| Renal cell carcinoma is urinary malignant tumor which incidence after the bladder cancer. Although the molecular biological research on the pathogenesis of renal cell carcinoma has made great progress, but the existing clinical chemotherapy and expensive targeted therapy still have considerable toxicity and multi drug resistance problem, so the current treatment is very limited. Recent studies reported that Notch signaling pathway is an important target for regulation of renal cell carcinoma tumor growth, and PI3K/Akt signaling pathway is a key target for malignant tumor cell apoptosis. Previous research team found, Ruanjian Sanjie capsule can inhibit the growth of animal tumor cells, so the topic based around what kind of role in Notch and PI3K/Akt play of Ruanjian Sanjie capsule, then what the internal connection and mechanism, at the molecular level to explore whether this Notch and PI3K/Akt interactions can regulate apoptosis in renal cell carcinoma. Opened a new way for traditional Chinese and Western medicine treatment for renal cell modern research.Objective:This study investigates the effects of Ruanjian Sanjie capsule medicated serum and pangolin protein extract on human renal clear cell carcinoma 7860 cells inhibits proliferation and induce apoptosis, the research from the molecular biology, Ruanjian Sanjie capsule medicated serum and pangolin protein extracts by regulating Notch and PI3K/Akt signaling pathway promotes cell apoptosis, and the mechanism of interaction between Notch and PI3K/Akt signal pathway inhibition cell growth.Methods:The experiment is divided into four parts:(1) With Ruanjian Sanjie capsule rats serum was obtained after 7 days, with CCK8 method observe the effect of inhibitory to cell proliferation that serum containing different concentration on human renal clear cell carcinoma in 7860 and renal proximal tubule epithelial cell line HKC cells 24h,48h,72h; effect of different concentration of Ruanjian Sanjie capsule medicated serum on apoptosis and cycle of 7860 and HKC by flow cytometry; observe 7860 apoptosis morphology by AnnexinV-FITC/PI double staining with confocal laser scanning microscopy.(2) The different doses of Ruanjian Sanjie capsule containing serum for 7860 48h cells, Western blot and RT-PCR detection of Notchl, NICD, Akt, p-Akt, Bcl-2, Bax, protein and mRNA; through confocal laser scanning microscopy observation of Bcl-2, Bax protein fluorescence intensity and nuclear displacement.(3) We block Notch and PI3K/Akt signal pathway, Western blot and RT-PCR detection change of Notchl, NICD, Akt, p-Akt, Bcl-2, Bax protein and mRNA, then effect of 7860 cell apoptosis by flow cytometry.(4) Observation inhibition of cell proliferation that different concentrations of pangolin protein extracts obtained 7860 cells for 24h,48h,72h by CCK8; detection effect on apoptosis and cell cycle by flow cytometry; with confocal laser scanning microscopy observation 7860 apoptosis morphology by AnnexinV-FITC/PI double staining method; 100 ug/ml pangolin protein extract obtained 7860 cells for 48h, Western blot and RT-PCR detection of Notchl, NICD, Akt, p-Akt, Bcl-2, Bax protein and mRNA; through the confocal laser scanning microscopy observation of Bcl-2, Bax protein fluorescence intensity and nuclear displacement.Results:1. The first experiment(1) CCK8 method was used to detect the cell proliferation activity, effect of proliferation of various doses of Ruanjian Sanjie capsule serum containing in renal clear cell carcinoma 7860 cells for 24,48,72 hours, inhibition of 7860 cells with obvious time and dose dependence.(2) Cell apoptosis rate by flow cytometry, effect of the doses of Ruanjian Sanjie capsule serum containing 7860 cells for 48h, the apoptosis rate was significantly higher than the control group (P< 0.05).(3) Cell cycle by flow cytometry, detect the doses of Ruanjian Sanjie capsule serum containing 7860 cells for 48h, the ratio of G0/G1 cell stage increased significantly than the negative control group (P< 0.05), cell differentiation was arrested in G0/G1 phase.