Cotransplantation of NSCs and ethyl stearate promotes synaptic plasticity in PD rats by Drd1/ERK/AP-1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine views kidney shortage as a significant contributor to the aetiology of Parkinson's disease (PD), a neurodegenerative condition that is closely linked to aging. In clinical, patients with Parkinson's disease are often treated with Testudinis Carapax et Plastrum (Plastrum Testudinis, PT), a traditional Chinese medication that tonifies the kidney. Previous research has demonstrated that ethyl stearate (PubChem CID: 8122), an active component of Plastrum Testudinis Extracted with ethyl acetate (PTE), may encourage neural stem cells (NSCs) development into dopaminergic (DAergic) neurons. However, the effectiveness and mechanism of cotransplantation of ethyl stearate and NSCs in treating PD model rats still require further investigation. AIM OF THE STUDY: PD is a neurodegenerative condition marked by the loss and degradation of dopaminergic neurons in the substantia nigra of the midbrain. Synaptic damage is also a critical pathology in PD. Because of their self-renewal, minimal immunogenicity, and capacity to differentiate into dopaminergic (DAergic) neurons, NSCs are a prospective treatment option for Parkinson's disease cell transplantation therapy. However, encouraging transplanted NSCs to differentiate into dopaminergic neurons and enhancing synaptic plasticity in vivo remains a significant challenge in improving the efficacy of NSCs transplantation for PD. This investigation seeks to examine the efficacy of cotransplantation of NSCs and ethyl stearate in PD model rats and its mechanism related to synaptic plasticity. MATERIALS AND METHODS: On 6-hydroxydopamine-induced PD model rats, we performed NSCs transplantation therapy and cotransplantation therapy involving ethyl stearate and NSCs. Rotating behavior induced by apomorphine (APO) and pole climbing tests were used to evaluate behavioral changes. Using a variety of methods, including Western blotting (WB), immunofluorescence analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qRT-PCR), we examined the function and potential molecular mechanisms of ethyl stearate in combined NSCs transplantation therapy. RESULTS: In the rat PD model, cotransplantation of ethyl stearate with NSCs dramatically reduced motor dysfunction, restored TH protein levels, and boosted dopamine levels in the striatum, according to our findings. Furthermore, the expression levels of SYN1 and PSD95, markers of synaptic plasticity, and BDNF, closely related to synaptic plasticity, were significantly increased. Cotransplantation with ethyl stearate and NSCs also increased the expression levels of Dopamine Receptor D1 (Drd1), an important receptor in the dopamine neural circuit, accompanied by an increase in MMP9 levels, ERK1/2 phosphorylation levels, and c-fos protein levels. CONCLUSIONS: According to the results of our investigation, cotransplantation of ethyl stearate and NSCs significantly improves the condition of PD model rats. We found that cotransplantation of ethyl stearate and NSCs may promote the expression of MMP9 by regulating the Drd1-ERK-AP-1 pathway, thus improving synaptic plasticity after NSCs transplantation. These findings provide new experimental support for the treatment of PD with the kidney tonifying Chinese medicine Plastrum Testudinis and suggest a potential therapeutic strategy for PD based on cotransplantation therapy.

Neural stem cells transplantation combined with ethyl stearate improve PD rats motor behavior by promoting NSCs migration and differentiation.

BACKGROUND: In recent years, the ability of neural stem cells (NSCs) transplantation to treat Parkinson's disease (PD) has attracted attention. However, it is still a challenge to promote the migration of NSCs to the lesion site and their directional differentiation into dopaminergic neurons in PD. C-C motif chemokine ligand 5 (CCL5) and C-C motif chemokine receptor 5 (CCR5) are expressed in the brain and are important regulators of cell migration. It has been reported that ethyl stearate (PubChem CID: 8122) has a protective effect in 6-OHDA-induced PD rats. METHODS: Parkinson's disease rats were injected with 6-hydroxydopamine (6-OHDA) into the right substantia nigra, and striatum followed by 8 µL of an NSC cell suspension containing 100 µM ethyl stearate and 8 × 105 cells in the right striatum. The effect of transplantation NSCs combined with ethyl stearate was assessed by evaluating apomorphine (APO)-induced turning behavior and performance in the pole test. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting (WB), and immunofluorescence staining were also performed. RESULTS: NSCs transplantation combined with ethyl stearate ameliorated the behavioral deficits of PD rats. PD rats that received transplantation NSCs combined with ethyl stearate exhibited increased expression of tyrosine hydroxylase (TH) and an increased number of green fluorescent protein (GFP)-positive cells. Furthermore, GFP-positive cells migrated into the substantia nigra and differentiated into dopaminergic neurons. The expression of CCL5 and CCR5 was significantly increased after transplantation NSCs combined with ethyl stearate. CONCLUSIONS: These findings suggest that NSCs transplantation combined with ethyl stearate can improve the motor behavioral performance of PD rats by promoting NSCs migration from the striatum to the substantia nigra via CCL5/CCR5 and promoting the differentiation of NSCs into dopaminergic neurons.

