Clinical Study Of Erhuang Decoction In The Treatment Of Non-dialysis Patients With Acute Kidney Injury And The Mechanism Of Erhuang Decoction In The Treatment Of Acute Kidney Injury Based On Network Pharmacology

OBJECTIVE1 Observe the clinical efficacy and safety of Erhuang Decoction(EHD)in the treatment of acute kidney injury(AKI)non-dialysis patients with "spleen-kidney qi deficiency,damp-heat stasis syndrome",and summarize the medication characteristics and addition and subtraction rules of EHD enema in the treatment of AKI non-dialysis patients.2 Use network pharmacology to explore the main mechanisms and potential target pathways of EHD in the treatment of AKI.3 Use in vitro experiments to verify the potential mechanism and target pathways of EHD in the treatment of AKI shown in network pharmacology.METHOD1 Looking back at the hospitalized patients with AKI in Sichuan Provincial Hospital of Traditional Chinese Medicine from December 2012 to December 2020,according to the inclusion criteria and exclusion criteria,the patients who meet the "spleen and kidney Qi deficiency,damp-heat stasis syndrome" were classified according to the actual treatment situation,and in the treatment group(supportive treatment + enema treatment with EHD as the main prescription)and control group(supportive treatment).The supportive treatment was limited by the supportive treatment part of the KDIGO guideline.The enema prescription must contain the main medicine of EHD Rhubarb,Astragalus,Danshen,Safflower,herbal thick decoction 100 ? 150 ml,completed by the decocting room of Sichuan Provincial Hospital of Traditional Chinese Medicine,rectally instilled in 2 to 3 times.The observation period starts from the diagnosis of AKI and the corresponding treatment to the end of the observation termination index.The renal function,electrolytes,60-day re-admission,prognosis,and safety indicators of patients with AKI non-renal replacement therapy "spleen and kidney qi deficiency,damp-heat stasis syndrome" were recorded.And EHD enema prescription and dosage.2 Use the TCMSP database to retrieve the main active ingredients and related targets of EHD.Use TTD and OMIM database to obtain AKI-related disease targets.The Uniprot database converts related targets into standard gene names.Cytoscape 3.7.2software was used to construct protein-protein interaction network(PPI).DAVID database performed GO enrichment and KEGG enrichment analysis to predict the potential mechanism and target pathway of EHD in the treatment of AKI.3 Eight rabbits were randomly divided into EHD group and blank group.The EHD group received an enema at 9.617ml/kg/d,and the blank group received an enema with the same amount of distilled water,3 times a day,continuous 3 days.Anesthetize with 3% pentobarbital sodium 30mg/kg after enema,take blood from inferior vena cava,and prepare blank serum,25% EHD medicated serum,50% EHD medicated serum,75% EHD medicated serum.Using the airtight method to simulate the process of hypoxia/reoxygenation(H/R),the human renal tubular epithelial cell line(HK-2cells)was divided into blank group(conventional culture)and an airtight group(sealed with tape),ROS fluorescent probe detects the reactive oxygen species(ROS)level of HK-2 cells at 0h,1h,2h,3h,4h,5h to determine the best confinement time.HK-2 cells are divided into normal group,model group,25% EHD group,50% EHD group,75% EHD group.The normal group is routinely cultured,and the rest of the group uses a closed method to establish an H/R model and uses blank serum,25%EHD containing Treated with medicated serum,50% EHD medicated serum,and 75%EHD medicated serum,complete the experimental grouping.Cell Counting Kit-8(CCK8)detected the viability of HK-2 cells in each group at 0h,4h,12 h,and 24h;Flow cytometry detected the apoptosis/mortality of HK-2 cells in each group at 0h,4h,12 h,and 24h;CFSE/ PI double staining was used to observe cell apoptosis in each group at 4h and 12h;Quantitative Real Time-PCR(RT-q PCR)was used to detect Bax,Bcl-2,I???,NF-?B m RNA expression in each group of HK-2 cells at 0h,4h,12 h,and 24 h.RESULT1 According to the inclusion and exclusion criteria,a total of 95 patients with non-renal replacement therapy for acute kidney injury who met the diagnosis of "spleen and kidney qi deficiency,damp-heat stasis syndrome" were included.According to the actual treatment,they were divided into treatment group with 44 cases and control group with 51 cases.There was no significant difference in general data between the two groups,P>0.05.After treatment,the Scr,BUN,Cys-C,and serum K of the treatment group were significantly lower than those of the control group(P<0.05).There were fewer patients with renal replacement therapy indications than the control group(P<0.05),and more patients with improved renal function than the control group In the treatment group(P<0.05),the renal function recovery time of patients in the treatment group was shorter than that in the control group(P<0.05).After treatment,there was no significant statistical difference between the treatment group and the control group in the 60-day readmission,average hospital stay,and renal function deterioration(P>0.05).After treatment,there was no significant difference in safety indicators(WBC,RBC,PLT,AST,ALT,ALP)between the two groups(P>0.05).According to the standard of 1 dose divided into 2 enemas,the main doses of the patient's single infusion are rhubarb(15.67±3.51),astragalus(17.67±5.01),salvia(9.56 ± 2.31),safflower(10.03 ± 1.56),rhubarb : Astragalus: Salvia Miltiorrhiza: The dosage ratio of safflower is close to 1.5:1.5:1:1.In