Longa Score Research Articles

3D biological scaffold delivers Bergenin to reduce neuroinflammation in rats with cerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH) is a severe form of stroke characterized by high incidence and mortality rates. Currently, there is a significant lack of effective treatments aimed at improving clinical outcomes. Our research team has developed a three-dimensional (3D) biological scaffold that incorporates Bergenin, allowing for the sustained release of the compound.MethodsThis 3D biological scaffold was fabricated using a combination of photoinitiator, GEMA, silk fibroin, and decellularized brain matrix (dECM) to encapsulate Bergenin through advanced 3D bioprinting techniques. The kinetics of drug release were evaluated through both in vivo and in vitro studies. A cerebral hemorrhage model was established, and a 3D biological scaffold containing Bergenin was transplanted in situ. Levels of inflammatory response, oxidative stress, and apoptosis were quantified. The neurological function of rats with cerebral hemorrhage was assessed on days 1, 3, and 5 using the turning test, forelimb placement test, Longa score, and Bederson score.ResultsThe 3D biological scaffold incorporating Bergenin significantly enhances the maintenance of drug concentration in the bloodstream, leading to a marked reduction in inflammatory markers such as IL-6, iNOS, and COX-2 levels in a cerebral hemorrhage model, primarily through the inhibition of the NF-κB pathway. Additionally, the scaffold effectively reduces the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in primary cultured astrocytes, which in turn decreases the production of reactive oxygen species (ROS) and inhibits IL-6 production induced by hemin. Subsequent experiments reveal that the 3D biological scaffold containing Bergenin promotes the activation of the Nrf-2/HO-1 signaling pathway, both in vivo and in vitro, thereby preventing cell death. Moreover, the application of this 3D biological scaffold has been demonstrated to improve drug retention in the bloodstream.ConclusionThis strategy effectively mitigates inflammation, oxidative stress, and cell death in rats with cerebral hemorrhage by inhibiting the NF-κB pathway while concurrently activating the Nrf-2/HO-1 pathway.

Synergism of salvianolic acid B and ginsenoside Rg1 magnifies the therapeutic potency against ischemic stroke.

Even though considerable progress has been made to reduce insult, ischemic stroke is still a significant cause of mortality and morbidity in the world, and new therapeutic strategies are urgently needed. In the present study, the magnesium salt of salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) combination as a multicomponent strategy against stroke was evaluated. The synergistic effect of Sa1B and Rg1 was evaluated by Bliss independence analysis on the middle cerebral artery occlusion model. The infarct volume, neuroethology, cerebral structure, and neurocyte number were evaluated by 3,5-triphenyltetrazolium chloride staining, Longa score, Garcia score, hematoxylin-eosin staining, and Nissl staining, respectively. Metabolomics was used to search for potential biomarkers and explore the mechanism of Sa1B/Rg1. First, the superior effects of SalB/Rg1 than SalB or Rg1 at the same dose were evaluated. Compared with SalB ( P < 0.001) or Rg1 ( P < 0.01), SalB/Rg1 significantly decreased infarct volume through 3,5-triphenyltetrazolium chloride staining and protected the structural integrity of cortex and striatum. The superior effect of SalB/Rg1 on neurological behavior was also detected compared with SalB or Rg1 significantly. Accompanying behavioral improvement, a considerable increase of SalB/Rg1 on neurons detected by Nissl staining was found on the cortex compared with SalB ( P < 0.05) or Rg1 ( P < 0.01). Second, the synergistic effect between SalB and Rg1 was strictly verified by Bliss independence analysis ( P < 0.01) based on infarct volume. Finally, alleviation of cerebral metabolic disorders may be the possible mechanism of SalB/Rg1. Our study provided a multicomponent strategy against ischemic stroke, with not only dose reduction but also improved efficacy relative to single agents.

