Based On Medical Cases To Explore The Core Drugs Of Xiaolin Tong In The Treatment Of DKD And To Explore The Mechanism Of Action

Diabetic kidney disease(DKD)is a common microvascular complication of diabetes.As the global incidence of diabetes continues to rise,the incidence of DKD is on the rise globally,is the leading cause of end-stage renal disease,and is the strongest predictor of mortality in patients with diabetes.Therefore,actively preventing the occurrence and development of DKD is the focus and difficulty of modern medicine.Modern medicine still lacks effective treatment for DKD.How to carry out effective treatment in the early stage of DKD,delay the progression of DKD and protect renal function,is the current research hotspot.In the progress of DKD,various pathological changes have occurred in the body’s microcirculation,glomeruli and cells in the glomerulus.Recently,along with the development of science and the establishment of podocyte lines,growing research showed that increased severity of DKD,renal microcirculation disturbance,and many changes and damage to podocyte morphology and function are the main reasons for the occurrence of DKD proteinuria.Long-term clinical practice and related studies have shown that TCM has the feature of multi-target and multi-channel intervention in diseases,which can exert overall regulation and synergistic effects,and provide new means and strategies for the prevention and treatment of DKD.According to many years of clinical experience in the treatment of DKD,Xiao lin Tong proposed the diagnosis and treatment ideas of nourishing qi,promoting blood circulation and dredging collaterals.Preliminary clinical research of the research group has preliminarily proved the efficacy and safety of nourishing qi,promoting blood circulation and dredging collaterals method in the treatment of DKD.Thus,according to the previous research,this paper explores the core drug of nourishing qi,promoting blood circulation and dredging collaterals method,and discusses its action mechanism,in order to provide a scientific basis for Xiaolin Tong’s academic thought of nourishing qi,promoting blood circulation to remove meridian in the treatment of DKD under the guidance of "prevention of disease" and "collateral disease" theory.ObjectiveExploring the core drug composition of the representative prescription(Shenzhuo formula)for DKD treated by the tutor with the method of nourishing qi,promoting blood circulation and dredging collaterals,and exploring the material basis of Shenzhuo formula,as well as its possible target and potential mechanism of action in the treatment of DKD.Methods1 Clinical research section1.1 DKD patients who were treated in the outpatient clinic of Xiaolin Tong of Guang’anmen Hospital of China Academy of Chinese Medical Sciences from June 2006 to September 2021 were screened and included.The information in the medical records of Xiaolin Tong’s clinical treatment of DKD in the past 15 years was organized,and the information of the first diagnosis and treatment of DKD patients is entered,or the first diagnosis and treatment information of patients with type 2 diabetes has progressed to DKD as the disease progresses.1.2 According to "Internal Medicine of Traditional Chinese Medicine","Diagnostics",and "Diagnostics of Traditional Chinese Medicine",etc.,the entered clinical symptoms,comorbidities,physicochemical indicators,and prescription drugs were handled in a unified and standardized manner.1.3 Descriptive analysis of the frequency of drugs,four qi,five flavors,meridians,clinical symptoms and comorbidities.Using association rules and complex network analysis methods,the core drugs for the treatment of DKD by Xiaolin Tong were mined.In addition,association rule analysis and cluster analysis were performed on drug-drug and drug-symptom.2.Experimental research part2.1 The main components of Shenzhuo formula were analyzed based on UHPLC/Q-TOF-MS technology.2.2 