Natural Isoquinoline Alkaloid Research Articles

Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells.

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR.

Exploration of biocompatible AIEgens from natural resources.

Luminogens with aggregation-induced emission (AIEgens) characteristics have been well developed and applied in various areas such as bio-imaging, theranostics, organic photoelectronics and chemo/bio sensors. However, most of the reported AIEgens suffer from the disadvantages of complex organic synthesis and high cost, as well as being environmentally unfriendly and hard to degrade, which have largely limited their real applications. In this work, we discovered berberine chloride, a natural isoquinoline alkaloid isolated from Chinese herbal plants, as an unconventional rotor-free AIEgen with bright solid-state emission and water-soluble characteristics. Single crystal structure analysis and optical property, viscosity, and host-guest interaction studies suggested that intramolecular vibration and twisted intramolecular charge transfer were responsible for the AIE phenomenon of berberine chloride. Moreover, berberine chloride was biocompatible and could specifically target lipid droplets in a fluorescence turn-on and wash-free manner, demonstrating the great potential of natural products as promising AIE probes.

Structuring polarity-inverted TBA to G-quadruplex for selective recognition of planarity of natural isoquinoline alkaloids.

Efficient structuring of DNA by small molecules is very crucial in developing DNA-based novel switches with an ideal performance. In this work, we found that inverting only the polarity of the 3' terminal guanine of the thrombin-binding aptamer (3iTBA) totally eradicates the original TBA G-quadruplex (G4) structure in K+. The unstructured 3iTBA can be further refolded upon specifically interacting with small molecules of natural isoquinoline alkaloids (IAs) due to their fruitful binding patterns with variant nucleic acid structures. We identified that 3iTBA can serve as a topology selector for planar IAs. Nitidine (NIT), owing to the planar aromatic ring and coplanar substituents, is the most efficient to restructure the 3iTBA random coil toward the anti-parallel G4 conformation. However, common metal ions can't realize this structuring. The topology selector competency of 3iTBA toward IAs' planarity can be visualized using gold nanoparticles (AuNPs) as the chromogenic readout. Our work expands the G4 repertoire by exploring the polarity inversion regulation and provides a new approach to switch nucleic acid structures toward a small molecule structure-sensitive sensor.

Lycorine possesses notable anticancer potentials in on-small cell lung carcinoma cells via blocking Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT)

Lycorine, an natural isoquinoline alkaloid has reportedly that possesses multi-anticancer activity. However, to date, the anticancer feature of lycorine in non-small cell lung carcinoma (NSCLC) has still not fully been spelled out. The present study mainly focused on the molecular mechanism of lycorine against NSCLC in vitro and vivo. The results showed that lycorine evidently inhibited proliferation of A549 and H460 with IC50 values were 10.83 ± 1.14 μM and 12.35 ± 1.13 μM, while caused slight cytotoxicity in normal pulmonary epithelial Beas-2B cells, and arrested cell cycle in G0/G1 phase. Hoechst DNA-binding staining showed that typical characteristics of nuclear morphology apoptosis, AnnexinV-FITC/PI staining revealed the early-period apoptosis and the dissipation of mitochondrial membrane potential (Δψm) were also captured after lycorine treatment. Moreover, lycorine effectively repressed the Wnt/β-catenin signaling and reversed epithelial-mesenchymal transition (EMT). In addition, lycorine also intervened the caspase-mediated mitochondrial apoptosis pathway. Furthermore, A549/Luc tumor xenograft model was also corroborated that lycorine significantly suppressed the growth and metastasis of the lung tumor. These data highlight the significance of lycorine as potential anti-neoplastic agents to combat NSCLC.

Berberine inhibits the MexXY-OprM efflux pump to reverse imipenem resistance in a clinical carbapenem-resistant Pseudomonas aeruginosa isolate in a planktonic state.