The mechanism of hypoxia-inducible factor-1α enhancing the transcriptional activity of transferrin ferroportin 1 and regulating the Nrf2/HO-1 pathway in ferroptosis after cerebral ischemic injury

Cerebral ischemic/reperfusion (I/R) injury has high disability and morbidity. Hypoxia-inducible factor-1α (HIF-1α) may enhance the transcriptional activity of transferrin ferroportin 1 (FPN1) in regulating ferroptosis after cerebral ischemia injury (CII). In this study, cerebral I/R injury rat models were established and treated with pcDNA3.1-HIF-1α, pcDNA3.1-NC lentiviral plasmid, or ML385 (a specific Nrf2 inhibitor). Additionally, oxygen-glucose deprivation/reoxygenation (OGD/R) exposed PC12 cells were used as an in vitro model of cerebral ischemia and treated with pcDNA3.1-HIF-1α, si-FPN1, or ML385. The results elicited that cerebral I/R injury rats exhibited increased Longa scores, TUNEL and NeuN co-positive cells, Fe2+ concentration, ROS and HIF-1α levels, and MDA content, while reduced cell density and number, GSH content, and GPX4 protein level. Morphologically abnormal and disordered hippocampal neurons were also observed in CII rats. HIF-1α inhibited brain neuron ferroptosis and ameliorated I/R injury. HIF-1α alleviated OGD-induced PC12 cell ferroptosis. OGD/R decreased FPN1 protein level in PC12 cells, and HIF-1α enhanced FPN1 transcriptional activity. FPN1 knockdown reversed HIF-1α-mediated alleviation of OGD/R-induced ferroptosis. HIF-1α activated the Nrf2/HO-1 pathway by enhancing FPN1 expression and alleviating OGD/R-induced ferroptosis. Conjointly, HIF-1α enhanced the transcriptional activity of FPN1, activated the Nrf2/HO-1 pathway, and inhibited ferroptosis of brain neurons, thereby improving I/R injury in CII rats.

Glycine Receptor Beta Subunit (GlyR-β) Promotes Potential Angiogenesis and Neurological Regeneration during Early-Stage Recovery after Cerebral Ischemia Stroke/Reperfusion in Mice.

Ischemic stroke is mainly caused by cerebral artery thrombosis. This study investigated the role of glycine receptor beta subunit (GlyR-β) in the recovery from cerebral ischemia stroke/reperfusion. The oxygen glucose deprivation and recovery (OGD/R) bEnd3 cell model and the middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model were used in this study. Expression of both the GlyR-β gene and vascular endothelial growth factor (Vegf), cell proliferation, and tube formation ability was decreased in bEnd3 cells after OGD/R, and was reversed by overexpression of GlyR-β. Neurological function, asindicated by Zea Longa scores, area of cerebral ischemia, and pathological changes were increased in mice after MCAO/R, and were ameliorated by overexpression of the glycine receptor beta (Glrb) gene at 24 h and 7 d after MCAO/R. Expression of GlyR-β and Gap-43 was decreased, and the expression of CD34, Vegf, and Bdnf, and cell growth as determined by a bromodeoxyuridine (BrdU) assay, increased in the affected brain tissue of MCAO/R mice in a time-dependent manner. GlyR-β overexpression resulted in enhanced expression of CD34, Vegf, Growth association protein 43 (Gap-43), and brain-derived neurotrophic factor (Bdnf) and cell growth in affected brain tissue of MCAO/R mice in a time-dependent manner. GlyR-β promoted potential angiogenesis and neurological regeneration in affected brain tissue, thus promoting recovery from cerebral ischemia stroke/reperfusion.

Fingolimod Alleviates Inflammation after Cerebral Ischemia via HMGB1/TLR4/NF-κB Signaling Pathway.

Clinically, ischemic reperfusion injury is the main cause of stroke injury. This study aimed to assess the effectiveness of fingolimod in suppressing inflammation caused by ischemic brain injury and explore its pharmacological mechanisms. In total, 75 male Sprague-Dawley rats were randomly and equally assigned to five distinct groups: sham, middle cerebral artery occlusion/reperfusion (MCAO/R) surgery, fingolimod low-dose (F-L), fingolimod medium-dose (F-M), and fingolimod high-dose (F-H). Neurobehavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and the brain tissue drying-wet method were conducted to evaluate neurological impairment, cerebral infarction size, and brain water content. Enzyme-linked immunosorbent assay was employed to quantify pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) protein levels. Western blotting and immunohistochemical staining were performed to assess high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and nuclear factor kappa-B p65 (NF-κBp65) levels. Rats in the F-L, F-M, and F-H groups exhibited lower Longa scores, reduced infarction volumes, and decreased brain edema than those in the MCAO/R group. Additionally, the F-L, F-M, and F-H groups exhibited lower serum levels of IL-1β, IL-6, and TNF-α than those of the MCAO/R group. Additionally, F-L, F-M, and F-H treatments resulted in decreased HMGB1, TLR4, and NF-κBp65 protein expression levels in the hippocampus of MCAO/R rats. Fingolimod was found to reduce ischemic brain injury in a dose-dependent manner. Moreover, it was also found to alleviate inflammation following ischemic brain injury via the HMGB1/TLR4/NF‑κB signaling pathway.

