Antitoxin MqsA decreases antibiotic susceptibility through the global regulator AgtR in Pseudomonas fluorescens.

Type II toxin-antitoxin systems are highly prevalent in bacterial genomes and play crucial roles in the general stress response. Previously, we demonstrated that the type II antitoxin PfMqsA regulates biofilm formation through the global regulator AgtR in Pseudomonas fluorescens. Here, we found that both the C-terminal DNA-binding domain of PfMqsA and AgtR are involved in bacterial antibiotic susceptibility. Electrophoretic mobility shift assay (EMSA) analyses revealed that AgtR, rather than PfMqsA, binds to the intergenic region of emhABC-emhR, in which emhABC encodes an resistance-nodulation-cell division efflux pump and emhR encodes a repressor. Through quantitative real-time reverse-transcription PCR and EMSA analysis, we showed that AgtR directly activates the expression of the emhR by binding to the DNA motif [5´-CTAAGAAATATACTTAC-3´], leading to repression of the emhABC. Furthermore, we demonstrated that PfMqsA modulates the expression of EmhABC and EmhR. These findings enhance our understanding of the mechanism by which antitoxin PfMqsA contributes to antibiotic susceptibility.

Steroidal alkaloids isolated from Veratrum grandiflorum Loes. as novel Smoothened inhibitors with anti-proliferation effects on DAOY medulloblastoma cells.

Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿5 (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿5 (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 â†’ 2)-ß-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and ß1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC50 values of 0.72-14.31 µM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while ß1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC50 value of 5.35 µM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.

[Diterpenoids as PPARγ agonists from Siegesbeckia pubescens and their anti-inflammatory effects in vitro].

This study aims to investigate the PPARγ agonists isolated from the aqueous extract of Siegesbeckia pubescens( SPA) and their anti-inflammatory activities in vitro. The 293 T cells transfected transiently with PPARγ recombinant plasmid were used as a screening model to guide the isolation of PPARγ activitating components,and then PPARγ activities were measured by double luciferase reporter gene assay. The chemical structures were identified by chromatography or spectroscopic techniques. Furthermore,a UC inflammatory model in vitro was established on HT-29 cells by stimulating with TNF-α． The mRNA levels and secretion of proinflammatory cytokines on HT-29 cells,such as IL-1ß,TNF-α,IL-8,were detected by RT-PCR and ELISA. The results showed that five diterpenoids were obtained from the fraction D_(50) with the strongest PPARγ activity among others in SPA,and determined as kirenol( 1),darutigenol( 2),enantiomeric-2-ketone-15,16,19-three hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 3),darutoside( 4),enantiomeric-2-ß,15,16,19-four hydroxypinomane-8( 14)-ene-19-O-ß-D-glucoside( 5),respectively. All the compounds exhibited active effects on PPARγ in a concentration-dependent manner( P<0. 01). In addition,compound 1 significantly inhibited the expression of IL-1ß mRNA and secretion of IL-8 on HT-29 cells inflammation model( P<0. 001); both compounds 2 and 3 effectively inhibited the expression of IL-1ß,TNF-α,IL-8 mRNA and secretion of IL-8( P<0. 01 or P<0. 001),although at different extent; compound 4 significantly inhibited the expression of IL-1ß and TNF-α mRNA( P<0. 01 or P<0. 001),while compound 5 inhibited the expression of IL-1ß mRNA obviously( P<0. 001). In conclusion,the diterpenoids 1-5 isolated from S. pubescens have the PPARγ activation activities and potential effects of anti-UC in vitro.

The Loss of a Sugar Chain at C(3) Position Enhances Stemucronatoside K-Induced Apoptosis, Cell Cycle Arrest, and Hedgehog Pathway Inhibition in HT-29 Cells.

