| Purpose and significance:The previous clinical research of our group confirmed that tonifying kidney method can reduce the level of ROS,improve the state of oxidative stress,improve the ability of antioxidation of oocytes and improve the quality of oocytes.It was also shown that repeated COS could decrease the activity of antioxidant enzyme SOD,GSH-Px and increase the oxidative stress products in the ovary of mice,resulting in oxidative stress and affecting the quality of oocytes.After intervention by Bushen Tiaojing recipe,the activity of SOD,GSH-Px was improved,the oxidative stress state of mouse ovary was improved,the quality of oocytes and the ability of anti-oxidation were improved,and the conditions for fertilization and embryo development were created.Nuclear factor E2 phase Critical factor is the main regulatory factor of cell redox reaction.When cells are stimulated by ROS,Nrf2 is activated to play its role in antioxidant damage.In addition to ROS,PKC is currently recognized as a kinase with Nrf2 activation both inside and outside cells.The damage to oocytes induced by oxidative stress was used as the target,and in vitro cell experiment was used to study the effects of oxidative stress on oocytes.To study the effect of Bushen Tiaojing recipe(Wuzi Yan Pill combined with Yangjing Zongyu decoction)on the oxidative ability of mouse oocytes under oxidative stress and on the regulation of antioxidant stress pathway of PKC/Nrf2,in order to reveal that the method of tonifying kidney can improving oocyte quality.The mechanism of regulating menstrual seeds enriches the scientific connotation of kidney and reproduction,and finally guides clinical practice.Methods:Thirty female SD rats aged 6-7 weeks were administered with BushenTiaojing recipe(2ml/100g),high dose containing crude drug 5.4 g / ml,low dose containing 2.7 g / ml of crude drug,(high and low dose being 20,10 times of clinical dosage).Two times a day for 4 days.Blood was collected from femoral artery of rats at 1 hour after the last administration.Resting at room temperature for 2 hours,centrifugation(3000rpmm)for 10 min,absorption of supernatant,56 ℃ water bath 30 min inactivated complement,0.22μm filtration sterilization,obtained Bushen Tiaojing prescription containing drug serum,stored at-80 ℃.Choose 120,D24-26 female Kunming mice,weight 22-28 g.They were randomly divided into 6 groups: normal group,H2O2 group,high dose group(H2O2 combined with high dose serum of Bushen Tiaojing recipe),low dose group(H2O2 combined with low dose serum of Bushen Tiaojing recipe),antioxidant NAC group,PKC blocker group(H2O2,Bushen Tiaojing recipe and PKC blocker),15 rats in each group.Each mouse was injected with PMSG(10IU)to induce ovulation 48 hours later,the mice were killed by cervical dislocation,75% alcohol was disinfected,the whole ovary was separated by dissection and injected with 1 ml.The larger antral follicles of the ovary were punctured by the tip of the emitter under a pose microscope,and the oocytes in the GV stage were dissociated from the ovary.The oocytes of phase M Ⅱ were collected by centrifugation after washing in M2 operation solution for 2-3 times and cultured in 35 mm culture dish for 6 h and 14 h.The first polar body efflux rate of oocytes was observed by inverted microscope.Detection of Antioxidant enzyme SOD activity by Chemical method.m RNA expression of antioxidant enzyme GSH-Px,SOD and m RNA expression of PKC,Keap1,Nrf2 in PKC/Nrf2 signaling pathway were detected by RT-PCR.Results:1.Compared with normal group,hydrogen peroxide group,kidney tonifying group and antioxidant NAC group decreased the first polar body excretion rate(P<0.05),the difference has statistical significance.Compared with hydrogen peroxide group,the first polar body excretion rate of high dose group and antioxidant NAC group was significantly higher than that ofhydrogen peroxide group(P<0.05),the difference has statistical significance.2.Compared with the normal group,the activity of antioxidant enzyme SOD in the low dose group and the hydrogen peroxide group was significantly lower than that in the control group(P<0.05),and the difference has statistical significance in the activity of antioxidant enzyme between the two groups(P<0.05).There was no significant difference in the activity of antioxidant SOD between the high dose group of tonifying kidney and the group of antioxidant NAC compared with the normal group(P>0.05).Compared with the hydrogen peroxide group,the activity of antioxidant SOD in the high dose group of tonifying kidney,antioxidant NAC group and normal group was higher than that in the control group(P<0.05),the difference has statistical significance.3.RT-PCR :The expression of antioxidant enzyme SOD,GSH-pxm RNA in hydrogen peroxide group,kidney tonifying group and antioxidant NAC group was lower than that in normal group(P<0.05),the difference has statistical significance.Compared with hydrogen peroxide group,antioxidant enzyme SOD,GSH-pxm RNA expression in kidney tonifying group and antioxidant NAC group was significantly higher than that in hydrogen peroxide group(P<0.05),the difference has statistical significance.4.RT-PCR: Compared with the normal group,the expression of,PKC m RNA in hydrogen peroxide group,kidney tonifying group,antioxidant NAC group and PKC inhibitor group was significantly higher than that in hydrogen peroxide group(P < 0.05),and the expression of PKC m RNA in kidney tonifying group and antioxidant NAC group was significantly higher than that in hydrogen peroxide group(P < 0.05),and the expression of PKC m RNA in kidney tonifying group and antioxidant NAC group was significantly higher than that in hydrogen peroxide group(P < 0.05).There was no statistical significance between PKC inhibitor group and hydrogen peroxide group.Keapl m RNA expression: there was no statistical significance in the expression of Keap1 between the two groups.Nrf2 m RNA expression:compared with normal group,the expression of Nrf2 m RNA in hydrogenperoxide group,kidney tonifying group,antioxidant NAC group and PKC inhibitor group was significantly higher than that in hydrogen peroxide group(P < 0.05),and the expression of Nrf2 m RNA in kidney tonifying group and antioxidant NAC group was significantly higher than that in hydrogen peroxide group(P < 0.05).There was no significant difference between PKC inhibitor group and hydrogen peroxide group.The expression of PKC,Nrf2 m RNA in high dose group of tonifying kidney was higher than that of tonifying kidney.Low dose group(P < 0.05).Conclusion:1.The results suggest that the method of tonifying the kidney can increase the first polar body excretion rate of mouse oocytes cultured in vitro.2.The antioxidant enzyme activity of oocyte in oxidative stress mice was decreased,and Bushen Tiaojing recipe increased the activity of antioxidant enzyme SOD.It is suggested that the method of tonifying the kidney can improve the antioxidation ability of oocytes.3.The expression of antioxidant enzymes in oocytes of oxidative stress mice was decreased,and the expression of antioxidant enzymes SOD and GSH-px was increased by Bushen Tiaojing decoction.The high dose group of tonifying kidney was superior to the low dose group.4.Bushen Tiaojing Fang upregulated the expression of PKC and Nrf2 in PKC/Nrf2 signaling pathway,and the high dose group was better than the low dose group. |
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