Immunofluorescence Staining Assay Research Articles

Ginkgolide A improves the pleiotropic function and reinforces the neuroprotective effects by mesenchymal stem cell-derived exosomes in 6-OHDA-induced cell model of Parkinson's disease.

Parkinson's disease (PD) is a common disorder attributed to the loss of midbrain dopamine (mDA) neurons and reduced dopamine secretion. Currently, the treatment regimes for PD comprise deep brain stimulations, however, it attenuates the PD progression marginally and does not improve neuronal cell death. We investigated the function of Ginkgolide A (GA) to reinforce Wharton's Jelly-derived mesenchymal stem cells (WJMSCs) for treating the in vitro model of PD. GA enhanced the self-renewal, proliferation, and cell homing function of WJMSCs as assessed by MTT and transwell co-culture assay with a neuroblastoma cell line. GA pre-treated WJMSCs can restore 6-hydroxydopamine (6-OHDA)-induced cell death in a co-culture assay. Furthermore, exosomes isolated from GA pre-treated WJMSCs significantly rescued 6-OHDA-induced cell death as determined by MTT assay, flow cytometry, and TUNEL assay. Western blotting showed that apoptosis-related proteins were decreased following GA-WJMSCs exosomal treatment which further improved mitochondrial dysfunction. We further demonstrated that exosomes isolated from GA-WJMSCs could restore autophagy using immunofluorescence staining and immunoblotting assay. Finally, we used the alpha-synuclein recombinant protein and found that exosomes derived from GA-WJMSCs led to the reduced aggregation of alpha-synuclein compared to that in control. Our results suggested that GA could be a potential candidate for strengthening stem cell and exosome therapy for PD.

USP21 Promotes the Progression of Nasopharyngeal Carcinoma by Regulating FOXM1.

The purpose of this work was to explore the molecular mechanisms by which USP21 regulates nasopharyngeal carcinoma tumor growth and cancer cell stemness. In this study, the USP21 transcript data was obtained from TCGA database. Then, qPCR and western blot tests revealed that, in contrast to normal tissue or normal nasopharyngeal epithelial cells, the expression of USP21 was greater in nasopharyngeal carcinoma tissues or cell lines, respectively. CCK-8 and EdU immunofluorescent staining assays revealed that USP21 promoted the proliferation of nasopharyngeal carcinoma cells. Meanwhile, scratch and transwell assays showed that USP21 facilitated migration and invasion of nasopharyngeal carcinoma cells. Sphere formation assay was performed on nasopharyngeal carcinoma cells after knockdown of USP21, which revealed that knockdown of USP21 inhibited the stemness profiles of nasopharyngeal carcinoma cells. Then, the western blot assays indicated that knockdown of USP21 in nasopharyngeal carcinoma cells would inhibit FOXM1 expression, and overexpression of FOXM1 could reverse the cell proliferation ability, cell migration and invasion ability, and cell stemness profiles. Finally, a nasopharyngeal xenograft model suggested that USP21 facilitated tumor growth in mice. These findings proved that USP21 promoted tumor growth and cancer cell stemness in nasopharyngeal carcinoma by regulating FOXM1.

DOCK8 interference alleviates Aβ‑induced damage of BV2 cells by inhibiting STAT3/NLRP3/NF‑κB signaling.