Linkage and Stereochemistry Characters of Phenolic Antioxidant Product Formation.

The present study developed a smart and novel strategy to elucidate the linkage and stereochemistry characters during phenolic antioxidant product formation. A series of phenolic isomers or analogues were treated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical, to create 16 antioxidant dimerization reactions in aqueous solution. The products were rapidly identified by ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass-spectrometry. Through a systematic function-structure relationship analysis of these reactions and theoretical calculations, it is concluded that the phenolic antioxidant product is formed via linear linkage or furanocyclic linkage. The linear linkage is fulfilled via a radical coupling and controlled by the O-O linkage exclusion, meta-linkage exclusion, and catechol-activated principles. However, when an exocyclic π-bond conjugates with the phenolic core and is affixed at the -OH para-position, the furanocyclic linkage may occur via a subsequent intramolecular Michael addition. The intramolecular addition always lacks Re-attack to show "α,ß diastereoselectivity." The α,ß diastereoselectivity is the stereochemistry character of furanocyclic linkage during phenolic antioxidant product formation. All these novel findings can benefit not only the field food science but also other fields as well.

Ferroptosis-Inhibitory Difference between Chebulagic Acid and Chebulinic Acid Indicates Beneficial Role of HHDP.

The search for a safe and effective inhibitor of ferroptosis, a recently described cell death pathway, has attracted increasing interest from scientists. Two hydrolyzable tannins, chebulagic acid and chebulinic acid, were selected for the study. Their optimized conformations were calculated using computational chemistry at the B3LYP-D3(BJ)/6-31G and B3LYP-D3(BJ)/6-311 + G(d,p) levels. The results suggested that (1) chebulagic acid presented a chair conformation, while chebulinic acid presented a skew-boat conformation; (2) the formation of chebulagic acid requires 762.1729 kcal/mol more molecular energy than chebulinic acid; and (3) the 3,6-HHDP (hexahydroxydiphenoyl) moiety was shown to be in an (R)- absolute stereoconfiguration. Subsequently, the ferroptosis inhibition of both tannins was determined using a erastin-treated bone marrow-derived mesenchymal stem cells (bmMSCs) model and compared to that of ferrostatin-1 (Fer-1). The relative inhibitory levels decreased in the following order: Fer-1 > chebulagic acid > chebulinic acid, as also revealed by the in vitro antioxidant assays. The UHPLC-ESI-Q-TOF-MS analysis suggested that, when treated with 16-(2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy free radicals, Fer-1 generated dimeric products, whereas the two acids did not. In conclusion, two hydrolyzable tannins, chebulagic acid and chebulinic acid, can act as natural ferroptosis inhibitors. Their ferroptosis inhibition is mediated by regular antioxidant pathways (ROS scavenging and iron chelation), rather than the redox-based catalytic recycling pathway exhibited by Fer-1. Through antioxidant pathways, the HHDP moiety in chebulagic acid enables ferroptosis-inhibitory action of hydrolyzable tannins.

Protective effect of plastrum testudinis extract on dopaminergic neurons in a Parkinson's disease model through DNMT1 nuclear translocation and SNCA's methylation.

The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.

Comparison of Ferroptosis-Inhibitory Mechanisms between Ferrostatin-1 and Dietary Stilbenes (Piceatannol and Astringin).

