal stem cells (MSCs). However, there are several limitations to the use of MSCs, such as low rates of survival, limited homing capacity, and unclear differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy of AKI. We established a renal ischemia/reperfusion injury model and treated mice with ATRA-pretreated MSCs via tail vein injection. We found that AKI mice treated with ATRA-MSCs significantly improved renal function compared with DMSO-MSCs treatment. RNA sequencing screened that hyaluronic acid (HA) production from MSCs promoted by ATRA. Further validation by chromatin immunoprecipitation experiments verified that retinoic acid receptor RARα/RXRγ was a potential transcription factor for hyaluronic acid synthase 2. Additionally, an in vitro hypoxia/reoxygenation model was established using human proximal tubular epithelial cells (HK-2). After co-culturing HK-2 cells with ATRA-pretreated MSCs, we observed that HA binds to cluster determinant 44 (CD44) and activates the PI3K/AKT pathway, which enhances the anti-inflammatory, anti-apoptotic, and proliferative repair effects of MSCs in AKI. Inhibition of the HA/CD44 axis effectively reverses the renal repair effect of ATRA-pretreated MSCs. Taken together, our study suggests that ATRA pretreatment promotes HA production by MSCs and activates the PI3K/AKT pathway in renal tubular epithelial cells, thereby enhancing the efficacy of MSCs against AKI.

Hepatoprotective effects of baicalein against liver ischemia-reperfusion injury and partial hepatectomy in a rat model.

BACKGROUND: Baicalein is the main active flavonoid in Scutellariae Radix and is included in shosaikoto, a Kampo formula used for treating hepatitis and jaundice. However, little is known about its hepatoprotective effects against hepatic ischemia-reperfusion injury (HIRI), a severe clinical condition directly caused by interventional procedures. We aimed to investigate the hepatoprotective effects of baicalein against HIRI and partial hepatectomy (HIRI + PH) and its potential underlying mechanisms. METHODS AND RESULTS: Male Sprague-Dawley rats received either baicalein (5 mg/kg) or saline intraperitoneally and underwent a 70% hepatectomy 15 min after hepatic ischemia. After reperfusion, liver and blood samples were collected. Survival was monitored 30 min after hepatic ischemia and hepatectomy. In interleukin 1ß (IL-1ß)-treated primary cultured rat hepatocytes, the influence of baicalein on inflammatory mediator production and the associated signaling pathway was analyzed. Baicalein suppressed apoptosis and neutrophil infiltration, which are the features of HIRI + PH treatment-induced histological injury. Baicalein also reduced the mRNA expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α). In addition, HIRI + PH treatment induced liver enzyme deviations in the serum and hypertrophy of the remnant liver, which were suppressed by baicalein. In the lethal HIRI + PH treatment group, baicalein significantly reduced mortality. In IL-1ß-treated rat hepatocytes, baicalein suppressed TNF-α and chemokine mRNA expression as well as the activation of nuclear factor-kappa B (NF-κB) and Akt. CONCLUSIONS: Baicalein treatment attenuates HIRI + PH-induced liver injury and may promote survival. This potential hepatoprotection may be partly related to suppressing inflammatory gene induction through the inhibition of NF-κB activity and Akt signaling in hepatocytes.

Gallic acid attenuates torsion/detorsion-induced testicular injury in rats through suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress.

It was aimed to evaluate whether gallic acid (GA) have a beneficial effect in the testicular ischemia/reperfusion injury (IRI) model in rats for the first time. Testicular malondialdehyde, 8-hydroxy-2'-deoxyguanosine, superoxide dismutase, catalase, high mobility group box 1 protein, nuclear factor kappa B, tumor necrosis factoralpha, interleukin-6, myeloperoxidase, 78-kDa glucose-regulated protein, activating transcription factor 6, CCAAT-enhancer-binding protein homologous protein and caspase-3 levels were determined using colorimetric methods. The oxidative stress, inflammation, endoplasmic reticulum stress and apoptosis levels increased statistically significantly in the IRI group compared with the sham operated group (p < 0.05). GA application improved these damage significantly (p < 0.05). Moreover, it was found that the results of histological examinations supported the biochemical results to a statistically significant extent. Our findings suggested that GA may be evaluated as a protective agent against testicular IRI.

Effects of ketamine on penile tissues in an experimental priapism model in rats.

