Research On The Mechanism Of Wuzi Yanzong Pill In The Prevention And Control Of Neural Tube Defects Based On PI3K/Akt Signaling Pathway

Objective:This subject intends to determine the content of folic acid(FA)in Wuzi Yanzong Pill(WYP),eliminate the interference of inherent folic acid,study the prevention and control effects of Wuzi Yanzong Pill on neural tube defects(NTDs),and compared folic acid with Wuzi Yanzong Pill which eliminate the interference of folic acid,research on the mechanism of Wuzi Yanzong Pill excluding folic acid interference in the prevention and control of neural tube defects from animal level and cell level,provide a scientific theoretical basis for the future application of WYP to prevent NTDs.Methods:LC-MS was used to determine the content of FA in WYP Water Honey Pills and Decoction Free Granules.Ninety C57BL/6 mice was caged according to the female:male =2:1.Those with vaginal embolism detected were pregnant mice,and they were randomly divided into normal group,model group,folic acid group(FA group),Wuzi Yanzong pill group(WYP group)and the same dose of folic acid and Wuzi Yanzong pill group(FA+WYP group),each group has 12 mice,and drug intervention was given at the same time.All-trans retinoic acid(atRA)was used to establish NTDs animal models(except for the normal group)at 7.5 days of pregnancy,the pregnant mice were killed at 11.5 days of pregnancy,the uterus was taken out,the embryos were separated,and the neural tube deformity was identified under a stereo microscope,and the malformation rate was counted.HE staining was used to observe the closure of neural tube in mice;TUNEL staining was used to detect the apoptosis rate of tissue sections.CHO cells and CHO/dhFrcells were divided into normal group,model group,FA group,WYP group and FA+WYP group.The FA group was added with a medium containing FA solution,the WYP group was added with a medium containing WYP solution,the FA+WYP group was added with a medium containing both FA and WYP solutions,the normal group and model group were added with the same volume of medium as the other groups.After 24 hours,except for the normal group,the other four groups were added with atRA solution-containing medium and the final concentration was 20 μmol/L,the normal group was added with the same volume of medium as the other groups.Flow cytometry was used to detect the apoptosis rate.Based on the PI3K/Akt signaling pathway,the expression levels of pathway indicators PI3 K,Akt,P-Akt and Nrf2,oxidative stress indicators SOD,CAT,GSH and GSH-Px,and apoptosis-related indicators Bax and Bcl-2 were detected.Results:1.Use LC-MS to determine the content of FA in WYPObserving the LC-MS spectrum and comparing the three graphs,the retention time of main characteristic peak of FA was 3.14 min,and the WYP Water Honey Pills and Decoction Free Granules did not show a peak at 3.14 min,indicating that FA was not detected in WYP.It can be determined that the prevention and control effect of WYP on NTDs was not related to FA.2.The effect of WYP on NTDs animal models2.1 The effect of WYP on NTDs embryo morphology2.1.1 Identify neural tube deformities under a stereo microscope.There were no uterine hyperemia in pregnant mice in the normal group,and absorptive fetuses were rare.The embryonic neural tube was well closed under the microscope,the brain vesicles were intact,and there were fetal heart beats;The uterine of pregnant mice were congestive in the model group,its embryos present NTDs,can appear cranial spina bifida malformation,eyeless deformity,single-eye malformation,facial malformation,etc.,absorptive fetuses were more common.Observed under the microscope,their development is mostly incomplete,and the development of embryos are slower than normal embryos.Among them,embryos with cranial spina bifida have not seen complete brain vesicles.2.1.2 HE staining to observe the closure of embryonic neural tube.The embryonic neural tube of the normal group was completely closed and the neuroepithelium layer developed well.The neuroepithelium layer,marginal layer and mantle layer were visible under the microscope,and the structure was clear and complete,the top plate was closed;The embryonic neural tube of the model group was incompletely closed,the neuroepithelium layer was thin,and the marginal layer was disappeared,the structure was blurred,and the top plate was not closed.2.2 The effect of WYP on the PI3K/Akt signaling pathway in NTDs embryosWestern Blot showed that,compared with the normal group,the protein expression of PI3 K,P-Akt and Nrf2 in the model group was significantly reduced(p<0.01);compared with the model group,the protein expression of PI3 K,P-Akt and Nrf2 in the FA group,WYP group and FA+WYP group increased in different degrees(p<0.05;p<0.01);compared with FA group,the protein expression of PI3 K,P-Akt and Nrf2 in WYP group was significantly increased(p<0.05;P<0.01);compared with WYP group,there was no statistical difference in the protein expression of PI3 K,P-Akt and