Albiflorin alleviates neuroinflammation of rats after MCAO via PGK1/Nrf2/HO-1 signaling pathway

Ischemic stroke is acknowledged as one of the most frequent causes of death and disability, in which neuroinflammation plays a critical role. Emerging evidence supports that the PGK1/Nrf2/HO-1 signaling can modulate inflammation and oxidative injury. Albiflorin (ALB), a main component of Radix paeoniae Alba, possesses anti-inflammatory and antioxidative properties. However, how it exerts a protective role still needs further exploration. In our study, the middle cerebral artery occlusion (MCAO) model was established, and the Longa score was applied to investigate the degree of neurological impairment. Dihydroethidium (DHE) staining and Malondialdehyde (MDA) assay were used to detect the level of lipid peroxidation. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was used to measure the infarct area. Evans blue staining was employed to observe the integrality of the blood–brain barrier (BBB). The injury of brain tissue in each group was observed via HE staining. Immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and western blot assay were used for the measurement of inflammatory factors and protein levels. We finally observed that ALB relieved cerebral infarction symptoms, attenuated oxidative damage in brain tissues, and reduced neuroinflammation and cell injury in MCAO rats. The overexpression of PGK1 abrogated the protective effect of ALB after experimental cerebral infarction. ALB promoted PGK1 degradation and induced Nrf2 signaling cascade activation for subsequent anti-inflammatory and antioxidant damage. Generally speaking, ALB exerted a protective role in treating cerebral ischemia, and it might target at PGK1/Nrf2/HO-1 signaling. Thus, ALB might be a potential therapeutic agent to alleviate neuroinflammation and protect brain cells after cerebral infarction.

Maresin1 alleviates neuroinflammation by inhibiting caspase-3/ GSDME-mediated pyroptosis in mice cerebral ischemia-reperfusion model

ObjectiveTo explore the mechanism of Maresin1 in reducing cerebral ischemia-reperfusion injury. Materials and methodsMale C57BL/6 mice were randomly divided (n = 5 in each group), and focal middle cerebral artery occlusion (MCAO) model was used to simulate cerebral ischemia/reperfusion injury. TTC and the Longa score were used to detect the degree of neurological deficits. Western blot was used to detect the expression levels of GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in cerebral ischemic penumbra tissue, and immunofluorescence was used to detect the expression levels of GSDME-N. The mRNA expression levels of GSDME and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) were detected by RT-PCR. ResultsCompared with sham group, GSDME mRNA levels in MCAO group were significantly increased at 12 h and 24 h after reperfusion, and GSDME and GSDME-N significantly increased at 6–48 h after reperfusion. Compared with sham group, the percentage of infarct size, the Longa score, the mRNA expression levels of IL-1β, IL-6 and TNF-α, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 in MCAO group was significantly increased. Then, the percentage of infarct size and the Longa score significantly decreased after MaR1 administration, the mRNA expression levels of IL-1β and IL-6 downregulated, and GSDME, GSDME-N, caspase-3 and cleaved caspase-3 were also reduced. After administration of Z-DEVD-FMK(ZDF), the expression of caspase-3, cleaved caspase-3 and GSDME-N was decreased, which in MCAO+MaR1+ZDF group was not statistically significant compared with MCAO+ ZDF group. ConclusionMaresin1 alleviates cerebral ischemia/reperfusion injury by inhibiting pyroptosis mediated by caspase-3/GSDME pathway and alleviating neuroinflammation.