Based on the compounds identified by Shenzhuo formula,the network pharmacology method was used to screen the possible targets of Shenzhuo formula in the treatment of DKD,and to predict the signal pathways,possible mechanism of action.2.3 Spontaneous diabetic kidney disease mice C57BLKS/J db/db mice and syngeneic non-diabetic kidney disease control mice db/m mice were used as experimental subjects.After modeling,db/db mice were randomly divided into model group(db/db),Shenzhuo formula group(db/db+SZF)and positive drug group(db/db+Irb),db/m was blank control Group.During the experiment,urine albumin/creatinine(UACR),fasting blood glucose were measured every 4 weeks.and body weight was measured every week.After 16 weeks of administration,the serum renal function,liver function,and renal injury factor-related indicators of the mice in each group were detected,and the urine UACR and renal injury factors of the mice collected in different time periods were uniformly detected.The staining of HE,PAS,and Masson were used to evaluate the degree of renal pathological changes,and transmission electron microscopy to evaluate the degree of glomerular podocyte injury,and around podocyte apoptosis,the TUNEL staining,immunohistochemistry(IHC),western blot(WB)and Real time-PCR(RT-PCR)were used to tested the expression level of maker protein related to renal cell apoptosis,podocyte injury and apoptosis,and the corresponding mRNA expression level.The above comprehensive evaluation of the efficacy and mechanism of Shenzhuo formula in the treatment of DKD.2.4 Taking transgenic vascular green fluorescent FLI-1 zebrafish as the experimental object,the zebrafish vascular endothelial injury thrombus model was established.The zebrafish was given low,medium and high concentrations of Shenzhuo formula in water solution.Meanwhile,the model control group,positive control group,and normal control group were setted,with 30 tails in each group.After the experiment:(1)10 zebrafish in each group were randomly selected and photographed under the fluorescence microscope.The data were analyzed and collected with NIS elements D 3.20 advanced image processing software to evaluate the improvement effect of Shenzhuo formula on internode vessels.(2)10 zebrafish were randomly selected from each group to be photographed under a dissecting microscope,and the incidence of thrombosis in each group was counted to evaluate the efficacy of Shenzhuo formula in preventing thrombosis.(3)Ten zebrafish in each group were randomly selected and placed in the heartbeat blood flow analysis system to record zebrafish blood flow video.Zebrafish blood flow velocity and cardiac output were analyzed with zebra blood 3.4 blood flow analysis software to evaluate the effect of Shenzhuo formula on hemodynamics.Result1 Clinical study results1.1 Basic informationA total of 975 DKD patients meeting the requirements were enrolled,including 542 DKD STAGE Ⅲ patients(56%),168 DKD stage Ⅳ patients(17%),and 265 DKD STAGE Ⅴ patients(27%).There were 320,106 and 178 males,there were 222,62 and 87 females respectively from microalbuminuria to kidney failure.From microalbuminuria to kidney failure,the average ages of patients were(53.78±13.08)years,(55.95±14.39)years,and(58.06±12.59)years;the average body mass index(BMI)was(24.99±3.64)Kg/m2,(25.23± 3.44)Kg/m2,(24.96± 3.14)Kg/m2;the average disease duration was(8.75± 5.48)years,(11.16±7.33)years,(6.98± 3.01)years,respectively.1.2 Frequency analysis of four diagnostic information1.2.1 Clinical symptom analysisThere were 146,190 and 182 clinical symptoms from microalbuminuria to kidney failure,respectively.The top 10 symptoms with frequency≥15 in different stages were urine foam,fatigue,blurred vision,poor sleep,limb numbness,lower limb edema and dry stool.In addition,microalbuminuria was prone to sweating,dizziness and fear of cold,macroalbuminuria was dry mouth,limb tingling and lumbago,and kidney failure was fear of cold,abdominal