Dual role of Nrf2/HO-1 pathway in Z-ligustilide-induced ferroptosis against AML cells.

BACKGROUND: The scarcity of drugs targeting AML cells poses a significant challenge in AML management. Z-Ligustilide (Z-LIG), a phthalide compound, shows promising pharmacological potential as a candidate for AML therapy. However, its precise selective mechanism remains unclear. PURPOSE: In order to assess the selective inducement effects of Z-LIG on ferroptosis in AML cells and explore the possible involvement of the Nrf2/HO-1 pathway in the regulation of ferroptosis. METHODS: Through in vitro cell proliferation and in vivo tumor growth tests, the evaluation of Z-LIG's anticancer activity was conducted. Ferroptosis was determined by the measurement of ROS and lipid peroxide levels using flow cytometry, as well as the observation of mitochondrial morphology. To analyze the iron-related factors, western blot analysis was employed. The up-regulation of the Nrf2/HO-1 axis was confirmed through various experimental techniques, including CRISPR/Cas9 gene knockout, fluorescent probe staining, and flow cytometry. The efficacy of Z-LIG in inducing ferroptosis was further validated in a xenograft nude mouse model. RESULTS: Our study revealed that Z-LIG specifically triggered lipid peroxidation-driven cell death in AML cells. Z-LIG downregulated the total protein and nuclear entrance levels of IRP2, resulting in upregulation of FTH1 and downregulation of TFR1. Z-LIG significantly increased the susceptibility to ferroptosis by upregulating ACSL4 levels and simultaneously suppressing the activity of GPX4. Notably, the Nrf2/HO-1 pathway displayed a twofold impact in the ferroptosis induced by Z-LIG. Mild activation suppressed ferroptosis, while excessive activation promoted it, mainly driven by ROS-induced labile iron pool (LIP) accumulation in AML cells, which was not observed in normal human cells. Additionally, Nrf2 knockout and HO-1 knockdown reversed iron imbalance and mitochondrial damage induced by Z-LIG in HL-60 cells. Z-LIG effectively inhibited the growth of AML xenografts in mice, and Nrf2 knockout partially weakened its antitumor effect by inhibiting ferroptosis. CONCLUSION: Our study presents biological proof indicating that the selective initiation of ferroptosis in leukemia cells is credited to the excessive activation of the Nrf2/HO-1 pathway triggered by Z-LIG.

Periplaneta Americana (L.) extract activates the ERK/CREB/BDNF pathway to promote post-stroke neuroregeneration and recovery of neurological functions in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Periplaneta americana (L.) (PA) has been used in traditional Chinese medicine for thousands of years for the effect of invigorating blood circulation and removing blood stasis. Modern pharmacological research shown that PA extract exhibits promising effects in promoting wound healing and regeneration, as well as in brain diseases such as Parkinson's disease (PD). However, whether it is effective for neuroregeneration and neurological function recovery after stroke still unknown. AIM OF THE STUDY: This study aims to investigate the potential effect of PA extract to promote brain remodeling through the activation of endogenous neurogenesis and angiogenesis, in addition, preliminary exploration of its regulatory mechanism. METHODS: Firstly, BrdU proliferation assay and immunofluorescence (IF) staining were used to evaluate the effect of PA extract on the neurogenesis and angiogenesis in vitro and in vivo. Subsequently, the effects of PA extract on brain injury in stroke rats were assessed by TTC and HE. While mNSS score, adhesive removal test, rota-rod test, and morris water maze test were used to assess the impact of PA extract on neurological function in post-stroke rats. Finally, the molecular mechanisms of PA extract regulation were explored by RNA-Seq and western blotting. RESULTS: The number of BrdU+ cells in C17.2 cells, NSCs and BMECs dramatically increased, as well as the expression of astrocyte marker protein GFAP and neuronal marker protein Tuj-1 in C17.2 and NSCs. Moreover, PA extract also increased the number of BrdU+DCX+, BrdU+GFAP+, BrdU+CD31+ cells in the SGZ area of transient middle cerebral artery occlusion model (tMCAO) rats. TTC and HE staining revealed that PA extract significantly reduced the infarction volume and ameliorated the pathological damage. Behavioral tests demonstrated that treatment with PA extract reduced the mNSS score and the time required to remove adhesive tape, while increasing the time spent on the rotarod. Additionally, in the morris water maze test, the frequency of crossing platform and the time spent in the platform quadrant increased. Finally, RNA-Seq and Western blot revealed that PA extract increased the expression of p-ERK, p-CREB and BDNF. Importantly, PA extract mediated proliferation and differentiation of C17.2 and NSCs reversed by the ERK inhibitor SCH772984 and the BDNF inhibitor ANA-12, respectively. CONCLUSION: Our study demonstrated that PA extract promoted neurogenesis and angiogenesis by activating the CREB/ERK signaling pathway and upregulating BDNF expression, thereby recovering neurological dysfunction in post-stroke.

