Inhibitory Effect Of Flavonoids From Semen Cuscutae On Rat Germ Cell Apoptosis In Vitro

Backgroud The quality and quantity of male semens have fallen dramatically over half century. Many aspects have been proved associated with environmental pollution. Germ cells are triggered into apoptosis by a wide variety of stimuli. How to abate or block the damage of male fertility by pollution became a vital problem. Studying of germ cell apoptosis has been transferred from death receptor pathway to mitochondrial pathway. Free radical medicine develop rapidly along with the highly concern of the mitocondrial pathway involved in apoptosis. Studying of germ cell apoptosis develop quickly along with the mecalism of fudmental theories. The key function of mitochondria is energy production through oxidative phosphorylation (OxPhos) and lipid oxidation. Furthermore,the mitochondrion is emerging as a crucial control point in the induction of apoptosis. As flavonoids from Semen Cuscutae promote fertility along with delay aging, suggest flavonoidsfrom Semen Cuscutae protect germ cell from apoptosis maybe involved in eliminate ROS and protect mitochondria. Since the traditional methods in study of Chinese herbs are complication, we applied seminiferous tissue culture in vitro to detect the antioxidant-mediated protection of apoptosis. The outcome of our experiment will provide new theories in explaining traditional functions of flavonoids from Semen Cuscutae.Objective 1) Establishing seminiferous epithelium culture system;2)Studying the inhibitory effect of flavonoids from Semen Cuscuta on germ cells apoptosis via pathological and molecular biological methods;3) Investigating the relationship among anti-oxidant action, maintenance of mitocondria function and inhibitory effect of flavonoids from Semen Cuscutae on germ cell apoptosis.Material and Methods Adult male Sprague-Dawley rats at 2-3 months of age were used and they were housed in a standard animal facility with constant temperature(22C)and photoperiod(12h: 12h),with free access to water and food. Use microdissection techniques, seminiferous tubules of rats were cultured.testis chocks of male BALB/c mouses were used in tissue blocks culture. The samples were divided into high dose concentration, low dose concentration, control and solvent. Each group was incubated fordifferent lengths of time(24, 48, and 72h n=12). The samples were fixed at the checkpoint, and the histology of the sample were observed by HE stain. The apoptotic germ cells were detected by TUNEL (TdT-mediated dUDP-Xnick end labeling) method, FACS(Fluorescence-Activated Cell Sorting), AO/EB double staining and DNA ladder ;The viability of germ cells were measured by MTS and PCNA (proliferating cell nuclear antigen); The anti-oxidant ability of germ cells were evaluated by MDA, ROS( reactive oxygen), T-AOC (total anti-oxidant capacity) and SOD;The mitochondrial transmembrane potential (MTP) change of germ cells were recorded by mitocondrial probe Rhol23.ResultsGerm cell apoptosis:There was significant difference in the number of apoptotic cells between treated groups and control groups (P<0.01). The differences in apoptotic cell numbers between the high dose treated group and low dose treated group are significant (P<0. 01).Anti-oxidative ability: There was significant difference in anti-oxidative ability between treated groups and control groups (P<0. 01).PCNA expression: There was significant difference in PI betweentreated groups and control groups (P<0.01).MTS anlysis: There was significant difference in viability between treated groups and control groups (P<0. 01).Conclusion1)semniferous epithelium cultured in medium without serum, which could induce oxidative activation and increase permeability of mitochondrial membrane. The apoptosis of germ cell may partially via mitochondrial pathway.2)Flavonoids from Semen Cuscutae are effective antioxidant, which can inhibit germ celhs apoptosis and protect the mitochondria failure.3)Flavonoids from Semen Cuscutae can elevate germ cells viability and promote proliferation of germ cells.