Synthetic arylamines and dietary phytophenolics could inhibit ferroptosis, a recently discovered regulated cell death process. However, no study indicates whether their inhibitory mechanisms are inherently different. Herein, the ferroptosis-inhibitory mechanisms of selected ferrostatin-1 (Fer-1) and two dietary stilbenes (piceatannol and astringin) were compared. Cellular assays suggested that the ferroptosis-inhibitory and electron-transfer potential levels decreased as follows: Fer-1 >> piceatannol > astringin; however, the hydrogen-donating potential had an order different from that observed by the antioxidant experiments and quantum chemistry calculations. Quantum calculations suggested that Fer-1 has a much lower ionization potential than the two stilbenes, and the aromatic N-atoms were surrounded by the largest electron clouds. By comparison, the C4'O-H groups in the two stilbenes exhibited the lowest bond disassociation enthalpies. Finally, the three were found to produce corresponding dimer peaks through ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis. In conclusion, Fer-1 mainly depends on the electron transfer of aromatic N-atoms to construct a redox recycle. However, piceatannol and astringin preferentially donate hydrogen atoms at the 4'-OH position to mediate the conventional antioxidant mechanism that inhibits ferroptosis, and to ultimately form dimers. These results suggest that dietary phytophenols may be safer ferroptosis inhibitors for balancing normal and ferroptotic cells than arylamines with high electron-transfer potential.

Gallic Acid Alleviates Gouty Arthritis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis Through Enhancing Nrf2 Signaling.

Gallic acid is an active phenolic acid widely distributed in plants, and there is compelling evidence to prove its anti-inflammatory effects. NLRP3 inflammasome dysregulation is closely linked to many inflammatory diseases. However, how gallic acid affects the NLRP3 inflammasome remains unclear. Therefore, in the present study, we investigated the mechanisms underlying the effects of gallic acid on the NLRP3 inflammasome and pyroptosis, as well as its effect on gouty arthritis in mice. The results showed that gallic acid inhibited lactate dehydrogenase (LDH) release and pyroptosis in lipopolysaccharide (LPS)-primed and ATP-, nigericin-, or monosodium urate (MSU) crystal-stimulated macrophages. Additionally, gallic acid blocked NLRP3 inflammasome activation and inhibited the subsequent activation of caspase-1 and secretion of IL-1ß. Gallic acid exerted its inhibitory effect by blocking NLRP3-NEK7 interaction and ASC oligomerization, thereby limiting inflammasome assembly. Moreover, gallic acid promoted the expression of nuclear factor E2-related factor 2 (Nrf2) and reduced the production of mitochondrial ROS (mtROS). Importantly, the inhibitory effect of gallic acid could be reversed by treatment with the Nrf2 inhibitor ML385. NRF2 siRNA also abolished the inhibitory effect of gallic acid on IL-1ß secretion. The results further showed that gallic acid could mitigate MSU-induced joint swelling and inhibit IL-1ß and caspase 1 (p20) production in mice. Moreover, gallic acid could moderate MSU-induced macrophages and neutrophils migration into joint synovitis. In summary, we found that gallic acid suppresses ROS generation, thereby limiting NLRP3 inflammasome activation and pyroptosis dependent on Nrf2 signaling, suggesting that gallic acid possesses therapeutic potential for the treatment of gouty arthritis.

Wedelolactone facilitates Ser/Thr phosphorylation of NLRP3 dependent on PKA signalling to block inflammasome activation and pyroptosis.

OBJECTIVES: Wedelolactone exhibits regulatory effects on some inflammatory diseases. However, the anti-inflammatory mechanism of wedelolactone has not been entirely unravelled. Therefore, the present study focuses on investigating the mechanism of wedelolactone on NLRP3 inflammasome in macrophages and its influence on MSU-induced inflammation. MATERIALS AND METHODS: BMDM, J774A.1 and PMA-differentiated THP-1 macrophages were primed with LPS and then stimulated with ATP or nigericin or MSU crystal in the presence or absence of wedelolactone. The cell lysates and supernatants were collected to detect NLRP3 inflammasome components such as NLRP3, ASC and caspase 1, as well as pyroptosis and IL-1ß production. In addition, the anti-inflammatory effects of wedelolactone on MSU-induced peritonitis and arthritis mice were also evaluated. RESULTS: We found that wedelolactone broadly inhibited NLRP3 inflammasome activation and pyroptosis and IL-1ß secretion. Wedelolactone also block ASC oligomerization and speck formation. The inhibitory effects of wedelolactone were abrogated by PKA inhibitor H89, which also attenuated wedelolactone-enhanced Ser/Thr phosphorylation of NLRP3 at PKA-specific sites. Importantly, wedelolactone could abate MSU-induced IL-1ß production and neutrophils migration into peritoneal cavity, and reduced caspase 1 (p20) and IL-1ß expression in the joint tissue of MSU-induced arthritis. CONCLUSION: Our results indicate that wedelolactone promotes the Ser/Thr phosphorylation of NLRP3 to inhibit inflammasome activation and pyroptosis partly through potentiating PKA signalling, thus identifying its potential use for treating MSU-induced peritonitis and gouty arthritis.

