Based On PI3K/Akt To Study The Mechanism Of Shengqing Jiangzhuo Capsules And Its Monomers Against Renal Fibrosis

Clinical research PurposeChronic kidney disease(CKD)is one of the diseases that seriously endanger human health.At present,the global incidence rate is about 10-15%,and the domestic incidence rate is about 10.8%,and it is on the rise.Therefore,how to prevent and treat CKD has become the need of the global medical system.The huge challenge facing."Shengqing Jiangzhuo Capsule" is one of our hospital’s in-hospital preparations.It mainly treats chronic renal insufficiency.In this study,Shengqing Jiangzhuo Capsules were used to treat CKD 3-5non-dialysis patients,and the improvement of Shengqing Jiangzhuo Capsules on renal function,nutritional status and TCM symptoms of CKD patients was observed to clarify the clinical efficacy of Shengqing Jiangzhuo Capsules.MethodUsing prospective research methods,84 cases of CKD 3-5 non-dialysis patients were enrolled,and they were randomly divided into treatment group and control group by number table,with 42 cases in each group.There was no statistical difference between the two groups in the specific CKD stage distribution,gender,age and other baselines.The control group was given conventional symptomatic treatment,and the treatment group was given Shengqing Jiangzhuo capsule treatment on the basis of the control group.Both groups were treated for 3 months continuously.Analyze the TCM syndrome scores and laboratory indicators(Creatinine(Cr),blood urea nitrogen(BUN),estimated glomerular filtration rate(Estimated Glomerular Filtration Rate,e GFR)before and after treatment in the two groups of patients),albumin(Albumin,ALB),hemoglobin(Hemoglobin,HGB),blood uric acid(Uric acid,UA),etc.Result1.Efficacy of TCM syndromes: Before treatment,the main TCM clinical symptoms and TCM syndrome scores were not statistically different between the two groups.After treatment,the main TCM clinical symptoms and TCM syndrome scores in the treatment group were significantly improved compared to before treatment,and the difference was statistically significant(P <0.05),while the control group did not improve significantly compared with the same group before treatment;comparison between groups,treatment Compared with the control group,the TCM clinical symptoms and TCM syndrome scores were significantly improved in the group,and the difference was statistically significant(P<0.05).2.Laboratory indicators: Before treatment,there was no statistical difference between the two groups in terms of Cr,BUN,e GFR,ALB,HGB,and UA.After treatment,the above indicators of the treatment group were improved compared with the same group before treatment(P <0.05),and the above indicators of the control group were improved compared with the same group before treatment,but only two indicators of UA and HGB improved significantly(P <0.05);Comparison between the groups,the treatment group has significantly improved the above indicators compared with the control group,and the difference is statistically significant(P <0.05).Conclusion:Shengqing Jiangzhuo Capsules can significantly improve the renal function of non-dialysis patients in CKD 3-5 stages,reduce their Cr,BUN,UA,and increase e GFR;it can significantly improve their nutritional status and clinical symptoms of TCM,and reduce TCM syndrome scores.Significantly improve the efficacy of TCM syndromes,so as to protect the residual renal function and delay the progress of CKD.Experimental StudyPurposeThe pathogenesis of CKD is complex,including immune inflammatory response,oxidative stress,lipid metabolism disorders,etc.The specific mechanism is still unclear.Renal interstitial fibrosis(RIF)is a common pathological feature of various CKD progression to end-stage renal disease.How to reduce RIF is of great significance to delay the progression of CKD.This study first screened the effective monomeric component of Shengqing Jiangzhuo Capsules in the treatment of CKD through network pharmacology,quercetin,which can regulate the PI3K/Akt signaling pathway after combining with PI3K;further inducing CKD model mice by adenine,it is confirmed that Shengqing Jiangzhuo Capsules can protect the kidney function of CKD mice and resist RIF.Finally,by detecting the expression of various proteins in the PI3K/Akt signaling pathway,it is demonstrated that Shengqing Jiangzhuo Capsules are effective monomeric quercetin,which can inhibit PI3K/The Akt signaling pathway reduces RIF.Method1.Using the network pharmacology method,search the main active ingredients of Shengqing Jiangzhuo Capsules through the TCMSP database,and cross the corresponding ingredient targets with the CKD disease targets downloaded from the Genecard database to construct a network of traditional Chinese medicine-components-targets and draw Venn diagram: Use STRING software to construct a protein interaction(PPI)network,and screen out PIK3R1 as a candidate gene.Using Cytoscape Clue GO software,91 targets were analyzed for GO analysis and KEGG pathway analysis,and the enrichment results were visualized,and effective monomer components,signal pathways and targets were screened out.2.Establish a CKD rat model by gavage of 200mg/(kg·d)of adenine solution.25 male SD rats were randomly divided into normal group(n=5),model group(n=5),Shengqingjiangzhuo capsule low-dose treatment group(n=5),Shengqingjiangzhuo capsule middle-dose treatment group(N=5),Shengqing Jiangzhuo capsule high-dose treatment group(n=5),the last four groups were given empty capsules,Shengqingjiangzhuo capsules0.25g/d,Shengqingjiangzhuo capsules 0.5g/d,Shengqingjiangzhuo capsules Qingjiangzhuo Capsule 1g/d was given by intragastric administration for a total of 8 weeks.The body weight of rats in 5 groups was measured,and venous blood of rats was collected to detect Cr and BUN.HE staining and MASSON staining were used to observe the pathological changes of the kidney.Western blot was used to detect the expression of P13 K,AKT,NLRP3,Caspase-1,IL-1β,SGK-1,and NF-κB.3.Randomly divide 25 CKD model mice into normal group(n=5),model group(n=5),low-dose quercetin group(n=5),and middle-dose quercetin