| Objective:In this paper,the basic concepts and theoretical origins of the method of tonifying the kidney and promoting blood and phlegm circulation were analyzed,and the effects of Bushen Huoxue Huatan Decoction on aging and dementia were analyzed.At the same time,the related literatures to the Alzheimer’s disease(Alzheimer’s disease,referred to as AD,the main type of Alzheimer’s disease),with the pathology process of Aβ(amyloid-βPeptide)protein accumulation and oxidative damage were sorted and analyzed.On the basis of the preliminary research foundation of the Supervisor’s team,the aging and the senile dementia were used as the starting point,and the Liuwei DiHuang Wan was used as the representative drug of tonifyingthe kidney method.On behalf of the drugs with effect of Huoxue huayu,the drug Sanqi was added as LiuWei DiHuang Wan plus Sanqi group(referred to as Bushen Huoxue group),LiuWei DiHuang Wan plus ErChen Decoction(referred to as Bushen Huatan group),Liu Wei Di Huang Wan plus Erchen and Sanqi,N2 and transgenic AD model(CL4176)were used to study the effect of oxidative level regulation,A β protein toxicity alleviation,aging related generegulation,and so on.This study was carried out to analyze and explain the mechanism of Bushen Huoxue Huatan Method on the prevention and treatment of dementia and anti-aging,and lay the solid foundation for the development of anti-AD drugs.Method:1.Theoretical Study: Collating literatures of effect on tonifying kidney,promoting blood and phlegm circulation,and discussing the relationship of attenuating antioxidation and Aβtoxicity in the C.elegans,in order to evaluate the method of tonifying kidney,promoting blood and phlegm circulation on the AD and aging,and the preliminary judgment on the the mechanism.2.Experimental study: Experimental study was carried out using N2(wild type)and CL4176(transgenic AD model)nematodes as animal model.Among them,CL4176 nematodes due to muscle cells in the expression of Aβ the the phenotype appeared on the symptoms of convulsions,with AD-related symptoms and pathological changes.In this paper,a paralysised experiment was carried out to screen the active drugs and the effectiveconcentration of the drug.At the same time,through the paraquat oxidative stress experiment,the antioxidative drug groups and effective concentration were screened out.And then through comparative analysis of the antioxidant effect of drugs and the alleviate the relationship between Aβ toxicity,to determine the anti-oxidant and can relieve Aβtoxicity of drug group and its effective concentration.On the basis of this,the following series of experiments were carried out: lifespan,heat stress,reproductive capacity,bending capacity and other aging related experiments to analyze the impact of active drugs on aging:reactive oxygen species(ROS),SOD and CAT antioxidantenzymes Activity and MDA(malondialdehyde)were measured to investigate the effect of active drugs on oxidative stress.The oxidative stress-related genes skn-1/Nrf2 and the heat shock protein family genes hsp-16.2、hsp-60 were analyzed by fluorescence quantitative PCR To investigate the mechanism of drug prevention and the treatment of AD and the mechanism of anti-aging.Aβprotein was used to collect Aβprotein in vitro to detect the effect of drugs on aggregation of Aβ protein.Through the above experiments,to explore the mechanism of renal function and the relationship between the role of AD and lifespan.Results:1.Effect on the toxicity of Aβin the C.elegans: the effect of the drug on the paralysis of the nematode can reflect the effect of the drug on the Aβtoxicity of the C.elegans.Through the induction of paralysis after the nematode counted and analysised,the Bushen group,Bushen Huoxue group,Bushen Huatan group and Bushen Huoxue Huatan group have been proved to alleviate the role of Aβ protein toxicity,including Bushen group,Bushen Huoxue group and the effective effect of the best concentration of Bushen Huatan group was 50 mg/ml,the best concentration of Bushen Huoxue Huatan group was 100 mg/ml.By further analysis of the effects of four treatments on Aβ toxicity,it was found that the effect of Bushen Huoxue group was the most obvious active drug,and the others were Bushen Huoxue Huatan group,Bushen Huatan group and Bushen group.This experiment was carried out on solid medium,so the optimal concentration of the drug obtained was the experimental concentration of the subsequent solid culture method.2.Effects of Antioxidant Effects in the C.elegans: Antioxidantexperiments include paraquat induced oxidative stress assay,In vivo ROS Levels,SOD and CAT Activities and MDA Content Detection.Through the modeling of paraquat oxidative stress,the survival time of the nematodes were observed to reflect the antioxidant capacity of the drug.The results showed that compared with the blank group,the drug group could significantly improve the survival rates of the nematode under paraquat stress.Among the0-120 h,the anti-oxidative stress of Bushen Huoxue Huatan group was the most significant,and after 120 h The best concentration was 40 mg/ml,the concentration of Bushen Huoxue group was 60 mg/ml,the concentration of Bushen Huatan group was 40 mg/ml,and the concentration of Bushen Huoxue Huatan group was 60 mg/ml.This experiment was carried out in liquid culture,so that the determined effective concentration was the concentration of the liquid culture method.Through the detection of ROS level,it was found that the levels of ROS in the nematode were significantly