Cytokine Profiling and Intra-Articular Injection of Autologous Platelet-Rich Plasma in Knee Osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease leading to joint pain and stiffness. Due to lack of effective treatments, physical and psychological disabilities caused by OA have a detrimental impact on the patient’s quality of life. Emerging evidence suggests that intra-articular injection of platelet-rich plasma (PRP) may provide favorable results since PRP comprises not only a high level of platelets but also a huge amount of cytokines, chemokines, and growth factors. However, the precise mechanism and standardization method remain uncertain. This study aimed to examine cytokine profiling in both PRP and platelet-poor plasma (PPP) of knee OA patients and to determine the effects of PRP on OA chondrocytes and knee OA patients. PRP contained a wide variety of cytokines, chemokines, growth factors, and autologous intra-articular PRP injection resulted in favorable outcomes in knee OA patients. Significant increases in levels of IL-1, IL-2, IL-7, IL-8, IL-9, IL-12, TNF-α, IL-17, PDGF-BB, bFGF, and MIP-1β were detected in PRP compared to PPP (p < 0.001). An in vitro study showed a marked increase in proliferation in OA chondrocytes cultured with PRP, compared to PPP and fetal bovine serum (p < 0.001). In a clinical study, knee OA patients treated with PRP showed improvement of physical function and pain, assessed by physical performance, Western Ontario and McMaster Universities Arthritis Index and visual analog scale. Our findings from both in vitro and clinical studies suggest that intra-articular PRP injection in knee OA patients may be a potential therapeutic strategy for alleviating knee pain and delaying the need for surgery.     

Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects’ treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-β and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.     

Blocking the Function of Inflammatory Cytokines and Mediators by Using IL-10 and TGF-β: A Potential Biological Immunotherapy for Intervertebral Disc Degeneration in a Beagle Model

The debilitating effects of lower back pain are a major health issue worldwide. A variety of factors contribute to this, and oftentimes intervertebral disk degeneration (IDD) is an underlying cause of this disorder. Inflammation contributes to IDD, and inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play key roles in the pathology of IDD. Therefore, the development of treatments that inhibit the expression and/or effects of TNF-α and IL-1β in IDD patients should be a promising therapeutic approach to consider. This study characterized the potential to suppress inflammatory cytokine production in degenerative intervertebral disc (NP) cells by treatment with IL-10 and TGF-β in a canine model of IDD. IDD was induced surgically in six male beagles, and degenerative NP cells were isolated and cultured for in vitro studies on cytokine production. Cultured degenerative NP cells were divided into four experimental treatment groups: untreated control, IL-10-treated, TGF-β-treated, and IL-10- plus TGF-β-treated cells. Cultured normal NP cells served as a control group. TNF-α expression was evaluated by fluorescence activated cell sorting (FACS) analysis and enzyme-linked immunosorbent assay (ELISA); moreover, ELISA and real-time PCR were also performed to evaluate the effect of IL-10 and TGF-β on NP cell cytokine expression in vitro. Our results demonstrated that IL-10 and TGF-β treatment suppressed the expression of IL-1β and TNF-α and inhibited the development of inflammatory responses. These data suggest that IL-10 and TGF-β should be evaluated as therapeutic approaches for the treatment of lower back pain mediated by IDD.     

A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 10⁶ or 2 × 10⁶ hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 μL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.     

Proinflammatory Cytokines Perturb Mouse and Human Pancreatic Islet Circadian Rhythmicity and Induce Uncoordinated β-Cell Clock Gene Expression via Nitric Oxide,Lysine Deacetylases,and Immunoproteasomal Activity

Pancreatic β-cell-specific clock knockout mice develop β-cell oxidative-stress and failure, as well as glucose-intolerance. How inflammatory stress affects the cellular clock is under-investigated. Real-time recording of Per2:luciferase reporter activity in murine and human pancreatic islets demonstrated that the proinflammatory cytokine interleukin-1β (IL-1β) lengthened the circadian period. qPCR-profiling of core clock gene expression in insulin-producing cells suggested that the combination of the proinflammatory cytokines IL-1β and interferon-γ (IFN-γ) caused pronounced but uncoordinated increases in mRNA levels of multiple core clock genes, in particular of reverse-erythroblastosis virus α (Rev-erbα), in a dose- and time-dependent manner. The REV-ERBα/β agonist SR9009, used to mimic cytokine-mediated Rev-erbα induction, reduced constitutive and cytokine-induced brain and muscle arnt-like 1 (Bmal1) mRNA levels in INS-1 cells as expected. SR9009 induced reactive oxygen species (ROS), reduced insulin-1/2 (Ins-1/2) mRNA and accumulated- and glucose-stimulated insulin secretion, reduced cell viability, and increased apoptosis levels, reminiscent of cytokine toxicity. In contrast, low (<5,0 μM) concentrations of SR9009 increased Ins-1 mRNA and accumulated insulin-secretion without affecting INS-1 cell viability, mirroring low-concentration IL-1β mediated β-cell stimulation. Inhibiting nitric oxide (NO) synthesis, the lysine deacetylase HDAC3 and the immunoproteasome reduced cytokine-mediated increases in clock gene expression. In conclusion, the cytokine-combination perturbed the intrinsic clocks operative in mouse and human pancreatic islets and induced uncoordinated clock gene expression in INS-1 cells, the latter effect associated with NO, HDAC3, and immunoproteasome activity.     