Stemucronatoside K (SMK) and its aglycone stephanthraniline A (STA) are the most representative of a series of novel C21 steriodal compounds that we have previously isolated from Asclepiadaceae plants. The objectives of this study were to investigate the antitumor activity of SMK and STA, and clarify the effect of the sugar chain at the C(3) position. Our results showed that both SMK and STA decreased the growth of HT-29 cells in a dose- and time-dependent manner. Meanwhile, STA showed much stronger inhibitory effect than SMK. Treatment of HT-29 cells with STA increased the apoptotic cell numbers and the protein expression of cleaved caspase 3 and cleaved-PARP. G1 phase cell cycle arrest and decreased expression of cyclin D1 and cyclin-dependent kinases 4 were also observed after STA treatment. Furthermore, STA reduced the mRNA levels of four Hedgehog pathway components (GLI1, GLI2, GLI3, and PTCH1) and suppressed Shh-induced Hedgehog pathway activation in a concentration-dependent manner. These results indicated that SMK and STA could inhibit the growth of HT-29 cells by inducing apoptosis, cell cycle arrest, and hedgehog pathway inhibition. The loss of sugar chain at C(3) position could enhance SMK's activity. This study is beneficial to understand the use of natural C21 steroids as antitumor lead compounds.

Advanced Lung Adenocarcinoma with EGFR 19-del Mutation Transformed into SCC after EGFR-tyrosine Kinase inhibitors Treatment: A Case report.

BACKGROUND: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) significantly improve the survival of patients with Epidermal growth factor receptor (EGFR) sensitive mutations in non-small cell lung cancer (NSCLC). CASE SUMMARY: A 67-year-old female patient in advanced lung adenocarcinoma suffered from drug resistance after EGFR-TKIs treatment. Secondary pathological tissue biopsy confirmed squamous cell carcinoma (SCC) transformation. Patients inevitably encountered drug resistance issues after receiving EGFR-TKIs treatment for a certain period of time, while EGFR-TKIs can significantly improve the survival of patients with EGFR-sensitive mutations in NSCLC. Notably, EGFR-TKIs resistance includes primary and acquired. Pathological transformation is one of the mechanisms of acquired resistance in EGFR-TKIs, with SCC transformation being relatively rare. Our results provide more detailed results of the patient's diagnosis and treatment process on SCC transformation after EGFR-TKIs treatment for lung adenocarcinoma. CONCLUSION: Squamous cell carcinoma transformation is one of the acquired resistance mechanisms of EGFR-TKIs in advanced lung adenocarcinoma with EGFR mutations.

[Clinical efficacy of shenfu injection in treating severe sepsis and its effects on serum levels of interleukin-6 and interleukin-10].

OBJECTIVE: To observe the therapeutic efficacy of Shenfu Injection (SFI) on patients with severe sepsis and its effects on serum levels of interleukin-6 (IL-6) and interleukin-10 (IL-10). METHODS: Sixty-eight patients with severe sepsis were randomly assigned to the SFI group (36 cases, treated by SFI + routine therapy) and the control group (32 cases, treated by routine therapy). The acute physiology and chronic health evaluation II (APACHE II) score and Marshall score were observed before treatment, 3 and 7days after treatment. The therapeutic efficacy was assessed, and the 28th-day mortality rates were compared. The serum levels of IL-6 and IL-10 were determined by enzyme-labeled immunosorbent assay (ELISA) before and after treatment. C-reactive protein (CRP) was determined by immunoturbidimetric assay. RESULTS: There was no significant difference in the APACHE II score, Marshall score, IL-6, IL-10, or CRP between the two groups before treatment (P>0.05). APACHE II score and Marshall score of all patients decreased after treatment, with more obvious decrease shown in the SFI group (P<0.05). The mortality rate in the SFI group and the control group was 25.0% (9/36) and 37.5% (12/32) respectively, with no significant difference shown between the two groups (P>0.05). The serum levels of IL-6 and CRP obviously decreased after 7 days of treatment (P<0.05). But more decrement was shown in the SFI group, showing significant difference when compared with the control group (P<0.05). There was no significant difference in the serum IL-10 level between the two groups before and after treatment (P>0.05). CONCLUSION: SFI could lower the serum IL-6 level, regulate the equilibrium of proinflammatory factors and anti-inflammatory cytokines in severe sepsis patients, thus playing a role in improving the therapeutic efficacy.

