Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway.

Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.

KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy.

BACKGROUND: Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC. METHODS: We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer. RESULTS: Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage. CONCLUSION: Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

Tetrahydrocannabinols: potential cannabimimetic agents for cancer therapy.

Tetrahydrocannabinols (THCs) antagonize the CB1 and CB2 cannabinoid receptors, whose signaling to the endocannabinoid system is essential for controlling cell survival and proliferation as well as psychoactive effects. Most tumor cells express a much higher level of CB1 and CB2; THCs have been investigated as potential cancer therapeutic due to their cannabimimetic properties. To date, THCs have been prescribed as palliative medicine to cancer patients but not as an anticancer modality. Growing evidence of preclinical research demonstrates that THCs reduce tumor progression by stimulating apoptosis and autophagy and inhibiting two significant hallmarks of cancer pathogenesis: metastasis and angiogenesis. However, the degree of their anticancer effects depends on the origin of the tumor site, the expression of cannabinoid receptors on tumor cells, and the dosages and types of THC. This review summarizes the current state of knowledge on the molecular processes that THCs target for their anticancer effects. It also emphasizes the substantial knowledge gaps that should be of concern in future studies. We also discuss the therapeutic effects of THCs and the problems that will need to be addressed in the future. Clarifying unanswered queries is a prerequisite to translating the THCs into an effective anticancer regime.

Dental Pulp-Derived Stem Cells Preserve Astrocyte Health During Induced Gliosis by Modulating Mitochondrial Activity and Functions.

Astrocytes have been implicated in the onset and complication of various central nervous system (CNS) injuries and disorders. Uncontrolled astrogliosis (gliosis), while a necessary process for recovery after CNS trauma, also causes impairments in CNS performance and functions. The ability to preserve astrocyte health and better regulate the gliosis process could play a major role in controlling damage in the aftermath of acute insults and during chronic dysfunction. Here in, we demonstrate the ability of dental pulp-derived stem cells (DPSCs) in protecting the health of astrocytes during induced gliosis. First of all, we have characterized the expression of genes in primary astrocytes that are relevant to the pathological conditions of CNS by inducing gliosis. Subsequently, we found that astrocytes co-cultured with DPSCs reduced ROS production, NRF2 and GCLM expressions, mitochondrial membrane potential, and mitochondrial functions compared to the astrocytes that were not co-cultured with DPSCs in gliosis condition. In addition, hyperactive autophagy was also decreased in astrocytes that were co-cultured with DPSCs compared to the astrocytes that were not co-cultured with DPSCs during gliosis. This reversal and mitigation of gliosis in astrocytes were partly due to induction of neurogenesis in DPSCs through enhanced expressions of the neuronal genes like GFAP, NeuN, and Synapsin in DPSCs and by secretion of higher amounts of neurotropic factors, such as BDNF, GDNF, and TIMP-2. Protein-Protein docking analysis suggested that BDNF and GDNF can bind with CSPG4 and block the downstream signaling. Together these findings demonstrate novel functions of DPSCs to preserve astrocyte health during gliosis.

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway.

Background: Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC℗) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods: The U87-MG cells were cultured with DPSC℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results: DPSC℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC℗. Additionally, DPSC℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion: Our findings lend the first evidence of the inhibitory effects of DPSC℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.

Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions.

A natural plant product, epigallocatechin-3-gallate (EGCG), was evaluated for its effectiveness in the regulation of osteoclastogenesis. We found that EGCG inhibited the osteoclast (OC) differentiation in vitro, and in primary bone marrow cells in a dose-dependent manner. Quantitative RT-PCR studies showed that the EGCG reduced the expression of OC differentiation markers. DCFDA, MitoSOX, and JC-1 staining revealed that the EGCG attenuated the reactive oxygen species (ROS), and mitochondrial membrane potential; and flux analysis corroborated the effect of EGCG. We further found that the EGCG inhibited mRNA and protein expressions of mitophagy-related molecules. We confirmed that the OC differentiation was inhibited by EGCG by modulating mitophagy through AKT and p38MAPK pathways. Furthermore, in silico analysis revealed that the binding of RANK and RANKL was blocked by EGCG. Overall, we defined the mechanisms of osteoclastogenesis during arthritis for developing a new therapy using a natural compound besides the existing therapeutics.

Dental Pulp-Derived Stem Cells Reduce Inflammation, Accelerate Wound Healing and Mediate M2 Polarization of Myeloid Cells.

This work aimed to validate the potential use of dental pulp-derived stem cells (DPSCs) for the treatment of inflammation by defining their mechanisms of action. We planned to investigate whether priming of DPSC with proinflammatory molecules had any impact on their behavior and function. In the first step of our validation in vitro, we showed that priming of DPSCs with the bioactive agents LPS, TNF-α, or IFN-γ altered DPSCs' immunologic properties by increasing their expression levels of IL-10, HGF, IDO, and IL-4 and by decreasing their mitochondrial functions. Moreover, DPSCs induced accelerated wound healing irrespective of priming, as determined by using a gut epithelial cell line in a scratch wound assay. Wound healing of gut epithelial cells was mediated by regulating the expressions of AKT, NF-κB, and ERK1/2 proteins compared to the control epithelial cells. In addition, primed DPSCs altered monocyte polarization toward an immuno-suppressive phenotype (M2), where monocytes expressed higher levels of IL-4R, IL-6, Arg1, and YM-1 compared to monocytes cultured with control DPSCs. In silico analysis revealed that this was accomplished in part by the interaction between kynurenine and PPARγ, which regulated the expression of M2 differentiation-related genes. Collectively, these data provided evidence that the DPSCs reduced inflammation, induced M2 polarization of myeloid cells, and healed damaged gut epithelial cells through inactivation of inflammation and modulating constitutively active signaling pathways.

Ferutinin induces osteoblast differentiation of DPSCs via induction of KLF2 and autophagy/mitophagy.

Osteoblast differentiation is critically reduced in various bone-related pathogenesis, including arthritis and osteoporosis. For future development of effective regenerative therapeutics, herein, we reveal the involved molecular mechanisms of a phytoestrogen, ferutinin-induced initiation of osteoblast differentiation from dental pulp-derived stem cell (DPSC). We demonstrate the significantly increased expression level of a transcription factor, Kruppel-like factor 2 (KLF2) along with autophagy-related molecules in DPSCs after induction with ferutinin. The loss-of-function and the gain-of-function approaches of KLF2 confirmed that the ferutinin-induced KLF2 modulated autophagic and OB differentiation-related molecules. Further, knockdown of the autophagic molecule (ATG7 or BECN1) from DPSC resulted not only in a decreased level of KLF2 but also in the reduced levels of OB differentiation-related molecules. Moreover, mitochondrial membrane potential-related molecules were increased and induction of mitophagy was observed in DPSCs after the addition of ferutinin. The reduction of mitochondrial as well as total ROS generations; and induction of intracellular Ca2+ production were also observed in ferutinin-treated DPSCs. To test the mitochondrial respiration in DPSCs, we found that the cells treated with ferutinin showed a reduced extracellular acidification rate (ECAR) than that of their vehicle-treated counterparts. Furthermore, mechanistically, chromatin immunoprecipitation (ChIP) analysis revealed that the addition of ferutinin in DPSCs not only induced the level of KLF2, but also induced the transcriptionally active epigenetic marks (H3K27Ac and H3K4me3) on the promoter region of the autophagic molecule ATG7. These results provide strong evidence that ferutinin stimulates OB differentiation via induction of KLF2-mediated autophagy/mitophagy.