Electroacupuncture Ameliorates Cerebral Ischemia-reperfusion Injury by Inhibiting Pyroptosis through the Sirtuin-1 Pathway

Abstract Objective: NOD-like receptor protein 3 (NLRP3)-mediated pyroptosis is pivotal in the pathological development of cerebral ischemia/reperfusion injury (CIRI). Although previous research has shown that electroacupuncture (EA) can alleviate CIRI through sirtuin-1 (SIRT1), the mechanism has not been well elucidated. Our study aimed to clarify whether the neuroprotective functions of EA are related to the reduction in NLRP3-mediated pyroptosis through the SIRT1 pathway. Materials and Methods: Rats received daily pretreatment with EA for 5 consecutive days before undergoing middle cerebral artery occlusion surgery. The Longa score was used to assess neurologic function. Infarct volume and morphological alterations were analyzed using 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin staining. In addition, neuronal pyroptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling/caspase-1 and neuronal nuclear antigen/caspase-1 immunofluorescence double staining. Levels of expression of pyroptosis markers were assessed by Western blotting and enzyme-linked immunosorbent assay. Results: EA improved deficits in neurologic function and minimized cerebral infarct volume. Mechanistically, a number of neuronal pyroptotic cells and protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD, and gasdermin D in the cerebral cortex were markedly reduced by EA treatment, and conversely, SIRT1 levels were increased. Notably, the specific SIRT1 inhibitor, EX527, reversed the effects of EA. Conclusions: EA potentially exerts a neuroprotective effect against CIRI through the SIRT1 pathway in NLRP3-mediated pyroptosis.

Tongqiao Huoxue Decoction inhibits ferroptosis by facilitating ACSL4 ubiquitination degradation for neuroprotection against cerebral ischemia-reperfusion injury

BackgroundCerebral ischemia-reperfusion injury (CIRI) refers to brain tissue injury caused by the temporary interruption of cerebral blood flow ischemia followed by the restoration of reperfusion, which is the main cause of post-stroke brain injury. A traditional Chinese herbal preparation called Tongqiao Huoxue Decoction (TQHX) has shown promise in reducing CIRI in rats. However, the mechanism of this herbal preparation for CIRI remains unclear. PurposeThis study aimed to evaluate the therapeutic effect of TQHX extract on rats with CIRI and to further explore the underlying mechanisms. MethodsThe active ingredients of TQHX extract were quantified by the high-performance liquid chromatography (HPLC) condition. We conducted thorough investigations to assess the effects of TQHX on CIRI and ferroptosis using oxygen-glucose deprivation/reperfusion (OGD/R)-treated PC12 cells as an in vitro model and transient middle cerebral artery occlusion (tMCAO) animals as an in vivo model. The neurological score assessment was performed to evaluate the neuroprotective effects of TQHX extract on tMCAO rats. Using histologic methods to study the extent of cerebral infarction, blood-brain barrier, and rat brain tissue. We examined the impact of TQHX on ferroptosis-related markers of Fe2+, superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissue. In addition, the expression of key proteins and markers of ferroptosis, as well as key factors associated with Acyl-CoA synthetase long-chain family member 4 (ACSL4) were detected by Western blot and quantitative real-time PCR (RT-qPCR). ResultsTQHX extract could decrease the Longa score and extent of cerebral infarction of tMCAO rats, which exerted the function of neuroprotection. Additionally, TQHX treatment efficiently decreased levels of MDA and ROS while increasing the expression of SOD and ferroptosis-related proteins including ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) at the transcription and translation level. Meanwhile, TQHX provided strong protection against oxidative stress and ferritin accumulation by increasing the ubiquitination and degradation of ACSL4. The injection of OE-ACSL4 reversed the effects of TQHX on neuroprotection and ferroptosis inhibition in PC12 cells. The injection of shACSL4 reversely validate the crucial role of ACSL4 in CIRI rat treatment. ConclusionThis work shows that TQHX promotes the ubiquitination-mediated degradation of ACSL4, which improves oxidative stress and inhibits the beginning of ferroptosis in cells. TQHX provides a possible path for additional research in CIRI therapies, advancing translational investigations.

Effect of electroacupuncture on proBDNF/mBDNF and synaptic plasticity in rats with learning and memory impairment after cerebral ischemia-reperfusion.