Chemical composition and antioxidant activities of different polysaccharides from the roots of Angelica dahurica.

The total crude polysaccharides (CADPs), isolated from the roots of Angelica dahurica by H(2) O extraction, EtOH precipitation, and dialysis, and the four fractions ADP1, ADP2, ADP3, and ADP4, obtained by gel filtration of the CADPs, were analyzed to characterize their composition and evaluated for their antioxidant activity using different in vitro tests such as the malondialdehyde (MDA)-production, the ferrous ion (Fe(2+) )-chelating, and the HO(.) radical-scavenging assays. The predominant neutral monosaccharides in the four fractions were identified as arabinose, galactose, and glucose, while the composition and ratio of the monosaccharides were different between the fractions. The CADPs and its fractions were found to significantly inhibit lipid peroxidation, chelate Fe(2+) , and scavenge HO(.) radicals, indicating that these polysaccharides possessed antioxidant activity. Among the four fractions, ADP4 exhibited the strongest antioxidant activity, which was stronger than that of the control antioxidant vitamin E (Vit E). Taken together, the chemical composition of these polysaccharides might affect their antioxidant activity, and ADP4 could be explored as a source of potential novel natural antioxidants for food and pharmaceutical purposes.

Stemucronatoside L, a pregnane glycoside from the roots of Stephanotis mucronata, inhibits Th1/Th2 immune responses in vitro.

Stemucronatoside L (SML), isolated from Stephanotis mucronata, could suppress the activation of T cells in vitro. However, the mechanisms responsible for its immunosuppressive activity remain poorly understood. The purpose of this study was to investigate whether SML could suppress Th1/Th2 immune responses and to characterize the cellular mechanisms involved. Effects of SML on T-lymphocyte subsets and the production of Th1 cytokines IL-2 and IFN-gamma, and Th2 cytokines IL-4 and IL-10 from ConA-stimulated mice splenocytes were detected by flow-cytometric analysis and ELISA method, respectively. Furthermore, effects of SML on mRNA expression level of Th1/Th2 cytokines and transcription factors T-bet and GATA-3 were evaluated by RT-PCR analysis. SML not only significantly decreased the percentage of CD4(+) T cells and the CD4(+)/CD8(+) ratio, but reduced the production of Th1/Th2 cytokines in a concentration-dependent manner. The mRNA expression levels of Th1/Th2 cytokines and transcription factors (T-bet and GATA-3) were also suppressed by SML. These results suggested that SML could simultaneously inhibit Th1/Th2 immune responses by suppressing gene expression of Th1/Th2 cytokines and transcription factors.

Astilbotriterpenic acid induces growth arrest and apoptosis in HeLa cells through mitochondria-related pathways and reactive oxygen species (ROS) production.

Astilbotriterpenic acid, a novel ursane-type triterpenoid from the rhizomes of Astilbe chinensis, has cytostatic properties in several cancer cell lines and induces apoptosis in HeLa cell. The aim of this study was to investigate the mechanisms by which such properties are exerted, with special reference to the anti-proliferative and apoptotic potential on HeLa cells. Compound 1 showed a marked concentration- and time-dependent inhibition of HeLa cell proliferation, and induced G(0)/G(1) phase arrest of HeLa cell in a dose-dependent manner. There was a quick attenuation of mitochondrial membrane potential (Deltapsi(m)) with the alterations of Bcl-2/Bax mRNA express value in 1-treated HeLa, indicating the participation of a mitochondria-related mechanism. Pretreatment of a pan-caspase inhibitor (benzyloxycarbonyl)-Val-Ala-Asp-(fluoromethyl) ketone (z-VAD-fmk) significantly increases the viability of 1-treated HeLa cells implied that the participation of caspase; Western-blot analysis showed the activation of initiator caspase-8 and caspase-9, and executor caspase-3. Meanwhile, treatment with 1 stimulates the generation of reactive oxygen species (ROS) in HeLa cell. Taken together, our data showed that compound 1 induced growth arrest and apoptosis in HeLa cells through mitochondria-related pathways and ROS production, and may be further evaluated as a chemotherapeutic agent for human cancer.