distension and dry mouth.1.2.2 Tongue and pulse analysisThere are 8,9 and 10 tongue images involved in microalbuminuria～kidney failure phase respectively.Tongue images in different stages are dark red tongue,sluggish tongue bottom,jagged tongue,fat tongue,quivering tongue,light red tongue,tender tongue,cracked tongue.In addition,macroalbuminuria is tongue stasis,and kidney failure are tongue stasis and purple dark tongue.There are 10,9,and 9 kinds of tongue coating involved,respectively.The tongue coating contained in different stages is greasy coating,rotten coating,thick coating,yellow and white coating,less coating,and thin coating.In addition,microalbuminuria was white fur,exfoliating fur,water-sliding fur,and dry fur,macroalbuminuria was yellow fur,powdery fur,and peeling fur,and kidney failure stage was yellow fur,pink fur,and dry fur.There are 9,9 and 13 kinds of pulse conditions involved.The pulse conditions contained in different stages are chord pulse,hard pulse,sink pulse,astringent pulse,slow pulse,solid pulse,weak pulse,number pulse and smooth pulse.In addition,kidney failure also contains intermittent pulse,slow and intermittent pulse,slow pulse and virtual pulse.1.2.3 Comorbidities analysisThere are 208,96,and 123 of comorbidities involved form microalbuminuria to kidney failure,respectively,and the top 10 with frequency≥15 are hypertension,hyperlipidemia,diabetic peripheral neuropathy,diabetic retinopathy,obesity,diabetic dyshidrosis,metabolic syndrome,diabetic pruritus.In addition,microalbuminuria is fatty liver and hyperuricemia,macroalbuminuria is fatty liver and diabetic erectile dysfunction,and kidney failure is renal anemia and hyperuricemia.1.2.4 Drug analysisThe drugs involved from microalbuminuria to kidney failure are 234,163 and 163,respectively,and the top 10 with a frequency of≥15 are Hirudo,Radix et Rhizoma Rhei,Radix Astragali seu Hedysari,and Radix Salviae Miltiorrhizae.Both microalbuminuria and macroalbuminuria contain Rhizoma Coptidis,Rhizoma Zingiberis Recens,Rhizoma Anemarrhenae,Caulis Spatholobi,and hongqu.In addition,microalbuminuria is Radix Notoginseng,macroalbuminuria is Poria,and kidney failure is Herba Leonuri,Poria,Radix Aconiti Lateralis Preparata,Fructus Rosae Laevigatae,Semen Cuscutae,and Herba Epimedii.1.3 High-frequency drug dose analysisIn different stages of DKD,the top 10 high-frequency drugs with a frequency of≥15 were subjected to dose analysis.The results showed that the commonly used doses of Hirudo were 3 or 6 g,3 or 6 g,6 g;the commonly used doses of Radix et Rhizoma Rhei were 3 or 6 g,6 or 9 g,9 or 15 g;the usual doses of Radix Astragali seu Hedysari are 15 or 30 g,30 or 45 g,60 or 90 g;the usual doses of Radix Salviae Miltiorrhizae are 15 g,15 or 30 g,30 g from microalbuminuria to kidney failure,respectively.1.4 Analysis of four characters,five tastes,and channel distributions of drugsThe prescription drugs from microalbuminuria to kidney failure are mainly flat,warm,and cold.The medicinal taste is mainly bitter,acrid and sweet.The meridian of traditional Chinese medicine is mainly liver meridian,spleen meridian and stomach meridian,followed by lung meridian,kidney meridian,heart meridian and large intestine meridian.1.5 Core drug analysismicroalbuminuria～kidney failure,the most connected drugs in different disease stages are Hirudo,Radix et Rhizoma Rhei,Radix Salviae Miltiorrhizae,and Radix Astragali seu Hedysari,indicating that the above traditional Chinese medicines play an important role in the treatment of DKD,and are the core drugs for the treatment of DKD.1.6 Association rule analysisAccording to the "drug-drug" association rule,the support degree is≥10%and the confidence degree is≥90%.The first association rules of each order of microalbuminuria are Radix et Rhizoma Rhei-Hirudo,Radix Astragali seu Hedysari+Radix Salviae Miltiorrhizae-Hirudo,Radix Astragali seu Hedysari+Radix Salviae Miltiorrhizae+Radix et Rhizoma