ABSTRACT
1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.
ABSTRACT
Objective This study focused on the secondary metabolites of endophytic fungus Alternaria alternate in Paeonia lactiflora. Methods Compounds were isolated from the EtOAc extract by chromatography technology and their structures were elucidated on the basis of comprehensive spectroscopic analysis. Results A total of 15 compounds were isolated and their structures were identified as methyl 4-acetamido-3-hydroxybenzoate (1), (E)-methyl5-hydroxy-3-methylpent-2-eno (2), isobenzofuranone A (3), 4-hydroxyacetophenone (4), 6-hydroxy-isosclerone (5), talarojlavone (6), 7-hydroxy-2-hydroxymethyl-5-methyl-4H-chromen-4-one (7), alternarienonic acid (8), 7-hydroxy-2,5-dimethyl-4H-1-benzopyran-4-one (9), 5-hydroxy-epialtenuene (10), alternariol (11), methyl 3-hydroxybenzoate (12), stemphyperylenol (13), altenusin (14), and altenuene (15). Conclusion Compounds 1, 3, 5-10, 12, and 13 are isolated from Alternaria alternatefor the first time.
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Objective To study the secondary metabolites of endophytic fungus Fusarium oxysporum form Paeonia ostii. Methods The fermentation liquor of the fungal strain F. oxysporum were isolated and purified using various chromatographic methods. The structures of the compounds were identified by spectrum analysis. Results Twenty-three compounds were isolated from the fungal and their structures were identified as (2S)-proline (1), (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (2), cyclo-(S-Pro-S-Leu) (3), cyclo-(L-Ala-L-Pro) (4), cyclo-(Val-Pro) (5), cyclo-(R-Pro-S-Phe) (6), cyclo-(D-cis-Hyp-L-Phe) (7), cyclo-(trans-4-hydroxy- L-Pro-L-Phe) (8), cyclo-(Gla-Tyr) (9), cyclo-(trans-4-hydroxy-L-Pro-L-Leu) (10), L,L-cyclo-(Trp-Pro) (11), cyclo-(L-Leu-Gly) (12), N-(3-(1H-indol-3-yl)propyl)acetamide (13), indole-3-acetic acid (14), 2-piperidone (15), 2-pyrrolidinone (16), thymidine (17), (5S)-5-[(4-hydroxyphenyl)methyl]-2,4-imidazolidinedione (18), (3S,6S)-3-(butan-2-yl)-6-(1-hydroxyethyl) piperazine-2,5-dione (19), cyclo-(Ala-Leu) (20), cyclo-(S-Pro-S-Phe) (21), cyclo-(Tyr-Pro) (22), and cyclo-(L-Phe-L-Tyr) (23). Conclusion With the exception of compounds 2, 6, and 14, the other compounds are isolated from the fermentation liquor of the fungal strain F. oxysporum for the first time.
ABSTRACT
To investigate the effect of the total flavonoids in Scutellaria barbata(TF-SB) against autophagy in tumor cells in vivo, and further determine whether the mechanism is correlated with the PI3K/AKT/mTOR pathway, which lead to autophagy-induced tumor cell death. Melanoma-bearing mice were prepared and divided into control group, rapamycin group (Rap 1.5 mg•kg⁻¹), and high, middle and low-dose TF-SB (200, 100, 50 mg•kg⁻¹) groups. The groups were given drugs once a day for successively 11 days. The inhibitory effect of TF-SB on the growth of melanoma was determined by measuring tumor volume and tumor inhibition rate. TUNEL method was used to detect the apoptosis of tumor cells to further verify the antitumor activity of TF-SB. The protein expressions of LC3-Ⅰ and LC3-Ⅱ were detected by Western blot, and the relative expression of LC3-Ⅱ was calculated based on LC3-Ⅱ/LC3-Ⅰ. In addition, the effect of TF-SB on autophagy of tumor cells, the underlying molecular mechanism of TF-SB in inducing autophagy and PI3K/AKT/mTOR pathway marker protein phosphorylation were also studied. According to the results, TF-SB effectively inhibited melanoma growth in mice, reduced tumor volume, increased the tumor inhibition rate, and significantly increased tumor cell apoptosis index and the ratio of LC3-Ⅱ/LC3-I (P<0.05, P<0.01 or P<0.001). The protein expressions of p-PI3K, p-AKT and p-mTOR were also suppressed dramatically compared with those in control group (P<0.05, P<0.01 or P<0.001). In conclusion, the total flavonoids in S. barbata could inhibit the growth of melanoma in vivo by inducing autophagy and apoptosis of tumor cells, which may be correlated with suppression of PI3K/AKT/mTOR pathway.
