Shengxue Busui Decoction activates the PI3K/Akt and VEGF pathways, enhancing vascular function and inhibiting osteocyte apoptosis to combat steroid-induced femoral head necrosis

Frontiers in Pharmacology · 2025-01-24 · 6 citations

Introduction Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating condition with no specific treatment. Inhibiting osteocyte apoptosis may be a promising therapeutic approach. Shengxue Busui Decoction (SBD) has shown protective effects against SONFH, but its mechanisms are not fully understood. This study aims to investigate the effects of SBD on SONFH in rats, identifying its key active components and regulatory mechanisms using network pharmacology, bioinformatics, machine learning, and experimental validation. Methods Key active components and disease targets of SBD were identified through network pharmacology and bioinformatics. GO/KEGG enrichment and ssGSEA analyses were performed to identify critical pathways. Cytoscape and machine learning (SVM) were used for target prediction and molecular docking validation. A dexamethasone (Dex)-induced SONFH rat model was established, and SBD was administered for 60 days. Histological changes were assessed via HE staining, osteoclast activity through TRAP staining, apoptosis levels with TUNEL assays, and vascular function through hematological tests. ELISA was used to measure ALP and OCN levels. In vitro , Dex-induced osteoblast apoptosis in MC3T3-E1 cells was examined to assess SBD’s effect on osteoblast proliferation, apoptosis, and signaling. Western blotting analyzed Caspase-9, Caspase-3, Bax, Bcl-2, and pathway-related proteins. ALP and Alizarin Red staining evaluated osteoblast differentiation and mineralization. Results Network pharmacology identified curcumin, berberine, and diosgenin as key active components of SBD, with the PI3K/Akt and VEGFR pathways as critical targets, and RAF1, FOXO3, and BRAF as hub genes. In vivo , SBD intervention significantly reduced bone structural damage and apoptosis, decreasing the rate of empty bone lacunae. SBD also increased osteogenic markers ALP and OCN in SONFH rats. In vitro , SBD inhibited osteoblast apoptosis, promoted PI3K/Akt and VEGF pathway expression, and enhanced osteoblast differentiation and mineralization. Conclusion This study integrates network pharmacology with experimental validation, showing that SBD protects against SONFH by inhibiting osteoblast apoptosis via PI3K/Akt and VEGFR pathways. SBD promotes osteoblast differentiation and mineralization, improving bone structure and vascular function. Curcumin, berberine, and diosgenin are likely key contributors to these effects, highlighting SBD as a potential therapeutic strategy for SONFH.

The Myristoylated Alanine-rich C-kinase Substrate (MARCKS) as a Host Defense Protein Against SARS-CoV-2 Infection Via an Extracellular Vesicle (EV) Mediated Mechanism

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Introduction: Identifying host proteins with antiviral properties is crucial for combating SARS-CoV-2 infection and preparing for potential future pandemics. The myristoylated alanine-rich C-kinase substrate (MARCKS) is recognized for its roles in cell shape regulation, motility, and actin cytoskeleton dynamics through interactions with the plasma membrane and actin filaments. Despite MARCKS’ significant involvement in inflammatory lung diseases, its role in infectious disease has not been studied. This research investigates MARCKS as a potential antiviral protein against SARS-CoV-2, with a focus on its functional domains and their impact on extracellular vesicle (EV) biogenesis. Methods: We used human cell lines and primary human epithelial cells to overexpress or knock down MARCKS, examining its effect on SARS-CoV-2 infection. Functional domain analyses included testing MARCKS phosphorylation using PKC inhibitors, a MARCKS phosphorylation antagonist peptide-MPS, and non-phosphorylated MARCKS variants. The role of the N-terminal myristoylation domain was tested by using MARCKS-G2A mutation. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to quantify EV production. These EVs were then tested against SARS-CoV-2 pseudo-spike virus and live SARS-CoV-2 virus to evaluate antiviral activity. Results: MARCKS overexpression significantly decreased SARS-CoV-2 protein levels and plaque formation in both cell lines and primary epithelial cells. Conversely, MARCKS knockdown increased viral protein, RNA, and plaque formation, confirming its antiviral role. Functional analyses showed that MARCKS's antiviral effect is phosphorylation-independent, as PKC inhibitors and the MPS peptide had no significant impact. However, the antiviral activity partially depended on the N-terminal myristoylation domain, as the MARCKS-G2A mutation greatly reduced antiviral function. Additionally, MARCKS overexpression, including its non-phosphorylated MARCKS variants, enhanced EV biogenesis, while those effects were partially lost in MARCKS-G2A mutants, aligning with its antiviral role. Furthermore, MARCKS driven ACE2-containing EVs also showed potent antiviral function against SARS-CoV-2. Conclusions: MARCKS functions as an effective antiviral protein against SARS-CoV-2 through an EV-mediated mechanism that relies on N-terminal myristoylation but is independent of phosphorylation. These findings position MARCKS as a promising therapeutic target for SARS-CoV-2 and suggest a broader role for MARCKS in host-virus interactions and EV-mediated antiviral responses.