Rhei-Hirudo;The first association rules of each order in macroalbuminuria are:Hirudo-Radix Astragali seu Hedysari,Radix et Rhizoma Rhei+Radix Astragali seu Hedysari-Hirudo,Radix Salviae Miltiorrhizae+Radix et Rhizoma Rhei+Radix Astragali seu Hedysari-Hirudo;the first order of association rules in kidney failure are Radix Salviae Miltiorrhizae-Hirudo,Radix Salviae Miltiorrhizae+Radix et Rhizoma Rhei-Hirudo,Radix Salviae Miltiorrhizae+Radix et Rhizoma Rhei+Hirudo-Radix Astragali seu Hedysari.According to the "drug-symptom" association rule,the support degree is≥10%and the confidence degree is greater or equal to 80%.The first rank of the association rules of each order of microalbuminuria are Hirudo-foam in urine,Hirudo+Radix Astragali seu Hedysari-foam in urine,Hirudo+Radix Salviae Miltiorrhizae+Radix Astragali seu Hedysari-foamy urine;macroalbuminuria association rules of each order are Hirudo-foam in urine,Hirudo+Radix et Rhizoma Rhei-foam in urine,and Hirudo+Hirudo+Radix Salviae Miltiorrhizae+Radix et Rhizoma Rhei-foam in urine;kidney failure association rules of each order are Hirudo-lower extremity edema,Hirudo+Radix et Rhizoma Rhei-lower extremity edema,Radix Astragali seu Hedysari+Radix et Rhizoma Rhei+Hirudo-lower extremity edema.1.7 Cluster analysisThe symptoms of DKD patients in different stages are mainly clustered into the following categories:dry stool,abdominal distension,poor sleep,fatigue,poor appetite;Low back pain,low back pain,endless urination and frequent urination;Blurred vision,numbness and tingling of limbs are grouped into one category;Urination foam,lower limb edema together.Fear of cold,tinnitus and frequent urination at night;Poor sleep,dreaminess and night sweats.The above symptoms suggest that the pathological states of DKD patients are mainly deficiency(qi deficiency,Yin deficiency and Yang deficiency),stasis(stasis of collateral arteries),turbidness(water dampness and turbidness)and cold(deficiency of kidney Yang).The drugs were mainly clustered into the following categories:Rhizoma Pinelliae Preparata,Rhizoma Coptidis,and Fructus Trichosanthis,Radix Scutellariae,Radix Puerariae,Rhizoma Coptidis,and Rhizoma Zingiberis;Hirudo and Radix et Rhizoma Rhei;Poria and Rhizoma Alismatis,Radix et Rhizoma Rhei and Radix Aconiti Lateralis Preparata,Ramulus Cinnamomi,Caulis Spatholobi,and Radix Glycyrrhizae,Rhizoma Anemarrhenae and Cortex Phellodendri.The prescriptions involved are mainly Xiaoxianxiong decoction,Gegen Qinlian decoction,Ganjianghuangqinhuanglian decoction,Didang decoction,Fulingzexie decoction,Dahuangfuzi decoction,Huangqiguizhiwuwu decoction,and Zhibodihuang decoction.2.Experimental Research ResultsPart 1:Analysis of the main components of Shenzhuo prescription based on UHPLC/Q-TOF-MS technology1.The fingerprints of the 12 batches of Shenzhuo formula samples were compared with the generated control spectrum,and the similarity of each sample was between 0.886 and 0.995,indicating that the quality of different batches of Shenzhuo formula samples was stable and controllable.2.A total of 152 compounds were identified in Shenzhuo formula,of which 29 components were identified by the reference substance.The 152 compounds identified can be assigned to single herbs,and these components are mainly from Radix et Rhizoma Rhei,Radix Astragali seu Hedysari,and Radix Salviae Miltiorrhizae.Among them,53 compounds were identified in positive ion mode and 99 compounds were identified in negative ion mode.Part 2:Based on network pharmacology to explore the potential mechanism of Shenzhuo formula in the treatment of DKD1.According to the compounds identified by the research on the medicinal components of Shenzhuo formula,354 drug targets were screened,of which 94 were potential targets for the treatment of DKD.2.GO and KEGG analysis of drug-disease targets showed that Shenzhuo Recipe in the treatment of DKD is mainly through VEGF signaling pathway,Apoptosis signaling pathway,MAPK