Dementia is defined as memory loss and other cognitive decline and it severely influences daily life. Alzheimer's disease (AD) is the most common cause of dementia. Dedicator of cytokinesis 8 (DOCK8) is reported to be involved in neurological diseases. The present study focused on investigating the role that DOCK8 serves in AD and addressing its hidden regulatory mechanism. Initially, Aβ1-42 (Aβ) was applied for the administration of BV2 cells. Subsequently, the mRNA and protein expression levels of DOCK8 were evaluated utilizing reverse transcription-quantitative PCR (RT-qPCR) and western blotting. After the DOCK8 silencing, immunofluorescence staining (IF), ELISA, wound healing and Transwell assays were applied to assess ionized calcium binding adapter molecule-1 (IBA-1) expression, release of inflammatory factors, migration and invasion in Aβ-induced BV2 cells. IF was used to evaluate cluster of differentiation (CD)11b expression. RT-qPCR and western blotting were to analyze the levels of M1 cell markers inducible nitric oxide synthase (iNOS) and CD86. The expression of STAT3/NLR family pyrin domain containing 3 (NLRP3)/NF-κB signaling-related proteins were determined by western blotting. Finally, the viability and apoptosis in hippocampal HT22 cells with DOCK8 depletion were estimated. Results revealed that Aβ induction greatly stimulated the expression levels of IBA-1 and DOCK8. DOCK8 silencing suppressed Aβ-induced inflammation, migration and invasion of BV2 cells. Additionally, DOCK8 deficiency conspicuously decreased the expression levels of CD11b, iNOS and CD86. The expression of phosphorylated (p-)STAT3, NLRP3, ASC, caspase1 and p-p65 was downregulated in Aβ-induced BV2 cells after DOCK8 depletion. STAT3 activator Colivelin reversed the effects of DOCK8 knockdown on IBA-1 expression, inflammation, migration, invasion and M1 cell polarization. In addition, the viability and apoptosis in hippocampal HT22 cells stimulated by neuroinflammatory release of BV2 cells were repressed following DOCK8 deletion. Collectively, DOCK8 interference alleviated Aβ-induced damage of BV2 cells by inhibiting STAT3/NLRP3/NF-κB signaling.

SAG, a sonic hedgehog signaling agonist, alleviates anxiety behavior in high-fat diet-fed mice

Anxiety is a prevalent and disabling psychiatric disorder. Mitochondrial dysfunction due to the high-fat diet (HFD) was regarded as a risk factor in the pathogenesis of anxiety. The Sonic hedgehog (SHH) pathway was known to improve mitochondrial dysfunction through antioxidant and anti-apoptotic effects on some neurological diseases. Nonetheless, its effect on anxiety has not been well studied. In this study, we aimed to explore whether SHH signaling pathway plays a protective role in anxiety by regulating mitochondrial homeostasis. SAG, a typical SHH signaling agonist, was administered intraperitoneally in HFD-fed mice. HFD-induced anxiety-like behavior in mice was confirmed using the open field and elevated plus maze tests. Immunofluorescence staining and Western blotting assays showed that the SHH signaling was downregulated in the prefrontal cortex neurons from HFD-fed mice. Electron microscopy results showed the mitochondria in the prefrontal cortex of HFD-fed mice were fragmented, which appeared small and spherical, and the area, perimeter and circularity of mitochondria were decreased. Mitofusin2 (Mfn2) and dynamin-related protein 1 (Drp1) were the key proteins involved in mitochondrial division and fusion. SAG treatment could rectify the imbalanced expression of Mfn2 and Drp1 in the prefrontal cortex of the HFD-fed mice, and alleviate the mitochondrial fragmentation. Furthermore, SAG decreased anxiety-like behavior in the HFD-fed mice. These findings suggested that SHH signal was neuroprotective in obesity and SAG relieved anxiety-like behavior through reducing mitochondrial fragmentation.

Targeting of MALT1 May Improve Functional Recovery and Attenuate Microglia M1 Polarization-Mediated Neuroinflammation During Spinal Cord Injury.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is involved in neural injury, neuroinflammation, microglia activation, and polarization, while its function in spinal cord injury (SCI) remains unclear. Thus, this study aimed to evaluate the role of MALT1 modification on SCI recovery and its underlying mechanism. SCI surgery or sham surgery was performed in Sprague-Dawley rats. Then, MALT1 knockdown or negative control lentivirus was injected into SCI rats. Subsequently, MALT1 expression, locomotor capability, neural injury, markers for microglia activation and polarization, inflammatory cytokine expressions, and nuclear factor (NF)-κB pathway were detected. SCI rats exhibited higher MALT1 expression, microglia activation and M1 polarization, neuroinflammation, and NF-κB pathway activation, while worse locomotor capacity compared to sham rats (all P < 0.05). In SCI rats, MALT1 knockdown alleviated Basso, Beattie, and Bresnahan score from 10 to 28days and attenuated HE staining reflected neural injury (all P < 0.05). Besides, MALT1 knockdown declined the number of IBA1+ cells, IBA1+ iNOS+ cells, and IBA1+ CD86+ cells, while enhanced the number of IBA1+ Arg1+ cells and IBA1+ CD206+ cells in SCI rats (all P < 0.05). Meanwhile, MALT1 knockdown declined the expressions of IL-1β, IL-6, and TNF-α in SCI (all P < 0.05), but did not affect IL-10 expression (P > 0.05). Furthermore, MALT1 knockdown suppressed NF-κB pathway activation validated by immunofluorescence staining and western blot assays (all P < 0.05). MALT1 knockdown improves functional recovery, attenuates microglia activation, M1 polarization, and neuroinflammation via inhibiting NF-κB pathway in SCI.