Pseudomonas (P.) aeruginosa is an ubiquitous and metabolically versatile opportunistic pathogen and may cause various life-threatening diseases. Due to increasing emergence of resistance to carbapenems, novel drugs with improved antibacterial activities compared with those of traditional antibiotics are required. In the present study, berberine (BEB), a natural isoquinoline alkaloid, was used in combination with imipenem (IMP), a commonly-used carbapenem, to investigate their antibacterial activities against a clinical P. aeruginosa isolate PA012 and the potential mechanism. Screening revealed that the minimum inhibitory concentrations (MICs) of BEB and IMP were 512 and 256 µg/ml, respectively. The combination of BEB (1/4 MIC) and IMP (1/8 MIC) exhibited a synergistic effect with a fractional inhibitory concentration index of 0.375. The synergism of BEB and IMP was also demonstrated in a time-kill test and by scanning electron microscopic observation. Treatment with BEB at ¼ MIC in combination with IMP at 1/16, 1/8, 1/4 and ½ MIC revealed a concentration-dependent promoting effect of IMP on the intracellular accumulation of BEB and inhibition of bacterial adhesion. Further analysis of gene expression revealed that BEB (1/4 MIC) combined with IMP (1/8 MIC) decreased MexZ, MexX, MexY and outer membrane protein (Opr)M by 38, 35, 46 and 49% in PA012. In conclusion, these results suggested that IMP had synergistic effects with BEB against the clinical isolate PA012 via inhibition of the MexXY-OprM efflux pump.

Berberine could inhibit thyroid carcinoma cells by inducing mitochondrial apoptosis, G0/G1 cell cycle arrest and suppressing migration via PI3K-AKT and MAPK signaling pathways

Berberine, an important natural isoquinoline alkaloid from traditional Chinese medicine, is reported to exhibit multiple pharmacological properties, including anti-microbial, anti-diabetes, anti-obesity, anti-inflammatory and anti-carcinogenic activities. Although studies have shown that a wide range of carcinoma cells could be inhibited by berberine, few studies involved thyroid carcinoma. We therefore examined the effect of berberine on papillary thyroid carcinoma (PTC, the most common subtype) and anaplastic thyroid carcinoma (ATC, the most malignant and aggressive subtype). Three thyroid carcinoma cell lines with different aberrant genotypes and one normal thyroid cell line were selected, including C643 (ATC, with H-RAS mutation), OCUT1 (ATC, with BRAFV600E and PIK3CA mutations), TPC1 (PTC, with RET/PTC1 rearrangement) and Htori3 (normal). We found that berberine inhibited the proliferation of C643, OCUT1 and TPC1 thyroid carcinoma cells in a dose- and time-dependent manner (p<0.01, compared with the control), while normal human thyroid cells showed less sensitivity to the cytotoxicity of berberine. Berberine-induced significant mitochondrial apoptosis, G0/G1 cell cycle arrest and inhibitive migration in thyroid carcinoma cells were also observed (p<0.05 or p<0.01, compared with the control). Increased Bax/Bcl-2, cleaved Caspase 3, P21 and decreased Cyclin E1, CDK2 and Vimentin were verified by western blot. Additionally, berberine caused markedly decreased p-AKT1 expression and disturbances in classic ERK MAPK as well as P38 and JNK MAPK pathways in diverse thyroid carcinoma cells. Therefore, berberine could modulate PI3K-AKT and MAPK signaling pathways in thyroid carcinoma cells, which leads to mitochondrial apoptosis, G0/G1 cell cycle arrest and suppressive migration. Berberine may represent a promising chemotherapy for the treatment of thyroid carcinoma.

