Clopidogrel with indobufen or aspirin in minor ischemic stroke or high-risk transient ischemic attack: a randomized controlled clinical study.

BACKGROUND: Ischemic stroke and transient ischemic attack (TIA) are the most prevalent cerebrovascular diseases. The conventional antiplatelet drugs are associated with an inherent bleeding risk, while indobufen is a new antiplatelet drug and has the similar mechanism of antiplatelet aggregation as aspirin with more safety profile. However, there have been no studies evaluating the combination therapy of indobufen and clopidogrel for antiplatelet therapy in cerebrovascular diseases. OBJECTIVE: The CARMIA study aims to investigate the effectiveness and safety of a new dual antiplatelet therapy consisting of indobufen and clopidogrel comparing with the conventional dual antiplatelet therapy consisting of aspirin and clopidogrel in patients with minor ischemic stroke or high-risk TIA. METHODS: An open-label randomized controlled clinical trial was conducted at a clinical center. We randomly assigned patients who had experienced a minor stroke or transient ischemic attack (TIA) within 72 h of onset, or within 1 month if they had intracranial stenosis (IS), to receive either indobufen 100 mg twice daily or aspirin 100 mg once daily for 21 days. For patients with IS, the treatment duration was extended to 3 months. All patients received a loading dose of 300 mg clopidogrel orally on the first day, followed by 75 mg once daily from the second day to 1 year. We collected prospective data using paper-based case report forms, and followed up on enrolled patients was conducted to assess the incidence of recurrent ischemic stroke or TIA, mRS score, NIHSS (National Institutes of Health Stroke Scale) score, and any bleeding events occurring within 3 month after onset. RESULTS: We enrolled 202 patients diagnosed with ischemic stroke or transient ischemic attack. After applying the criteria, 182 patients were eligible for data analysis. Endpoint events (recurrence of ischemic stroke/TIA, myocardial infarction, or death) were observed in 6 patients (6.5%) receiving aspirin and clopidogrel, including 4 (4.3%) with stroke recurrence, 1 (1.1%) with TIA recurrence, and 1 (1%) with death. In contrast, no endpoint events were reported in the indobufen and clopidogrel group (P = 0.029). The group of patients receiving indobufen and clopidogrel exhibited significantly lower modified Rankin Scale (mRS) score. (scores range from 0 to 6, with higher scores indicating more severe disability) compared to the aspirin and clopidogrel group (common odds ratio 3.629, 95% CI 1.874-7.036, P < 0.0001). Although the improvement rate of NIHSS score in the indobufen and clopidogrel group was higher than that in the aspirin and clopidogrel group, the difference was not statistically significant (P > 0.05). Bleeding events were observed in 8 patients (8.6%) receiving aspirin and clopidogrel, including 4 (4.3%) with skin bleeding, 2 (2.2%) with gingival bleeding, 1 (1.1%) with gastrointestinal bleeding, and 1 (1.1%) with urinary system bleeding. On the other hand, only 1 patient (1.1%) in the indobufen and clopidogrel group experienced skin bleeding (P = 0.035). CONCLUSION: The combination of indobufen and clopidogrel has shown non-inferior and potentially superior effectiveness and safety compared to aspirin combined with clopidogrel in patients with minor ischemic stroke and high-risk TIA in the CARMIA study (registered under chictr.org.cn with registration number ChiCTR2100043087 in 01/02/2021).

Medium-intensity statin with ezetimibe versus high-intensity statin in acute ischemic cerebrovascular disease (MESIA): A randomized clinical trial.