Isobavachalcone inhibits acute myeloid leukemia: Potential role for ROS-dependent mitochondrial apoptosis and differentiation.

Isobavachalcone (IBC) has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. Herein, IBC exhibited significant inhibition on the cell viability, proliferation, and the colony formation ability of AML cells. Moreover, IBC induced mitochondrial apoptosis evidenced by reduced mitochondrial membrane potential, increased Bax level, decreased Bcl-2, Bcl-xL, and Mcl-1 levels, elevated cytochrome c level in the cytosol and increased cleavage of caspase-9, caspase-3, and PARP. Furthermore, IBC obviously promoted the differentiation of AML cells, accompanied by the increase of the phosphorylation of MEK and ERK and the C/EBPα expression as well as the C/EBPß LAP/LIP isoform ratio, which was significantly reversed by U0126, a specific inhibitor of MEK. Notably, IBC enhanced the intracellular ROS level. More importantly, IBC-induced apoptosis and differentiation of HL-60 cells were significantly mitigated by NAC. In addition, IBC also exhibited an obvious anti-AML effect in NOD/SCID mice with the engraftment of HL-60 cells. Together, our study suggests that the ROS-medicated signaling pathway is highly involved in IBC-induced apoptosis and differentiation of AML cells.

Z-Ligustilide Selectively Targets AML by Restoring Nuclear Receptors Nur77 and NOR-1-mediated Apoptosis and Differentiation.

BACKGROUND: Acute myeloid leukemia (AML) is a devastating hematologic malignancy with a high mortality. The nuclear receptors Nur77 and NOR-1 are commonly downregulated in human AML blasts and have emerged as key therapeutic targets for AML. METHODS: This study aimed to identify Z-ligustilide (Z-LIG), the main phthalide of Rhizoma Chuanxiong, as a potential agent that can selectively target AML. The anti-AML activity of Z-LIG was evaluated in vitro and in vivo, and the effect and underlying mechanisms of Z-LIG on the restoration of Nur77 and NOR-1 was determined. Moreover, the role of Nur77 and NOR-1 in the regulation of Z-LIG-induced apoptosis and differentiation of AML cells was explored. RESULTS: Z-LIG preferentially inhibited the viability of human AML cells, as well as suppressed the proliferation and colony formation ability. Notably, a concentration-dependent dual effect of Z-LIG was observed in AML cells: inducing apoptosis at relatively high concentrations (25 µM to 100 µM) and promoting differentiation at relatively low concentrations (10 µM and 25 µM). Importantly, Z-LIG restored Nur77 and NOR-1 expression in AML cells by increasing Ace-H3 (lys9/14) enrichment in their promoters. Meanwhile, Z-LIG enhanced the recruitment of p300 and reduced the recruitment of HDAC1, HDAC4/5/7, and MTA1 in the Nur77 promoter and enhanced the recruitment of p-CREB and reduced HDAC1 and HDAC3 in the NOR-1 promoter. Furthermore, Z-LIG-induced apoptosis was shown to be correlated with the mitochondria localization of Nur77/NOR-1 and subsequent Bcl-2 conformational change, converting Bcl-2 from a cyto-protective phenotype into a cyto-destructive phenotype. Z-LIG-promoted differentiation was found to be related to Nur77/NOR-1-mediated myeloid differentiation-associated transcription factors Jun B, c-Jun, and C/EBPß. Finally, silencing of Nur77 and NOR-1 attenuated anti-AML activity of Z-LIG in NOD/SCID mice. CONCLUSIONS: Our study suggests that Z-LIG may serve as a novel bifunctional agent for AML by restoring Nur77/NOR-1-mediated apoptosis and differentiation.

