Spatiotemporal Cofilin Signaling, Microglial Activation, Neuroinflammation, and Cognitive Impairment Following Hemorrhagic Brain Injury.

Intracerebral hemorrhage (ICH) is a significant health concern associated with high mortality. Cofilin plays a crucial role in stress conditions, but its signaling following ICH in a longitudinal study is yet to be ascertained. In the present study, we examined the cofilin expression in human ICH autopsy brains. Then, the spatiotemporal cofilin signaling, microglia activation, and neurobehavioral outcomes were investigated in a mouse model of ICH. Human autopsy brain sections from ICH patients showed increased intracellular cofilin localization within microglia in the perihematomal area, possibly associated with microglial activation and morphological changes. Various cohorts of mice were subjected to intrastriatal collagenase injection and sacrificed at time points of 1, 3, 7, 14, 21, and 28 days. Mice suffered from severe neurobehavioral deficits after ICH, lasting for 7 days, followed by a gradual improvement. Mice suffered post-stroke cognitive impairment (PSCI) both acutely and in the chronic phase. Hematoma volume increased from day 1 to 3, whereas ventricle size increased from day 21 to 28. Cofilin protein expression increased in the ipsilateral striatum on days 1 and 3 and then decreased from days 7 to 28. An increase in activated microglia was observed around the hematoma on days 1 to 7, followed by a gradual reduction up to day 28. Around the hematoma, activated microglia showed morphological changes from ramified to amoeboid. mRNA levels of inflammatory [tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), and interleukin-6 (IL-6) and anti-inflammatory markers [interleukin-10 (IL-10), transforming growth factor-ß TGF-ß, and arginase I (Arg1)] increased during the acute phase and decreased in the chronic phase. Blood cofilin levels increased on day 3 and matched the increase in chemokine levels. slingshot protein phosphatase 1 (SSH1) protein, which activates cofilin, was increased from day 1 to 7. These results suggest that microglial activation might be the sequel of cofilin overactivation following ICH, leading to widespread neuroinflammation and consequent PSCI.

Cofilin Inhibitor Protects against Traumatic Brain Injury-Induced Oxidative Stress and Neuroinflammation.

Microglial activation and failure of the antioxidant defense mechanisms are major hallmarks in different brain injuries, particularly traumatic brain injury (TBI). Cofilin is a cytoskeleton-associated protein involved in actin binding and severing. In our previous studies, we identified the putative role of cofilin in mediating microglial activation and apoptosis in ischemic and hemorrhagic conditions. Others have highlighted the involvement of cofilin in ROS production and the resultant neuronal death; however, more studies are needed to delineate the role of cofilin in oxidative stress conditions. The present study aims to investigate the cellular and molecular effects of cofilin in TBI using both in vitro and in vivo models as well as the first-in-class small-molecule cofilin inhibitor (CI). An in vitro H2O2-induced oxidative stress model was used in two different types of cells, human neuroblastoma (SH-SY5Y) and microglia (HMC3), along with an in vivo controlled cortical impact model of TBI. Our results show that treatment with H2O2 increases the expression of cofilin and slingshot-1 (SSH-1), an upstream regulator of cofilin, in microglial cells, which was significantly reduced in the CI-treated group. Cofilin inhibition significantly attenuated H2O2-induced microglial activation by reducing the release of proinflammatory mediators. Furthermore, we demonstrate that CI protects against H2O2-induced ROS accumulation and neuronal cytotoxicity, activates the AKT signaling pathway by increasing its phosphorylation, and modulates mitochondrial-related apoptogenic factors. The expression of NF-E2-related factor 2 (Nrf2) and its associated antioxidant enzymes were also increased in CI-treated SY-SY5Y. In the mice model of TBI, CI significantly activated the Nrf2 and reduced the expression of oxidative/nitrosative stress markers at the protein and gene levels. Together, our data suggest that cofilin inhibition provides a neuroprotective effect in in vitro and in vivo TBI mice models by inhibiting oxidative stress and inflammatory responses, the pivotal mechanisms involved in TBI-induced brain damage.

Synthesis and Pharmacological Evaluation of Novel Triazole-Pyrimidine Hybrids as Potential Neuroprotective and Anti-neuroinflammatory Agents.

