miRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1.

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated ß-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

Analysis of Clinical Characteristic and Risk Factors for Short-Term Prognosis of Moyamoya Disease with Intraventricular Hemorrhage in Adults.

BACKGROUND: Intraventricular hemorrhage (IVH) is the most common type of hemorrhage in moyamoya disease (MMD) with intracerebral hemorrhage (ICH), but the risk factors affecting the short-term prognosis of MMD with IVH in adults are still unclear. METHODS: We retrospectively analyzed patients of MMD with IVH between January 1, 2018 and January 31, 2020 in the First Affiliated Hospital of Zhengzhou University. According to the modified Rankin Scale (mRS) score at 3 months after discharge, the patients were divided into mRS score ≤2 (good prognosis) group and mRS score >2 (poor prognosis) groups. Univariate and multivariate logistics regression analysis was used to analyze the risk factors affecting the short-term prognosis of adult MMD with IVH. RESULTS: Univariable analyses showed that patients in the poor prognosis group had a significantly older age of onset (48.48 ± 8.34 vs. 43.74 ± 5.44 years; P = 0.002), a higher percentage of hypertension (57.97% vs. 33.33%; P = 0.014), a higher percentage of tracheotomy (23.19% vs. 2.56%; P = 0.005), a lower Glasgow Coma Scale (GCS) score (7.90 ± 3.58 vs. 11.19 ± 2.56; P = 0.000), a higher Graeb score (7.46 ± 4.04 vs. 5.23 ± 1.93; P = 0.002), and treatment methods (P = 0.000). Multiple logistic regression analysis showed that the lower GCS score (odds ratio [OR], 1.761; P = 0.001) and higher Graeb score (OR, 1.767; P = 0.002) were independently associated with the poor prognosis of MMD with IVH, and surgery treatment (OR, 0.032; P = 0.000) was independently related to the good prognosis of MMD with IVH. CONCLUSIONS: Among patients with MMD with IVH, the lower GCS score and higher Graeb score are independent risk factors for poor prognosis, whereas in patients with MMD with IVH, surgery treatment acts as a protective factor.

A positive feedback loop of miR-30a-5p-WWP1-NF-κB in the regulation of glioma development.

Previous studies demonstrated that miR-30a-5p promotes glioma cell growth and invasion. Furthermore, WWP1 (WW domain containing E3 ubiquitin protein ligase 1) inhibits NF-κB activation that is strongly correlated with gliomagenesis. Using the GEO database and bioinformatics analyses, we identified WWP1 was downregulated in glioma tissues and might be a putative target for miR-30a-5p. Hence, this study aims to explore the interaction among miR-30a-5p, WWP1, and NF-κB and their roles in the regulation of glioma development. We found decreased WWP and increased miR-30a-5p expression and p65 phosphorylation in glioma tissues. Furthermore, WWP1 mRNA level was negatively correlated with miR-30a-5p expression in glioma tissues. Interestingly, miR-30a-5p targeted WWP1 expression. Additionally, NF-κB p65 overexpression increased miR-30a-5p expression through direct binding of NF-κB RelA subunit to the promoter of miR-30a-5p. We also confirmed that WWP1 overexpression decreased phosphorylation of NF-κB p65. Importantly, miR-30a-5p promoted glioma cell proliferation, migration, and invasion via targeting WWP1. Furthermore, NF-κB p65 overexpression inhibited WWP1 expression and promoted glioma cell malignant behaviors via inducing miR-30a-5p transcription. Moreover, WWP1 overexpression decreased miR-30a-5p expression and inhibited glioma cell malignant behaviors via inhibiting NF-κB p65. Our further assay showed that WWP1 inhibited in vivo growth of xenograft tumors of glioma cells, accompanied with a decrease in miR-30-5p expression and phosphorylation of NF-κB p65. In conclusion, there is a "miR-30a-5p-WWP1-NF-κB" positive feedback loop, which plays an important role in regulating glioma development and might provide a potential therapeutic strategy for treating glioma.

AG490 ameliorates early brain injury via inhibition of JAK2/STAT3-mediated regulation of HMGB1 in subarachnoid hemorrhage.

