Highly oxygenated germacrane-type sesquiterpenoids from the whole plant of Salvia cavaleriei H.Lév. and their biological activities.

The entire plant Salvia cavaleriei H.Lév. (Lamiaceae) is used as a traditional Chinese herbal medicine. Its leaves are edible, and the flowers can be soaked in water to make a health-care tea. In an effort to find natural bioactive chemical components, twelve undescribed germacrane-type sesquiterpenoids, salcavalins A-L, were isolated from the whole plant of S. cavaleriei and were identified as analogs. This is the first study to isolate highly oxygenated germacrane-type sesquiterpenoids from this plant. The structures of these undescribed compounds were elucidated by various spectroscopic methods, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis with Cu Kα radiation and electronic circular dichroism calculations. The biological activity of these undescribed compounds on the production of tumor necrosis factor-alpha in lipopolysaccharide induced NR8383 cells was evaluated, and salcavalins I and K showed anti-inflammatory activity to some extent. Salcavalins A-C, F and L were found to be neuroprotective with antiparkinsonic potential in a nematode (Caenorhabditis elegans) model. In addition, salcavalins F and I displayed marked phytotoxic activity against radish seeds at a low concentration of 50 ppm. Our findings provide scientific justification to show that bioactive sesquiterpenoids from the edible herb have anti-inflammatory in vitro, neuroprotective and phytotoxic activities.

Bioactive diterpenoids and sesquiterpenoids with different skeletons from Salvia digitaloides Diels.

Salvia has been regarded as a beneficial healing herb in ancient Egypt, Rome and Greece, and is listed as an official medicine in the pharmacopoeias of many countries worldwide. Currently, Salvia is widely used to flavor and preserve food. Here, two undescribed norabietane-type diterpenoids, sadigitaloides A and B, two undescribed germacrane-type sesquiterpenoids, sadigitaloides C and D, five undescribed guaiane-type sesquiterpenoid lactones, sadigitaloides E-I, two undescribed noreudesmane-type sesquiterpenoids, sadigitaloides J and K, one known diterpenoid, three known sesquiterpenoids, and three other types of known compounds were isolated from the 95% ethanol extract of the whole plants of Salvia digitaloides. Their structures and absolute configurations were characterized using 1D and 2D NMR spectroscopic techniques, electronic circular dichroism (ECD) calculations, HRESIMS experiments, and single-crystal X-ray diffraction analysis. Some compounds were evaluated for their anti-inflammatory activities against lipopolysaccharide (LPS)-induced TNF-α production in rat macrophage NR8383 cells. Sadigitaloide A showed noticeable anti-inflammatory activity at a concentration of 100.0 µM. At a concentration of 60 µM, sadigitaloide B exhibited better protection of dopaminergic neurons than the positive control n-butylidenephthalide in the Caenorhabditis elegans model injured by 6-OHDA. The phytotoxic activities of some compounds were attributed to considerable inhibitory effects on the growth of the roots and hypocotyls of Raphanus sativus L seedlings, especially cis, trans-abscisic acid, whose inhibition rates were much higher than those of glyphosate at concentrations ranging from 50 to 400 ppm. These results indicated that abietane-type diterpenoids possessed excellent anti-inflammatory and neuroprotective activities and further suggested that the low-molecular-weight compounds exhibited outstanding phytotoxic activities.

[Effect of Coixenolide on Foxp3+ CD4+ CD25+ Regulatory T Cells in Collagen-induced Arthritis Mice].

OBJECTIVE: To study the effect of coixenolide on Foxp3+ CD4+ CD25+ regulatory T cells (Treg) in collagen induced arthritis (CIA) mice, and to explore its possible mechanism for treating rheumatiol arthritis. METHODS: Five mice were recruited as a normal control group from 25 mice, and the rest 20 were used in CIA modeling. After successful modeling they were randomly divided in the model control group and the coixenolide group, 10 in each group. Coixenolide injection at 25 mL/kg was intraperitoneally injected to mice in the coixenolide group, while normal saline at 25 mL/kg was intraperitoneally injected to mice in the normal control group and the model control group. The injection lasted for 21 days. Scoring for CIA was performed after injection and arthritis index was calculated. The peripheral blood Foxp3+ CD4+ CD25+ Treg ratio was determined by flow cytometry (FCM). RESULTS: Compared with the normal control group, the arthritis index obviously increased in the model control group (P < 0.01). The arthritis index obviously decreased more in the coixenolide group than in the model control group (P < 0.01). Foxp3+ CD4+ CD25+ Treg levels obviously decreased more in the model control group than in the normal control group (P < 0.01 ). Foxp3+ CD4+ CD25+ Treg levels obviously increased more in the coixenolide control group than in the model control group (P < 0.01). CONCLUSION: Coixenolide could up-regulate Foxp3+ CD4+ CD25+ Treg ratios in CIA mice, which might play certain immunoregulation roles in the incidence of CIA.

