Zhonglin Liu

Delivery of cationic polymer-siRNA nanoparticles for gene therapies in neural regeneration.

The therapeutic applications of neural stem cells (NSCs) have potential to promote recovery in many obstinate diseases in central nervous system. Regulation of certain gene expressions using siRNA may have significant influence on the fate of NSC. To achieve the optimum gene silencing effect of siRNA, non-viral vector polyethylene glycol-polyethyleneimine (PEG-PEI) was investigated in the delivery of siRNA to NSCs. The characteristics of PEG-PEI/siRNA polyplexes were detected by scanning electron microscopy (SEM). The effects of nanoparticles on cell viability were measured via CCK-8 assay. In addition, the transfection efficiency was evaluated by fluorescence microscope and flow cytometry, and real-time PCR and Western Blot were employed to detect the gene inhibition effect of siRNA delivered by PEG-PEI. The SEM micrographs showed that PEG-PEI could condense siRNA to form diffuse and spherical nanoparticles. The cytotoxicity of PEG-PEI/siRNA nanocomplexes (N/P=15) was significantly lower when compared with that of Lipofectamine 2000/siRNA (P<0.05). Moreover, the highest transfection efficiency of PEG-PEI/siRNA nanoparticles was obtained at an N/P ratio of 15, which was better than that achieved in the transfection using Lipofectamine 2000 (P<0.05). Finally, the gene knockdown effect of PEG-PEI/siRNA nanoparticles was verified at the levels of mRNA and protein. These results suggest that PEG-PEI may potentially be used as a siRNA delivery vector for neural regeneration therapy.

Investigation of the performance of PEG-PEI/ROCK-II-siRNA complexes for Alzheimer's disease in vitro.

Recent studies have showed inhibiting ROCK promoted axonal regeneration and suppressing ROCK-II decreased Aβ formation, suggesting ROCK is a potential target for the treatment of Alzheimers disease. Because ROCK-II mRNA is abundantly expressed in brain, we targeted ROCK-II mRNA using a siRNA approach. To suppress ROCK-II mRNA expression, we synthesized PEG-PEI/ROCK-II-siRNA complexes and transfected C17.2 neural stem cells in vitro. The characteristics of the complexes were tested using a gel retardation assay. Particle size and zeta potential were examined using dynamic light scattering and the morphology of the complexes were observed by transmission electron microscopy. The toxicity was detected by an MTT assay and transfection efficiency was determined by flow cytometry. Laser confocal microscopy was employed to investigate the cell uptake of the complexes. RT-PCR and western blotting were used to verify the effect of gene silencing. Our results indicated that the characteristics of the complexes depended on the N/P ratios. At a high N/P ratio, PEG-PEI could completely condense the siRNA into small-sized uniform particles. However, high N/P ratios are accompanied with high cytotoxicity. Because of high transfection efficiency and low cytotoxicity, N/P=50 was chosen to transfect C17.2 cells in vitro. Laser confocal microscopy showed that ROCK-II-siRNA with green fluorescence was mainly distributed in the cytoplasm and synapses. Moreover, ROCK-II-siRNA was successfully released from the lysosome. RT-PCR and western blotting demonstrated effective gene silencing. These results indicated that PEG-PEI/ROCK-II-siRNA complexes effectively suppressed ROCK-II mRNA expression, providing the basis for future research in vivo.

Neuroprotective effect of astaxanthin against glutamate-induced cytotoxicity in HT22 cells: Involvement of the Akt/GSK-3β pathway

Oxidative stress (OS) mediated the pathogenesis of Alzheimers disease (AD). Astaxanthin (ATX) has been reported to exert antioxidant activities as well as neuroprotective effects in vivo and in vitro. But it is still unknown whether the Akt/glycogen synthase kinase-3β (GSK-3β) signaling mediated the neuroprotective effect of ATX in HT22 cells. Flow cytometric analysis was used to evaluate reactive oxygen species (ROS) generation. Caspase and PARP activity was measured. The expressions of heme oxygenase-1 (HO-1), nuclear factor-E2-related factor 2 (Nrf2), Bcl-2, Bax, apoptosis-inducing factor (AIF), cytochrome-c (Cyto-c), p-Akt and p-GSK-3β were evaluated to elucidate the underlying mechanism. Our results showed that ATX significantly attenuated glutamate-induced cell viability loss and lactate dehydrogenase (LDH) release, decreased the expression of caspase-3/8/9 activity and cleaved PARP, and suppressed the intracellular accumulation of ROS in HT22 cells after exposure to glutamate. ATX also increased the mitochondrial expression of AIF, Cyto-c as well as Bax while decreased Bcl-2. Moreover, ATX also induced the HO-1 expression in a dose and time-dependent manner, increased the antioxidant-responsive element (ARE) activity and nuclear Nrf2 expression. Furthermore, treatment with ATX restored the p-Akt and p-GSK-3β (Ser9) as well as HO-1 expression reduced by glutamate. This protective effect was partially blocked by the inhibitors lithium chloride treatment in HT22, indicating the involvement of Akt/GSK-3β inactivation during the neuroprotective effect of ATX. Our results provide the first evidence that ATX can protect glutamate-induced cytotoxicity in HT22 via attenuating caspase activation and mitochondrial dysfunction and modulating the Akt/GSK-3β signaling, indicating ATX may be useful for the treatment of neurodegenerative disorders such as AD.

