| ObjectiveTo investigate the level of serum superoxide dismutase(SOD),renal malondialdehyde(MDA),renal tissue Klotho gene and protein expression,and Klotho gene methylation level in kidney tissue induced by H2O2 in oxidative damage induced by H2O2.The impact of Klotho/FGF-23 signaling pathway on the molecular mechanism of Qiongyu Paste(composed of four components:rehmannia,honey,alfalfa,and ginseng)in delaying aging of zebrafish,and opening up a new field of research on the molecular mechanism of Qiongyu Paste anti-aging.MethodsModel:Three-month-old adult zebrafish with H2O2 stimulation was selected to detect 4-week survival rate and oxidative stress level.At the same time,the effects of 7 different concentrations of Qiongyu paste on the survival rate of zebrafish were observed.We determine the optimal H202 concentration,set the high,medium and low concentrations of Qiongyu paste for subsequent research,and determine the concentration of specific Qiongyu paste in the high,medium and low concentrations of the formal experiment.Group:5 groups in total,A:normal control group,B:model group(200?M H2O2),C:low concentration Qiongyu paste group(100 ?g g/ml Qiongyu paste+ 200 ?MH2O2),D:medium concentration Qiongyu paste group(200 ? g/ml Qiongyu paste+200 ?M H2O2),E:high concentration Qiongyu paste group(400 ?g/ml Qiongyu paste+ 200 ?M H202),20 zebrafish(each male and female)in each group.Feeding and administration methods:Adult fish are concentrated and cultured at 28° C constant temperature system,PH=7,2?7,6,water conductivity 550?650 ? s/cm,flowing water,alternating ratio of light and dark is 14H:10H.Feed feeding:twice a day,hatching shelled brine shrimp.Method of administration:the model group zebrafish was incubated with pure aquaculture water for 8 hours from 8:00 am to 16:00 pm,and incubated with 200 ?M hydrogen peroxide culture water for 16 hours from 16:00 pm to 8:00 am the next day for 6 weeks;low,medium,The high concentration Qiongyu paste group zebrafish was incubated with 100 ?g/ml,200 ?g/ml,400 ?g/ml Qiongyu paste culture water for 8h every day from 8:00am to 16:00pm,and used for 200 ?M peroxidation from 16:00pm to 8:00am.Hydrogen culture water was incubated for 16 h for 6 weeks.Detection indicators and corresponding methods:(1)biochemical kit detection of superoxide dismutase(SOD),malondialdehyde(MDA)levels;(2)real time quantitative PCR(Real time PCR)detection of Klotho gene expression levels;(3)Western blotting was used to detect Klotho protein expression level;(4)Methylmion specific PCR(MSP)was used to detect Klotho gene methylation level.Results1.Pre-experimental results of Qiongyu paste concentration gradient:0 p g/ml,100 ?g/ml,200 ?g/ml,400 ?g/ml group,3 weeks zebrafish 4 weeks survival rate is 100%;Qiongyu paste concentration is 800 ?g/ml At 1600 ?g/ml and 3200 ?g/ml,the 4-week survival rate of zebrafish at 3 months was gradually decreased with the increase of Qiongyu paste concentration.From the data analysis,we determined that the concentration gradient of Qiongyu paste was:100 ?g/ml for the low concentration Qiongyu paste group,200 ?g/ml for the medium concentration Qiongyu paste group,and 400 ?g/ml for the high concentration Qiongyu paste group.The pre-experiment of H202 concentration gradient showed that the 4-week survival rate of zebrafish was 100%when the concentration of H202 was below 200 ?M.The 4-week survival rate of zebrafish decreased with the increase of H202 concentration to 400 ?M and 800 ?M.From the results analysis,we determined the concentration of 200 u M H202 as the oxidant for modeling aging zebrafish.2.Serum SOD results of zebrafish:The difference between groups was statistically significant(P=0.000,P<0.01).The Dunett test between the control group and the experimental group showed that the model group vs the normal control group,P=0.000;the low concentration Qiongyu paste group vs the normal control group,P=0.000;the medium concentration Qiongyu paste group vs the normal control Group,P=0.000;Evs normal control group,P=0.090;the above data showed that the SOD value of low concentration Qiongyu paste group and medium concentration Qiongyu paste group was significantly lower than that of normal control group,and the difference was statistically significant.P<0.01;while the SOD value of the high concentration Qiongyu paste group was almost close to the SOD value of the normal control group,the difference was not statistically significant,P>0.05,reflecting the high concentration of Qiongyu paste,the antioxidant enzyme SOD in the aging zebrafish almost Close to normal values,the antioxidant effect is more accurate.The Bonferroni test between the model group and the experimental group showed:model group vs low concentration Qiongyu paste group,P=1.000;model group vs medium concentration Qiongyu paste group,P=0.000;model group vs high concentration Yufu group,P=0.000;low concentration Qiongyu paste group vs medium concentration Qiongyu paste group,P=0.001;low concentration Qiongyu paste group vs high concentration Qiongyu paste group,P=0.000;medium concentration Qiongyu paste group Vs high concentration Qiongyu paste group,P=:0.000;the above data showed that low concentration Qiongyu paste had no significant effect on increasing the SOD value of oxidized aging zebrafish,the difference was not statistically significant,P>0.05;but the medium concentration Qiongyu paste The group and the high concentration Qiongyu paste group had obvious effects on increasing the SOD value of oxidized aging zebrafish,and the high concentration of Qiongyu paste was better than the medium concentration Qiongyu paste.The difference was statistically significant(P<0.01).3.MDA