Berberine ameliorates methotrexate-induced liver injury by activating Nrf2/HO-1 pathway and PPARγ, and suppressing oxidative stress and apoptosis in rats

Berberine (BBR) is a natural isoquinoline alkaloid with very impressive health benefits. It is one of the most effective natural supplements available; however, its ameliorative mechanism against methotrexate (MTX)-induced liver injury is not well defined. This study investigated the protective effect of BBR against MTX hepatotoxicity, focusing on its ability to attenuate oxidative stress and apoptosis and to activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling and peroxisome proliferator activated receptor gamma (PPARγ). Rats received BBR (25 and 50mg/kg) orally for 7days before MTX injection. Other groups received MTX followed by BBR (25 and 50mg/kg) orally for 7 days. MTX-induced rats showed significant body weight loss, increased serum liver function marker enzymes, bilirubin and tumor necrosis factor alpha (TNF-α). Liver lipid peroxidation, nitric oxide (NO) and caspase-3 were significantly increased following MTX administration. BBR supplemented either before or after MTX significantly ameliorated body weight, liver function markers, TNF-α, lipid peroxidation, NO and caspase-3. BBR increased serum albumin and liver antioxidant defenses in MTX-induced rats. Histological and immunohistochemical examination showed improved histological structure and decreased expression of Bax in liver of MTX-induced rats treated with BBR. In addition, BBR up-regulated Nrf2, HO-1 and PPARγ expression in the liver of MTX-induced rats. In conclusion, BBR attenuated MTX-induced oxidative stress and apoptosis, possibly through up-regulating Nrf2/HO-1 pathway and PPARγ. Therefore, BBR can protect against MTX-induced liver injury.

Preventive effect of berberine against DMBA-induced breast cancer in female Sprague Dawley rats

Breast cancer is the prime cause for cancer mortality in women worldwide. The importance of diverse natural and dietary agents to reduce the risk of developing breast cancer is well established. Berberine, a natural isoquinoline alkaloid found in many medicinal plants is widely used in traditional Indian and Chinese medicine. Because of its capability to seize the cell cycle and induce apoptosis of numerous malignant cells, berberine has received considerable attention as a potential anticancer agent. In the present study, breast cancer was induced in Sprague Dawley (SD) rats by intragastric administration of 7, 12-dimethylbenz[a]anthracene (DMBA) at a dose of 80mg/kg of body weight. Treatment of berberine (50mg/kg BW) to breast tumor bearing rats was found to be effective against DMBA induced mammary carcinoma. The increased levels of lipid peroxide (malonaldehyde), pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), enzymatic antioxidants (SOD and CAT), non-enzymatic antioxidants (GSH and vitamin C) and transcription factor NF-κB were decreased significantly by administration of berberine. Furthermore, RT-PCR and western blot analysis showed the down-regulation of NF-κB and PCNA in breast tumors. Histopathological studies validated that berberine is effective against DMBA induced ductal carcinoma & invasive carcinoma. Altogether, these findings demonstrate the preventive role of berberine against DMBA induced mammary carcinoma in SD rats.

Berberine improves cognitive impairment by promoting autophagic clearance and inhibiting production of β-amyloid in APP/tau/PS1 mouse model of Alzheimer's disease

This study investigates the neuroprotective properties of berberine (a natural isoquinoline alkaloid isolated from the Rhizoma coptidis) and finds that berberine could promote β-amyloid (Aβ) clearance and inhibit Aβ production in the triple-transgenic mouse model of Alzheimer's disease (3×Tg-AD). During the study, berberine was first administrated to treat 3×Tg-AD mice and primary neurons. Morris water maze assay, western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining and histological analysis, transmission electron microscopic analysis were then used to evaluate the effects of the berberine administration. The result showed that berberine significantly improved 3×Tg-AD mice's spatial learning capacity and memory retention, promoted autophagy activity identified by the enhancement of brain LC3-II, beclin-1, hVps34, and Cathepsin-D levels as well as the reduction of brain P62 and Bcl-2 levels in AD mice, facilitated reduction of Aβ and APP levels, reduced Aβ plaque deposition in the hippocampus of AD mice, and inhibited b-site APP cleavage enzyme 1 (BACE1) expression. Similar results were also found in 3×Tg-AD primary hippocampal neurons: berbernine treatment decreased the levels of extracellular and intracellular Aβ1-42, increased the protein levels of LC3-II, beclin-1, hVps34, and Cathepsin-D, and decreased the levels of P62, Bcl-2, APP and BACE1 levels. In summary, berberine shows neuroprotective effects on 3×Tg-AD mice and may be a promising multitarget drug in the preventionand protection against AD.