Multi-functional carboxymethyl chitosan/sericin protein/halloysite composite sponge with efficient antibacterial and hemostatic properties for accelerating wound healing

The development of wound dressings with hemostatic and antibacterial properties has attracted great attention. In this study, we prepared a multi-functional natural substance sponge (CMC/Ser-Ag/HNT) composed of carboxymethyl chitosan (CMC), sericin-silver nanoparticle (Ser-Ag), and halloysite (HNT). CMC/Ser-Ag/HNT sponge was demonstrated to bear desired hygroscopicity, porosity, compressive strength and compressive stability, cytocompatibility, and hemocompatibility. The mechanical properties (compressive strength of 100 kPa) and hemostatic capacity (hemostasis time of 15 ± 3 s in the liver injury model and 12 ± 3 s in the caudal injury model) were enhanced by introducing HNT into the CMC sponge. Ser-Ag was synthesized in situ via the redox nature of tyrosine residues in sericin in a “one-step, green” way to enhance the antibacterial activity of the hybrid sponge against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). In addition, the rat full-thickness skin defect model experiments demonstrated that the CMC/Ser-Ag/HNT4 sponge significantly promoted epithelialization and collagen formation. Immunofluorescence staining assays revealed that the composite sponge reduced inflammation by downregulating the expression of IL-6 and enhanced angiogenesis by upregulating VEGF expression. All the findings demonstrated the great potential of CMC/Ser-Ag/HNT sponge as versatile clinical wound dressing, especially for hemorrhagic and infected wounds.

SDF-1α Promotes Chondrocyte Autophagy through CXCR4/mTOR Signaling Axis

SDF-1α, the most common isoform of stromal cell-derived factor 1, has shown vital effects in regulating chondrocyte proliferation, maturation, and chondrogenesis. Autophagy is a highly conserved biological process to help chondrocytes survive in harsh environments. However, the effect of SDF-1α on chondrocyte autophagy is still unknown. This study aims to investigate the effect of SDF-1α on chondrocyte autophagy and the underlying biomechanism. Transmission electron microscope assays and mRFP-GFP-LC3 adenovirus double label transfection assays were performed to detect the autophagic flux of chondrocytes. Western blots and immunofluorescence staining assays were used to detect the expression of autophagy-related proteins in chondrocytes. RNA sequencing and qPCR were conducted to assess changes in autophagy-related mRNA expression. SDF-1α upregulated the number of autophagosomes and autolysosomes in chondrocytes. It also increased the expression of autophagy-related proteins including ULK-1, Beclin-1 and LC3B, and decreased the expression of p62, an autophagy substrate protein. SDF-1α-mediated autophagy of chondrocytes required the participation of receptor CXCR4. Moreover, SDF-1α-enhanced autophagy of chondrocytes was through the inhibition of phosphorylation of mTOR signaling on the upstream of autophagy. Knockdown by siRNA and inhibition by signaling inhibitor further confirmed the importance of the CXCR4/mTOR signaling axis in SDF-1α-induced autophagy of chondrocytes. For the first time, this study elucidated that SDF-1α promotes chondrocyte autophagy through the CXCR4/mTOR signaling axis.

PRKX down-regulates TAK1/IRF7 signaling in the antiviral innate immunity of black carp Mylopharyngodon piceus.