lower than those in the other groups,and the levels of ROS were the lowest in the Bushen Huoxue group,followed by the Bushen Huoxue Huatan group,Bushen Huatan group and Bushen group Low,therefore,the antioxidant effect of drugs from large to small followed by Bushen Huoxue group,Bushen Huoxue Huatan group,Bushen Huatan group and Bushen group;compared to the blank control group,Bushen group and other drug groups can be significantly enhanced(P <0.01),followed by Bushen Huoxue Huatan group(p <0.05),Bushen Huatan group(p<0.05),Bushen Huishen group(P <0.05),Bushen Huoxue group Group(p <0.05)..The patients were treated with Bushen Huoxue group(p<0.05),Bushen Huatan group(p <0.05)and Bushen group(p <0.05).,followed by Bushen Huatan group(p <0.05),Bushen Huoxue group(p<0.05),and the content of MDA in the nematode was significantly lower than that in the control group(p <0.05).The results showed that there was no significant decrease in MDA content.Bushen Huoxue Huatan method can be through the antioxidant damage,relieve Aβtoxicity,protect the neurons,reflecting the Bushen Huoxue Huatan method in the fight against the effect of the beautiful nematode Aβ play a major role in the process.3.The effect on the lifespan of the nematode: life-related experiments,including solid life experiments,thermal stress experiments,productiveexperiments,movement capacity experiments.The concentration of the Bushen,the Bushen Huatan and Bushen Huoxue were 50mg/ml,the concentration of Bushen Huoxue Huatan group was100mg/ml,the result of life experiment was the same as that of blank(P<0.05),the average life expectancy of the Bushen group was21.842±0.19 days(P<0.05),the average life span of the blank group was 18.975±0.175 days,the average life expectancy of the bushen group was 21.842±0.190 days,the the average life expectancy of the kidney group was 22.943±0.194 days(p<0.05).The average lifespan of Bushen Huoxue Huatan group was 25.651±0.106 days(p<0.001),and the results were statistically significant.The results of four kinds of anti-aging methods were compared,the effect of anti-aging effect of Bushen Huoxue Huatan group was the best,followed by Bushen Huoxue group,followed by Bushen Huatan group and finally Bushen group.The results of thermal stress test were compared with blank group(P <0.001),the effect of heat stress on the other groups was significantly higher than that of the control group(P <0.001),and the difference was statistically significant(P <0.001).The results of fecundity test showed thatthe number of progeny in the peak of reproductive group was 168.7±25,2 in the control group,and the difference between the two groups(P <0.05)50mg/ml)244.7±22.8(p<0.001),Bushen Huoxue group was 280.6±20.7(p<0.001),Bushen Huatan group 235.7±19.1(p<0.001),compared with the control group,the drug group were significantly improved the beautiful nematode reproductive ability(P <0.01),which is more significant role in the kidney and blood circulation group,the other drugs The results were followed by Bushen Huoxue Huatan group,Bushen group,Bushen Huatan group,the difference was the results showed that the effect of tonifying kidney and activating blood circulation on exercise ability was better than that of other groups,followed by Bushen Huoxue Huatan group,Bushen group,and Bushen Huatan group.4.The effects of Bushen Huoxue Huatan method on Antioxidant genes and major heat shock protein family genes:The relative expression level of mRNA of the related gene in the beautiful nematode was expressed by the2-ΔΔCT value of Real-Time PCR.The expression of skn-1 mRNA was significantly up-regulated compared with act-1 m RNA,indicating that the mechanism of Bushen Huoxue Huatan Decoction to alleviate the toxicity of Aβ may be related to the anti-oxidative pathway of skn-1/Nrf2.The expression of hsp-16.2 and hsp-60 genes was studied by using the method of RT-PCR.The results showed that the expression of hsp-16.2 and hsp-60 genes in the heat shock protein family was studied.The results showed that the AD model nematode The expression of hsp-16.2 m RNA and hsp-60 m RNA were not up-regulated or down-regulated by phlegm method.5.Effects of Bushen Huoxue Huatan Method on In vitroAggregation of Aβ Protein:The results showed that the drug was incubated with Aβ but did not affect the vitro aggregation of Aβ protein.Therefore,the results showed that the effect of Bushen Huoxue Huatan on the removal of Aβ in nematode was not achieved by direct competitive binding of Aβ protein May be related to some of the stress in the body.Conclusion:1.Bushen Huoxue Huatan method for delaying Aβ toxicity and delay life have a significant role in its treatment is significantly better thanBushen、Bushen Huoxue and Bushen Huoxue Huatan and blood and phlegm,anti-oxidation effect is the most significant kidney and blood circulation.2.Bushen Huoxue Huatan method can alleviate the role of Aβtoxic drugs more antioxidant activity,and has a certain degree of extension on lifespan,indicating that anti-AD drugs on the health of life has a positive effect.3.Bushen Huoxue Huatan method on AD nematode mainly through the body of antioxidant stress,and this effect has anti-aging,improve the role of antioxidant enzymes and other effects.4.Bushen Huoxue Huatan method relieves the mechanism of Aβ toxicity protein toxicity,not through the regulation of heat shock protein family gene or the space structure,but mostly related to the skn-1/Nrf2 mediated antioxidant mechanism. |
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