Hydrogen Peroxide-induced Cytotoxicity Research Articles

The Metabolite of γ-Tocopherol, 2,7,8-Trimethyl-2-(2′-Carboxyethyl)-6-Hydroxychroman, Exerts Intracellular Antioxidant Activity via Up-Regulation of Heme Oxygenase-1 in Hepatocytes

γ-Tocopherol (γT) is the major form of vitamin E contained in plants and seed oils. Although it is readily metabolized in the liver, the function of the metabolites is not fully understood. This study investigated the antioxidant activities of the γT metabolite 2,7,8-trimethyl-2-(2′-carboxyethyl)-6-hydroxychroman (γCEHC) in comparison to its parent compound. The pretreatment of mouse hepatoma Hepa1c1c7 cells with γCEHC showed a cytoprotective effect on the hydrogen peroxide-induced cytotoxicity to a lesser extent than that of γT. A mechanistic investigation revealed that both γ-CEHC and γT significantly up-regulated the gene and protein expressions of heme oxygenase-1 (HO-1) via the promotion of the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the combination of γCEHC and γT significantly increased the gene and protein levels of HO-1 and the nuclear translocation of Nrf2, suggesting that it was an additive effect. Tin protoporphyrin IX (SnPP), a representative HO-1 inhibitor, significantly impaired the cytoprotection of γCEHC and γT against the hydrogen peroxide-induced cytotoxicity. These results suggested that not only γT but also its metabolite, γCEHC, are a promising cytoprotective factor against oxidative stress-induced cytotoxicity and that the cytoprotective effect is attributable to the cooperation of both compounds.

Enzymatic Acrolein Production System and Its Impact on Human Cells.

Acrolein is an environmental toxicant and is also generated by microbial metabolism in the intestinal tract. Aqueous acrolein rapidly dissipates from standard human cell culture media with nondetectable levels after 8 h, hindering cell-based studies to understand its biological impacts. Thus, we developed an extracellular acrolein biosynthesis system to continuously produce acrolein compatible with human cell culture conditions. The approach uses spermine as a precursor, amine oxidase found in fetal calf serum, and catalase to remove the hydrogen peroxide byproduct. We confirmed amine oxidase activity of calf serum using a colorimetric assay and further tested the requirement for catalase in the system to mitigate hydrogen peroxide-induced cytotoxicity. We calibrated responses of human colon cells to this enzymatic acrolein production system by comparing transcriptional responses, DNA adduct formation and cytotoxicity responses to either this system or pure acrolein exposures in a human colon cell line. Several genes related to oxidative stress including HMOX1, and the colorectal cancer-related gene SEMA4A were upregulated similarly between the enzymatic acrolein production system or pure acrolein. The acrolein-DNA adduct γ-OH-Acr-dG increased in a dose-dependent manner with spermine in the enzymatic acrolein production system, producing a maximum of 1065 adducts per 108 nucleosides when 400 μM spermine was used. This biosynthetic production method provides a relevant model for controlled acrolein exposure in cultured human cells and overcomes current limitations due to its physical properties and limited availability.

Electrical Pulse Stimulation Protects C2C12 Myotubes against Hydrogen Peroxide-Induced Cytotoxicity via Nrf2/Antioxidant Pathway.

Skeletal muscle contraction evokes numerous biochemical alterations that underpin exercise benefits. This present study aimed to elucidate the mechanism for electrical pulse stimulation (EPS)-induced antioxidant adaptation in C2C12 myotubes. We found that EPS significantly upregulated Nrf2 and a broad array of downstream antioxidant enzymes involved in multiple antioxidant systems. These effects were completely abolished by pretreatment with a ROS scavenger, N-acetylcysteine. MitoSOX-Red, CM-H2DCFDA, and EPR spectroscopy revealed a significantly higher ROS level in mitochondria and cytosol in EPS cells compared to non-stimulated cells. Seahorse and Oroboros revealed that EPS significantly increased the maximal mitochondrial oxygen consumption rate, along with an upregulated protein expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor. A post-stimulation time-course experiment demonstrated that upregulated NQO1 and GSTA2 last at least 24 h following the cessation of EPS, whereas elevated ROS declines immediately. These findings suggest an antioxidant preconditioning effect in the EPS cells. A cell viability study suggested that the EPS cells displayed 11- and 36-fold higher survival rates compared to the control cells in response to 2 and 4 mM H2O2 treatment, respectively. In summary, we found that EPS upregulated a large group of antioxidant enzymes in C2C12 myotubes via a contraction-mitochondrial-ROS-Nrf2 pathway. This antioxidant adaptation protects cells against oxidative stress-associated cytotoxicity.