Meta-analysis on the efficacy and tolerability of the augmentation of antidepressants with atypical antipsychotics in patients with major depressive disorder

We assessed the efficacy and tolerability of the augmentation of antidepressants (ATDs) with atypical antipsychotics (AAPs) to treat patients with major depressive disorder. A retrograde study to identify relevant patient data included databases of PubMed, EMBASE, Cochrane Central Register of Controlled Trials, and Database of Abstracts of Reviews of Effects. Data from 17 trials, involving 3807 participants, were identified. The remission rate (RR) and overall response rate (ORR) of adjunctive treatment with AAPs were significantly higher than placebo treatment: RR=1.90 (95%CI=1.61-2.23, z=7.74, P<0.00001) and ORR=1.68 (95%CI=1.45-1.94, z=7.07, P<0.00001). We found that the short-term (4 weeks) treatment [ORR=1.70 (95%CI=0.98-2.95, Z=1.89, P=0.06)] was significantly different from the long-term (6-12 weeks) treatment [ORR=1.68 (95%CI=1.45-1.94, z=7.07, P<0.00001)]. No significant difference in ORR was observed between groups with or without sedative drugs. The discontinuation rate due to adverse effects was higher for adjunctive treatment with AAPs: ORR=3.32 (95%CI=2.35-4.70, z=6.78, P<0.00001). These results demonstrate that the augmentation of ATDs with AAPs (olanzapine, quetiapine, aripiprazole, and risperidone) was more effective than a placebo in improving response and remission rates, although associated with a higher discontinuation rate due to adverse effects.

Characterization of Polyethylene Glycol‐Polyethyleneimine as a Vector for Alpha‐Synuclein siRNA Delivery to PC12 Cells for Parkinson's Disease

Gene therapy targeting the SNCA gene yields promising results in the treatment of Parkinsons disease (PD). The most challenging issue of the RNAi gene therapy strategy is maintaining efficient delivery without inducing significant toxicity and other adverse effects. This study aimed to characterize polyethylene glycol‐polyethyleneimine as a vector for alpha‐synuclein siRNA delivery to PC12 cells for Parkinsons disease.

Intracranial injection of PEG-PEI/ROCK II-siRNA improves cognitive impairment in a mouse model of Alzheimer's disease

Purpose: A plenty of studies have demonstrated that the Rho/ROCK pathway is involved in the neuronal loss and inhibition of axonal regeneration observed in Alzheimers disease (AD). Therefore, we conducted this study to evaluate whether intracranial injection of PEG-PEI/ROCK II siRNA (PPRS) would improve the cognitive impairments in a senescence-accelerated mouse (SAM) model of AD. Materials and Methods: Five male senescence-resistant inbred strain (SAMR1) mice and 15 male senescence-accelerated mouse prone-8 (SAMP8) strain mice were divided into the following three groups:PPRS group, PEG-PEI/ ROCK II-Scramble (PPRScr) siRNA group, and normal group (SAMR1). Total volumes of 2.3 μl of nanoparticles or saline were intracranially injected under the guidance of a stereotaxic apparatus. The injections were performed every three days and lasted for two weeks. Four weeks after injection, the Morris water maze (MWM) was used to evaluate the spatial learning and memory functions of the mice. Choline acetyltransferase (ChAT) activity was detected by immunohistochemistry. Results: Mice in the PPRS-treated group exhibited decreases in escape latencies over the three successive days of navigating the test and crossing the target quadrant during the spatial probe test more frequently than did the mice in the PPRScr-treated group. Analyses of ChAT activity revealed that greater numbers of ChAT-positive cells were present in the hippocampal regions of the PPRS-treated mice than in the PPRScr group. Conclusions: Intracranial injection of PPRS improved the cognitive impairments of SAM mice, and this improvement may have been mediated by enhancement of ChAT activity in the hippocampus.

