Hefeng Zhou

Transcriptome analysis of leaves, roots and flowers of Panax notoginseng identifies genes involved in ginsenoside and alkaloid biosynthesis

BackgroundPanax notoginseng (Burk.) F.H. Chen is one of the most highly valued medicinal plants in the world. The major bioactive molecules are triterpene saponins, which are also known as ginsenosides. However, its large genome size has hindered the assembly of a draft genome by whole genome sequencing. Hence, genomic and transcriptomic details about P. notoginseng, especially its biosynthetic pathways and gene expression in different parts of the plant, have remained largely unknown until now.ResultsIn this study, RNA sequencing of three different P. notoginseng tissues was performed using next generation DNA sequencing. After assembling the high quality sequencing reads into 107,340 unigenes, biochemical pathways were predicted and 9,908 unigenes were assigned to 135 KEGG pathways. Among them, 270 unigenes were identified to be involved in triterpene saponin biosynthesis. In addition, 350 and 342 unigenes were predicted to encode cytochrome P450s and glycosyltransferases, respectively, based on the annotation results, some of which encode enzymes responsible for the conversion of the triterpene saponin backbone into different ginsenosides. In particular, one unigene predominately expressed in the root was annotated as CYP716A53v2, which probably participates in the formation of protopanaxatriol from protopanaxadiol in P. notoginseng. The differential expression of this gene was further confirmed by real-time PCR.ConclusionsWe have established a global transcriptome dataset for P. notoginseng and provided additional genetic information for further genome-wide research and analyses. Candidate genes involved in ginsenoside biosynthesis, including putative cytochrome P450s and glycosyltransferases were obtained. The transcriptomes in different plant tissues also provide invaluable resources for future study of the differences in physiological processes and secondary metabolites in different parts of P. notoginseng.

The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides

Abstract Protopalythoa is a zoanthid that, together with thousands of predominantly marine species, such as hydra, jellyfish, and sea anemones, composes the oldest eumetazoan phylum, i.e., the Cnidaria. Some of these species, such as sea wasps and sea anemones, are highly venomous organisms that can produce deadly toxins for preying, for defense or for territorial disputes. Despite the fact that hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, practically nothing is known about the toxin repertoire in zoanthids. Here, based on a transcriptome analysis of the zoanthid Protopalythoa variabilis, numerous predicted polypeptides with canonical venom protein features are identified. These polypeptides comprise putative proteins from different toxin families: neurotoxic peptides, hemostatic and hemorrhagic toxins, membrane-active (pore-forming) proteins, protease inhibitors, mixed-function venom enzymes, and venom auxiliary proteins. The synthesis and functional analysis of two of these predicted toxin products, one related to the ShK/Aurelin family and the other to a recently discovered anthozoan toxin, displayed potent in vivo neurotoxicity that impaired swimming in larval zebrafish. Altogether, the complex array of venom-related transcripts that are identified in P. variabilis, some of which are first reported in Cnidaria, provides novel insight into the toxin distribution among species and might contribute to the understanding of composition and evolution of venom polypeptides in toxiferous animals.

Discovery of Novel ROCK1 Inhibitors via Integrated Virtual Screening Strategy and Bioassays

Rho-associated kinases (ROCKs) have been regarded as promising drug targets for the treatment of cardiovascular diseases, nervous system diseases and cancers. In this study, a novel integrated virtual screening protocol by combining molecular docking and pharmacophore mapping based on multiple ROCK1 crystal structures was utilized to screen the ChemBridge database for discovering potential inhibitors of ROCK1. Among the 38 tested compounds, seven of them exhibited significant inhibitory activities of ROCK1 (IC50 < 10 μM) and the most potent one (compound TS-f22) with the novel scaffold of 4-Phenyl-1H-pyrrolo [2,3-b] pyridine had an IC50 of 480 nM. Then, the structure-activity relationships of 41 analogues of TS-f22 were examined. Two potent inhibitors were proven effective in inhibiting the phosphorylation of the downstream target in the ROCK signaling pathway in vitro and protecting atorvastatin-induced cerebral hemorrhage in vivo. The high hit rate (28.95%) suggested that the integrated virtual screening strategy was quite reliable and could be used as a powerful tool for identifying promising active compounds for targets of interest.