BACKGROUND: High-risk stroke patients are recommended to receive high-intensity statin therapy to reduce the risk of stroke recurrence. However, doubling the dosage of statin drugs did not increase the achievement rate of LDL-C target or provide additional clinical benefits, but significantly increased the risk of adverse reactions. Statins and ezetimibe work through different mechanisms and the combined use of statins and ezetimibe significantly improves outcomes with comparable safety profiles. We tested the hypothesis that moderate-intensity statin with ezetimibe may offer advantages over the conventional high-intensity statin regimen in terms of efficacy and safety. METHODS: We conducted a randomized controlled trial. Eligible participants were aged 18 years or older with acute ischemic cerebrovascular disease. We randomly assigned (1:1) participants within the acute phase of ischemic stroke, i.e., within 1 week after the onset of mild ischemic stroke (NIHSS score ≤ 5), within 1 month for severe cases (NIHSS score ≥ 16), and within 2 weeks for the rest, as well as patients with TIA within 1 week of symptom onset, to receive either moderate-intensity statin with ezetimibe (either 10-20 mg atorvastatin calcium tablets plus a 10 mg ezetimibe tablet, or 5-10 mg rosuvastatin calcium tablets once per day plus a 10 mg ezetimibe tablet once per day) or high-intensity statin (40 mg atorvastatin calcium tablets or 20 mg rosuvastatin calcium tablets once per day) for 3 months. Randomization was performed using a random number table method. The primary efficacy outcome was the level and achievement rate of LDL-C after 3 months of treatment, specifically LDL-C ≤ 1.8 mmol/L or a reduction in LDL-C ≥ 50 %. The secondary outcome was the incidence of new stroke or transient ischemic attack (TIA) within 3 months. The safety outcome was liver and renal function tests, and the occurrence of statin-related muscle events within 3 months. FINDINGS: This trial took place between March 15, 2022, and March 7, 2023. Among 382 patients screened, 150 patients were randomly assigned to receive either medium-intensity statins with ezetimibe (n = 75) or high-intensity statins (n = 75). Median age was 60.0 years (IQR 52.75-70.25); 49 (36.6 %) were women and 85 (63.4 %) were men. The target achievement of LDL-C at 3 months occurred in 62 (89.86 %) of 69 patients in the medium-intensity statin with ezetimibe group and 46 (70.77 %) of 65 patients in the high-intensity statin group (P=0.005, OR=0.273, 95 % CI: 0.106, 0.705). The reduction magnitude of LDL-C in moderate-intensity statin with ezetimibe group was significantly higher (-56.540 % vs -47.995 %, P=0.001). Moderate-intensity statin with ezetimibe group showing a trend of a greater reduction in LDL-C absolute value than high-intensity statin group but without statistical significance (-1.77±0.90 vs -1.50±0.89, P=0.077). New AIS or TIA within 3 months, liver and renal function tests, and the occurrence of statin-related muscle events within 3 months were also statistically insignificant. Multivariate logistic regression analysis showed that both gender and lipid-lowering regimen as independent risk factors influencing the rate of LDL-C achievement in individuals diagnosed with acute ischemic cerebrovascular disease, but only lipid-lowering regimen had predictive value. INTERPRETATION: Compared to guideline-recommended high-intensity statin therapy, moderate-intensity statin with ezetimibe further improved the achievement rate of LDL-C in patients with acute ischemic cerebrovascular disease, with a higher reduction magnitude in LDL-C. In terms of safety, there was no significant difference between the two regimens, suggesting that moderate-intensity statin with ezetimibe can also be considered as an initial treatment option for patients with acute ischemic cerebrovascular disease.

Silencing Celsr2 inhibits the proliferation and migration of Schwann cells through suppressing the Wnt/ß-catenin signaling pathway.

