Molecular force-induced liberation of transforming growth factor-beta remodels the spleen for ectopic liver regeneration.

BACKGROUND & AIMS: Ectopic liver regeneration in the spleen is a promising alternative to organ transplantation for treating liver failure. To accommodate transplanted liver cells, the splenic tissue must undergo structural changes to increase extracellular matrix content, demanding a safe and efficient approach for tissue remodelling. METHODS: We synthesised sulphated hyaluronic acid (sHA) with an affinity for the latent complex of transforming growth factor-ß (TGF-ß) and cross-linked it into a gel network (sHA-X) via click chemistry. We injected this glycan into the spleens of mice to induce splenic tissue remodelling via supraphysiological activation of endogenous TGF-ß. RESULTS: sHA-X efficiently bound to the abundant latent TGF-ß in the spleen. It provided the molecular force to liberate the active TGF-ß dimers from their latent complex, mimicking the 'bind-and-pull' mechanism required for physiological activation of TGF-ß and reshaping the splenic tissue to support liver cell growth. Hepatocytes transplanted into the remodelled spleen developed into liver tissue with sufficient volume to rescue animals with a metabolic liver disorder (Fah-/- transgenic model) or following 90% hepatectomy, with no adverse effects observed and no additional drugs required. CONCLUSION: Our findings highlight the efficacy and translational potential of using sHA-X to remodel a specific organ by mechanically activating one single cytokine, representing a novel strategy for the design of biomaterials-based therapies for organ regeneration. IMPACT AND IMPLICATIONS: Cell transplantation may provide a lifeline to millions of patients with end-stage liver diseases, but their severely damaged livers being unable to accommodate the transplanted cells is a crucial hurdle. Herein, we report an approach to restore liver functions in another organ - the spleen - by activating one single growth factor in situ. This approach, based on a chemically designed polysaccharide that can mechanically liberate the active transforming growth factor-ß to an unusually high level, promotes the function of abundant allogenic liver cells in the spleen, rescuing animals from lethal models of liver diseases and showing a high potential for clinical translation.

Enzymatic Protein Immobilization for Nanobody Array.

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody's anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields.

Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis.

BACKGROUND: Osthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited. PURPOSE: This study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model. METHODS: Network pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model. RESULTS: The network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats. CONCLUSION: Our study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.

Integrating metabolomics and network pharmacology to investigate Panax japonicus prevents kidney injury in HFD/STZ-induced diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax japonicus C. A. Meye (PJ) has unique effects on diseases by "qi" stagnation and blood stasis in ancient. Modern studies have shown that PJ can treat diabetic kidney disease (DKD) caused by deficiency and blood stasis. AIM OF THE STUDY: This study evaluated the potential effects of PJ on DKD, a microvascular complication, and investigated its possible mechanisms. MATERIALS AND METHODS: In this study, the chemical constituents of PJ were analyzed by HPLC. In vivo studies, we constructed a diabetic mice model by HDF combined with STZ, then administered PJ to diabetic mice for 6 weeks. Blood lipid, BUN, 24h urine protein, and renal tissue HE staining were detected to comprehensively evaluate the protective effect of PJ on DKD. Metabolomics investigated the metabolic pathways influenced by PJ in the treatment of DKD. Moreover, the potential targets and signal pathways were investigated using network pharmacology. Finally, molecular docking predicts affinity of active compounds and core targets, and western blotting was used to detect core target expression levels. RESULTS: In vivo study, PJ can reduce hyperlipidemia, serum BUN, and 24-h urinary protein in diabetic mice, and protect the pathological changes in renal tissue. Metabolomics results showed that PJ had significant regulatory effect on unsaturated fatty acids, glycerophospholipid metabolism, and purine metabolism. Network pharmacology showed that MAPK1, MAPK8, Bcl-2, and Caspase 3 were the core targets in PJ against DKD. Molecular docking revealed that Bcl-2 and Caspase 3 have a strong affinity for Chikusetsusaponin Iva, Ginsenoside Rb1, and Ginsenoside Rg1. Moreover, when compared to the model group, the PJ group had higher levels of anti-apoptosis protein Bcl-2 and lower levels of pro-apoptosis protein Caspase 3. CONCLUSION: PJ can reduce blood lipids, regulate the biosynthesis of unsaturated fatty acids and purine metabolism, thereby alleviating the renal injury of diabetic mice. Moreover, it can regulate the Bcl-2/caspase 3 apoptosis signaling pathway to prevent the apoptosis of renal cells and protect the renal function of diabetic mice.