NADPH oxidase-dependent formation of reactive oxygen species contributes to transforming growth factor ß1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells, and the role of astragalus intervention.

OBJECTIVE: To investigate the role of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidasedependent formation of reactive oxygen species (ROS) in the transforming growth factor ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) in rat peritoneal mesothelial cells (RPMCs), and the effect of Astragalus injection (AGI) intervention. METHODS: Primary RPMCs were cultured to the second generation in vitro. After synchronization for 24 h, the cells were randomly assigned to the following groups: control (Group A), AGI (2 g/mL; Group B), TGF-ß1 (10 ng/mL; Group C), TGF-ß1 (10 ng/mL) + AGI (2 g/mL; Group D; pretreated for 1 h with AGI before TGF-ß1 stimulation). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to evaluate the mRNA and protein expression of the NADPH oxidase subunit p67phox, α-smooth muscle actin (α-SMA) and E-cadherin. The dichlorofluorescein-sensitive cellular ROS levels were measured by a fluorometric assay and confocal microscopy. RESULTS: TGF-ß1 significantly induced NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, as well as inducing the production of intracellular ROS. AGI inhibited this TGF-ß1-induced up-regulation by 39.3% and 47.8%, respectively (P<0.05), as well as inhibiting the TGF-ß1-induced ROS generation by 56.3% (P<0.05). TGF-ß1 also induced α-SMA mRNA and protein expression, and down-regulated E-cadherin mRNA and protein expression (P<0.05). This effect was suppressed by AGI (P<0.05). CONCLUSIONS: NADPH oxidase-dependent formation of ROS may mediate the TGF-ß1-dependent EMT in RPMCs. AGI could inhibit this process, providing a theoretical basis for AGI in the prevention of peritoneal fibrosis.

Systemic lupus erythematosus complicated with femoral head ischemic necrosis treated by Chinese medicine therapy for activating blood and dredging collaterals method.

OBJECTIVE: To observe the effect and mechanism of Chinese medicine therapy for activating blood and dredging collaterals (ABDC) on treating systemic lupus erythematosus complicated with avascular necrosis of the femoral head (SLE-ANFH). METHODS: Thirty-four patients (51 joints) with SLE-ANFH were assigned by a random number table to two groups: 22 patients (32 joints) in the treatment group and 12 patients (19 joints) in the control group. All received Western medical conventional treatment for anti-inflammation and immunosuppression, but an additional Chinese medicine decoction prescribed based on ABDC principle was administered to patients in the treatment group. The observation on the patients' condition and therapeutic effect lasted for 3 years. RESULTS: The patients' conditions in the two groups, as assessed by Association for Research Circulation Osseous (ARCO) staging, were similar before treatment. After treatment, comparison between groups showed significant difference (P<0.05), and the raised Harris functional scores in the treatment group were higher than that in the control group (P<0.01). The post-treatment symptom improving rate in the treated group was 72.73%, which was higher than that in the control group (50.00%, P<0.05). Moreover, the former was superior in improving hematologic and hemorrheologic parameters in terms of prolonging activated partial thromboplastin time, lowering whole blood middle/low shear viscosity, and plasma viscosity (P<0.05 or P<0.01). Two patients in the control group but none in the treatment group received hip joint replacement operation during the observation period. CONCLUSIONS: Chinese medicine ABDC therapy could effectively alleviate clinical symptoms and improve joint function of patients with SLE-ANFH. The mechanism may be related to its effects on improving high coagulation manner and trend for getting embolism.

[A new type of iridium (III) phenylpyrazole complexes: synthesis, photophysical characterization].