After a peripheral nerve injury, the remaining Schwann cells undergo proliferation and adopt a migratory phenotype to prepare for the regeneration of nerves. Celsr2 has been reported to play an important role in the development and maintenance of the function of the nervous system. However, the role and mechanism of Celsr2 during peripheral nerve regeneration remain unknown. Here, we showed that after sciatic nerve injury, Celsr2 mRNA and protein were significantly increased in nerve tissues. In addition, silencing Celsr2 decreased the ki67-positve portion and the migration distance of Schwann cells in vivo. In vitro, the results of MTT and EdU staining, transwell and wound healing assays indicated that Celsr2 siRNA-transfected primary Schwann cells showed significant decrease in proliferation and migration compared to that seen in negative control (NC)-transfected cells. Furthermore, we found that Wnt/ß-catenin luciferase activity was reduced, as were the expression of ß-catenin in the nucleus and the mRNA levels of its downstream genes Cyclin D1 and MMP-7 in Celsr2 siRNA-transfected primary Schwann cells. Further investigations showed that silencing Celsr2 inhibited the phosphorylation of GSK3ß. Moreover, specific activators of the Wnt/ß-catenin pathway, LiCl or mutant ß-catenin (S33Y), partially reversed the inhibitory effect of Celsr2 siRNA. Taken together, our data indicated that silencing Celsr2 inhibited Schwann cells migration and proliferation through the suppressing Wnt/ß-catenin pathway, providing a potential target for peripheral nerve regeneration.

Circ_016719 plays a critical role in neuron cell apoptosis induced by I/R via targeting miR-29c/Map2k6.

BACKGROUND: Stroke is a leading cause of mortality worldwide. Rac-MAPK kinase 6 (Map2k6) plays important roles in cell proliferation and apoptosis. However, the role played by Map2k6 in stroke injury and the underlying mechanism of action remain unknown. METHODS: Mice received cerebral ischemia/reperfusion (I/R) injuries by transient middle cerebral artery occlusion. HT22 cells were subjected to oxygen glucose deprivation and reoxygenation (OGD/R) to simulate an I/R injury. Subsequently, the levels of circ_016719, miR-29c and Map2k6 expression were determined, and their interactions were examined by luciferase assays. Circ_016719 knockdown, miR-29c inhibition or Map2k6 overexpression was induced in HT22 cells; after which, the cells were examined for their viability, apoptosis, autophagy and proliferation, as well their levels of Map2k6, p38, p53, LC3B-I, LC3B-II, Beclin 1, and p62 expression. RESULTS: Significantly increased levels of circ_016719 and Map2k6, and decreased levels of miR-29c were observed in both in vivo and in vitro I/R injury models. In HT22 cells, circ_016719 knockdown significantly increased miR-29c expression and cell proliferation, but decreased Map2k6 expression and cell apoptosis. Additionally, significant increases in LC3B-I and p62 levels and decreased LC3B-II levels were observed, indicating that circ_016719 knockdown had significantly inhibited autophagy. Furthermore, additional inhibition of miR-29c markedly suppressed the effects of circ_016719 knockdown; however, that suppression was significantly attenuated by Map2k6 overexpression. Additionally, Map2k6 was identified as a direct target of miR-29c, which in turn, might be sponged by circ_016719. CONCLUSIONS: Our results suggest that circ_016719 directly targets miR-29c, and thereby regulates the expression and functions of Map2k6, which significantly contributes to the pro-apoptotic role of circ_016719.

Identification of cholest-4-ene-3,6-dione as a Novel Neuroprotectant in Ischemic Stroke and Its Lipidomics.

Purpose: Stroke is a leading cause of disability and death globally. However, there are few clinical drugs for stroke therapy. Novel and effective neuroprotectants are called on the way. Methods: In this study, 93 steroids from a constructed steroidal library were randomly numbered and blindly evaluated in an L-glutamate-induced HT-22 oxidative stress model. The neuroprotective effects of 5 candidates were further investigated in potassium deprivation-induced apoptosis of cerebellar granule neurons (CGNs), D-glutamate-induced excitotoxicity of CGNs, and cortical neuron (CN) models. Results: Interestingly, unblinding revealed that cholest-4-ene-3,6-dione (78), a cholesterol derivative, was first found to have comprehensive neuroprotective effects in all cell models. 78 administration also decreased the infarction volume and improved motor function in middle cerebral artery occlusion (MCAO) model rats. Additionally, 78 treatment decreased intercellular reactive oxygen species (ROS) and NO production in the HT-22 cell model. Finally, lipidomics and molecular docking results showed that 78 may exert its neuroprotective effects by increasing platelet-activating factor (PAF) analog 1-(9Z-pentadecenoyl)-glycero-3-phosphocholine production. Conclusion: This study indicates that 78, a novel neuroprotectant, is a promising therapeutic candidate with comprehensive neuroprotective effects for the treatment of ischemic stroke by decreasing ROS/reactive nitrogen species (RNS) levels and increasing 1-(9Z-pentadecenoyl)-glycero-3-phospho-choline production.

