Inhibition of MAPK pathway-induced apoptosis in renal cancer cells by ethanol extraction of Dysosma versipellis / 中国药理学通报

Aim To explore the effects of ethanol extraction of Dysosma versipellis on the proliferation and apoptosis of renal clear cell carcinoma OS-RC-2 cells and the underlying mechanism. Methods After treated with Dysosma versipellis, the proliferation of OS-RC-2 cells was detected by the CCK-8 assay and clone formation assay. The migration rate of cells was detected by thewound healing assay and Transwell assay. The level of ROS was detected by the reactive oxygen detection kit. The common targets between Dysosma versipellis and ROS were obtained by the network pharmacology. The above common targets were analyzed by KEGG. The apoptosis rate and cell cycle were detected by the flow cytometry, and the key proteins of MAPK signaling pathway and the levels of apoptosis related proteins were measured by Western blot. Results The results showed that Dysosma versipellis significantly inhibited the cell viability and migration ability of 0S-RC-2 cells, and up-regulated the level of ROS. Network pharmacology analysis showed a total of 165 common targets between Dysosma versipellis and ROS. KEGG analysis of the common targets revealed that there were significant changes in the MAPK signaling pathway. The results of Western blot showed that after treated with Dysosma versipellis, the protein level of JNK and the ratio of p-ERK/ERK were down-regulated. Besides, the protein level of caspase-9 and Bcl-2 declined, while the levels of cleaved caspase-9 and Bax were promoted. The flow cytometry results showed that Dysosma versipellis could significantly promote the apoptosis rate,down-regulate the cells in Gl-phase,while up-regulate the cells in G2/M-phase. The results of the rescue experiment showed that co-administration of NAC and Dysosma versipellis could significantly reverse the cell viability and apoptosis rate, the level of apop-totic related proteins, as well as the protein levels of MAPK pathway,when compared to treated with Dysosma versipellis alone in OS-RC-2. Conclusion In summary, Dysosma versipellis may inhibit the MAPK signaling pathway via the changes in ROS,further promoting apoptosis rate and decline cell proliferation in OS-RC-2 cell line.

Maternal hypothyroidism during pregnancy alters the function of the retinol-binding protein 4-mediated mitochondrial permeability conversion pore in the kidneys of offspring rats

OBJECTIVES To determine the role of the RBP4/PiC/SIRT3 signaling pathway in the opening of the mitochondria permeability transition pore (mPTP) in offspring rats with hypothyroidism during pregnancy. METHODS Sixty Sprague-Dawley (SD) rats were employed in this study. Pregnancy was deemed successful when a sperm was found in the uterus. After one week of pregnancy, offspring rats were divided into the following groups: overall hypothyroidism group (OH group), subclinical hypothyroidism group (SCH group), and normal control group (CON group). The establishment of the hypothyroidism model was confirmed when the serum thyroid stimulating hormone (TSH) levels were higher than normal value and TT4 level was within the normal range. The renal mitochondria of offspring rats were extracted on the 14th postnatal day (P14) and 35th postnatal day (P35). RESULTS At P14, no significant differences in the degree of mPTP opening and expression of phosphoric acid carrier vector (PiC) were detected between the rats in the OH group and the SCH group. However, the expression level of silent mating-type information regulation 3 homolog (SIRT3) was markedly reduced. Retinol-binding protein 4 (RBP4) expression increased in the rats from the OH group, relative to that in those from the SCH group. At P35, the degree of mPTP opening and the expression levels of PiC and RBP4 in the OH group were higher than those in the SCH group. However, SIRT3 expression in the OH group was lower than that observed in the SCH group. CONCLUSION RBP4 plays an important role in early renal mitochondrial damage and renal impairment in rats suffering from hypothyroidism during pregnancy. The RBP4/PiC/SIRT3 pathway is thus involved in the opening of the renal mPTP in offspring rats with hyperthyroidism.

Effects of transfection of hGM-CSF gene into HepG2 cells mediated by hydroxyapatite nanoparticles on the growth of HepG2 cells / 肿瘤

Objective: To observe the impact of transfection of human granulocyte macrophage colony stimulating factor (hGMCSF) mediated by hydroxyapatite(HA) nanoparticles on the growth of HepG2 cells in order to lay the foundation for further studying gene-modified tumor vaccines of hepatoma in clinic. Methods: The proliferation of HepG2 cells was measured by MTT assay. The plasmids containing hGM-CSF gene were transfected into HepG2 cells mediated by HA nanoparticles. Subsequently, resistant cells were selected by G418 screening. The monoclonal cells were selected by limiting dilution method. RT-PCR was used to identify the integration of hGM-CSF into HepG2 cells and transcription of hGM-CSF. ELISA was performed to detect the level of secreted hGM-CSF in cultured medium and the lasting time. The effect of hGM-CSF on cell cycle and apoptosis were assessed by flow cytometry (FCM) analysis. Results: MTT assay demonstrated that Nano-HA suspension liquid had no significant effects on the growth of HepG2 cells at the concentration below 80 μg/mL. RT-PCR demonstrated the hGM-CSF gene was successfully integrated and steadily expressed in the transfected HepG2 cells. ELISA indicated stable secretion of hGM-CSF from the stable transfected HepG2 cells [mean (216.22 ± 45.78) ng/106 cells per 24 h]. FCM analysis showed that hGM-CSF had no significant effects on the cell cycle and apoptosis of HepG2 cells. Conclusion: hGM-CSF gene could be safely transfected and stably expressed in HepG2 cells mediated by HA nanoparticles. Transfection of hGM-CSF gene mediated by HA nanoparticles has no effects on the growth of HepG2 cells. This study may lay a foundation for the further study of gene-modified hepatoma vaccines.