The protective effect of venlafaxine on hydrogen peroxide-induced cytotoxicity in C6 glioma cells

Aim: Neurodegeneration is the progressive loss and structural deterioration of neuronal cells. Hydrogen peroxide (H2O2) is formed by dismutation and causes oxidative stress in neuronal cells. Venlafaxine is a drug that increases both serotonin and noradrenaline in the synaptic gap.In this study, the effect of venlafaxine on H2O2-induced cytotoxicity in C6 cells was investigated. Methods: First of all, different doses of venlafaxine (25, 50, and 100 µM) were tried to find the appropriate dose in C6 glioma cells. Then, the effect of venlafaxine on H2O2-induced cytotoxicity in the cells was investigated. For this purpose, cell viability rate, proinflammatory markers IL-1β and TNF-α, and NO and iNOS levels were examined by ELISA kits. Results: H2O2-treated caused cytotoxicity in the C6 glioma cells; when venlafaxine 25, 50, and 100 μM doses were evaluated in terms of cell viability, it was observed that the 100 μM venlafaxine applied group significantly increased cell viability compared to the other groups. When we look at the levels of IL-1β and TNF-α, it is observed that there is an increase in the H2O2 applied group and a significant decrease in the venlafaxine (100 μM) applied group. It was observed that NO and iNOS levels increased in the H2O2 applied group compared to the other groups. It was observed that Venlafaxine treatment reduced the increased NO and iNOS levels caused by H2O2. Conclusion: The study results showed that venlafaxine may have a protective effect on H2O2-induced cytotoxicity in C6 glioma cells.

An anthocyanin-rich extract from Zea mays L. var. ceratina alleviates neuronal cell death caused by hydrogen peroxide-induced cytotoxicity in SH-SY5Y cells

The incidence of dementia is rising, with neuronal cell death from oxidative stress and apoptosis recognized as a significant contributor to its development. However, effective strategies to combat this condition are lacking, necessitating further investigation. This study aimed to assess the potential of an anthocyanin-rich extract from Zea mays L. var. ceratina (AZC) in alleviating neuronal cell death.Neurotoxicity was induced in SH-SY5Y cells using hydrogen peroxide (H2O2) at a concentration of 200 µM. Cells were pretreated with varying doses (31.25 and 62.5 µg/mL) of AZC. Cell viability was assessed using the MTT assay, and molecular mechanisms including reactive oxygen species (ROS) levels, antioxidant enzyme activities (catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)), malondialdehyde (MDA) levels for oxidative stress, and the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), cAMP response element-binding protein (CREB), and apoptotic factors (B-cell lymphoma 2 (Bcl-2), caspase 3) were explored.Results showed that AZC significantly improved cell viability, reduced ROS production and MDA levels, and downregulated caspase 3 expression. It enhanced CAT, SOD, and GSH-Px activities, activated ERK1/2 and CREB, and upregulated Bcl-2 expression. These findings support the neuroprotective effects of AZC, suggesting it activates ERK1/2, leading to CREB activation and subsequent upregulation of Bcl-2 expression while suppressing caspase 3. AZC may mitigate neuronal cell death by reducing ROS levels through enhanced scavenging enzyme activities.In conclusion, this study underscores the potential of AZC as a neuroprotective agent against neuronal cell death. However, further investigations including toxicity assessments, in vivo studies, and clinical trials are necessary to validate its benefits in neuroprotection.

