Artemiprinolides A-M, thirteen undescribed sesquiterpenoid dimers from Artemisia princeps and their antihepatoma activity.

Bioassay-guided investigation of the active fraction of Artemisia princeps led to 13 undescribed sesquiterpenoid dimers, artemiprinolides A-M (1-13), together with 11 known ones (14-24). Their structures were elucidated by comprehensive spectroscopic data and absolute configurations were assigned based on single crystal X-ray diffraction data and ECD calculations. Structurally, all compounds were postulated to be derived from the Diels-Alder cycloaddition. The isolated dimers except 11 and 15 were assayed for their cytotoxicity against HepG2, Huh7, and SK-Hep-1 cell lines, of which four compounds (3, 13, 17, 18) exhibited obvious cytotoxicity with IC50 values ranging from 8.8 to 20.1 µM. Interestingly, the most active compounds 1 and 16 manifested significant cytotoxicity on the three tested hepatoma cell lines with IC50 values of 5.4, 4.1 (HepG2), 7.7, 5.6 (Huh7), and 11.8, 15.7 µM (SK-Hep-1), respectively, which were better than sorafenib. Compound 1 dose-dependently inhibited cell migration and invasion, and significantly induced the HepG2 cell arrest in G2/M phase by downregulating cdc2 and pcdc2 and upregulating cyclinB1; and induced apoptosis by downregulating Bcl-2 expression and upregulating Bax level. The molecular docking study implied that the carbonyl at the C-12' of 1 had a strong binding affinity with PRKACA.

Artemidubolides A-T, cytotoxic unreported guaiane-type sesquiterpenoid dimers against three hepatoma cell lines from Artemisia dubia.

A random bioassay revealed that the EtOH extract and EtOAc fraction of Artemisia dubia Wall. (Asteraceae) exhibited cytotoxic activity against HepG2 cells with inhibitory ratios of 57.1% and 84.2% at a concentration of 100.0 µg/mL. Bio-guided isolation combined by LC-MS-IT-TOF analyses of the active fractions led to the isolation of 20 previously undescribed guaiane-type sesquiterpenoid dimers named artemidubolides A-T (1-20). Their structures and the absolute configurations were determined by comprehensive spectral analyses, comparison of the experimental and calculated ECD spectra, and seven compounds (artemidubolides A, B, D, F, K, O and R) were confirmed unequivocally by single crystal X-ray diffraction analysis. Structurally, artemidubolides A-Q were [4 + 2] Diels-Alder adducts of two monomeric guaianolides, and artemidubolides R-T were linked though an ester bond. All the isolated compounds were evaluated for their hepatomatic cytotoxicity against HepG2, Huh7, and SK-Hep-1 cell lines to demonstrate that 18 compounds exhibited obvious cytotoxicity against three tested hepatoma cell lines with IC50 values in the range of 5.4-87.6 µM. Importantly, artemidubolides B, D, and M exhibited hepatoma cytotoxicity with IC50 values of 5.4, 5.7, and 9.7 (HepG2), 8.2, 4.3, and 12.2 (Huh7), and 13.4, 8.4, and 12.9 µM (SK-Hep-1), respectively. Mechanism investigation in HepG2 cells suggested the most active artemidubolide D dose-dependently inhibited cell migration and invasion, induced G1/M cell cycle arrest by down-regulating proteins CDK4, CDK6 and CyclinD1 and up-regulating the level of protein P21; and induced apoptosis by down-regulated of PARP-1 and BCL-2 expression and up-regulating Bax and cleaved PARP-1 levels.

Effect of Electroacupuncture on Spermatogenesis in Rats with Oligozoospermia of Insufficiency of Shen (Kidney) Essence Syndrome.

OBJECTIVE: To assess the effect of electroacupuncture (EA) on expression of cytoskeletal proteins from Sertoli cells (SCs) and spermatogenesis in rats with oligozoospermia of insufficiency of Shen (Kidney) essence syndrome (OIKES). METHODS: Twenty healthy male Sprague-Dawley rats were randomly assigned to four groups using a random number table: control, tripterygium glycosides (TG) treatment, sham and EA groups (n=5 in each group). A rat model of OIKES was established by oral gavage with TG. The EA group was treated with TG and received EA at Shenshu (BL 23) and Zusanli (ST 36) acupoints for 20 min, once daily for 30 days, while the sham group received EA at identical acupoints with skin penetration without stimulation. After 30 days, the final body weight and coefficients for the testis and epididymis were calculated and sperm parameters were measured. Immunohistochemical analyses were performed to detect expression of vimentin and α-tubulin in SCs and proliferating cell nuclear antigen (PCNA) immunoreactivity in germ cells. Apoptosis in germ cells was quantified by the transferase biotin-dUTP nick end labeling assay. RESULTS: Compared with the control group, the final body weight and testis/epididymis coefficients of rats in the TG-treated group were not significantly different, but the sperm count and motility were lower (P<0.05). Expressions of vimentin and α-tubulin were also significantly weaker (P<0.01). The PCNA immunoreactivity of germ cells was decreased (P=0.059), whereas the apoptotic index of germ cells was increased significantly (P<0.01). In contrast, EA at BL 23 and ST 36 acupoints significantly improved the final body weight as well as the sperm count, concentration and motility (P<0.01 or P<0.05). EA increased expression of vimentin and α-tubulin in SCs markedly, and significantly enhanced PCNA immunoreactivity with decreased apoptosis in germ cells (P<0.01 or P<0.05). CONCLUSIONS: EA at BL 23 and ST 36 acupoints has protective effects on spermatogenesis in rats with OIKES. This effect seems to be achieved by attenuating TG-induced disruption of cytoskeletal protein in SCs.