results of zebrafish kidney tissue:The difference between groups was statistically significant(P=0.000,P<0.01).The Dunett test between the control group and the experimental group showed that the model group vs the normal control group.P=0.000;low concentration Qiongyu paste group vs normal control group,P=0.000;medium concentration Qiongyu paste group vs normal control group,P=0.000;high concentration Qiongyu paste group vs normal control group,P=0.018;The data showed that the MDA values of the low,medium and high concentrations of Qiongyu paste group were significantly different from those of the normal control group,P<0.05,reflecting that the low,medium and high concentrations of Qiongyu paste group failed to make the intervention.Free radical MDA values in aging zebrafish have dropped to near normal levels.The Bonferroni test between the model group and the experimental group showed:model group vs low concentration Qiongyu paste group,P=1.000;model group vs medium concentration Qiongyu paste group,P=0.001;model group vs high concentration Yufu group,P=0.000;low concentration Qiongyu paste group vs medium concentration Qiongyu paste group,P=0.002;low concentration Qiongyu paste group vs high concentration Qiongyu paste group,P=0.000;medium concentration Qiongyu paste group Vs high concentration Qiongyu paste group,P=0.056;the above data showed that there was no significant difference between the MDA value of the low concentration Qiongyu paste group and the MDA value of the model group,P>0.05,reflecting the low concentration of Qiongyu paste to reduce the aging zebra The effect of fish MDA was not obvious;the medium concentration Qiongyu paste group and the high concentration Qiongyu paste group were more effective in reducing the MDA value of aging zebrafish,the difference was statistically significant,P<0.01,but the medium concentration Qiongyu paste group There was no significant difference between the effect and the high concentration Qiongyu paste group,P>0.05.This indicates that after the concentration of Qiongyu paste reaches 200 ?g/ml,increasing the concentration of Qiongyu paste can not increase the effect of reducing the MDA value of aging zebrafish.4.Real time PCR reaction system was used to detect Klotho gene expression in renal tissues:the difference between groups was statistically significant(P=0.000,P<0.01),and the Dunett test between the control group and the experimental group showed:Group vs normal control group,P=0.000,low concentration Qiongyu paste group vs normal control group,P=0.000,medium concentration Qiongyu paste group vs normal control group,P=0.000,high concentration Qiongyu paste group vs normal control group P=0.209;the above data showed that the expression level of Klotho gene in the low concentration Qiongyu paste group and the medium concentration Qiongyu paste group was significantly different from that in the normal control group,P<0.01,while the high concentration Qiongyu paste group There was no significant difference between the expression level of Klotho gene in the kidney and the normal control group,P>0.05,which indicated that the low concentration of Qiongyu paste and medium concentration Qiongyu paste could not increase the expression of Klotho gene in kidney tissue,but the concentration of high concentration Qiongyu paste increased.The expression of Klotho gene in kidney tissue is more accurate.The Bonferroni test between the model group and the experimental group showed:model group vs low concentration Qiongyu paste group,P=1.000;model group vs medium concentration Qiongyu paste group,P=0.000;model group vs high concentration Yufu group,P=0.000;low concentration Qiongyu paste group vs medium concentration Qiongyu paste group,P=0.000;low concentration Qiongyu paste group vs high concentration Qiongyu paste group,P:0.000;medium concentration Qiongyu paste group Vs high concentration Qiongyu paste group,P=0.000;the above data showed that low concentration Qiongyu paste had no significant effect on promoting the expression of Klotho gene in aging zebrafish,the difference was not statistically significant,P>0.05,but medium concentration Jade cream and high-concentration Qiongyu paste have obvious effects on promoting the expression of Klotho gene in kidneys that are easy to cause aging zebrafish,and the high concentration of Qiongyu paste group has significant statistical significance due to the medium concentration Qiongyu paste group,P<0.01.5.Westen blotting was used to detect the expression of Klotho protein in renal tissues.The difference between the two groups was statistically significant(P=0.000,P<0.01).The Dunett test between the control group and the experimental group showed that the model group was normal.In the control group,P=0.000,low concentration Qiongyu paste group vs normal control group,P=0.000,medium concentration Qiongyu paste group vs normal control group,P=0.002,high concentration Qiongyu paste group vs normal control group,P=0.095;The above data showed that the expression levels of Klotho protein in the low and medium concentration Qiongyu paste group were significantly different from those in the normal control group,P<0.01,but the high concentration of Klotho protein in Qiongyu paste group There was no significant difference in the expression of Klotho protein between the level and the normal control group,P>0.05,indicating that only the high concentration of Qiongyu paste can make the Klotho protein expression level of senile zebrafish kidney close to normal level.The Bonferroni test between the model group and the experimental group showed:model group vs low concentration Qiongyu paste group,P=1.000;model group vs