Immunomodulatory and antitumor activity of triterpenoid fractions from the rhizomes of Astilbe chinensis.

AIM OF THE STUDY: The objectives of this study were to evaluate the in vivo antitumor potential of the triterpenoid fraction from the rhizomes of Astilbe chinensis (Saxifragaceae) (Saxifragaceae) (ATF) and to elucidate its immunological mechanisms by determining its effects on the growth of mouse transplanted tumors and the immune response in naïve and tumor-bearing mice. MATERIALS AND METHODS: The mice inoculated with mouse tumor cell lines were treated per os with ATF at the doses of 20, 40, 60 mg/kg for 10 days. The effects of ATF on the growth of transplantable tumor, splenocyte proliferation, the activity of natural killer (NK) cells, and production of interleukin-2 (IL-2) from splenocytes in tumor-bearing mice were measured. Meanwhile, the effects of ATF on 2,4-dinitrofluorobenzene (DNFB)-induced delayed type hypersensitivity (DTH) reaction and the sheep red blood cell (SRBC)-induced antibody response in naïve mice were also studied. RESULTS: ATF could not only significantly inhibit the growth of mice transplantable tumor, but also remarkably increase splenocytes proliferation, NK cells activity, and the level of IL-2 secreted by splenocytes in tumor-bearing mice, promote the DTH reaction and enhance anti-SRBC antibody level in naïve mice, which indicated that the ATF could improve both specific and non-specific cellular and humoral immune response. CONCLUSIONS: The antitumor activity of ATF might be achieved by improving immune response, and ATF could act as antitumor agent with immunomodulatory activity.

[Saponins from roots of Stephanotis mucronata].

OBJECTIVE: To study the chemical constituents from n-BuOH fraction of the roots of Stephanotis mucronata. METHOD: The compounds were separated by chromatographic methods. A combination of UV, MS, and NMR spectroscopic methods was applied to identify structure of these compounds. RESULT: Four oleane saponins were isolated and identified as sitakisoside VII (1), sitakisoside VI (2), sitakisoside II (3), and sitakisoside I (4). CONCLUSION: These four compounds were obtained for the first time from this plant.

Cynanbungeigenin C and D, a pair of novel epimers from Cynanchum bungei, suppress hedgehog pathway-dependent medulloblastoma by blocking signaling at the level of Gli.

Uncontrolled excessive activation of Hedgehog (Hh) signaling pathway is linked to a number of human malignant tumorigenesis. To obtain valuable Hh pathway inhibitors from natural product, in present study, a pair of novel epimers, Cynanbungeigenin C (CBC) and D (CBD) from the plant Cynanchum bungei Decne were chemically characterized by multiple spectroscopic data and chemical derivatization, and evaluated for their inhibition on Hh pathway. Mechanistically, CBC and CBD block Hh pathway signaling not through targeting Smo and Sufu, but at the level of Gli. In addition, both eipmers significantly suppress Hh pathway-dependent Ptch+/-; p53-/- medulloblastoma in vitro and in vivo. Furthermore, both CBC and CBD inhibited two Smo mutants induced Hh pathway activation, which suggested that they are potential compounds for the treatment of medulloblastoma with primary or acquired resistance to current Smo inhibitors. These results highlight the potential of CBC and CBD as effective lead compounds in the treatment of medulloblastoma and other Hh-dependent malignancy.

Immunological adjuvant effect of ginsenoside Rh4 from the roots of Panax notoginseng on specific antibody and cellular response to ovalbumin in mice.