Erythrocarpines I[sbnd]N, new limonoids from the barks of Chisocheton erythrocarpus and their neuroprotective effects against hydrogen peroxide in NG108–15 cells

A phytochemical study on the bark of Chisocheton erythrocarpus Hiern (Meliaceae) has led to the isolation of six new phragmalin-type limonoids named erythrocarpines I – N (1–6) along with one known limonoid, erythrocarpine F (7). Their structures were fully characterized by spectroscopic methods. The pre-treatment of NG108–15 cells with 1–5, 7 (2 h) demonstrated low to good protective effects against H2O2 exposure; 1 (83.77% ± 1.84 at 12.5 μM), 2 (69.07 ± 2.01 at 12.5 μM), 3 (80.38 ± 2.1 at 12.5 μM), 4 (62.33 ± 1.95 at 25 μM),5 (58.67 ± 1.85 at 50 μM) and 7 (66.07 ± 2.03 at 12.5 μM). Interestingly, 1 and 3 demonstrated comparable protective effects to positive control epigallocatechin gallate (EGCG) with similar cell viability capacity (≈ 80%), having achieved that at lower concentration (12.5 μM) than EGCG (50 μM). Collectively, the results suggested the promising use of 1 and 3 as potential neuroprotective agents against hydrogen peroxide-induced cytotoxicity in neuronal model.

The Cytotoxic Effect of Thymoquinone Enhance on HepG2 Cell Line due to Induction of Fenton Reaction by Hydrogen Peroxide: An In Vitro and In Silico Study

Thymoquinone (TQ) is a component derived from the volatile oil of Nigella sativa. Fenton reaction induction is a well-known strategy to prevent the growth of cancer cells which can stimulate by hydrogen peroxide. This study was designed to investigate the TQ effects on hydrogen peroxide-induced cytotoxicity. In this study, HepG2 cell survival, reactive oxygen species (ROS) production, cell membrane integrity, and changes of superoxide dismutase (SOD)/ catalase (CAT) activity were evaluated following incubation of HepG2 cells with 31 μM hydrogen peroxide and different concentrations of TQ (18.5, 37 and 75 μM). In addition, molecular docking studies on the interference of TQ with CAT/SOD enzymes were investigated. Our findings showed that TQ low concentration can increase the survival of HepG2 cells when exposed to hydrogen peroxide, and on the contrary, its high concentration can potentiate cytotoxicity induced by hydrogen peroxide. The TQ alongside hydrogen peroxide increased the production of ROS, which was related to increase CAT and SOD activity in the HepG2 cells. Molecular docking findings showed that TQ effects on the formation of free radicals were not related to its chemical interference with the structure of the SOD/CAT molecules. Fenton reaction induction may increase the effectiveness of TQ in preventing HepG2 cells proliferation.

The neuroprotective effects of the combined extract of mulberry fruit and mulberry leaf against hydrogen peroxide-induced cytotoxicity in SH-SY5Y Cells

The prevalence of dementia is increasing, and most of the causes are related to neuronal cell death. Unfortunately, no effective strategy is available for protecting against this condition. Based on the use of the synergistic concept together with the positive modulation effect of both mulberry fruit and mulberry leaf on dementia, we hypothesized that the combined extract of mulberry fruit and mulberry leaf (MFML) should mitigate neuronal cell death. Neuronal cell damage was induced in SH-SY5Y cells by exposure to hydrogen peroxide at a dose of 200 μM. SH-SY5Y cells were given MFML at doses of 62.5 and 125 μg/mL before induced cytotoxicity. Then, the cell viability was determined via MTT assay, and the possible underlying mechanisms were investigated via the alterations of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nuclear factor-κB (NF-κB), and tumor necrosis factor-alpha (TNF-α), together with apoptotic factors including (B-cell lymphoma 2) BCL2, Casapase-3 and Caspase-9. The results showed that MFML significantly enhanced cell viability. It also significantly decreased MDA level, NF-κB, TNF-α, Casapase-3, Caspase-9, but increased SOD, GSH-Px and BCL2. These data demonstrated the neuroprotective effect of MFML. The possible underlying mechanisms might occur partly via the improvement of the inappropriate apoptotic mechanisms via BCL2, Casapase-3 and Caspase-9 together with the decrease in neurodegeneration induced by the reduction of inflammation and oxidative stress. In conclusion, MFML is a potential neuroprotectant candidate against neuronal cell injury. However, toxicity, animal studies, and clinical trials are essential to confirm these benefits.