Autologous Precision-Cut Lung Slice Co-Culture Models for Studying Macrophage-Driven Fibrosis

American Journal of Respiratory and Critical Care Medicine · 2025-05-01

Abstract Rationale : Precision-cut lung slices (PCLS) are commonly used as an ex vivo model to study lung fibrosis; however, traditional models lack immune cell infiltration, including the recruitment of monocytes and macrophages, which are critical for inflammation and fibrosis. To address this limitation, we developed novel autologous PCLS-immune co-culture models that better replicate the processes of inflammation, repair, and immune cell recruitment associated with fibrosis. Methods: We evaluated fibrotic responses to nicotine, cigarette smoke extract (CSE), and a fibrosis-inducing cocktail (FCK) in mouse PCLS. To investigate the role of macrophages, we developed PCLS-immune co-culture models, including a direct co-culture that simulates the repair phase of fibrosis and an indirect co-culture that mimics blood vessel function, allowing for the observation of immune cell recruitment to injury sites. Results: Immunofluorescence and Western blotting confirmed upregulation of α-SMA-expressing fibroblasts in response to these inducers. Sirius Red staining showed increased collagen deposition in PCLS exposed to these agents, demonstrating their impact on fibrosis development. Chemotactic studies revealed enhanced migration and infiltration of bone marrow-derived macrophages (BMDMs) toward CSE-injured PCLS. Direct co-culture with autologous BMDMs further increased collagen deposition, indicating that this effect is specific to autologous interactions in the fibrotic response. Conclusion: These findings demonstrate the utility of our novel PCLS co-culture models in elucidating macrophage involvement in fibrosis and suggest potential targets for macrophage-focused therapies in pulmonary fibrosis.

Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma

European Journal of Pharmacology · 2024-08-05 · 2 citations

Growth and Differentiation of Tracheobronchial Epithelial Cells

2024-10-18 · 3 citations

In a broad sense, airway diseases are a result related to a failure of airway epithelium to perform a homeostatic role in airway lumen. At least three features of the “epithelial failure” have been characterized. The first feature involves an uncontrolled cell proliferation in certain epithelial cell types that leads to bronchogenic neoplasm development. Four main histopathological types of lung cancer—adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma—have been described (1–3). The cell type origins of these neoplasms are still poorly understood, even though some cancer cells in these lung tumors often retain many characteristics of normal airway epithelial cells; for instance, adenocarcinoma cells exhibit secretory features of mucous cell type, squamous carcinoma cells exhibit high tonofilament features resembling the basal cell type, and small cell carcinomas maintain some neuroendocrine characteristics. Reasons for this poor understanding are many. One problem is lack of a reproducible and predictable carcinogenesis model for manipulating both the initiation and the progression of bronchogenic tumors. The other reason is related to a plasticity of airway epithelium which can modify their intrinsic function (4). It is common to see a diverse histological appearance in the same neoplasm. This phenomenon has led to the interpretation that all cancer cell types and tumor types may derive from a common stem cell. Aberrations in the tracheobronchial epithelial stem cell could account for the heterogeneity and diversity in lung cancers. Clearly, identification of the stem cell population in tracheobronchial epithelium and elucidation of the pathway of cell differentiation and its regulation in these stem cells will lead us to a better understanding of neoplastic development in the lung (5).