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.

3 Beta-hydroxyolean-12-en-27-oic acid: a cytotoxic, apoptosis-inducing natural drug against COLO-205 cancer cells.

3Beta-hydroxyolean-12-en-27-oic acid (1), a biologically active, pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis, was found to be considerably more cytotoxic toward human colorectal carcinoma (COLO-205) and human cervical squamous carcinoma (HeLa) cells than its congener oleanolic acid (4). This suggests that the position of the COOH group significantly affects the cytotoxicity of oleanane-type pentacyclic triterpene carboxylic acids. To elucidate the underlying biological mechanism responsible for the cytotoxicity of 1, we investigated its growth-inhibitory effect on COLO-205 cells. Compound 1 induced a marked concentration- and time-dependent inhibition of cell proliferation, with the typical morphological characteristics of apoptosis, and under formation of DNA ladders in agarose-gel electrophoresis. Flow-cytometric analysis showed that the cell cycle of COLO-205 cells exposed to 1 was arrested in the G0/G1 phase. Also, 1 increased and decreased the expression of Bax and Bcl-2 proteins, respectively, and lowered the mitochondrial transmembrane potential (delta psi(m)). The peptidic caspase-3 inhibitor NH2-Asp-Glu-Val-Asp-CHO (at 1 microM) could increase the viability of COLO-205 cells previously treated with 1. These results indicate that 1 induces efficient cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering delta psi(m), and by activating the caspase-3 pathway.

Haemolytic activity and adjuvant effect of notoginsenoside K from the roots of Panax notoginseng.

Notoginsenoside K (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD50 value) being 318+/-13 microg/ml, on a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1, especially at a dose of 25 mug compared to an OVA control group (P<0.001). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (AlOH gel; P<0.01). These results suggest that 1 exhibits a slight haemolytic activity and a significant adjuvant effect on specific antibody and cellular response against OVA in mice.

Ginsenoside Rd from Panax notoginseng is cytotoxic towards HeLa cancer cells and induces apoptosis.

The saponin ginsenoside Rd (1), isolated from Panax notoginseng, is used for the treatment of cardiovascular diseases, inflammation, different body pains, trauma, and internal and external bleeding due to injury. In this study, we report that 1 inhibits the cell growth of human cervical cancer (HeLa) cells in a concentration- and time-dependent manner, with an IC(50) value of 150.5+/-0.8 mcirog/ml after 48 h of incubation. The drug-treated cells displayed features of apoptosis, including typical morphological characteristics and formation of DNA ladders, as evident from agarose-gel electrophoresis. Flow-cytometric analysis showed that the cell-cycle distribution of HeLa cells exposed to 1 is characterized by a decrease of the G(0)/G(1)-phase and an increase of the S-phase cells, respectively, in a dose-dependent manner. The apoptotic rate of HeLa cells treated for 48 h with 210 microg/ml of 1 was 35.8%. Further, 1 was found to increase the expression of Bax and to decrease the expression of Bcl-2 proteins, respectively, and to lower the mitochondrial transmembrane potential of HeLa cells. The caspase-3 inhibitor DEVD-CHO (at 2 microM) increased the viability of HeLa cells treated with 1. Taken together, our study suggests that ginsenoside Rd (1) significantly inhibits HeLa cell proliferation, and induces cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering the mitochondrial transmembrane potential, and activating the caspase-3 pathway. Thus, 1 could serve as a lead to develop novel chemotherapeutic or chemopreventive agents against human cervical cancer.

Induction of apoptosis in HeLa cells by 3beta-hydroxyurs-12-en-27-oic acid.

