Graphdiyne nanoplatforms for photothermal-ferroptosis combination therapy against glioblastoma.

Glioblastoma (GBM) is one of the most malignant tumors of the central nervous system and has a poor prognosis. GBM cells are highly sensitive to ferroptosis and heat, suggesting thermotherapy-ferroptosis as a new strategy for GBM treatment. With its biocompatibility and photothermal conversion efficiency, graphdiyne (GDY) has become a high-profile nanomaterial. Here, the ferroptosis inducer FIN56 was employed to construct GDY-FIN56-RAP (GFR) polymer self-assembled nanoplatforms against GBM. GDY could effectively load FIN56 and FIN56 released from GFR in a pH-dependent manner. The GFR nanoplatforms possessed the advantages of penetrating the BBB and acidic environment-induced in situ FIN56 release. Moreover, GFR nanoplatforms induced GBM cell ferroptosis by inhibiting GPX4 expression, and 808 nm irradiation reinforced GFR-mediated ferroptosis by elevating the temperature and promoting FIN56 release from GFR. In addition, the GFR nanoplatforms were inclined to locate in tumor tissue, inhibit GBM growth, and prolong lifespan by inducing GPX4-mediated ferroptosis in an orthotopic xenograft mouse model of GBM; meanwhile, 808 nm irradiation further improved these GFR-mediated effects. Hence, GFR may be a potential nanomedicine for cancer therapy, and GFR combined with photothermal therapy may be a promising strategy against GBM.

Kai-Xin-San Inhibits Tau Pathology and Neuronal Apoptosis in Aged SAMP8 Mice.

Alzheimer's disease (AD) is an age-related neurological disorder. Currently, there is no effective cure for AD due to its complexity in pathogenesis. In light of the complex pathogenesis of AD, the traditional Chinese medicine (TCM) formula Kai-Xin-San (KXS), which was used for amnesia treatment, has been proved to improve cognitive function in AD animal models. However, the active ingredients and the mechanism of KXS have not yet been clearly elucidated. In this study, network pharmacology analysis predicts that KXS yields 168 candidate compounds acting on 863 potential targets, 30 of which are associated with AD. Enrichment analysis revealed that the therapeutic mechanisms of KXS for AD are associated with the inhibition of Tau protein hyperphosphorylation, inflammation, and apoptosis. Therefore, we chose 7-month-old senescence-accelerated mouse prone 8 (SAMP8) mice as AD mouse model, which harbors the behavioral and pathological hallmarks of AD. Subsequently, the potential underlying action mechanisms of KXS on AD predicted by the network pharmacology analyses were experimentally validated in SAMP8 mice after intragastric administration of KXS for 3 months. We observed that KXS upregulated AKT phosphorylation, suppressed GSK3ß and CDK5 activation, and inhibited the TLR4/MyD88/NF-κB signaling pathway to attenuate Tau hyperphosphorylation and neuroinflammation, thus suppressing neuronal apoptosis and improving the cognitive impairment of aged SAMP8 mice. Taken together, our findings reveal a multi-component and multi-target therapeutic mechanism of KXS for attenuating the progression of AD, contributing to the future development of TCM modernization, including KXS, and broader clinical application.

New anti-inflammatory withanolides from Physalis pubescens fruit.

Physalis pubescens L. is a medicinal plant widely cultivated in northeast of China. Investigation on the extract of P. pubescens fruit led to the isolation and identification of four new withanolides, namely, physapubescins J-M (1, 2, 4 and 5), together with four known analogues (3, 6-8) and fifteen other compounds. Their structures were elucidated on the basis of comprehensive NMR, MS, and ECD spectroscopic data analysis. Among isolates, physapubescin J (1) contained an unusual sulphide linkage, and four withanolides (3, 5, 7 and 8) showed anti-inflammatory potential in LPS-induced RAW264.7 cells. This study supports P. pubescens fruit could be a valuable source of withanolides. Further studies to investigate anti-inflammatory activities of isolated withanolides using in vivo models are warranted.

