Study On The Mechanism Of Erzhi Pill In Nourishing Yin And Regulating Water Metabolisim To Anti-Aging Based On The Theory Of "Kidney Governing Water"

Objective:Based on the theory of"kidney governing water",this project explores the changes of water metabolism in the process of renal aging by constructing a natural aging rat model in vivo and an in vitro induced aging kidney cell model.The ultimate goal is to provide a scientific basis for the use of Erzhi Pill to nourish Yin and delay the aging process.Methods:1.Pharmacological efficacy study of Erzhi Pill intervention in naturally aging ratsThe 20-month-old naturally senescent rats were randomly divided into four group:Erzhi Pill low-dose group（EL,n=7）,Erzhi Pill medium-dose group（EM,n=8）,Erzhi Pill high-dose group（EH,n=8）and natural aging model group（K,n=7）.Rats in the Erzhi Pill groups were fed diets containing the prepared Erzhi Pill at 8%,16%,and 32%respectively for the low,medium,and high-dose groups.The rats in the model group were fed 20 g each per day until 24 months of age.The 6-month-old SD rats were used as the youth control group（Q,n=10）and the 6-week-old SD rats were used as the juvenile group（Y,n=10）,and the rats in the natural aging group were fed with normal diet.The changes of body mass,morphological characteristics and survival ratio of naturally aged were recorded before and after drug administration.Bioelectrical impedance detection of body composition,blood gas analyzer for blood gas indexes,HE pathological sections,SA-β-galactosidase staining for organ physiopathological changes,kits for SOD and MDA antioxidant indexes and biochemical indexes,q PCR for telomere length,and ELISA for telomerase activity,aldosterone,and antidiuretic hormone were used to analyze the physiological changes before and after administration of Erzhi Pill.2.Investigation of the potential mechanism of action of Erzhi Pill in delaying aging based on metabolomicsNaturally aged rat plasma and urine samples collected by UPLC-Q-TOF/MS were metabolomic analyzed,by Progeneisi QI software to extract the peak area,retention time,m/z value and other information of each group,and OPLS-DA analysis was performed by SIMCA software,and substances with VIP>1 and P<0.05 were screened as potential differential metabolites,and differential metabolites and their related metabolic pathways were identified and analyzed by HMDB,Metabo Analyst database and KEGG database.From the perspective of substance metabolism,the potential mechanism of action of Erzhi Pill to delay aging was discussed.3.Effect of Erzhi Pill on the viability of senescent HK-2 cellsThe extract of Erzhi Pill was prepared according to the pharmacopoeia method,and the serum containing the drug was prepared by gavaging rats with a conversion factor of 6.17 times the equivalent dose in rats and humans for 7 days.D-gal was selected to construct an in vitro HK-2 cell senescence model,and the effects of different concentrations of D-gal(0,5,10,20,30,40 mg·ml^-1)on the survival rate of HK-2 cells at different times（24,48,72 h）were examined by the cck-8 method to screen out the optimal modeling dose concentration and time of D-gal.The effects of different concentrations of Erzhi Pill drug-containing serum（5%,10%,and 15%）on the viability of HK-2 cells were observed to determine the subsequent in vitro administration concentration.4.Protective effect of Erzhi Pill on senescent HK-2 cellsWith reference to the above administration concentrations,blank control group（Control）,model control group（Model）,low dose Erzhi Pill group（E-5%）,medium dose Erzhi Pill group（E-10%）and high dose Erzhi Pill group（E-15%）were set up to observe the effects of Erzhi Pill on senescent HK-2cells by usingβ-galactosidase assay and ELISA.The effects of Erzhi Pill on apoptosis and cell membrane permeability of senescent HK-2 cells were detected by flow cytometry and the effects of Erzhi Pill on the oxidation levels of SOD and MDA of senescent HK-2 cells were detected by ELISA.5.Probing the mechanism of aging retarding effect of Erzhi Pill based on water channel protein expressionThe protein of naturally aged rat kidney tissues and senescent HK-2 cells were extracted,and the changes in the expression of key water channel proteins AQP1 and AQP2 were detected by Western blot assay.This was done to further investigate the mechanism of action of Erzhi Pill in nourishing Yin and regulating water-liquid metabolism to slow down