Downregulation of ROCK2 through Nanocomplex Sensitizes the Cytotoxic Effect of Temozolomide in U251 Glioma Cells

Objective Rho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells. Methods Glycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration. Results Our results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone. Conclusions In conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.

Overexpression of NTRK1 Promotes Differentiation of Neural Stem Cells into Cholinergic Neurons

Neurotrophic tyrosine kinase type 1 (NTRK1) plays critical roles in proliferation, differentiation, and survival of cholinergic neurons; however, it remains unknown whether enhanced expression of NTRK1 in neural stem cells (NSCs) can promote their differentiation into mature neurons. In this study, a plasmid encoding the rat NTRK1 gene was constructed and transfected into C17.2 mouse neural stem cells (NSCs). NTRK1 overexpression in C17.2 cells was confirmed by western blot. The NSCs overexpressing NTRK1 and the C17.2 NSCs transfected by an empty plasmid vector were treated with or without 100 ng/mL nerve growth factor (NGF) for 7 days. Expression of the cholinergic cell marker, choline acetyltransferase (ChAT), was detected by florescent immunocytochemistry (ICC). In the presence of NGF induction, the NSCs overexpressing NTRK1 differentiated into ChAT-immunopositive cells at 3-fold higher than the NSCs transfected by the plasmid vector (26% versus 9%, P < 0.05). The data suggest that elevated NTRK1 expression increases differentiation of NSCs into cholinergic neurons under stimulation of NGF. The approach also represents an efficient strategy for generation of cholinergic neurons.

A meta-analysis of effects of selective serotonin reuptake inhibitors on blood pressure in depression treatment: outcomes from placebo and serotonin and noradrenaline reuptake inhibitor controlled trials

Background Selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs) have been commonly prescribed for depression treatment. However, their effects on blood pressure are unclear. Materials and methods Effects on blood pressure of depressive patients in two groups (SSRIs versus placebo and SSRIs versus SNRIs) were evaluated. A search was conducted for double-blind, randomized controlled trials (RCTs) in PubMed, EMBASE, ISI Web of Science, PsycNET, CCRCT, and DARE (up to March 2017). The outcomes were systolic blood pressure (SBP) changes and diastolic blood pressure (DBP) changes from baseline to endpoint or to a certain period of treatment duration. Weighted mean differences (WMDs) and 95% CIs were calculated and pooled using random effects models. The χ2 test and I2 statistics were used to assess heterogeneity. Funnel plots, Begg’s test, and Egger’s test were used to estimate publication bias. Results A total of 23 RCTs involving 13,285 participants were included. Patients on SSRIs showed no significant differences in blood pressure changes compared with placebo. In the group of SSRIs versus SNRIs, overall SBP changes and DBP changes revealed statistical significances (WMD 1.5 mmHg, 95% CI −2.15, −0.84, Z=4.46, P<0.00001 and WMD 1.34 mmHg, 95% CI −1.92, −0.75, Z=6.18, P<0.00001). Subgroup analyses on treatment duration and age further evidenced these findings. Conclusion It was established that SSRIs did not affect blood pressure, while SNRIs led to a modest increase in SBP and DBP with statistical significance compared with SSRIs.

Astaxanthin acts via LRP-1 to inhibit inflammation and reverse lipopolysaccharide-induced M1/M2 polarization of microglial cells

Microglia become activated during neuroinflammation and produce neurotoxic and neurotrophic factors, depending on whether they acquire M1 proinflammatory or M2 anti-inflammatory phenotypes. Astaxanthin (ATX), a natural carotenoid, has anti-inflammatory and neuroprotective effects. We investigated whether ATX could reverse M1/M2 polarization and suppress neuroinflammation via low-density lipoprotein receptor-related protein-1 (LRP-1). We observed increased expression of M1 (TNF-α, IL-1β, and CD86) and decreased expression of M2 (Arg-1, IL-10, and CD206) markers in BV2 microglial cells stimulated with lipopolysaccharide (LPS). These alterations were reversed by pretreating the cells with ATX. Activation of the NF-κB and JNK pathways was observed upon LPS stimulation, which was reversed by ATX. ATX-induced M2 polarization was attenuated by inhibition of NF-κB and JNK. Pretreatment of LPS-stimulated BV2 cells with ATX resulted in increased LRP-1 expression. The addition of receptor-associated protein, an LRP-1 antagonist, ameliorated ATX-induced inactivation of NF-κB and JNK signaling, and M2 polarization. ATX promotes M2 polarization to suppress neuroinflammation via LRP-1 by inhibiting NF-κB and JNK signaling. This novel mechanism may suppress neuroinflammation in diseases such as Alzheimer’s disease.