Inhibitory Effect and Mechanism of Action of Quercetin and Quercetin Diels-Alder anti-Dimer on Erastin-Induced Ferroptosis in Bone Marrow-Derived Mesenchymal Stem Cells.

In this study, the anti-ferroptosis effects of catecholic flavonol quercetin and its metabolite quercetin Diels-Alder anti-dimer (QDAD) were studied using an erastin-treated bone marrow-derived mesenchymal stem cell (bmMSCs) model. Quercetin exhibited higher anti-ferroptosis levels than QDAD, as indicated by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY), 2',7'-dichlorodihydrofluoroscein diacetate (H2DCFDA), lactate dehydrogenase (LDH) release, cell counting kit-8 (CCK-8), and flow cytometric assays. To understand the possible pathways involved, the reaction product of quercetin with the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH●) was measured using ultra-performance liquid-chromatography coupled with electrospray-ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS). Quercetin was found to produce the same clusters of molecular ion peaks and fragments as standard QDAD. Furthermore, the antioxidant effects of quercetin and QDAD were compared by determining their 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical-scavenging, Cu2+-reducing, Fe3+-reducing, lipid peroxidation-scavenging, and DPPH●-scavenging activities. Quercetin consistently showed lower IC50 values than QDAD. These findings indicate that quercetin and QDAD can protect bmMSCs from erastin-induced ferroptosis, possibly through the antioxidant pathway. The antioxidant pathway can convert quercetin into QDAD-an inferior ferroptosis-inhibitor and antioxidant. The weakening has highlighted a rule for predicting the relative anti-ferroptosis and antioxidant effects of catecholic flavonols and their Diels-Alder dimer metabolites.

Simultaneous Study of Anti-Ferroptosis and Antioxidant Mechanisms of Butein and (S)-Butin.

To elucidate the mechanism of anti-ferroptosis and examine structural optimization in natural phenolics, cellular and chemical assays were performed with 2'-hydroxy chalcone butein and dihydroflavone (S)-butin. C11-BODIPY staining and flow cytometric assays suggest that butein more effectively inhibits ferroptosis in erastin-treated bone marrow-derived mesenchymal stem cells than (S)-butin. Butein also exhibited higher antioxidant percentages than (S)-butin in five antioxidant assays: linoleic acid emulsion assay, Fe3+-reducing antioxidant power assay, Cu2+-reducing antioxidant power assay, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-trapping assay, and α,α-diphenyl-ß-picrylhydrazyl radical (DPPH•)-trapping assay. Their reaction products with DPPH• were further analyzed using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS). Butein and (S)-butin produced a butein 5,5-dimer (m/z 542, 271, 253, 225, 135, and 91) and a (S)-butin 5',5'-dimer (m/z 542, 389, 269, 253, and 151), respectively. Interestingly, butein forms a cross dimer with (S)-butin (m/z 542, 523, 433, 419, 415, 406, and 375). Therefore, we conclude that butein and (S)-butin exert anti-ferroptotic action via an antioxidant pathway (especially the hydrogen atom transfer pathway). Following this pathway, butein and (S)-butin yield both self-dimers and cross dimers. Butein displays superior antioxidant or anti-ferroptosis action to (S)-butin. This can be attributed the decrease in π-π conjugation in butein due to saturation of its α,ß-double bond and loss of its 2'-hydroxy group upon biocatalytical isomerization.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

Steric Effect of Antioxidant Diels-Alder-Type Adducts: A Comparison of Sanggenon C with Sanggenon D.