High mobility group box 1 (HMGB1) is a classic damage-associated molecular pattern that has an important role in the pathological inflammatory response. In vitro studies have demonstrated that the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the regulation of HMGB1 expression, mediating the inflammatory response. Therefore, the purpose of the present study was to evaluate JAK2/STAT3 pathway involvement in the subarachnoid hemorrhage (SAH)-dependent regulation of HMGB1, using an in vivo rat model. A SAH model was established by endovascular perforation. Western blotting, immunohistochemistry and immunofluorescence were used to analyze HMGB1 expression after SAH. In addition, the effects of AG490 after SAH on JAK2/STAT3 phosphorylation, HMGB1 expression and brain damage were evaluated. The results of the present study demonstrated that JAK2/STAT3 was significantly phosphorylated (P<0.05) and the total HMGB1 protein level was significantly increased (P<0.05) after SAH. In addition, the cytosolic HMGB1 level after SAH demonstrated an initial increase followed by a decrease to the control level, while the nuclear HMGB1 level after SAH demonstrated the opposite trend, with an initial decrease and subsequent increase. AG490 administration after SAH significantly inhibited JAK2/STAT3 phosphorylation (P<0.05), suppressed the expression and translocation of HMGB1, reduced cortical apoptosis, brain edema and neurological deficits. These results demonstrated the involvement of the JAK2/STAT3 pathway in HMGB1 regulation after SAH.

Role of the AMPK signaling pathway in early brain injury after subarachnoid hemorrhage in rats.

BACKGROUND: AMP-activated protein kinase (AMPK) is a key metabolic and stress sensor/effector. Few investigations have been performed to study the role of AMPK in subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was undertaken to investigate the time course of AMPK activation in the early stage of SAH and to evaluate the influence of AICAR (which is known to mimic AMP and activates AMPK) and compound C (a commonly used AMPK inhibitor) on EBI in rats following SAH. METHODS: Adult male rats were divided into six groups: control, sham, SAH, SAH + vehicle, SAH + AICAR and SAH + compound C. SAHs were induced by a modified endovascular perforation method. Immunohistochemistry, real-time PCR and Western blot were used to detect the spatial and dynamic expression of AMPK after SAH. Cortical apoptosis and the expressions of apoptosis-related proteins such as FOXO3a (forkhead box, class O, 3a) and Bim (Bcl-2-interacting mediator of cell death) were detected after different drug interventions. RESULTS: We found SAH induced prolonged activation of AMPK. Treatment with AICAR markedly induced overactivation of AMPK and upregulation of FOXO3a and Bim. AICAR also significantly exacerbated cerebral apoptosis and neurological impairment following SAH. On the other hand, pre-administration of compound C attenuated EBI in this SAH model by modulating cerebral apoptosis by inhibiting FOXO3a and Bim. CONCLUSIONS: Our findings suggest that the AMPK pathway may play an important role in SAH-induced neuronal apoptosis, and the use of AMPK inhibitors can provide neuroprotection in EBI after SAH.

Advances in research of the neuroprotective mechanisms of cerebral ischemic postconditioning.

Ischemic postconditioning refers to controlling reperfusion blood flow during reperfusion after ischemia, which can induce an endogenous neuroprotective effect and reduce ischemia-reperfusion injury. Activation of endogenous neuroprotective mechanisms plays a key role in protecting against brain ischemia-reperfusion injury. The mechanisms of cerebral ischemic postconditioning are not completely clear, and the following aspects may be involved: downregulation of oxidative stress, attenuating mitochondrial dysfunction, attenuating endoplasmic reticulum stress, accelerating the elimination of glutamate, increasing rCBF, inhibiting apoptosis, inhibiting autophagy, and regulating signal transduction.

The role of rosiglitazone in the proliferation of vascular smooth muscle cells after experimental subarachnoid hemorrhage.