TGF-β-activated kinase-1 (TAK1), tightly related to innate immunity, is phosphorylated and activated by X-linked protein kinase (PRKX) in humans and mammals, which belongs to the c-AMP-dependent protein kinase family. However, the relationship between PRKX and TAK1 remains unknown in teleost. It has been reported in vertebrates for the first time that TAK1 of black carp (bcTAK1) interacts with bcIRF7 and is capable to up-regulate bcIRF7-mediated IFN signaling in our previous study. In this study, the role of PRKX homologue of black carp (Mylopharyngodon piceus) (bcPRKX) in bcTAK1/IFN signaling has been explored. Overexpression of bcPRKX suppressed the transcription of interferon promoters but enhanced the transcription of NF-κB promoter. Mylopharyngodon piceus kidney (MPK) cells transfected with shRNA targeting bcPRKX gene presented enhanced antiviral activity against spring viremia of carp virus (SVCV), in which the mRNA levels of the antiviral proteins were increased, including MX1, Viperin and PKR. Overexpressed bcPRKX dampened bcTAK1/bcIRF7/IFN signaling in the luciferase reporter assay and plaque assay. The interaction between bcTAK1 and bcPRKX has been identified by the immunofluorescence (IF) staining and co-immunoprecipitation (co-IP) assay. In addition, we found that bcPRKX can trigger the degradation of bcTAK1. However, the lysosome inhibitor chloroquine, but not the proteasome inhibitor MG-132, prevented the bcTAK1 degradation mediated by bcPRKX. Thus, we conclude that bcPRKX inhibits bcTAK1/bcIRF7/IFN signaling during the innate immune activation by targeting bcTAK1 and triggers lysosome-dependent degradation of bcTAK1.

Adipose-Derived Mesenchymal Stem Cells Inhibit JNK-Mediated Mitochondrial Retrograde Pathway to Alleviate Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) is the major cause of drug-induced liver injury, with limited treatment options. APAP overdose invokes excessive oxidative stress that triggers mitochondria-to-nucleus retrograde pathways, contributing to APAP-induced liver injury (AILI). Mesenchymal stem cell therapy is a promising tool for acute liver failure. Therefore, the purpose of this study was to investigate the beneficial effects of adipose-derived mesenchymal stem cell (AMSC) therapy on AILI and reveal the potential therapeutic mechanisms. C57BL/6 mice were used as the animal model and AML12 normal murine hepatocytes as the cellular model of APAP overdose. Immunohistochemical staining, Western blotting, immunofluorescence staining, and RNA sequencing assays were used for assessing the efficacy and validating mechanisms of AMSC therapy. We found AMSC therapy effectively ameliorated AILI, while delayed AMSC injection lost its efficacy related to the c-Jun N-terminal kinase (JNK)-mediated mitochondrial retrograde pathways. We further found that AMSC therapy inhibited JNK activation and mitochondrial translocation, reducing APAP-induced mitochondrial damage. The downregulation of activated ataxia telangiectasia-mutated (ATM) and DNA damage response proteins in AMSC-treated mouse liver indicated AMSCs blocked the JNK-ATM pathway. Overall, AMSCs may be an effective treatment for AILI by inhibiting the JNK-ATM mitochondrial retrograde pathway, which improves APAP-induced mitochondrial dysfunction and liver injury.