Biphasic Dose-Response of Components From Coptis chinensis on Feeding and Detoxification Enzymes of Spodoptera litura Larvae.

Due to long-term coevolution, secondary metabolites present in plants apparently function as chemical defense against insect feeding, while various detoxification enzymes in insects are adaptively induced as a prosurvival mechanism. Coptis chinensis, a medicinal plant used in traditional Chinese medicine for a thousand years, was found to be less prey to insects in our earlier field observations. Herein, 4 crude extracts obtained from sequential partition of aqueous extract of Rhizoma coptidis with petroleum ether, ethyl acetate, and n-butanol exhibited antifeedant activity against Spodoptera litura (Fabricius) larvae at high doses and inducing activity at low doses. Furthermore, a similar biphasic dose-response of the antifeedant activity against S litura larvae was also observed for jateorhizine, palmatine, and obakunone in Coptis chinensis. Notably, the enzyme activities of glutathione-S-transferase and carboxyl esterase in S litura larvae affected by the different components (jateorhizine, palmatine, obakunone, berberine, and coptisine) of C chinensis also showed a biphasic dose-response with an increasing trend at low doses and a decreasing trend at high doses. Together, our study suggests that the components of C chinensis may play a chemical defensive role against S litura larvae in a hormetic manner.

Ethyl acetate extract of Caesalpinia sappan L. inhibited acute myeloid leukemia via ROS-mediated apoptosis and differentiation.

BACKGROUND: The dried heartwood of Caesalpinia sappan L. is traditionally prescribed in the formula of traditional Chinese medicine (TCM) for the treatment of acute myeloid leukemia (AML), while nothing is yet known of the active fractions and the underlying mechanisms. PURPOSE: This study aims to investigate the effect of the ethyl acetate extract of the dried heartwood of Caesalpinia sappan L. (C-A-E) on induction of apoptosis and promotion of differentiation in vitro and anti-AML activity in vivo. STUDY DESIGN/METHODS: The aqueous extract was sequentially separated with solvents of increasing polarity and the active fraction was determined through the inhibition potency. The inhibition of the active fraction on cell viability, proliferation and colony formation was performed in different AML cells. Induction of apoptosis and the promotion of differentiation were further determined. Then, the level of the reactive oxygen species (ROS) and its potential role were assessed. Finally, anti-AML activity was evaluated in NOD/SCID mice. RESULTS: C-A-E exhibited the highest inhibition on the cell viability of HL-60 cells. Meanwhile, C-A-E significantly suppressed the proliferation and the colony formation ability of HL-60 and Kasumi-1 cells. Moreover, C-A-E significantly induced the apoptosis, which was partially reversed by Z-VAD-FMK. C-A-E also reduced the level of mitochondrial membrane potential, promoted the release of cytochrome C, decreased the Bcl-2/Bax ratio, and promoted the cleavage of caspase-9 and -3. In addition, Mdivi-1 (mitochondrial fission blocker) remarkably reduced the apoptosis caused by C-A-E. Meanwhile, C-A-E also induced the expression of Mff and Fis1 and increased the location of Drp1 in mitochondria. Furthermore, C-A-E obviously promoted the differentiation of AML cells characterized by the typic morphological changes, the increased NBT positive cells, as well as the increased CD11b and CD14 levels. Notably, C-A-E significantly enhanced the intracellular ROS level. Moreimportantly, C-A-E-mediated apoptosis and differentiation of HL-60 cells was significantly mitigated by NAC. Additionally, C-A-E also exhibited an obvious anti-AML effect in NOD/SCID mice with the injection of HL-60 cells. CONCLUSIONS: C-A-E exhibited an inhibitory effect on AML cells by inducing mitochondrial apoptosis and promoting differentiation, both of which were highly correlated to the activation of ROS.

Z-Ligustilide Exerted Hormetic Effect on Growth and Detoxification Enzymes of Spodoptera litura Larvae.

