Effects Of The Prescription Of Invigorating Kidney And Resolving Phlegm And Removing Blood Stasis On Follicular Development And Testicular Morphology In TX Mice With Wilson Disease

1 Objective1.1 To observe the effects of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on follicular growth and development,and ovarian local factor BDNF in TX mice with Wilson’s disease;and further to investigate the correlation between ovarian local regulator BDNF and follicular cell proliferation and apoptosis.1.2 To explore the relationship between the improvement of follicular dysplasia and BDNF in TX mice by the herbs of invigorating kidney and resolving phlegm and removing blood stasis,and to elucidate the role of BDNF in the proliferation and apoptosis of follicular cells,so as to provide some experimental basis for the treatment of women with Wilson’s disease with abnormal reproductive symptoms by the herbs of invigorating kidney and resolving phlegm and removing blood stasis.1.3 Use animal experiments to explore whether the testicular tissue of TX mice was damaged in morphology and structure,and the herbs of invigorating kidney and resolving phlegm and removing blood stasis was used to intervene the phenomenon in order to clarify whether the herbs of invigorating kidney and resolving phlegm and removing blood stasis had protective effect on testicular tissue of TX mice,and to provide evidence for the treatment of male with Wilson disease with abnormal reproductive symptoms.2 Methods2.1 Genotype DetectionGenome was extracted from toes of 7-10 days mice,and purified DNA was obtained.The samples were amplified by PCR according to relevant primers.The amplified products were obtained by pre-denaturation,denaturation,annealing,extension,and repair.The amplified products were purified and recovered by gel cutting and verified by Sanger sequencing.2.2 Experimental Research on Female MiceSeventy-five SPF grade C3He-ATP7b^tx-j female mice（001576）were fed for one week and were randomly divided into five groups（n=15）:blank group,Wilson disease model group,DMSA group,the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,and DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group.The dosage of mice was converted by body surface area,and the ratio was 1:9 according to the daily dosage of adults.Mice in each medication group were intragastrically administered with 20 ml of liquid/kg body weight in the morning,while mice in the blank group and Wilson disease model group were intragastrically administered with physiological saline of equal volume once a day for two weeks.On the morning of the 15th day,all mice were injected with PMSG,BrdU 24 hours later and hCG 48 hours later.HE staining was used to observe the development of follicles at all levels;ultrastructural changes of granulosa cells and follicular cells in ovarian tissue were observed by electron microscopy;and the correlation between BDNF and follicular cell proliferation and apoptosis was analyzed by immunohistochemistry combined with fluorescence and TUNEL.2.3 Experimental Research on Male MiceFifty SPF grade C3He-ATP7b^tx-j male mice（001576）were fed for one week and were randomly divided into five groups（n=10）:blank group,Wilson disease model group,DMSA group,the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,and DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group.The dosage of mice was converted by body surface area,and the ratio was 1:9 according to the daily dosage of adults.Mice in each medication group were intragastrically administered with 20 ml of liquid/kg body weight in the morning,while mice in the blank group and Wilson disease model group were intragastrically administered with physiological saline of equal volume once a day for two weeks.At the end of the gavage cycle,the mice were anesthetized with 10%chloral hydrate the next day.The mice were sacrificed after cervical dislocation.HE staining was used to observe the changes of testicular tissue structure in mice;ultrastructure of testicular tissue was observed by electronmicroscopy;and the expression of 3β-HSD in testicular tissue was detected by immunohistochemistry.3.Results3.1 Genotype Detection Results of MiceSanger sequencing was used to detect the genotype of newborn mice.The single-peak map G of wild type mice was not mutated,the double-peak map heterozygous mice and pure-sum mice were mutated to base A.3.2 Experimental Results of Female Mice3.2.1 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on follicular development in TX miceCompared with the blank group,the number of normal secondary follicles and antral follicles in the ovarian tissue of Wilson disease model group decreased significantly（P<0.01）,and the number of atresia follicles increased significantly（P<0.01）.Compared with Wilson’s disease model group,the number of normal secondary follicles and antral follicles in the ovaries of DMSA group and the prescription of invigorating kidney and resolving phlegm and removing blood stasis group increased,and the number of atresia follicles decreased（P<0.01）.Compared with DMSA group,the number of normal secondary follicles and antral follicles increased（P<0.01）,atresia follicles decreased（P<0.05）in the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,and the number of normal secondary follicles and antral follicles increased（P<0.01）and atresia follicles decreased（P<0.01）in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group.3.2.2 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on ultrastructure of ovarian granular cells and oocytes in TX miceCompared with the blank group,abnormal degenerative vacuolated mitochondria were significantly increased in the ovarian tissue of Wilson disease model mice（P<0.01）.Compared with Wilson disease model mice,the number of normal mitochondria increased,the number of abnormal degeneration and vacuolation mitochondria decreased in DMSA group and the prescription of invigorating kidney and resolving phlegm and removing blood stasis group（P<0.05）,while the number of apoptotic granulosa cells decreased,the number of normal mitochondria increased,the number of abnormal degeneration and vacuolation mitochondria decreased in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group（P<0.01）.Compared with DMSA group,the apoptotic phenomena of granulosa cells in ovaries of the prescription of invigorating kidney and resolving phlegm and removing blood stasis group and DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group were reduced,the number of normal mitochondria increased,the number of abnormal degeneration and vacuolation mitochondria decreased（P<0.05）.3.2.3 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on the expression of BDNF and the proliferation of follicular cells in TX miceCompared with the blank group,the expression of BDNF in ovarian tissue and the average optical density of positive proliferating cells in follicles of Wilson disease model mice decreased,and the positive intensity decreased（P<0.01）.Compared with Wilson disease