To observe the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the rats' behavior and the transforming precursor of brain-derived neurotrophic factor (proBDNF) into mature brain-derived neurotrophic factor (mBDNF) in the hippocampus of rats with learning and memory impairment induced by cerebral ischemia-reperfusion (IR), so as to explore its mechanisms underlying improvement of learning and memory ability. SD rats were randomly divided into blank, sham operation, model, and EA groups, with 6 rats in each group. The model of IR was established by occlusion of the middle cerebral artery. EA (1 Hz/20 Hz) was applied to GV24 and GV20 for 30 min, once daily for 14 days. The neurological function was evaluated according to the Zea Longa's score criteria 24 h after modeling and after intervention. Morris water maze test was used to detect the learning and memory function of the rats. TTC staining was used to evaluate the cerebral infarction volume on the affected side. The protein expression levels of proBDNF, mBDNF, tissue plasminogen activator (tPA), tyrosine kinase receptor B (TrkB) and p75 neurotrophin receptor (p75NTR) in hippocampal tissue were detected by Western blot. Compared with the sham operation group, the neurological function score, the percentage of cerebral infarction volume and the expression levels of proBDNF and p75NTR protein in hippocampus were increased (P<0.01), while the times of crossing the original platform and the total distance in the target quadrant, the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were decreased (P<0.01, P<0.05) in the model group. Compared with the model group, the neurological function score, the percentage of cerebral infarction volume, and the expression levels of proBDNF and p75NTR protein in hippocampus were decreased (P<0.01, P<0.05), while the times of crossing the original platform, the total distance in the target quadrant, and the expression levels of mBDNF, TrkB and tPA protein and the ratio of mBDNF/proBDNF were increased (P<0.05, P<0.01) in the EA group. EA can alleviate learning and memory impairment in IR rats, which may be related to its function in up-regulating the expression of tPA protein and promoting the transformation of proBDNF to mBDNF, thus improving the synaptic plasticity.

Morphine Preconditioning Alleviates Ischemia/Reperfusion-induced Caspase-8-dependent Neuronal Apoptosis through cPKCγ-NF-κB-cFLIPL Pathway.

Perioperative cerebral ischemia/reperfusion injury is a major contributor to postoperative death and cognitive dysfunction in patients. It was reported that morphine preconditioning (MP) can mimic ischemia/hypoxia preconditioning to protect against ischemia/reperfusion injury. However, the mechanism of MP on the ischemia/reperfusion-induced neuronal apoptosis has not been fully clarified. The middle cerebral artery occlusion/reperfusion (MCAO/R) model of mice and the oxygen-glucose deprivation/reoxygenation (OGD/R) model in primary cortical neurons were used to mimic ischemic stroke. In vivo, the infarct size was measured by using TTC staining; NDSS, Longa score system, and beam balance test were performed to evaluate the neurological deficits of mice; the expression of the protein was detected by using a western blot. In vitro, the viability of neurons was determined by using CCK-8 assay; the expression of protein and mRNA were assessed by using western blot, RT-qPCR, and immunofluorescent staining; the level of apoptosis was detected by using TUNEL staining. MP can improve the neurological functions of mice following MCAO/R (P<0.001, n=10 per group). MP can decrease the infarct size (P<0.001, n=10 per group) and the level of cleaved-caspase-3 of mice following MCAO/R (P<0.01 or 0.001, n=6 per group). MP can increase the levels of cPKCγ membrane translocation, p-p65, and cFLIPL, and decrease the levels of cleaved-caspase-8, 3 in neurons after OGD/R or MCAO/R 1d (P<0.05, 0.01 or 0.001, n=6 per group). In addition, MP could alleviate OGD/R-induced cell apoptosis (P<0.001, n=6 per group). MP alleviates ischemia/reperfusion-induced Caspase 8-dependent neuronal apoptosis through the cPKCγ-NF-κB-cFLIPL pathway.

Exosomes derived from human umbilical cord mesenchymal stem cells decrease neuroinflammation and facilitate the restoration of nerve function in rats suffering from intracerebral hemorrhage.

Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-ex) have become a hopeful substitute for whole-cell therapy due to their minimal immunogenicity and tumorigenicity. The present study aimed to investigate the hypothesis that hUCMSC-ex can alleviate excessive inflammation resulting from intracerebral hemorrhage (ICH) and facilitate the rehabilitation of the nervous system in rats. In vivo, hemorrhagic stroke was induced by injecting collagenase IV into the striatum of rats using stereotactic techniques. hUCMSC-ex were injected via the tail vein at 6h after ICH model establishment at a dosage of 200µg. In vitro, astrocytes were pretreated with hUCMSC-ex and then stimulated with hemin (20μmol/mL) to establish an ICH cell model. The expression of TLR4/NF-κB signaling pathway proteins and inflammatory factors, including TNF-α, IL-1β, and IL-10, was assessed both in vivo and in vitro to investigate the impact of hUCMSC-ex on inflammation. The neurological function of the ICH rats was evaluated using the corner turn test, forelimb placement test, Longa score, and Bederson score on the 1st, 3rd, and 5th day. Additionally, RT-PCR was employed to examine the mRNA expression of TLR4 following hUCMSC-ex treatment. The findings demonstrated that hUCMSC-ex downregulated the protein expression of TLR4, NF-κB/P65, and p-P65, reduced the levels of pro-inflammatory cytokines TNF-α and IL-1β, and increased the expression of the anti-inflammatory cytokine IL-10. Ultimately, the administration of hUCMSC-ex improved the behavioral performance of the ICH rats. However, the results of PT-PCR indicated that hUCMSC-ex did not affect the expression of TLR4 mRNA induced by ICH, suggesting that hUCMSCs-ex may inhibit TLR4 translation rather than transcription, thereby suppressing the TLR4/NF-κB signaling pathway. We can conclude that hUCMSC-ex mitigates hyperinflammation following ICH by inhibiting the TLR4/NF-κB signaling pathway. This study provides preclinical evidence for the potential future application of hUCMSC-ex in the treatment of cerebral injury.

Suppression of cerebral ischemia injury induced blood brain barrier breakdown by dexmedetomidine via promoting CCN1.

Blood-brain barrier (BBB) could aggravate cerebral ischemia injury. Dexmedetomidine (Dex) has been believed to play a protective role in cerebral ischemia injury-induced BBB injury. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) models were established to simulate cerebral ischemia injury. Animal experiments included 4 groups, Sham, MCAO, MCAO+Dex, MCAO+Dex+sh-CCN1. Generally applicable gene set enrichment analysis was performed to analyze gene expression difference. Total collagen content and Evans blue staining were performed to measure infarct ratio and BBB breakdown, respectively. The cell apoptosis, mRNA and protein expression were measured through flow cytometry, PCR, and western blotting, respectively. The levels of IL-1β, TNF-α, and IL-6 in serum were measured with commercial ELISA kits. Dex greatly promoted the expression level of CCN1. Dex suppressed cerebral ischemia injury, increased tight junction protein expression, improved the memory ability and neurological function of MCAO rats through targeting CCN1. The significant increase of inflammatory factors in the serum of MCAO rats were suppressed by Dex. Dex suppressed OGD induced increase of HRP permeability and promoting tight junction protein expression in vitro through regulating CCN1. The neurological function evaluation was performed with Neurological Severity Score (NSS) and Longa Score Scale. Dex could remarkably alleviate cerebral ischemia injury by inhibiting BBB breakdown, inflammatory response, and promoting neurological function and tight junction protein expression via up-regulating CCN1. This study might provide a novel therapeutic target for the prevention and treatment of cerebral ischemia injury-induced BBB.

Tetrahydropiperine, a natural alkaloid with neuroprotective effects in ischemic stroke

BackgroundIschemic stroke (IS) is a life-threatening neurological disease with various pathological mechanisms. Tetrahydropiperine (THP) is a natural alkaloid with protective effects against multiple diseases, such as seizure, and pain. This study was to examine the impact of THP on IS and investigate its potential mechanism. Material and methodsWe employed network pharmacology and molecular docking techniques to identify the target proteins of THP for intervention in IS. Adult male Sprague-Dawley rats were used to create a permanent middle cerebral artery occlusion model. PC-12 cells were chosen to establish an oxygen–glucose deprivation (OGD) cell model. Disease modeling followed by nimodipine (NIMO); 3-methyladenine (3-MA) and rapamycin (RAP) interventions. Open field test, Longa score, balance beam test, and forelimb grip test were used to measure motor and neurological functions. The degree of neurological damage recovery was assessed through behavioral analysis, and cerebral infarction volume was determined using TTC staining. Morphological changes were examined through HE and Nissl staining, and ultrastructural changes in neurons were observed using transmission electron microscopy. The protein expression of autophagy and related pathways was analyzed through Western blot (WB). The appropriate hypoxia time and drug concentration were determined using CCK-8 assay, which also measured cell survival rate. ResultsThe network pharmacology findings indicated that the impact of THP on IS was enhanced in the PI3K/Akt signaling pathway. THP demonstrated robust docking capability with proteins associated with the autophagy and PI3K/Akt/mTOR, as indicated by the molecular docking outcomes. THP significantly improved behavioral damage, reduced the area of cerebral infarction, ameliorated histopathological damage from ischemia, increase neuronal survival, and alleviated ultrastructural damage in neurons (P < 0.05). THP enhanced the survival of PC-12 cells induced by OGD and ameliorated the morphological harm to the cells (P < 0.05). THP was found to elevate the quantities of P62, LC3-Ⅰ, PI3K, P-AKt/Akt, and P-mTOR/mTOR proteins while reducing the levels of Atg7 and Beclin1 proteins. The results of transmission electron microscopy showed no autophagosomes in the THP, 3-MA, and 3-MA + THP groups. ConclusionThe activation of the PI3K/Akt/mTOR signaling pathway by THP inhibits autophagy and provides relief from neurological damage in IS.