Sanggenons C and D are two Diels-Alder-type adducts from Chinese crude drug Sang-bai-pi. Structurally, both sanggenons construct stereoisomers. In the study, they were comparatively determined using four antioxidant assays, including ferric ion reducing antioxidant power (FRAP) assay, Cu2+-reducing assay, 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging assay, and 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid radical (ABTS•⁺)-scavenging assay. Their Fe2+-binding reactions were explored using UV-Vis spectra. Finally, their cytoprotective effects were evaluated using flow cytometry. In electron transfer (ET)-based FRAP and Cu2+-reducing assays, sanggenon D was found to have lower IC50 values than sanggenon C; however, in multi-pathway-based DPPH•-scavenging and ABTS•⁺-scavenging assays, sanggenon C possessed lower IC50 values than sanggenon D. UV-Vis spectra suggested that sanggenon C generated a bathochromic-shift (286 nm → 302 nm) and displayed stronger UV absorption than sanggenon D. In flow cytometry, sanggenon C and sanggenon D, respectively, exhibited 31.1% and 42.0% early apoptosis-percentages towards oxidative-stressed mesenchymal stem cells (MSCs). In conclusion, both sanggenons may undergo multiple pathways (e.g., ET and Fe2+-binding) to protect MSCs against oxidative stress. In the mere ET aspect, sanggenon D possesses a higher level than sanggenon C, while in multi-pathway-based radical-scavenging, Fe2+-binding, and cytoprotection aspects, sanggenon C is more active than sanggenon D. These discrepancies can conclusively be attributed to the steric effect.

Preconditioning Enhances the Therapeutic Effects of Mesenchymal Stem Cells on Colitis Through PGE2-Mediated T-Cell Modulation.

Mesenchymal stem cell (MSC)-based cell therapy has been demonstrated as a promising strategy in the treatment of inflammatory bowel disease (IBD), which is considered an immune disease. While the exact mechanisms underlying the therapeutic effect of MSCs are still unclear, MSCs display anti-inflammatory and immunomodulatory effects by interacting with various immunoregulatory cells. Our previous studies have shown that MSCs can be preconditioned and deconditioned with enhanced cell survival, differentiation and migration. In this study, we evaluated the effect of preconditioning on the immunoregulatory function of human umbilical cord-derived MSCs (hUCMSCs) and their therapeutic effect on treating IBD. Our results show that intraperitoneal administration of deconditioned hUCMSCs (De-hUCMSCs) reduces the disease activity index (DAI), histological colitis score and destruction of the epithelial barrier, and increases the body weight recovery more intensively than that of un-manipulated hUCMSCs. In addition, De-hUCMSCs but not hUCMSCs elicit anti-apoptotic effects via induction of the ERK pathway during the early stage of IBD development. In vitro co-culture studies indicate that De-hUCMSCs suppress T-cell proliferation and activation more markedly than hUCMSCs. Moreover, De-hUCMSCs block the induction of inflammatory cytokines such as tumor necrosis factor (TNF)α and interleukin (IL)-2, while promoting the secretion of the anti-inflammatory cytokine IL-10 in T-cells. Mechanically, we find that prostaglandin E2 (PGE2) secretion is significantly increased in De-hUCMSCs, the suppression of which dramatically abrogates the inhibitory effect of De-hUCMSCs on T-cell activation, implying that the crosstalk between De-hUCMSCs and T-cells is mediated by PGE2. Together, we have demonstrated that preconditioning enhances the immunosuppressive and therapeutic effects of hUCMSCs on treating IBD via increased secretion of PGE2.

Protective Mechanism of the Antioxidant Baicalein toward Hydroxyl Radical-Treated Bone Marrow-Derived Mesenchymal Stem Cells.