addition to the main symptoms of spleen and kidney qi deficiency and damp-heat stasis,AKI non-renal replacement therapy patients have concurrent symptoms including kidney qi and yin deficiency(34.2%),spleen and kidney yang deficiency(24.4%),spleen and kidney yin and yang deficiency(13.1%),Phlegm turbidity syndrome(33.9%),water-gas syndrome(24.2%);non-primary drugs with a frequency of >50% include Puhuang(81.20%),Chuanxiong(80.10%),oyster(76.30%),and dandelion(70.10%),Aconite(73.10%),Scutellaria(68.50%),etc.2 Based on network pharmacology,123 effective active ingredients of Erhuang Decoction,163 related targets,and 43 AKI-related disease targets were screened.After Cytoscape 3.7.2 screening,206 targets of Erhuang Decoction for the treatment of acute kidney injury were obtained.According to the "Degree" ranking,BCL2,IKBKB,TP53,etc.are the key core targets.The GO enrichment and KEGG enrichment analysis of the core targets showed that the targets of Erhuang Decoction for the treatment of acute kidney injury were mainly enriched in pathway related with cancer,apoptosis,and chronic myeloid leukemia.chronic myeloid leukemia)and other related pathways.3 Compared with the blank group,the ROS fluorescence intensity of the HK-2 cells after sealing for 5 hours increased,and the ROS level increased sharply,indicating that sealing for 5 hours was a suitable sealing time.After 5 hours,the sealing was released,normal oxygen supply was restored,and the modeling was completed.Compared with the normal group,the cell viability of the model group was significantly decreased at 4h and 12h(P>0.05);compared with the model group,the cell viability of each group of EHD-containing serum was significantly increased(P<0.05),and the effect of the EHD(50%)group The most significant.The HK-2cells of the model group showed obvious apoptosis at 4h and 12 h after modeling(P<0.05),while the apoptosis of each group of EHD-containing serum was significantly inhibited(P<0.05),with EHD(50%)The group effect is the most significant.At 4h and 12 h,compared with the model group,the Bax m RNA level in each EHD drug-containing serum group was significantly down-regulated(P<0.05),and the Bcl-2 m RNA expression was significantly up-regulated(P<0.05).At 24 h,compared with the model group,the expression of Bax and Bcl-2 m RNA in HK-2cells in the EHD(75%)drug-containing serum group was still statistically significant(P<0.05).From 0h to 24 h,the ratio of Bax/Bcl-2 of HK-2 cells in the EHD-containing serum group was significantly lower than that of the model group,which was close to 1/5(P<0.05).12 h after modeling,the EHD(25%)group showed an increase in IKK? m RNA level(P<0.05).24 h after modeling,the IKK? m RNA level in the EHD(50%)group and the EHD(25%)group was significantly increased.No high expression of IKK? m RNA in the EHD(75%)group was detected within the point.12 h after modeling,the expression of NF-?B m RNA in the EHD(50%)group and EHD(75%)group was significantly up-regulated(P<0.05),24 h after modeling,the expression of NF-?B m RNA in each EHD-containing serum group was up-regulated(P<0.05),which was positively correlated with dose.CONCLUSION1 EHD enema has a positive effect on promoting renal function,serum K recovery,and reducing deterioration of renal function in patients with acute kidney injury without renal replacement therapy,and is safe and reliable.The main medicine of EHD is rhubarb: Astragalus: Danshen: Safflower with a ratio close to 1.5:1.5:1:1."Spleen and kidney qi deficiency,damp-heat stasis" type of acute kidney injury non-renal replacement therapy patients with concurrent syndromes include spleen and kidney yang deficiency,spleen and kidney qi and yin deficiency,spleen and kidney yin and yang deficiency,phlegm turbidity,and water-qi syndrome.Guidance of clinical addition and subtraction of medicines for concurrent syndromes,such as the combination of Qi and Yin deficiency often add Codonopsis,Radix Rehmanniae,Radix Scutellaria,etc.;the combination of Yang deficiency add Aconite,dried ginger,and Guizhi;those with both Yin and Yang deficiency add raw Rehmannia,Aconite,Oyster,etc.Keel,etc.;cassia twig,tuckahoe,dried ginger,pot belly bark for those with moisture;add cardamom,tangerine peel,magnolia,etc.for those with turbid phlegm;add Glauber's salt,dandelion,scutellaria,etc.for hot and severe;add scutellaria,etc.for wet and severe Grass fruit,gardenia;for severe blood stasis,add cattail yellow,bitter almond,chuanxiong and so on.2 EHD based on network pharmacology has 206 main targets for the treatment of acute kidney injury,among which BCL2,IKBKB,TP53,etc.are the key core targets.Apoptosis-related targets and pathways are thought to be related to the treatment of acute kidney injury by EHD.3 EHD medicated serum can significantly increase the apoptosis of H/R-damaged HK-2 cells.This effect is related to the inhibition of H/R-damaged HK-2 cell apoptosis by EHD medicated serum.The protective effect of EHD medicated serum on H/R injury of HK-2 cells is related to the regulation of Bax / Bcl-2 and IKK? /NF-?B signaling pathways.The regulation of Bax / Bcl-2 pathway in H/R-damaged HK-2 cells by EHD-containing serum began to be significant at 4 hours after modeling,and was dose-dependent.The regulation of IKK? / NF-?B pathway by EHD medicated serum began to be significant at 12 h after modeling,and the regulation of IKK? by EHD was the most obvious in EHD(25%),and the regulation of NF-?B was the most obvious in the EHD(75%)group.It shows that the high expression of NF-?B induced by EHD medicated serum does not completely depend on the up-regulation of IKK?.