Betulinaldehyde exhibits effective anti-tumor effects in A549 cells by regulating intracellular autophagy.

It is of great significance to find new effective drugs for an adjuvant therapy targeting lung cancer to improve the survival rate and prognosis of patients with the disease. Previous studies have confirmed that certain Chinese herbal extracts have clear anti-tumor effects, and in our preliminary study, betulinaldehyde was screened for its potential anti-tumor effects. The current study thus aimed to confirm the anti-tumor effect of betulinaldehyde, using in vitro experiments to explore its underlying molecular mechanism. It was found that betulinaldehyde treatment significantly inhibited the viability, proliferation, and migration of A549 cells in a dose-dependent manner. In addition, betulinaldehyde inhibited the activation of Akt, MAPK, and STAT3 signaling pathways in A549 cells in a time-dependent manner. More importantly, betulinaldehyde also decreased the expression level of SQSTM1 protein, increased the expression level of LC3 II, and increased the autophagy flux in A549 cells. The pretreatment of A549 cells with the autophagy inhibitor, 3-methyladenine, could partially negate the anti-tumor effects of betulinaldehyde. These findings suggest that betulinaldehyde could significantly inhibit the oncological activity of A549 cells by regulating the intracellular autophagy level, making it a potentially effective option for the adjuvant therapy used to treat lung cancer in the future.

Chondrocyte secretome enriched microparticles encapsulated with the chondrocyte membrane to facilitate the chondrogenesis of BMSCs and reduce hypertrophy.

Co-culture of chondrocytes and mesenchymal stem cells (MSCs) represents an effective way to stimulate the chondrogenesis of MSCs and reduce hypertrophy, but the limited donor site supply and the requirement of two-stage operations are among the major barriers of using autologous chondrocytes in clinical settings. With recent evidence indicating that the chondrogenic effects of the above co-culture mainly lied on the paracrine secretion, and that cell membranes also played crucial roles during the chondrocyte-MSC interaction, we fabricated a multifunctional design of "artificial chondrocytes", which consist of chondrocyte secretome enriched PLGA microparticles with the encapsulation of chondrocytes' membrane fragments. The artificial chondrocytes had shown a similar diameter and surface electrical charge to natural chondrocytes, with the preserved key chondrocyte membrane surface proteins and sustainedly released chondrogenic cytokines from the chondrocyte secretome to extend their effects in vivo. Consequently, the co-culture studies of artificial chondrocytes and bone marrow MSCs had shown the beneficial effects from both chondrocyte secretome and membrane fragments, which also synergistically facilitated the cell proliferation, chondrogenic gene expression, cartilaginous matrix production, and reduced phenotypic hypertrophy in vitro and in vivo. Together, this study has successfully developed the proof-of-concept design of "artificial chondrocytes", which could potentially conquer many major barriers of using natural chondrocytes and provided a novel synthetic-cell approach to current therapeutical strategies towards the functional regeneration of articular cartilage.

Comparison of chondro-inductivity between collagen and hyaluronic acid hydrogel based on chemical/physical microenvironment.

Achieving chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) successfully is crucial for cartilage regeneration. To date, various hydrogels with different chemical microenvironment have been used to modulate chondrogenic differentiation of BMSCs, especially collagen and hyaluronic acid hydrogel. However, the chondro-inductive ability of collagen and hyaluronic acid hydrogel has not been evaluated yet and the different chemical and physical microenvironment of these two hydrogels increase the difficulty of comparison. In this study, three different hydrogels based on collagen and hyaluronic acid (self-assembled collagen hydrogel (Col), self-assembled collagen hydrogel cross-linked with genipin (Cgp), and methacrylated hyaluronic acid hydrogel (HA)) were prepared and their chondro-inductive ability on the encapsulated BMSCs was evaluated. Col and Cgp have the same chemical composition and similar microstructure, but are different from HA, while Cgp and HA hydrogels have the same mechanical strength. It was found that chemical and physical microenvironments of the hydrogels combined to influence cell condensation. Thanks to cell condensation was more likely to occur in collagen hydrogels in the early stage, the cartilage-induced ability was in the order of Col > Cgp > HA. However, the severe shrinkage of Col and Cgp resulted in no enough space for cell proliferation within hydrogels in the later stage. In contrast, relatively stable physical microenvironment of HA helped to maintain continuous production of cartilage-related matrix in the later stage. Overall, these results revealed that the chondro-inductive ability of collagen and hyaluronic acid hydrogel with different chemical and physical microenvironment cannot be evaluated by a particular time period. However, it provided important information for optimization and design of the future hydrogels towards successful repair of articular cartilage.