Network Pharmacology-Based Analysis on the Effects and Mechanism of the Wang-Bi Capsule for Rheumatoid Arthritis and Osteoarthritis.

Wang-Bi capsule (WB) is a traditional Chinese medicine (TCM)-based herbal formula, and it has been used in the treatment of rheumatoid arthritis (RA) in China for many years. Additionally, WB is also used as a supplement to the treatment of osteoarthritis (OA) in clinical practice. Our research aimed to reveal the therapeutic effects and underling mechanism of WB on RA and OA through computational system pharmacology analysis and experimental study. Based on network pharmacology analysis, a total of 173 bioactive compounds interacted with 417 common gene targets related to WB, RA, and OA, which mainly involved the PI3K-Akt signaling pathway. In addition, the serine-threonine protein kinase 1 (AKT1) might be a core gene protein for the action of WB, which was further emphasized by molecular docking. Moreover, the anti-inflammatory activity of WB in vitro was confirmed by reducing NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells. The anti-RA and OA effects of WB in vivo were confirmed by ameliorating the disease symptoms of collagen II-induced RA (CIA) and monosodium iodoacetate-induced OA (MIA) in rats, respectively. Furthermore, the role of the PI3K-Akt pathway in the action of WB was preliminarily verified by western blot analysis. In conclusion, our study elucidated that WB is a potentially effective strategy for the treatment of RA and OA, which might be achieved by regulating the PI3K-Akt pathway. It provides us with systematic insights into the effects and mechanism of WB on RA and OA.

Redox-Sensitive Ultrashort Peptide Hydrogel with Tunable Mechanical Properties for Anti-Tumor Drug Delivery.

In this study, we report a new ultrashort peptide (LOC), which forms a redox-sensitive hydrogel after cross-linking with the mild oxidant H2 O2 and used it for tumor-targeted delivery of doxorubicin hydrochloride (DOX). LOC gelled within a few minutes in low-concentration H2 O2 solution. The concentration of H2 O2 significantly altered the gelation time and mechanical properties of the hydrogel. The in vitro micromorphology, secondary structure and rheology characterization of cross-linked hydrogels confirmed the sensitivity and injectability to reducing agent. The cross-linked hydrogel had a strong drug loading capacity, and the drug was released in a GSH concentration-dependent manner, following the Fick diffusion model. In addition, the cross-linked hydrogel showed no cytotoxicity to normal fibroblasts, and no damage to the subcutaneous tissue of mice was observed. In vitro cytotoxicity experiments showed that the DOX-hydrogel system exhibited good anti-cancer efficacy. In vivo studies using 4T1 tumor-bearing mice showed that the DOX-hydrogel system had a significant inhibitory effect on tumors. Therefore, the newly designed redox-sensitive hydrogel can effectively enhance the therapeutic efficacy of DOX and reduce toxicity, making it an attractive biological material.

Flp, a Fis-like protein, contributes to the regulation of type III secretion and virulence processes in the phytopathogen Xanthomonas campestris pv. campestris.

The ability of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) to cause disease is dependent on its ability to adapt quickly to the host environment during infection. Like most bacterial pathogens, Xcc has evolved complex regulatory networks that ensure expression and regulation of their virulence genes. Here, we describe the identification and characterization of a Fis-like protein (named Flp), which plays an important role in virulence and type III secretion system (T3SS) gene expression in Xcc. Deletion of flp caused reduced virulence and hypersensitive response (HR) induction of Xcc and alterations in stress tolerance. Global transcriptome analyses revealed the Flp had a broad regulatory role and that most T3SS HR and pathogenicity (hrp) genes were down-regulated in the flp mutant. ß-glucuronidase activity assays implied that Flp regulates the expression of hrp genes via controlling the expression of hrpX. More assays confirmed that Flp binds to the promoter of hrpX and affected the transcription of hrpX directly. Interestingly, the constitutive expression of hrpX in the flp mutant restored the HR phenotype but not full virulence. Taken together, the findings describe the unrecognized regulatory role of Flp protein that controls hrp gene expression and pathogenesis in Xcc.