Neuroblasts migration under control of reactive astrocyte-derived BDNF: a promising therapy in late neurogenesis after traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a disease with high mortality and morbidity, which leads to severe neurological dysfunction. Neurogenesis has provided therapeutic options for treating TBI. Brain derived neurotrophic factor (BDNF) plays a key role in neuroblasts migration. We aimed to investigate to the key regulating principle of BDNF in endogenous neuroblasts migration in a mouse TBI model.MethodsIn this study, controlled cortical impact (CCI) mice (C57BL/6J) model was established to mimic TBI. The sham mice served as control. Immunofluorescence staining and enzyme-linked immunosorbent assay were performed on the CCI groups (day 1, 3, 7, 14 and 21 after CCI) and the sham group. All the data were analyzed with Student’s t-test or one-way or two-way analysis of variance followed by Tukey’s post hoc test.ResultsOur results revealed that neuroblasts migration initiated as early as day 1, peaking at day 7, and persisted till day 21. The spatiotemporal profile of BDNF expression was similar to that of neuroblasts migration, and BDNF level following CCI was consistently higher in injured cortex than in subventricular zone (SVZ). Reactive astrocytes account for the major resource of BDNF along the migrating path, localized with neuroblasts in proximity. Moreover, injection of exogenous CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, at random sites promoted neuroblasts migration and astrocytic BDNF expression in both normal and CCI mice (day 28). These provoked neuroblasts can also differentiate into mature neurons. CC chemokine ligand receptor 2 antagonist can restrain the neuroblasts migration after TBI.ConclusionsNeuroblasts migrated along the activated astrocytic tunnel, directed by BDNF gradient between SVZ and injured cortex after TBI. CCL2 might be a key regulator in the above endogenous neuroblasts migration. Moreover, delayed CCL2 administration may provide a promising therapeutic strategy for late neurogenesis post-trauma.

Curcumol Exerts Antitumor Effect via Inhibiting EGFR-Akt-Mcl-1 Signaling.

Dysfunction of epidermal growth factor receptor (EGFR) signaling plays a critical role in the tumorigenesis of oral squamous cell carcinoma (OSCC). In the present study, the data analysis results of immunohistochemistry and the TCGA database verified that the expression of EGFR is significantly upregulated in OSCC tumor tissues, and depletion of EGFR inhibits the growth of OSCC cells in vitro and in vivo. Moreover, these results showed that the natural compound, curcumol, exhibited a profound antitumor effect on OSCC cells. Western blotting, MTS, and immunofluorescent staining assays indicated that curcumol inhibited cell proliferation and induced intrinsic apoptosis in OSCC cells via downregulating myeloid cell leukemia 1 (Mcl-1). A mechanistic study revealed that curcumol inhibited the EGFR-Akt signal pathway, which activated GSK-3[Formula: see text]-mediated Mcl-1 phosphorylation. Further research showed that curcumol-induced Mcl-1 Ser159 phosphorylation is required to disrupt the interaction between deubiquitinase JOSD1 and Mcl-1 and eventually induce Mcl-1 ubiquitination and degradation. In addition, curcumol administration can effectively inhibit CAL27 and SCC25 xenograft tumor growth and is well-tolerated in vivo. Finally, we demonstrated that Mcl-1 is upregulated and positively correlates with p-EGFR and p-Akt in OSCC tumor tissues. Collectively, the present results provide new insights into the antitumor mechanism of curcumol, identifying it as an attractive therapeutic agent that reduces Mcl-1 expression and inhibits OSCC growth. Targeting EGFR/Akt/Mcl-1 signaling could be a promising option in the clinical treatment of OSCC.

Detection of Ferroptosis in Models of Brain Diseases.

Ferroptosis is a regulated form of non-apoptotic cell death driven by iron-dependent lipid peroxidation. In the past decade, ferroptosis has been reported to be involved in the pathological role in the central nervous system degenerative diseases (e.g., Alzheimer's disease, Huntington's disease, and Parkinson's disease), stroke, traumatic brain injury, and brain tumor. However, how to reliably detect and classify ferroptosis from other cell death in pathological conditions remains a great challenge, especially in primary brain cells and brain tissues. Here, we summarize the methods and protocols (such as real-time PCR, western blotting, immunofluorescence staining, lipid peroxidation assay kits and probe, immunofluorescence staining, GPX activity and glutathione depletion assay kits, iron detection, and TEM) used in the present study to detect and classify ferroptosis in the brain.

A functional study of zinc-titanium coatings and exploration of the intrinsic correlation between angiogenesis and osteogenesis.

