Inhibition of immunoproteasome promotes angiogenesis via enhancing hypoxia-inducible factor-1α abundance in rats following focal cerebral ischaemia.

Angiogenesis after ischemic stroke contributes to the restoration of blood supply in the ischemic zone. Strategies to improve angiogenesis may facilitate the function recovery after stroke. Growing evidence shows that proteasome inhibitors enhance angioneurogenesis and induces a long-term neuroprotection after cerebral ischemia in rodents' models. We have previously reported that inhibition of the immunoproteasome subunit low molecular mass peptide 2 (LMP2) offers a strong neuroprotection in ischemic stroke rats. However, there are no data available to show the relationship between immunoproteasome and angiogenesis under ischemia stroke context. In this study, we identified that inhibition of immunoproteasome LMP2 was able to enhance angiogenesis and facilitate neurological functional recovery in rats after focal cerebral ischemia/reperfusion. In vitro, oxygen-glucose deprivation and reperfusion (OGD/R) significantly enhanced the expression of immunoproteasome LMP2 and proteasome activities in primary culture astrocytes, but these beneficial effects were abolished by knockdown of LMP2 with siRNA transfection. Along with this, protein abundance of HIF-1α was significantly increased by inhibition LMP2 in vivo and in vitro and was associated with angiogenesis and cell fates. However, these beneficial effects were partly abolished by HIF-1α inhibitor 2-methoxyestradiol (2ME). Taken together; this study highlights an important role for inhibition of LMP2 in promoting angiogenesis events in ischemic stroke, and point to HIF-1α as a key mediator of this response, suggesting that immunoproteasome inhibitors may be a promising strategy for stroke treatment.

Plasma Immunoproteasome Predicts Early Hemorrhagic Transformation in Acute Ischemic Stroke Patients.

BACKGROUND AND PURPOSE: Currently, blood biomarkers associated with an increased hemorrhagic transformation (HT) risk remain uncertain. We aimed to determine the significance of immunoproteasome as predictors of early HT in acute ischemic stroke patients. METHODS: This study enrolled 316 patients with ischemic stroke. HT was assessed by computed tomography examination performed on day 5 ± 2 after stroke onset or immediately in case of clinical deterioration (CD). Plasma immunoproteasome subunits low molecular mass peptide 2 (LMP2), multicatalytic endopeptidase complex-like 1 (MECL-1), LMP7, interleukin-1ß (IL1ß), and high-sensitivity C-reactive protein (Hs-CRP) were measured with quantitative sandwich enzyme-linked immunosorbent assay kits. Factors associated with HT were analyzed using a multivariate logistic regression analysis. RESULTS: There were 42 (13.3%, 42 of 316) patients who experienced HT. Compared with those patients without HT, plasma LMP2, MECL-1, LMP7, IL1ß, and Hs-CRP concentrations on admission were significantly increased in patients with subsequent HT (P < .001). These protein concentrations increased with hemorrhage severity. Patients with CD caused by HT had the highest levels of LMP2 (1679.5 [1394.6-136.6] pg/mL), MECL-1 (992.5 [849.7-1075.8] pg/mL), LMP7 (822.6 [748.6-1009.5] pg/mL), IL1ß (113.2 [90.6-194.5] pg/mL), and Hs-CRP (30.0 [12.8-75.6] mg/L) (P < .05). Logistic regression analysis showed cardioembolism, LMP2, MECL-1, and LMP7 as independent predictors of HT (P < .05). Receiver operating characteristic curve analysis demonstrated LMP2 ≥ 988.3 pg/mL, MECL-1 ≥ 584.7 pg/mL, and LMP7 ≥ 509.0 pg/mL as independent factors associated with HT (P < .001). CONCLUSION: Evaluation of plasma levels of immunoproteasome could be helpful in the early prediction of HT in acute ischemic stroke patients.

Scutellarin attenuates hypertension-induced expression of brain Toll-like receptor 4/nuclear factor kappa B.

Hypertension is associated with low-grade inflammation, and Toll-like receptor 4 (TLR4) has been shown to be linked to the development and maintenance of hypertension. This study aimed to investigate the effects of scutellarin (administered by oral gavage daily for 2 weeks) on brain TLR4/nuclear factor kappa B-(NF- κ B-) mediated inflammation and blood pressure in renovascular hypertensive (using the 2-kidney, 2-clip method) rats. Immunofluorescence and western immunoblot analyses revealed that hypertension contributed to the activation of TLR4 and NF- κ B, accompanied by significantly enhanced expression of proinflammatory mediators, such as tumor necrosis factor- α (TNF- α ), interleukin-1 ß (IL-1 ß ), and interleukin-18 (IL-18). Furthermore, expression of the antiapoptotic protein, myeloid cell leukemia-1 (Mcl1), was decreased, and the pro-apoptotic proteins, Bax and cleavedcaspase-3 p17 were increased in combined cerebral cortical/striatal soluble lysates. Scutellarin significantly lowered blood pressure and attenuated the number of activated microglia and macrophages in brains of hypertensive rats. Furthermore, scutellarin significantly reduced the expression of TLR4, NF- κ B p65, TNF- α , IL-1 ß , IL-18, Bax and cleaved-caspase-3 p17, and increased the expression of Mcl1. Overall, these results revealed that scutellarin exhibits anti-inflammatory and anti-apoptotic properties and decreases blood pressure in hypertensive rats. Therefore, scutellarin may be a potential therapeutic agent in hypertension-associated diseases.

[Expression of matrix metalloproteinase MMP-9 in the plasma and hematoma fluid of intracerebral hemorrhage patients].

OBJECTIVE: To investigate the matrix metalloproteinase (MMP)-9 played in secondary brain injury following intracerebral hemorrhage (ICH). METHODS: Hematoma fluid and peripheral blood samples were collected from 60 ICH patients, 34 males and 26 females, aged 60 +/- 13 (37 - 81) n the days 1, 4, and 7 after evacuation of hematoma. Peripheral blood samples were collected form. 30 sex, and age-matched healthy adults as normal controls. Cerebrospinal fluid (SCF) samples were collected from 10 sex, and age-matched patients to undergo operation during lumbar anesthesia. ELISA was used to detect the content of MMP-9. Tada formula was used to calculate the perihematomal edema volume. The National Institutes of Health Stroke Scale (NIHHS) and Glasgow Coma Score (GCS) were used to assess the condition of patients. RESULTS: (1) The MMP-9 levels in the plasma and hematoma fluid of the ICH patients at all time points were all significantly higher than those of the normal controls (all P < 0.01). MMP-9 was not found in the normal CSF. (2) The plasma and hematoma fluid MMP-9 levels were increased already in the day 1, peaked in the day 4, and then kept at a high level until the day 7. (3) The MMP-9 levels in hematoma fluid t all time points were all significantly higher than those in the plasma (all P < 0.01). (4) The MMP-9 level was positively correlated with the hematoma volume and NIHSS score, and negatively correlated with the GCS score (both P < 0.01). CONCLUSIONS: MMP-9 may takes part in the secondary injury after ICH, and its change is correlated with the hydrocephalus of patients. The dynamical change of the plasma MMP-9 level is consistent with the hematoma fluid MMP-9 level after ICH. There is a positive correlation among the MMP-9 level, perihematomal edema volume, and severity of ICH.