Berberine mitigates methotrexate-induced oxidative stress and inflammation in the cerebrum of rats

Berberine is a natural isoquinoline alkaloid with multiple beneficial therapeutic effects. This study was designed to evaluate the protective effect of berberine against methotrexate (MTX)-induced oxidative stress and inflammation in the brain of rats. Rats received a single intraperitoneal injection of MTX (20 mg/kg) and orally administered 25 mg/kg and 50 mg/kg body weight berberine for 7 days. MTX-induced rats showed significantly increased lipid peroxidation and nitric oxide levels in the cerebrum. Treatment of the MTXinduced rats with berberine produced a significant decrease in cerebral levels of lipid peroxidation and nitric oxide. In addition, berberine induced a significant increase in reduced glutathione, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase in the cerebrum of MTX-induced rats. Rats received MTX showed a significant up-regulation of nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β) expression in the cerebrum, an effect that was significantly reversed following treatment with berberine. In conclusion, berberine protects against MTXinduced neurotoxicity through attenuating oxidative stress and inflammation, and boosting the antioxidant defenses.

Berberine Depresses Contraction of Smooth Muscle via Inhibiting Myosin Light-chain Kinase.

Background:Berberine is a natural isoquinoline alkaloid possessing various pharmacological effects, particularly apparent in the treatment of diarrhea, but the underlying mechanism remains unclear. Smooth muscle myosin light-chain kinase (MLCK) plays a crucial role in the smooth muscle relaxation-contraction events, and it is well known that berberine can effectively depress the contraction of smooth muscle. Hence, whether berberine could inhibit MLCK and then depress the smooth muscle contractility might be researched.Objective:The purpose of this study is to investigate the effects of berberine on MLCK. Based on this, the contractility of gastro-intestine, catalysis activity of MLCK, and molecular docking are going to be evaluated.Materials and Methods:The experiment of smooth muscle contraction was directly monitored the contractions of the isolated gastrointestine by frequency and amplitude at different concentration of berberine. The effects of berberine on MLCK were measured in the presence of Ca2+-calmodulin, using the activities of 20 kDa myosin light chain (MLC20) phosphorylation, and myosin Mg2+-ATPase induced by MLCK. The docking study was conducted with expert software in the meantime.Results:The phosphorylation of myosin and the Mg2+-ATPase activity is reduced in the presence of berberine. Moreover, berberine could inhibit the contractibility of isolated gastric intestine smooth muscle. Berberine could bind to the ATP binding site of MLCK through hydrophobic effect and hydrogen bonding according to the docking study.Conclusion:The present work gives a deep insight into the molecular mechanism for the treatment of diarrhea with berberine, i.e., berberine could suppress the contractility of smooth muscle through binding to MLCK and depressing the catalysis activity of MLCK.SUMMARY Berberine significantly reduced the amplitude of contraction in isolated duodenum and gastric strips in ratsBerberine inhibited the phosphorylated extents of MLC20 and Mg2+-ATPase activity of phosphorylated myosin induced by MLCKBerberine binds to the ATP binding site of MLCK by hydrophobic effect and hydrogen bondingBerberine may modulate contraction of smooth muscle by inhibiting MLCK. Abbreviations used: MLCK: Myosin light chain kinase; MLC20: 20 KDa regulating myosin light chain; CaM: Calmodulin.

