The Study On Mechanism Of Jia Kangning Capsule In TSHR Signal Transduction Pathways For FRTL-5Cells

Objective:1. To explore the effects of Jia Kangning on the TSHR intracellular signal transduction pathway.2. To investigat the effects of Jia Kangning on apoptosis.Methods:1. We use MTT method to detect the different concentration and time of Jia Kangning effects on FRTL-5cells, in order to determine the concentration and time of Jia Kangning.2. We used an inverted fluorescence microscope to see the form of FRTL-5cells after Jia Kangning intervented, and then used CCK-8to know FRTL-5cells proliferation after Jia Kangning intervented.3. We used flow cytometry to detect the apoptosis of FRTL-5cells after Jia Kangning intervented.4. We used the radioimmunoassay to detect the content of cAMP in the FRTL-5cells after Jia Kangning intervented.5. We used Western Blot to detect TSHR signal transduction pathway of FRTL-5cells after Jia Kangning intervented.Results:1.A dose-and time-dependent manner in Jia KangningThe Jia Kangning inhibit cell proliferation, with the more increasing concentration and extension of time, the stronger the reaction. Methimazole no use of inhibition of cell proliferation. Thus, we selected Jia Kangning and methimazole concentration and reaction time was3mg/ml of Jia Kangning, lOug/ml of methimazole, and the reaction time was24h.2. The effect of Jia Kangning on the proliferation of FRTL-5cells①The FRTL-5cells morphology after Jia Kangning intervented:baseline:The number of cells became small; slightly smaller volume;The died cell floated in culture medium. model group:The number of cells increased significantly; large cell volume, cell outline a clear, full shape; visible pseudopodia-like cytoplasmic processes; visible black dot-like particles in the cytoplasm; higher magnification clearly visible nucleus, nuclear cytoplasm ratio, the nucleus of a circle shape, you could see the nucleolus. Number of hyperthyroidism Ning cells reduced cell shrink-age significantly, smaller size; intracytoplasmic vacuoles appear; does not clearly distinguish between the nucleus and nucleolus. The number of cells was significantly more than methimazole; large cell volume, cell outline a clear, full shape; visible pseudopodia-like cytoplasmic processes; visible black dot-like particles in the cytoplasm; higher magnification clearly visible nucleus, nuclear cytoplasm ratio, nuclear circular, could see nucleoli.②The influence of Jia Kangning on the proliferation of FRTL-5cells. We used CCK-8method to detecte OD value in FRTL-5cells in each group:The model group (1.76±0.22) was significantly higher than the baseline group (0.41±0.04),P<0.01; the Jia Kangning group (0.12±0.01) was lower than the model group (1.76±0.22),P<0.01; the Jia Kangning group(0.12±0.01) was lower than the methimazole group (1.45±0.26),P<0.01; the difference of the methimazole group and the model group had no statistical significance (P>0.05).3. The effect of Jia Kangning on the apoptosis of FRTL-5cellsFlow cytometry was used to study the apoptosis of FRTL-5cells. According to the kit, the apoptosis can be divided into the early apoptosis and late apoptosis. In the early apoptosis rate, the model group (1.52±0.34) was lower than the baseline group (3.97±0.62),P<0.01; the Jia Kangning group (14.55±1.80) was significantly higher than the model group (1.52±0.34),P<0.01; the Jia Kangning group (14.55±1.80) was higher than the methimazole group(2.14±0.68),P<0.01; The difference of the methimazole group and the model group had no statistical significance (P>0.05). In the late apoptotic rate, no statistically significant difference between the various groups (P>0.05). In the total cell apoptosis rate, the Jia Kangning group (35.63±8.70) was significantly higher than the model group (10.28±2.52)、the methimazole group (9.05±1.96),P<0.01; the difference of the methimazole group and the model group had no statistical significance (P>0.05).4. The effect of Jia Kangning on the cAMP of FRTL-5cellsBy the method of radioimmunoassay in cAMP group of FRTL-5in supernatant was detected, the model group(2.80±0.13pmol/ml) was higher than the baseline group (1.88±0.09pmol/ml),P<0.01; the Jia Kangning group (2.54±0.10pmol/ml) was lower than the model group (2.80±0.13pmol/ml),P<0.05; the Jia Kangning (2.54±0.10pmol/ml) was lower than the methimazole group(2.79±0.07pmol/ml), P<0.05; the difference of the methimazole group and the model group had no statistical significance (P>0.05).5. The effect of Jia Kangning on TSHR signal transduction pathway for FRTL-5cells(1)The effect of Jia Kangning on PKAc expression for FRTL-5cells:We used Western Blot to detect the expression of p-PKAc/PKAc protein in FRTL-5cells in each group, the model group (0.91±0.03) was significantly higher than the baseline group (0.43±0.15), P<0.01; the Jia Kangning group (0.56±0.10) was lower than the model group (0.91±0.03), P<0.01; the methimazole group (0.60±0.14) was lower than that of model group (0.91±0.03) P<0.05.(2)The effect of Jia Kangning on Erkl/2expression for FRTL-5cells:We used Western Blot to detect the expression of p-Erkl/2/Erkl/2protein in FRTL-5cells in each group, the model group (0.86±0.05) was significantly higher than the baseline group (0.36±0.17), P<0.01; the Jia Kangning group (0.28±0.13) was significantly lower than the model group (0.86±0.05), P<0.01; the Jia Kangning group (0.28±0.13) was lower than the methimazole group (0.82±0.07), P<0.01; the difference of the methimazole group and the model group had no statistical significance (P>0.05).③The effect of Jia Kangning on PKCδ expression for FRTL-5cells:We used Western Blot to detect the expression of p-PKCδ/PKC δ protein in FRTL-5cells in each group, the model group (0.88±0.13) was significantly higher than the baseline group (0.47±0.33), P<0.01; the Jia Kangning group (0.56±0.16) was lower than the model group (0.88±0.13), P<0.05; the difference of the methimazole group and the model group had no statistical significance (P>0.05).④The effect of Jia Kangning on Akt expression for FRTL-5cells:We used Western Blot to detect the expression of p-Akt/Akt protein in FRTL-5cells in each group, the model group (0.73±0.12) was significantly higher than the baseline group (0.30±0.12), P<0.01; the Jia Kangning group (0.30±0.12) was significantly lower than the model group (0.73±0.12), P<0.01; the Jia Kangning group (0.30±0.12) was lower than the methimazole group (0.68±0.13), P<0.01; the difference of the methimazole group and the model group had no statistical significance (P> 0.05).⑤The effect of Jia Kangning on mTOR expression for FRTL-5cells:We used Western Blot to detect the expression of p-Akt/Akt protein in FRTL-5cells in each group, the model group (0.75±0.08) was significantly higher than the baseline group (0.26±0.16), P<0.01; the Jia Kangning group (0.35±0.21) was lower than the model group (0.75±0.08), P<0.05; the difference of the methimazole group and the model group had no statistical significance (P>0.05).Conclusion:1. Jia Kangning can down regulate the TSHR signal transduction pathway, such like PKA、Erk1/2、PKC8、Akt and mTOR, which may be a key factor in Graves’ disease.2. Jia Kangning can promote apoptosis significantly, and it is needed further study in the relationship of TSHR signal transduction pathways and apoptosis.