Benefits of bone marrow mesenchymal stem cells compared to their conditioned medium in valproic acid-induced autism in rats.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behaviors, a limited range of activities, and deficiencies in social communications. Bone marrow mesenchymal stem cells (BM-MSCs), which secrete factors that stimulate surrounding microenvironment, and BM-MSCs conditioned medium (BM-MSCs-CM), which contains cell-secreted products, have been speculated to hold potential as a therapy for ASD. This study aimed to compare the therapeutic effects of BM-MSCs and BM-MSCs-CM on behavioral and microglial changes in an animal model of autism induced by valproic acid (VPA). METHODS AND RESULTS: Pregnant Wistar rats were administered by VPA at a dose of 600 mg/kg at 12.5 days post-conception. After birth, male pups were included in the study. At 6 weeks of age, one group of rats received intranasal administration of BM-MSCs, while another group received BM-MSCs-CM. The rats were allowed to recover for 2 weeks. Behavioral tests, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry were performed. Both BM-MSCs and BM-MSCs-CM administration significantly improved some behavioral deficits. Furthermore, these treatments notably reduced Iba-1 marker associated with microgliosis. Additionally, there was a significant reduction in the expression of pro-inflammatory cytokines IL-1ß and IL-6, and an increase in the levels of the anti-inflammatory cytokine IL-10 in rats administered by BM-MSCs and BM-MSCs-CM. CONCLUSIONS: Post-developmental administration of BM-MSCs and BM-MSCs-CM can ameliorate prenatal neurodevelopmental deficits, restore cognitive and social behaviors, and modulate microglial and inflammatory markers. Results indicated that the improvement rate was higher in the BM-MSCs group than BM-MSCs-CM group.

Novel Identification of Cysteinyl Derivatives of Toxic Clozapine Nitrenium Ions and the Effect of Valproic Acid on Metabolite Formation: A Study Using Reprocessed High-Resolution Mass Spectra of Analyzed Therapeutic Drug Monitoring Samples.

BACKGROUND: Clozapine (CLZ) use is hampered by the risk of granulocyte toxicity, which is associated with the formation of nitrenium ions and the concurrent use of valproic acid (VPA). These highly reactive nitrenium ions cannot be measured in vivo. Instead, deactivated cysteinyl conjugates may potentially be detected. The aim of this study was to develop a novel method for identifying cysteinylated derivates of CLZ nitrenium ions to investigate the effect of VPA on their formation using therapeutic drug monitoring data. METHODS: A population comprising 93 VPA comedicated and 162 control patients from a therapeutic drug monitoring (TDM) service in Oslo, Norway, was included. Reprocessing of ultraperformance liquid chromatography high-resolution mass spectra (UHPLC-HR-MS) of previously analyzed TDM samples, combined with the assessment of MS/MS fragmentation patterns, was performed to identify the CLZ cysteinyl conjugates. Smoking, which induces CLZ metabolism, was assessed by detecting cotinine in the reprocessed mass spectra. RESULTS: By reprocessing the UHPLC-HR-MS files of the TDM analyses and reviewing the MS/MS fragment profiles, four cysteinyl conjugates of CLZ were identified. The formations of CLZ cysteinyl (CLZ-Cys 1+ ) and CLZ- N -oxide cysteinyl (CLZ-NOX-Cys 1+ ) were 1.5-fold ( P = 0.038) and 2.1-fold ( P < 0.001) higher in VPA-treated patients than those in the controls. In agreement with previous studies, a 45% reduction in N -desmethylclozapine formation was observed in VPA-treated patients ( P < 0.001). CONCLUSIONS: A novel method for detecting cysteinyl conjugates of CLZ was developed. Application of this method indicated that VPA significantly increased the formation of CLZ-Cys 1+ metabolites, which might explain the granulocyte toxicity reported after adding VPA to CLZ treatment. The developed method opens new avenues for investigating CLZ toxicity, e.g. by correlating cysteinyl conjugates and granulocyte counts in patients.

