The Protective Effect And Mechanism Of Saponins In Raw And Steamed Panax Notoginseng On Oxidative Damage Of Nerve Cells Via Drug Metabolizing Enzymes

Due to the aggravation of population aging,the incidence rate and mortality of neurodegenerative diseases are increasing,and there is still a lack of effective treatment.The pathogenesis of neurodegenerative diseases is complex,in which oxidative stress can lead to the dysfunction of nerve cells,manifested as memory dysfunction,which is an important pathogenesis.Therefore,the search for antioxidant substances to maintain redox balance in the body has become a new strategy to treat neurodegenerative diseases caused by oxidative stress injury.Panax notoginseng（Burk）F.H.Chen is a commonly used Chinese medicinal herb belonging to the genus Ginseng,which has antioxidant activity and plays a role in the treatment of diseases such as stroke,Alzheimer’s disease,tremor paralysis,etc.Traditionally,P.notoginseng was used raw and steamed differently.After processing,the main saponins in P.notoginseng are converted into rare saponins,which might lead to different antioxidant activities due to significant differences in their components.By comparing the antioxidant activities of raw and steamed P.notoginseng,the antioxidant markers were explored,so as to elucidate the intrinsic protective mechanism of raw and steamed P.notoginseng in improving nerve damage with antioxidant activity.Therefore,this paper combined with a variety of in vitro antioxidant activity methods to investigate the antioxidant activity of raw and steamed P.notoginseng.With H₂O₂-induced oxidative damage in PC12 cells as a model,the neuroprotective effect of raw and steamed P.notoginseng was studied.Based on metabolic enzymes,in order to explore the mechanism of neuroprotection of saponin-mediated oxidative stress response and elucidate the protective effect and related internal mechanism of raw and steamed P.notoginseng on H₂O₂-induced oxidative damage in PC12 cells.It improves the possibility of rational application and development of P.notoginseng,provides a theoretical basis for the development of new drugs for oxidative stress-related diseases,expands new field directions,provides new targets for the treatment of clinical diseases,and directly provides new scientific targets in the field of clinical treatment.The main research contents and results are as follows:1.The content changes of saponins in raw and steamed P.notoginseng and the effect of free radical scavengingHPLC method was used to analyze 13 saponin components in raw and steamed P.notoginseng.The results showed that the content of the main saponins（R₁,Rg₁,Re,Rb₁,Rd）in raw P.notoginseng decreased with the increase of the number of steaming times,while the content of rare saponins（20（S）-Rh₁,20（R）-Rh₁,20（S）-Rg₃,20（R）-Rg₃,Rk₃,Rh₄,Rk₁,Rg₅）in steamed P.notoginseng increased.Four kinds of antioxidant models were established in vitro using the principle of free radical scavenging and it was found that the antioxidant activity of steamed P.notoginseng was better than that of raw P.notoginseng.The correlation between saponins content and antioxidant activity in raw and steamed P.notoginseng was determined by Pearson method.The results showed that the scavenging rates of ABTS and DPPH were negatively correlated with the content of saponins in raw P.notoginseng,and positively correlated with the content of saponins in steamed P.notoginseng,indicating that the antioxidant activity of steamed P.notoginseng was related to the increase of rare saponin content.2.The neuroprotective of saponins in raw and steamed P.notoginseng on H₂O₂- induced oxidative stress damage in PC12 cellsThe oxidative damage model was established in PC12 cells treated with H₂O₂.The neuroprotective effects of raw and steamed P.notoginseng and 13 saponins were evaluated by measuring cell survival rate and ROS levels in cells.The results showed that steamed P.notoginseng,which were steamed three times,and ginsenoside Rk₃significantly improved cell survival and decreased ROS levels.Fluorescence microscopy was used to observe the fluorescence intensity of ROS in cells.It was intuitively evident that steamed P.notoginseng,which were steamed three times,and ginsenoside Rk₃,significantly reduced the accumulation of ROS in cells after H₂O₂damage.The effects of raw and steamed P.notoginseng saponins on intracellular oxidative stress indexes were further explored,and the results showed that raw and steamed P.notoginseng saponins could increase the levels of T-SOD,CAT,GSH,T-AOC and reduce LDH levels,among which the antioxidant stress damage effect of steamed P.notoginseng was better than that of raw P.notoginseng.Hoechst 33342/PI staining was used to detect apoptosis,and the results showed that the apoptosis cells were significantly reduced from cell morphology after raw and steamed P.notoginseng saponin treatment,and the apoptosis effect of steamed P.notoginseng was better than that of raw P.notoginseng.The results of western blotting showed that raw and steamed P.notoginseng saponins inhibited the release of pro-apoptotic proteins Bax,Caspase-3and Caspase-9 in cells,and promoted the production of anti-apoptotic protein Bcl-2,thereby reducing the apoptosis of nerve cells.Nrf2 is the most critical transcription factor in the antioxidant defense system.Raw and steamed P.notoginseng saponins activated Keap1-Nrf2/HO-1 pathway and promoted nuclear translocation of Nrf2,and activated antioxidant stress pathways,thereby reducing the oxidative damage of H₂O₂-stimulated PC12 cells.Further studies had shown that the active ingredient Rk₃ in steamed P.notoginseng also inhibited oxidative stress by activating Nrf2 and promoting nuclear translocation of Nrf2,thereby inhibiting the apoptosis of nerve cells and exerting neuroprotective effects.3.The molecular mechanism of saponins in raw and steamed P.notoginseng to mediate oxidative stress reactions and exert neuroprotective effects based on metabolic enzymesDrug metabolizing enzymes are important targets for drug production.Total saponins of raw and steamed P.notoginseng and ginsenoside Rk₃ had different degrees of regulatory effects on phase I metabolizing enzymes（CYP2E1,CYP1A2,CYP3A4）,phase II metabolizing enzymes（SULT1A1,SULT1A3,UGT1A6）and effector transporter（BCRP）.These metabolic enzymes were closely related to oxidative stress response.Further studies showed that the total saponins of raw and steamed P.notoginseng and ginsenoside Rk₃ had inhibitory effects on the protein expression levels of phase I metabolic enzymes（CYP1A1 and CYP1A2）in H₂O₂-induced PC12 cells.It can induce the expression level of phase I metabolic enzymes（CYP2E1,CYP3A4）,phase II metabolic enzymes（UGT1A6,SULT1A3）and transporter BCRP protein.The total saponins of raw and steamed P.notoginseng and active compound Rk₃ might affect the metabolism of drugs in vivo by regulating the activity of metabolic enzymes,so as to enhance the antioxidant effect.Conclusion:Both raw and steamed P.notoginseng had antioxidant activity,and steamed P.notoginseng was superior to raw P.notoginseng.The main reason for the difference in the activity of raw and steamed P.notoginseng might inhibit oxidative stress response and apoptosis of nerve cells by regulating metabolic enzymes to varying degrees,activating Nrf2,and thus had neuroprotective effect.