Plants have evolved a variety of phytochemicals to defense insect feeding, whereas insects have also evolved diverse detoxification enzymes, which are adaptively induced as a prosurvival mechanism. Herein, Z-ligustilide in Ligusticum chuanxiong Hort. was found to exhibit a similar trend in the accumulation from December to May as the occurrence of Spodoptera litura (Fabricius) larvae. Importantly, S. litura larvae feeding enhanced Z-ligustilide level in the stem and leaf (p < 0.01). Moreover, Z-ligustilide ranging from 1 to 5 mg·g-1 exhibited remarkable larvicidal activity, antifeedant activity, and growth inhibition against S. litura larvae. The LC50 values of larvicidal activity for phthalides in L. chuanxiong were compared as follows: Z-ligustilide > levistilide A > senkyunolide A > 3-butylidenephthalide > senkyunolide I, implicating the critical role of conjugated structure. Notably, there was a biphasic dose response for glutathione S-transferase (GST), cytochrome P450 (CYP) 450, Acetylcholinesterase (AChE), and Carboxylesterase (CarE) activities and GSTs1, cytochrome P450 (CYP) 4S9, and CYP4M14 mRNA expression. Particularly, low dose (0.1 mg·g-1) of Z-ligustilide conferred the resistance of S. litura larvae against chlorpyrifos (p < 0.05). Together, our data suggest that Z-ligustilide may function in a hormetic way in the chemical defense of L. chuanxiong against S. litura larvae.

Cellular stress response mechanisms as therapeutic targets of ginsenosides.

Ginseng, one of the most widely used traditional herbal medicines and dietary supplements, has historically been recognized as a tonic herb and adaptogen that can enhance the body's tolerance to various adversities. Ginsenosides are a diverse group of steroidal saponins that comprise the major secondary metabolites of ginseng and are responsible for its multiple pharmacological effects. Emerging evidence suggests that hormetic phytochemicals produced by environmentally stressed plants can activate the moderate cellular stress response mechanisms at a subtoxic level in humans, which may enhance tolerance against severe dysfunction or disease. In this review, we initially describe the role of ginsenosides in the chemical defense of plants from the genus Panax suffering from biotic and abiotic stress. Next, we summarize the diverse evolutionarily conserved cellular stress response pathways regulated by ginsenosides and the subsequent stress tolerance against various dysfunctions or diseases. Finally, the structure-activity relationship involved in the effect of ginsenosides is also analyzed. The evidence presented in this review implicates that ginseng as "the King of all herbs" could be regarded as a well-characterized example of the critical role of cellular stress response mechanisms in understanding the health benefits provided by herbal medicines from an evolutionary and ecological perspective.

Z-ligustilide restores tamoxifen sensitivity of ERa negative breast cancer cells by reversing MTA1/IFI16/HDACs complex mediated epigenetic repression of ERa.

Emerging evidence indicates epigenetic modification represses estrogen receptor α (ERα) and contributes to the resistance to tamoxifen in aggressive ERα-negative (ERα-) breast cancer. Z-ligustilide is a major compound in Radix Angelica sinensis, an herb from traditional Chinese medicine (TCM) most frequently prescribed for breast cancer. However, the role of Z-ligustilide in ERα- breast cancer and epigenetic modification remains largely unknown. Herein we showed, for the first time, that Z-ligustilide restored the growth inhibition of tamoxifen on ERα- breast cancer cells. Apoptosis and S and G2/M phases cell cycle arrest were induced by combinatorial Z-ligustilide and tamoxifen. Importantly, Z-ligustilide reactivated the ERα expression and transcriptional activity, which is proved to be indispensable for restoring the sensitivity to tamoxifen. Interestingly, Z-ligustilide increased Ace-H3 (lys9/14) enrichment in the ERα promoter. Moreover, Z-ligustilide dramatically reduced the enrichment of metastasis-associated protein 1 (MTA1) as well as IFN-γ-inducible protein 16 (IFI16) and histone deacetylases (HDACs) onto the ERα promoter. Meanwhile, Z-ligustilide downregulated MTA1, IFI16 and HDACs, which caused destabilization of the corepressor complex. Collectively, our study not only highlights Z-ligustilide as a novel epigenetic modulator, but also opens new possibilities from TCM for treating aggressive tamoxifen-resistant breast cancer.

Intranasal Pretreatment with Z-Ligustilide, the Main Volatile Component of Rhizoma Chuanxiong, Confers Prophylaxis against Cerebral Ischemia via Nrf2 and HSP70 Signaling Pathways.