Paeoniflorin promotes angiogenesis in a vascular insufficiency model of zebrafish in vivo and in human umbilical vein endothelial cells in vitro

ObjectiveTo investigate the pro-angiogenic effects of paeoniflorin (PF) in a vascular insufficiency model of zebrafish and in human umbilical vein endothelial cells (HUVECs).MethodsIn vivo, the pro-angiogenic effects of PF were tested in a vascular insufficiency model in the Tg(fli-1:EGFP)y1 transgenic zebrafish. The 24 h post fertilization (hpf) embryos were pretreated with vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor II (VRI) for 3 h to establish the vascular insufficiency model and then post-treated with PF for 24 h. The formation of intersegmental vessels (ISVs) was observed with a fluorescence microscope. The mRNA expression of fms-like tyrosine kinase-1 (flt-1), kinase insert domain receptor (kdr), kinase insert domain receptor like (kdrl) and von Willebrand factor (vWF) were analyzed by real-time polymerase chain reaction (PCR). In vitro, the pro-angiogenic effects of PF were observed in HUVECs in which cell proliferation, migration and tube formation were assessed.ResultsPF (6.25–100 μmol/L) could rescue VRI-induced blood vessel loss in zebrafish and PF (25–100 μmol/L), thereby restoring the mRNA expressions of flt-1, kdr, kdrl and vWF, which were down-regulated by VRI treatment. In addition, PF (0.001–0.03 μmol/L) could promote the proliferation of HUVECs while PF stimulated HUVECs migration at 1.0–10 μmol/L and tube formation at 0.3 μmol/L.ConclusionPF could promote angiogenesis in a vascular insufficiency model of zebrafish in vivo and in HUVECs in vitro.

A New Danshensu Derivative Protects Against 6-Hydroxydopamine-Induced Neurotoxicity In Vitro and In Vivo

We previously reported a novel danshensu derivative ([Formula: see text])-(3,5,6-Trimethylpyrazinyl) methyl-2-acetoxy-3-(3,4-diacetoxyphenyl) propanoate (ADTM), which conferred cardioprotective and anti-thrombotic effects in vitro and in vivo. Here, we examined the neuroprotective actions of ADTM on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in PC12 cells 1 in vitro and zebrafish in vivo. Pretreatment with ADTM significantly inhibited 6-OHDA-induced cytotoxicity and production of reactive oxygen species (ROS) in PC12 cells through Akt signaling. Moreover, treatment with ADTM also inhibited expression of inducible nitric oxide synthase (iNOS) and production of intracellular nitric oxide (NO), which are associated with inflammation. In addition, ADTM exhibited significant protection against 6-OHDA-induced loss of tyrosine hydroxylase-positive dopaminergic neurons in a zebrafish model. Taken together, our findings suggest that ADTM is also a potential effective therapeutic agent for neurodegenerative conditions such as Parkinsons disease (PD) through anti-oxidant cytoprotective and anti-inflammatory actions.

Inhibitory Effects of Betulinic Acid on LPS-Induced Neuroinflammation Involve M2 Microglial Polarization via CaMKKβ-Dependent AMPK Activation

In response to the microenvironment, microglia may polarize into either an M1 pro-inflammatory phenotype, exacerbating neurotoxicity, or an M2 anti-inflammatory phenotype, conferring neuroprotection. Betulinic acid (BA) is a naturally pentacyclic triterpenoid with considerable anti-inflammatory properties. Here, we aim to investigate the potential effects of BA on microglial phenotype polarization and to reveal the underlying mechanisms of action. First, we confirmed that BA promoted M2 polarization and inhibited M1 polarization in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Then, we demonstrated that the effect of BA on microglial polarization was dependent on AMP-activated protein kinase (AMPK) activation, as evidenced by the fact that both AMPK inhibitor compound C and AMPK siRNA abolished the M2 polarization promoted by BA. Moreover, we found that calmodulin-dependent protein kinase kinase β (CaMKKβ), but not liver kinase B1, was the upstream kinase required for BA-mediated AMPK activation and microglial M2 polarization, via the use of both the CaMKKβ inhibitor STO-609 and CaMKKβ siRNA. Finally, BA enhanced AMPK phosphorylation and promoted M2 microglial polarization in the cerebral cortex of LPS-injected mice brains, which was attenuated by pre-administration of the AMPK inhibitor. This study demonstrated that BA promoted M2 polarization of microglia, thus conferring anti-neuroinflammatory effects via CaMKKβ-dependent AMPK activation.

Evaluation in zebrafish model of the toxicity of rhodamine B‐conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics

Crotamine is defensin‐like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom‐derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B‐conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB‐crotamine was as low as 4 μM, which effectively kill zebrafish larvae in less than 10 min. With non‐lethal concentrations (<1 μM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB‐crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles.