New heteroleptic iridium(III) complexes (ppz)2Ir(LX), which consist of two cyclometalated ligands ppz(1-phenylpyrazole) together with an ancillary ligand LX (LX= 2-(2'-hydroxylphenyl)benzothiazole (BTZ), 2-(3'-methyl-2'-hydroxylphenyl) benzothiazole (3-MeBTZ), 2-(4'-methyl-2'-hydroxylphenyl) benzothiazole (4-MeBTZ) and 2-(4'-Trifluoromethyl-2'hydroxylphenyl) benzothiazole (4-tfmBTZ)), were synthesized and characterized. The molecular structures and photophysical properties were characterized and analyzed comparatively. The results show that the four complexes have basically similar UV-Vis absorption spectra, fluorescence excitation and emission spectra. Their maximum emission peaks are located at 583-615 nm, and accompanied by a lower intensity emission band around 400 nm. The weak emissions around 400 nm are ascribed to the radi ation transition of single state excition from ancillary ligand BTZ perturbed by metallic ion, and light emission around long-wave-length to the radiation transition of 3MLCT of Ir(BTZ) fragment. While the triplet state 3 MLCT of Ir(ppz)2 fragment might be quenched at room temperature. For all complexes, the excitations with maximum efficiency are located at 250-310 nm, which indicates that main contributor to light emitting is ligand-centered absorption (1pi-pi*) of ppz and BTZ rather than 3MLCT transitions, and thus provides a striking evidence that there is intersystem crossing from 1pi-pi* state to 3MLCT state in these complexes. Compared with Ir(ppz)3, these complexes not only have stronger phosphorescence at room temperature but also their emission color can be tuned by modifying ancillary ligand.

5-AZA-2'-deoxycytidine (5-AZA-CdR) leads to down-regulation of Dnmt1o and gene expression in preimplantation mouse embryos.

5-AZA-2'-deoxycytidine (5-AZA-CdR) is a demethylating, teratogenic agent and a mutagen, which causes defects in the developing mouse and rat after implantation. Our previous data indicated that 5-AZA-CdR (0.2 and 1.0 muM) inhibited the development of mouse preimplantation embryos. Pronuclear embryos exposed to 5-AZA-CdR at the pronuclear stage were unable to form 8-cell embryos, while 2-cell-stage embryos exposed to 5-AZA-CdR only developed into uncompacted 8-cell-stage embryos. And there was no formation of blastocysts when 4-cell embryos cultured in 5-AZA-CdR. In our present study, we detected Dnmt1o protein and some developmental gene expression in order to find the reasons for the developmental arrest. Dnmt1o could not traffic to 8-cell nuclei as control when embryos were exposed to 5-AZA-CdR. Dnmt1o was in cytoplasm at 2-cell and 4-cell stages before and after treated with 5-AZA-CdR. Gene expression changes were also detected in this research. Our data indicated that connexin 31 (Cx31), connexin 43 (Cx43), connexin 45 (Cx45), E-cadherin (Cdh1) and beta-catenin (Ctnnb1) were all downregulated by 5-AZA-CdR. Cx31, Cx43 and Cx45 are members of connexins family, which have a central role in gap junctions. Cdh1 and Ctnnb1 are necessary for the foundation of tight junctions. Therefore, developmental arrest induced by 5-AZA-CdR may be caused by the failure of Dnmt1o cytoplasmic-nuclear traffic and the down-regulation of developmental gene expression. Normal compaction and blastocoel cavitation need Dnmt1o traffic to 8-cell nuclei and the right gene expression, especially the correlative genes in gap junctions and tight junctions.

Regulatory effect of Langchuang serial recipes on T-lymphocyte subsets Th and Tc in patients with systemic lupus erythematosus.

