Effect Of Rat Spermatogenic Cell Mitochondrial Energy Metabolism By UU Infected And Intervention Roll Of Zhibai Dihuang Pill

There are three parts in this study:Part 1 Impacts on the quality of semen and oxidative stress by UU infected and intervention of Zhibai-Dihuang pillObjective:Through the experimental study on rat sperm quality and oxidative stress correlation and the effects of zhibai-dihuang pill UU infection on this part of the pathogenesis of UU infection of infertility.Methods:16-20 week old SPF male SD 90 rats were randomly selected in 75 rabbits,after intravesical injection of Ureaplasma urealyticum isolates suspension,establishment of rat model of UU infection,the remaining 15 rats were injected into the bladder.In 0.9% Sodium Chloride Injection 10 d after inoculation were taken randomly in 5 model rats.Death,epididymal under aseptic chopped tissue in UU medium,cultured UU,showed that the model was successful.The successful model of survival after 64 UU animal models were randomly divided into testicular zhibai-dihuang pill infection treatment group（Chinese medicine group）,doxycycline treatment group（Western medicine group）,zhibai-dihuang pill + doxycycline treatment group（combined therapy group）,model group,16 rats in each group rats in sham operation group,set up at the same time,15 rats in each group,second days in the animal model after the success of the medicine was given,the sham operation group,model group with 0.9% Sodium Chloride Injection gavage treatment.The treatment group was given corresponding drugs The intragastric administration of Zhibai-dihuang pill group.The dosage was2ml/d（0.94 g crude drug /ml,according to the surface area of equivalent 60 kg adult dosage）,doxycycline group given drug dosage was 2ml/d（doxycycline2mg/m L）,Zhibai-dihuang pill and doxycycline and control group were given intragastric administration,1 times / day,each group of continuous irrigation gastric 21 d.SD rats were killed by cervical dislocation after preparation of sperm suspension.Using conventional methods of semen quality,sperm mitochondrial membrane potential were detected by flow cytometry（MMP）,reactive oxygen species（ROS）level,ELISA was used to detect the sperm cell Cyt C content.Results : After UU infection,the sperm density and activity rate,the percentage of a+b grade sperm decreased,the sperm mitochondrial membrane potential（MMP）decreased,the level of mitochondrial ROS increased,and the content of Cyt C increased.By Zhibai-dihuang pill intervention,UU infection and semen quality were significantly improved,such as sperm density,sperm motility,the percentage of grade a+b sperm increased,the detection rate of UU was significantly decreased,sperm ROS level decreased,sperm MMP increased,decreased sperm Cyt C content,the western group improved significantly.Conclusions : Zhibai-dihuang pill can inhibit UU infection of rat spermatogenic cells oxidative stress,reduce the production of ROS MMP less decline,mitochondrial structure and function of repair and protection,reduce the release of Cyt C from mitochondria,apoptosis of sperm cells decreased,thus improves the survival rate of sperm,UU infection on semen quality of rats improvement of rat sperm density increased.This may be one of the mechanisms of infertility of Zhibai-dihuang pill in the treatment of Ureaplasma urealyticum infection.Part 2 The intervention effects on sperm mitochondrial respiratory function in rats and zhibai-dihuangwan Ureaplasma urealyticum infectionObjective:Through the experimental study on the effects of UU infection on mitochondrial respiratory function in rats and Zhibai-dihuang pill,further elaborates the pathogenesis of UU infection infertility.Methods: Modeling,grouping,administration,preparation of sperm suspension,with the first part.Using spectrophotometry Methods to detect the respiratory chain complexes,Na⁺-K⁺-ATP and Ca²⁺-Mg²⁺-ATP in rat sperm mitochondria Enzyme activity was used to detect the mitochondrial NADH dehydrogenase of rat sperm RT-PCR（mRNA）,Expression of ATP synthase mRNA.Results : Rat sperm mitochondrial respiratory complex UU infection（I-IV）activity decreased,NADH dehydrogenase mRNA,ATP synthase mRNA expression decreased,Na⁺-K⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATP enzyme decreased.After Zhibai-dihuang pill intervention,UU infection rat sperm mitochondrial respiratory complex mRNA activity increased,NADH dehydrogenase,ATP synthase mRNA expression elevated levels of Na⁺-K⁺-ATP enzyme activity of Ca²⁺-Mg²⁺-ATP increased,the combined therapy group improved the most obvious.Conclusion:Zhibaidihuang pill may enhance the activity of mitochondrial respiratory complex UU infection in rats,Upregulation of NADH mRNA dehydrogenase,ATP synthase mRNA expression enhanced mitochondrial Na⁺-K⁺-ATP enzyme,Ca²⁺-Mg²⁺-ATP enzyme activity,to maintain the integrity of sperm mitochondrial respiratory chain function,increase sperm oxidative phosphorylation,promote the synthesis of mitochondrial ATP,improve sperm energy supply,so that the sperm motility,viability and survival rate can be improved.Effectively improve the semen quality of UU infected rats.This may be Zhibai-dihuang pill by improving rat spermatogenic cell mitochondrial respiratory function to treat one of the mechanism of UU infection in infertility.Part 3 The intervention effect on sperm mitochondrial permeability in rats and effects of Zhibai-dihuang pill of Ureaplasma urealyticumObjective : Through the experimental study of UU infection intervention effect on mitochondrial permeability in rats and effects of Zhibai-dihuang pill on further elucidate the pathogenesis of infertility Ureaplasma urealyticum infection.Methods : Modeling,grouping,administration,preparation of sperm suspension,with the first part.Spectrophotometry was used to detect rat sperm mitochondrial m PTP opening,the determination of ANT₄ protein in rat sperm mitochondrial VDAC₂,the expression level of Western by using blot method,RT-PCR method was used to detect the expression of VDAC₂ mRNA and ANT₄ mRNA sperm mitochondria of rats by HPLC measurement of rat sperm ATP,adenosine phosphate（ADP）,two adenosine monophosphate（AMP）content.Results : UU m PTP activity in sperm mitochondria increased in rats,increasing openness,rat sperm mitochondrial VDAC₂,the expression level of ANT₄ protein decreased,the expression level of VDAC₂ mRNA and ANT₄ mRNA decreased,ATP,ATP/ADP and EC decreased.By Zhibai-dihuang pill intervention,UU m PTP activity in rat sperm mitochondria decreased,the degree of openness is reduced,rat sperm VDAC₂,ANT₄ protein expression level increased,increased the expression level of VDAC₂ mRNA,ANT₄ mRNA,ATP,ATP/ADP and EC value increased,the western group improved significantly.Conclusion:Zhibai-dihuang pill may regulate UU infection rat sperm m PTP open,the regulation of ANT/VDAC protein complex transport function,to ensure that the ATP,ADP effective transport in mitochondria and cytoplasm between the maintenance of sperm mitochondria of rat respiratory chain coupling and normal cell function,provided the energy supply or sperm,so that exercise capacity and the sperm can be improved.This is probably Zhibai-dihuang pill by improving rat spermatogenic cell mitochondrial energy metabolism to treat another mechanism of UU infection in infertility.