Sex-biased single-cell genetic landscape in mice with autism spectrum disorder.

Autistic spectrum disorder (ASD) is a male-biased, heterogeneous neurodevelopmental disorder that affects approximately 1%-2% of the population. Prenatal exposure to valproic acid (VPA) is a recognized risk factor for ASD, but the cellular and molecular basis of VPA-induced ASD at the single-cell resolution is unclear. Here, we aim to compare the cellular and molecular differences in the hippocampus between male and female prenatal mice with ASD at the single-cell transcriptomic level. The transcriptomes of more than 45,000 cells are assigned to 12 major cell types, including neurons, glial cells, vascular cells, and immune cells. Cell type-specific genes with altered expression after prenatal VPA exposure are analyzed, and the largest number of differentially expressed genes (DEGs) are found in neurons, choroid plexus epithelial cells, and microglia. In microglia, several pathways related to inflammation are found in both males and females, including the tumor necrosis factor (TNF), nuclear factor kappa B (NF-κB), toll-like receptor (TLR), and mitogen-activated protein kinase (MAPK) signaling pathways, which are important for the induction of autistic-like behavior. Additionally, we note that several X-linked genes, including Bex1, Bex3, and Gria3, were among the male-specific DEGs of neurons. This pioneering study describes the landscape of the transcriptome in the hippocampus of autistic mice. The elucidation of sexual differences could provide innovative strategies for the prevention and treatment of ASD.

Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells.

BACKGROUND: Cardiovascular diseases remain a major cause of death globally. Cardiac cells once damaged, cannot resume the normal functioning of the heart. Bone marrow derived mesenchymal stem cells (BM-MSCs) have shown the potential to differentiate into cardiac cells. Epigenetic modifications determine cell identity during embryo development via regulation of tissue specific gene expression. The major epigenetic mechanisms that control cell fate and biological functions are DNA methylation and histone modifications. However, epigenetic modifiers alone are not sufficient to generate mature cardiac cells. Various small molecules such as ascorbic acid (AA) and salvianolic acid B (SA) are known for their cardiomyogenic potential. Therefore, this study is aimed to examine the synergistic effects of epigenetic modifiers, valproic acid (VPA) and 5-azacytidine (5-aza) with cardiomyogenic molecules, AA and SA in the cardiac differentiation of MSCs. METHODS AND RESULTS: BM-MSCs were isolated, propagated, characterized, and then treated with an optimized dose of VPA or 5-aza for 24 h. MSCs were maintained in a medium containing AA and SA for 21 days. All groups were assessed for the expression of cardiac genes and proteins through q-PCR and immunocytochemistry, respectively. Results show that epigenetic modifiers VPA or 5-aza in combination with AA and SA significantly upregulate the expression of cardiac genes MEF2C, Nkx2.5, cMHC, Tbx20, and GATA-4. In addition, VPA or 5-aza pretreatment along with AA and SA enhanced the expression of the cardiac proteins connexin-43, GATA-4, cTnI, and Nkx2.5. CONCLUSION: These findings suggest that epigenetic modifiers valproic acid and 5-azacytidine in combination with ascorbic acid and salvianolic acid B promote cardiac differentiation of MSCs. This pretreatment strategy can be exploited for designing future stem cell based therapeutic strategies for cardiovascular diseases.

NFκB and Cyclic AMP Response Element Sites Mediate the Valproic Acid and UL138 Responsiveness of the Human Cytomegalovirus Major Immediate Early Enhancer and Promoter.

