Study On The Intervention Of Cyanidin-3-O-glucoside On The Reproduction Poison Of Heavy Metal Cd In R2C Cells

According to the experiment in vitro, heavy metal Cd was used to establish male reproductive damage model on R2C cells, and then explore the intervention effect of Cyanidin-3-O-glucoside (C3G) on male toxicity caused by heavy metal Cd, and this study can provide basis for the development and utilization of Cyanidin-3-O-glucoside.In this study, the MTT method was used to determine the half inhibiting concentration of CdSO4 on R2C cells, and then different concentration of C3G were selected by MTT method to detect the influence of C3G on R2C cells exposed to heavy metal cadmium. Fluorescence microscopy was used to observe the morphological changes in R2C cells. Chemiluminescence (CL) was used to detect the changes of ROS levels in R2C cells. Flow cytometry (FCM) was selected to detect changes of mitochondrial membrane potential in R2C cells. The protein expression levels related with progesterone synthesis and antioxidation was detected by Western Blotting assay. Radioimmunoassav (RIA) was used to measure progesterone synthesis of R2C cells. Results showed that:Heavy metal Cd could inhibit the proliferation of R2C, and the half inhibitory concentration was (44.80±0.0479) ?mol/L. C3G could inhibit the decline of proliferation ability of R2C cells exposed to heavy metal Cd, and it presented a dose-response relationship when the concentration of C3G was between 5-80 ?mol/L. Microscope observation results showed that Cd could affect morphology of R2C cells. When R2C cells were stimulated by Cd for 24 h, cells becomes bright, round and small, the C3G could reduce the morphological damage of R2C cells exposed to heavy metal cadmium. ROS results showed that heavy metal Cd can improve production of ROS in R2C cells, and ROS production increased along with the extension of time, while the C3G could decrease the production of ROS in R2C cells exposed to heavy metal Cd, and the ROS would not increase with the extension of time. Flow cytometry instrument testing results showed that the heavy metal Cd could significantly induce mitochondrial damage, and C3G played a inhibitory effect to the damage of mitochondria in R2C cells exposed to heavy metal Cd. Western Blotting results showed that the StAR protein expression level of R2C cells only added heavy decreased, compared with the damage group, StAR protein expression level in R2C cells preliminary protected by C3G quantity increased. Protein SOD2 related with antioxidant was also tested, compared with normal group, SOD2 protein expression in R2C cells exposed to heavy metal Cd rised significantly, and after added the anthocyanins, SOD2 protein expression of the R2C cells is lower, and there were significant differences above comparison.C3G could remove excess reactive oxygen species(ROS) in R2C cells induced by heavy metal Cd. The drop rate of mitochondrial membrane potential could be lower according to the decrease of ROS in R2C cells. The results proved that C3G could inhibit the damage of mitochondria. C3G could inhibit the decrease of StAR (a key protein associated with progesterone synthesis pathways) protein expression level. Increasing the expression of StAR protein could promote the ability to transrer cholesterol from mitochondrial membrane outside to internal of mitochondrial membrane. and finnaly increased the amount of progesterone synthesis.