Effect Of Kidney-tonifying Method On Enhancing Antioxidant Ability Of Oocytes Cultured In Vitro And Regulating ERK5-Keap1-Nrf2 Signaling Pathway

OBJECTIVE:During the in vitro culture,oocytes are susceptible to oxidative stress due to the influence of culture environment,resulting in a decrease in quality.In the previous study,the research team confirmed that the tonifying kidney method can improve the ovarian antioxidant capacity and improve the mitochondrial function of oocytes,and study the effect of tonifying kidney method on the antioxidant capacity of oocytes cultured in vitro and its ERK5-Keap1-Nrf2 signaling pathway.The regulation effect,in order to reveal the role and mechanism of kidney-tonifying method to improve the antioxidant capacity of oocytes in vitro,enrich the theory of"kidney reproductive"in traditional Chinese medicine.Methods:1.Preparation of rat drug-containing serumTwenty 6-week-old healthy female SD rats were used to prepare the serum containing Bushen Tiaojing.2.In vitro culture of mouse oocytes32 female Kunming mice using D24-26,Mouse oocytes were randomly divided into 4 groups:normal group,model group,tonifying kidney group and NAC group.The ovaries were separated by mechanical method,and the oocytes were collected in an incubator at 37°C,5%CO ₂,100%humidity for14h:the PB1 excretion rate of the oocytes was observed under an inverted microscope,and the morphology of the spindle was observed by Oosight software,respectively,using MDA.The SOD and GST activities of oocytes were detected by SOD and GST kits,respectively.45 female Kunming mice of D24-26 were randomly divided into 5groups:normal group,model group,kidney group,NAC group and ERK5inhibitor group.The oocyte collection and culture methods were as follows.After culture for 6 hours,the ERK5,Nrf2 and Keap1 mRNAs of oocytes were detected by real-time fluorescent quantitative PCR,and the expressions of SOD and CAT mRNA of Nrf2 downstream antioxidant enzymes were detected.Result:1.Comparison of PB1 excretion rate of oocytes in each groupCompared with the normal group,the PB1 excretion rate of the oocytes in the model group was significantly lower（P<0.01）.There was no significant difference in the PB1 excretion rate between the kidney and NAC groups（P>0.05）.Compared with the model group,the kidney group The PB1excretion rate of oocytes in NAC group was significantly increased（P<0.01）.There was no significant difference between NAC group and Bushen group（P>0.05）.2.Comparison of spindle morphology of oocytes in each groupNormal group oocytes have normal barrel-shaped spindle morphology;model group oocytes have mild shrinkage or irregular changes,and abnormal narrow spindles are observed;kidney group and NAC group have normal spindles Body shape.3.Comparison of MDA content,SOD activity and GST activity in oocytes of each groupMDA content:Compared with the normal group,the MDA content in the model group was significantly increased（P<0.01）.There was no significant difference between the kidney-reinforcing group,the NAC group and the normal group（P>0.05）.Compared with the model group,the MDA content in the NCA group was compared.The decrease of MDA content in the kidney group was significantly lower（P<0.05）.There was no significant difference in MDA between the kidney and NAC groups（P>0.05）.SOD activity:Compared with the normal group,the SOD activity of the model group was significantly decreased（P<0.01）.There was no significant difference between the kidney-reinforcing group,the NAC group and the normal group（P>0.05）.Compared with the model group,the kidney-reinforcing group and the NAC group.The SOD content increased significantly（P<0.01）.There was no significant difference in SOD between the kidney and NAC groups（P>0.05）.GST activity:Compared with the normal group,the GST content in the model group was significantly lower（P<0.01）.There was no significant difference between the kidney-reinforcing group,the NAC group and the normal group（P>0.05）.Compared with the model group,the kidney group There was no significant difference in GST content between the kidney and NAC groups（P>0.05）.4.Comparison of ERK5,Keap1 and Nrf2 mRNA expression in oocytes of each groupERK5 mRNA expression:Compared with the normal group,the expression of ERK5 mRNA in the model group was increased（P<0.05）,and the expression in the kidney and NAC groups was significantly increased（P<0.01）.There was no significant difference between the inhibitor group and the normal group（P<0.05）;Compared with the model group,the expression of the kidney and NAC groups was increased（P<0.05）,and the expression of the inhibitor group was decreased（P<0.05）.Compared with the inhibitor group,the expression of the kidney and NAC groups was significantly increased.（P<0.01）;there was no significant difference between the kidney-reinforcing group and the NAC group（P>0.05）.Keap1 mRNA expression:There was no significant difference in the expression of Keap1 mRNA between the normal group,the model group,the tonifying kidney group,the NAC group and the inhibitor group（P>0.05）.Nrf2 mRNA expression:Compared with the normal group,the expression of model group,tonifying kidney group,NAC group and inhibitor group was significantly increased（P<0.01）.Compared with the model group,the expression of kidney and NAC group was significantly increased（P<0.01）.The expression of the inhibitor group was increased（P<0.05）.Compared with the kidney-reinforcing group and the NAC group,the expression of the inhibitor group was decreased（P<0.05）.There was no significant difference between the kidney-reinforcing group and the NAC group（P>0.05）.5.Comparison of MnSOD and CAT mRNA expression in oocytes of each groupMn SOD mRNA statistical results:compared with the normal group,the expression of kidney,NAC,inhibitor group was significantly increased（P<0.01）,the model group increased（P<0.05）;compared with the model group,kidney group,NAC group The expression was significantly increased（P<0.01）,and the expression of the inhibitor group was increased（P<0.05）.Compared with the kidney and NAC groups,the expression of the inhibitor group was decreased（P<0.05）.There was no statistical relationship between the kidney-reinforcing group and the NAC group.Learning differences（P>0.05）.CAT mRNA statistics:Compared with the normal group,the expression of CAT mRNA in the model group,the inhibitor group,the kidney-enhanced group and the NAC group was significantly increased（P<0.01）.Compared with the model group,the CAT mRNA in the kidney-reinforcing group and the NAC group was significantly increased.（P<0.01）,there was no significant difference between the inhibitor group and the model group（P>0.05）.Compared with the kidney and NAC groups,the expression of the inhibitor group was decreased（P<0.05）.There was no statistical relationship between the kidney group and the NAC group.Learning differences（P>0.05）.Conclusion:1.Adding H202 in cultured oocytes in vitro will reduce the PB1excretion rate,affect the assembly of spindles during oocyte meiosis,increase the content of MDA in oocytes,reduce the activity of antioxidant enzymes SOD and GST,and cause eggs.Oocyte oxidative stress damage.2.Bushen Tiaojing and NAC can increase the PB1 excretion rate of oocytes,improve spindle assembly,reduce the MDA content of oocytes,increase the activity of antioxidant enzymes SOD and GST,improve the antioxidant capacity of mouse oocytes,and improve eggs.Mother cell mass.3.In the early stage of oxidative stress,H2O2,NAC,and tonifying kidney can up-regulate the anti-oxidative stress signaling pathway of ERK5-Nrf2 in oocytes.4.The mechanism of tonifying kidney to improve oxidative stress in oocytes cultured in vitro may be related to the regulation of ERK5-Keap1-Nrf2 anti-oxidative stress pathway.And the tonifying kidney method to improve the antioxidant capacity of oocytes may also have other protein kinases to activate the Nrf2 pathway.