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)-1β, 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-1β, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P&lt;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-1β, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P&lt;0.05 or P&lt;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.

Faecal Microbiota Transplantation Alleviates Ferroptosis after Ischaemic Stroke

Ischaemic stroke can induce changes in the abundance of gut microbiota constituents, and the outcome of stroke may also be influenced by the gut microbiota. This study aimed to determine whether gut microbiota transplantation could rescue changes in the gut microbiota and reduce ferroptosis after stroke in rats. Male Sprague-Dawley rats (6 weeks of age) were subjected to ischaemic stroke by middle cerebral artery occlusion (MCAO). Fecal samples were collected for 16S ribosomal RNA (rRNA) sequencing to analyze the effects of FMT on the gut microbiota. Neurological deficits were evaluated using the Longa score. triphenyl tetrazolium chloride (TTC) staining was performed in the brain, and kits were used to measure malondialdehyde (MDA), iron, and glutathione (GSH) levels in the ipsilateral brain of rats. Western blotting was used to detect the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and the transferrin receptor 2 (TFR2) in the ipsilateral brain of rats. Stroke induced significant changes in the gut microbiota, and FMT ameliorated these changes. TTC staining results showed that FMT reduced cerebral infarct volume. In addition, FMT diminished MDA and iron levels and elevated GSH levels in the ipsilateral brain. Western blot analysis showed that FMT increased GPX4 and SLC7A11 protein expression and decreased TFR2 protein expression in the ipsilateral brain after stroke. FMT can reverse gut microbiota dysbiosis, reduce cerebellar infarct volume, and decrease ferroptosis after stroke.

Validation of a C57/BL6J mouse model of focal cerebral ischemia established by electrocoagulation of the middle cerebral artery

To modify the method for establishing mouse models of middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia using electrocoagulation. Forty-six C57/BL6J male mice were divided into MCAO model group (n=34) and sham-operated group (n=12). In the model group, MCAO was induced by permanent coagulation of the right middle cerebral artery (MCA) using a coagulator, and cerebral blood flow perfusion was monitored before and at 20 min and 1 day after modeling. Neurological deficits of the mice at 1, 7, and 14 days after modeling were evaluated using Longa score, mNSS score, beam walking test, cylinder test and corner test. TTC staining was used to measure the cerebral infarct size, and Western blotting was performed to detect the expressions of BDNF, GFAP and DCX proteins in the ischemic cortex. The mice in the model group showed significantly reduced cerebral blood flow in the MCA on the ischemic side and obvious neurological deficits with increased forelimb use asymmetry on days 1, 7 and 14 after modeling (P < 0.05). In the cerebral cortex on the ischemic side of the model mice, the expressions of GFAP and DCX increased significantly at 1, 7, and 14 days (P < 0.05) and the expression of BDNF increased at 1 day after modeling ischemia (P < 0.05). We successfully prepared mouse models of MCAO using a modified method by changing the electrocoagulation location from the distal location of the junction between the MCA and the inferior cerebral vein to a 2 mm segment medial to the junction between the MCA and the olfactory bundle.

Ultrasound-Targeted Microbubble Destruction Increases BBB Permeability and Promotes Stem Cell-Induced Regeneration of Stroke by Downregulating MMP8.