group(n=5)In the quercetin high-dose group(n=5),the model group was intragastrically administered with normal saline every day,and the three dose groups were intragastrically administered with quercetin solution at doses of 0.75,1.5 and 3g/(kg·d)each day for continuous 8 weeks.The levels of Cr,BUN and UA in 5 groups of rats were detected.HE staining was used to observe kidney pathological changes,flow cytometry was used to detect cell apoptosis,immunofluorescence staining was used to detect the expression of NLRP3,caspase1,AQP1,AQP2,and Western blot to detect the expression of NLRP3,caspase1,p-PI3 k,p-Akt,AQP1,and AQP2,ELISA to detect the expression of α-SMA and COL IV protein in rat kidney tissue,and q PCR to detect the expression of α-SMA and COL IV m RNA in rat kidney tissue.Result1.Obtain 44 main active ingredients of Shengqing Jiangzhuo Capsules and 298 target points through network pharmacology.A total of 1270 CKD disease targets were sorted out.The two crossed to obtain 91 overlapping genes;GO function enrichment analysis and KEGG Path analysis obtained 35 GO entries and 49 signal paths,of which PIK3R1 was located in the center with the most edges.The four main components of Shengqing Jiangzhuo Capsules Polygonum cuspidatum,Smilax chinensis,Sophora japonicus,and Astragalus all contain quercetin,which may interact with multiple target genes.2.The body weight of rats in the model group and the treatment group was lower than that of the normal group,but the body weight of the treatment group was higher than that of the model group.Compared with the normal group,the levels of Cr,BUN and UA in the model group were significantly increased(P<0.01).Compared with the model group,the levels of Cr,BUN and UA in the three treatment groups were significantly reduced after administration(P<0.01),and the high-dose group had the best effect.In HE staining,the glomeruli in the model group showed segmental or spherical sclerosis,adhesion of the balloon wall,and partially thickened basement membrane.In the three treatment groups,some glomerular capillary loops reopened,some glomerular mesangium showed mild proliferation,and the mesangial matrix increased slightly.The pathological changes of tubulointerstitium were the slightest and the closest to the normal group was the high-dose treatment group.In MASSON staining,the number of glomeruli in the kidneys of the model group was significantly reduced with severe atrophy,a large number of inflammatory cells infiltrated and deposited in the glomeruli,and interstitial collagen fibers were ring-shaped and deposited near the basement membrane area and epithelial tissues..In the high-dose treatment group,the renal tubular interstitial collagen deposition was significantly reduced compared with the model group,and the deposition area became smaller.Westen Blot results showed that the expression of P13 K,AKT,NLRP3,Caspase-1,IL-1β,SGK-1,NF-κB and other proteins in the three treatment groups was lower than that in the model group.Among them,the decrease in protein expression of PI3 K,Akt and SGK-1 was the most significant in the high-dose treatment group,and the decrease in protein expression of NLRP3,Caspase-1,IL-1β,and NF-κB was the most significant in the middle-dose treatment group.3.Compared with the model group,the levels of Cr,BUN and UA in the middle and high dose groups of quercetin were significantly reduced after administration(P<0.01).In HE staining,rats in the model group showed typical renal tubular necrosis in the cortex and outer medulla.In the three quercetin treatment groups,the degree of renal tubular necrosis was milder than that in the model group,and only mild edema occurred in the renal interstitium.The infiltration of inflammatory cells is not obvious.Flow cytometry showed that there were a lot of apoptosis in the model group,and the apoptotic rate was significantly higher than that in the normal group.The apoptosis rate of the quercetin treatment group with different doses was significantly lower than that of the model group,and the high-dose group had the most obvious effect.Immunofluorescence detection indicated that the average fluorescence intensity ratio of NLRP3 and caspase1 in the kidney tissue of the model group was significantly higher than that of the normal group.Compared with the model group,the average fluorescence intensity ratio of the quercetin treatment group was significantly lower.The average fluorescence intensity ratio of AQP1 and AQP2 in the model group was significantly lower than that of the normal group,while the average fluorescence intensity ratio of AQP1 and AQP2 in the quercetin group increased.The results of Western blot and immunofluorescence showed a consistent trend.Conclusion1.Shengqing Jiangzhuo Capsule can significantly improve the process of CKD in rats,and has a definite therapeutic effect on CKD treatment.The curative effect is related to the inhibitory effect of the preparation on renal tubular interstitial fibrosis.2.Using network pharmacology methods to analyze the effective monomers,signal pathways and target points of Shengqing Jiangzhuo Capsules,it was found that one of its effective monomer components was quercetin,and it is recommended to study the signal pathway as PI3K/Akt.3.Further studies have found that quercetin can bind to PI3 K,inhibit the PI3k/Akt pathway to reduce renal fibrosis and cell apoptosis in CKD rats,and effectively alleviate the process of renal tubular interstitial fibrosis by regulating AQP1 and AQP2.Symptoms of uremia.We conclude that Shengqing Jiangzhuo Capsule has an inhibitory effect on renal tubulointerstitial fibrosis in the process of CKD,and this regulatory effect may be through the combination of its active ingredient quercetin and PI3 K,which mediates the effects of PI3 k and Akt.Phosphorylation inhibits the activation of PI3K/Akt signaling pathway,thereby reducing renal interstitial fibrosis and cell apoptosis in CKD rats.On the other hand,inhibiting the PI3K/Akt signaling pathway can also regulate AQP1 and AQP2 aquaporins,effectively reducing CKD uremic toxin accumulation and water and sodium retention,and alleviating CKD symptoms.