Screening a specific Zn(ii)-binding peptide for improving the cognitive decline of Alzheimer's disease in APP/PS1 transgenic mice by inhibiting Zn2+-mediated amyloid protein aggregation and neurotoxicity.

Zn2+ has been implicated in the progression of Alzheimer's disease (AD), as amyloid-ß protein (Aß) aggregation and neurotoxicity are mediated by zinc ions. Therefore, development of metal chelators for inhibiting and regulating metal-triggered Aß aggregation has received attention as a strategy for treating AD. Here, we used an approach based on phage display to screen for a Zn(ii)-binding peptide that specifically blocks Zn-triggered Aß aggregation. A fixed Zn(ii) resin was prepared using Ni-IDA affinity resin, and the target Zn(ii) was screened by interaction with a heptapeptide phage library. After negative biopanning against IDA and four rounds of positive biopanning against Zn(ii), high specificity Zn(ii)-binding phages were obtained. Through DNA sequencing and ELISA, 15 sets of Zn(ii)-binding peptides with high histidine contents were identified. We chose a highly specific peptide against Zn(ii) with the sequence of H-M-Q-T-N-H-H, and its abilities to chelate Zn2+ and inhibit Zn2+-mediated Aß aggregation were assessed in vitro. We loaded the Zn(ii)-binding peptide onto PEG-modified chitosan nanoparticles (NPs) to improve the stability and the bioavailability of the Zn(ii) binding peptide. PEG-modified chitosan NPs loaded with Zn(ii)-binding peptide (PEG/PZn-CS NPs) reduced Zn2+ concentrations and Aß secretion in mouse neuroblastoma (N)2a cells stably over-expressing the APP Swedish mutation (N2aswe). Zn2+-Induced neurotoxicity, oxidative stress, and apoptosis were attenuated by PEG/PZn-CS NPs. Intranasal administration of PEG/PZn-CS NPs improved the cognitive ability of APPswe/PS1d9 (APP/PS1) double-transgenic mice and reduced Aß plaques in the mouse brain. This study indicated that a Zn(ii)-binding peptide and its NPs have promise as a potential anti-AD agent.

Paricalcitol accelerates BACE1 lysosomal degradation and inhibits calpain-1 dependent neuronal loss in APP/PS1 transgenic mice.

BACKGROUND: Recent studies have revealed that vitamin D deficiency may increase the risk of Alzheimer's disease, and vitamin D supplementation may be effective strategy to ameliorate the neurodegenerative process in Alzheimer's disease patients. Paricalcitol (PAL), a low-calcemic vitamin D receptor agonist, is clinically used to treat secondary hyperparathyroidism. However, the potential application of PAL for treating neurodegenerative disorders remains unexplored. METHODS: The APP/PS1 mice were intraperitoneally injected with PAL or vehicle every other day for 15 weeks. The ß-amyloid (Aß) production was confirmed using immunostaining and enzyme linked immunosorbent assay. The underlying mechanism was verified by western blot and immunostaining in vivo and in vitro. FINDINGS: Long-term PAL treatment clearly reduced ß-amyloid (Aß) generation and neuronal loss in APP/PS1 transgenic mouse brains. PAL stimulated the expression of low-density lipoprotein receptor-related protein 1 (LRP1) possibly through inhibiting sterol regulatory element binding protein-2 (SREBP2); PAL also promoted LRP1-mediated ß-site APP cleavage enzyme 1 (BACE1) transport to late endosomes, thus increasing the lysosomal degradation of BACE1. Furthermore, PAL diminished 8-hydroxyguanosine (8-OHdG) generation in neuronal mitochondria via enhancing base excision repair (BER), resulting in the attenuation of calpain-1-mediated neuronal loss. INTERPRETATION: The present data demonstrate that PAL can reduce Aß generation through accelerating BACE1 lysosomal degradation and can inhibit neuronal loss through suppressing mitochondrial 8-OHdG generation. Hence, PAL might be a promising agent for treating Alzheimer's disease. FUND: This study was financially supported by the Natural Science Foundation of China (U1608282).

Dihydromyricetin and Salvianolic acid B inhibit alpha-synuclein aggregation and enhance chaperone-mediated autophagy.

