| Purpose:Under the guidance of the theory of "salty injury to the kidney" of traditional Chinese medicine,explore the regulatory effect of high salt on renal sodium in salt sensitive hypertension,and explore the renal protective mechanism of Buyang Huanwu Decoction in interfering with ENaC ubiquitination to regulate renal sodium metabolism.This experiment first set salt sensitive hypertension as the research target with the network pharmacological method,and used Buyang Huanwu Decoction as the research carrier to build the interaction network of "Buyang Huanwu Decoction-drug target-salt sensitive hypertension-disease target",and carried out enrichment analysis and biological function analysis on these targets to predict the molecular biological mechanism of Buyang Huanwu Decoction in treating salt sensitive hypertension.Secondly,through animal experiments,Dahl salt sensitive rats induced by high salt diet were used as hypertension models,and Buyang Huanwu Decoction was administered by gavage to observe the renal protective effects of traditional Chinese medicine on salt sensitive hypertension,such as blood pressure,renal function,urine sodium,renal morphology,fibrosis status,glomerular basement membrane,apoptosis,etc.Finally,through the cell experiment,the renal tubular epithelial cells were selected to observe the effects of high salt culture medium on the growth morphology,proliferation,ROS,apoptosis,and secretion of inflammatory factors of HK2 cells and the cytoprotective effect of Buyang Huanwu Decoction,and explained the modern molecular biological mechanism of Buyang Huanwu Decoction in treating ENaC ubiquitination related signal pathway,aiming to clarify the mechanism of action of Buyang Huanwu Decoction on abnormal sodium metabolism in kidney related to salt sensitive hypertension caused by "salt injured kidney",It is helpful to understand the pathogenesis of salt sensitive hypertension,and can also provide a theoretical basis for finding new therapeutic targets from traditional Chinese medicine.Material and method:1 Study on the Potential Mechanism of Buyang Huanwu Decoction in Treating Salt Sensitive Hypertension Based on Network PharmacologyAccording to the ingredient information of Buyang Huanwu Decoction,screen the effective ingredients in the TCMSP database,set the oral availability OB value ≥ 30%,and the drug like property index DL value ≥ 0.18.After the effective ingredients are screened,the corresponding targets of the effective ingredients are found in the database.After finding effective targets,target proteins are used as research objects to transform target proteins into target gene names in Uni Prot database(an internationally recognized and standardized comprehensive resource database on protein sequence and annotation data).Subsequently,with Gene Cards online database,OMIM database,TTD database,Pharm Gkb database,Drug Bank database as the research scope,salt sensitive hypertension(Salt sensitive hypertension)as the keyword search,the corresponding target genes of salt sensitive hypertension disease were collected,and the median was used as the basis for target screening.Intersect the effective ingredient target of Buyang Huanwu Decoction with the target of salt sensitive hypertension,and draw the Wayne diagram.The effective components of traditional Chinese medicine can be inferred from the intersection targets,and a network of effective components of traditional Chinese medicine-intersection targets can be constructed.The key components of Buyang Huanwu Decoction in the treatment of salt sensitive hypertension can be selected according to the connection between the effective components of traditional Chinese medicine and the intersection targets.PPI analysis was conducted to construct protein interaction network.GO analysis and KEGG pathway analysis were carried out for the intersection protein,and the biological mechanism of Buyang Huanwu Decoction in treating salt sensitive hypertension was speculated to guide the follow-up experimental research.2 Study on the Protective Mechanism of Buyang Huanwu Decoction on the Kidney of Salt Sensitive Hypertensive Rats60 Dahl salt sensitive rats,7 weeks old.They were randomly divided into normal group,model group,low dose,medium dose,high dose and hydrochlorothiazide group.The normal group was fed with 0.3% low salt diet,and the other