Spectral Analysis of Nanodiamond-Berberine Complex Interaction with Living Cells for Nanoparticle Mediated Drug Delivery

The natural isoquinoline alkaloid berberine has been demonstrated for its significant activity against a variety of deceases caused by bacterial and parasite infections. Nanodiamond (ND) has also been proven to have great potential for drug delivery. In this work, the complex of ND with berberine is constructed through surface functionalized ND, aiming for drug delivery applications. Spectroscopic analysis was performed on the complex interaction with human lung alveolar carcinoma epithelial cell (A549) and red blood cell (RBC) in comparison with berberine in solution. The studied cells were treated with carboxylated ND, ND-berberine complex and berberine, respectively. Their distributions in cells structures are visualized using ND and berberine’s fluorescence signals for detection. The distribution of ND and ND-berberine complex differs with the distribution of berberine introduced in solution form. The results show that the ND with attached drug molecules can act as a drug carrier allowing controllable localization of the drug into the cell. This result can have implication in drug delivery, release and delivery tracing applications.

Synthesis of 2-(2-hydroxyethyl)-1-(2-hydroxyphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline and pseudosymmetry in its crystal structure.

Natural and synthetic isoquinoline alkaloids display a wide variety of potent biological activities. The title 1-aryl-2-hydroxyethyl-1,2,3,4-tetrahydroisoquinoline, C19H23NO4, crystallizes with two molecules in the asymmetric unit related by pseudo-translation but differing only slightly in conformation. The pseudosymmetry is also reflected in the diffraction pattern. The subset of reflections corresponding to the smaller cell and average structure are on average twice as intense as those subtending the larger cell. Tentative refinement in the subcell leads to a disordered structural model with satisfactory agreement factors and, after appropriate use of restraints, acceptable molecular geometry but significantly larger and more anisotropic displacement parameters. In the correct unit cell, the independent molecules differ with respect to the orientation of the hydroxyethyl group. Intramolecular hydrogen bonding occurs between the hydroxyphenyl group and the N atom.

Natural isoquinoline alkaloids: binding aspects to functional proteins, serum albumins, hemoglobin, and lysozyme.

The putative anticancer alkaloids berberine, palmatine, jatrorrhizine, and sanguinarine are known to bind to nucleic acids. To develop them as potential drugs for therapeutic use, their binding affinity to functional proteins and mode of transport in the circulatory system need to be clearly understood. Towards this, many studies on their binding aspects to proteins have been reported and a considerable amount of data, mostly of biophysical nature, exists in the literature. The importance of these natural isoquinoline alkaloids and the recent literature on their interaction phenomena with functional proteins, serum albumins, hemoglobin, and lysozyme are presented in this review.

The Role of Berberine in the Multi-Target Treatment of Senile Dementia.

Dementia is an acquired progressive cognitive dysfunction, affecting the content of consciousness rather than the level of consciousness. Most dementia is senile dementia, accounting for the majority of dementia. The most important and common form of senile dementia is refractory dementia, which includes Alzheimer's disease (AD) and vascular dementia (VD). Due to the diversity and uncertainty of the pathogenic mechanism of VD and AD, no significantly effective drug currently exists. Berberine is a natural isoquinoline alkaloid extracted from Coptis chinensis that has a wide range of pharmacological effects. studies in recent years have also found that berberine can additionally treat senile dementia by affecting neurotransmitter, anti-oxidative stress, metabolism and other multi-target pathways. Multi-target treatment of senile dementia by berberine is a potential avenue of study for senile dementia treatment.