The major immediate early enhancer and promoter (MIEP) of human cytomegalovirus (HCMV) drives the transcription of the immediate early one (IE1) and IE2 genes, whose encoded proteins stimulate productive, lytic replication. The MIEP is activated by the virally encoded and tegument-delivered pp71 protein at the start of de novo lytic infections of fully differentiated cells. Conversely, the MIEP is silenced at the start of de novo latent infections within incompletely differentiated myeloid cells in part because tegument-delivered pp71 is sequestered in the cytoplasm in these cells, but also by viral factors that repress transcription from this locus, including the UL138 protein. During both modes of infection, MIEP activity can be increased by the histone deacetylase inhibitor valproic acid (VPA); however, UL138 inhibits the VPA-responsiveness of the MIEP. Here, we show that two families of cellular transcription factors, NF-κB and cAMP response element-binding protein (CREB), together control the VPA-mediated activation and UL138-mediated repression of the HCMV MIEP. IMPORTANCE Artificial regulation of the HCMV MIEP, either activation or repression, is an attractive potential means to target the latent reservoirs of virus for which there is currently no available intervention. The MIEP could be repressed to prevent latency reactivation or induced to drive the virus into the lytic stage that is visible to the immune system and inhibited by multiple small-molecule antiviral drugs. Understanding how the MIEP is regulated is a critical part of designing and implementing either strategy. Our revelation here that NF-κB and CREB control the responsiveness of the MIEP to the viral UL138 protein and the FDA-approved drug VPA could help in the formulation and execution of promoter regulatory strategies against latent HCMV.

Kidney tubular injury induced by valproic acid: systematic literature review.

BACKGROUND: Valproic acid is prescribed for epilepsy and as prophylaxis for bipolar disorder and migraine headaches. It has also been implicated as a cause of a kidney tubular injury. METHODS: We undertook a review of the literature to characterize the biochemical and histopathological features of the overt kidney tubular injury and to evaluate the possible existence of a pauci-symptomatic injury. The pre-registered review (CRD42022360357) was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology. Searches were conducted in Excerpta Medica, the National Library of Medicine, and Web of Science. The gray literature was also considered. RESULTS: For the final analysis, we retained 36 articles: 28 case reports documented 48 individuals with epilepsy on valproic acid for 7 months or more and presenting with features consistent with an overt kidney tubular injury. The following disturbances were noted: hypophosphatemia (N = 46), normoglycemic glycosuria (N = 46), total proteinuria (N = 45), metabolic acidosis (N = 36), hypouricemia (N = 27), tubular proteinuria (N = 27), hypokalemia (N = 23), and hypocalcemia (N = 8). A biopsy, obtained in six cases, disclosed altered proximal tubular cells with giant and dysmorphic mitochondria. Eight case series addressed the existence of a pauci- or even asymptomatic kidney injury. In the reported 285 subjects on valproic acid for 7 months or more, an isolated tubular proteinuria, mostly N-acetyl-ß-glucosaminidase, was often noted. CONCLUSIONS: Valproic acid may induce an overt kidney tubular injury, which is associated with a proximal tubular mitochondrial toxicity. Treatment for 7 months or more is often associated with a pauci- or oligosymptomatic kidney tubular injury. A higher resolution version of the Graphical abstract is available as Supplementary information.

Effect of Moringa oleifera leaf extract on valproate-induced oxidative damage in muscle.

Valproic acid (VPA) is a drug used for the treatment of epilepsy worldwide. Depending on usage, it can cause complications such as coagulopathies, hepatotoxicity, and encephalopathy. Moringa oleifera has been shown to have antitumor, anti-inflammatory, antiulcer, antispasmodic, diuretic, antihypertensive, antidiabetic, and hepatoprotective activities. The current study investigated the effects of Moringa leaves extract (70% ethanol) on antioxidant systems against valproate-induced oxidative damage in muscle tissues of rats. Female Sprague Dawley rats were randomly divided into four groups. Group I: control group; Group II: animals given only Moringa extract; Group III: animals that received only sodium valproate; Group IV: animals administered with sodium valproate + Moringa extract. Moringa extract and sodium valproate were administered orally. Muscle tissues were collected after sacrificing the animals. Biochemical analysis of muscle tissue homogenates of the valproate group revealed elevated levels/activities of lipid peroxidation, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, catalase, glutathione reductase, glutathione-S-transferase, reactive oxygen species, total oxidant status, oxidative stress index, glucose-6-phosphate dehydrogenase, sialic acid, protein carbonyl, nitric oxide, and myeloperoxidase. While glutathione, superoxide dismutase, glutathione peroxidase, total antioxidant status, aryl esterase and sodium/potassium ATPase were decreased. The administration of Moringa extract reversed these biochemical changes. These results indicate that Moringa leaves extract had a protective effect on muscle tissues against valproate-induced damage.