With the development of implant applications, osseointegration has become a criterion for implant success. A good blood supply is essential for successful osseointegration. To improve the pro-angiogenic ability of the implants, in this experiment we introduced zinc into the titanium coating. The physical morphology, biocompatibility, pro-angiogenic ability, and osteogenic effect of the zinc-containing titanium coatings were investigated. The pro-angiogenic effect of zinc ions was determined, and the intrinsic link between angiogenesis and osteogenesis under the effect of zinc ions was investigated. Zinc-containing titanium coating was prepared using a micro-arc oxidation (MAO) technique. The physical properties of the coating materials were determined by analyzing the microstructure, roughness, hydrophilic properties, constituent elements, and ionic release of the coating. The biocompatibility of the coating materials was examined using apoptosis and proliferation assays of human umbilical vein endothelial cells (HUVECs). The pro-angiogenic function and osteogenic ability of the zinc-containing coating were investigated by CD31 immunofluorescence staining and quantitative polymerase chain reaction (q-PCR) assay. The optimal concentration of zinc ions for pro-angiogenesis was screened by ion assay. Conditioned media (CM) were prepared for the experiments. The intrinsic link between angiogenesis and osteogenesis was determined by q-PCR to detect the expression of genes related to HUVECs and BMSCs after culture in CM. The prepared Zn-containing micro-arc oxide coatings were shown to have good physical properties, stable Zn2+ release ability, and biocompatibility, as well as good angiogenic and osteogenic potential. In addition, ion experiments confirmed that 60 μM Zn2+ exhibited the best angiogenic effect; more importantly, a mutual promotion between angiogenesis and osteogenesis regeneration in the Zn2+ microenvironment was also found. The introduction of Zn2+ improved the implants' functionality and laid the foundation for the clinical application of Zn2+ pro-angiogenesis.

Photobiomodulation attenuates oligodendrocyte dysfunction and prevents adverse neurological consequences in a rat model of early life adversity.

Rationale: Adverse experiences in early life including abuse, trauma and neglect, have been linked to poor physical and mental health outcomes. Emerging evidence implies that those who experienced early life adversity (ELA) are more likely to develop cognitive dysfunction and depressive-like symptoms in adulthood. The molecular mechanisms responsible for the negative consequences of ELA, however, remain unclear. In the absence of effective management options, anticipatory guidance is the mainstay of ELA prevention. Furthermore, there is no available treatment that prevents or alleviates the neurologic sequelae of ELA, especially traumatic stress. Hence, the present study aims to investigate the mechanisms for these associations and evaluate whether photobiomodulation (PBM), a non-invasive therapeutic procedure, can prevent the negative cognitive and behavioral manifestations of ELA in later life. Methods: ELA was induced by repeated inescapable electric foot shock of rats from postnatal day 21 to 26. On the day immediately following the last foot shock, 2-min daily PBM treatment was applied transcranially for 7 consecutive days. Cognitive dysfunction and depression-like behaviors were measured by a battery of behavioral tests in adulthood. Subsequently, oligodendrocyte progenitor cells (OPCs) differentiation, the proliferation and apoptosis of oligodendrocyte lineage cells (OLs), mature oligodendrocyte, myelinating oligodendrocyte, the level of oxidative damage, reactive oxygen species (ROS) and total antioxidant capacity were measured and analyzed using immunofluorescence staining, capillary-based immunoassay (ProteinSimple®) and antioxidant assay kit. Results: The rats exposed to ELA exhibited obvious oligodendrocyte dysfunction, including a reduction in OPCs differentiation, diminished generation and survival of OLs, decreased OLs, and decreased matured oligodendrocyte. Furthermore, a deficit in myelinating oligodendrocytes was observed, in conjunction with an imbalance in redox homeostasis and accumulated oxidative damage. These alternations were concomitant with cognitive dysfunction and depression-like behaviors. Importantly, we found that early PBM treatment largely prevented these pathologies and reversed the neurologic sequelae resulting from ELA. Conclusions: Collectively, these findings provide new insights into the mechanism by which ELA affects neurological outcomes. Moreover, our findings support that PBM may be a promising strategy to prevent ELA-induced neurologic sequelae that develops later in life.

Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Borneol-driven meningeal lymphatic drainage clears amyloid-β peptide to attenuate Alzheimer-like phenotype in mice.