Z-Ligustilide (Z-LIG) is a major component in Rhizoma Chuanxiong, which has been traditionally used as a health food supplement for the prevention of cerebrovascular disease in China. This study investigates the ability of intranasal Z-LIG pretreatment to enhance protection against neuronal damage in rats with middle cerebral artery occlusion (MCAO) and the role of cellular stress response mechanisms Nrf2 and HSP70. Z-LIG significantly mitigated infarct volume, neurological dysfunction, blood-brain barrier disruption, and brain edema (p < 0.01). Moreover, Z-LIG prevented the loss of collagen IV, occludin, and ZO-1 (p < 0.05) and decreased MMP-2 and -9 levels (p < 0.01). Meanwhile, Z-LIG up-regulated NQO1 and HSP70. Notably, blockage of Nrf2-driven transcription or down-regulation of HSP70 remarkably attenuated the preventive effect of Z-LIG (p < 0.05). Together, intranasal delivery of Z-LIG enhanced protection against ischemic injury via Nrf2 and HSP70 signaling pathways and has prophylactic potential in the population at high risk of stroke.

Anti-platelet activity of panaxatriol saponins is mediated by suppression of intracellular calcium mobilization and ERK2/p38 activation.

BACKGROUND: Increased platelet aggregation is implicated in the pathogenesis of ischemic stroke and anti-platelet strategy may contribute to its therapy. Panaxatriol saponin (PTS), the main components extracted from Panax notoginseng, has been shown to be efficacious in the prevention and treatment of ischemic stroke in China. The aim of this study is to determine the anti-platelet activity and explore the underlying mechanisms. METHODS: Inhibitory effect of PTS and its main ginsenosides on agonists-induced platelet aggregation was determined using rabbit or human platelets. Intracellular Ca(2+) concentration ([Ca(2+)]i) mobilization was detected with fura-2/AM probe. MAPKs phosphorylation was determined by Western blotting. RESULTS: Our results showed PTS inhibited the rabbit platelet aggregation induced by various agonists (collagen, thrombin and ADP). The three main ginsenosides (Rg1, Re and R1) existing in PTS also showed anti-platelet activity, while their combination exhibited no synergistic effect on rabbit platelet aggregation. Further study demonstrated that PTS and its main ginsenosides also exhibited inhibitory effect on human platelet aggregation. Mechanism study demonstrated that pre-treatment with PTS inhibited the agonists-induced intracellular calcium mobilization. Moreover, PTS significantly suppressed the activation of both ERK2 and p38 by the agonists via reducing the phosphorylation of ERK2 and p38. CONCLUSION: We proved that PTS is effective in anti-platelet aggregation, which may, at least in part, be related to the suppression of intracellular calcium mobilization and ERK2/p38 activation. This study may provide one reasonable explanation for the efficacy of PTS on the prevention and treatment of ischemic stroke.

Curcumol Suppresses Breast Cancer Cell Metastasis by Inhibiting MMP-9 Via JNK1/2 and Akt-Dependent NF-κB Signaling Pathways.

Curcumolhas been reported to possess antitumor activity. However, its effect and mechanisms against tumor metastasis are still unclear. This study is to investigate the inhibitory effect of curcumol on breast cancer cell metastasis and elucidate the underlying molecular mechanisms. Our results showed that noncytotoxicity was caused by curcumol within 10 to 40 µg/mL in MDA-MB-231 and 4T1 cells for 24 hours, whereas sustained treatment with curcumol for 14 days significantly suppressed the clonogenic activity of cells. Importantly, curcumol at noncytotoxic concentrations suppressed the migration ability of both MDA-MB-231 and 4T1 cells. Moreover, curcumol suppressed the migration and invasion of MDA-MB-231 cells in the Boyden chamber migration and invasion assay and inhibited the adhesion of MDA-MB-231 cells onto the matrigel. Further investigations revealed that curcumol decreased the enzyme activity and protein expression of matrix metalloproteinase (MMP-9) in MDA-MB-231 cells. Moreover, curcumol inhibited the activation of c-Jun N-terminal kinase (JNK) 1/2 and Akt (Ser473). Meanwhile, it also inhibited the nuclear translocation and transcriptional activity of nuclear factor κB (NF-κB). Furthermore, JNK inhibitor SP600125 and Akt (Ser473) inhibitor LY294002 enhanced the inhibition of curcumol on NF-κB p65 nuclear translocation. Finally, supplementation with SP600125, LY294002, or NF-κB inhibitor Ammonium pyrrolidinedithiocarbamate (PDTC) significantly enhanced the inhibitory effect of curcumol on MMP-9 expression and cell migration, invasion, and adhesion in MDA-MB-231 cells. Our findings provide evidence for the suppression of breast cancer cell metastasis by curcumol and suggest that the inhibition of MMP-9 via JNK1/2 and Akt (Ser473)-dependent NF-κB signaling pathways may be the underlying mechanisms.

