ABSTRACT
ObjectiveTo investigate the effect of Yiqi Jiedu prescription-containing serum on the proliferation of medullary thymic epithelial cells （mTEC） and regulatory T （Treg） cells in myasthenia gravis （MG） patients with thymus hyperplasia. MethodAccording to serological methods，35 SD rats were adaptively fed for one week and randomized into the low-，medium-， and high-dose Yiqi Jiedu prescription groups，control group， and prednisone group，with seven rats in each group， which were then gavaged with the corresponding drugs for one week for preparing the drug-containing serum. The effect of Yiqi Jiedu prescription-containing serum at different concentrations on the proliferation of mTEC and Treg cells were determined by cell counting kit-8 （CCK-8） assay. Besides， the effect of mTEC and Yiqi Jiedu prescription-containing serum on Treg cell proliferation were observed through co-culture. ResultThymocytes were cultured for a period of time. Their mean positive rate revealed by flow cytometry using mTEC characteristic marker Ulex europaeus agglutinin Ⅰ （UEAI） was 92.54%. Treg cells were sorted by magnetic beads. The purity of Treg cells after repeated magnetic bead sorting was as high as 92%. mTEC and Treg cells showed high positive expression rates，and their cell purity met the requirements of subsequent experiments. When the concentration of Yiqi Jiedu prescription-containing serum was 2.5%-15%，it exhibited an inhibitory effect against mTEC and Treg cells. When the concentration was equal to or greater than 20%，it promoted cell proliferation，which was further enhanced with the extension of action time. The results after 48 h of culture showed that compared with the control group，prednisone and low-dose Yiqi Jiedu prescription had no significant effect on the proliferation of these two kinds of cells，but the medium- and high-dose Yiqi Jiedu prescription remarkably reduced their proliferation inhibition rate （P<0.01）. After co-culture with mTEC， the control group was not significantly different from the prednisone group and the low-dose Yiqi Jiedu prescription-containing serum group in the proliferation of Treg cells，while the medium- and high-dose Yiqi Jiedu prescription-containing serum groups significantly lowered the proliferation inhibition rate （P<0.01）. ConclusionYiqi Jiedu prescription-containing serum affects the proliferation of mTEC and Treg cells in MG patients with thymus hyperplasia. Compared with the solely cultured Treg cells isolated from MG patients，the Treg cells co-cultured with mTEC exhibit enhanced proliferation in MG patients，suggesting that mTEC can regulate the proliferation of Treg cells. This effect becomes more obvious after the intervention with Yiqi Jiedu prescription-containing serum，indicating that intervention effect of Yiqi Jiedu prescription on Treg cells can be produced during its treatment of mTEC， which may be one of the mechanisms of Yiqi Jiedu prescription-containing serum in alleviating MG.
ABSTRACT
Objective To observe the effects of Yiqi Jiedu prescription drug serum on the apoptosis of human renal cell carcinoma ACHN cells and Notch1 gene in Notch signal pathway. Methods The healthy SD female rats were selected, which were divided into 4 groups, 8 rats in each group: the rats were fed with the normal saline 10 ml·kg-1·d-1 (the blank serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 50 g·kg-1·d-1 (the high-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 25 g·kg -1·d -1 (the medium-concentration of Yiqi Jiedu prescription serum group); the rats were fed with the Yiqi Jiedu prescription decoctum of 12.5 g·kg-1·d-1 (the low-concentration medicated of Yiqi Jiedu prescription serum group); then the serum would be extracted 10 days later in each group. ACHN cells at exponential phase were cultured in the above 4 groups. Theproliferation of ACHN cells in each group was observed by using CCK-8 method. The apoptosis of ACHN cells was detected by using flow cytometry (FCM). The expression levels of Notch1 mRNA of ACHN cells in each groups were detected by using real time fluorescence quantitative polymerase chain reaction (RT-PCR). Results The inhibition rates of ACHN cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the blank serum group 24 h later were (12.34±4.25)％, (7.47±1.40)％, (2.52±0.62)％, (1.05±0.31)％, respectively (F= 15.04, P< 0.01); after 48 h, the inhibition rates were (24.20 ±2.41)％, (14.23 ±1.56)％, (5.08 ±1.34)％, (1.16 ±0.14)％, respectively (F=227.36, P<0.01);after 72 h, the inhibition rates were (32.16±2.45)％, (25.05±3.69)％, (10.29±2.76)％, (1.07± 0.71)％, respectively (F=110.51, P<0.01). The results showed that Yiqi Jiedu prescription drug serum could significantly inhibit the proliferation of ACHN cells in a dose-dependent manner. Besides, the inhibition rate differences at all time points of the high-concentration serum group (F= 31.44, P< 0.01), the medium-concentration serum group (F= 49.61, P< 0.01) and the low-concentration serum group (F= 68.78, P<0.01) were all statistically significant, and they were in a time-dependent manner. The apoptosis rate of cells in the high-concentration group, the medium-concentration group, the low-concentration group of Yiqi Jiedu prescription and the control serum group was (34.5±1.5)％, (24.4±1.5)％and (13.1±0.5)％, (5.2±0.3)％, respectively, and the differences were statistically significant (F = 1153.36, P < 0.01). The relative expression level of Notch1 mRNA of cells in the high-concentration group, the medium-concentration group, the low-concentration serum group of Yiqi Jiedu prescription and the control serum group was 0.213 ±0.032, 0.432 ±0.049, 0.781 ±0.018, 1.013 ±0.047, respectively, and the differences were statistically significant (F=270.60, P<0.01). Conclusion Yiqi Jiedu prescription drug serum can induce apoptosis of ACHN cells, and its mechanism may be related to the down-regulation of the expression level of Notch1 receptors.