The role of epigenetic modifiers in the hepatic differentiation of human umbilical cord derived mesenchymal stem cells.

Human umbilical cord (hUC) derived mesenchymal stem cells (MSCs) can be progressively differentiated into multiple lineages including hepatic lineages, and thus provide an excellent in vitro model system for the study of hepatic differentiation. At present, hepatic differentiation protocols are based on the use of soluble chemicals in the culture medium and provide immature hepatic like cells. Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) are two important epigenetic modifiers that regulate stem cell differentiation. Therefore, this study aimed to investigate the role of HDACi, valproic acid (VPA) and DNMTi,5-azacytidine (5-aza) along with a hepatic inducer in the hepatic differentiation of hUC-MSCs. hUC-MSCs were characterized via immunocytochemistry and flow cytometry. The final concentrations of VPA and 5-aza were optimized via MTT cytotoxicity assay. All treated groups were assessed for the presence of hepatic genes and proteins through qPCR and immunocytochemistry, respectively. The results showed that the pretreatment of epigenetic modifiers not only increased the hepatic genes but also increased the expression of the hepatic proteins. VPA induces hepatic differentiation in hUC-MSCs with significant gene expression of hepatic markers i.e., FOXA2 and CK8. Moreover, VPA pretreatment enhanced the expression of hepatic proteins AFP and TAT. The pretreatment of 5-aza shows significant gene expression of hepatic marker LDL-R. However, 5-aza treatment failed to induce hepatic protein expression. The results of the current study highlighted the effectiveness of epigenetic modifiers in the hepatic differentiation of hUC-MSCs. These differentiated cells can be employed in cell-based therapeutics for hepatic diseases in future.

Induction of Periodontal Ligament-like Cells by Coculture of Dental Pulp Cells, Dedifferentiated Cells Generated from Epithelial Cell Rests of Malassez, and Umbilical Vein Endothelial Cells.

INTRODUCTION: Apart from the epithelial cell rests of Malassez (ERMs), dental pulp (DP) contains the same types of mesenchymal cells as the periodontal ligament (PDL). ERMs may affect the characteristics of the mesenchymal cells in the PDL. The aim of this study was to examine whether DP cells cultured with ERMs and human umbilical vein endothelial cells (HUVECs) could transform into PDL-like cells. METHODS: Progenitor-dedifferentiated into stem-like cells (Pro-DSLCs) were produced by the induction of ERMs with 5-Azacytidine and valproic acid. DP cells were cultured in mesenchymal stem cell medium for 1 week under the following conditions: DP cells alone (controls); PDL cells alone; coculture of DP cells and ERMs (DP + ERM) or Pro-DSLCs (DP + Pro-DSLC); and coculture of DP cells, HUVECs, and ERMs (DP + ERM + HUVEC) or Pro-DSLCs (DP + Pro-DSLC + HUVEC). Quantitative real-time reverse transcription polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, and flow cytometry were performed. RESULTS: The expression levels of PDL-related markers Msx1, Msx2, Ncam1, Postn, and S100a4 and mesenchymal stem cell-positive markers Cd29, Cd90, and Cd105 were significantly higher in the PDL cells and DP + Pro-DSLC + HUVEC cultures than in the controls (P < .05). The DNA methylation levels of Msx1 and Cd29 in the PDL cells and the DP + Pro-DSLC + HUVEC culture were significantly lower than in the controls (P < .01). We found a significant increase in the number of cells stained with MSX1 (P < .05) and CD29 (P < .01) in the DP + Pro-DSLC + HUVEC culture than in the controls. CONCLUSIONS: Coculture of DP cells with Pro-DSLCs and HUVECs induced their transformation into PDL-like cells. This method may prove to be useful for periodontal regeneration via tissue engineering.

