Effects Of Tonifying Kidney On Mitochondrial Function And The Relationship With Nrf2 Signal Pathway

With the development of the society and the change of policy,the reproductive age of women has been postponed,and the morbidity of infertility is also rised.IVF-ET?in vitro fertilization and embryo transfer?become an important treatment method.Zygotes come from the combination of egg and sperm,the quality of oocyte is very important for the outcome of IVF-ET.COS?control ovarian stimulation?is an essential procedure of IVF-ET.The low pregnancy rate makes patients need more than once COS procedure.Besides,as the largest number of organelles in the cytoplasm,the mitochondria provide energy for oocytes and embryos.After repeated COS,the decline of oocytes' quality and mitochondrial function has already been proved.An important factor affecting mitochondrial function is the generation of oxidative stress.Previous studies have found that tonifying the kidney can regulate oocyte secretory factors expression,improve the quality of oocytes,and can improve the ovarian antioxidant capacity in a rat model of anovulation,reduce the level of oxidation products,improve ovarian function.This study intends to observe whether COS could cause oxidative stress on ovary,the impact and the mechanism of tonifying kidney on oocytes after repeated COS.To reveal the possible mechanism of tonifying kidney in improving the quality of oocytes.Enrich the scientific connotation of “kidney dominating reproduction”.Part one Impact of Tonifying Kidney on antioxidant capacity in mouse ovarian after repeated COSObjective: To study the impact of repeated ROS on antioxidant enzyme activity and T-AOC in mouse ovary.To study whether repeated ROS cause oxidative damage to ovarian proteins,lipids and nucleic acids.To observe effects of Tonifying Kidney on the above indexes.Methods:Kunming female mice between 8 and 10 weeks of age.Vaginal smear to observe estrous cycle by everyday.Randomly divided into 4 groups: Bushen high group,Bushen low group,Normal group,Model group.Model group,Bushen high group and Bushen low group intragastric administration of distilled water and BuShenTiaoJing recipe,1mL/100 g weight,last for 10 days.In the 11 th,cycles of OS were initiated in mice that received 10 IU i.p.injections of PMSG followed 48 h later by 10 IU i.p.of hCG.3 cycles of intragastric and ovarian stimulation were performed using a 4 days interval between each cycle.Mices were killed by cervical vertebra.Mices' ovarian in Model and Bushen groups were obtained 46 h post-PMSG injection in the 3rd cycle.Mices' ovarian in Normal group were obtained 10 h after oestrus.Ovary placed in liquid nitrogen and then stored at-80?.Ovary tissues homogenate on ice in PBS when applied.The levels of 8-OHd G,AOPP,MDA,SOD,GSH-Px and T-AOC were detected by ELISA Kit.Results:1 Comparison of 8-OH-dG content in ovary of each groupCompared with the Normal group,no significant difference between the content of 8-OHdG in ovarian tissue of Bushen high group?P>0.05?,the content of 8-OHdG in Bushen low group was increased?P<0.05?,Model group 8-OHdG content was significantly increased?P<0.01?;compared with the Model group,8-OHdG content in Bushen high group and Normal group decreased significantly?P<0.01?,8-OHdG content in Bushen low group has no statistically difference?P>0.05?;comparison between high and low doses of Bushen group,with statistical difference of 8-OHdG content?P<0.05?,and high dose group is better.2 Comparison of AOPP content in ovary of each groupCompared with the Normal group,the AOPP content in Bushen high and low dose group increased?P<0.05?,the AOPP content in Model group increased significantly?P<0.01?;compared with the Model group,the AOPP content in Bushen high and low dose group and Normal group decreased significantly?P<0.01?.There was no significant difference between the Bushen high and low dose groups?P>0.05?.3 Comparison of MDA content in ovary of each groupCompared with the Normal group,the content of MDA showed no significant difference in Bushen high and low dose group?P>0.05?,MDA of Model group was significantly higher than the Normal group?P<0.01?;compared with the Model group,MDA content in Bushen high and low dose and Normal group significantly reduced?P<0.01?;compare the MDA content between Bushen high and low dose group,the difference was not statistically different?P>0.05?.4 Comparison of SOD activity in ovary of each groupCompared with the Normal group,the activity of SOD in ovarian tissue showed no significant difference in Bushen high and low dose group?P>0.05?,decreased significantly in Model group?P<0.01?;compared with the model group,the activity of SOD in ovarian tissue was increased in Bushen groups and Normal group?P<0.01?,SOD activity in Bushen high group higher than low dose group?P<0.05?.5 Comparison of GSH-Px activity in ovary of each groupCompared with the