Caspofungin Suppresses Brain Cell Necroptosis in Ischemic Stroke Rats via Up-Regulation of Pellino3.

PURPOSE: Pellino3, an ubiquitin E3 ligase, prevents the formation of the death-induced signaling complex in response to TNF-α by targeting receptor-interacting protein kinase 1 (RIPK1), and bioinformatics analysis predicted an interaction between Pellino3 and caspofungin, a common antifungal drug used in clinics. This study aimed to explore the effect of caspofungin on brain injury in ischemic stroke and the underlying mechanisms. METHODS: Ischemic stroke injury was induced in Sprague Dawley rats by occlusion of the middle cerebral artery (MCA) for 2 h, followed by 24 h reperfusion. PC12 cells were deprived of both oxygen and glucose for 8 h and then were cultured for 24 h with oxygen and glucose to mimic an ischemic stroke in vitro. RESULTS: Animal experiments showed brain injury (increase in neurological deficit score and infarct volume) concomitant with a downregulation of Pellino3, a decreased ubiquitination of RIPK1, and an up-regulation of necroptosis-associated proteins [RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), p-RIPK1, p-RIPK3, and p-MLKL]. Administration of caspofungin (6 mg/kg, i.m.) at 1 h and 6 h after ischemia significantly improved neurological function, reduced infarct volume, up-regulated Pellino3 levels, increased RIPK1 ubiquitination, and down-regulated protein levels of RIPK1, p-RIPK1, p-RIPK3, and p-MLKL. PC12 cells deprived of oxygen/glucose developed signs of cellular injury (LDH release and necroptosis) concomitant with downregulation of Pellino3, decreased ubiquitination of RIPK1, and elevated necroptosis-associated proteins. These changes were reversed by overexpression of Pellino3. CONCLUSION: We conclude that Pellino3 has an important role in counteracting necroptosis via ubiquitination of RIPK1 and caspofungin can suppress the brain cell necroptosis in ischemic stroke through upregulation of Pellino3.

TIMP-1 polymorphisms in a Chinese Han population with intracerebral hemorrhage.

Remodeling of extracellular matrix (ECM) and breakdown of blood-brain barrier (BBB) are crucial events in the pathogenesis of intracerebral hemorrhage (ICH). Matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, are the most important degrading enzymes in the ECM and BBB. These proteolytic effects are controlled predominantly by tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 is the main endogenous inhibitor of MMP-9. Two polymorphisms in the TIMP-1 gene (rs4898 and rs2070584) were selected through a literature review and successfully genotyped in a study sample of 410 ICH patients and 305 controls. Differences in genotype and allele frequencies of identified polymorphisms were determined. Furthermore, the serum levels of TIMP-1 were measured in a subgroup of 96 ICH patients on days 1 after ICH onset and 76 controls. Analyses showed that C allele of rs2070584 was significantly associated with the development of ICH in male subjects (p = 0.037, OR = 1.535, 95%CI 1.025-2.300). Multiple logistic regression analysis under three genetic models demonstrated both rs4898 and rs2070584 were not risk factors for ICH in female subjects. Furthermore, serum levels of TIMP-1 were significantly higher in ICH patients than those in normal controls. However, the serum levels of TIMP-1 showed a nonsignificant decrease, depending on the alleles and genotypes of rs2070584 both in male and female cases. In conclusion, this is the first association study of the TIMP-1 gene variants with ICH. Our data suggest that C allele of rs2070584 is a risk factor for ICH development in the Chinese male population. However, the precise function of this variant needs further investigation.

The Protective Effect of Vitexin Compound B-1 on Rat Cerebral I/R Injury through a Mechanism Involving Modulation of miR-92b/NOX4 Pathway.

BACKGROUND: Recent studies have uncovered that vitexin compound B-1 (VB-1) can protect neurons against hypoxia/reoxygenation (H/R)-induced oxidative injury through suppressing NOX4 expression. OBJECTIVE: The aims of this study are to investigate whether VB-1 can protect the rat brain against ischemia/ reperfusion (I/R) injury and whether its effect on NOX4 expression is related to modulation of certain miRNAs expression. METHODS: Rats were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion to establish an I/R injury model, which showed an increase in neurological deficit score and infarct volume concomitant with an upregulation of NOX4 expression, increase in NOX activity, and downregulation of miR-92b. RESULTS: Administration of VB-1 reduced I/R cerebral injury accompanied by a reverse in NOX4 and miR-92b expression. Similar results were achieved in a neuron H/R injury model. Next, we evaluated the association of miR-92b with NOX4 by its mimics in the H/R model. H/R treatment increased neurons apoptosis concomitant with an upregulation of NOX4 and NOX activity while downregulation of miR-92b. All these effects were reversed in the presence of miR-92b mimics, confirming the function of miR-92b in suppressing NOX4 expression. CONCLUSION: We conclude the protective effect of VB-1 against rat cerebral I/R injury through a mechanism involving modulation of miR-92b/NOX4 pathway.

Polymyxin B Reduces Brain Injury in Ischemic Stroke Rat Through a Mechanism Involving Targeting ESCRT-III Machinery and RIPK1/RIPK3/MLKL Pathway.