Our study explores the antioxidant and cytoprotective effects of baicalein and further discusses the possible mechanisms. A methyl thiazolyl tetrazolium (MTT) assay revealed that baicalein could considerably enhance the viability of hydroxyl radical-treated bone marrow-mesenchymal stem cells (bmMSCs) at 37-370 µM. The highest viability rate was 120.4%. In subsequent studies, baicalein was observed to effectively scavenge hydroxyl radical and PTIO• radicals, reducing Fe3+ and Cu2+ ions. In the Fe2+-chelating UV-vis spectra, mixing of baicalein with Fe2+ yielded two evident redshifts (275 → 279 nm and 324 → 352 nm) and a broad absorption peak (λmax ≈ 650 nm, ε = 1.6 × 10³ L mol-1·cm-1). Finally, we compared the Fe2+-chelating UV-vis spectra of baicalein and its analogues, including 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, catechol, pyrogallol, and chrysin. This analysis revealed that the 4-keto group of the C-ring played a role. The 5,6,7-trihydroxy-group (pyrogallol group) in the A-ring served as an auxochrome, enhancing the absorbance of the UV-vis spectra and deepening the color of the Fe2+-complex. We concluded that baicalein, as an effective hydroxyl radical-scavenger, can protect bmMSCs from hydroxyl radical-mediated oxidative stress. Its hydroxyl radical-scavenging effects are likely exerted via two pathways: direct scavenging of hydroxyl radicals, possibly through electron transfer, and indirect inhibition of hydroxyl radical generation via Fe2+ chelation through the 4-keto-5,6,7-trihydroxy groups.

Antioxidant and Cytoprotective Effects of the Di-O-Caffeoylquinic Acid Family: The Mechanism, Structure-Activity Relationship, and Conformational Effect.

In this study, a series of di-O-caffeoylquinic acids (di-COQs) were systematically investigated for their antioxidant and cytoprotective effects towards •OH-damaged bone marrow-derived mesenchymal stem cells (bmMSCs). Five di-COQs were measured using a set of antioxidant assays. The results show that adjacent 4,5-Di-O-caffeoylquinic acid (4,5-COQ) and 3,4-di-O-caffeoylquinic acid (3,4-COQ) always gave lower IC50 values than did non-adjacent di-COQs. In the Fe2+-chelating assay, 4,5-COQ and 3,4-COQ presented greater UV-Vis spectra and darker colors than did non-adjacent di-COQs. In the UPLC-ESI-MS/MS analysis, no corresponding radical adduct formation (RAF) peak was found in the reaction products of di-COQs with PTIO•. In the MTT assay, all di-COQs (especially 1,5-COQ, 1,3-COQ, and 4,5-COQ) dose-dependently increased the cellular viabilities of •OH-damaged bmMSCs. Based on this evidence, we conclude that the five antioxidant di-COQs can protect bmMSCs from •OH-induced damage. Their antioxidant mechanisms may include electron-transfer (ET), H⁺-transfer, and Fe2+-chelating, except for RAF. Two adjacent di-COQs (4,5-COQ and 3,4-COQ) always possessed a higher antioxidant ability than the non-adjacent di-COQs (1,3-COQ, 1,5-COQ, and 3,5-COQ) in chemical models. However, non-adjacent 1,3-COQ and 1,5-COQ exhibited a higher cytoprotective effect than did adjacent di-COQs. These differences can be attributed to the relative positions of two caffeoyl moieties and, ultimately, to the conformational effect from the cyclohexane skeleton.

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-Oxide (PTIO•) Radical Scavenging: A New and Simple Antioxidant Assay In Vitro.

Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent results. The study develops a new antioxidant assay using the 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•). After the investigation of various factors, the experimental protocol was briefly recommended as follows: PTIO• and the sample solution were added to phosphate buffer (pH 7.4, 50 mM), incubated at 37 °C for 2 h, and then spectrophotometrically measured at 557 nm. The validation test based on 20 pure compounds and 30 lyophilized aqueous extracts suggested that PTIO• scavenging had a good linear relationship, stability, and reproducibility. In the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, PTIO• was observed to give m/z 234 when encountering l-ascorbic acid. As an antioxidant assay, PTIO• scavenging possesses four advantages, i.e., oxygen-centered radical, physiological aqueous solution, simple and direct measurement, and less interference from the tested sample. It can also satisfactorily analyze the antioxidant structure-activity relationship. PTIO• scavenging has no stereospecificity and is at least involved in H+ transfer.

Role of the p-Coumaroyl Moiety in the Antioxidant and Cytoprotective Effects of Flavonoid Glycosides: Comparison of Astragalin and Tiliroside.