Normal group,the activity of GSH-Px in ovarian tissue in Bushen high and low dose group showed no significant difference?P>0.05?,decreased significantly in Model group?P<0.01?;compared with the Model group,the activity of GSH-Px in ovarian tissue was increased in Bushen groups and Normal group?P<0.01?,compare the effects of Bushen high and low dose group,difference was not statistically significant?P>0.05?.6 Comparison of T-AOC level in ovary of each groupCompared with the Normal group,T-AOC of Bushen groups has no statistical difference?P>0.05?,Model group decreased significantly?P<0.01?;compared with the Model group,T-AOC of Bushen groups and Normal group increased?P<0.05?.There was no significant difference between the Bushen high and low dose groups?P>0.05?.Summary: 1 Repeated COS can increase the level of oxidative damage of nucleic acid,protein and lipid in the ovary of mice,and it will affect the activity of antioxidant enzymes in the ovary of mice,and cause the oxidative stress in mice.2 BuShenTiaoJing recipe can reduce 8-OHdG,AOPP,MDA level in ovary after repeated COS,improve the activity of antioxidant enzymes in mouse ovary,helps to eliminate the oxidative stress caused by repeated COS of mouse ovary.3 Repeated COS may affect the development of oocytes by affecting the oxidative stress and decrease the quality of oocytes.While BuShenTiaoJing recipe could eliminate ovarian oxidative stress,improve oocyte quality.Its mechanism deserves further study.Part two Effect of Tonifying Kidney on the mitochondrial function of repeated COS mouse oocytesObjective: Study on the influence of repeated COS on the ROS level and mitochondria function in mouse oocyte,and observe the effect of BuShenTiaoJing recipe on repeated COS mouse oocytes on the above indexes.Methods:Kunming female mice between 8 and 10 weeks of age were used for all oocyte collection.Mices divided into 4 groups randomly: Bushen high group,Bushen low group,Normal group,Model group.Model group,Bushen high group and Bushen low group intragastric administration of distilled water and BuShenTiaoJing recipe,1mL/100 g weight,last for 10 days.In the 11 th,cycles of OS were initiated in mice that received 10 IU i.p.injections of PMSG followed 48 h later by 10 IU i.p.of hCG.3 cycles of intragastric and ovarian stimulation were performed using a 4 days interval between each cycle.Mices were killed 14-16 h later by cervical dislocation in Model group,after Bushen groups received HCG i.p.injection.Mices in Normal group were killed 10 h after oestrus.Separated the fallopian tube from abdominal cavity.Pierced the ampulla of fallopian tube with needle,collected COCs,digested the COCs with hyaluronidase.Gather the oocyes in Ep tubes.Frozen in liquid nitrogen or send to detected immediately.The level of ROS was observed by confocal microscopy after DCFH-DA staining.Mito Tracker Red staining and confocal microscope were used to observe the mitochondrial distribution of oocytes.The oocyte ATP was detected by luciferase assay.The oocyte membrane potential?MMP?was observed by JC-1 staining and confocal microscopy.The mt DNA copy number of oocytes was detected by real-time quantitative PCR.Results:1 Comparison of ROS levels in each groupsCompared with the Normal group,ROS levels in Bushen groups had no statistical difference,the level of ROS significantly increased in Model group?P<0.01?;compared with the model group,ROS levels in Bushen low group was decreased?P<0.05?,Bushen high group decreased significantly?P<0.01?;compare the effects of Bushen high and low dose group,high dose group ROS level was lower,with statistical difference?P<0.05?.2 Comparison of mtDNA copies in each groupsCompared with the Normal group,mtDNA copies in Bushen groups had no statistical difference?P>0.05?,mtDNA copies significantly decreased in Model group?P<0.01?;compared with the Model group,mtDNA copies of Bushen high group increased significantly?P<0.01?,and Bushen low group and Normal group mt DNA copies increased?P<0.05?;copy number of mt DNA between Bushen high and low group had no significant difference?P>0.05?.3 Comparison of ATP in each groupsCompared with the normal group,ATP in Bushen groups had no statistical difference?P>0.05?,ATP significantly decreased in Model group?P<0.01?;compared with the Model group,ATP of Bushen high group increased significantly?P<0.01?,and Bushen low group and Normal group ATP increased?P<0.05?;ATP content of oocytes between Bushen high and low group had no significant difference?P>0.05?.4 Comparison of MMP in each groupsCompared with the Normal group,MMP in Bushen groups had no statistical difference?P>0.05?,MMP intensity in Model group significantly decreased?P<0.01?;compared with the Model group,MMP intensity of Bushen high group increased significantly?P<0.01?,and Bushen low group and Normal group MMP increased?P<0.05?;MMP average intensity in high group of was higher