BACKGROUND: Progressive accumulation of α-synuclein is a key step in the pathological development of Parkinson's disease. Impaired protein degradation and increased levels of α-synuclein may trigger a pathological aggregation in vitro and in vivo. The chaperone-mediated autophagy (CMA) pathway is involved in the intracellular degradation processes of α-synuclein. Dysfunction of the CMA pathway impairs α-synuclein degradation and causes cytotoxicity. RESULTS: In the present study, we investigated the effects on the CMA pathway and α-synuclein aggregation using bioactive ingredients (Dihydromyricetin (DHM) and Salvianolic acid B (Sal B)) extracted from natural medicinal plants. In both cell-free and cellular models of α-synuclein aggregation, after administration of DHM and Sal B, we observed significant inhibition of α-synuclein accumulation and aggregation. Cells were co-transfected with a C-terminal modified α-synuclein (SynT) and synphilin-1, and then treated with DHM (10 µM) and Sal B (50 µM) 16 hours after transfection; levels of α-synuclein aggregation decreased significantly (68% for DHM and 75% for Sal B). Concomitantly, we detected increased levels of LAMP-1 (a marker of lysosomal homeostasis) and LAMP-2A (a key marker of CMA). Immunofluorescence analyses showed increased colocalization between LAMP-1 and LAMP-2A with α-synuclein inclusions after treatment with DHM and Sal B. We also found increased levels of LAMP-1 and LAMP-2A both in vitro and in vivo, along with decreased levels of α-synuclein. Moreover, DHM and Sal B treatments exhibited anti-inflammatory activities, preventing astroglia- and microglia-mediated neuroinflammation in BAC-α-syn-GFP transgenic mice. CONCLUSIONS: Our data indicate that DHM and Sal B are effective in modulating α-synuclein accumulation and aggregate formation and augmenting activation of CMA, holding potential for the treatment of Parkinson's disease.

α-Lipoic acid improves abnormal behavior by mitigation of oxidative stress, inflammation, ferroptosis, and tauopathy in P301S Tau transgenic mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease and is characterized by neurofibrillary tangles (NFTs) composed of Tau protein. α-Lipoic acid (LA) has been found to stabilize the cognitive function of AD patients, and animal study findings have confirmed its anti-amyloidogenic properties. However, the underlying mechanisms remain unclear, especially with respect to the ability of LA to control Tau pathology and neuronal damage. Here, we found that LA supplementation effectively inhibited the hyperphosphorylation of Tau at several AD-related sites, accompanied by reduced cognitive decline in P301S Tau transgenic mice. Furthermore, we found that LA not only inhibited the activity of calpain1, which has been associated with tauopathy development and neurodegeneration via modulating the activity of several kinases, but also significantly decreased the calcium content of brain tissue in LA-treated mice. Next, we screened for various modes of neural cell death in the brain tissue of LA-treated mice. We found that caspase-dependent apoptosis was potently inhibited, whereas autophagy did not show significant changes after LA supplementation. Interestingly, Tau-induced iron overload, lipid peroxidation, and inflammation, which are involved in ferroptosis, were significantly blocked by LA administration. These results provide compelling evidence that LA plays a role in inhibiting Tau hyperphosphorylation and neuronal loss, including ferroptosis, through several pathways, suggesting that LA may be a potential therapy for tauopathies.

Anti-Inflammatory Effects, SAR, and Action Mechanism of Monoterpenoids from Radix Paeoniae Alba on LPS-Stimulated RAW 264.7 Cells.