aging.Result:1.Pharmacological efficacy study of Erzhi Pill intervention in naturally aging ratsCompared with the young and young control rats,the rats in the natural aging model group showed obvious aging symptoms and gradual weight loss.The intervention of Erzhi Pill with medicated feed alleviated the aging symptoms of the natural aging model to a certain extent,and the body mass rebounded showed an increasing trend.The results of body composition analysis showed that compared with juvenile and young controls,TBW was significantly lower in naturally senescent rats（P<0.01）,while ECF showed an increasing trend,ICF showed a decreasing trend,but there was no statistical difference（P<0.05）,FM accounted for an increasing trend（P<0.01）and FFM accounted for a decreasing trend（P<0.01）.In the blood gas results,p O2decreased significantly（P<0.01）and p CO2 increased in the rats in the natural aging model group compared with the young and youthful groups,while arterial blood p CO2 showed a decreasing trend and p O2,s O2 showed an increasing trend after drug administration compared with the rats in the natural aging model group;in the liver and kidney function tests,except for UA in the low-dose administration group,the remaining indexes compared with the elderly model group,ALT,In comparison with the aged model group,the remaining indexes showed a decreasing trend in ALT,AST,UA and CRE in each dosing group;in the HE pathological staining of renal tissues,compared with the juvenile and youth control groups,the renal cystic space in the aged model group was significantly enlarged,and the tubular lumen of the renal tubules was dilated;while compared with the natural aging model group,the renal cystic space in the Erzhi Pill dosing group was relatively narrow,and the epithelial cells were more neatly arranged.The results ofβ-gal staining showed that a large area of the kidney in the aged model group was stained bright blue,and the area of senescence decreased in a dose-dependent manner after Erzhi Pill administration.Oxidative indexes showed that SOD activity decreased and MDA content increased in the natural senescence model group compared with the young and youth control rats（P<0.05）.Compared with the natural senescence model group,MDA content in the high dose group showed a significant downward trend after Erzhi pill intervention,and SOD in both the middle and high dose groups showed an upward trend,but there was no statistical difference（P>0.05）.The telomere results showed that there was a significant difference in telomerase activity between the natural aging model group and the dosing group compared with the juvenile control group（P<0.05）,but there was no significant difference in telomere length（P>0.05）,and compared with the natural aging model group,there was a tendency for telomerase activity to increase in the low-dose group and high-dose group of Erzhi Pills（P>0.05）.The analysis of ADH and ALD results showed that compared with the juvenile control group,the youth control group and the natural aging model group were significantly elevated（P<0.05）,and there was no statistical different with the ALD groups（P>0.05）,compared with the natural aging model group,only the juvenile group had a statistical difference in ADH（P<0.05）,but ADH showed a downward trend after administration,and ALD only had a pullback trend compared with the natural aging model group in the high-dose group and the natural aging model group.2.Investigation of the potential mechanism of action of Erzhi Pill in delaying aging based on metabolomicsIn the plasma metabolic pathway,a total of 18 metabolic differences were identified in positive and negative ion modes,including tetrahydrocortisol,phosphorylcholine,hemolytic lecithin,etc.,and a total of16 pathways were enriched,and a total of 14 pathways were enriched after the intervention of Erzhi Pill administration group.In the urine metabolic pathway,a total of 15 metabolic differences,including estradiol,pantheophylline 4’-phosphate,N-acetylasone glutamate,etc.,were identified in positive and negative ion modes,and a total of 12 pathways were enriched after the intervention of Erzhi Pill administration group.After the intervention of Erzhi Pill,nine core pathways such as steroid hormone biosynthesis,glycerophospholipid metabolism,primary bile acid biosynthesis,pantothenic acid and coenzyme A biosynthesis played a role in delaying aging,among which the differential metabolites related to