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This paper was aimed to investigate the relationship between autophagy and NLRP3 inflammasome activation by studying the effect oftotal flavonoids in Scutellaria barbata (TF-SB) on autophagy in tumor cells and NLRP3 inflammasome, and to provide experimental evidence for further study of the anti-tumor mechanism of TF-SB. Mielanoma models were established by inoculating B16-F1 cell line to mice, and then were randomly divided into 5 groups (n=10 in each group): model control, positive control control(Rap, 1.5 mg•kg⁻¹), and TF-SB low, middle and high groups (50, 100 and 200 mg•kg⁻¹). Meanwhile, healthy C57BL/6J mice were used as normal control group (n=10). The drugs were given once daily for 2 weeks consecutively. Thirty minutes after last treatment, the determinations at endpoint were performed; pathological changes of tumor tissue were evaluated by using HE staining; protein expressions of LC3-II/LC3-I or NLRP3inflammasome/caspase-1/IL-1β and IL-18 in tumor tissues were detected by using Western-blot; and serum levels of IL-1β and IL-18 were detected by using Elisa kit. The results showed that the tumor cells in model group showed obvious atypia and malignant proliferation; the invasion of tumor tissue was significantly reduced, the tumor necrosis area was significantly increased, and the inflammatory reaction was significantly alleviated in positive control group and various TF-SB groups. As compared with model control group, LC3-II/LC3-I was significantly increased, while NLRP3/caspase-1/IL-1βand IL-18 protein expressions were significantly decreased in positive control group and TF-SB groups. Serum IL-1β and IL-18 levels in model control group were found higher than those in control group (P<0.001), but they were significantly lowered in positive control group and TF-SB groups (P<0.05, P<0.01 or P<0.001). Taken together, total flavonoids in S. barbata could effectively alter the tumor growth micro-environment by inhibiting the expression of NLRP3 inflammasome, and its anti-tumor effect may be associated with the induction of tumor cell autophagy.
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<p><b>OBJECTIVES</b>To investigate the protective effects of Sapindus saponins in spontaneously hypertensive rats, and the possible cellular and molecular mechanisms.</p><p><b>METHODS</b>Thirty-two 16-week-old spontaneously hypertensive rats were randomly divided into four groups (8 in each group): model group (placebo), positive control group (27 mg/kg of Captopril Tablets), Sapindus saponins groups (27 mg/kg and 108 mg/kg, respectively). Another 8 healthy Wistar-Kyoto strain (WKY) rats were used as the normal group. The animals were treated for 8 weeks. Blood pressure of rats was determined by non-invasive blood pressure meter (BP-6). Furthermore, the contents of angiotensin II (Ang II) in plasma and myocardial tissue were determined by enzyme-linked immunosorbent assay (ELISA), the gene expression of receptor angiotensin type 1 (AT1R) in aorta was determined by quantitative realtime polymerase chain reaction (qRT-PCR). The protein expression of transforming growth factor-β1 (TGF-β1) and AT1R in heart was determined by immunohistochemical staining. The protein expression of p-phosphorylation of p38 mitogen-activated protein kinase (p-p38MAPK) was determined by Western blotting. The contents of interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) in serum were determined by radioimmunoassay. And the histopathological and morphological changes of aorta and heart tissue samples were assessed semi-quantitatively by hematoxylin-eosin (HE) or Masson staining.</p><p><b>RESULTS</b>Thirty minutes after single or continuous treatment, systolic blood pressure (SBP) was reduced significantly in Sapindus saponins groups. And the contents of AngII, IL-1, IL-6 and TNF-α in serum, the expression of AT1R mRNA, p-p38MAPK and TGF-β1 were significantly suppressed dose-dependently (P<0.05 or P<0.01). With the Sapindus saponins treatment, compared with those of the model group, the cardiac and aortic pathological changes were ameliorated significantly.</p><p><b>CONCLUSIONS</b>Our findings suggest that Sapindus saponins might have protective effects in spontaneously hypertensive rats, the cellular and molecular mechanisms of which might be relevant to the regulation of inflammatory responses mediated by p-p38MAPK signal pathway based on activated Ang II and AT1R.</p>