Preliminary Study On The Anti-apoptotic Mechanism Of Astragaloside ? On Radiationinduced Brain Cells

Natural medicines have multiple targets and low cytotoxicity.Therefore,it can be used as potential neuroprotective agents.The neuroprotective effects of saponins on nervous system diseases and injuries are increasingly reported:they promote neural network regeneration by directly promoting cell survival,improving neurite outgrowth,and restoring axonal and synaptic activity.Radiation causes an increase in oxidative stress levels and inflammatory responses in the brain,which affects cognitive function and neuronal structure,and ultimately leads to abnormal changes in neurogenesis,differentiation,and apoptosis.Astragaloside??AS-??is one of the main active constituents of astragalus.It is known for its antioxidant,antihypertensive,antidiabetic,anti-infarction,anti-inflammatory,anti-apoptotic and wound healing,angiogenesis and other protective effects.A large number of literatures have shown that the anti-apoptotic effect of AS-?is related to the improvement of various diseases of the central nervous system,but there are few reports on the protective mechanism of AS-?on radiation-induced neuronal apoptosis.In this study,the mechanism of AS-IV resistance to radiation-induced apoptosis of brain cells in vitro and in vivo was explored by radiation modeling,which provided a theoretical basis for the development of anti-radiation Chinese herbal active molecules and brain health products.Method:1.In vivo test,the mice were randomly divided into 5 groups,20 in each group,blank control group?Control?,solvent group?DMSO?,solvent+radiation group?DMSO+R?,low concentration of AS-?+radiation group?AS-?-L+R?,high concentration of AS-?+radiation group?AS-?-H+R?.The test was administered by intraperitoneal injection once a day.The Control group was given normal saline,DMSO group and DMSO+R group were given DMSO,and the final concentration could not exceed 0.01%.The doses of AS-?-L+R group and AS-?-H+R group were 20 mg/kg and 40 mg/kg,respectively.After one month of administration,8 Gy⁶⁰Co?-rays were used for one-time uniform radiation.After one week of radiation,brain tissue and molecular samples were collected.Brain sections were subjected to TUNEL staining to show apoptosis,and Western Blotting was used to detect changes in the expression of apoptosis-related proteins in mouse brain tissue.2.In vitro experiments,with neuron-like cells PC12 as the research object,divided into 5 groups,blank control group?Control?,solvent group?DMSO?,solvent+radiation group?DMSO+R?,low concentration of AS-?+radiation group?AS-?-L+R?,high concentration of AS-?+radiation group?AS-?-H+R?.The Control group did not do any treatment,DMSO group and DMSO+R group were given DMSO,and the final concentration could not exceed 0.01%.The doses of AS-?-L+R group and AS-?-H+R group were 25?g/mL and 50?g/mL,respectively.After the cells were attached to the wall,the drug was added and 70%⁸0%was used for radiation treatment.It is 6.5 J/cm².After the end of the irradiation,the cultivation was continued for 24hours.Apoptosis of PC12 cells was detected by flow cytometry,and the expression of apoptosis-related proteins in PC12 cells was detected by Western Blotting.Result:1.TUNEL staining of brain slices of in vivo mice showed that the apoptosis rate in the DMSO+R group was significantly higher than that in the Control group?P<0.001?,compared with the DMSO+R group,the apoptosis rate of the AS-?-H+R group decreased significantly?P<0.05?.The results of Western Blotting showed that the protein phosphorylation levels of JNK and p38 in the DMSO+R group showed a significant increase compared with the Control group?P<0.05,P<0.01?.The protein expression levels of p53,Caspase-9 and Caspase-3 and the ratio of Bax and Bcl-2showed a significant upward trend?P<0.05,P<0.001,P<0.001,P<0.001?.There was no significant difference in the protein expression levels of JNK and p38?P>0.05?.Compared with the DMSO+R group,the protein phosphorylation levels of JNK and p38 in the AS-?-L+R group showed a significant decrease?P<0.05?.The protein expression levels of p53,Caspase-9 and Caspase-3 and the ratio of Bax and Bcl-2showed a significant downward trend?P<0.05,P<0.001,P<0.001,P<0.01?.There was no significant difference in protein expression levels between JNK and p38?P>0.05?.2.Flow cytometry results in vitro showed that the apoptosis rate in the DMSO+R group was significantly higher than that in the Control group?P<0.001?,compared with the DMSO+R group,the apoptosis rate of the AS-?-H+R group decreased significantly?P<0.01?.The results of Western Blotting showed that the phosphorylation levels of JNK and p38 in the DMSO+R group showed a significant increase compared with the Control group?P<0.001?.The protein expression levels of p53,Caspase-9 and Caspase-3 and the ratio of Bax and Bcl-2 showed a significant upward trend?P<0.001,P<0.001,P<0.001,P<0.01?.There was no significant difference in protein expression levels between JNK and p38?P>0.05?.Compared with the DMSO+R group,the protein phosphorylation levels of JNK and p38 in the AS-?-L+R group showed a significant decrease?P<0.01?.The protein expression levels of p53,Caspase-9 and Caspase-3 and the ratio of Bax and Bcl-2 showed a significant downward trend?P<0.001,P<0.001,P<0.01,P<0.01?.There was no significant difference in protein expression levels between JNK and p38?P>0.05?.Conclusion:1.AS-?can effectively reduce radiation-induced apoptosis of brain cells.2.The mechanism of AS-?on radiation-induced brain cell anti-apoptosis may be related to the phosphorylation regulation mechanism of JNK-p38.