Nrf2 in FA+WYP group(p>0.05).2.3 The effect of WYP on oxidative stress in NTDs embryosCompared with the normal group,the GSH content,CAT protein expression,SOD and GSH-Px activity of the model group was significantly reduced(p<0.01;p<0.001);compared with the model group,the GSH content,CAT protein expression,SOD and GSH-Px activity of the FA group,WYP group and FA+WYP group incressed in different degrees(p<0.05;p<0.01);compared with FA group,the GSH content,CAT protein expression,SOD and GSH-Px activity in the WYP group was increased(p<0.05);compared with WYP group,FA+WYP group had no significant difference in GSH content,CAT protein expression,SOD and GSH-Px activity(p>0.05).2.4 The effect of WYP on cell apoptosis in NTDs embryos2.4.1 TUNEL staining to detect the apoptosis rate of tissue sections.Compared with the normal group,the apoptosis rate of the model group was significantly increased(P<0.01);compared with the model group,the apoptosis rate of the FA group,WYP group and FA+WYP group decreased in different degrees(p<0.05;p<0.01);compared with FA group,the apoptosis rate in WYP group was reduced(p<0.05);compared with WYP group,the apoptosis rate in FA+WYP group was not significantly different(p>0.05).2.4.2 The protein expression levels of Bcl-2 and Bax,which are related to apoptosis.Western Blot showed that,compared with the normal group,the protein expression of Bcl-2 in the model group was significantly reduced,the protein expression of Bax was significantly increased,and the ratio of Bcl-2/Bax was reduced(P<0.01;p<0.001);compared with the model group,the protein expression of Bcl-2 increased,the protein expression of Bax decreased,and the ratio of Bcl-2/Bax increased in FA group,WYP group and FA+WYP group(p<0.05;p<0.01);compared with FA group,the protein expression of Bcl-2 was increased,the protein expression of Bax was decreased,and the ratio of Bcl-2/Bax increased in WYP group(p<0.05;p<0.01);compared with WYP group,there was no statistically significant difference in the protein levels of Bcl-2,Bax and the ratio of Bcl-2/Bax in FA+WYP group(p>0.05).3.The effect of WYP on CHO cells and CHO/dhFr-cells3.1 The effect of WYP on oxidative stress in CHO cells and CHO/dhFr-cellsCompared with the normal group,the GSH content,SOD,CAT and GSH-Px activity of model group was significantly reduced(p<0.01);compared with the model group,the GSH content,SOD,CAT and GSH-Px activity in the FA group,WYP group and FA+WYP group increased in different degrees in CHO cells(p<0.05;p<0.01),the GSH content,SOD,CAT and GSH-Px activity in WYP group and FA+WYP group were significantly increased in CHO/dhFr-cells(p<0.05),but there was no difference in FA group;compared with FA group,the activity of SOD,CAT in WYP group increased(p<0.05),but the GSH content,GSH-Px activity of WYP group was no difference in CHO cells(p>0.05);and the GSH content,SOD,CAT and GSH-Px activity of WYP group was significantly increased in CHO/dhFr-cells(p<0.05);compared with WYP group,there was no statistical difference in the GSH content,SOD,CAT and GSH-Px activity in FA+WYP group(p>0.05).3.2 The effect of WYP on cell apoptosis in CHO cells and CHO/dhFr-cellsAnnexin V-FITC/PI double staining method to detect cell apoptosis rate.Compared with the normal group,the apoptosis rate of model group was significantly increased(p<0.01);compared with the model group,the apoptosis rate in the FA group,WYP group and FA+WYP group decreased to very degrees in CHO cells(p<0.05),the apoptosis rate in WYP group and FA+WYP group were significantly reduced in CHO/dhFr-cells(p<0.05),there was no difference in FA group;compared with FA group,the apoptosis rate of WYP group was reduced(p<0.05);compared with WYP group,the apoptosis rate of FA+WYP group was not significantly different(p>0.05).Conclusion:1.FA was not detected in WYP Water Honey Pills and Decoction Free Granules and interference of FA inherent in WYP can be eliminated.2.WYP has a good preventive effect on atRA-induced NTDs mice.WYP can significantly improve the closure of mouse embryonic neural tubes and effectively reduce the incidence of NTDs,and the preventive effect of WYP is better than FA.3.WYP regulates the PI3K/Akt signaling pathway,phosphates Akt,activates the downstream transcription factor Nrf2,and WYP has a better effect than FA,thus playing a role in antioxidant stress and anti-apoptosis.4.WYP can improve the oxidative stress state of NTDs mice,CHO cell models and CHO/dhFr-cell models induced by atRA,increase the content of GSH,elevate the activity of SOD and GSH-Px,up-regulate the expression of CAT,and the effect of WYP is better than FA,thereby mitigating body and cell damage.5.WYP can inhibit apoptosis pathways of NTDs mice,CHO cell models and CHO/dhFr-cell models induced by atRA,effectively reduce apoptosis rate,increase the protein expression of Bcl-2,reduce the protein expression of Bax,and increase the ratio of Bcl-2/Bax,and WYP has a better effect than FA,thereby reducing the excessive apoptosis of nerve cells.