The objective of this study was to evaluate the feasibility, safety, and effectiveness of intravenous stem cell delivery utilizing ultrasound-targeted microbubble destruction (UTMD) in a rat model of middle cerebral artery occlusion (MCAO), while investigating the underlying mechanisms. Acute cerebral infarction (ACI) was induced surgically in adult rats to create the MCAO rat model. Intravenous injection of SonoVue microbubbles and bone marrow-derived mesenchymal stem cells (BMSC) was performed concurrently, with or without ultrasound targeting the stroke. The animals were divided into four groups: sham-operated group, ACI-MCAO rats treated with phosphate-buffered saline (ACI+PBS), rats receiving intravenous delivery of BMSC expressing green fluorescent protein (GFP-BMSC; ACI+BMSC), and rats receiving intravenous GFP-BMSC with simultaneous UTMD exposure (ACI+BMSC+UTMD). The efficacy of the treatments was assessed by evaluating the animals' neurological function using the Longa score and examining histopathological changes such as cerebral infarct volume, cerebral edema, and cell apoptosis. A rat cytokine array was utilized to identify the potential cytokines that may be responsible for the therapeutic effect of UTMD-mediated BMSC treatment. Optimal UTMD parameters resulted in an increase in blood-brain barrier (BBB) permeability after 30 min, which returned to baseline 72 h later without causing any residual injury. UTMD application significantly increased the homing of intravenously delivered BMSC, resulting in a 2.2-fold increase in GFP-BMSC cell count on day 3 and a 2.6-fold increase on day 7 compared with intravenous delivery alone. This effect persisted for up to 6 weeks after injection. Intravenous BMSC delivery significantly reduced the volume of cerebral infarct and decreased cerebral edema, leading to a lower Longa score. Furthermore, this effect was further enhanced by UTMD. Acute cerebral infarction induced by MCAO led to elevated matrix metalloproteinase 8 (MMP8) levels in the cerebrospinal fluid, which were significantly reduced following UTMD-mediated BMSC treatment. Ultrasound-targeted microbubble destruction facilitates the migration and homing of BMSC into the brain, possibly by transiently increasing blood-brain barrier (BBB) permeability, thereby improving therapeutic outcomes in an ACI rat model. The observed effect may be partly attributed to modulation of MMP8 levels.Advances in knowledge: UTMD-mediated intravenously delivered BMSC transplantation led to a significant increase in cell homing and reduction of MMP8 levels, resulting in increased therapeutic effect in an acute ischemic cerebral infarction model.

Morroniside improves the symptoms of post-stroke depression in mice through the BDNF signaling pathway mediated by MiR-409-3p

BackgroundPost-stroke depression (PSD) is a common psychiatric symptom after a stroke. Morroniside, an iridoid glycoside found in Cornus officinalis, has garnered significant attention for its potential to alleviate symptoms associated with depression. PurposeThis study aims to highlight the potential use of morroniside in the treatment of PSD and elucidate the underlying molecular mechanisms. MethodsTo establish a reliable PSD model, male C57BL/6 mice were subjected to brief MCAO in conjunction with CUMS. Post-morroniside administration, neuronal viability, and hippocampal cell apoptosis were evaluated by Nissl staining and TUNEL detection, respectively. Depression-like behaviors were evaluated using SPT, TST, and FST. The Longa score and cylinder test were used to evaluate the effect of morroniside on motor function. Furthermore, to investigate the underlying molecular mechanisms, bioinformatic analysis and the dual luciferase assay were performed to investigate the MiR-409-3p-BDNF interaction. In addition, subsequent to MiR-409-3p overexpression via AAV virus, we assessed mRNA expression and protein levels of key components within the BDNF/TrkB signaling pathway using RT-qPCR, immunohistochemistry, and western blot analysis. ResultsThe observed decrease in apoptosis and amelioration of depression-like behaviors strongly indicate the potential of morroniside as a therapeutic agent for PSD. Furthermore, the upregulation of key proteins within the BDNF/TrkB signaling pathway in the cortex suggests that morroniside activates this pathway. Through bioinformatics analysis, MiR-409-3p was identified and found to bind to the BDNF gene, resulting in the inhibition of BDNF expression. Importantly, we demonstrate that morroniside mitigates this inhibitory effect of MiR-409-3p on BDNF, thereby facilitating the activation of the BDNF/TrkB signaling pathway. ConclusionThe findings suggest that morroniside demonstrates the ability to improve PSD symptoms through the BDNF/TrkB signaling pathway mediated by MiR-409-3p. These results emphasize the importance of the BDNF signaling pathway in improving PSD symptoms and provide a possible mechanism for morroniside to treat PSD.

Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.