[Differentially expressed proteins in the penile tissue of rats with compound stress-induced ED after intervention with Yimusake Tablets: A bioinformatics analysis].

OBJECTIVE: To search for specific protein makers and target proteins for intervention with Yimusake Tablets (YT) in the penile tissue of rats with ED induced by compound cold stress and explore the molecular mechanisms underlying the development and progression of ED. METHODS: Eighty adult male rats were screened and divided into three groups, normal control (n = 10), ED model control (n = 15) and YT intervention (n = 15). The model of compound cold stress-induced ED was established in the latter two groups, and meanwhile the animals in the YT intervention group were treated with oral YT for 2 weeks. After that, proteins were extracted from the penile tissues of the rats for screening and identification by iTRAQ labeling combined with LC-MS-MS proteomics, and the IPA bioinformatics software was used for analysis of differentially expressed proteins. RESULTS: A total of 48 differentially expressed proteins were identified from the penile tissue of the ED model controls, of which 18 were associated with endothelial function, 5 with smooth muscle activity and 4 with inflammation, involving the biological processes of glucose metabolism and alcohol catabolism and the signaling pathways of glucose metabolism, calcium and RXR activation. In comparison, 29 differentially expressed proteins were identified from the rats in the YT intervention group, of which 5 were associated with endothelial function, 1 with smooth muscle activity and 4 with inflammation, involving the biological processes of glucose metabolism, vasodilation and acute-phase response and the signaling pathways glucose metabolism, RXR activation and acute-phase response. Seven ED-associated candidate biomarkers were obtained from the differentially expressed proteins in the ED model control and YT intervention group, including Collagen alpha-1(III) chain（COL3α1）, Collagen alpha-1(I) chain（COL1α1）, Collagen alpha-2(I) chain（COL1α2）, Glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, T-kininogen 1（MAP1）,Biglycan（BGN）, and Myosin-11（MYH11）. CONCLUSIONS: Changes of vascular endothelial and smooth muscle functions in the penile tissue are likely to be the key mechanisms underlying the development and progression of compound stress-induced ED, which is also associated with inflammation as well as the interaction of the identified differentially expressed proteins and their participation in the relevant signaling pathways. The 7 proteins obtained can be used as the markers of compound stress-induced ED in the rat penile tissue, of which MAP1, GAPDH, BGN and MYH11 may serve as target proteins for YT intervention.

Effects of brucine on vascular endothelial growth factor expression and microvessel density in a nude mouse model of bone metastasis due to breast cancer.

OBJECTIVE: To study the effects of brucine on vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in a nude mouse model of bone metastasis due to breast cancer, and to assess the possible antitumor mechanism of brucine. METHODS: A syringe needle was used to directly inject 0.2 mL monoplast suspension (with 2×10(5) human breast cancer cells contained) into the bony femoral cortex of the right hind leg for modeling. Twenty-five nude mice were randomized into five groups and administered with an intraperitoneal injection of saline or drug for 8 consecutive days: model group (0.2 mL normal saline), low-dose brucine group (1.73 mg·kg(-1)), medium-dose brucine group (3.45 mg·kg(-1)), high-dose brucine group (6.90 mg·kg(-1)), and thalidomide group (200 mg·kg(-1)). Diet and activity were recorded, and the tumors were harvested 5 weeks later. The percentage of VEGF-positive cells was determined with hematoxylin and eosin staining and immunohistochemical staining, and MVD expression was determined by optical microscopy. RESULTS: The VEGF expressions in brucine- or thalidomide-treated mice were significantly reduced as compared with mice in the model group (P <0.01). There were no significant difference between the high-dose brucine group and the thalidomide group (P >0.05). Significant difference was between the high- and low-dose brucine group P<0.05). Further, VEGF expression was significantly increased in the low- and medium-dose brucine groups compared with the thalidomide group (P <0.05). The MVD values in the three brucine and thalidomide groups were significantly lower than that in the model group (P <0.01). The MVD values in the medium- and high-dose brucine groups were not significantly different from those in the thalidomide group (P >0.05), while the MVD value showed a significant increase in the low-dose group compared with the thalidomide group (P <0.05). CONCLUSION: Brucine could inhibit the growth of breast cancer to bone metastases, possibly by inhibiting tumor angiogenesis.