Epigallocatechin gallate protects the hydrogen peroxide-induced cytotoxicity and oxidative stress in tenocytes

Nonsteroidal anti-inflammatory drugs (NSAIDs) and periodic corticosteroid injections are frequently used to treat tendinopathy. However, prior research clinical studies revealed that the role of these medications in tendon rupture is still debatable. Research is needed to reduce the corticosteroids and NSAIDs associated with toxicity in the emerging field of tendinopathy. The present study was designed to explore the clinical efficacy of epigallocatechin gallate (EGCG) against H2O2-mediated inflammatory response in tenocytes derived from human bone marrow stem cells. To attenuate the H2O2-mediated toxicity, the tenocyte cells were supplemented with EGCG at a concentration of 3.125–100 µM. The study's findings explored that the supplementation of EGCG (100 µM) effectively suppressed the H202 mediated cytotoxicity and modulated the expression of apoptotic (Bax, caspase-3 and caspase-7) and inflammatory associated protein expression (Inducible nitric oxide synthase (iNOS), nuclear factor kappa B (NF-κB), and cyclooxygenase-2 (COX-2) along with collagen expression (collagen type I and COL3A1). It was confirmed by western blot and molecular docking analysis. This study suggests that the optimal concentration (100 µM) of EGCG has great potential for tendon healing and restoration in H2O2-mediated cytotoxicity.

Cycloartenyl Ferulate Is the Predominant Compound in Brown Rice Conferring Cytoprotective Potential against Oxidative Stress-Induced Cytotoxicity.

Since brown rice extract is a rich source of biologically active compounds, the present study is aimed to quantify the major compounds in brown rice and to compare their cytoprotective potential against oxidative stress. The content of the main hydrophobic compounds in brown rice followed the order of cycloartenyl ferulate (CAF) (89.00 ± 8.07 nmol/g) >> α-tocopherol (αT) (19.73 ± 2.28 nmol/g) > γ-tocotrienol (γT3) (18.24 ± 1.41 nmol/g) > α-tocotrienol (αT3) (16.02 ± 1.29 nmol/g) > γ-tocopherol (γT) (3.81 ± 0.40 nmol/g). However, the percent contribution of CAF to the radical scavenging activity of one gram of whole brown rice was similar to those of αT, αT3, and γT3 because of its weaker antioxidant activity. The CAF pretreatment displayed a significant cytoprotective effect on the hydrogen peroxide-induced cytotoxicity from 10 µM, which is lower than the minimal concentrations of αT and γT required for a significant protection. CAF also enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation coincided with the enhancement of the heme oxygenase-1 (HO-1) mRNA level. An HO-1 inhibitor, tin protoporphyrin IX (SnPP), significantly impaired the cytoprotection of CAF. The cytoprotective potential of CAF is attributable to its cycloartenyl moiety besides the ferulyl moiety. These results suggested that CAF is the predominant cytoprotector in brown rice against hydrogen peroxide-induced cytotoxicity.