aqueous metabolism were21-hydroxypregnenolone,corticosterone and estradiol,and the enriched pathway was the steroid hormone biosynthesis pathway.3.Effect of Erzhi Pill on Vitality of Senescent HK-2 CellsThe cell proliferation viability was significantly reduced when the concentration was≥20 mg·m L^-1and the intervention time was≥48 h.There was no statistical difference between the different concentrations and time of D-gal intervention on HK-2 cells,and the cell proliferation viability was not inhibited.After combining the intervention concentrations and time factors,the 5,10 and 15%concentrations of the drug-containing serum were selected as the low,medium and high dose groups of Erzhi Pill administration to interfere with senescent HK-2 cells for 48 h.4.Protective effect of Erzhi Pill on senescent HK-2 cellsAfter the induction of senescence in HK-2 cells,the MDA content increased significantly（P<0.01）and SOD activity decreased significantly（P<0.01）compared with the blank control group;there was a tendency for MDA to decrease after administration,and it was dose-dependent,and compared with the model control group,MDA decreased significantly（P<0.01）in the Erzhi Pill high-dose group,and SOD activity both had a tendency to be retuned,and the high-dose in the administration group.The highest SOD activity was observed in the high dose group,but there was no statistical difference;the positive rate of SA-β-gal staining was the highest in the model control group,and there was a significant difference（P<0.01）in the middle and high dose groups of Erzhi Pill compared with them,and the number of nuclei ofβ-gal gradually decreased as the concentration of Erzhi Pill increased;the apoptosis rate was the highest in the model control group,and there was a highly.The apoptosis rate of the control group was the highest,and there was a statistically significant difference（P<0.001）between the two groups,and the overall apoptosis rate showed a decreasing trend and was dose-dependent.The permeability of cell membrane showed that there was a trend of pullback between the concentration group of two to pills and the model control group,but only the normal control group and the model control group had a significant statistical difference（P<0.01）,and there was no statistical difference between the remaining groups（P>0.05）.5.Probing the mechanism of aging retarding effect of Erzhi Pill based on water channel protein expressionThe levels of AQP1 and AQP2 in kidney tissues and senescent HK-2cells of naturally senescent rats showed a decreasing trend compared to the normal group,while in vivo experiments showed an increasing trend in AQP1and AQP2 in the Erzhi Pill administration group compared to the model control group,and only the high dose group showed a statistical difference（P<0.05）,and in vitro experiments showed an increasing trend in AQP1 in the Erzhi pill administration group compared to the model control group,while AQP2 showed an increasing trend in the Erzhi Pill administration group compared to the model control group.AQP2 showed an increasing trend only in the high dose group,but there was no statistical difference between all groups（P>0.05）.Conclusion:1.Erzhi Pill can improve the aging characteristics of natural aging rats,protect the liver and kidney tissues,which correspond to the liver and kidney meridians.Additionally,it can enhance antioxidant capacity,p O2,ADH and ALD level,especially in the high dose group.However,the effect on telomere length and telomerase activity was not evident in this experiment.2.Erzhi Pill can regulate the level of differential metabolites in natural aging rats,and its anti-aging effect may be related to metabolic pathways such as steroid hormone biosynthesis and glycerophospholipid metabolism,among which the pathway closely related to aqueous metabolism is the steroid hormone biosynthesis pathway.3.In vitro experiments showed that the Erzhi Pill,which contains medicinal serum,has a protective effect on aging HK-2 cells.It can enhance the antioxidant capacity of HK-2 cells,reduce free radical accumulation,and lower the apoptosis level.Moreover,it can delay aging and,to some extent,reduce the permeability of cell membranes.4.After the administration of the Erzhi Pill to intervene in natural aging rats and aging HK-2 cells,the expression of AQP1 and AQP2 increased.This suggests that the Erzhi Pill may increase the expression level of aquaporin by nourishing kidney yin.