Autophagy Activation Protects Ocular Surface from Inflammation in a Dry Eye Model In Vitro

Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases.     

Transient Receptor Potential Ankyrin 1 (TRPA1) Is Involved in Upregulating Interleukin-6 Expression in Osteoarthritic Chondrocyte Models

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.     

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer.     

A Shortage of FTH Induces ROS and Sensitizes RAS-Proficient Neuroblastoma N2A Cells to Ferroptosis

Ferroptosis, an iron-dependent form of programmed cell death, has excellent potential as an anti-cancer therapeutic strategy in different types of tumors, especially in RAS-mutated ones. However, the function of ferroptosis for inhibiting neuroblastoma, a common child malignant tumor with minimal treatment, is unclear. This study investigated the anti-cancer function of ferroptosis inducer Erastin or RSL3 in neuroblastoma N2A cells. Our results show that Erastin or RSL3 induces ROS level and cell death and, therefore, reduces the viability of RAS-proficient N2A cells. Importantly, inhibitors to ferroptosis, but not apoptosis, ameliorate the high ROS level and viability defect in Erastin- or RSL3-treated cells. In addition, our data also show that N2A cells are much more sensitive to ferroptosis inducers than primary mouse cortical neural stem cells (NSCs) or neurons. Moreover, a higher level of ROS and PARylation is evidenced in N2A, but not NSCs. Mechanically, ferritin heavy chain 1 (Fth), the ferroxidase function to oxidate redox-active Fe²⁺ to redox-inactive Fe³⁺, is likely responsible for the hypersensitivity of N2A to ferroptosis induction since its expression is lower in N2A compared to NSCs; ectopic expression of Fth reduces ROS levels and cell death, and induces expression of GPX4 and cell viability in N2A cells. Most importantly, neuroblastoma cell lines express a significantly low level of Fth than almost all other types of cancer cell lines. All these data suggest that Erastin or RSL3 induce ferroptosis cell death in neuroblastoma N2A cells, but not normal neural cells, regardless of RAS mutations, due to inadequate FTH. This study, therefore, provides new evidence that ferroptosis could be a promising therapeutic target for neuroblastoma.     

Regulation of Neuroinflammatory Signaling by PPARγ Agonist in Mouse Model of Diabetes

Many relevant studies, as well as clinical practice, confirm that untreated diabetes predisposes the development of neuroinflammation and cognitive impairment. Having regard for the fact that PPARγ are widely distributed in the brain and PPARγ ligands may regulate the inflammatory process, the anti-inflammatory potential of the PPARγ agonist, pioglitazone, was assessed in a mouse model of neuroinflammation related with diabetes. In this regard, the biochemical and molecular indicators of neuroinflammation were determined in the hippocampus and prefrontal cortex of diabetes mice. The levels of cytokines (IL-1β, IL-6, and TNF) and the expression of genes (Tnfrsf1a and Cav1) were measured. In addition, behavioral tests such as the open field test, the hole-board test, and the novel object recognition test were conducted. A 14-day treatment with pioglitazone significantly decreased IL-6 and TNFα levels in the prefrontal cortex and led to the downregulation of Tnfrsf1a expression and the upregulation of Cav1 expression in both brain regions of diabetic mice. Pioglitazone, by targeting neuroinflammatory signaling, improved memory and exploratory activity in behavioral tests. The present study provided a potential theoretical basis and therapeutic target for the treatment of neuroinflammation associated with diabetes. Pioglitazone may provide a promising therapeutic strategy in diabetes patients with muffled of behavioral activity.     

Identification of an IL-22-Dependent Gene Signature as a Pharmacodynamic Biomarker

Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1β. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1β suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.     