groups were fed with 8% high salt diet.After 5 weeks,the blood pressure of salt sensitive hypertensive rats increased significantly,and they were identified as hypertensive models.During the modeling period,the traditional Chinese medicine group was treated with different concentrations of Buyang Huanwu Decoction by gavage,while the western medicine group was treated with hydrochlorothiazide by gavage for 4 weeks.Observe the blood pressure and urine volume of rats in each group.After the experiment,the serum and urine of rats were collected,the kidneys were fixed,and the renal tubules and collecting ducts were collected.The renal function and urine sodium of rats were detected.Masson staining was used to detect renal interstitial fibrosis,PAS staining was used to observe the difference of glomerulosclerosis,Tunel staining was used to observe apoptosis,HE staining was used to observe the morphological difference of rat kidneys,q PCR was used to detect the expression of SGK1,NEDD4-2,ENaC mRNA in rat renal tubules.3 Study on the Protective Effect of Buyang Huanwu Decoction on Renal Tubular Epithelial Cells Based on ENaC UbiquitinationHK2 was selected for cell culture experiment.F12 medium was selected for cell culture.1% penicillin/streptomycin solution,10% fetal bovine serum and 1m Mol glutamine were added to the medium.The cell culture environment was 37 ℃,and the CO2 concentration was5%.The experimental Chinese medicine was treated with Buyang Huanwu Decoction,and the prepared Chinese medicine granules were mixed evenly.After they were completely melted,they were left to stand for 10 minutes,and the Chinese medicine solution was rotated,evaporated and concentrated.The reaction condition is 200 revolutions per minute,and the temperature is 50 ℃.After drug concentration,it is freeze-dried in a vacuum condensation dryer for 48 hours and ground into powder.After preparing the freeze-dried powder,use ultra pure water to dissolve the freeze-dried powder to prepare the mother liquor for standby.Select a sufficient number of HK2 cells growing in logarithmic phase,and divide them into five groups:blank group,high salt group,low dose group of Buyang Huanwu Decoction,medium dose group of Buyang Huanwu Decoction,and high dose group of Buyang Huanwu Decoction.After the cells adhere to the wall,the model is made for treatment.During modeling,except the blank group,the culture medium was changed normally,the cells of other groups were intervened for 24 hours by adding 150 m Mol Na Cl solution in F12 medium.Subsequently,the treatment group was treated with Buyang Huanwu Decoction of different concentrations for24h(the concentrations of crude drugs in the culture medium were 5mg/ml,10mg/ml and20mg/ml respectively).After the treatment,CCK-8 experiment verified the protective effect of Buyang Huanwu Decoction on HK2 cell damage.High connotation analysis system was used to observe the number of cells,fluorescent quantitative PCR was used to detect the expression of SGK1,NEDD4-2,ENaC mRNA in HK2 cells,Western blot was used to detect the content of SGK1,NEDD4-2,ENaC protein,flow cytometry was used to detect apoptosis and ROS expression,and Elisa was used to detect IL-1 in cell culture medium β And IL-18 content.Results:1 Network Pharmacological Study of Buyang Huanwu Decoction in Intervention of Salt Sensitive HypertensionIn this study,we collected 1402 of 123 chemical components and drug action targets,779 salt sensitive hypertension targets,and 93 intersection targets.Through biological function analysis,the results showed that it mainly involved cell proliferation,apoptosis,angiogenesis,etc.The pathway enrichment analysis found that Buyang Huanwu Decoction might affect RAS signal pathway The signal pathway of sodium reabsorption regulated by aldosterone involved in renin secretion is related to the signal pathway of sodium reabsorption.The main indicators are: IR,PLA,RAS,NEDD4-2,PDK,etc.2 Protective effect of buyang huanwu decoction on kidney in salt sensitive hypertensive rats2.1 Effect of Buyang Huanwu Decoction on blood pressure in salt sensitive hypertensive ratsThe results of animal experiment showed that compared with the blank group,the systolic blood pressure and diastolic blood pressure of the model group rats were significantly higher(P<0.05);Buyang Huanwu Decoction can significantly reduce the systolic and diastolic