Sustained release of sulforaphane by bioactive extracellular vesicles for neuroprotective effect on chick model.

Novel studies have shown neurological treatment possibilities with extracellular vesicles (EVs) as natural particles with a special composition that are produced by different cell types. Their stability, natural structure, composition, and bioavailability make them good candidates as drug vehicles. Here, EVs were isolated from amniotic fluid (AF) through differential centrifugation, and characterized for size (<200 nm), structure, and composition, their effectiveness on the human PC12 cell line, and brain of chick embryos exposed to sodium valproate (animal autistic model). Sulforaphane (SFN) was employed as a bioactive compound and then encapsulated into Evs using three methods including passive (incubation), active (sonication), and active-passive (sonication-incubation). Further, the loading and in vitro releases of SFN fitted the Korsmeyer-Peppas (R2  = 0.99) kinetic model by non-Fickian diffusion case II (n = 0.44, passive loading) and Fickian diffusion case I (n = 0.41, active and active-passive loading). SFN-loaded EVs (SFN@EVs; 11 µM: 103 nM) stimulated hPC-12 cell proliferation. The gene expression analysis revealed that SFN@EVs could upregulate Nrf2 and reduce IL-6 expression. Eventually, histopathological results of the coronal cross-section of the chick embryos brain showed treatment with SFN@EVs. This treatment illustrated normality in the gray and white matter and the orientation of the bipolar neurons. Our findings showed EVs' potentially acting as a gene expression regulator in autism spectrum disorder.

Valproic acid counteracts polycyclic aromatic hydrocarbons (PAHs)-induced tumorigenic effects by regulating the polarization of macrophages.

Polycyclic aromatic hydrocarbons (PAHs) are common persistent organic pollutants that are carcinogenic, teratogenic and mutagenic, causing a variety of harm to human health. In this study, we investigated the mechanism of how valproic acid (VPA) interferes with the carcinogenesis of PAHs protect normal tissues via the regulation of macrophages' function. Using the established model of transformed malignant breast cancer by 7,12-dimethylbenz[a]anthracene (DMBA), a representative PAH carcinogen, we discovered VPA induces the polarization of macrophages toward the M1 phenotype in the tumor tissues, facilitates the expression of pro-inflammatory cytokines such as IFN-γ, IL-12 and TNF-α, activates CD8+ T cells to secret Granzyme B thus to promote the apoptosis of tumor cells and suppresses the viability of vascular endothelial cells in tissue stroma of tumor. Surprisingly, VPA selectively induces macrophages to polarize towards the M2 phenotype in normal tissues and promotes the expression of anti-inflammatory cytokines such as IL-10 to enhance cell proliferation. Additionally, at the cellular level, VPA can directly regulate the polarization of macrophages to affect the growth of vascular endothelial cells by simulating the living conditions of tumor and normal cells. Collectively, VPA exerts an interventional effect on tumor growth and a protective effect on normal tissues by regulation of selective macrophages' polarization in their microenvironment.

Syringic acid and silymarin concurrent administration inhibits sodium valproate-induced liver injury in rats.