than Bushen low group?P<0.05?.5 Comparison of mtDNA distribution in each groupsThe ratio of the distribution of mitochondria in the Bushen high/low group,the model group and the normal group was 79.2%,73.9%,69% and 80.9%,respectively.Chi square test results showed that although the high and low dose group of the Bushen and the Normal group were higher than the Model group,the difference between the four groups was not statistically significant?P>0.05?.Summary:1 Repeated COS affected the oocytes' ROS level,mito-chondrial mt DNA copy number and MMP intensity of oocytes,affected the level of oocyte ATP,reduce the quality of oocyte.2 BuShenTiaoJing recipe can lower the level of ROS after repeated COS,increased the mt DNA copy number,mitochondrial MMP intensity and ATP level of oocyte,improve the quality of oocytes after repeated COS,and the effect of Bushen high group is better in improving the strength of mitochondrial MMP.3 BuShenTiaoJing recipe could improve the quality of oocyte and mitochondrial function of oocyte after repeated COS,can be used as adjuvant to decrease oocyte quality after repeated COS treatment.Part three The morphological changes of mouse oocytes cultured in vitro and the effect of Tonifying Kidney on ROS level andPB1 excretion rate in vitro under OS stateObjectives: To study oocyte morphology changes during oocyte development in vitro,to study the effect of H₂O₂ on oocyte ROS level and PB1,and to observe the effect of BuShenTiaoJing recipe on the above indexes.Methods:SD female rats between 6 and 7 weeks old were used to prepare for drug containing serum.Intragastric administration of BuShenTiaoJing recipe 2 times a day,1mL/200 g weight?equivalent to clinical dose equivalent?for 3 consecutive days.On the 4th day,intraperitoneal injection with chloral hydrate,then collected femoral artery blood.After centrifuge and filtration,the serum reserved in-80? until use.Kunming female mice between 24 and 26 days of age were used for all oocyte collection.Every mice killed by cervical dislocation 44 h after injection of PMSG 5IU for ovulation induction.Separated the ovary from abdominal cavity.Pierced the ovary with needle,collected COCs with 3-5 layers of cumulus cell,cultured in 35 mm dishes with in vitro matured culture media??-MEM +10%FBS+100mIU/mL rFSH+1.5 U/mLhCG +3ng/mL EGF+100IU/rnL Penicillin+100? g/mL Streptomycin?.Divided the oocyes into 3 groups randomly,?1?Normal group: cultured with IVM culture media to 14-16h;?2?Bushen group:cultured 2h with IVM culture media with 10% serum containing BuShenTiaoJing recipe,then given H₂O₂,cultured to14-16h;?3?H₂O₂ group:cultured 2h with IVM culture media,then adding H₂O₂ in 100 M concentration,cultured to 14-16 h.The morphological changes of COCs during development were observed by inverted microscope.Digested COCs,counted PB1 emission rate under inverted microscope.DCFH-DA staining and confocal microscopy were used to observe the level of ROS.Results:1 The morphological changes of COCs during in vitro maturationMultilayer cumulus cell was visible in GV COCs,cumulus cell layer was compact,zona pellucida was visible.In vitro matured for 16-18 h,COCs increase in size,the cumulus cells grown and expanded in loose structure.After digested in 100U/mL hyaluronidase,the first polar body was not visible in M ? oocytes,perivitelline space is visible in the M ? oocytes,the perivitelline space showed the first polar body.2 Comparison of ROS levels of oocytes in each groupCompared with the Normal group,ROS level in the Bushen group was not statistically significant?P>0.05?.ROS level in the H₂O₂ group compared with Normal group was significantly increased?P<0.01?;Bushen group compared with H₂O₂ group,the level of ROS oocytes was significantly decreased?P<0.01?.3 Comparison of PB1 excretion rate of oocytes in each groupThe statistical results showed that compared with Normal group,the PB1 excretion rate of Bushen group,no significant difference?P>0.05?,PB1 excretion rate in H₂O₂ group oocytes was significantly lower than the Normal group?P<0.01?;Bushen group compared with H₂O₂ group,the oocyte PB1 excretion rate increased significantly?P<0.01?.Summary:1 Added H₂O₂ to the culture medium of the oocytes in vitro maturation can cause the increase of ROS and the decrease of PB1 emission rate in the oocytes,which leads to the oxidative stress state.2 Added serum containing BuShenTiaoJing recipe can reduce the ROS level caused by accumulation of H₂O₂ in oocytes cultured in vitro,improve oocyte PB1 excretion rate,suggesting that BuShenTiaoJing recipe could attenuate oocyte oxidative stress,improve oocyte maturation rate.Part four Effect of BuShenTiaoJing recipe on Nrf2 signal pathway in mouse oocytes under oxidative stress matured in vitroObjective: Study on the changes of Nrf2 signaling pathway and its downstream antioxidant enzymes mRNA in the initial stage of OS state of mouse oocytes,influence