Rationale: The accumulation and clearance of amyloid-β (Aβ) peptides play a crucial role in the pathogenesis of Alzheimer's disease (AD). The (re)discovery of meningeal lymphatic vessels in recent years has focused attention on the lymphatic clearance of Aβ and has become a promising therapeutic target for such diseases. However, there is a lack of small molecular compounds that could clearly regulate meningeal lymphatic drainage to remove Aβ from the brain. Methods: We investigated the effect of borneol on meningeal lymphatic clearance of macromolecules with different molecular weights (including Aβ) in the brain. To further investigate the mechanism of borneol regulating meningeal lymphatic drainage, immunofluorescence staining, western blotting, ELISA, RT-qPCR, and Nitric Oxide assay kits were used. The cognitive function of AD mice after borneol treatment was evaluated using two behavioral tests: open field (OF) and Morris water maze (MWM). Results: This study discovered that borneol could accelerate the lymphatic clearance of Aβ from the brain by enhancing meningeal lymphatic drainage. Preliminary mechanism analysis revealed that borneol could improve the permeability and inner diameter of lymphatic vessels, allowing macromolecules to drain into the cervical lymph nodes (CLNS) and then be transported to the lymphatic circulation. To speed up the clearance of macromolecules, borneol also stimulated lymphatic constriction by lowering the level of nitric oxide in the meninges. In addition, borneol stimulated lymphangiogenesis by increasing the levels of FOXC2, VEGFC, and LYVE-1 in the meninges, which promoted the clearance rates of macromolecules. Borneol improved meningeal lymphatic clearance not only for Aβ but also for other macromolecular polymers (molecular weight in the range of 2 KD - 45 KD. Borneol ameliorated cognitive deficits and alleviated brain Aβ burden in Aβ-injected mice. Conclusions: Our findings not only provide a strategy to regulate lymphatic clearance pathways of macromolecules in the brain, but also new targets and ideas for treating neurodegenerative diseases like AD. Furthermore, our findings indicate that borneol is a promising therapeutic drug for AD.

Evaluating the Efficacy of Polyglycolic Acid-Loading Tetrandrine Nanoparticles in the Treatment of Dry Eye

Introduction: Dry eye disease (DED) is a multifactor-induced disease accompanied by increased osmolarity of the tear film and inflammation of the ocular surface. Traditional anti-inflammation agent corticosteroids applied in DED treatment could result in high intraocular pressure, especially in long-term treatment. Therefore, we explored a nano drug that aimed to block the formation pathway of DED which had anti-inflammatory, sustained release, and good biocompatibility characteristics in this study. Methods: We prepared a novel nanomedicine (Tet-ATS@PLGA) by the thin film dispersion-hydration ultrasonic method and detected its nanostructure, particle size, and zeta potential. Flow cytometry was used to detect the cell survival rate of each group after 24 h of drug treatment on inflammed Statens Seruminstitut Rabbit Corneal (SIRC) cells. Observed and recorded corneal epithelial staining, tear film rupture time, and Schirmer test to detect tear secretion on the ocular surface of rabbits. The corneal epithelial thickness, morphology, and number of bulbar conjunctival goblet cells were recorded by H&E staining. Finally, we detected the expression of VEGF, IL-1β, PGE2, and TNF-α by cellular immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). Results: The encapsulation efficiency and drug loading of Tet-ATS@PLGA were 79.85% and 32.47%, respectively. At eye surface temperature, Tet can easily release from Tet-ATS@PLGA while that it was difficult to release at storage temperature and room temperature. After 2 weeks medication, Tet-ATS@PLGA can effectively improve the tear film rupture time and tear secretion time in a DED model (p <0.05). Compared with the normal group (62.34 ± 4.86 mm), the thickness of corneal epithelium in ATS (29.47 ± 3.21 mm), Tet-ATS (46.23 ± 2.87 mm), and Tet-ATS@PLGA (55.76 ± 3.95 mm) gradually increased. Furthermore, the flow cytometry indicated that Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory SIRC cells, and the cellular immunofluorescence and ELISA experiments showed that the expression intensity of inflammatory factors such as VEGF, IL-1β, PGE2, and TNF-α decreased in this process. Interestingly, Tet also had the effect of reducing intraocular pressure. Conclusion: Tet-ATS@PLGA can effectively promote the apoptosis of inflammatory corneal epithelial cells, thus inhibiting the expression of inflammatory factors to block the formation of DED and improve the secretion of tear on the ocular surface.

