Network analysis, in vivo, and in vitro experiments identified the mechanisms by which Piper longum L. [Piperaceae] alleviates cartilage destruction, joint inflammation, and arthritic pain

Osteoarthritis (OA) is characterized by irreversible joint destruction, pain, and dysfunction. Piper longum L. [Piperaceae] (PL) is an East Asian herbal medicine with reported anti-inflammatory, analgesic, antioxidant, anti-stress, and anti-osteoporotic effects. This study aimed to evaluate the efficacy of PL in inhibiting pain and progressive joint destruction in OA based on its anti-inflammatory activity, and to explore its potential mechanisms using in vivo and in vitro models of OA. We predicted the potential hub targets and signaling pathways of PL through network analysis and molecular docking. Network analysis results showed that the possible hub targets of PL against OA were F2R, F3, MMP1, MMP2, MMP9, and PTGS2. The molecular docking results predicted strong binding affinities for the core compounds in PL: piperlongumine, piperlonguminine, and piperine. In vitro experiments showed that PL inhibited the expression of LPS-induced pro-inflammatory factors, such as F2R, F3, IL-1β, IL-6, IL-17A, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, NOS2, PTGS2, PGE2, and TNF-β. These mechanisms and effects were dose-dependent in vivo models. Furthermore, PL inhibited cartilage degradation in an OA-induced rat model. Thus, this study demonstrated that multiple components of PL may inhibit the multilayered pathology of OA by acting on multiple targets and pathways. These findings highlight the potential of PL as a disease-modifying OA drug candidate, which warrants further investigation

Highly sensitive near-infrared SERS nanoprobes for in vivo imaging using gold-assembled silica nanoparticles with controllable nanogaps

Abstract Background To take advantages, such as multiplex capacity, non-photobleaching property, and high sensitivity, of surface-enhanced Raman scattering (SERS)-based in vivo imaging, development of highly enhanced SERS nanoprobes in near-infrared (NIR) region is needed. A well-controlled morphology and biocompatibility are essential features of NIR SERS nanoprobes. Gold (Au)-assembled nanostructures with controllable nanogaps with highly enhanced SERS signals within multiple hotspots could be a breakthrough. Results Au-assembled silica (SiO2) nanoparticles (NPs) (SiO2@Au@Au NPs) as NIR SERS nanoprobes are synthesized using the seed-mediated growth method. SiO2@Au@Au NPs using six different sizes of Au NPs (SiO2@Au@Au50–SiO2@Au@Au500) were prepared by controlling the concentration of Au precursor in the growth step. The nanogaps between Au NPs on the SiO2 surface could be controlled from 4.16 to 0.98nm by adjusting the concentration of Au precursor (hence increasing Au NP sizes), which resulted in the formation of effective SERS hotspots. SiO2@Au@Au500 NPs with a 0.98-nm gap showed a high SERS enhancement factor of approximately 3.8 × 106 under 785-nm photoexcitation. SiO2@Au@Au500 nanoprobes showed detectable in vivo SERS signals at a concentration of 16μg/mL in animal tissue specimen at a depth of 7mm. SiO2@Au@Au500 NPs with 14 different Raman label compounds exhibited distinct SERS signals upon subcutaneous injection into nude mice. Conclusions SiO2@Au@Au NPs showed high potential for in vivo applications as multiplex nanoprobes with high SERS sensitivity in the NIR region. Graphical Abstrac

Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of <i>Bifidobacterium longum</i> subsp. <i>suis</i>

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics

Chaperone-mediated autophagy dysregulation during aging impairs hepatic fatty acid oxidation via accumulation of NCoR1

Objective: Alterations in lipid metabolism are associated with aging and age-related diseases. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process involved in specific protein degradation. Heat shock cognate 71 kDa protein (Hsc70) recognizes cytosolic proteins with KFERQ motif and allows them to enter the lysosome via lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A). CMA deficiency is associated with dysregulated lipid metabolism in the liver. In this study, we examined the effect of CMA on lipid metabolism in the aged liver. Methods: 12-week-old and 88-week-old mice were employed to assess the effect of aging on hepatic CMA activity. We generated CMA-deficient mouse primary hepatocytes using siRNA for Lamp2a and liver-specific LAMP2A knockdown mice via adeno-associated viruses expressing short hairpin RNAs to investigate the influence of CMA on lipid metabolism. Results: We noted aging-induced progression toward fatty liver and a decrease in LAMP2A levels in total protein and lysosomes. The expression of genes associated with fatty acid oxidation was markedly downregulated in the aged liver, as verified in CMA-deficient mouse primary hepatocytes. In addition, the aged liver accumulated nuclear receptor corepressor 1 (NCoR1), a negative regulator of peroxisome proliferator-activated receptor α (PPARα). We found that Hsc70 binds to NCoR1 via the KFERQ motif. Lamp2a siRNA treatment accumulated NCoR1 and decreased the fatty acid oxidation rate. Pharmacological activation of CMA by AR7 treatment increased LAMP2A expression, leading to NCoR1 degradation. A liver-specific LAMP2A knockdown via adeno-associated viruses expressing short hairpin RNAs caused NCoR1 accumulation, inactivated PPARα, downregulated the expression of fatty acid oxidation-related genes and significantly increased liver triglyceride levels. Conclusions: Our results elucidated a novel PPARα regulatory mechanism involving CMA-mediated NCoR1 degradation during aging. These findings demonstrate that CMA dysregulation is crucial for the progression of aging-related fatty liver diseases

βPix Guanine Nucleotide Exchange Factor Regulates Regeneration of Injured Peripheral Axons

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify βPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of βPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, βPix is localized in the peripheral domain of the growth cone. Using βPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of βPix are specifically removed, we demonstrate that βPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in βPix NIKO mice, supporting the local action of βPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the βPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the βPix NIKO neurons. Altogether, these data indicate that βPix plays an important role in axonal regrowth during peripheral nerve regeneration

Incorporation of SKI-G-801, a Novel AXL Inhibitor, With Anti-PD-1 Plus Chemotherapy Improves Anti-Tumor Activity and Survival by Enhancing T Cell Immunity

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86(+) macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.N