Hongyi Qi

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.

Protective HSP70 Induction by Z-Ligustilide against Oxygen–Glucose Deprivation Injury via Activation of the MAPK Pathway but Not of HSF1

Heat-shock protein 70 (HSP70) is known to function as a protective molecular chaperone that is massively induced in response to misfolded proteins following cerebral ischemia. The objective of this study was to characterize HSP70 induction by Z-ligustilide and explore its potential role in protection against cerebral ischemia-reperfusion injury. Our results demonstrated that the intranasal administration of Z-ligustilide reduced infarct volume and improved neurological function in a rat stroke model. Meanwhile, Z-ligustilide enhanced the cell viability of PC12 cells insulted by oxygen-glucose deprivation-reoxygenation (OGD-Reoxy) and decreased apoptotic and necrotic cell death. Importantly, Z-ligustilide induced HSP70 expression both in vitro and in vivo. Although heat-shock factor 1 (HSF1) nuclear translocation was promoted by Z-ligustilide, HSP70-based heat-shock element (HSE)-binding luciferase activity was not activated, and HSP70 expression responsive to Z-ligustilide was not attenuated by HSE decoy oligonucleotides. However, Z-ligustilide significantly activated the phosphorylation of mitogen-activated protein kinases (MAPKs). Further inhibition of MAPK activity by specific inhibitors attenuated HSP70 induction by Z-ligustilide. Meanwhile, downregulation of HSP70 using KNK437, an HSP70 synthesis inhibitor, or small hairpin RNA (shRNA) significantly attenuated the protection of Z-ligustilide against OGD-Reoxy-induced injury. Moreover, the application of specific inhibitors of MAPKs also achieved similar results. Finally, Z-ligustilide alleviated the accumulation of ubiquitinated proteins induced by OGD-Reoxy, which was inhibited by HSP70-shRNA. Taken together, our results demonstrated that Z-ligustilide may induce protective HSP70 expression via the activation of the MAPK pathway, but not canonical HSF1 transcription. HSP70 plays a key role in the protection of Z-ligustilide against OGD-Reoxy-induced injury.

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.

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

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.

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, THSWDs 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.

Ginsenoside 20(S)-Rh2 Induces Apoptosis and Differentiation of Acute Myeloid Leukemia Cells: Role of Orphan Nuclear Receptor Nur77

Ginsenoside 20(S)-Rh2 has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. In our study, 20(S)-Rh2 induced the expression of orphan nuclear receptor Nur77 and death receptor proteins Fas, FasL, DR5, and TRAIL, as well as the cleavage of caspase 8 and caspase 3 in HL-60 cells. Importantly, shNur77 attenuated 20(S)-Rh2-induced apoptosis and Fas and DR5 expression. Meanwhile, 20(S)-Rh2 promoted Nur77 translocation from the nucleus to mitochondria and enhanced the interaction between Nur77 and Bcl-2, resulting in the exposure of the BH3 domain of Bcl-2 and activation of Bax. Furthermore, 20(S)-Rh2 promoted the differentiation of HL-60 cells as evidenced by Wright-Giemsa staining, NBT reduction assay, and detection of the myeloid differentiation marker CD11b by flow cytometry. Notably, shNur77 reversed 20(S)-Rh2-mediated HL-60 differentiation. Additionally, 20(S)-Rh2 also exhibited an antileukemic effect and induced Nur77 expression in NOD/SCID mice with the injection of HL-60 cells into the tail vein. Together, our studies suggest that the Nur77-mediated signaling pathway is highly involved in 20(S)-Rh2-induced apoptosis and differentiation of AML cells.

β-Asarone Inhibits Invasion and EMT in Human Glioma U251 Cells by Suppressing Splicing Factor HnRNP A2/B1

