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Skin aging is characterized by reactive oxygen species (ROS) accumulation, principal players in triggering events associated with aging. Our recent data on the ability of an innovative poly-component formulation (KARISMA Rh Collagen® FACE: K formulation) to suppress the biomolecular events associated with oxidative stress-induced aging prompted us to deepen the mechanisms underlying the observed effects on aged human dermal fibroblasts (HDFs). Here, we evaluated K’s ability to perform a direct free radical-scavenging action and modulate anti-oxidant systems by counteracting the inflammatory process in an H2O2-induced cellular senescence model. Standard methods were used to measure scavenging capacity and enzymatic anti-oxidant system activities. Nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappa-B (NF-κB) levels were analyzed by Western blot. We assessed pro-inflammatory cytokines, matrix metalloproteinases (MMPs), and advanced glycation end-products (AGEs). Our results show that K counteracted stress-induced aging in a dose-dependent manner by exerting a direct scavenging action and increasing anti-oxidant systems, such as superoxide dismutase (SOD) and catalase (CAT) up to control values. These findings could be associated with increased phospho-Nrf2 (p-Nrf2) expression, generally reduced in aged HDFs following exposure to different concentrations of K formulation. Moreover, K formulation caused a reduction of pro-inflammatory cytokines, interleukin-1β and -6, MMP-1 and -9, and AGE levels, events related to a downregulation of p-NF-κB level. The results indicate that K formulation re-established the normal physiology of HDFs by reducing p-NF-κB expression and restoring Nrf2 activation, thus supporting its efficacious reparative and regenerative action in treating skin aging.

Skin aging is influenced by intrinsic and extrinsic factors that progressively impair skin functionality over time. Investigating the skin aging process requires thorough research using innovative technologies. This review explores the use of in vitro human 3D culture models, serving as valuable alternatives to animal ones, in skin aging research. The aim is to highlight the benefits and necessity of improving the methodology in analyzing the molecular mechanisms underlying human skin aging. Traditional 2D models, including monolayers of keratinocytes, fibroblasts, or melanocytes, even if providing cost-effective and straightforward methods to study critical processes such as extracellular matrix degradation, pigmentation, and the effects of secretome on skin cells, fail to replicate the complex tissue architecture with its intricated interactions. Advanced 3D models (organoid cultures, “skin-on-chip” technologies, reconstructed human skin, and 3D bioprinting) considerably enhance the physiological relevance, enabling a more accurate representation of skin aging and its peculiar features. By reporting the advantages and limitations of 3D models, this review highlights the importance of using advanced in vitro systems to develop practical anti-aging preventive and reparative approaches and improve human translational research in this field. Further exploration of these technologies will provide new opportunities for previously unexplored knowledge on skin aging.

The mechanism underlying intestinal fibrosis, the main complication of inflammatory bowel disease (IBD), is not yet fully understood, and there is no therapy to prevent or reverse fibrosis. We evaluated, in in vitro cellular models, the ability of different classes of drugs currently used in IBD to counteract two pivotal processes of intestinal fibrosis, the differentiation of intestinal fibroblasts to activated myofibroblasts using CCD-18Co cells, and the epithelial-to-mesenchymal transition (EMT) of intestinal epithelial cells using Caco-2 cells (IEC), both being processes induced by transforming growth factor-β1 (TGF-β1). The drugs tested included mesalamine, azathioprine, methotrexate, prednisone, methylprednisolone, budesonide, infliximab, and adalimumab. The expression of fibrosis and EMT markers (collagen-I, α-SMA, pSmad2/3, occludin) was assessed by Western blot analysis and by immunofluorescence. Of the drugs used, only prednisone, methylprednisolone, budesonide, and adalimumab were able to antagonize the pro-fibrotic effects induced by TGF-β1 on CCD-18Co cells, reducing the fibrosis marker expression. Methylprednisolone, budesonide, and adalimumab were also able to significantly counteract the TGF-β1-induced EMT process on Caco-2 IEC by increasing occludin and decreasing α-SMA expression. This is the first study that evaluates, using in vitro cellular models, the direct antifibrotic effects of drugs currently used in IBD, highlighting which drugs have potential antifibrotic effects.

Glioblastoma (GBM) is characterized by an immunosuppressive tumor microenvironment (TME) strictly associated with therapy resistance. Cyclooxygenase-2 (COX-2) fuels GBM proliferation, stemness, and chemoresistance. We previously reported that COX-2 upregulation induced by temozolomide (TMZ) supported chemoresistance. Also, COX-2 transfer by extracellular vesicles released by T98G promoted M2 polarization in macrophages, whereas COX-2 inhibition counteracted these effects. Here, we investigated the COX-2 role in the stemness potential and modulation of the GBM immunosuppressive microenvironment. The presence of macrophages U937 within tumorspheres derived from GBM cell lines and primary cultures exposed to celecoxib (COX-2 inhibitor) with or without TMZ was studied by confocal microscopy. M2 polarization was analyzed by TGFβ-1 and CD206 levels. Osteopontin (OPN), a crucial player within the TME by driving the macrophages’ infiltration, and CD44 expression was assessed by Western blot. TMZ strongly enhanced tumorsphere size and induced the M2 polarization of infiltrating macrophages. In macrophage-infiltrated tumorspheres, TMZ upregulated OPN and CD44 expression. These TMZ effects were counteracted by the concurrent addition of CXB. Remarkably, exogenous prostaglandin-E2 restored OPN and CD44, highlighting the COX-2 pivotal role in the protumor macrophages’ state promotion. COX-2 inhibition interfered with TMZ’s ability to induce M2-polarization and counteracted the development of an immunosuppressive TME.

Skin aging is a dynamic process that determines structural alterations in ECM and reduction in dermal fibroblasts. The recent availability on the market of an innovative polycomponent formulation (KARISMA Rh Collagen® FACE, K) containing noncrosslinked high-molecular-weight hyaluronic acid (HMW-HA), a human recombinant polypeptide of collagen-1 alpha chain, and carboxymethyl cellulose (CMC), attracted our scientific interest in evaluating its biomolecular effects on human dermal adult and aged fibroblasts. After treatment with increasing K concentrations, cell proliferation, collagen I, prolyl 4-hydroxylase (P4HA1), an essential protein in collagen biosynthesis, and α-SMA levels were assessed. The fibroblast contractility, TGF-β1 levels, and oxidative stress markers were also evaluated. K formulation exposure led to a significant and dose-dependent increase in the proliferation and migration of adult fibroblasts. Of note, the K exposure counteracted the H2O2-induced aging by promoting cell proliferation, reducing β-galactosidase activity, and neutralizing the aging-associated oxidative damage. Moreover, an increase in collagen I, P4HA1, α-SMA, TGF-β1 levels, and improved contractility of adult and aged fibroblasts were observed after treatment. Overall, our results show evidence that the K treatment is efficacious in improving biological functions in adult fibroblasts and suppressing the biomolecular events associated with H2O2-induced cellular aging, thus supporting the regenerative and bio-revitalizing action of the K formulation helpful in preventing or treating skin aging.