BACKGROUND: Recent evidence has demonstrated that rosiglitazone can attenuate cerebral vasospasm following subarachnoid hemorrhage (SAH). Some studies have shown that rosiglitazone can suppress inflammation and immune responses after SAH. However, the precise molecular mechanisms by which cerebral vasospasm is attenuated is not clear. METHODS: In this study, SAH was created using a "double hemorrhage" injection rat model. Rats were randomly divided into three groups and treated with saline (control group), untreated (SAH group), or treated with rosiglitazone. Using immunocytochemistry, hematoxylin and eosin (HE) staining, and measurement of the basilar artery, we investigated the formation of pathologic changes in the basilar artery, measured the expression of caveolin-1 and proliferating cell nuclear antigen (PCNA), and investigated the role of rosiglitazone in vascular smooth muscle cell (VSMC) proliferation in the basilar artery after SAH. RESULTS: In this study, we observed significant pathologic changes in the basilar artery after experimental SAH. The level of vasospasm gradually increased with time during the 1st week, peaked on day 7, and almost recovered on day 14. After rosiglitazone treatment, the level of vasospasm was significantly attenuated in comparison with the SAH group. Immunocytochemistry staining showed that caveolin-1 expression was significantly increased in the rosiglitazone group, compared with the SAH group. Inversely, the expression of PCNA showed a notable decrease after rosiglitazone treatment. CONCLUSIONS: The results indicate that rosiglitazone can attenuate cerebral vasospasm following SAH. Up-regulation of caveolin-1 by rosiglitazone may be a new molecular mechanism for this response, which is to inhibit proliferation of VSMCs after SAH, and this study may provide a novel insight to prevent delayed cerebral vasospasm (DCVS).

Dynamic expression of the suppressor of cytokine signaling-3 and cytokines in the cerebral basilar artery of rats with subarachnoid hemorrhage, and the effect of acetylcholine.

BACKGROUND: There are complex interactions between acetylcholine (ACh), the suppressor of cytokine signaling-3 (SOCS-3), and cytokines, however, little is known about their dynamic expression or their effects on cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). Therefore, we aimed to describe and clarify the dynamic expression of SOCS-3 and cytokines after SAH, as well as the relationships between the levels of SOCS-3, cytokines, and ACh. METHODS: The rat model of single cisterna magna injection was used to mimic acute SAH. The degree of CVS was indicated by lumen diameter and artery wall thickness under H&E staining. A semi-quantitative immunohistochemical analysis method was used to clarify the role of SOCS-3 in the CVS after SAH. We also measured the content of IL-6 and IL-10 in cerebrospinal fluid. RESULTS: We found that SOCS-3 expression levels increased rapidly within 12 h after SAH, more slowly after 12 h, and did not reach a peak within 48 h. Interleukin 6 (IL-6) levels rapidly increased within 24 h after SAH, reached a peak 24 h after SAH, and decreased slightly at 48 h. IL-10 levels increased during the first 6 h after SAH, after which this increase tapered off. ACh treatment reduced IL-6 levels and resulted in elevated levels of SOCS-3, but had no effect on IL-10 expression. Furthermore, ACh treatment relieved basilar arterial vasospasm, whereas mecamylamine pretreatment counteracted the activity of ACh. CONCLUSIONS: Taken together, these data indicate that SOCS-3 was involved in vasospasm via an IL-6- and IL-10-related mechanism, and that CVS following SAH could be reversed by the intraventricular injection of ACh.

The roles of MMP-9/TIMP-1 in cerebral edema following experimental acute cerebral infarction in rats.

Matrix metalloproteinases 9 (MMP-9) and its endogenous inhibitor, tissue inhibitor of metalloproteinases 1 (TIMP-1), regulate homeostasis and turnover of the extra cellular matrix (ECM). They play important roles in acute cerebral infarction (ACI). The contributions of MMP-9 and TIMP-1 to the early stages of ACI are not completely understood. This study investigates the time course of MMP-9 and TIMP-1 and their relations to edema after ACI in rats. Serum concentrations of MMP-9 and TIMP-1 protein were measured using ELISA and mRNA level were measured using real-time PCR. Brain samples were harvested and the brain water content (BWC) was measured. Results revealed that MMP-9 concentration increased fast during the first 12 h after ACI, while after 12 h the increase was much slower. The MMP-9 protein concentration was elevated earlier than the mRNA level. BWC increased starting at 6 h after ACI to reach a peak at 12 h and decreased back to normal levels at 72 h. Both the MMP-9 protein and its mRNA were positively correlated with BWC, however no correlation was found between TIMP-1 levels and BWC. The MMP-9/TIMP-1 protein ratio was more closely correlated with BWC than the MMP-9 concentration. These results indicate that brain edema induced by ACI is associated with increased MMP-9 levels and MMP-9/TIMP-1 ratio in serum.

Role of Akt signaling pathway in delayed cerebral vasospasm after subarachnoid hemorrhage in rats.