Endosomal sorting complex required for transport III (ESCRT-III) machinery is a key component to counteract the mixed lineage kinase domain-like pseudokinase (MLKL)-induced plasma membrane broken in cells undergoing necroptosis. Based on the bioinformatics analysis, polymyxin B, a polypeptide antibiotic, is predicted to simultaneously interact with ESCRT-III subunits and necroptosis-relevant proteins. This study aims to explore whether polymyxin B could reduce necroptosis in the stroke rat brain via enhancing the ESCRT-III machinery and/or suppressing the RIPK1/RIPK3/MLKL pathway. The stroke rats showed evident brain injury, concomitant with the downregulation of ESCRT-III subunits and the upregulation of necroptosis-relevant proteins. Post-ischemic administration of polymyxin B could alleviate the brain injury, accompanied by restoration of the levels of ESCRT-III subunits and suppression of necroptosis-relevant proteins. And, polymyxin B exerted similar effects in hypoxia-treated HT22 cells. We conclude that polymyxin B can reduce necroptosis in the stroke rat brain via enhancing the ESCRT-III machinery and suppressing the RIPK1/RIPK3/MLKL pathway simultaneously.

Dipsacoside B Exerts a Beneficial Effect on Brain Injury in the Ischemic Stroke Rat through Inhibition of Mitochondrial E3 Ubiquitin Ligase 1.

BACKGROUND: Upregulation of mitochondrial E3 ubiquitin ligase 1 (Mul1) contributes to brain injury in ischemic stroke due to disturbance of mitochondrial dynamics, and bioinformatics analysis predicts that Mul1 is a potential target of Dipsacoside B. OBJECTIVE: The aim of the study was to explore whether Dipsacoside B can exert a beneficial effect on brain injury in the ischemic stroke rat via targeting Mul1. METHODS: The SD rat brains or PC12 cells were subjected to 2 h-ischemia or 8 h-hypoxia plus 24 h-reperfusion or 24 h-reoxygenation to establish the ischemic stroke rat model in vivo or in vitro, which were treated with Dipsacoside B at different dosages. The brain or PC12 cell injury, relevant protein levels and mitochondrial functions were measured by methods of biochemistry, flow cytometry or Western blot. RESULTS: The neurological dysfunction and brain injury (such as infarction and apoptosis) observed in the ischemic stroke rats were accompanied by increases in Mul1 and Dynamin-related protein 1 (Drp1) levels along with decreases in mitofusin 2 (Mfn2) level and ATP production. These effects were attenuated by Dipsacoside B. Consistently, cell injury (necroptosis and apoptosis) occurred in the PC12 cells exposed to hypoxia concomitant with the upregulation of Mul1 and Drp1 along with downregulation of Mfn2 and mitochondrial functions (such as increases in reactive oxygen species production and mitochondrial fission and decreases in mitochondrial membrane potential and ATP production).These phenomena were reversed in the presence of Dipsacoside B. CONCLUSION: Dipsacoside B can protect the rat brain against ischemic injury via inhibition of Mul1 due to the improvement of mitochondrial function.

[Association of single nucleotide polymorphisms of kallikrein 1 gene with cerebral hemorrhage in Changsha Han Chinese].

OBJECTIVE: To explore the association between single nucleotide polymorphisms (SNPs) of KLK1 gene and cerebral hemorrhage in Changsha Han Chinese. METHODS: Two hundred and seventy-three cerebral hemorrhage (CH) patients and 140 healthy controls were collected. The SNPs of rs5516 and rs5517 loci of KLK1 gene were analyzed by SNaPshot methods and direct sequencing. RESULTS: (1)Genotype and allele frequencies in rs5516 locus had no difference between the CH patients and controls (P> 0.05). However, the A allele frequency of the rs5517 locus in CH patients was higher than that in the control group (0.419, 0.321 respectively, P< 0.05). (2)In the control group,the levels of diastolic blood pressure (DBP) of the GA and AA genotype carriers of the rs5517 locus were significantly higher than those of the GG genotype (P< 0.05), while the levels of blood pressure were not significantly different among different genotypes of the rs5516 polymorphism in both CH patients and the control group(P> 0.05). CONCLUSION: Author's preliminary results suggested that the rs5517 polymorphism was associated with cerebral hemorrhage, while the rs5516 polymorphism was not in Changsha Han Chinese.

[The association of apolipoprotein B gene polymorphism with cerebral infarction with positive family history and its effect on plasma lipid levels].