Synthesis, antioxidant and anticancer screenings of berberine–indole conjugates

A variety of heterocyclic nitrogen cores in the form of indole moieties were linked to the natural isoquinoline alkaloid molecule berberine to achieve anticipated antioxidant and anticancer properties. An efficient synthetic pathway afforded final compounds 5a–j, which were tested in vitro for antioxidant potency using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical (ABTS) bioassays, and for anticancer activity using sulforhodamine B (SRB) assay against HeLa and Caski cancer cell lines. Moreover, the toxic nature of the resultant molecules was investigated using Madin–Darby canine kidney cells. The therapeutic indices of 5a–j were more appreciable against the Caski than HeLa cell line, in which compounds with electron-releasing alkyl or alkoxy functional group on indole entity as well as azaindole derivative performed well. In addition, these compounds were well endowed with antioxidant properties, in addition to the equal antioxidant effect of the compound with electron-withdrawing chlorine atom within indole entity. Adequate confirmation of the structure of the final analogues was achieved using Fourier-transform infrared (FT-IR), 1H nuclear magnetic resonance (NMR), and mass spectroscopy and elemental (CHN) analysis.

Induction of G₂/M Arrest by Berberine via Activation of PI3K/Akt and p38 in Human Chondrosarcoma Cell Line.

Berberine is a clinically important natural isoquinoline alkaloid found in many medicinal herbs. Berberine has been shown to have many pharmacological effects including antimicrobial, antitumor, and anti-inflammatory activities. However, the effects and mechanism of action of berberine have not been studied in chondrosarcoma. Therefore, the effects of berberine on proliferation in a human chondrosarcoma cell line (HTB-94) were investigated. Berberine inhibited cell proliferation in a concentration-dependent manner. We also determined that inhibition of cell proliferation by berberine occurred via G2/M phase arrest in HTB-94 cells. Berberine induced cell cycle arrest at the G2/M phase by upregulation of p53 and p21 expression and suppressed cyclin B1, cyclin-dependent kinase 1 (cdc2), cdc25c, and phosphorylated retinoblastoma tumor-suppressor protein (pRb) expression. In addition, berberine stimulated phosphorylation of protein kinase B (Akt) and p38 kinase. Inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt with LY294002 (LY) and p38 kinase with SB203580 (SB), respectively, decreased berberine-induced p53 and p21 expression and restored cell proliferation and expression of cyclin B1, cdc2, cdc25c, and pRb cell cycle progression proteins. These results suggest that berberine-induced inhibition of cell proliferation by cell cycle arrest at the G2/M phases was regulated through PI3K/Akt and p38 kinase pathways in HTB-94 chondrosarcoma cells.

Antiangiogenic and antitumor activities of berberine derivative NAX014 compound in a transgenic murine model of HER2/neu-positive mammary carcinoma.

Berberine (BBR) is a natural isoquinoline alkaloid with proven antiangiogenic and anticancer activities. We recently demonstrated that BBR and its synthetic derivative 13-(4-chlorophenylethyl)berberine iodide, NAX014, exert antiproliferative activity against HER2-overexpressing breast cancer cells, inducing apoptosis, modulating the expression of cell cycle checkpoint molecules involved in cell senescence, and reducing both HER2 expression and phosphorylation on tumor cells. In this study, we examined the anticancer properties of BBR and NAX014 in a transgenic mouse model which spontaneously develops HER2-positive mammary tumors. Repeated intraperitoneal injections of a safety dose (2.5mg/kg) of NAX014 delayed the development of tumors, reducing both the number and size of tumor masses. In vivo sidestream dark field videomicroscopy revealed a significant lower vessel density in mammary tumors from NAX014-treated mice in comparison with the control group. Immunohistochemical evaluation using CD34 antibody confirmed the reduced vessel density in NAX014 group. Statistically significant increase of senescence associated β-galactosidase and p16 expression, and reduced expression of heparanase were observed in tumors from NAX014-treated mice than in tumors from control animals. Finally, NAX014 treatment decreased the level of perforine and granzyme mRNA in mammary tumors. Berberine did not show any statistically significant modulation in comparison with control mice. The results of the present study indicate that NAX014 is more effective than BBR in exerting anticancer activity delaying the development of mammary tumors in mice transgenic for the HER-2/neu oncogene. The antitumor efficacy of NAX014 is mainly related to its effect on tumor vascular network and on induction of tumor cell senescence.

