OBJECTIVE:To investigate the long-term toxicity of repeated intramuscular injection of Escherichia coli O157∶H7 polysaccharide-conjugated vaccine(O157) in rats so as to provide safety evidence for clinical trials.METHODS:A total of 48 SD rats were randomly assigned to receive either 0.5 mL vaccine(containing 25 ?g polysaccharides) (immunization group,n=24) or phosphate buffered solution (PBS,control group,n=24) with the same volume for 3 times at a dose interval of two weeks.Sacrifice of 6 rats in each group were scheduled at 2 weeks after first immunization,and at 1,3,and 5 weeks after the third immunization,respectively for observation and determination of hematological and biochemical parameters,histopathology,specific antibody,myeloid tissue,the tissues in injection sites,etc.RESULTS:Compared with control group,immunization group showed no significant pathological change except the dynamic regular change of some hematological parameters induced by the immunization,and no immunologic system damage was observed.CONCLUSION:Repeated intramuscular injection with O157 vaccine in rats wouldn't cause overt toxicity and local irritation.

Paris saponin VII (PSVII), a bioactive constituent extracted from Trillium tschonoskii Maxim., is cytotoxic to several cancer types. This study was designed to explore whether PSVII prevents non-small-cell lung cancer (NSCLC) proliferation and to investigate its molecular target. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. In cultured human NSCLC cell lines, PSVII induces autophagy by activating AMPK and inhibiting mTOR signaling. Furthermore, PSVII-induced autophagy activation was reversed by the AMPK inhibitor compound C. Computational docking analysis showed that PSVII directly interacted with the allosteric drug and metabolite site of AMPK to stabilize its activation. Microscale thermophoresis assay and drug affinity responsive target stability assay further confirmed the high affinity between PSVII and AMPK. In summary, PSVII acts as a direct AMPK activator to induce cell autophagy, which inhibits the growth of NSCLC cells. In the future, PSVII therapy should be applied to treat patients with NSCLC.

This study aims to investigate the molecular mechanism of polyphyllin Ⅰ(PPⅠ) inhibiting proliferation of human breast cancer cells. Human breast cancer BT474 and MDA-MB-436 cells were treated with different concentrations of PPⅠ, and then the effect of PPⅠ on cell proliferation was detected by MTT assay, trypan blue dye exclusion assay, real-time cell analysis, and clone forming assay, respectively. The apoptosis was detected by Annexin V-FITC/PI staining and then analyzed by flow cytometry. The change of mitochondrial membrane potential was detected by flow cytometry after fluorescent probe JC-1 staining. Western blot was used to detect protein expression and phosphorylation. Molecular docking was performed to detect the binding between PPⅠ and EGFR. The affinity between PPⅠ and EGFR was determined by drug affinity responsive target stability assay. The results indicated that PPⅠ inhibited the proliferation and colony formation of BT474 and MDA-MB-436 cells in a time-and concentration-dependent manner. The PPⅠ treatment group showed significantly increased apoptosis rate and significantly decreased mitochondrial membrane potential. PPⅠ down-regulated the expression of pro-caspase-3 protein, promoted the cleavage of PARP, and significantly reduced the phosphorylation levels of EGFR, Akt, and ERK. Molecular docking showed that PPⅠ bound to the extracellular domain of EGFR and formed hydrogen bond with Gln366 residue. Drug affinity responsive target stability assay confirmed that PPⅠ significantly prevented pronase from hydrolyzing EGFR, indicating that PPⅠ and EGFR have a direct binding effect. In conclusion, PPⅠ inhibited the proliferation and induced apoptosis of breast cancer cells by targeting EGFR to block its downstream signaling pathway. This study lays a foundation for the further development of PPⅠ-targeted drugs against breast cancer.
Apoptosis ; Breast Neoplasms/genetics* ; Cell Line, Tumor ; Cell Proliferation ; Diosgenin/analogs & derivatives* ; ErbB Receptors ; Female ; Humans ; Molecular Docking Simulation