The aim of this study was to explore the role of p-coumaroyl in the antioxidant and cytoprotective effects of flavonoid glycosides. The antioxidant effects of astragalin and tiliroside were compared using ferric ion reducing antioxidant power, DPPH• scavenging, ABTS•⁺ scavenging, •O2- scavenging, and Fe2+-chelating assays. The results of these assays revealed that astragalin and tiliroside both exhibited dose-dependent activities; however, tiliroside exhibited lower IC50 values than astragalin. In the Fe2+-chelating assay, tiliroside gave a larger shoulder-peak at 510 nm than astragalin, and was also found to be darker in color. Both of these compounds were subsequently evaluated in a Fenton-induced mesenchymal stem cell (MSC) damaged assay, where tiliroside performed more effectively as a cytoprotective agent than astragalin. Tiliroside bearing a 6''-O-p-coumaroyl moiety exhibits higher antioxidant and cytoprotective effects than astragalin. The 6''-O-p-coumaroyl moiety of tiliroside not only enhances the possibility of electron-transfer and hydrogen-atom-transfer-based multi-pathways, but also enhances the likelihood of Fe-chelating. The p-coumaroylation of the 6"-OH position could therefore be regarded as a potential approach for improving the antioxidant and cytoprotective effects of flavonoid glycosides in MSC implantation therapy.

Lyophilized aqueous extracts of Mori Fructus and Mori Ramulus protect Mesenchymal stem cells from •OH-treated damage: bioassay and antioxidant mechanism.

BACKGROUND: Mori Fructus and Mori Ramulus are two traditional Chinese herbal medicines from mulberries. The present work explores their beneficial effects on •OH-treated mesenchymal stem cells (MSCs) and discusses possible mechanisms. METHODS: Lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR) were prepared and analyzed using HPLC. LAMF and LAMR (along with morin) were further investigated for their effects on •OH-treated MSCs using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The direct antioxidation mechanisms were studied using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO•)-scavenging, 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS+•)-scavenging and 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging, as well as Cu2+-reducing and Fe3+-reducing antioxidant power. Finally, the indirect antioxidant mechanism was investigated based on the UV-vis spectra of Fe2+-chelation. RESULTS: In each LAMF and LAMR, seven phytophenols were successfully measured by HPLC, including five flavonoids (morin, rutin, astragalin, isoquercitrin and luteolin) and two non-flavonoids (chlorogenic acid and maclurin). MTT assays revealed that LAMF, LAMR and morin could effectively increase the survival of •OH-treated MSCs at 10-100 µg/mL, and could effectively scavenge PTIO• (IC 50 6609.7 ± 756.6, 4286.9 ± 84.9 and 103.4 ± 0.9 µg/mL, respectively), DPPH• (IC 50 208.7 ± 3.0, 97.3 ± 3.1 and 8.2 ± 0.7 µg/mL, respectively) and ABTS+• (IC 50 73.5 ± 5.8, 34.4 ± 0.1 and 4.2 ± 0.2 µg/mL, respectively), and reduce Cu2+ (IC 50 212.5 ± 7.0, 123.2 ± 0.9 and 14.1 ± 0.04 µg/mL, respectively) & Fe3+ (IC 50 277.0 ± 3.1, 191.9 ± 5.2 and 5.0 ± 0.2 µg/mL, respectively). In the Fe2+-chelating assay, the five flavonoids produced much stronger shoulder-peaks than the two non-flavonoids within 420-850 nm. CONCLUSION: Mori Fructus and Mori Ramulus, can protect MSCs from •OH-induced damage. Such beneficial effects can mainly be attributed to the antioxidant action of phytophenols, which occurs via direct (ROS-scavenging) and indirect mechanism (Fe2+-chelating). The ROS-scavenging mechanism, however, include at least a H+-transfer and an electron-transfer (ET), and possibly includes a hydrogen-atom-transfer (HAT). In the Fe2+-chelating, flavonoids are more effective than non-flavonoids. This can be attributed to several adjacent planar chelating-sites between the 3-OH and 4-C = O, between the 4-C = O and 5-OH, or between the 3'-OH and 4'-OH in flavonoids. Such multiple-Fe2+-chelating reactions cause overlap in the UV-vis absorptions to deepen the complex color, enhance the peak strength, and form shoulder-peaks. By comparison, two non-flavonoids with catechol moiety produce only a weak single peak.

The mechanism of (+) taxifolin's protective antioxidant effect for •OH-treated bone marrow-derived mesenchymal stem cells.