Necroptosis plays a crucial role in the exacerbation of retinal injury after blunt ocular trauma.

Retinal injury after blunt ocular trauma may directly affect prognosis and lead to vision loss. To investigate the pathological changes and molecular mechanisms involved in retinal injury after blunt ocular trauma, we established a weight drop injury model of blunt ocular trauma in male Beagle dogs. Hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL assays were performed to investigate retinal injury within 14 days after blunt ocular trauma. Compared with the control group, the thicknesses of the inner and outer nuclear layers, as well as the number of retinal ganglion cells, gradually decreased within 14 days after injury. The number of bipolar cells in the inner nuclear layer began to decrease 1 day after injury, while the numbers of cholinergic and amacrine cells in the inner nuclear layer did not decrease until 7 days after injury. Moreover, retinal cell necroptosis increased with time after injury; it progressed from the ganglion cell layer to the outer nuclear layer. Visual electrophysiological findings indicated that visual impairment began on the first day after injury and worsened over time. Additionally, blunt ocular trauma induced nerve regeneration and Müller glial hyperplasia; it also resulted in the recruitment of microglia to the retina and polarization of those microglia to the M1 phenotype. These findings suggest that necroptosis plays an important role in exacerbating retinal injury after blunt ocular trauma via gliosis and neuroinflammation. Such a role has important implications for the development of therapeutic strategies.

Spliceosomal GTPase Eftud2 regulates microglial activation and polarization.

Elongation factor Tu GTP binding domain protein 2 (Eftud2) is a spliceosomal GTPase that serves as an innate immune modulator restricting virus infection. Microglia are the resident innate immune cells and the key players of immune response in the central nervous system. However, the role of Eftud2 in microglia has not been reported. In this study, we performed immunofluorescent staining and western blot assay and found that Eftud2 was upregulated in microglia of a 5xFAD transgenic mouse model of Alzheimer's disease. Next, we generated an inducible microglia-specific Eftud2 conditional knockout mouse line (CX3CR1-CreER; Eftud2f/f cKO) via Cre/loxP recombination and found that Eftud2 deficiency resulted in abnormal proliferation and promoted anti-inflammatory phenotype activation of microglia. Furthermore, we knocked down Eftud2 in BV2 microglia with siRNA specifically targeting Eftud2 and found that Eftud2-mediated regulation of microglial proinflammatory/anti-inflammatory phenotype activation in response to inflammation might be dependent on the NF-κB signaling pathway. Our findings suggest that Eftud2 plays a key role in regulating microglial polarization and homeostasis possibly through the NF-κB signaling pathway.

Chronic spinal cord compression associated with intervertebral disc degeneration in SPARC-null mice.

Chronic spinal cord compression (CSCC) is induced by disc herniation and other reasons, leading to movement and sensation dysfunction, with a serious impact on quality of life. Spontaneous disc herniation rarely occurs in rodents, and therefore establishing a chronic spinal cord compression (CSCC) animal model is of crucial importance to explore the pathogenesis and treatment of CSCC. The absence of secreted protein, acidic, and rich in cysteine (SPARC) leads to spontaneous intervertebral disc degeneration in mice, which resembles human disc degeneration. In this study, we evaluated whether SPARC-null mice may serve as an animal model for CSCC. We performed rod rotation test, pain threshold test, gait analysis, and Basso Mouse Scale score. Our results showed that the motor function of SPARC-null mice was weakened, and magnetic resonance images revealed compression at different spinal cord levels, particularly in the lumbar segments. Immunofluorescence staining and western blot assay showed that the absence of SPARC induced apoptosis of neurons and oligodendrocytes, activation of microglia/macrophages with M1/M2 phenotype and astrocytes with A1/A2 phenotype; it also activated the expression of the NOD-like receptor protein 3 inflammasome and inhibited brain-derived neurotrophic factor/tyrosine kinase B signaling pathway. Notably, these findings are characteristics of CSCC. Therefore, we propose that SPARC-null mice may be an animal model for studying CSCC caused by disc herniation.