OBJECTIVE: To explore the relationship between apolipoprotein B (apoB) gene G12669A polymorphism and cerebral infarction with family history, and to evaluate the effect of G12669A polymorphism on plasma lipid levels. METHODS: Peripheral blood samples were collected from 147 members of 15 cerebral infarction families, including 47 cerebral infarction patients with positive family history (CIFH-P), 43 first-degree relatives (CIFH-I), 28 second-degree relatives (CIFH-II), and 29 third-degree relatives (CIFH-III), 83 sporadic cerebral infarction (SCI) patients, and 100 healthy controls. Polymerase chain reaction- restriction fragment length polymorphism was used to detect the apoB gene G12669A polymorphism. Oxidase method was used to detect the levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The serum levels of lipoprotein (a) [LP (a)], apoB-100, and apoAI were determined by immune method. RESULTS: (1) The frequencies of A allele in the CIFH-P, CIFH-I, CIFH-II, CIFH-III, and SCI groups patients and control group were 0.106, 0.081, 0.036, 0.034, 0.090, and 0.045 respectively, that of the CIFH-P group being significantly higher than that of the control group (P < 0.05), and those of the CIFH-I, CIFH-II, CIFH-III, and SCI groups not being significantly different from that of the control group. (2) In both CIFH-P and SCI groups, the TC and LDL-C levels of the patients with G/A gene type were significantly higher than those of the G/G gene type, while the HDL-C level of the patients with G/A gene type was significantly lower than that of the G/G gene type (all P < 0.05). CONCLUSION: A allele in G12669A polymorphism may be one of the genetic factors influencing the susceptibility to CI in the individuals with a positive family history, and it may play its role through its influence on the blood lipid levels.

[Effect of apoB polymorphism on plasma lipid levels and cerebral hemorrhage in Changsha Han Chinese].

OBJECTIVE: To explore the effect of apoB polymorphism on plasma lipid levels and cerebral hemorrhage in (CH) Changsha Han Chinese. METHODS: One hundred thirty CH patients and 100 normal people were involved. C7673T polymorphism of apoprotein B was analyzed by PCR-restriction fragment length polymorphism (PCR-PFLP); and the triglyceride(TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL), and low density lipoprotein-cholesterol(LDL) levels were examined by oxidase method. The serum level of lipoprotein(a) was determined by immunology method. RESULTS: (1) Allele T frequencies of apoB C7673T in CH patients and the control group were 0.108 and 0.040, respectively. Allele T frequencies of apoB C7673T in the CH patients were significantly higher than those in the control group (P< 0.01). (2) In the CH patients, the levels of TC and LDLjC of the T/C gene type were significantly higher than those of the C/C gene type, while the levels of HDLjC of the T/C gene type were significantly lower than those of the C/C gene type (P< 0.05). CONCLUSION: ApoB C7673T polymorphism may be related to cerebral hemorrhage, and the changing blood lipid level may increase the susceptibility of CH.

[Effect of willed movement therapy on the expression of neurotrophin 3 and growth-associated protein 43 in rats with cerebral ischemia reperfusion].

OBJECTIVE: To observe the effect of willed movement therapy on the expression of neurotrophin 3 (NT-3) and growth associated protein 43 (GAP-43) in rats with cerebral ischemia-reperfusion (IR) and investigate the neuroprotective mechanism of willed movement therapy in nerve regeneration and repair. METHODS: Cerebral IR model was established by middle cerebral artery occlusion (MCAO) in SD rats. The rats were randomly divided into MCAO group, environment modification group (EM group) and willed movement therapy group (WM group). The rats were evaluated for neurological deficits and decapitated on days 3, 7 and 15 after the reperfusion to examine the expressions of NT-3 and GAP-43 in the ischemic brain tissues by immunohistochemistry. RESULTS: Compared with MCAO and EM groups, the rats in WM group showed significantly lowered grade of neurological deficits (P<0.05) at 15 days and significantly increased the expressions of NT-3 and GAP-43 (P<0.05) at 7 and 15 days after the reperfusion. No significant difference was found in the expression of NT-3 and GAP-43 between MCAO and EM groups (P>0.05). The expression of NT-3 was positively correlated to that of GAP-43 in the ischemic tissues. CONCLUSIONS: Willed movement therapy increases the expression of NT-3 and GAP-43 in the ischemic brain area in rats with cerebral ischemia-reperfusion, which is probably related to nerve regeneration and repair.

[Effect of willed movement therapy on GFAP and SYP expression in rats with cerebral ischemia-reperfusion].

OBJECTIVE: To determine the effect of willed movement on the expression of glial fibrillary acidic protein (GFAP) and synaptophysin (SYP) in adult rats with cerebral ischemia-reperfusion, and explore the mechanism of willed movement in promoting nerve repair and regeneration. METHODS: Adult rat models of cerebral ischemia-reperfusion injury were established by middle cerebral artery occlusion (MCAO) for 2 h followed by a 24-h reperfusion. The models were then divided randomly into 3 groups, namely the model group, environmental modification (EM) group, and willed movement (WM) group. In each group, neurological deficits were evaluated at 3, 7 and 15 days after reperfusion. Immunohistochemistry and immunofluorescence assay were employed to examine the expression of GFAP and SYP in the brain tissue near the ischemic foci. RESULTS: The rats in WM group showed lessened neurological deficits at 15 days and lowered expression of GFAP and SYP at 7 and 15 days after reperfusion compared with the model and EM groups (P<0.05). No significant difference was found in the expression of GFAP or SYP between the model group and EM group at any time points. CONCLUSION: Willed movement can promote the functional recovery of neurological deficits following cerebral ischemia-reperfusion probably in relation to enhanced GFAP and SYP expressions in the ischemic brain tissues.