Digital gene expression analysis of Microsporum canis exposed to berberine chloride.

Berberine, a natural isoquinoline alkaloid of many medicinal herbs, has an active function against a variety of microbial infections including Microsporum canis (M. canis). However, the underlying mechanisms are poorly understood. To study the effect of berberine chloride on M. canis infection, a Digital Gene Expression (DGE) tag profiling was constructed and a transcriptome analysis of the M. canis cellular responses upon berberine treatment was performed. Illimina/Hisseq sequencing technique was used to generate the data of gene expression profile, and the following enrichment analysis of Gene Ontology (GO) and Pathway function were conducted based on the data of transcriptome. The results of DGE showed that there were 8476945, 14256722, 7708575, 5669955, 6565513 and 9303468 tags respectively, which was obtained from M. canis incubated with berberine or control DMSO. 8,783 genes were totally mapped, and 1,890 genes have shown significant changes between the two groups. 1,030 genes were up-regulated and 860 genes were down-regulated (P<0.05) in berberine treated group compared to the control group. Besides, twenty-three GO terms were identified by Gene Ontology functional enrichment analysis, such as calcium-transporting ATPase activity, 2-oxoglutarate metabolic process, valine catabolic process, peroxisome and unfolded protein binding. Pathway significant enrichment analysis indicated 6 signaling pathways that are significant, including steroid biosynthesis, steroid hormone biosynthesis, Parkinson’s disease, 2,4-Dichlorobenzoate degradation, and tropane, piperidine and Isoquinoline alkaloid biosynthesis. Among these, eleven selected genes were further verified by qRT-PCR. Our findings provide a comprehensive view on the gene expression profile of M. canis upon berberine treatment, and shed light on its complicated effects on M. canis.

Selective lighting up of epiberberine alkaloid fluorescence by fluorophore-switching aptamer and stoichiometric targeting of human telomeric DNA G-quadruplex multimer.

Aptamers, that exist naturally in living cells as functional elements and can switch nonfluorescent natural targets to fluorophores, are very useful in developing highly sensitive and selective biosensors and screening functional agents. This work demonstrates that human telomeric G-quadruplex (HTG) can serve as a potential fluorophore-switching aptamer (FSA) to target a natural isoquinoline alkaloid. We found that, among the G-quadruplexes studied here and the various structurally similar alkaloids including epiberberine (EPI), berberine (BER), palmatine (PAL), jatrorrhizine (JAT), coptisine (COP), worenine (WOR), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT), only the HTG DNA, especially with a 5'-TA-3' residue at the 5' end of the G-quadruplex tetrad (5'-TAG3(TTAG3)3-3', TA[Q]) as the minimal sequence, is the most efficient FSA to selectively light up the EPI fluorescence. Compared to the 5' end flanking sequences, the 3' end flanking sequences of the tetrad contribute significantly less to the recognition of EPI. The binding affinity of EPI to TA[Q] (K(d) = 37 nM) is at least 20 times tighter than those of the other alkaloids. The steady-state absorption, steady-state/time-resolved fluorescence, and NMR studies demonstrate that EPI most likely interact with the 5' end flanking sequence substructure beyond the core [Q] and the G-quadruplex tetrad in a much more specific manner than the other alkaloids. The highly selective and tight binding of EPI with the FSA and significantly enhanced fluorescence suggest the potential development of a selective EPI sensor (detection limit of 10 nM). More importantly, EPI, as the brightest FSA emitter among the alkaloids, can also serve as an efficient conformation probe for HTG DNA and discriminate the DNA G-quadruplex from the RNA counterpart. Furthermore, EPI can bind stoichiometrically to each G-quadruplex unit of long HTG DNA multimer with the most significant fluorescence enhancement, which has not been achieved by the previously reported probes. Our work suggests the potential use of EPI as a bioimaging probe and a therapeutic DNA binder.