Volatile Oil of Acori Graminei Rhizoma-Induced Apoptosis and Autophagy are dependent on p53 Status in Human Glioma Cells.

Acori Graminei Rhizoma is well known for the beneficial effects on CNS disorders in traditional medicine. Though it is frequently prescribed in formulations for brain tumors, the anti-glioma effect has not been examined. We used volatile oil of Acori Graminei Rhizoma (VOA) and human glioblastoma multiforme (GBM) cells in this study. We found that VOA exhibited greater growth suppression in p53 wild-type cells than p53 mutant cells and very low effect on fibroblasts and human glial HEB cells. Apoptosis was triggered by VOA with a caspase-dependent way in p53 wild-type A172 cells, while a caspase-independent way in p53 mutant U251 cells. Meanwhile, both A172 and U251 cells treated by VOA displayed autophagic features. Furthermore, p53 decrease was observed along with VOA-induced apoptosis and autophagy in A172 cells. VOA-induced autophagy was mediated through a p53/AMPK/mTOR signaling pathway in A172 cells, while an mTOR-independent signaling pathway in U251 cells. Finally, blockage of autophagy potentiated the proapoptotic effect in both A172 and U251 cells, indicating a protective role of autophagy in VOA-induced cell death. Together, VOA exhibited anti-tumor activity in human GBM cells and induced apoptotic cell death and protective autophagy, which is cell type specific and dependent on p53 status.

Proteomic analysis of ß-asarone induced cytotoxicity in human glioblastoma U251 cells.

Though rhizoma acori graminei (RAG) is frequently prescribed in formulations for brain tumor in traditional Chinese medicine, the potential mechanisms are still unclear. The aim of this study is to determine the effect of ß-asarone, a major component in the volatile oil of RAG, against brain tumor and elucidate the underlying molecular mechanisms. The results showed that ß-asarone significantly inhibited the cell viability of human glioblastoma U251 cells. Moreover, YO-PRO-1/PI staining revealed that cells treated with ß-asarone underwent apoptotic and necrotic death. Then, the two-dimensional gel electrophoresis (2-DE)-based proteomics was applied to investigate the different protein profiles of U251 cells treated with vehicle or ß-asarone. Sixteen proteins affected by ß-asarone were successfully identified by MALDI-TOF/TOF mass spectrometry. Gene ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. Importantly, four proteins (heterogeneous nuclear ribonucleoprotein H1 (H), isoform CRA_b, heterogeneous nuclear ribonucleoprotein A2/B1, isoform CRA_a, ubiquitin carboxyl-terminal hydrolase isozyme L1 and cathepsin D) acting as either oncoproteins or tumor suppressors draw our special attention. Finally, the effect of ß-asarone on these four genes was confirmed at transcriptional level by semi-quantitative RT-PCR. Collectively, a variety of proteins affected by ß-asarone were identified by 2-DE coupled with MALDI-TOF/TOF MS/MS analysis. Four potential protein targets were proposed, which will enable a better understanding of the anti-tumor activity of ß-asarone.

Chinese herbal medicine formula tao hong si wu decoction protects against cerebral ischemia-reperfusion injury via PI3K/Akt and the Nrf2 signaling pathway.