A novel compound DT-010 protects against doxorubicin-induced cardiotoxicity in zebrafish and H9c2 cells by inhibiting reactive oxygen species-mediated apoptotic and autophagic pathways

ABSTRACT Doxorubicin (Dox) is an effective anti‐cancer agent but limited by its cardiotoxicity, thus the search for pharmacological agents for enhancing anti‐cancer activities and protecting against cardiotoxicity has been a subject of great interest. We have previously reported the synergistic anti‐cancer effects of a novel compound DT‐010. In the present study, we further investigated the cardioprotective effects of DT‐010 in zebrafish embryos in vivo and the molecular underlying mechanisms in H9c2 cardiomyocytes in vitro. We showed that DT‐010 prevented the Dox‐induced morphological distortions in the zebrafish heart and the associated cardiac impairments, and especially improved ventricular functions. By using H9c2 cells model, we showed that DT‐010 directly inhibited the generation of reactive oxygen species by Dox and protected cell death and cellular damage. We further observed that DT‐010 protected against Dox‐induced myocardiopathy via inhibiting downstream molecular pathways in response to oxidative stress, including reactive oxygen species‐mediated MAPK signaling pathways ERK and JNK, and apoptotic pathways involving the activation of caspase 3, caspase 7, and PARP signaling. Recent studies also suggest the importance of alterations in cardiac autophagy in Dox cardiotoxicity. We further showed that DT‐010 could inhibit the induction of autophagosomes formation by Dox via regulating the upstream Akt/AMPK/mTOR signaling. Since Dox‐induced cardiotoxicity is multifactorial, our results suggest that multi‐functional agent such as DT‐010 might be an effective therapeutic agent for combating cardiotoxicity associated with chemotherapeutic agents such as Dox.

A novel agent attenuates cardiotoxicity and improves antitumor activity of doxorubicin in breast cancer cells: ZHANG et al.

Doxorubicin (Dox) is a well‐known chemotherapeutic agent used in the treatment of various cancers. However, Dox‐induced cardiotoxicity limits its further clinical use. We have previously reported a small molecular named biotin‐conjugated ADTM analog (BAA) that exhibits cytoprotective effects against oxidative stress–induced cell injury in cardiomyoblast H9c2 cells. Here, the protective effects of BAA, indexed by attenuation of the cardiotoxicity induced by Dox as well as synergistic antitumor activity that increases the chemotherapeutic efficacy of Dox were investigated. Our results demonstrated that BAA significantly ameliorated Dox‐induced toxicity in the H9c2 cells and zebrafish models. In addition, BAA attenuated Dox‐induced endoplasmic reticulum (ER) stress in H9c2 cells. An ER stress inhibitor, 4‐phenylbutyric acid, reversed the protective effect of BAA in H9c2 cells. In contrast, in human breast tumor MDA‐MB‐231 cells, BAA significantly enhanced Dox‐induced cytotoxicity through upregulating Dox‐induced ER stress response. Taken together, our findings indicate that Dox combined with BAA can significantly enhance its antitumor activity in breast cancer cells and reduce its cardiotoxicity, at least in part, by mediating ER stress activation.

BHDPC is a novel neuroprotectant that provides anti-neuroinflammatory and neuroprotective effects by inactivating NF-κB and activating PKA/CREB

Microglia-mediated neuroinflammatory responses are inevitable and important pathological processes in several kinds of disorder of the central nervous system (CNS). Therefore, alleviating activated microglia-induced inflammatory process might be a valuable therapeutic approach to neuroinflammation-related diseases. In the present study, we investigated BHDPC, a novel neuroprotectant discovered in our previous study that had anti-inflammatory effects under neuroinflammatory conditions. First, we found that BHDPC could inhibit neuroinflammatory responses and promote microglial M2 phenotype polarization in both lipopolysaccharide (LPS)-activated BV-2 microglia l cells. Furthermore, BHDPC provided protective actions against neuroinflammation-induced neurotoxicity in HT22 mouse hippocampal cells co-cultured with activated BV-2 microglia. Further experiments demonstrated that BHDPC could suppress LPS-induced activation of transcription factor nuclear factor kappa B (NF-κB) via interfering with the degradation of the inhibitor of kappa B (IκB) and phosphorylation of IκB, the IκB kinase (IKK). Moreover, we also found that BHDPC could induce phosphorylation of cAMP-dependent protein kinase A (PKA) and cAMP-response element-binding protein (CREB) in BV-2 microglial cells. Also, using the PKA-specific inhibitor, we found that BHDPC-induced CREB phosphorylation was dependent on PKA, which also contributed to BHDPC-mediated anti-inflammation and neuroprotection.