Nine monoterpenoids from Radix Paeoniae Alba, including paeoniflorin derivatives, paeoniflorin (PF), 4-O-methylpaeoniflorin (MPF), 4-O-methylbenzoylpaeoniflorin (MBPF); paeonidanin derivatives, paeonidanin (PD), paeonidanin A (PDA), albiflorin derivatives, albiflorin (AF), benzoylalbiflorin (BAF), galloylalbiflorin (GAF), and debenzoylalbiflorin (DAF), were obtained in our previous phytochemistry investigations. Their anti-inflammatory effects were determined in the present study. The expression and production of pro-inflammatory cytokines in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells were measured using an Elisa assay and nitric oxide (NO) release was determined using the Griess method. The results demonstrated that the most of the monoterpenoids suppressed the LPS-induced production of NO, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α). The anti-inflammatory activities of these monoterpenoids were closely related to their structural characteristics. Paeoniflorins and paeonidanins presented stronger anti-inflammatory activities than those of albiflorin derivatives. Furthermore, the action mechanisms of MBPF, having a strong anti-inflammatory effect, were investigated using quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot methods. The results indicated that MBPF could down-regulate the mRNA and protein expression level of inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW 264.7 cells. The mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor κB (NF-κB) signaling pathways are involved in mediating the role of MBPF in suppressing the expression and production of pro-inflammatory cytokines in RAW 264.7 cells.

[Acupotomy and acupuncture in the treatment of avascular necrosis of femoral head at the early and middle stages:a clinical randomized controlled trial].

OBJECTIVE: To compare the efficacy difference between acupotomy and acupuncture in the treatment of avascular necrosis of femoral head at the early and middle stages. METHODS: The randomized controlled prospective study method was adopted. Sixty cases of avascular necrosis of femoral head at Ficat-ArletⅠto Ⅱ stages were randomized into an acupotomy group (32 cases) and an acupuncture group (28 cases) by the third part. In the acupotomy group, the acupotomy was adopted for the loose solution at the treatment sites of hip joint, once every two weeks, totally for 3 times. In the acupuncture group, ashi points around the hip joint were selected and stimulated with warm acupuncture therapy, once every day, for 6 weeks. Harris hip score was observed before and after treatment. The efficacy was evaluated in the two groups. RESULTS: Harris hip score was improved significantly after treatment in the two groups (both P<0.05). The result in acupotomy group was better than that in the acupuncture group (P<0.05). The effective rate was 90.6% (29/32) in the acupotomy group, better than 75.0% (21/28) in the acupuncture group after treatment (P<0.05). CONCLUSIONS: Harris hip score and the effective rate in the acupotomy group are better than those in the treatment with routine acupuncture for avascular necrosis of femoral head at the early and middle stages.

Panax notoginseng saponins promote wound repair of anterior cruciate ligament through phosphorylation of PI3K, AKT and ERK.

Panax notoginseng saponins (PNS) are components derived from Chinese herb panax notoginseng and play important roles in the cure of wounds. However, how PNS plays this function is still unclear. In this study, we used MTT assay, wound healing assay, western blot, quantitative real time PCR and enzyme-linked immunosorbent assay to detect the effects of PNS on the proliferation, migration and expression of collagen and fibronectin of anterior cruciate ligament (ACL) fibroblasts as well as the underlying mechanism. We found that PNS promoted the proliferation and migration of ACL fibroblasts and increased the expression levels of collagen and fibronectin. Further mechanism study indicates that PNS might play its function through the phosphorylation of PI3K, AKT and ERK. This study provides a possible mechanism for the function of PNS and lays foundation for further study on the function of panax notoginseng.

Zinc modulates high glucose-induced apoptosis by suppressing oxidative stress in renal tubular epithelial cells.

Hyperglycemia is a characteristic of diabetic nephropathy, inducing renal tubular cell apoptosis by eliciting oxidative stress and inflammation. Zinc (Zn) is known as an essential trace element in many enzymes and proteins involved in antioxidant defenses, electron transport, and exerting antiapoptotic or cytoprotective effects. In this study, the underlying mechanisms involved in the protective effects of Zn on high glucose-induced cytotoxicity were explored using cultured renal tubular epithelial cells (NRK-52E). The authors discovered that Zn supplementation inhibited high glucose (HG)-induced NRK-52E cell apoptosis by attenuating reactive oxygen species production, inhibiting HG-induced caspase-3 and caspase-9 activation, and inhibiting the release of cytochrome c from mitochondria to the cytosol. Further analysis revealed that Zn supplementation facilitated cell survival through increasing nuclear translocation of NF-E2-related factor 2 (Nrf2), leading to increased regulation of levels of two antioxidant enzymes, hemeoxygenase-1 and glutamate cysteine ligase, which provided an adaptive survival response against the HG-induced oxidative cytotoxicity. Moreover, the Zn-mediated increases in Nrf2 activity were suppressed by the pharmacological inhibition of Akt or extracellular signal-regulated kinase 1/2. Taken together, these findings suggest that Zn antiapoptosis capacity through the activation of Akt and ERK signal pathways leads to Nrf2 activation and, subsequently, Nrf2 target gene induction, thereby protecting the NRK-52E cells from HG-induced apoptosis.

Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain.

Prior work has shown that iron interacts with hyperphosphorylated tau, which contributes to the formation of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD), whereas iron chelator desferrioxamine (DFO) slows down the clinical progression of the cognitive decline associated with this disease. However, the effects of DFO on tau phosphorylation in the presence or absence of iron have yet to be determined. Using amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mouse brain as a model system, we investigated the effects and potential mechanisms of intranasal administration of DFO on iron induced abnormal tau phosphorylation. High-dose iron treatment markedly increased the levels of tau phosphorylation at the sites of Thr205, Thr231 and Ser396, whereas highly induced tau phosphorylation was abolished by intranasal administration of DFO in APP/PS1 transgenic mice. Moreover, DFO intranasal administration also decreases Fe-induced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3ß (GSK3ß), which in turn suppressing tau phosphorylation. Cumulatively, our data show that intranasal DFO treatment exerts its suppressive effects on iron induced tau phosphorylation via CDK5 and GSK3ß pathways. More importantly, elucidation of DFO mechanism in suppressing tau phosphorylation may provide insights for developing therapeutic strategies to combat AD.

Intranasal deferoxamine reverses iron-induced memory deficits and inhibits amyloidogenic APP processing in a transgenic mouse model of Alzheimer's disease.

Increasing evidence indicates that a disturbance of normal iron homeostasis and an amyloid-ß (Aß)-iron interaction may contribute to the pathology of Alzheimer's disease (AD), whereas iron chelation could be an effective therapeutic intervention. In the present study, transgenic mice expressing amyloid precursor protein (APP) and presenilin 1 and watered with high-dose iron served as a model of AD. We evaluated the effects of intranasal administration of the high-affinity iron chelator deferoxamine (DFO) on Aß neuropathology and spatial learning and memory deficits created in this AD model. The effects of Fe, DFO, and combined treatments were also evaluated in vitro using SHSY-5Y cells overexpressing the human APP Swedish mutation. In vivo, no significant differences in the brain concentrations of iron, copper, or zinc were found among the treatment groups. We found that high-dose iron (deionized water containing 10 mg/mL FeCl(3)) administered to transgenic mice increased protein expression and phosphorylation of APP695, enhanced amyloidogenic APP cleavage and Aß deposition, and impaired spatial learning and memory. Chelation of iron via intranasal administration of DFO (200 mg/kg once every other day for 90 days) inhibited iron-induced amyloidogenic APP processing and reversed behavioral alterations. DFO treatment reduced the expression and phosphorylation of APP protein by shifting the processing of APP to the nonamyloidogenic pathway, and the reduction was accompanied by attenuating the Aß burden, and then significantly promoted memory retention in APP/PS1 mice. The effects of DFO on iron-induced amyloidogenic APP cleavage were further confirmed in vitro. Collectively, the present data suggest that intranasal DFO treatment may be useful in AD, and amelioration of iron homeostasis is a potential strategy for prevention and treatment of this disease.

Huperzine A activates Wnt/ß-catenin signaling and enhances the nonamyloidogenic pathway in an Alzheimer transgenic mouse model.

Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to ß-amyloid (Aß) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aß levels and Aß burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/ß activity, and enhanced the ß-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/ß-catenin signaling pathway in AD brain.

Zinc overload enhances APP cleavage and Aß deposition in the Alzheimer mouse brain.

BACKGROUND: Abnormal zinc homeostasis is involved in ß-amyloid (Aß) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aß deposition has not been well established in AD animal models in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aß deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro. CONCLUSIONS/SIGNIFICANCE: The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography.

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.