model group,the expression of BDNF in ovarian tissue and the average optical density of positive proliferating cells in follicles of mice in DMSA group and the prescription of invigorating kidney and resolving phlegm and removing blood stasis group increased（P<0.05）,and the average optical density of mice in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group increased significantly（P<0.01）.Compared with DMSA group,the expression of BDNF in ovarian tissue and the average optical density of positive proliferating cells in follicles of mice in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group increased,and the positive intensity increased（P<0.05）.3.2.4 Effect of the prescription on the expression of BDNF and the apoptosis of follicular cells in TX miceCompared with the blank group,the number of apoptotic cells increased,the expression of BDNF protein decreased and the positive intensity decreased in the Wilson disease model group（P<0.01）.Compared with Wilson disease model group,the number of apoptotic granulosa cells in ovarian tissue of mice in DMSA group and the prescription of invigorating kidney and resolving phlegm and removing blood stasis group decreased（P<0.01）,the expression of BDNF protein increased（P<0.05）,the number of apoptotic cells in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group decreased significantly（P<0.01）,and the expression of BDNF protein increased（P<0.01）.Compared with DMSA group,the number of apoptotic cells in ovarian tissue of mice in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group decreased significantly（P<0.01）,and the expression of BDNF protein increased（P<0.05）.3.3 Experimental Results of Male Mice3.3.1 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on histomorphology of testis in TX miceCompared with the blank group,JTBS score of testicular tissue in Wilson disease model group decreased significantly（P<0.01）,and the diameter of seminiferous tubules decreased,but there was no statistical difference（P>0.05）.Compared with Wilson disease model group,JTBS score increased in the three groups（P<0.05）,while the diameter of seminiferous tubules increased slightly in the three groups（P>0.05）.Compared with DMSA group,JTBS score increased in the DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group（P>0.05）.3.3.2 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on ultrastructure of sperm,Leydig cells and Sertoli cells in testicular tissue of TX miceUltrastructure of sperm in testicular tissue of mice showed complete sperm structure in blank group,abnormal sperm and mitochondria in Wilson disease model group,swelling of sperm structure and vacuolar degeneration of mitochondria around them.Normal sperm structure was observed in DMSA group and the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,but mild vacuolar degeneration of mitochondria still existed around them.In DMSA+the rescription of invigorating kidney and resolving phlegm and removing blood stasis group,sperm arranged neatly and mitochondria with vacuolar degeneration were decreased.The observation of nuclear membrane and mitochondrial structure of Leydig cells in blank group showed that the chromatin in the nucleus was evenly distributed.The mitochondria structure was normal,and there were tubular cristae mitochondria without swelling.The model group of Wilson disease showed that the number of mitochondria in Leydig cells decreased,and a large number of vacuolar degenerated mitochondria were observed,and the chromatin nucleus appeared margin gathering phenomenon.DMSA group showed that the structure of Leydig cells was acceptable.In the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,a small amount of lysosomes and mitochondrial structure were restored,while in the DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,the structure of mitochondria was normal,the chromatin edge collection of nucleus was reduced,and the morphological structure was significantly improved compared with the Wilson disease model group.The Sertoli cells in blank group had clear outline,complete structure,and uniform distribution of nucleus and cytoplasm,and no nuclear condensation and chromatin aggregation.Wilson disease model group showed that the structure of Sertoli cells was destroyed,and nuclear membrane was destroyed accompanied by chromosome aggregation and nuclear condensation.The Sertoli cells in DMSA group,the prescription of invigorating kidney and resolving phlegm and removing blood stasis group,and DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group had no chromatin aggregation and chromatin aggregation,and normal mitochondrial structure were observed.3.3.3 Effect of the prescription of invigorating kidney and resolving phlegm and removing blood stasis on the expression of 3β-HSD in testicular tissue of TX mice Compared with the blank group,the expression level of 3β-HSD in testicular tissue of Wilson disease model group decreased,and the average optical density decreased（P<0.01）.Compared with Wilson disease model group,the expression level of 3β-HSD and the average optical density in the DMSA group,prescription of invigorating kidney and resolving phlegm and removing blood stasis group,and DMSA+the rescription of invigorating kidney and resolving phlegm and removing blood stasis group increased（P<0.01）.Compared with DMSA group,the expression level of 3β-HSD in DMSA+the prescription of invigorating kidney and resolving phlegm and removing blood stasis group increased,and the average optical density increased（P<0.05）.4 Conclusions4.1 The prescription of invigorating kidney and resolving phlegm and removing blood stasis can improve the follicular development of TX mice with Wilson’s disease and reduce the damage of oocyte structure,decrease the number of abnormal mitochondria and increase the number of normal mitochondria.4.2 The prescription of invigorating kidney and resolving phlegm and removing blood stasis can increase the number of BDNF positive cells and proliferating follicular cells in TX mice with Wilson disease,which indicates that BDNF is positively correlated with the proliferation of follicular cells,and BDNF can affect the proliferation of follicular cells.4.3 The prescription of invigorating kidney and resolving phlegm and removing blood stasis can increase the number of BDNF positive cells and decrease the number of apoptotic follicular cells in TX mice with Wilson’s disease,indicating that BDNF is negatively correlated with follicular cell apoptosis,and BDNF can affect follicular cell apoptosis.4.4 The prescription of invigorating kidney and resolving phlegm and removing blood stasis may further affect the proliferation and apoptosis of granulosa cells in follicles by regulating the expression of BDNF,thus promoting the development of follicular cells.4.5 The prescription of invigorating kidney and resolving phlegm and removing blood stasis can alleviate the damage of testicular tissue in mice and might promote spermatogenesis by increasing the expression of 3β-HSD in testicular tissue.