BACKGROUND: This study aimed to evaluate the histopathological and biochemical effects of ketamine on penile tissues following ischemia-reperfusion injury induced by priapism. METHODS: Twenty-four male rats were randomized into three groups. Group 1 served as the control group. Group 2 underwent the priapism model to induce ischemia-reperfusion injury. Group 3, the treatment group, experienced a similar ischemia-reperfusion model as Group 2; additionally, 50 mg/kg of ketamine was administered intraperitoneally just before reperfusion. Blood biochemical analyses and penile histopathological evaluations were performed. RESULTS: In Group 3, significant improvements were observed in all histopathological scores, including desquamation, edema, inflammation, and vasocongestion compared to Group 2 (p<0.001). Blood biochemical analyses showed that the malondialdehyde (MDA) levels were recorded as 10 in Group 2, with a significant decrease in Group 3 (p=0.013). Similarly, proinflammatory cytokine levels, including interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), were found to be suppressed in Group 3 compared to Group 2 (p=0.003, p=0.022, and p=0.028, respectively). Antioxidant enzyme activities, such as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), were higher in Group 3 compared to Group 2 (p=0.016 and p=0.024, respec-tively). CONCLUSION: Ketamine is an effective anesthetic agent in alleviating the effects of penile ischemia-reperfusion injury.

Naodesheng Pills Ameliorate Cerebral Ischemia Reperfusion-Induced Ferroptosis via Inhibition of the ERK1/2 Signaling Pathway.

Background: Ferroptosis plays a crucial role in the occurrence and development of cerebral ischemia-reperfusion (I/R) injury and is regulated by mitogen-activated protein kinase 1/2 (ERK1/2). In China, Naodesheng Pills (NDSP) are prescribed to prevent and treat cerebrosclerosis and stroke. However, the protective effects and mechanism of action of NDSP against cerebral I/R-induced ferroptosis remain unclear. We investigated whether NDSP exerts its protective effects against I/R injury by regulating ferroptosis and aimed to elucidate the underlying mechanisms. Methods: The efficacy of NDSP was evaluated using a Sprague-Dawley rat model of middle cerebral artery occlusion and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model. Brain injury was assessed using 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin staining, Nissl staining, and neurological scoring. Western blotting was performed to determine the expression levels of glutathione peroxidase 4 (GPX4), divalent metal-ion transporter-1 (DMT1), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor 1 (TFR1). Iron levels, oxidative stress, and mitochondrial morphology were also evaluated. Network pharmacology was used to assess the associated mechanisms. Results: NDSP (1.08 g/kg) significantly improved cerebral infarct area, cerebral water content, neurological scores, and cerebral tissue damage. Furthermore, NDSP inhibited I/R- and OGD/R-induced ferroptosis, as evidenced by the increased protein expression of GPX4 and SLC7A11, suppression of TFR1 and DMT1, and an overall reduction in oxidative stress and Fe2+ levels. The protective effects of NDSP in vitro were abolished by the GPX4 inhibitor RSL3. Network pharmacology analysis revealed that ERK1/2 was the core target gene and that NDSP reduced the amount of phosphorylated ERK1/2. Conclusion: NDSP exerts its protective effects against I/R by inhibiting cerebral I/R-induced ferroptosis, and this mechanism is associated with the regulation of ferroptosis via the ERK1/2 signaling pathway.