β-asarone, the main component in the volatile oil of Acori tatarinowii Rhizoma, has been found to possess antitumor activity. However, its effect and mechanisms against tumor invasion and epithelial–mesenchymal transition (EMT) are still unclear. In this study, no or less cytotoxicity was caused by β-asarone within 0–120 μM in human glioma U251 cells for 48 h. β-asarone (30 and 60 μM) inhibited the migration of U251 cells in the wound healing assay, suppressed the invasion of U251 cells in the Boyden chamber invasion assay, and inhibited the adhesion of U251 cells onto the Matrigel. Moreover, β-asarone suppressed EMT with the up-regulation of E-cadherin and the down-regulation of vimentin. HnRNP A2/B1, a well-characterized oncogenic protein, was shown at a high basal level in U251 cells and β-asarone reduced hnRNP A2/B1 expression in a concentration and time-dependent way. Importantly, hnRNP A2/B1 overexpression significantly counteracted the inhibition of β-asarone on the migration, invasion, and adhesion of U251 cells and reversed the modulation of EMT markers by β-asarone. Additionally, β-asarone decreased the MMP-9 and p-STAT3 in U251 cells, which was also reversed by hnRNP A2/B1 overexpression. Together, our results suggest that hnRNP A2/B1 may be a potential molecular target underlying the inhibitory effect of β-asarone on invasion and EMT in glioma cells.

β-Asarone Induces Apoptosis and Cell Cycle Arrest of Human Glioma U251 Cells via Suppression of HnRNP A2/B1-Mediated Pathway In Vitro and In Vivo

HnRNP A2/B1 has been found to be an oncogenic protein strongly related to the growth of human glioma cells. Herein, β-asarone, the main component in the volatile oil of Acori tatarinowii Rhizoma, inhibited the cell viability, proliferation, and colony formation ability of U251 cells. Moreover, β-asarone induced apoptosis and cell cycle arrest at the G1 phase. Notably, β-asarone suppressed the expression of hnRNP A2/B1 and hnRNPA2/B1 overexpression remarkably reversed β-asarone-mediated apoptosis and cell cycle arrest. Importantly, β-asarone promoted the alternative splicing of Bcl-x by enhancing the ratio of Bcl-xS/Bcl-xL. Meanwhile, hnRNPA2/B1 overexpression mitigated the promoting effect of β-asarone on the alternative splicing of Bcl-x. β-asarone also regulated the level of the key proteins involved in the death receptor pathway and mitochondrial apoptosis pathway. Additionally, β-asarone modulated the cell cycle-related proteins p21, p27, Cdc25A, cyclin D, cyclin E, and CDK2. Finally, β-asarone inhibited tumor growth and induced apoptosis in nude mice bearing U251 tumor xenografts. β-asarone also suppressed the hnRNP A2/B1 expression, enhanced the expression of cleaved-caspase 3 and p27 and the ratio of Bcl-xS/Bcl-xL, and reduced the expression of CDK2 in U251 xenografts. Together, β-asarone-induced apoptosis and cell cycle arrest of U251 cells may be related to the suppression of hnRNPA2/B1-mediated signaling pathway.

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.

Sensitization of tamoxifen-resistant breast cancer cells by Z-ligustilide through inhibiting autophagy and accumulating DNA damages

Autophagy plays a pro-survival role in the tamoxifen-resistant breast cancer cells. Herein we found that autophagy was concomitantly induced in tamoxifen-resistant MCF-7 (MCF-7TR5) cells through the dissociation of Bcl-2 from Beclin 1 and subsequent enhancement of interaction among the ATG14-Beclin1-PI3KC3 complex. Moreover, higher level of DNA damage was observed in MCF-7TR5 cells with the decreased BRCA1 and RAD51 level and the increased Ku80 level. Interestingly, Nur77 was selectively degraded by autophagy, which causes the release of Ku80 from the Nur77-Ku80 complex, resulting in the increase of the DNA binding of Ku80 and DNA-PKcs. Meanwhile, Z-ligustilide, a phthalide compound from Radix Angelica sinensis, was shown to inhibit the autophagic flux by blocking the autophagosome-lysosome fusion. Importantly, Z-ligustilide-mediated autophagy inhibition restored Nur77 expression in MCF-7TR5 cells. Furthermore, Z-ligustilide promoted the interaction of Nur77 with Ku80 and thereby abolished the association of DNA-PKcs with DNA ends. Moreover, Z-ligustilide sensitized MCF-7TR5 cells in a caspase-independent cell death and enhanced the DNA damage caused by tamoxifen, which was found to be attenuated by shNur77. Together, these findings not only provide important insights into the formation of tamoxifen resistance in breast cancer cells, but also suggest Z-ligustilide may function as a novel autophagy inhibitor to overcome chemoresistance.