The Chinese herbal medicine formula Tao Hong Si Wu decoction (THSWD) is traditionally used for the prevention and treatment of ischemic stroke. Transcription factor NF-E2-related factor 2 (Nrf2) regulates a battery of phase II enzymes and is known as the major mechanism of cellular defense against oxidative stress. The present study aimed to explore the potential effect of THSWD on the Nrf2 signaling pathway and the consequent effect during cerebral ischemia-reperfusion (I/R) injury. We found that THSWD reduced infarct volume and improved neurological function in a rat stroke model induced by middle cerebral artery occlusion (MCAO). Additionally, heme oxygenase 1 (HO-1), a key endogenous antioxidant enzyme regulated by Nrf2, was significantly further induced by THSWD in this in vivo model. In neuronal-like PC12 cells, THSWD remarkably up-regulated HO-1 expression and promoted Nrf2 nuclear translocation. Furthermore, phosphatidylinositol 3-kinase (PI3K)/Akt kinase was found to be involved in the upstream of Nrf2 regulation. In an in vitro oxygen-glucose deprivation/reperfusion (OGD-Rep) model, THSWD treatment significantly reduced cell death induced by OGD-Rep insult. Importantly, the protective action was attenuated while PI3K activity was inhibited by a specific inhibitor, LY294002, and the Nrf2 signaling pathway was blocked by antioxidant response element (ARE) decoy oligonucleotides. Collectively, these results demonstrated that THSWD exhibited notable neuroprotective properties in vitro and in vivo and activation of PI3K/Akt and the Nrf2 signaling pathway may be, at least in part, responsible for the protection. This study provides a better understanding of the molecular mechanism underlying the traditional use of the Chinese herbal medicine formula THSWD.

Proteomic Identification of Nrf2-Mediated Phase II Enzymes Critical for Protection of Tao Hong Si Wu Decoction against Oxygen Glucose Deprivation Injury in PC12 Cells.

Chinese herbal medicine formula Tao Hong Si Wu decoction (THSWD) is traditionally used in China for cerebrovascular diseases. However, the molecular mechanisms of THSWD associated with the cerebral ischemia reperfusion injury are largely unknown. The current study applied the two-dimensional gel electrophoresis-based proteomics to investigate the different protein profiles in PC12 cells with and without the treatment of THSWD. Twenty-six proteins affected by THSWD were identified by MALDI-TOF mass spectrometry. Gene ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. In particular, six of them were found to be phase II antioxidant enzymes, which were regulated by NF-E2-related factor-2 (Nrf2). Quantitative PCR further confirmed a dose-dependent induction of the six phase II enzymes by THSWD at the transcription level. Moreover, the individual ingredients of THSWD were discovered to synergistically contribute to the induction of phase II enzymes. Importantly, THSWD's protection against oxygen-glucose deprivation-reperfusion (OGD-Rep) induced cell death was significantly attenuated by antioxidant response element (ARE) decoy oligonucleotides, suggesting the protection of THSWD may be likely regulated at least in part by Nrf2-mediated phase II enzymes. Thus, our data will help to elucidate the molecular mechanisms underlying the neuroprotective effect of THSWD.

[Xenohormesis: understanding biological effects of traditional Chinese medicine from an evolutionary and ecological perspective].

Xenohormesis explains how bioactive secondary metabolites produced by environmentally stressed plants can confer stress resistance and survival benefits to animals that consume them. This principle holds that animals retain the ability to sense these stress-induced signaling molecules under nature selective pressure, which will activate the evolutionarily conserved cellular stress response and subsequently enhance their adaptation to adversity. In this review, we have introduced the concept and mechanisms of xenohormesis, analyzed and summarized the xenohormesis relationship between plants and insects or human beings. Based on these, we have elucidated the traditional Chinese medicine (TCM) xenohormesis with ginseng as an example, proposed that we could understand the essence of TCM biological effects from an evolutionary and ecological perspective with the help of xenohormesis principle and also pointed out it has very important value in the modernization of TCM.

HPLC-APCI-MS/MS method for the determination of catalpol in rat plasma and cerebrospinal fluid: application to an in vivo pharmacokinetic study.