Mechanism of Astragalus mongholicus Bunge ameliorating cerebral ischemia-reperfusion injury: Based on network pharmacology analysis and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus mongholicus Bunge (AMB) is a herb with wide application in traditional Chinese medicine, exerting a wealth of pharmacological effects. AMB has been proven to have an evident therapeutic effect on ischemic cerebrovascular diseases, including cerebral ischemia-reperfusion injury (CIRI). However, the specific mechanism underlying AMB in CIRI remains unclear. AIM OF THE STUDY: This study aimed to investigate the potential role of AMB in CIRI through a comprehensive approach of network pharmacology and in vivo experimental research. METHODS: The intersection genes of drugs and diseases were obtained through analysis of the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Gene Expression Omnibus (GEO) database. The protein-protein interaction (PPI) network was created through the string website. Meanwhile, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out using R studio, and thereafter the key genes were screened. Then, the molecular docking prediction was made between the main active ingredients and target genes, and hub genes with high binding energy were obtained. In addition, molecular dynamic (MD) simulation was used to validate the result of molecular docking. Based on the results of network pharmacology, we used animal experiments to verify the predicted hub genes. First, the rat middle cerebral artery occlusion and reperfusion (MACO/R) model was established and the effective dose of AMB in CIRI was determined by behavioral detection and 2,3,5-Triphenyltetrazolium chloride (TTC) staining. Then the target proteins corresponding to the hub genes were measured by Western blot. Moreover, the level of neuronal death was measured using hematoxylin and eosin (HE) and Nissl staining. RESULTS: Based on the analysis of the TCMSP database and GEO database, a total of 62 intersection target genes of diseases and drugs were obtained. The KEGG enrichment analysis showed that the therapeutic effect of AMB on CIRI might be realized through the advanced glycation endproduct-the receptor of advanced glycation endproduct (AGE-RAGE) signaling pathway in diabetic complications, nuclear factor kappa-B (NF-κB) signaling pathway and other pathways. Molecular docking results showed that the active ingredients of AMB had good binding potential with hub genes that included Prkcb, Ikbkb, Gsk3b, Fos and Rela. Animal experiments showed that AWE (60 g/kg) could alleviate CIRI by regulating the phosphorylation of PKCß, IKKß, GSK3ß, c-Fos and NF-κB p65 proteins. CONCLUSION: AMB exerts multi-target and multi-pathway effects against CIRI, and the underlying mechanism may be related to anti-apoptosis, anti-inflammation, anti-oxidative stress and inhibiting calcium overload.

Role of cAMP/pCREB and GSK-3ß/NF-κB p65 signaling pathways in the renoprotective effect of mirabegron against renal ischemia-reperfusion injury in rats.

Acute kidney injury and other renal disorders are thought to be primarily caused by renal ischemia-reperfusion (RIR). Cyclic adenosine monophosphate (cAMP) has plenty of physiological pleiotropic effects and preserves tissue integrity and functions. This research aimed to examine the potential protective effects of the ß3-adrenergic receptors agonist mirabegron in a rat model of RIR and its underlying mechanisms. Male rats enrolled in this work were given an oral dose of 30 mg/kg mirabegron for two days before surgical induction of RIR. Renal levels of kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), Interleukin-10 (IL-10), cAMP, cAMP-responsive element binding protein (pCREB), and glycogen synthase kinase-3 beta (GSK-3ß) were assessed along with blood urea nitrogen and serum creatinine. Additionally, caspase-3 and nuclear factor-kappa B (NF-κB) p65 were explored by immunohistochemical analysis. Renal specimens were inspected for histopathological changes. RIR led to renal tissue damage with elevated blood urea nitrogen and serum creatinine levels. The renal KIM-1, MCP-1, TNF-α, and GSK-3ß were significantly increased, while IL-10, cAMP, and pCREB levels were reduced. Moreover, upregulation of caspase-3 and NF-κB p65 protein expression was seen in RIR rats. Mirabegron significantly reduced kidney dysfunction, histological abnormalities, inflammation, and apoptosis in the rat renal tissues. Mechanistically, mirabegron mediated these effects via modulation of cAMP/pCREB and GSK-3ß/NF-κB p65 signaling pathways. Mirabegron administration could protect renal tissue and maintain renal function against RIR.

Triptolide alleviates cerebral ischemia/reperfusion injury via regulating the Fractalkine/CX3CR1 signaling pathway.