(4) By confocal laser scanning microscopy, Ruanjian Sanjie capsule serum containing 7860 cells for 48h,compared with the negative control group, FITC and PI were significantly high dye concentration, each dose group cells appeared apoptosis in different early, middle, late phase, a typical apoptotic morphology.(5) The no effect of inhibition of proliferation or induce apoptosis in doses of Ruanjian Sanjie capsule drug serum human kidney HKC cells.2. The second experiment(1) Ruanjian Sanjie capsule serum and elemene injection on 7860 cells for 48h, decrease the level of Notchl and Akt expression(P<0.05).(2) Ruanjian Sanjie capsule serum and elemene injection on 7860 cells for 48h, reduce the level of Notchl, NICD, p-Akt, Akt proteins expression significantly (P<0.05).(3) Ruanjian Sanjie capsule serum and elemene injection on 7860 cells for 48h, reduce the level of Bcl-2 gene expression, and regulation the level of Bax gene significantly (P<0.05).(4) Ruanjian Sanjie capsule serum and elemene injection on 7860 cells for 48h, decrease the levels of Bcl-2 protein expression (P<0.05), increase level of Bax and protein expression significantly (P<0.05).(5) By confocal laser scanning microscopy detection Ruanjian Sanjie capsule serum and elemene Injection on 7860 cells for 48h, with the increasing concentration dose, Bcl-2 protein fluorescence intensity gradually weakened, Bax protein increased gradually.3. The third experiment(1) The transfection of siNotchl or DAPT(the Notch pathway blocker) and Ruanjian Sanjie capsule serum on 7860 cells 48h, decrease level of p-Akt protein expression significantly than single use drug serum (P<0.05).(2) The transfection of siAkt or LY2940029(PI3K/Akt pathway blocker) and Ruanjian Sanjie capsule serum on 7860 cells 48h, decrease the levels of NICD protein expression significantly than single use of drug serum (P< 0.05).(3) The transfection of siNotchl, siAkt or DAPT, LY294002 and Ruanjian Sanjie capsule serum on 7860 cells 48h,the apoptosis increased significantly than single use serum.4. The forth experiment(1) Pangolin protein extract and elemene injection inhibit human renal clear cell carcinoma 7860 cell proliferation obviously time and dose dependence.(2) Cell apoptosis rate by flow cytometry, different concentrations of pangolin protein extract and elemene injection on 7860 cells for 48h, the apoptosis rate was significantly higher than the control group (P< 0.05).(3) By confocal laser scanning microscopy, with the increasing of pangolin and elemene medicine concentration, red, green fluorescence gradually increased, and changes of apoptosis morphology.(4) Pangolin extracts and elemene injection protein can significantly decrease the level of Notchl,Akt, Bcl-2 expression.(5) Pangolin extracts and elemene injection protein can significantly reduce the level of Notchl, NICD, p-Akt, Akt, Bcl-2 expression, increase Bax protein expression.(6) By confocal laser scanning microscopy detection pangolin protein extract and elemene Injection on 7860 cells for 48h, Bcl-2 protein fluorescence intensity gradually weakened, Bax protein increased gradually.Conclusion:1. Ruanjian Sanjie capsule medicated serum can inhibit the proliferation of human renal clear cell carcinoma 7860 cells and induced apoptosis, cell cycle arrest in G0/G1 phase.2. Ruanjian Sanjie capsule serum can inhibit Notch and PI3K/Akt signal pathway of 7860 cells, reduced Bcl-2, upregulated the levels of Bax expression, thus promoting apoptosis.3. Block Notch signal pathway can decrease the level of p-Akt protein expression, block PI3K/Akt signaling reduce the NICD protein, and block Notch or PI3K/Akt signal pathway can inhibition of cell proliferation, induce apoptosis.4. Notch and PI3K/Akt signal pathways can activate mutually.5. Pangolin protein extract and elemene injection can inhibit human renal clear cell carcinoma 7860 cell proliferation, induction of apoptosis, arrest of cell differentiation, and inhibition the activity of Notch and PI3K/Akt signaling pathway, lowered Bcl-2,upregulated the Bax expression, promote apoptosis of tumor cells.
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