OBJECTIVE: Neuroprotection is a precise target for the treatment of neurodegenerative diseases, ischemic stroke, and traumatic brain injury. Pyrimidine and its derivatives have been proven to use antiviral, anticancer, antioxidant, and antimicrobial activity prompting us to study the neuroprotection and anti-inflammatory activity of the triazole-pyrimidine hybrid on human microglia and neuronal cell model. METHODS: A series of novel triazole-pyrimidine-based compounds were designed, synthesized and characterized by mass spectra, 1HNMR, 13CNMR, and a single X-Ray diffraction analysis. Further, the neuroprotective, anti-neuroinflammatory activity was evaluated by cell viability assay (MTT), Elisa, qRT-PCR, western blotting, and molecular docking. RESULTS: The molecular results revealed that triazole-pyrimidine hybrid compounds have promising neuroprotective and anti-inflammatory properties. Among the 14 synthesized compounds, ZA3-ZA5, ZB2-ZB6, and intermediate S5 showed significant anti-neuroinflammatory properties through inhibition of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated human microglia cells. From 14 compounds, six (ZA2 to ZA6 and intermediate S5) exhibited promising neuroprotective activity by reduced expression of the endoplasmic reticulum (ER) chaperone, BIP, and apoptosis marker cleaved caspase-3 in human neuronal cells. Also, a molecular docking study showed that lead compounds have favorable interaction with active residues of ATF4 and NF-kB proteins. CONCLUSION: The possible mechanism of action was observed through the inhibition of ER stress, apoptosis, and the NF-kB inflammatory pathway. Thus, our study strongly indicates that the novel scaffolds of triazole-pyrimidine-based compounds can potentially be developed as neuroprotective and anti-neuroinflammatory agents.

Synthesis and Development of a Novel First-in-Class Cofilin Inhibitor for Neuroinflammation in Hemorrhagic Brain Injury.

Intracerebral hemorrhage (ICH) is devastating among stroke types with high mortality. To date, not a single therapeutic intervention has been successful. Cofilin plays a critical role in inflammation and cell death. In the current study, we embarked on designing and synthesizing a first-in-class small-molecule inhibitor of cofilin to target secondary complications of ICH, mainly neuroinflammation. A series of compounds were synthesized, and two lead compounds SZ-3 and SK-1-32 were selected for further studies. Neuronal and microglial viabilities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay using neuroblastoma (SHSY-5Y) and human microglial (HMC-3) cell lines, respectively. Lipopolysaccharide (LPS)-induced inflammation in HMC-3 cells was used for neurotoxicity assay. Other assays include nitric oxide (NO) by Griess reagent, cofilin inhibition by F-actin depolymerization, migration by scratch wound assay, tumor necrosis factor (TNF-α) by enzyme-linked immunosorbent assay (ELISA), protease-activated receptor-1 (PAR-1) by immunocytochemistry and Western blotting (WB), and protein expression levels of several proteins by WB. SK-1-32 increased neuronal/microglial survival, reduced NO, and prevented neurotoxicity. However, SZ-3 showed no effect on neuronal/microglial survival but prevented microglia from LPS-induced inflammation by decreasing NO and preventing neurotoxicity. Therefore, we selected SZ-3 for further molecular studies, as it showed potent anti-inflammatory activities. SZ-3 decreased cofilin severing activity, and its treatment of LPS-activated HMC-3 cells attenuated microglial activation and suppressed migration and proliferation. HMC-3 cells subjected to thrombin, as an in vitro model for hemorrhagic stroke, and treated with SZ-3 after 3 h showed significantly decreased NO and TNF-α, significantly increased protein expression of phosphocofilin, and decreased PAR-1. In addition, SZ-3-treated SHSY-5Y showed a significant increase in cell viability by significantly reducing nuclear factor-κ B (NF-κB), caspase-3, and high-temperature requirement (HtrA2). Together, our results support the novel idea of targeting cofilin to counter neuroinflammation during secondary injury following ICH.

A reappraisal of the 6-O-desmethylnaproxen-sulfating activity of the human cytosolic sulfotransferases.

In this study, we aimed to obtain a comprehensive account of the human cytosolic sulfotransferases (SULTs) that are capable of sulfating 6-O-desmethylnaproxen (O-DMN), a major metabolite of naproxen. Of the 13 known human SULTs tested, 7 (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, and SULT1E1) displayed O-DMN-sulfating activity, when analyzed using an elevated substrate concentration (500 µmol·L-1) together with 14 µmol·L-1 of the sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). At 10 µmol·L-1 O-DMN concentration, however, only SULT1A1 and SULT1A3 displayed detectable activity, with the former being nearly 2 orders of magnitude more active than the latter. A pH-dependence study indicated that SULT1A1 exhibited a broad pH optimum spanning pH 5.5-7. Kinetic parameters of the sulfation of O-DMN by SULT1A1 were determined. The production and release of sulfated O-DMN was demonstrated using cultured human HepG2 hepatoma cells and Caco-2 colon carcinoma cells. Moreover, assays using human organ specimens revealed that the O-DMN-sulfating activities present in the cytosols of liver and small intestine (at 502.5 and 497.2 pmol·min-1·(mg protein)-1, respectively) were much higher than those detected for the cytosols of lung and kidney. Taken together, these results provided relevant information concerning the sulfation of O-DMN both in vitro and in vivo.