Sodium valproate (SV) is a well-known anti-epileptic drug, also used to control convulsions, bipolar disorders and migraines. SV has been shown to induce liver toxicity in clinical subjects. Syringic acid (SA), a natural polyphenolic compound has potential antioxidant, anti-inflammatory and several beneficial effects. Therefore, in this study, we evaluated hepatoprotective effect of SA against SV-induced liver injury in rats. Wistar rats were treated with SV orally at a dose of 500 mg/kg, once daily, for 14 days. Another three groups of rats were administered with SV and concurrently treated with SA (40 and 80 mg/kg) and silymarin (SIL) (100 mg/kg) for 14 days. SV administration for 14 days caused significant (p < .001) elevation of liver transaminases and ALP in serum. Liver MDA level was significantly (p < .001) increased with a concomitant decrease (p < .001) in enzymic antioxidants activities in SV administered rats. SV administration also caused the upregulation of proinflammatory markers such as tumor necrosis factor α, c-Jun N-terminal kinase, nuclear factor kappa B, cyclooxygenase-2 and Interleukin 6 expressions in liver tissue. Histopathological studies also revealed the presence of inflammatory cell infiltration and hepatocellular necrosis upon SV administration. At both doses, concurrent administration of SA and SIL significantly (p < .001) inhibited the liver transaminase activities in serum, oxidative stress, and proinflammatory markers expression in liver tissue. Our current results suggest that SA can be a promising herbal drug that can inhibit SV-induced hepatotoxicity when administered together due its potential anti-inflammatory effects.

Enhancement of cordycepin production from Cordyceps militaris culture by epigenetic modification.

Cordycepin (3'-deoxyadenosine) is a nucleoside analogue and biosynthesised by Cordyceps militaris, an entomopathogenic fungus. In this study, an epigenetic modifier was applied to static liquid cultures to enhance cordycepin production. C. militaris was cultured in a static liquid culture, and valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was supplemented in order to modifying the epigenetic status. Gene regulatory network was explored to understand the molecular mechanisms underlying cordycepin production. 50 micromolar of VPA enhanced cordycepin production by 41.187% via the upregulation of 5'-nucleotidase, adenylate kinase, phosphorybosyltransferase, Cns1, Cns2, Cnsa3, and Cns4 of C. militaris for at least 2 days after VPA treatment. The maximum production of cordycepin was 2,835.32 ± 34.35 mg/L in 400 mL-working volume. A scaled-up culture was established with a working volume of 10 L, which led to the slight decrease of cordycepin production. This might due to multifactorial effects, for instance limited aeration and an uneven dispersion of nutrients in the culture system. This scaled-up culture was still needed further optimization. The modification of epigenetic status by VPA significantly enhanced cordycepin production by altering key gene regulatory network of C. militaris. The strategy established in this study might be applicable to other microorganism culture in order to improving the production of bioactive compounds. This work aimed to enhance the production of cordycepin by modifying the epigenetic status of C. militaris, in which subsequently altered gene regulatory network of cordycepin biosynthesis pathway. The weekly supplementation of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, significantly improve cordycepin production over 40%, compared to the untreated control, and the gene regulatory network of C. militaris was also adapted.

Valproic Acid-Induced CCN1 Promotes Osteogenic Differentiation by Increasing CCN1 Protein Stability through HDAC1 Inhibition in Tonsil-Derived Mesenchymal Stem Cells.

Our previous study found that the level of CCN1 increases as osteogenic differentiation progresses in tonsil-derived mesenchymal stem cells (TMSCs). This study investigated how CCN1 is regulated through HDAC inhibition in TMSCs and their relationship with osteogenesis. Valproic acid (VPA) (1-5 mM), a well-known histone deacetylase (HDAC) inhibitor, strongly inhibited TMSC proliferation without altering MSC-specific surface markers, CD14, 34, 45, 73, 90 and 105. However, CD146 expression increased at 5 mM VPA. VPA increased osteogenic differentiation of TMSCs but decreased adipogenesis and chondrogenesis, as evidenced by the cell-specific staining of differentiation. The former was validated by the increased osteocalcin (OCN). The changes in CCN1 by VPA was biphasic; it increased until 48 h and decreased thereafter. Knockdown of CCN1 by using siRNA inhibited the osteogenic effect of VPA. VPA had no effect on CCN1 mRNA expression, but inhibition of protein synthesis by cycloheximide showed that VPA slowed down the CCN1 protein degradation. Moreover, overexpression of HDAC1 completely inhibited VPA-induced CCN1. Our results indicate that VPA inhibits the HDAC1, inducing CCN1 protein stability rather than gene expression, thereby promoting osteogenic differentiation of TMSCs. These findings present the noble implication of VPA as an inhibitor of HDAC1 to facilitate CCN1-induced osteogenic differentiation of MSCs.