Maf1 Ameliorates Sepsis-Associated Encephalopathy by Suppressing the NF-kB/NLRP3 Inflammasome Signaling Pathway.

Background: The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome has been identified as an important mediator of blood-brain-barrier disruption in sepsis-associated encephalopathy (SAE). However, no information is available concerning the critical upstream regulators of SAE. Methods: Lipopolysaccharide (LPS) was used to establish an in vitro model of blood-brain barrier (BBB) disruption and an in vivo model of SAE. Disruption of BBB integrity was assessed by measuring the expression levels of tight-junction proteins. NLRP3 inflammasome activation, pro-inflammatory cytokines levels, and neuroapoptosis were measured using biochemical assays. Finally, the FITC-dextran Transwell assay and Evan's blue dye assay were used to assess the effect of Maf1 on LPS-induced endothelial permeability in vitro and in vivo. Results: We found that Maf1 significantly suppressed the brain inflammatory response and neuroapoptosis induced by LPS in vivo and in vitro. Notably, Maf1 downregulated activation of the NF-κB/p65-induced NLRP3 inflammasome and the expression of pro-inflammatory cytokines. In addition, we found that Maf1 and p65 directly bound to the NLRP3 gene promoter region and competitively regulated the function of NLRP3 in inflammations. Moreover, overexpression of NLRP3 reversed the effects of p65 on BBB integrity, apoptosis, and inflammation in response to LPS. Our study revealed novel role for Maf1 in regulating NF-κB-mediated inflammasome formation, which plays a prominent role in SAE. Conclusions: Regulation of Maf1 might be a therapeutic strategy for SAE and other neurodegenerative diseases associated with inflammation.

Eicosapentaenoic acid prevents inflammation induced by acute cerebral infarction through inhibition of NLRP3 inflammasome activation.

OBJECTIVE: Acute cerebral infarction (ACI) is the most common type of acute cerebrovascular diseases resulting in high rate of death and disability. Numerous evidences show that inflammation is the leading cause of ischemic brain injury, thus anti-inflammatory therapy is an attractive candidate for ischemic brain damage. Eicosapentaenoic acid (EPA) exerts anti-inflammatory activity in lots of human inflammatory diseases, whereas its effect in ACI is left to elucidate. METHOD: Nlpr3-/- mice, Gpr40-/-; Gpr120-/- mice and mice with right middle cerebral artery occlusion (MCAO) were used to detect NLR family pyrin domain containing 3 (NLRP3) inflammasome activation by Western Blot and the release of proinflammatory cytokines by ELISA. To estimate the acute ischemic condition in vitro, oxygen-glucose deprivation (OGD) was induced in BV2 microglia cells. Transfection of the shRNA targeting GPR40 and GPR120 mRNA into BV2 cells was also assessed. Apoptosis in ischemic cerebral tissues and BV2 cells was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry. RESULT: Here we show that EPA suppresses ACI-induced inflammatory responses through blocking NLRP3 inflammasome activation. In addition, EPA inhibits NLRP3 inflammasome activation through G protein-coupled receptor 40 (GPR40) and GPR120. Importantly, EPA ameliorates ACI-induced apoptosis. CONCLUSION: EPA exerts beneficial effect on ACI-induced inflammation through blocking NLRP3 inflammasome activation by GPR40 and GPR120. Our findings suggest the potential clinical use of EPA in ACI.

Positive correlation between cognitive impairment and renal microangiopathy in patients with type 2 diabetic nephropathy: a multicenter retrospective study.