medium concentration Qiongyu paste group,P=0.006;model group vs high concentration Yufu group,P=0.000;low concentration Qiongyu paste group vs medium concentration Qiongyu paste group,P=0.038;low concentration Qiongyu paste group vs high concentration Qiongyu paste group,P=0.001;medium concentration Qiongyu paste group Vs high concentration Qiongyu paste group,P=0.382;the above data showed that there was no significant difference between the expression of Klotho protein in the low concentration Qiongyu paste group and the expression of Klotho protein in the model group,P>0.05,while the medium and high concentration Qiong There was a statistically significant difference in the expression of Klotho protein between the jade cream group and the model group,P<0.05,reflecting that the low concentration of Qiongyu paste had no significant effect on increasing the expression level of Klotho protein in zebrafish kidney which is easy to cause aging.Ointment and high concentration of Qiongyu paste have obvious effects on increasing the expression level of Klotho protein in zebrafish kidney which is easy to cause aging.The high concentration of Qiongyu paste is better than the medium concentration Qiongyu paste,the difference is statistically significant,P<0.05.6.Methylmion specific PCR(MSP)was used to detect the methylation of Klotho gene in renal tissues:in normal:control group,it showed partial methylation,and in model group and low-dose group,it showed all-A The base was partially methylated in the high-dose Qiongyu paste group.ConclusionBased on the research on the anti-aging mechanism of Qiongyu paste in the past,the pre-experiment of the Qiongyu paste concentration gradient determination was continued on the new animal model zebrafish.By observing the survival rate of adult zebrafish,the final selection was 100 ?g/Ml was low concentration Qiongyu paste group,200 ?g/ml was medium concentration Qiongyu paste group,400 ?g/ml was high concentration Qiongyu paste group,and the appropriate H202 concentration for inducing zebrafish senescence was determined to be 200 ?M.In the study of antioxidant-related indicators of aging zebrafish serum SOD and renal MDA free radical production,it can be found that the medium concentration Qiongyu paste group and the high concentration Qiongyu paste group have significant effects on increasing the SOD value of oxidized aging zebrafish,and the high concentration The effect of Qiongyu paste is better than that of medium concentration Qiongyu paste.The high concentration of Qiongyu paste can increase the serum SOD of aging zebrafish to the level of normal control group,and the antioxidant effect is obvious.The medium concentration Qiongyu paste group and the high concentration Qiongyu paste group are more effective in reducing the MDA value of oxidized aging zebrafish.However,after the concentration of Qiongyu paste reaches the medium concentration of 200 ?g/ml,increasing the concentration of Qiongyu paste does not increase its aging zebra.The effect of fish MDA value,and the low,medium and high concentrations of Qiongyu paste group failed to reach the level of normal control group to reduce the MDA value of aging zebrafish.In general,Qiongyu paste can improve the antioxidant enzymes of aging zebrafish and remove a certain amount of free radicals,which proves that Qiongyu paste can resist aging by anti-oxidative stress.Based on the study of sirtl/p53 signaling pathway in the past,Qiongyu paste delayed the aging of zebrafish,this experiment innovatively studied the Klotho/FGF-23 signaling pathway of aging zebrafish,and found that Qiongyu paste can improve the Klotho gene and protein of aging zebrafish.The expression level,to some extent,reversed the methylation level of Klotho gene in senile zebrafish kidney tissue,and opened up a new field for the molecular mechanism of Qiongyu paste anti-aging.Regarding the mechanism of anti-aging of Qiongyu paste,the instructor and team of the teaching and research section of the author have done a lot of work in the past 10 years,and have rich research foundations for Qiongyu paste anti-aging egg genomics,proteomics and metabolomics.In the past experiments,Qiongyu paste was found to be simple in the prescription,but it has good anti-oxidation,anti-aging,beauty and other effects.From the mechanism of traditional Chinese medicine,Qiongyu paste can strengthen the spleen and tonify the kidney,and it can be used to replenish the innate.After the adjustment,the innate effect is nourished;from the modern medical mechanism,Qiongyu paste promotes the signalling of the first-pass signal by reversing the methylation and anti-aging of anti-aging genes and promoting the expression of anti-aging genes and proteins.Expression plays a role in delaying aging.This topic is also based on the instructor' s establishment on the basis of continuous innovation,under the guidance of the tutor' s "full health" concept,and constantly research Qiongyu paste,it is expected that through scientific research into health care products,supplies,etc.for people to be practical and convenient to use form.Anti-aging,health is our daily life,clothing,food and shelter.Qiongyu paste is a potential entry point for us to study specific health regimens such as diet,beauty and health,to meet people' s growing needs for health and beauty.As an ancient researcher,we should constantly innovate,innovate research methods and practical methods,and use the ancients and the present to play a greater role in the current aging society in China and the sustainable economic and social development of Guangdong,Hong Kong and Macao. |
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