blood pressure levels in salt sensitive hypertensive rats,with a statistically significant difference.2.2 Buyang Huanwu Decoction can protect renal function and promote urinary sodium excretion in salt sensitive hypertensive ratsThe experimental results showed that in the model group,the content of urinary protein in the urine was significantly increased,while the content of urinary white blood cells and epithelial cells was also significantly increased.The 24 h urine volume,urine sodium and serum UREA were significantly increased,and urine creatinine was decreased(P<0.05).It suggests that the rat kidney has inflammatory damage,and the renal tubules have begun to change.Buyang Huanwu Decoction can significantly reduce the serum UREA and urine protein of rats,reduce the excretion of white blood cells and epithelial cells in urine,increase urine volume,urine creatinine,and urine sodium excretion(P<0.05),and play a certain role in renal protection.2.3 Kidney protective effect of Buyang Huanwu Decoction on salt sensitive hypertensive ratsThe experimental results showed that the rats in the model group showed glomerular hypertrophy,mesangial cells and mesangial matrix proliferation,renal tubular swelling,epithelial cell vacuolar degeneration,basement membrane thickening,a large number of collagen fiber deposition,and increased apoptotic cells.Buyang Huanwu Decoction can significantly improve a series of manifestations such as kidney damage caused by high salt.2.4 Study on Molecular Mechanism of Buyang Huanwu Decoction in Regulating Sodium MetabolismThe results showed that compared with the blank group,the high salt diet significantly increased the mRNA expression of SGK1,ENaC,and decreased the mRNA expression of NEDD4-2(P<0.05);However,Buyang Huanwu Decoction could significantly reduce the mRNA expression of SGK1,ENaC,and increase the mRNA expression of NEDD4-2,with a statistically significant difference(P<0.05).The results showed that Buyang Huanwu Decoction could significantly reduce the content of SGK1 in rat renal tubules and increase the content of ubiquitin ligase NEDD4-2,thereby promoting the ubiquitination degradation of epithelial sodium channel ENaC,reducing the sodium reabsorption of HK2 cells,and playing a protective role in HK2 cells under high salt environment.3 Buyang Huanwu Decoction promotes ENaC ubiquitination and inhibits sodium ion reabsorption to play a renal protective role3.1 Buyang Huanwu Decoction can promote the proliferation of HK2 cellsThe results of CCK-8 showed that compared with the blank group,the activity of HK2 cells in the high salt group was significantly reduced,the number of cells observed under the microscope was significantly reduced,the morphology was abnormal,and the floating dead cells in the culture medium were increased;Compared with the high salt group,the addition of Buyang Huanwu Decoction can significantly enhance the cell activity,increase the number of cells,and protect the normal growth of cells(P<0.05).The results of high connotation experiment showed that high salt could significantly inhibit the proliferation of HK2 cells and destroy the normal growth rate of cells,while Buyang Huanwu Decoction could promote cell proliferation and reduce cell damage caused by high salt.The experimental results showed a dose effect dependent relationship.The medium dose is the best.3.2 Buyang Huanwu Decoction can inhibit the expression of ENaC ubiquitination related signal pathway in HK2 cellsThe results showed that compared with the blank group,150 mmol high salt stimulation significantly increased the mRNA and protein expression of SGK1,ENaC,and decreased the mRNA and protein expression of NEDD4-2(P<0.05);However,Buyang Huanwu Decoction could significantly reduce the mRNA and protein expression of SGK1,ENaC,and increase the mRNA and protein expression of NEDD4-2,with a statistically significant difference(P<0.05).The results showed that Buyang Huanwu Decoction could significantly reduce the content of SGK1 and increase the content of ubiquitin ligase NEDD4-2,thereby promoting the ubiquitination degradation of epithelial sodium channel ENaC,reducing the re absorption of sodium ions in HK2 cells,and playing a protective role in HK2 cells under high salt environment.3.3 Buyang Huanwu Decoction can inhibit the expression of F-actin and improve the morphology of HK2 cellsThe