OBJECTIVE: This study was performed to explore the correlation between cognitive impairment and renal microangiopathy in patients with type 2 diabetic nephropathy (T2DN) by detecting changes in cognitive function and cerebral metabolism in these patients with different stages of T2DN. METHODS: Prospectively maintained databases were reviewed from 2006 to 2017. Blood biochemical indexes and the urinary albumin excretion rate (UAER) were measured in all participants. Cognitive function was assessed by the Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment Scale (MoCA). Cognitive impairment was the primary endpoint. Renal microangiopathy was the secondary endpoint. Pearson correlation analysis was used to assess correlations. RESULTS: Two hundred sixteen patients with type 2 diabetes mellitus (T2DM) were divided into three groups according to their UAER: T2DM without nephropathy (n=72), early T2DM with nephropathy (n=74), and the clinical stage of early T2DM with nephropathy (n=70). Healthy participants were selected as the normal control group (n=70). Pearson correlation analysis demonstrated that the total MMSE and MoCA score was negatively correlated with the UAER (r=-0.327) and positively correlated with the estimated glomerular filtration rate (r=0.428) in patients with T2DN. CONCLUSIONS: The present study showed a positive correlation between cognitive impairment and renal microangiopathy in patients with T2DN.

MiR-375 attenuates injury of cerebral ischemia/reperfusion via targetting Ctgf.

Ischemic stroke is the leading cause of disability and deaths worldwide. MiRNAs have been shown to play an important role in development and pathogenesis of the nervous system. However, the precise function and mechanism of miRNAs are not fully understood in the brain injury induced by ischemia/reperfusion (I/R). Herein, our study showed that miR-375 expression was significantly down-regulated in the rat I/R brain. With the in vivo and in vitro I/R stroke models, we found that miR-375 mimic provides significant protection from injury to cerebral I/R, which is reflected by reduced infarct volumes and cell apoptosis, and increased proliferation and migration of PC12 cells. Mechanistically, our findings showed that miR-375 binds to 3'-UTR region of Ctgf mRNA, subsequently leading to the decreased expression of Ctgf in the I/R brain. Furthermore, we showed that miR-375/Ctgf-mediated protective effects are associated with p21/PI3K/Akt signaling pathways. Our findings thus provide a new insight into the mechanism of cerebral I/R injury and pave a potential new way for the therapy of cerebral I/R injury.

Ischemic stroke damages the intestinal mucosa and induces alteration of the intestinal lymphocytes and CCL19 mRNA in rats.

The immunoreaction has a pivotal effect on ischemic stroke. It has been demonstrated that intestinal lymphocytes infiltrate into the brain and aggravate tissue injury after stroke. However, less attention has been paid to the influence on the intestinal immunology as well as morphology. Here, we utilized a rat permanent middle cerebral artery occlusion (MCAO) model to investigate the influences on intestinal mucosa, lymphocytes of the gut-associated lymphoid tissue (GALT), and the intestinal expression of CCL25 mRNA and CCL19 mRNA of stroke. Rats were randomly divided into stroke, sham, and control groups. Stroke and sham groups were further divided into interval groups of 6h, 12h, and 24h after surgery. Intestinal pathophysiological changes were observed by hematoxylin-eosin (H&E) staining. The lymphocyte numbers were detected by flow cytometry. The expression of CCL25 mRNA and CCL19 mRNA was tested with the PCR technique. We found significant necrosis and shedding of the epithelium after stroke. Moreover, the lesion aggravated with time. In addition, there was a significant increase of T lymphocytes in Peyer's patches (PPs), especially at 12h and 24h after stroke, while no differences in the number of B lymphocytes and the intraepithelial lymphocytes (IELs) were found. The data displayed no alteration of CCL25 mRNA expression. In contrast, an upregulation of CCL19 mRNA expression was detected at 6h after stroke. This study showed that ischemic stroke significantly damaged the intestinal epithelium and activated intestinal immunity.