The microbiota catabolites of quercetin glycosides concertedly enhance the resistance against acetaldehyde-induced oxidative stress

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (OPAC) are the predominant catabolites of quercetin glycosides, such as quercetin 4′-O-β-glucoside from the onion, produced by intestinal microbiota. Although each catabolite has been reported to protect the cells from acetaldehyde-induced cytotoxicity, the effect of their combination remains to be clarified. The purpose of this study was to determine whether the combination of DOPAC and OPAC enhances the resistance against the acetaldehyde-induced oxidative stress in the cultured hepatocytes. The pretreatment of the combination of DOPAC (5 μM) and OPAC (5 μM) showed significant protection against the acetaldehyde- and hydrogen peroxide-induced cytotoxicity, even though each compound at the same concentration did not. This combination also significantly inhibited the intracellular dichlorofluorescin diacetate-detectable reactive oxygen species (ROS) level, whereas the solo treatment did slightly, suggesting that reducing mechanisms of ROS or compounds that enhance ROS production are involved in the cytoprotective effect. The combinatory treatment significantly enhanced the gene expression of not only the aldehyde dehydrogenases (ALDHs), but also glutamate-cysteine ligase, catalytic subunit, the first rate-limiting enzyme of glutathione (GSH) synthesis. Accordingly, both the intracellular GSH level and the total ALDH activity were enhanced by DOPAC plus OPAC. Involvement of GSH in the cytoprotection as well as ALDH up-regulation by the combination was confirmed by the experiments using a GSH biosynthesis inhibitor, buthionine sulfoximine. Taken together, the present results suggested that the quercetin microbiota catabolites concertedly protect the cells from acetaldehyde through a pre-enhanced resistance against oxidative stress by the GSH-dependent up-regulation of ALDHs.

White rice ethanol extract is qualitatively, but not quantitatively, equivalent to that of brown rice as an antioxidant source.

The purpose of this study is to compare the potentials to exhibit biologically active antioxidant actions between white rice (WR) and brown rice (BR) in in vitro assays and a cellular model. The Trolox equivalent (TE) per 1 mg ethanol extract of WR for the 1,1-diphenyl-2-picrylhydrazyl assay was slightly higher than that of BR, whereas the TE per 1 g whole WR was much lower than that for BR. This tendency was very comparable to those for the oxygen radical absorbance capacity and total polyphenol content. Both of the ethanol extracts also similarly suppressed the hydrogen peroxide-induced cytotoxicity and enhanced the gene expression of drug-metabolizing enzymes. Based on the α-tocopherol quantity, its contribution to the cytoprotective effect of the rice extracts is very limited. Taken together, the ethanol extract of WR might be a qualitatively, but not quantitatively, equivalent source of antioxidative phytochemicals to that of BR.

Application of Ethanol Extracts From Alnus sibirica Fisch. ex Turcz in Hair Growth Promotion.

Alnus sibirica Fisch. ex Turcz (ASFT), belonging to the family of Betulaceae, grows naturally in Asia, Europe, and America. The aims of this study are determining the efficacy of various biomarkers related to hair loss, evaluated by extracting the branch with 60% alcohol, and purely separating diarylheptanoid oregonin, an indicator and active substance, from 60% alcohol extract of the tree. To determine the preventive effects on hair loss, we investigated the anti-oxidative and anti-apoptotic effects on hydrogen peroxide-induced cytotoxicity on human hair dermal papilla cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Western blotting analysis for proving of apoptosis-related marker alteration, respectively. Moreover, we examined the ameliorative effects of 60% alcohol extract of the tree and oregonin against changes of oxidative stress-induced cytokine and testosterone-induced dihydrotestosterone production as crucial pathways of the hair loss mechanism. These results suggest that 60% alcohol extract of the tree and oregonin were available as novel natural materials for maintaining hair health in mammals.

Correction to: TIP60 recruits SUV39H1 to chromatin to maintain heterochromatin genome stability and resist hydrogen peroxide-induced cytotoxicity

The original version of this article, published on November 6, 2020, contained a mistake.

