Research On Anti-aging Effect And Mechanism Of Chikusetsu Saponin Ⅳa Through Regulating Oxidative Stress And Autophagy

Background: Aralia taibaiensis is a medical plant,which is widely distributed in the Qinba Mountains and their extensions in Western China.Saponins are active ingredients in Aralia taibaiensis that play a major pharmacological role.We found that the total saponins of Aralia taibaiensis showed good anti-aging effect in D-galactose induced aging model.Chikusetsu saponin IVa（CHS）,a major saponin with relatively high content in Aralia taibaiensis,exhibited significant antioxidant effect.A large number of studies have shown that oxidative stress is closely related to aging,so can CHS p lay an anti-aging role through its excellent antioxidant capacity? Moreover our previous studies have shown that CHS has the function of activating AMPK,which regulats autophagy.More and more evidences show that autophagy can affect aging.Based on these finding,we have reasons to speculate that CHS can regulate aging by modulating autophagy through activting AMPK.In order to make clear these questions,we established two kinds of cell senescence model to obseave the anti-aging effect of CHS from the views of oxidative stress and autophagy.The findings of this study may provide new data for the research and the exploitation of Aralia taibaiensis and its active ingredients.Objective: To study the anti-aging effect and mechanism of CHS,a major ingredient of Aralia taibaiensis by using hydrogen peroxide（H₂O₂）-induced mouse embryonic fibroblasts（MEFs）senescence and MEFs replicative senescence model.Methods: 1.MEFs were isolated from embryos of Balb/c mice for conventional cell culture.For H₂O₂-induced cell senescence,we treated the MEFs of 5th 8th passage with different concentrations of H₂O₂ with/without CHS.For the replicative senescence model,MEFs were cultured in a conventional manner to 15 th passage.MEFs of 5th,10 th and 15 th passage were harvested for further determination.In intervention experiment,MEFs were treated with different concentrations of CHS,NH4 Cl and/or Compound C.2.The effects of H₂O₂ and CHS on MEFs viability were detected by MTT assay respectively.3.The aging status of MEFs was detected by the senescence-related β galactosidase（SA-β-gal）staining.4.DCFH-DA fluorescent probe staining and Mito SOXTM Red mitochondrial superoxide probe staining were used to detect the total ROS and mitochondrial ROS respectively.5.The distribution and quantity of autophagic lysosomes in MEFs were observed under fluorescence microscopy after MDC staining.6.Using m RFP-GFP-LC3 adenovirus to transfect MEFs,fluorescence microscopy was used to observe the red and green fluorescence ratio as well as yellow fluorescence to evaluate the autophagic flux.7.The ultrastructural changes of autophagy and aging of MEFs in different passage number were observed by transmission electron microscopy.8.Western blot was used to detect proteins levels,such as p53,p21,p16,β-gal（as senescence marker proteins）,Nrf2 and its downstream target molecule HO-1,GCLM and GCLC,autophagy related proteins levels,such as LC3,p62,Lamp 1,Lamp 2,Beclin 1,LC3,Atg 7,Atg 5,Atg 12,and autophagy upstream regulators,such as AMPK,mTOR,ULK1 and their phosphorylation form p-AMPK（Thr172）,p-mTOR（Ser2448）,p-ULK1（Ser555）,p-ULK1（Ser757）.Results: 1.H₂O₂ induced senescence of MEFs.H₂O₂ effectively induced the senescence of MEFs,showing a higher positive rate of SA-β-gal staining,and higher protein levels of p53 and p21.Moreover,H₂O₂ stimulated cell ROS and mitochondrial ROS levels,as well as antioxidant transcription factor Nrf2 and its downstream targeted molecule protein expressions,such as HO-1,GCLM and GCLC.2.CHS protected form H₂O₂ induced MEFs senescence by its anti-oxidative stress effect.CHS was able to reduce the positive rate of SA-β-gal staining of the senescent cells induced by H₂O₂,as well as the expressions of p53 and p21.In addition,CHS inhibited the total ROS and mitochondrial ROS levels induced by H₂O₂.Moreover,CHS effectively inhibited the expressions of antioxidant transcription factor Nrf2 and its downstream targeted molecules,HO-1,GCLM and GCLC in H₂O₂ induced MEFs senescence.3.Establishment of MEFs replicative senescece model.MEFs showed an obvious senescece phenotype by continuous subculturing 15 passages,namely,the higher positive rate of SA-β-gal staining and the higher expression of β-gal,p21 and p16 protein,compared with the 5th passgage.4.The level of autophagy of MEFs decreased in replicative senescece model.With the increase of cell passage number,autophagy levels of MEFs decreased,showed by the lower level of Lamp 1,Lamp 2,Beclin 1,LC3,Atg 7,Atg 5 protein expression,and the higher level of p62 protein expression.Morever,the MDC staining decreased with autophagy flux weakened.5.CHS reduced MEFs replicative senescence phenotype.CHS could reduce the reproductive senescence phenotype of MEFs in a dose-dependent manner,such as the lowering the positive rate of SA-β-gal staining and the protein expression levels of p21 and p16.6.CHS attenuated the replicative senescence phenotype of MEFs by increasing the level of autophagy.CHS cuold improve the level of cell autophagy,characterized by higher LC3 II protein level and MDC staining,lower level of p62 protein level and higher level of autophagy flux.7.The anti-aging effect of CHS was achieved by improving autophagy through modulating AMPK / mTOR / ULK1 pathway In the process of MEFs replicative senescence,the 15 th passage control group comparing with the 5th passage control group,the levels of p-AMPK（Thr172）and p-ULK1（Ser555）decreased,but the levels of p-mTOR（Ser2448）and p-ULK1（Ser757）increased.CHS supplementation reversed these changes above.When the AMPK inhibitor Compound C was added,the antagonistic effect of CHS was inhibited.At the same time,the effects of CHS enhancing autophagy and autophagy flux,as well as reducing the senescence phenotype were inhibited.Conclusions: 1.CHS protects from H₂O₂ induced MEFs senescence by its anti-oxidative stress effect.On the base of research on H₂O₂ induced MEFs senescence model,we found that CHS,the major ingredient of Aralia taibaiensis,can effectively reduce oxidative stress,alleviate the cells senescence phenotype caused by H₂O₂.CHS has a remarkable antioxidant and anti-aging effect.2.CHS enhances autophagy to attenuate MEFs replicative senescence by activating AMPK / mTOR / ULK1 Pathway In the process of MEFs reproductive senescence,the level of aut ophagy decreases with the levels of p-AMPK（Thr172）and p-ULK1（Ser555）decreased,and the levels of p-mTOR（Ser2448）and p-ULK1（Ser757）increased.CHS can effectively increase the level of autophagy to protect cells from aging by activating the p-AMPK（Thr172）-p-ULK1（Ser555）pathway,and inhibiting the p-mTOR（Ser2448）-p-ULK1（Ser757）pathway.