Poncirus trifoliata Aqueous Extract Protects Cardiomyocytes against Doxorubicin-Induced Toxicity through Upregulation of NAD(P)H Dehydrogenase Quinone Acceptor Oxidoreductase 1.

Doxorubicin (DOX), an anthracycline-based chemotherapeutic agent, is widely used to treat various types of cancer; however, prolonged treatment induces cardiomyotoxicity. Although studies have been performed to overcome DOX-induced cardiotoxicity (DICT), no effective method is currently available. This study investigated the effects and potential mechanisms of Poncirus trifoliata aqueous extract (PTA) in DICT. Changes in cell survival were assessed in H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells. The C57BL/6 mice were treated with DOX to induce DICT in vivo, and alterations in electrophysiological characteristics, serum biomarkers, and histological features were examined. The PTA treatment inhibited DOX-induced decrease in H9c2 cell viability but did not affect the MDA-MB-231 cell viability. Additionally, the PTA restored the abnormal heart rate, R-R interval, QT interval, and ST segment and inhibited the decrease in serum cardiac and hepatic toxicity indicators in the DICT model. Moreover, the PTA administration protected against myocardial fibrosis and apoptosis in the heart tissue of mice with DICT. PTA treatment restored DOX-induced decrease in the expression of NAD(P)H dehydrogenase quinone acceptor oxidoreductase 1 in a PTA concentration-dependent manner. In conclusion, the PTA inhibitory effect on DICT is attributable to its antioxidant properties, suggesting the potential of PTA as a phytotherapeutic agent for DICT.

In vitro mitochondrial apoptosis of melanoma cells via immature Poncirus trifoliata fruit extract.

OBJECTIVE: Poncirus trifoliata (P. trifoliata) fruits exert phytotherapeutic effects, depending on their maturity level. However, the mechanism by which these phytotherapeutic effects are exerted remains undefined - especially in cancers. Therefore, in this study, we investigated the effects of the immature fruit extract of P. trifoliata on a B16 melanoma cell line. MATERIALS AND METHODS: The effect of immature P. trifoliata extract on B16 cells was evaluated by MTT assay, cell proliferation, FACScan analysis of cell cycles, confocal imaging analysis, nuclear (Hoechst) staining, apoptosis assay (Annexin V-fluorescein isothiocyanate/propidium iodide staining), and Western blot assay. The capacity of immature P. trifoliata extract to inhibit the invasion and migration of B16 cells was assessed using the scratch-wound assay and Matrigel migration assay. The effect of immature P. trifoliata extract on mitochondrial function was determined via the mitochondrial membrane potential assay, activity, and fraction and cytosol proteins. RESULTS: Treating B16 cells with a methanol extract of immature P. trifoliata (MEPT) significantly inhibited cell viability, migration, and invasiveness in a dose- (p<0.01) and time (p<0.01)- dependent manner. MEPT arrested the cells in the G1 phase of the cell cycle and led to the activation of the PI3K/AKT/p21 pathway. Furthermore, MEPT dose-dependently induced apoptosis in B16 cells by increasing the expression of the pro-apoptotic proteins Bax and Apaf-1, while decreasing the expression of the anti-apoptotic protein, Bcl-2. MEPT treatment also decreased mitochondrial membrane potential. CONCLUSIONS: Immature P. trifoliata extract inhibited the growth of melanoma cells by inducing cell apoptosis through mitochondrial pathways. Therefore, further research into immature P. trifoliata extract as a potential therapeutic compound for melanoma treatment is warranted.

Boron and calcium deficiency disturbing the growth of trifoliate rootstock seedlings (Poncirus trifoliate L.) by changing root architecture and cell wall.

Boron (B) and calcium (Ca) are essential elements for plant growth. Both deficiencies inhibit root growth. However, the mechanism of inhibition is not well clear. Morphological characteristics of roots and changes in root cell wall grown at different B and Ca deficiencies were examined by using a hydroponic culture system. Both B and Ca deficiencies caused reduced plant biomass and root growth. Ca deficiency significantly decreased the fresh weight of root, stem, and leaves by 47%, 50%, and 62%, respectively, while B deficiency only reduced root fresh weight. The PCA combined with Pearson correlation analysis showed that there was significant different correlation among root parameters under B and Ca deficiency treatments when compared to control. The results of observation of transmission electron microscope showed that Ca deficiency reduced but B deprivation increased the thickness of the cell wall. Combining these technologies like X-ray diffraction, fourier transform infrared spectroscopy, homogalacturonan epitopes (JIM5 and JIM7), we confirmed that those changes above may be due to different changes in the degree of methyl esterification of pectin and glycoprotein of the cell wall. Taken together, we concluded that B deficiency can promote the formation of more low methyl esterified pectin to increase cell wall thickness, and then affect the morphological development of root system, while the formation of more highly methyl esterified pectin to increase cell wall degradation under Ca deficiency, which inhibited root elongation and formation of root branches.