PURPOSE: To evaluate the potential efficacy of Triptolide (TP) on cerebral ischemia/reperfusion injury (CIRI) and to uncover the underlying mechanism through which TP regulates CIRI. METHODS: We constructed a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate CIRI, and established a lipopolysaccharide (LPS)-stimulated BV-2 cell model to mimic the inflammatory state during CIRI. The neurological deficits score (NS) of mice were measured for assessment of neurologic functions. Both the severity of cerebral infarction and the apoptosis level in mouse brain tissues or cells were respectively evaluated using corresponding techniques. The expression levels of Ionized calcium binding adapter molecule 1 (IBA-1), Inductible Nitric Oxide Synthase (iNOS), Arginase 1 (Arg-1), Tumor necrosis factor-α (TNF-α), Interleukin 1ß (IL-1ß), Cysteine histoproteinase S (CTSS), Fractalkine, chemokine C-X3-C motif receptor 1 (CX3CR1), BCL-2-associated X protein (BAX), and antiapoptotic proteins (Bcl-2) were detected using immunofluorescence, qRT-PCR as well as Western blot, respectively. RESULTS: Relative to the Sham group, treatment with TP attenuated the increased NS, infarct area and apoptosis levels observed in MCAO/R mice. Upregulated expression levels of IBA-1, iNOS, Arg-1, TNF-α and IL-1ß were found in MCAO/R mice, while TP suppressed iNOS, TNF-α and IL-1ß expression, and enhanced Arg-1 expression in both MCAO/R mice and LPS-stimulated BV-2 cells. Besides, TP inhibited the CTSS/Fractalkine/CX3CR1 pathway activation in both MCAO/R mice and LPS-induced BV-2 cells, while overexpression of CTSS reversed such effect. Co-culturing HT-22 cells with TP+LPS-treated BV-2 cells led to enhanced cell viability and decreased apoptosis levels. However, overexpression of CTSS further aggravated HT-22 cell injury. CONCLUSION: TP inhibits not only microglia polarization towards the M1 phenotype by suppressing the CTSS/Fractalkine/CX3CR1 pathway activation, but also HT-22 apoptosis by crosstalk with BV-2 cells, thereby ameliorating CIRI. These findings reveal a novel mechanism of TP in improving CIRI, and offer potential implications for addressing the preventive and therapeutic strategies of CIRI.

Synthesis and Neuroprotective Evaluation of Substituted Indanone/Benzofuranone and Piperidine Hybrids.

Based on the neuroprotection of butylphthalide and donepezil, a series of indanone/benzofuranone and piperidine hybrids were designed and synthesized for assessment of their neuroprotective activities, aiming to enhance the bioavailability and therapeutic efficacy of natural phthalide analogues. Within this study, it was observed that most indanone derivatives bearing 1-methylpiperidine in the tail segment demonstrated superior neuroprotective effects on the oxygen glucose deprivation/reperfusion (OGD/R)-induced rat primary neuronal cell injury model in vitro compared to benzofuranone compounds. Among the synthesized compounds, 11 (4, 14, 15, 22, 26, 35, 36, 37, 48, 49, and 52) displayed robust cell viabilities in the OGD/R model, along with favorable blood-brain barrier permeability as confirmed by the parallel artificial membrane permeability assay. Notably, compound 4 showed significant neuronal cell viabilities within the concentration range of 3.125 to 100 µM, without inducing cytotoxicity. Further results from in vivo middle cerebral artery occlusion/R experiments revealed that 4 effectively ameliorated ischemia-reperfusion injury, reducing the infarct volume to 18.45% at a dose of 40 mg/kg. This outcome suggested a superior neuroprotective effect compared to edaravone at 20 mg/kg, further highlighting the potential therapeutic efficacy of compound 4 in addressing neurological disorders.

[Effect of Naotaifang on microglial polarization and glial scar following cerebral ischemia reperfusion injury].

This study aims to investigate the effect of Naotaifang(NTF) on the proteins associated with microglial polarization and glial scar in the rat model of cerebral ischemia reperfusion injury(CIRI). The CIRI model was established by middle cerebral artery occlusion/reperfusion. The 48 successfully modeled rats were randomized into model 7 d, model 14 d, NTF 7 d, and NTF 14 d groups(n=12). In addition, 12 SD rats were selected as the sham group. The NTF group was administrated with NTF suspension at 27 g·kg~(-1)·d~(-1) by gavage, and the sham, model 7 d, and model 14 d groups were administrated with the same volume of normal saline every day by gavage for 7 and 14 days, respectively. After the intervention, Longa score was evaluated. The infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) staining. Morris water maze and open field tests were carried out to evaluate the spatial learning, memory, cognitive function, and anxiety degree of rats. Hematoxylin-eosin(HE) staining was employed to observe the morphological structure and damage of the brain tissue. The immunofluorescence assay was employed to measure the expression of glial fibrillary acidic protein(GFAP) and glial scar. Western blot was employed to determine the protein levels of GFAP, neurocan, phosphacan, CD206, arginase-1(Arg-1), interleukin(IL)-1ß, IL-6, and IL-4. Compared with the sham, model 7 d and model 14 d groups showed cerebral infarction of different degrees, severe pathological injury of cerebral cortex and hippocampus, neurological impairment, reduced spatial learning and memory, cognitive dysfunction, severe anxiety, astrocyte hyperplasia, thickening penumbra glial scar, and up-regulated protein levels of IL-1ß, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P<0.01). Compared with the model group, NTF 7 d and NTF 14 d groups improved spatial learning, memory, and cognitive function, reduced anxiety, improved nerve function, reduced cerebral infarction volume, reduced astrocyte hyperplasia, thinned penumbra glial scar, down-regulated the protein levels of GFAP, neurocan, phosphacan, IL-6, and IL-1ß, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P<0.05 or P<0.01). NTF exerts a neuroprotective effect on CIRI by inducing the M2 polarization of microglia, inhibiting inflammatory response, and reducing the formation of glial scar.