Catalpol is a Chinese herb ingredient with potential for the treatment of neurodegenerative disorders. A high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method was developed and validated to investigate the pharmacokinetics and biodistribution of catalpol in both the plasma and cerebrospinal fluid (CSF) of rats. Methanol was used to precipitate the protein from the biosamples, and the supernatant was collected for the assay. A Diamonsil C18 column (150 mm × 4.6 mm, 5 µm) with a mobile phase of methanol-ammonium acetate (20 mM) (50:50, v/v), a 0.6 mL/min flow rate and a total run time of 3 min was used to separate catalpol and aucubin (internal standard, IS). A triple-quadrupole tandem mass spectrometer with an atmospheric pressure chemical ionization (APCI) source in the positive ion mode was operated in the multiple reaction-monitoring (MRM) mode to determine the concentration of catalpol at m/z 380.0-165.0 and that of IS at m/z 364.0-148.9. The linear range was 10-50000 ng/mL for plasma samples and 20-5000 ng/mL for CSF samples. At both the lower limit of quantification (LLOQ) and three levels QC (quality control) concentrations, the RSD was less than 12.9%, and the bias was -10.0% to 7.1%. The extraction recoveries for catalpol ranged from 72.9% to 109.5% from both rat plasma and CSF. The catalpol was administered to rats in 6 mg/kg doses via intravenous (i.v.) injection, and the pharmacokinetics and biodistribution studies were performed in both plasma and CSF. The results demonstrated that catalpol could be transported into the CSF via the AUC(CSF)/AUC(plasma) of 5.8% with a half-life (t(1/2)) of 1.5 h. Overall, the established HPLC-APCI-MS/MS method is rapid and sensitive, and has been successfully used to quantify catalpol in both plasma and CSF.

Proteomic characterization of the cellular response to chemopreventive triterpenoid astragaloside IV in human hepatocellular carcinoma cell line HepG2.

Triterpenoids are implicated in the chemoprevention of various cancers. Current challenge is to define the molecular mechanism underlying the chemopreventive activity of triterpenoids. This study was designed to characterize the intracellular proteins regulated by astragaloside IV, the major active triterpenoid in Radix Astragali. Upon the treatment with astragaloside IV, human hepatocellular carcinoma HepG2 cells were evaluated for the colonogenic survival and anchorage-independent growth. The cellular proteins of treated and untreated cells were resolved by 2-D polyacrylamide gel electrophoresis. The protein spots mostly altered by drug treatment were identified by mass spectrometry and subsequently verified by Western blotting using specific antibodies and RT-PCR technique using specific DNA primers. We found that astragaloside IV attenuated the colonogenic survival and anchorage-independent growth of cancer cells. Based on the proteomic profiles, top 14 upregulated and 13 down-regulated protein spots were subjected to mass spectrometric analysis. As an example, Vav3.1 belongs to the oncogene Vav family, which is implicated in tumorigenesis. Vav3.1 expression was down-regulated by astragaloside IV in a dose- and time-dependent manner. Down-regulation of Vav3.1 was highly correlated with the suppression of cell malignant transform. Thus, astragaloside IV may elicit anticancer activity via down-regulating the expression of oncogenes such as Vav3.1.

Senkyunolides reduce hydrogen peroxide-induced oxidative damage in human liver HepG2 cells via induction of heme oxygenase-1.

Rhizoma Chuanxiong is widely used as folk medicine to treat the diseases caused by oxidative stress and inflammation. To delineate the underlying molecular mechanisms, we recently found that Rhizoma Chuanxiong extract significantly induced heme oxygenase-1 (HO-1), an enzyme that degrades intracellular heme into three bioactive products: biliverdin, carbon monoxide and free iron. The anti-inflammatory, antiapoptotic and antiproliferative actions of these products highlight HO-1 as a key endogenous antioxidant and cytoprotective gene. This study was designed to further characterize HO-1 induction of Rhizoma Chuanxiong through bioactivity-guided fractionation. All isolated fractions were assayed for HO-1 induction in human HepG2 cell line at mRNA and protein levels. Based on chromatographic profiling, nuclear magnetic resonance (NMR) and mass spectrometric analysis, the active compounds were identified as senkyunolide-H and its stereoisomer senkyunolide-I. Both senkyunolide isomers inhibited the formation of reactive oxygen species and lipid peroxidation and enhanced the cellular resistance to hydrogen peroxide-induced oxidative damage. Notably, heme oxygenase inhibitor tin protoporphyrin IX (SnPP) significantly suppressed the antioxidant activity of senkyunolide stereoisomers. Thus, this study demonstrated that senkyunolide-H and -I attenuated oxidative damage via activation of HO-1 pathway.