Methyl Brevifolincarboxylate Attenuates Free Fatty Acid-Induced Lipid Metabolism and Inflammation in Hepatocytes through AMPK/NF-κB Signaling Pathway

The prevalence of non-alcoholic fatty liver disease (NAFLD) is one of the leading causes of chronic liver diseases worldwide. This study examined the potential protective effects of a naturally occurring polyphenolic compound, methyl brevifolincarboxylate (MBC) on fatty liver injury in vitro. The results showed that MBC at its non-cytotoxic concentrations, reduced lipid droplet accumulation and triglyceride (TG) levels in the oleic acid (OA)-treated human hepatocarcinoma cell line, SK-HEP-1 and murine primary hepatocytes. In OA-treated SK-HEP-1 cells and primary murine hepatocytes, MBC attenuated the mRNA expression levels of the de novo lipogenesis molecules, acetyl-coenzyme A carboxylase (Acc1), fatty acid synthase (Fasn) and sterol regulatory element binding protein 1c (Srebp1c). MBC promoted the lipid oxidation factor peroxisome proliferator activated receptor-α (Pparα), and its target genes, carnitine palmitoyl transferase 1 (Cpt1) and acyl-coenzyme A oxidase 1 (Acox1) in both the SK-HEP-1 cells and primary murine hepatocytes. The mRNA results were further supported by the attenuated protein expression of lipogenesis and lipid oxidation molecules in OA-treated SK-HEP-1 cells. The MBC increased the expression of AMP activated protein kinase (AMPK) phosphorylation. On the other hand, MBC treatment dampened the inflammatory mediator’s, tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-8, and IL-1β secretion, and nuclear factor (NF)-κB expression (mRNA and protein) through reduced reactive oxygen species production in OA-treated SK-HEP-1 cells. Taken together, our results demonstrated that MBC possessed potential protective effects against NAFLD in vitro by amelioration of lipid metabolism and inflammatory markers through the AMPK/NF-κB signaling pathway.     

Ethyl Gallate Induces Apoptosis of HL-60 Cells by Promoting the Expression of Caspases-8, -9, -3, Apoptosis-Inducing Factor and Endonuclease G

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways.     

A Novel Method Facilitating the Simple and Low-Cost Preparation of Human Osteochondral Slice Explants for Large-Scale Native Tissue Analysis

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-β3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-β3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.     

Effects of High Glucose Concentration on Pericyte-Like Differentiated Human Adipose-Derived Mesenchymal Stem Cells

A pericyte-like differentiation of human adipose-derived mesenchymal stem cells (ASCs) was tested in in vitro experiments for possible therapeutic applications in cases of diabetic retinopathy (DR) to replace irreversibly lost pericytes. For this purpose, pericyte-like ASCs were obtained after their growth in a specific pericyte medium. They were then cultured in high glucose conditions to mimic the altered microenvironment of a diabetic eye. Several parameters were monitored, especially those particularly affected by disease progression: cell proliferation, viability and migration ability; reactive oxygen species (ROS) production; inflammation-related cytokines and angiogenic factors. Overall, encouraging results were obtained. In fact, even after glucose addition, ASCs pre-cultured in the pericyte medium (pmASCs) showed high proliferation rate, viability and migration ability. A considerable increase in mRNA expression levels of the anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1) and interleukin-10 (IL-10) was observed, associated with reduction in ROS production, and mRNA expression of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and angiogenic factors. Finally, a pmASC-induced better organization of tube-like formation by retinal endothelial cells was observed in three-dimensional co-culture. The pericyte-like ASCs obtained in these experiments represent a valuable tool for the treatment of retinal damages occurring in diabetic patients.     

Cannabidiol-Loaded Poly Lactic-Co-Glycolic Acid Nanoparticles with Improved Bioavailability as a Potential for Osteoarthritis Therapeutic

Cannabidiol (CBD) is a non-intoxicating cannabinoid from cannabis sativa that has demonstrated efficacious against inflammation, which can be considered as a potential drug for arthritis treatment. However, the poor solubility and low bioavailability limit its clinical application. Here, we report an effective strategy to fabricate Cannabidiol-loaded poly(lactic-co-glycolic acid) copolymer (CBD-PLGA) nanoparticles (NPs), with a spherical morphology and an average diameter of 238 nm. CBD was sustained release from CBD-PLGA-NPs, which improved the bioavailability of CBD. The CBD-PLGA-NPs effectively protect the damage of LPS to cell viability. We observed that CBD-PLGA-NPs significantly suppressed LPS-induced primary rat chondrocyte expression of inflammatory cytokines, including interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and matrix metalloproteinase 13 (MMP-13). Remarkably, CBD-PLGA-NPs also showed better therapeutic effects of inhibiting the degradation of the extracellular matrix of chondrocytes than equivalent CBD solution. In general, the fabrication CBD-PLGA-NPs showed good protection of primary chondrocytes in vitro and is a promising system for osteoarthritis treatment.