Dexmedetomidine protects the uterus against ischemia-reperfusion injury in rats.

OBJECTIVE: This study aimed to analyze the histopathological and biochemical effects of dexmedetomidine on the rat uteri exposed to experimental ischemia-reperfusion injury. MATERIALS AND METHODS: Twenty-four female rats were randomly divided into three groups. Group 1 was defined as the control group. An experimental uterine ischemia-reperfusion model was created in Group 2. Group 3 was assigned as the treatment group. Similar uterine ischemia-reperfusion models were created for the rats in Group 3, and then, unlike the other groups, 100 µg/kg of dexmedetomidine was administered intraperitoneally immediately after the onset of reperfusion. In blood biochemical analysis, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA), interleukin 1beta (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels were measured. In the histopathological analyses, endometrial epithelial glandular changes (leukocytosis, cell degeneration) and endometrial stromal changes (congestion, edema) were analyzed using the tissue damage scoring system. RESULTS: It was observed that IL-1ß, IL-6, and TNF-α levels were significantly suppressed in Group 3 compared to Group 2 (p=0.001, p<0.001 and p=0.001, respectively). MDA level was noted as the highest in Group 2. The MDA value in Group 3 was measured at 5.37±0.82, which was significantly decreased compared to Group 2 (p<0.001). An increase in antioxidant enzyme activities (SOD and GSH-PX) was observed in Group 3 compared to Group 2 (p=0.001 and p=0.006, respectively). In our histopathological analysis, a significant improvement in endometrial epithelial glandular and endometrial stromal changes was revealed in Group 3 compared to Group 2 (p<0.001). CONCLUSIONS: In our study, it has been documented that dexmedetomidine protects the uterine tissue against ischemia-reperfusion injury.

The protective effect and mechanism of glycosides of cistanche deserticola on rats in middle cerebral artery occlusion (MCAO) model.

Cerebral ischemia-reperfusion injury (CIRI) occurs frequently clinically as a complication following cardiovascular resuscitation resulting in neuronal damage specifically to the hippocampal CA1 region with consequent cognitive impairment. Apoptosis and oxidative stress were proposed as major risk factors associated with CIRI development. Previously, glycosides obtained from Cistanche deserticola (CGs) were shown to play a key role in counteracting CIRI; however, the underlying mechanisms remain to be determined. This study aimed to investigate the neuroprotective effect of CGs on subsequent CIRI in rats. The model of CIRI was established for 2 hr and reperfusion for 24 hr by middle cerebral artery occlusion (MCAO) model. The MCAO rats were used to measure the antioxidant and anti-apoptotic effects of CGs on CIRI. Neurological function was evaluated by the Longa neurological function score test. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to detect the area of cerebral infarction. Nissl staining was employed to observe neuronal morphology. TUNEL staining was used to detect neuronal apoptosis, while Western blot determined protein expression levels of factors for apoptosis-related and PI3K/AKT/Nrf2 signaling pathway. Data demonstrated that CGs treatment improved behavioral performance, brain injury, and enhanced antioxidant and anti-apoptosis in CIRI rats. In addition, CGs induced activation of PI3K/AKT/Nrf2 signaling pathway accompanied by inhibition of the expression of apoptosis-related factors. Evidence indicates that CGs amelioration of CIRI involves activation of the PI3K/AKT/Nrf2 signaling pathway associated with increased cellular viability suggesting these glycosides may be considered as an alternative compound for CIRI treatment.