Solubilized Cartilage ECM Facilitates the Recruitment and Chondrogenesis of Endogenous BMSCs in Collagen Scaffolds for Enhancing Microfracture Treatment.

Articular cartilage has very poor intrinsic healing ability and its repair remains a significant clinical challenge. To promote neocartilage regeneration, we fabricated two collagen (Col) scaffolds functionalized with a porcine decellularized extracellular matrix (dECM) in the forms of particle and solution named pE-Col and sE-Col, respectively. Their differences were systematically compared, including the biochemical compositions, scaffold properties, cell-material interactions, and in situ cartilage regeneration. While it is demonstrated that both forms of dECM could enhance the cell recruitment, proliferation, and chondrogenesis of bone marrow stem cells (BMSCs) in vitro, better performance was seen in the sE-Col group, which could quickly provide a more favorable chondrogenic microenvironment for endogenous BMSCs. The superiority of sE-Col was also proved by our in vivo study, which showed that the sE-Col scaffold achieved better structural hyaline-like neocartilage formation and subchondral bone repair compared to the pE-Col scaffold, according to the gross morphology, biological assessment, and micro-CT imaging analysis. Together, this study suggests that the sE-Col scaffold holds great potential in developing the one-step microfracture-based strategy for cartilage repair and also reminds us that despite dECM being a promising biomaterial in tissue engineering, the optimization of the proper processing methodology would be a crucial consideration in the future design of dECM-based scaffolds in articular cartilage regeneration.

Bazedoxifene Plays a Protective Role against Inflammatory Injury of Endothelial Cells by Targeting CD40.

The inflammatory response and oxidative stress play key roles in the formation and development of atherosclerosis. Bazedoxifene is a new IL6/GP130 inhibitor recommended by the FDA for clinical use as a selective estrogen receptor modulator. However, its role in cardiovascular diseases has been poorly studied. In our study, we explored the mechanism of bazedoxifene's protective effect against inflammatory injury of vascular endothelial cells (VECs) stimulated by TNF-α. Various methods were used to verify the effect of bazedoxifene on VECs, including a cell viability assay, a wound healing assay, immunofluorescence staining, and western blotting. Our results showed that TNF-α could induce inflammatory damage to VECs, which manifested as upregulated expression of CD40, increased production of ROS, enhanced adhesion of THP-1 cells to VECs, and impaired viability and migration of VECs, while bazedoxifene could significantly reduce the endothelial damage caused by TNF-α. In addition, we found that an siRNA targeting CD40 dramatically alleviated the VEC damage induced by TNF-α. Therefore, we explored the potential relationship between bazedoxifene and CD40. Our data suggest that bazedoxifene has a protective effect against VEC damage induced by TNF-α and that its underlying mechanism may be related to the regulation of CD40.

Bazedoxifene inhibits PDGF-BB induced VSMC phenotypic switch via regulating the autophagy level.

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.

The negatively charged microenvironment of collagen hydrogels regulates the chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro and in vivo.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) into functional chondrocytes is crucial for successful cartilage tissue engineering. Since the extracellular matrix (ECM) microenvironment can regulate the behaviours of BMSCs and guide their differentiation, it is important to simulate the natural cartilage ECM to induce the chondrogenesis of BMSCs. As the most abundant protein in the ECM, collagen hydrogels were found to provide a structural and chemical microenvironment for natural cartilage, and regulate the chondrogenic differentiation of BMSCs. However, as the negatively charged ECM microenvironment is crucial for chondrogenesis and homeostasis within cells in cartilage tissue, the electrical properties of collagen hydrogels need to be further optimized. In this study, three collagen hydrogels with different electrical properties were fabricated using methacrylic anhydride (MA) and succinic anhydride (SA) modification. The collagen hydrogels had a similar composition, storage modulus and integral triple helix structure of collagen, but their different negatively charged microenvironments significantly impacted the hydrophilicity, protein diffusion and binding, and consequently influenced BMSC adhesion and spreading on the surface of the hydrogels. Moreover, the BMSCs encapsulated in the collagen hydrogels also demonstrated improved sGAG secretion and chondrogenic and integrin gene expression with the increased negative charge in vitro. Similar results were also observed in subcutaneous implantation in vivo, where higher secretions of sGAG, SOX9 and collagen type II proteins were found in the collagen hydrogels with higher negative charge. Together, our results demonstrated that more negative charges introduced into the collagen hydrogel microenvironment would enhance the chondrogenic differentiation of BMSCs in vitro and in vivo. This revealed that the electrical properties are an important consideration in designing future collagen hydrogels for cartilage regeneration.