OBJECTIVE: To study the principle of clearing Fei (), cooling blood, and detoxification as well as nourishing yin and moisening Fei (abbr. as CCD-NM) in regulating the levels of peripheral T-lymphocyte subsets Th and Tc cells to explore its mechanism for lowering the incidence of infection in patients with systemic lupus erythematosus (SLE). METHODS: Sixty SLE patients without complicated infection were assigned to the treatment group and the control group, 30 in each group. The control group was treated with Western medicine alone, while the treatment group was treated with the same program of Western medicine, but additionally administered with either Langchuang No.1 (I) or 2 (II), serial concerted Chinese recipes, applied respectively in patients in the active stage or in the resting stage. The total time of treatment for both groups was 1 year. Further, a healthy control group was set up with 20 healthy subjects. The expressions of Th1, Th2, and Tc1 and Tc2 cells in peripheral blood were detected and compared with those in the healthy control group. RESULTS: (1) As compared with the healthy control group, ratios of Th1/Th2 and Tc1/Tc2 in SLE patients, whether complicated with infection or not, were significantly lower (P<0.05 or P<0.01). (2) Comparison between patients with complications and those uncomplicated with infection showed that the two ratios and Th1 expression were lower and Tc2 was higher in the former than those in the latter (all P<0.05). (3) Ratios of Th1/Th2 and Tc1/Tc2 increased after treatment in patients of both the treatment group and the control group (P<0.05 and P<0.01), but the changes in the treatment group were more significant (P<0.05). CONCLUSION: The principle of CCD-NM could regulate the Th and Tc subsets toward equilibrium in SLE patients, which might be one of the mechanisms of action for alleviating complicated infection.

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats.

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 alpha-hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.

[Chromosome changes of aged oocytes after ovulation].

In mouse, matured oocytes are arrested at metaphase- (M) after ovulation. If the M oocytes are not fertilized on time, they will increasingly become aged in the oviduct. The aging of oocytes can lead to abnormal development of embryos. So it is important to study the mechanism of oocyte aging. Herein, we studied the change of oocyte chromosome after ovulation into the oviduct in vivo. Results indicated that 65% of old oocytes (34 h post hCG) showed aberrant MII, with chromosome misalignment and dispersal, in contrast to the young oocytes (14 h post hCG). In addition, chromosome changes may be associated with the increase of acetylation of histone 3 and histone 4, at lysine 14 and lysine 16 (H3K14 and H4K16), respectively. On the other hand, the decrease of methylation of histone 3 at lysine 9 (H3K9) presumably facilitated aberrant chromosome formation.

[Clinical research on effect of TCM treatment beginning with Fei in treating systemic lupus erythematosus for reducing incidence of complicated infection].

OBJECTIVE: To observe the effect of TCM treatment beginning with Fei in reducing the incidence of complicated infection and the antibiotic utilization rate in patients with systemic lupus erythematosus (SLE). METHODS: One hundred and ten SLE patients were randomly assigned to 2 groups equally, the control group treated with the conventional Western medicinal treatment and the treated group treated with the same conventional treatment and SLE I formula (in active stage) or SLE II formula (in silent period) additionally. RESULTS: After 3-month and 6-month treatment, the total effective rate was 83.64% , 87.27% in the treated group, and 78.18%, 81.82% in the control group respectively, showing insignificant difference between the two groups. It lowered in both groups after 1-year treatment, however, which in the treated group (78.18%) was higher than that in the control group (60.00%, P < 0.05). But the difference became insignificant again after 2-year treatment, it being 87.27% in the treated group and 72.73% in the control group. The incidence of complicated infection and antibiotic utilization rate in the 2-year treatment was 23.6%, 55.0% respectively in the treated group, markedly lower than those (50.9% and 100%) in the control group respectively (P < 0.01). CONCLUSION: TCM treatment beginning with Fei could decrease the incidence of complicated infection and the antibiotic utilization rate in SLE patients.

Effects of species and cellular activity of oviductal epithelial cells on their dialogue with co-cultured mouse embryos.

An efficient co-culture system, especially with oviductal or uterine epithelial cells, is important not only for the production of high quality embryos, but also for the study of the molecular dialogue between embryos and their maternal environment. Although mouse embryos have been co-cultured successfully with oviductal epithelial cells (OECs) from several species, studies on the effects of species and functionality of OECs are few. Reports concerning the necessity of direct contact between the embryo and OECs and about the culture of mouse embryos in medium conditioned with heterologous OECs have been controversial. In this study, pronuclear embryos from Kunming mice, characterized by an obvious two-cell block in vitro, were co-cultured with mouse, goat, and chick OECs. The functionality of OECs was determined by analyzing the cell cycle, apoptosis, the numbers of mitochondria and cilia, and the ability both to support embryonic development and to remove hypoxanthine from the culture medium. The necessity of direct contact between OECs and embryos was studied by repeated renewal of culture medium with fresh conditioned medium, the culture of embryos in plastic wells connected by tunnels to wells with OEC monolayers, and the co-culture of embryos separated from OECs by a filter. Both goat and chick OECs supported mouse embryonic development, but their embryotrophic lifespan was shorter than that of the mouse OECs. Whereas media conditioned with mouse OECs supported mouse embryonic development satisfactorily, medium conditioned with goat OECs supported little development. Immediate dialogue between heterologous OECs and embryos was essential for efficient co-culture, whereas direct contact between the two cell types was not; neither dialogue nor contact was needed between isologous OECs and embryos. Embryotrophic activity and the ability to remove hypoxanthine from conditioned medium declined with time after confluence and number of passages of OECs, mainly because of apoptosis and dedifferentiation. Thus, the species and functionality of OECs have profound effects on their molecular dialogue with co-cultured embryos, and efficient co-culture depends upon both positive and negative conditioning.

Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.

Methods for cell cycle synchronization of mouse fetal fibroblast cells (MFFCs) were first selected and optimized. When MFFCs were cooled at 5 C for different periods of time, the highest percentage of cells at the G0/G1 phase (75.4+/-2.9%), with 3.5+/-0.3% of apoptotic cells, was achieved after 5 h of treatment. Extended cooling increased the number of apoptotic cells significantly. When MFFCs were treated with different concentrations of roscovitine (ROS) for different periods of time, the highest percentage of G0/G1 cells (83.5+/-1.8%), with 9.2+/-0.6% apoptotic cells, was obtained after exposure to 10 microM ROS for 24 h. When the cells were cooled at 5 C for 5 h followed by incubation in 10 microM ROS for 12 h, 83.6+/-1.9% were synchronized at the G0/G1 stage, with 3.6% undergoing apoptosis. Cell cycle progression was then observed after release of the MFFCs from different synchronization blocks. The highest percentages of S and G2/M cells (81% and 75%) were achieved at 12 and 20 h, respectively, after release of the MFFCs from the cooling plus ROS treatment, and these percentages were significantly higher than those obtained after release from the cooling or ROS alone blocks. Finally, MFFCs were transfected with pEGFP-N1 plasmid at the peak of the G0/G1, S, and G2/M phases, respectively, after release from the different blocks and both the transient and stable transfection efficiencies were determined. The GFP gene expression was greatly enhanced when transfection was performed at the time when most cells were at the G2/M stage after release from cooling, ROS alone, and cooling plus ROS treatments. Statistical analysis revealed a close correlation between the rate of G2/M cells and the transient and stable GFP gene expression efficiencies. Together, the results indicated that (a) the best protocol for cell cycle synchronization of MFFCs was a 5-h cooling at 5 C followed by incubation in 10 microM ROS for 12 h which produced both a high rate of synchronization in the G0/G1 phase with acceptable apoptosis and a high rate of G2/M cells after release; and (b) that the cell cycle status had marked effects on the efficiency of liposome-mediated transfection in MFFCs, with the highest transfection efficiency obtained in cells at the G2/M stage.

[Factors affecting liposome-mediated gene transfection of mouse somatic cells].

We studied the effects of the amount of liposome and plasmid, exposure time of cells to the liposome-plasmid complexes, number of cell passages and cell types on GFP gene transfection of mouse somatic cells. The maximal GFP transgene expression (30.7%) was achieved when mouse fetal fibroblast cells (MFFC) at 70%-90% confluence of passage 3 were exposed for 6 h to the complexes of 4 microg liposome (LipofectAMINE) and 0.3 microg plasmid (pEGFP-N1). Under these conditions, we compared the effect of the number (from primary to 15) of passages on the transfection efficiency of MFFC. The transfection efficiency of MFFC was 10.0%, 28.9% and 7.2% at the primary, 3rd and 15th passage, respectively, which indicated that the transfection efficiency decreased with passaging. When MFFC, mouse oviductal epithelial cells (MOEC) and mouse granulosa cells (MGC) were transfected at passage 3, the transfection efficiency was 27.8%, 13.7% and 14.2%, respectively, under the described transfection conditions. When the cell cycle stages of different cell types at transfection were examined, it was found that 17.2% of MFFC, 8.7% of MOEC and 9.9% of MGC were at M phases of the cell cycle. Examination of the cell cycle stages of MFFC at different passages showed that MFFC at the third passage had the highest percentage of M cells and the percentage decreased afterwards. This suggested that the transfection efficiency was correlated with the percentages of cells at M phase, and provided essential data for improvement of the transfection efficiency.