Effect of valproic acid on functional bleb morphology in a rabbit model of minimally invasive surgery.

PURPOSE: To determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS). METHODS: Nine New Zealand white rabbits were subjected to MIGS with intraoperative implantation of the PreserFlo MicroShunt. Rabbits were then administered with subconjunctival injections of phosphate buffered saline (PBS) (n=4) or with VPA (n=5). Bleb morphology was examined by slit-lamp biomicroscopy and in vivo confocal microscopy. Postoperative day 28 tissues were examined by immunohistochemical evaluation and label-free multiphoton microscopy to visualise the collagen matrix, by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay and immunofluorescent labelling for Ki67 expression to detect apoptosis and cell growth, and by real-time quantitative PCR to measure Col1a1, Fn, and Smad6 transcript expression. RESULTS: VPA-treated blebs were detectable on day 28, while the PBS-treated blebs were not detectable by day 14. VPA-treated blebs were diffuse, extended posteriorly with near normal conjunctival vascularity and featured a combination of reticular/blurred stromal pattern with evidence of relatively large stromal cysts. Instead of the deposition of thick, disorganised collagen fibres characteristic of the PBS bleb, the VPA bleb contained conspicuously thinner collagen fibres which were associated with similarly thinner fibronectin fibres. In corroboration, Col1a1 and Fn mRNA expression was reduced in the VPA blebs, while increased Smad6 expression implicated the disruption of the transforming growth factor beta pathway. Apoptosis and cell growth profiles appeared similar with both treatments. CONCLUSIONS: The results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity.

Effect of valproic acid on miRNAs affecting histone deacetylase in a model of anaplastic thyroid cancer.

BACKGROUND: Thyroid cancer is the most common malignant tumor of the endocrine system seen in the thyroid gland. More than 90% of thyroid cancers comprise papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although anaplastic thyroid carcinoma (ATC) accounts for less than 2% of thyroid cancer. But patients' lifespan after diagnosis is about 6 months. Surgical interventions, radioactive iodine use, and chemotherapy are not sufficient in the treatment of ATC, so alternative therapies are needed. METHODS AND RESULTS: The WST-1 assay test was performed to evaluate the anti-proliferative effects of Valproic acid (VPA). Also, the effect of VPA on miRNAs affecting histone deacetylase was determined by Quantitative RT-PCR. In the SW1736 cell line, IC50 dose for VPA was found 1.6 mg/ml. In our study, the level of oncogenic genes expression in cells treated with VPA, including miR-184, miR-222-5p, miR-124-3p, and miR-328-3p, decreased. Also, the expression of tumor inhibitory genes including miR-323-5p, miR-182-5p, miR-138-5p, miR-217, miR-15a-5p, miR-29b-3p, miR-324-5p and miR-101-5p increased significantly. CONCLUSIONS: VPA can ad-just countless gene expression patterns, including microRNAs (miRNAs), by targeting histone deacetylase (HDAC). However, further studies are required for more accurate results.

A valproic acid-modified platinum diimine complex as potential photosensitizer for photodynamic therapy.

Histone deacetylase inhibitors have often been used in combination treatment of various types of cancers due to their non-genotoxic epigenetic potential. Valproic acid (VPA) is a well-known histone deacetylase inhibitor. Conjugate of VPA with a phtoactive platinum diimine complex through an ester bond has been fabricated to potentiate the photocytotoxicity of the photosensitizer. Its capability to generate singlet oxygen, behavior in the presence of esterase, and photocytotoxicity in tumor cells have also been studied. The results revealed that the novel VPA-modified platinum diimine complex could produce singlet oxygen efficiently and release VPA in the presence of porcine liver esterase. The results also suggested that incorporation of VPA moiety into the platinum diimine complex might significantly enhance the cytotoxicity of the complex.

Regulation of valproic acid induced EMT by AKT/GSK3ß/ß-catenin signaling pathway in triple negative breast cancer.