The natural dihydroflavonol (+) taxifolin was investigated for its protective effect on Fenton reagent-treated bone marrow-derived mesenchymal stem cells (bmMSCs). Various antioxidant assays were used to determine the possible mechanism. These included •OH-scavenging, 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide radical-scavenging (PTIO•-scavenging), 1, 1-diphenyl-2-picryl-hydrazl radical-scavenging (DPPH•-scavenging), 2, 2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) radical-scavenging (ABTS+•-scavenging), Fe3+-reducing, and Cu2+-reducing assays. The Fe2+-binding reaction was also investigated using UV-Vis spectra. The results revealed that cell viability was fully restored, even increasing to 142.9 ± 9.3% after treatment with (+) taxifolin. In the antioxidant assays, (+) taxifolin was observed to efficiently scavenge •OH, DPPH• and ABTS+• radicals, and to increase the relative Cu2+- and Fe3+-reducing levels. In the PTIO•-scavenging assay, its IC50 values varied with pH. In the Fe2+-binding reaction, (+) taxifolin was found to yield a green solution with two UV-Vis absorbance peaks: λmax = 433 nm (ε =5.2 × 102 L mol-1 cm -1) and λmax = 721 nm (ε = 5.1 × 102 L mol-1 cm -1). These results indicate that (+) taxifolin can act as an effective •OH-scavenger, protecting bmMSCs from •OH-induced damage. Its •OH-scavenging action consists of direct and indirect antioxidant effects. Direct antioxidation occurs via multiple pathways, including ET, PCET or HAT. Indirect antioxidation involves binding to Fe2+.

Sarcandra glabra (Caoshanhu) protects mesenchymal stem cells from oxidative stress: a bioevaluation and mechanistic chemistry.

BACKGROUND: Sarcandra glabra (Caoshanhu) is a traditional Chinese herbal medicine used for treating various oxidative-stressed diseases. The present work evaluated its protective effect on mesenchymal stem cells (MSCs) from oxidative stress and then discussed possible mechanisms underlying this observation. METHODS: Ethanolic extract of S. glabra (ESG) was investigated by chemical methods for its content of total phenolics, rosmarinic acid, and astilbin. ESG, along with rosmarinic acid and astilbin, was investigated for the effect on the viability of Fenton-treated MSCs using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The observed cell protective effect was further explored by mechanistic chemistry using various antioxidant assays, including DNA protection, •OH-scavenging, •O2--scavenging, FRAP (ferric ion reducing antioxidant power), ABTS+•-scavenging, DPPH•-scavenging, and Fe2+-chelating assays. RESULTS: Analysis of ESG revealed a content of 46.31 ± 0.56 mg quercetin/g total phenolics, 0.78 ± 0.01 % rosmarinic acid, and 3.37 ± 0.01 % astilbin. Results from the MTT assay revealed that three compounds (rosmarinic acid>astilbin>ESG) could effectively increase the survival of Fenton-treated MSCs. Similarly, in •O2--scavenging, DPPH•-scavenging, and Fe2+-chelating assays, rosmarinic acid exhibited more activity than astilbin; while in FRAP, ABTS+•-scavenging assays, astilbin was stronger than rosmarinic acid. CONCLUSION: S. glabra can prevent MSCs from •OH-induced oxidative stress. Such protective effect can be attributed to its antioxidant ability and the presence of two kinds of phytophenols, i.e. caffeoyl derivatives and flavonoids. As the respective representatives of caffeoyl derivatives and flavonoids, rosmarinic acid and astilbin may exert the antioxidant action via direct ROS-scavenging and indirect ROS-scavenging (i.e. Fe2+-chelating). The direct ROS-scavenging ability involves hydrogen atom transfer (HAT) and/or electron transfer (ET) pathway. Astilbin engages the latter pathway more, which can be attributed to the larger planar conjugation in A/C fused rings. Rosmarinic acid, on the other hand, shows more HAT and Fe2+-chelating potential, which may be due to rosmarinic acid bearing one more catechol moiety whereas astilbin has steric-hindrance from 3-α-L-rhamnose and an H-bonding between 4,5 sites. The antioxidant features of rosmarinic acid can be generalized to other caffeoyl derivatives, while that of astilbin cannot be generalized to other flavonoids because of the difference in chemical structures.