of BuShenTiao Jing recipe in oocytes cultured in vitro with OS state on the above indexes.Methods:SD female rats between 6 and 7 weeks old were used to prepare for drug containing serum.The way of preparation of serum was the same with part three.Kunming female mice between 24 and 26 days of age were used for all oocyte collection.The way of separation way of oocyte was the same with part three.Divided the collected oocytes into 3 groups randomly,?1?Normal group: IVM cultured for 6 hours;?2?:Beshen group: cultured in IVM medium with 10% serum containing of BuShenTiaoJing recipe for 4h,then given H₂O₂,cultured to 6h;?3?H₂O₂ group: cultured in IVM medium for 4h,then add 100 M concentration H₂O₂,cultured to 6h.Collected COCs,collected oocytes after digested COCs with 100U/mL hyaluronidase,detected part by immunofluorescence,frozen part in-80?.Results: 1 Comparison of PKC,Keap1,Nrf2,SOD,GSH-Px mRNA in mouse oocytes 1.1 Comparison of PKC,Keap1 and Nrf2 mRNA in the oocytes of each groupCompared with the Normal group,the expression of PKC in H₂O₂ group and Bushen group were significantly higher?P<0.01?;Compared with H₂O₂ group,the expression of PKC mRNA in Bushen group was significantly higher?P<0.01?.There was no significant difference in the expression of Keap1 mRNA between Normal group,H₂O₂ group and Bushen group?P>0.05?.Compared with the Normal group,the expression of Nrf2 in H₂O₂ group and Bushen group was significantly higher?P<0.01?.Compared with H₂O₂ group,the expression of Nrf2 mRNA in Bushen group was higher?P<0.05?.1.2 Comparison of the expression of Mn SOD and GSH-Px mRNA in oocytes of each groupCompared with the Normal group,the expression of Mn SOD mRNA in the H₂O₂ group was higher?P<0.05?,Bushen group were significantly higher?P<0.01?;Compared with the H₂O₂ group,the expression of Mn SOD was significantly higher in the Bushen group?P<0.01?.Compared with the Normal group,the expression of GSH-Px in H₂O₂ group and Bushen group were significantly higher?P<0.01?.Compared with H₂O₂ group,the expression of GSH-Px mRNA in Bushen group was significantly higher?P<0.01?.2 Comparison of the expression of PKC,p-PKC,Keap1,Nrf2 and p-Nrf2 protein in mouse oocytes cultured in vitroThe expression of PKC protein in oocytes were not statistically different between the groups?P>0.05?.Compared with the Normal group,the expression of p-PKC protein in H₂O₂ group was increased?P<0.05?,protein expression were significantly higher in Bushen group?P<0.01?;Compared with the Normal group,p-PKC/PKC ratio in H₂O₂ group increased?P<0.05?,the Bushen group significantly increased?P<0.01?;compared with the H₂O₂ group,the Bushen group significantly increased?P<0.01?.There was no significant difference in the expression of Keap1 protein between each group?P>0.05?.There was no significant difference in the expression of Keap1 protein between each group?P>0.05?.Compared with the Normal group,the expression of Nrf2 protein in the H₂O₂ group and Bushen group was higher?P<0.05?;there was no significant difference between the H₂O₂ group and the Bushen group?P>0.05?.Compared with the Normal group,the expression of p-Nrf2 protein in H₂O₂ group and Bushen group was significantly higher?P<0.01?.Compared with the H₂O₂ group,the expression of p-Nrf2 protein in Bushen group was significantly higher?P<0.01?.Compared with the Normal group,the p-Nrf2/ Nrf2 ratio of H₂O₂ group and Bushen group was significantly higher?P<0.01?;compared with H₂O₂ group,the p-Nrf2/Nrf2 was significantly higher in the Bushen group?P<0.01?.Summary:1 Oxidative stress activates the Nrf2 signaling pathway,resulting in increased expression of Nrf2 mRNA and its downstream antioxidant enzymes Mn SOD,GSH-Px gene transcription.2 There was no significant relationship between the increase of Nrf2 protein activity and the expression level of Keap1 in the early stage of oxidative stress,which may be related to the increase of Nrf2 phosphorylation level by PKC and the dissociation of Keap1.3 Oocytes matured with BuShenTiaoJing recipe serum can reduce oxidative stress level of ROS,the mechanism may be related to the regulation of PKC-Nrf2 signaling pathways to regulate its downstream antioxidase gene transcription in early oxidative stress state.Conclusions: Repeated controlled ovarian stimulation can cause the decrease of antioxidant capacity,the decrease of antioxidant enzyme activity,the increase of oxidative damage products,and low down oocyte mitochondrial function and the quality of oocyte.Tonifying kidney could improve the antioxidant ability of ovary after repeated COS,improve the activity of antioxidant enzymes,reduce ovarian oxidative damage,and can improve the function of mitochondria in oocyte,its mechanism may be through the increased expression of PKC in oocytes,to activate the Nrf2 gene transcription,increase antioxidant enzymes activity to improve the antioxidant,improve the quality of oocytes.