BACKGROUND: Akt plays an important role in cell survival, proliferation, apoptosis and other activities. It also has been involved in maintaining smooth muscle cell contraction phenotypes in vitro and in vivo. Recent studies have focused on the inhibition of Akt in acute vasospasm and neuronal apoptosis after subarachnoid hemorrhage (SAH). However, its role in delayed cerebral vasospasm (DCVS) has not been reported. METHODS: In this study, using a "two-hemorrhage" rat model of SAH, we examined the expression of p-Akt and the formation of vasospasm in the basilar arteries. To investigate the possible role of Akt in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and proliferating cell nuclear antigen (PCNA), markers of smooth muscle phenotypic switching. RESULTS: We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became most severe on day 7 after SAH. Elevated protein expression of p-Akt was detected 4 days after SAH induction, peaked on day 7, and recovered on day 21, which was in a parallel time course to the development of DCVS. Moreover, results of immunohistochemical staining revealed enhanced expression of PCNA but gradual reduction in expression of SMα-actin from day 1 to day 7 after SAH; then, the expressions of PCNA and SMα-actin gradually recovered until day 21. CONCLUSIONS: These results support a novel mechanism in which the Akt signaling pathway plays an important role in the proliferation of smooth muscle cells (SMCs) rather than inducing phenotype switching in basilar arteries, which promotes the development of DCVS after SAH.

Potential contribution of SOCC to cerebral vasospasm after experimental subarachnoid hemorrhage in rats.

Cerebral vasospasm (CVS) is the most treatable component of subarachnoid hemorrhage (SAH), which can be reduced by endothelin receptor antagonists. Endothelin-evoked vasospasm is considered to be mediated by Ca(2+) influx in the smooth muscle through voltage-dependent Ca(2+) channel (VDCC) and nonselective cation channels (NSCC). Because VDCC antagonists such as nimodipine have been shown to be relatively less effective than the endothelin receptor antagonists, it is assumed that NSCC maybe a more important component in mediating Ca(2+) influx during CVS. In this study, we used the basilar arteries from a "two-hemorrhage" rat model of SAH to investigate expressions of transient receptor potential channel 1 (TRPC1), transient receptor potential channel 3 (TRPC3) and stromal interaction molecule 1 (STIM1), which are considered as the promising candidates constituting NSCC. To investigate the possible role of NSCC in phenotypic switching, we performed immunohistochemical staining to examine expressions of SMα-actin and PCNA, markers of smooth muscle phenotypic switching. We found that the basilar arteries exhibited vasospasm after SAH and that vasospasm became more severe on days 5 and 7 after SAH. Elevated mRNA and protein expressions of TRPC1 and STIM1 were detected after SAH and peaked on days 5 and 7, which was in a parallel time course to the development of cerebral vasospasm. The mRNA and protein expressions of TRPC3 were not changed in the SAH group when compared with those in the control. Results of immunohistochemical staining with anti-PCNA and anti-SMα-actin antibodies also showed enhanced expression of PCNA and disappearance of SMα-actin from day 1 to day 7. Taken together, the above results supported a novel mechanism that the components of store-operated calcium channels, TRPC1 and STIM1 mediated the Ca(2+) influx and phenotypic switching in smooth muscle cells, which promoted the development of vasospasm after SAH. TRPC3, which is a component of receptor-operated calcium channels, was not involved in the above-mentioned mechanism.

High expression of STIM1 in the early stages of diffuse axonal injury.

Increased intracellular calcium ([Ca(2+)](i)) is a key pathological mechanism involved in secondary neuronal injury and cell death due to diffuse axonal injury (DAI). To date, this increased [Ca(2+)](i) is believed to be mainly caused by dysfunction of voltage-gated sodium channels and mechanoporation of the plasma membrane. Store-operated calcium entry (SOCE) is another source of Ca(2+) influx, and stromal interaction molecule 1 (STIM1) is considered as a sensor and a regulator of SOCE. In this study, we established a DAI in vivo model in rats by lateral head rotation. Using immunohistochemistry, real-time RT-PCR and Western blot, we investigated STIM1 expression levels in the cerebral cortex of rats after lateral head rotational injury. Results revealed notably high STIM1 expression in neurons in the early stages (within 24 h) of DAI. STIM1 began to increase at 6 h post-injury (PI) peaked at 12 h PI, and then gradually decreased. At 2 days PI, STIM1 expression in the injury group showed no significant difference compared with that of the control group. These results indicate that abnormal SOCE may participate in Ca(2+) overload of neurons in the early stages after DAI via enhanced STIM1 expression.