TIP60 recruits SUV39H1 to chromatin to maintain heterochromatin genome stability and resist hydrogen peroxide-induced cytotoxicity

Tat interacting protein 60 (TIP60) is a histone acetyltransferase involved in chromatin remodeling and the DNA damage response. However, the role of TIP60 in maintaining genome stability is poorly understood. Here, we show that TIP60 can directly interact with suppressor of variegation 3–9 homologue 1 (SUV39H1), a methyltransferase that is responsible for histone H3 tri-methylation on Lys9 (H3K9me3). When we knocked down TIP60 or treated cells with hydrogen peroxide, a typical reactive oxygen species (ROS) generator that induces genome instability, SUV39H1 dissociated from chromatin but its acetylation levels remained unchanged. Consequently, H3K9me3 levels in the heterochromatin decreased, leading to a significant increase in the expression of satellite 2 (Sat2) and α-satellite (α-Sat), indicators of heterochromatin relaxation. Micrococcal nuclease sensitivity and colony formation assays further demonstrated that TIP60 knockdown or hydrogen peroxide treatment resulted in a relaxing of the heterochromatin and genome instability. Exogenous TIP60 could rescue the assembly of SUV39H1 on chromatin and ensure genome stability in response to hydrogen peroxide. This study is the first to describe a role for TIP60 in maintaining heterochromatin structure and genome stability by recruiting SUV39H1 to the chromatin upon oxidative stress, presenting TIP60 as a promising target to sensitize cancer cells to ROS-promoting therapies.

Hydrogen Peroxide Causes Cell Death via Increased Transcription of HOXB13 in Human Lung Epithelial A549 Cells.

Although homeobox protein B13 (HOXB13) is an oncogenic transcription factor, its role in stress response has rarely been examined. We previously reported that knockdown of HOXB13 reduces the cytotoxicity caused by various oxidative stress inducers. Here, we studied the role of HOXB13 in cytotoxicity caused by hydrogen peroxide in human lung epithelial A549 cells. The knockdown of HOXB13 reduced hydrogen peroxide-induced cytotoxicity; however, this phenomenon was largely absent in the presence of antioxidants (Trolox or N-acetyl cysteine (NAC)). This suggests that HOXB13 may be involved in the cytotoxicity caused by hydrogen peroxide via the production of reactive oxygen species (ROS). Hydrogen peroxide also increased both the mRNA and protein levels of HOXB13. However, these increases were rarely observed in the presence of a transcriptional inhibitor, which suggests that hydrogen peroxide increases protein levels via increased transcription of HOXB13. Furthermore, cell death occurred in A549 cells that highly expressed HOXB13. However, this cell death was mostly inhibited by treatment with antioxidants. Taken together, our findings indicate that HOXB13 may be a novel factor involved in the induction of oxidative stress, which causes cell death via intracellular ROS production.

UNG2 deacetylation confers cancer cell resistance to hydrogen peroxide-induced cytotoxicity

Cancer therapeutics produce reactive oxygen species (ROS) that damage the cancer genome and lead to cell death. However, cancer cells can resist ROS-induced cytotoxicity and survive. We show that nuclear-localized uracil-DNA N-glycosylase isoform 2 (UNG2) has a critical role in preventing ROS-induced DNA damage and enabling cancer-cell resistance. Under physiological conditions, UNG2 is targeted for rapid degradation via an interaction with the E3 ligase UHRF1. In response to ROS, however, UNG2 protein in cancer cells exhibits a remarkably extended half-life. Upon ROS exposure, UNG2 is deacetylated at lysine 78 by histone deacetylases, which prevents the UNG2–UHRF1 interaction. Accumulated UNG2 protein can thus excise the base damaged by ROS and enable the cell to survive these otherwise toxic conditions. Consequently, combining HDAC inhibitors (to permit UNG2 degradation) with genotoxic agents (to produce cytotoxic cellular levels of ROS) leads to a robust synergistic killing effect in cancer cells in vitro. Altogether, these data support the application of a novel approach to cancer treatment based on promoting UNG2 degradation by altering its acetylation status using an HDAC inhibitor.