Autophagy of OTUD5 destabilizes GPX4 to confer ferroptosis-dependent kidney injury

Nature Communications · 2023-12-18 · 71 citations

Ferroptosis is an iron-dependent programmed cell death associated with severe kidney diseases, linked to decreased glutathione peroxidase 4 (GPX4). However, the spatial distribution of renal GPX4-mediated ferroptosis and the molecular events causing GPX4 reduction during ischemia-reperfusion (I/R) remain largely unknown. Using spatial transcriptomics, we identify that GPX4 is situated at the interface of the inner cortex and outer medulla, a hyperactive ferroptosis site post-I/R injury. We further discover OTU deubiquitinase 5 (OTUD5) as a GPX4-binding protein that confers ferroptosis resistance by stabilizing GPX4. During I/R, ferroptosis is induced by mTORC1-mediated autophagy, causing OTUD5 degradation and subsequent GPX4 decay. Functionally, OTUD5 deletion intensifies renal tubular cell ferroptosis and exacerbates acute kidney injury, while AAV-mediated OTUD5 delivery mitigates ferroptosis and promotes renal function recovery from I/R injury. Overall, this study highlights a new autophagy-dependent ferroptosis module: hypoxia/ischemia-induced OTUD5 autophagy triggers GPX4 degradation, offering a potential therapeutic avenue for I/R-related kidney diseases.

Knockdown of Long Noncoding RNA CCAT2 Suppresses Malignant Phenotype in Human Laryngeal Squamous Cell Carcinoma

Bulletin of Experimental Biology and Medicine · 2023-09-01

The Role of MARCKS in Metastasis and Treatment Resistance of Solid Tumors

Cancers · 2022-10-08 · 35 citations

The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells. MARCKS plays important roles in multiple cellular processes, including cell adhesion and motility, mucin secretion, exocytosis, and inflammatory response. Aberrant MARCKS signaling has been observed in the development and progression of multiple cancer types. In addition, MARCKS facilitates cancer metastasis through modulating cancer cell migration and invasion. Moreover, MARCKS contributes to treatment resistance, likely by promoting cancer stem cell renewal as well as immunosuppression. In this review, we describe MARCKS protein structure, cellular localization, and biological functions. We then discuss the role of MARCKS in cancer metastasis as well as its mechanisms of action in solid tumors. Finally, we review recent advances in targeting MARCKS as a new therapeutic strategy in cancer management.

Targeting the phosphorylation site of myristoylated alanine‐rich C kinase substrate alleviates symptoms in a murine model of steroid‐resistant asthma

British Journal of Pharmacology · 2019-02-01 · 20 citations

BACKGROUND AND PURPOSE: Myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, facilitates mucus production and neutrophil migration. However, the effects of therapeutic procedures targeting the phosphorylation site of MARCKS on steroid-resistant asthma and the mechanisms underlying such effects have not yet been investigated. We designed a peptide that targets the MARCKS phosphorylation site (MPS peptide) and assessed its therapeutic potential against steroid-resistant asthma. EXPERIMENTAL APPROACH: Mice were sensitized with ovalbumin (OVA), alum, and challenged with aerosolized OVA five times a week for 1 month. The mice were intratracheally administered MPS peptides three times a week, 1 hr before OVA challenge. Asthma symptoms and cell profiles in the bronchoalveolar lavage were assessed, and key proteins were analysed using Western blotting. KEY RESULTS: Phosphorylated (p)-MARCKS was highly expressed in inflammatory and bronchial epithelial cells in OVA-immunized mice. MPS peptide reduced eosinophils, neutrophils, mucus production, collagen deposition, and airway hyper-responsiveness. Dexamethasone (Dexa) did not alleviate steroid-resistant asthma symptoms. MPS peptide caused a decrease in p-MARCKS, nitrotyrosine and the expression of oxidative stress enzymes, NADPH oxidase dual oxidase 1 and inducible NOS, in lung tissues. Compared to Dexa, MPS peptides inhibited C5a production and attenuated IL-17A and KC production in the airway more effectively, thus suppressing asthma symptoms. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that targeting MARCKS phosphorylation through MPS treatment may inhibit neutrophilic inflammation and relieve asthma symptoms, thereby highlighting its potential as a therapeutic agent for steroid-resistant asthma.

Tackling MARCKS‐PIP3 circuit attenuates fibroblast activation and fibrosis progression

The FASEB Journal · 2019-10-26 · 23 citations

Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5‐bisphosphate (PIP2)‐binding protein, myristoylated alanine‐rich C‐kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25‐mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α‐smooth muscle actin (α‐SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)‐trisphosphate pools and AKT activity occurred in MPS‐treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin‐treated mice in vivo. Surprisingly, we found that MPS peptide decreases α‐SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.—Yang, D. C., Li, J.‐M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.‐H. Tackling MARCKS‐PIP3 circuit attenuates fibroblast activation and fibrosis progression. FASEB J. 33, 14354‐14369 (2019). www.fasebj.org