experimental results showed that when the concentration of extracellular sodium ions was 150 mmol/l,compared with the normal group,the F-actin of endothelial cells in the high salt group was significantly increased,shortened and thickened,and condensed toward the center of the cell,and the cell morphology was significantly changed;Buyang Huanwu Decoction can significantly improve cell morphology,reduce the expression of F-actin,and promote cell morphology to return to normal.3.4 Buyang Huanwu Decoction can inhibit HK2 cell apoptosisThe experimental results showed that,compared with the blank group,the proportion of early apoptosis and late apoptosis in the high salt group increased significantly after adding150 mmol/l Na Cl(P<0.05);Compared with the high salt group,Buyang Huanwu Decoction treatment can significantly inhibit apoptosis,and the inhibition effect of apoptosis is dose dependent,among which the middle dose group has the most significant inhibition effect(P<0.05).3.5 Buyang Huanwu Decoction can inhibit ROS expression in HK2 cellsThe results showed that ROS expression of HK2 cells in high salt group increased significantly(P<0.05);Compared with the high salt group,Buyang Huanwu Decoction can significantly reduce the expression of ROS(P<0.05),and its therapeutic effect is dose dependent,with the effect of medium dose being the most significant.3.6 Buyang Huanwu Decoction can inhibit the inflammatory reaction of HK2 cells and reduce IL-1β and IL-18 in the supernatantThe experimental results showed that after 150mmol/l high salt intervention on HK2 cells,IL-1β and IL-18 content increased significantly in cell culture medium(P<0.05),suggesting that after inhibiting ubiquitination of epithelial sodium channel,high salt could induce apoptosis and release related inflammatory factors by reabsorption of large amount of sodium ions;Buyang Huanwu Decoction can significantly improve the inflammatory reaction induced by high salt,and reduce the release of inflammatory factors from cells into the cell culture medium.The intervention effect was dose dependent,and the middle dose effect was the most significant(P<0.05)Conclusion:1 Based on network pharmacology,this study preliminarily explored the main active components and potential molecular mechanism of Buyang Huanwu Decoction in the treatment of salt sensitive hypertension.The biological processes that it may interfere with may be closely related to nutritional level,oxidative stress,exogenous stimulation,cell response to chemical stress,lipopolysaccharide,oxygen and water equality;The signaling pathway involves lipid and atherosclerosis,fluid shear stress and atherosclerosis,RAGE signaling pathway in AGE diabetes complications,chemical carcinogenic reactive oxygen species,MAPK signaling pathway,HIF-1 signaling pathway,IL-17 signaling pathway and epithelial sodium channel ubiquitination related signaling pathway.2 It was confirmed by animal experiments that Buyang Huanwu Decoction had a significant protective effect on the kidney of salt sensitive hypertensive rats.It can improve the renal function of rats,reduce the reabsorption of sodium in urine of renal tubular cells,reduce glomerular basement membrane sclerosis,reduce renal fibrosis,and reduce apoptosis of renal tissue cells.Its therapeutic effect is closely related to its promotion of ENaC ubiquitination of epithelial sodium channel and reduction of sodium reabsorption.3 Cell experiment results show that Buyang Huanwu Decoction can reduce the content of SGK1 in HK2 cells,activate the expression of gene and protein of ubiquitin ligase NEDD4-2,promote the combination of NEDD4-2 with epithelial sodium channels on the surface of HK2 cells,make ENaC ubiquitin internalized by cells,thereby reducing the number of sodium ionophore channels on the surface of cell membrane,reducing the reabsorption of sodium ions,maintaining the stability of the internal environment of sodium ions,reducing the accumulation of ROS in cells,and reducing the number of apoptosis,It can inhibit the release of inflammatory factors from HK2 cells,improve the growth status of HK2 cells,and increase the proliferation of HK2 cells,which partly explains the renal protective mechanism of Buyang Huanwu Decoction in promoting ENaC ubiquitination and inhibiting sodium reabsorption. |
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