Valproic acid (VPA) is a selective histone deacetylation (HDAC) inhibitor and exerts anti-cancer properties in different types of cancer. The epithelial-to-mesenchymal transition (EMT) mediating by different signaling cascade can be a potential target in aggressive human cancers. Therefore, we aimed to clarified the unravel relationship between AKT/GSK3ß/ß-catenin signalling pathway and VPA-induced EMT in triple negative breast cancer (TNBC). The cytotoxicity of VPA in MDA-MB-231 TNBC and MCF-10A control cells was evaluated. Alterations in the expression levels of Snail, E-cadherin, AKT, GSK3ß, ß-catenin were analyzed by RT-PCR. Additionally, Annexin V, cell cycle and wound healing assays were performed. Our results showed that VPA remarkably inhibited the growth of TNBC cell and triggered apoptotic cell death through G0/G1 arrest. Furthermore, VPA increased cell migration and activated the EMT process through significantly increasing Snail expression and in turn downregulation of E-cadherin and GKS3ß levels. However, the level of AKT and ß-catenin was reduced after treatment of VPA. Our data showed that VPA induced EMT process and cell migration in TNBC cells. However, AKT/GSK3ß/ß-catenin signaling pathway did not mediate EMT activation.

Valproic acid, A Potential Inducer of Osteogenesis in Mouse Mesenchymal Stem Cells.

BACKGROUND: Recent reports have unveiled the potential of flavonoids to enhance bone formation and assuage bone resorption due to their involvement in cell signaling pathways. They also act as an effective alternative to circumvent the disadvantages associated with existing treatment methods, which has increased their scope in orthopedic research. Valproic acid (VA, 2-propylpentanoic acid) is one such flavonoid, obtained from an herbaceous plant, used in the treatment of epilepsy and various types of seizures. OBJECTIVE: In this study, the role of VA in osteogenesis and the molecular mechanisms underpinning its action in mouse mesenchymal stem cells (mMSCs) were determined. METHODS: Results: Cytotoxic studies validated VA's amiable nature in mMSCs. Alizarin red and von Kossa staining results showed an increased deposition of calcium phosphate in VA-treated mMSCs, which confirmed the occurrence of osteoblast differentiation and mineralization at a cellular level. At the molecular level, there were increased levels of expression of Runx2, a vital bone transcription factor, and other major osteoblast differentiation marker genes in the VA-treated mMSCs. Further, VA-treatment in mMSCs upregulated mir-21 and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway, which might be essential for the expression/activity of Runx2. CONCLUSION: Thus, the current study confirmed the osteoinductive nature of VA at the cellular and molecular levels, opening the possibility for its application in bone therapeutics with mir-21.

Valproic Acid: A Potential Therapeutic for Spinal Cord Injury.

The lack of an effective pharmaceutical agent for spinal cord injury (SCI) is a current problematic situation for clinicians, as the rate of motor vehicle accidents among young adults is on the rise. SCI contributes to the high disability rate. Presently, evidences detailing the precise pathological mechanisms in SCI are limited, compounding to the unavailability of an effective treatment method. Surgery, though not a complete curative method, is useful in managing some of the associated symptoms of secondary SCI. Autophagy and inflammation are contributive factors to both exacerbation and improvement of SCI. The mammalian target of rapamycin (mTOR) signaling pathway is a key player in the regulation of inflammatory response and autophagy. Valproic acid (VPA), a clinically used antiepileptic drug, has been suggested to improve neurological conditions, including SCI. This report reviewed the correlation between mTOR and autophagy, as well as autophagy's role and the therapeutic effects of VPA in SCI. VPA regulates autophagy by potentially inhibiting mTORC1, a complex of mTOR, while also hindering inflammatory response. Conclusively, an effective treatment for SCI could lie in the timely regulation of mTOR signaling pathway, and VPA could be the potential drug that improves SCI owing to its propensity to regulate the mTOR signaling pathway.