Effects On Rat Spermatogenic Cell Mitochondria By UU Infected And Intervention Roll Of Zhibai Dihuang Decoction

There are three parts in this study:Part1Impacts on the quality of semen by UU infected and interve-ntion of Zhibai DihuangDecoctionObjective:This study explores the effects of semen quality of rats and regulatory role of Zhibai Dihuang Decoction after UU infected. The sperm motility parameters are measured by color sperm dynamic detection system. And further confirm that UU infection will lead to a decline of sperm quality and Zhibai DihuangDecoction can treat it, for further revealing the related theoretical basis for the pathogenesis of male infertility by UU infected.Methods:From40SD rats of4-5months age, randomly selecte30and inoculate the bladder with UU suspension, to establish the animal model of infected testis by UU. The remaining10rats are synchronously injected normal saline as the control group.Seventh days after inoculation, detect UU of the testis puncture fluid by the culture method,to judge the model.If the model is successful,they are randomly divided into the model group,azithromycin group, Zhibai Dihuang Decoction group.the dosage of ZhibaiDihuang Decoction group was1g/kg·D (1ml/d), azithromycin group0.105g/kg·D (1ml/d).the control groupandthe model group are given the same dosage of normal saline.every group is administered for21d orally, once a day.The rats are sacrificed by cervical dislocation, take the epididymal tissue, remove epididymal fat tissue and the small blood vessels carefully by the ophthalmology operation scissor.isolating sperm cells by the mechanical method and determinating sperm motion parameters by the color sperm dynamic detection system.Results:A grade sperm,B grade sperm, linear velocity, mean velocity of the model group, are significantly lower than the control group.After treatment, A grade sperm, B grade sperm, linear velocity, curvilinear velocity, average velocity of Zhibai Dihuang Decoction group and azithromycin group improved comparing with the model group and the difference has statistical significance (P<0.05, P<0.01),but the linear velocity of two group not improve.A grade sperm, B grade sperm, curvilinear velocity, average velocity of Zhibai Dihuang Decoction group is better than azithromycin group and the difference has statistical significance (P<0.05, P<0.01)Conclusions:UU can reduce the level of A grade sperm, B grade sperm, linear velocity and average velocity.Zhibai Dihuang Decoction and azithromycin can treat it and Zhibai Dihuang Decoction better than azithromycin.Part2Effects of rat spermatogenic cell mitochondrial ultrastructure and mitochondria membrane potential after UU infected and intervention of Zhibai Dihuang DecoctionObjective:This study to explore the effects of rat spermatogenic cells ultrastructure and mitochondria membrane potential by UU infected and intervention effect of Zhibai Dihuang Decoction, using electron microscopy to observe the ultrastructure of mitochondria of spermatogenic cells,using flow cytometry to detecte the mitochondrial membrane potential,to reveal the mechanism of UU infection inducing sperm motility and intervention mechanism of Zhibai Dihuang DecoctionMethods:The rats are sacrificed by cervical dislocation, take the testis tissue, remove testis fat tissue and the small blood vessels carefully by the ophthalmology operation scissor, intercepte testicular fragments,isolate sperm cells by the mechanical method and measure mitochondrial membrane potential by Flow cytometric detection.Testis fragments were fixed in glutaraldehyde, and observate the spermatogenic cells mitochondrial structure by electron microscope.Results:The spermatogenic cell mitochondrial membrane potential of the model group is Significantly lower than the control group.After treatment, the spermatogenic cell mitochondrial membrane potential of Zhibai Dihuang Decoction group and azithromycin group significantly improve compared with the model group, and the difference has statistical significance (P<0.01).Even Zhibai Dihuang Decoction group is better than azithromycin group, and the difference was statistically significant (P<0.01).Conclusion:UU can reduce the spermatogenic cell mitochondrial membrane potential.Zhibai Dihuang Decoction and azithromycin can improve it, but Zhibaidihuang Decoction is better.Part3Effects of the rat spermatogenic cell mitochondrial respir-atory metabolism function by UU infected and intervention of Zhibai Dihuang DecoctionObjective:This study to explore the Effects of the rat spermatogenic cell mitochondrial respiratory metabolism function by UU infected and intervention of Zhibai Dihuang Decoction,using ELISA to detect the activity and content of SDH,Using immunohistochemical method (DAB method) to detect the activity of COX,using RT-PCR to detect the expression of SDH, COX I and II mRNA,to reveal the mechanism of mitochondrial respiratory capacity function by UU infected,to provide the theoretical basis for the Zhibai Dihuang Decoction improve sperm motility.Methods:The above method to take the testis,using ELISA to detect the activity and content of SDH,Using immunohistochemical method (DAB method) to detect the activity of COX,using RT-PCR to detect the expression of SDH, COX I and II mRNA.Results:The activity and content of SDH in the model group rats’testicular tissue were significantly lower than the control group and the difference has statistical significance(P<0.01). After the treatment,the content of SDH of Zhibai Dihuang Decoction group and azithromycin group both significantly increase, compared with the model group, the difference was statistically significant (P<0.01),but the activity of SDH of the azithromycin group does not enhance. And the activity and content of SDH in the two groups failed to be normal, compared with the normal group,P<0.01. The activity of COX in the model group rats’testis did not decline, compared with other groups and the difference was not statistically significant (P>0.05).The expression of SDH,COX I, COX II mRNA in the model group rats’testicular tissue was lower than the normal group.After treatment, only the expression of SDH mRNA of the azithromycin group improve.the expression of COX I mRNA of, Zhibai Dihuang Decoction group and azithromycin group increase, compared with the model group, the difference was statistically significant (P<0.05),But compared with each othe,the difference was no significant (P>0.05).Conclusion:UU can reduce the activity and content of SDH and the expression of SDH,COXI,COX ⅡmRNA, but can not reduce the activity of COX.After treatment, the content of SDH and the expression of COX I mRNA in both Zhibai Dihuang Decoction group and azithromycin group significantly increase, but the expression of COX Ⅱ mRNA not increase.Zhibai Dihuang Decoction can improve the activity of SDH, azithromycin can improve the expression of SDH mRNA in the testis tissue.Summary1.UU can reduce the level of A grade sperm, B grade sperm, linear velocity and average velocity.Zhibai Dihuang Decoction and azithromycin can treat it and Zhibai Dihuang Decoction better than azithromycin.2. UU can reduce the spermatogenic cell mitochondrial membrane potential and cause mitochondrial ultrastructure changes.Zhibai Dihuang Decoction can improve sperm motility by means of improving the mitochondrial membrane potential,reducing the structure of mitochondria damage, and repairing or protectiving the mitochondria.3. UU can reduce the activity and content of SDH, but can not reduce the activity of COX.It indicate that the decline in sperm motility caused by UU has nothing to do with the COX, may be mainly related to the activity and content of SDH.4. UU can reduce the expression of SDHmRNA.After treatment,the expression of SDH enzyme mRNA increased, while the activity of SDH did not improve,which revealed that the expression of SDHmRNA increasing may be independent of the activity of SDH or reduce the activity of SDH, may be related to the content of SDH.UU can reduce the expression of COXI,COX II mRNA.After treatment,the expression of COX I mRNA increased, the expression of COX II mRNA and the activity of COX did not increase, which reveal that the activity of COX may be related to the expression of COX II mRNA, and not to the expression of COX I mRNA.