Superior protective effects of in vitro propagated green garlic against hydrogen peroxide-induced cytotoxicity in human hepatoma cells

Garlic is a valuable source material for medicines due to its known antitumor, hypolipidaemic, antioxidant, and immunomodulatory effects. This study compares the protective effects of conventionally grown (CG) and in vitro propagated garlic (PG) against hydrogen peroxide-induced cytotoxicity in HepG2 cells and their antioxidant activity. Garlic used in this study was obtained by planting garlic cloves or by planting the transplants of PG directly in the field. At the end of the vegetation period, CG and PG were sampled and extracts prepared for the experiment. Compared to conventionally grown garlic bulbs, PG leafy part yielded significantly higher content of polyphenols, flavonoids and alliin, and also showed equal or higher antioxidant activity, measured by the cell viability test, GSH and ROS level. Moreover, PG can be produced in less time (shorter vegetation period) and with significantly less material (cloves). Significantly higher content of alliin, polyphenols, and flavonoids and significantly higher yield of plant biomass in PG has a great potential to become a new production model with improved garlic properties as a medicine material.

Circular RNA HIPK3 downregulation mediates hydrogen peroxide-induced cytotoxicity in human osteoblasts.

Hydrogen peroxide (H2O2) induces oxidative injury to human osteoblasts. The expression and potential function of circular RNA HIPK3 (circHIPK3) in H2O2-treated human osteoblasts were tested. We show that H2O2 significantly downregulated circHIPK3 in OB-6 cells and primary human osteoblasts. Furthermore, circHIPK3 levels were decreased in the necrotic femoral head tissues of dexamethasone-treated patients. In OB-6 osteoblastic cells and primary human osteoblasts, forced overexpression of circHIPK3 by a lentiviral construct alleviated H2O2-induced viability reduction, cell death and apoptosis. Contrarily, circHIPK3 silencing by targeted shRNA potentiated H2O2-induced cytotoxicity in OB-6 cells and primary human osteoblasts. Moreover, circHIPK3 downregulation by H2O2 induced miR-124 accumulation in OB-6 cells and primary human osteoblasts. On the contrary, miR-124 inhibition by transfection of the miR-124 inhibitor protected human osteoblasts from H2O2. Importantly, forced overexpression of miR-124 by transfection of the miR-124 mimic induced significant cytotoxicity in OB-6 cells and primary human osteoblasts. H2O2 downregulated miR-124’s targets, cyclin dependent kinase 6 and Rho-Associated Protein Kinase 1, in human osteoblasts. In conclusion circHIPK3 downregulation mediates H2O2-induced cytotoxicity in human osteoblasts.

Phytochemical Repurposing of Natural Molecule: Sabinene for Identification of Novel Therapeutic Benefits Using In Silico and In Vitro Approaches.

Repurposing of drugs/natural or synthetic chemicals is a promising approach to identify the new therapeutic indication/use and mode of action. In pharmaceuticals, this process is used to save the time and cost for the drug discovery process with reduced risk of failure. In the present studies, repurposing of a natural molecule: sabinene (major phytochemical in cardamom) was used to characterize the new biological activities using in silico as well as in vitro approaches. In silico similarity searching demonstrated that (+)-3-carene possessed the maximum structural similarity with sabinene. In vitro activities of (+)-3-carene were repurposed for sabinene based on similarity hypothesis (similar structures may have similar biological activities). In vitro studies demonstrated that sabinene is having antimicrobial activity and also showed concentration-dependent antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl scavenging assay. Sabinene treatment protected the yeast cells from hydrogen peroxide-induced cytotoxicity in 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, it was found that sabinene treatment decreased the generation of oxidative stress and also decreased the activities of antioxidant enzymes; glutathione S-transferase, catalase, and lipid peroxidase as compared with untreated yeast cells. Sabinene was also found to have angiostatic and antiangiogenic effects. These results were supported by molecular docking studies against antiangiogenic targets. Therefore, the results of these studies suggested that structurally similar molecules are having the same activity. The phytochemical repurposing using in silico similarity searching as well as in vitro approaches can also be applied for other phytochemicals whose activities are not/less known. Furthermore, this could also be useful in the novel lead/scaffold discovery and target fishing.