3Beta-hydroxyurs-12-en-27-oic acid (1), a pentacyclic triterpenoid isolated from the rhizomes of Astilbe chinensis, was structurally very similar to ursolic acid, with the only difference being the interchange of the COOH and Me group at C(14) and C(17). Ursane-type triterpene with a COOH group at C(14) is present in a limited number of natural resources. Compound 1 was found to exhibit more distinctive cytotoxicity toward human cervical squamous carcinoma (HeLa) cells than ursolic acid, suggesting that the position of the COOH group significantly affects the cytotoxicity of ursane-type pentacyclic triterpenes with a COOH group. To elucidate the underlying biological mechanism responsible for the cytotoxicity of 1, we investigated its growth-inhibitory and apoptosis-inducing effect on HeLa cells. Compound 1 induced a marked concentration- and time-dependent inhibition of cell proliferation with an IC50 value of 6.80+/-0.88 microg/ml following 48 h incubation. The drug-treated HeLa cells displayed typical morphological apoptotic characteristics and formation of DNA ladders in agarose-gel electrophoresis. Flow cytometric analysis showed that the cell cycle was arrested in G0/G1 phase by 1, and the apoptotic rate of HeLa cells treated for 48 h with 20 microg/ml of 1 was 21.08+/-2.14%. Also, 1 increased and decreased the expression of Bax and Bcl-2 proteins, respectively, and lowered the mitochondrial transmembrane potential (delta psi(m)). The peptidic caspase-3 inhibitor DEVD-CHO (NH2-Asp-Glu-Val-Asp-CHO, at 2 microM) could increase the viability of HeLa cells previously treated with 1. These results indicate that 1 induces efficient cell apoptosis through down-regulating Bcl-2 expression, up-regulating Bax expression, lowering delta psi(m), and by activating the caspase-3 pathway.

Two New Steroidal Aglycones from Roots of Cynanchun paniculatum.

Two new 13, 14/14, 15-disecopregnane-type skeleton C21 steroidal aglycones, neocynapanogenin G (1) and neocynapanogenin H (2), were isolated from the hydrolyzed extract of the CHCl3 soluble extract of the roots of Cynanchun paniculatum. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods, including 1D and 2D NMR spectroscopy. Compound 1 displayed signifidant inhibition of the Hedgehog signaling pathway in vitro.

Two New 8, 14-seco-pregnane Steroidal Aglycones froni Roots of Cynanchum bungei.

Two new 8, 14-seco skeleton C(21) steroidal aglycones, cynanbungeigenin A (1) and cynanbungeigenin B (2), were isolated from the hydrolyzed extract of the EtOAc soluble extract of the roots of Cynanchum bungei. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods, including 1D and 2D NMR spectroscopy.

Stephanthraniline A suppressed CD4(+) T cell-mediated immunological hepatitis through impairing PKCθ function.

Stephanthraniline A (STA), a C21 steroid isolated from Stephanotis mucronata (Blanco) Merr., was previously shown to inhibit T cells activation and proliferation in vitro and in vivo. The purpose of this study was to further evaluate the in vivo immunosuppressive activity of STA and to elucidate its potential mechanisms. The results showed that pretreatment with STA significantly attenuated concanavalin A (Con A)-induced hepatitis and reduced CD4(+) T cells activation and aggregation in hepatic tissue in mice. STA directly suppressed the activation and proliferation of Con A-induced CD4(+) T cells, and inhibited NFAT, NFκB and MAPK signaling cascades in activated CD4(+) T cells in vitro. Moreover, it was proved that STA inhibited T cells activation and proliferation through proximal T cell-receptor (TCR) signaling- and Ca(2+) signaling-independent way. The molecular docking studies predicted that STA could tight bind to PKCθ via five hydrogen. The further findings indicated STA directly inhibited PKCθ kinase activity, and its phosphorylation in activated CD4(+) T cells in vitro. Collectively, the present study indicated that STA could protect against CD4(+) T cell-mediated immunological hepatitis in mice through PKCθ and its downstream NFAT, NFκB and MAPK signaling cascades. These results highlight the potential of STA as an effective leading compound for use in the treatment of CD4(+) T cell-mediated inflammatory and autoimmune diseases.