Overexpression of a stress-responsive MYB transcription factor of Poncirus trifoliata confers enhanced dehydration tolerance and increases polyamine biosynthesis.

MYBs are an important family of transcription factors that play significant roles in plant development and stress response in plants. However, knowledge concerning the functions of MYBs in the non-model plants and the target genes is still limited. In this study, we isolated a stress-responsive R2R3-type MYB gene from trifoliate orange (Poncirus trifoliata (L.) Raf.), designated as PtsrMYB. PtsrMYB shares the highest degree of identity with AtMYB109. Subcellular localization using onion epidermal cells indicates that PtsrMYB is localized in the nucleus. Transcript levels of PtsrMYB were up-regulated by abiotic stresses such as dehydration, salt, cold and ABA treatment. Overexpression of PtsrMYB in tobacco confers enhanced dehydration tolerance, as indicated by less water loss, lower levels of malondialdehyde and reactive oxygen species. The transgenic tobacco lines displayed higher mRNA levels of two arginine decarboxylase (ADC) genes before and after dehydration treatment when compared with the wild type, concurrent with the greater levels of polyamines. Several MYB-recognizing cis-acting elements exist on the promoters of PtADC gene. Yeast one-hybrid assay demonstrated that PtsrMYB predominantly interact with two regions of the promoter, indicating the PtADC may be a target gene of PtsrMYB. Take together, PtsrMYB plays a positive role in dehydration tolerance, which may be, at least in part, due to the modulation of polyamine synthesis by regulating the ADC gene.

Anti-inflammatory effect of auraptene extracted from trifoliate orange (Poncirus trifoliate) on LPS-stimulated RAW 264.7 cells.

Poncirus trifoliate is a traditional Chinese medicinal plant used for treating inflammation-related diseases for a long time and trifoliate orange contains abundant auraptene. The present study was to evaluate auraptene as a potential anti-inflammatory agent and investigate the mechanism of auraptene against prostaglandins E2 (PGE2) and cyclooxygenase-2 (COX-2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells by comparing it with aspirin as a positive control group. The methods of enzyme-linked immunosorbent assay, reverse transcriptive polymerase chain reaction, real-time PCR, and western-blotting were used in the study. The results showed that auraptene exhibited better biocompatibility and lower cytotoxicity. At the same time, it significantly inhibited the production of PGE2 on LPS-stimulated macrophage cells. The auraptene-treated group had a higher COX-2 mRNA expression but relatively lower COX-2 protein level which implied that auraptene suppressed the post-transcriptional expression of COX-2 protein but not the transcriptional process. Compared with aspirin, the lower cytotoxicity of auraptene can make it a potential source for medicine that can benefit patients who are suffering from chronic inflammatory diseases and need long-term medication.

Cloning and sequence analysis of a low temperature-induced gene from trifoliate orange with unusual pre-mRNA processing.

Exposure of cold-hardy Rubidoux trifoliate orange [Poncirus trifoliata (L) Raf.] plants to temperatures from 28 degrees C to -5 degrees C enabled us to isolate and characterize a novel citrus low-temperature gene (CLT) with two transcripts, called CLTa and CLTb, from leaves and stems. CLTa was produced when plants were subjected to low temperatures (starting at 10 degrees C), while CLTb was constitutively expressed. Both CLTa and CLTb have the same open reading frame (ORF) of 165 nucleotides and encode a small (54 deduced amino acid) protein. However, CLTa has an additional 98 nucleotides in the 3'-untranslated region (UTR) that are absent in CLTb. Expression analysis using relative quantitative RT-PCR demonstrated that CLTa is expressed exclusively at low temperatures, while CLTb is expressed constitutively (expression verified from 33 degrees C to -5 degrees C). A GenBank database search identified 61 nucleotides inside of the ORF that are highly similar to low-temperature-responsive genes from Arabidopsis thaliana and Solanum tuberosum. The deduced amino acid sequence revealed similarity with low-temperature-responsive proteins from A. thaliana, Oryza sativa, and S. tuberosum of 77%, 81%, and 73.9%, respectively. A genomic clone was isolated, and the genome organization revealed the presence of three exons and two introns, the second of which is in the 3' UTR and participates in alternative 3' splice site selection. One of the 3' splice sites of the second intron was located immediately before the additional 98-bp non-coding fragment of CLTa, and the second at the very end of the 98-bp fragment. Additionally, the presence of the tetranucleotides TCTT and TTCT, which are involved in the regulation of transcript processing in animals and possibly also active in peach, was found in this intron. Competition for splicing sites on the pre-mRNA in the spliceosome, which is induced by low temperature, may be involved in the production of the two transcripts of the CLT gene.