signaling pathway,AGEs-RAGE signaling pathway and other signaling pathways,and the mechanism involved is mainly cell apoptosis,angiogenesis,advanced glycation end products,immune inflammatory response,regulation of cell adhesion.Part 3:Exploring the mechanism of Shenzhuo formula on db/db mice model with diabetic kidney disease1.Body weight:the db/m group showed a gradual upward trend from 0 to 16 weeks.The db/db group,db/db+SZF group and db/db+Irb group had the same trend of change,gradually increasing from 0 to 8 weeks,gradually decreasing from 8 to 16 weeks.Compared with db/db group,db/db+SZF group and db/db+Irb group had no significant effect on body weight(p>0.05).2.Fasting blood glucose:the db/m group showed a gradual upward trend from 0 to 16 weeks.The db/db group,db/db+SZF group and db/db+Irb group had the same trend of change,gradually increasing from 0 to 4 weeks,steady trend from 4 to 12 weeks,gradually decreasing from 12 to 16 weeks.Compared with db/db group,db/db+SZF group and db/db+Irb group had no significant effect on fasting blood glucose(p>0.05).3.UACR:compared with db/m group,db/db group increased significantly from 0 to 16 weeks(p<0.05).Compared with db/db group,db/db+SZF group and db/db+Irb group decreased significantly from 12 to 16 weeks(p<0.05).4.Urine NGAL:with the increase of the age of the mice,the NGAL in the urine of the mice in each group showed an upward trend.Compared with the db/m group,the NGAL of the db/db group was significantly increased from 0 to 16 weeks(p<0.01).Compared with db/db group,the NGAL of db/db+SZF group and db/db+Irb group decreased significantly from 4 to 16 weeks(p<0.01).5.Kidney weight/body weight ratio:compared with db/m group,db/db group was significantly higher(p<0.001),compared with db/db group,db/db+SZF group and db/db+Irb group were significantly decreased(p<0.001).6.Serum Cre-s,UA,UREA,and NGAL:compared with the db/m group,the db/db group were significantly increased(p<0.01),compared with the db/db group,the db/db+SZF group and db/db+Irb group were significantly decreased(p<0.05).There was no significant difference in AST and ALT among the groups(p>0.05).7.Renal pathology:compared with db/m group,the glomerular basement membrane thickening,mesangial cell proliferation,mesangial matrix accumulation,and collagen fiber deposition were significantly increased in db/db group.Compared with db/db group,the above renal pathological changes were significantly alleviated in db/db+SZF group and db/db+Irb group.8.Foot width,basement membrane thickness:compared with db/m group,db/db group significantly increased the width of the foot process and reduced GBM thickness(p<0.05);compared with db/db group,db/db+SZF group and db/db+Irb group significantly decreased the width of the foot process and GBM thickness(p<0.05).In addition,compared with the db/m group,the podocytes in the db/db group had severe edema,partial blurring of the cell membrane,sparse intracellular matrix,large areas of low electron density edema,swelling and disintegration of organelles,and large fusion of the foot processes,which were significantly different from those in the db/m group.Compared with the db/db group,the db/db+SZF group and the db/db+Irb group significantly alleviated the pathological changes of the above podocytes.9.TUNEL staining:compared with the db/m group,the apoptosis of kidney cells in the db/db group was significantly increased,and the intervention of Shenzhuo formula and irbesartan could significantly reduce the apoptosis of kidney cells.10.The WB results:compared with db/m group,the expressions of WT-1,Nephrin,Podocin,and BCL-2 in db/db group were obviously decreased(p<0.05),and the expression of Desmin,cleaved caspase 3,and cleaved PARP1 was significantly increased(p<0.05);After shenzhuo formula and Irbesartan intervention,the expression levels of WT-1,Nephrin,and Podocin were significantly increased(p<0.05),while the expression levels of Desmin,cleaved caspase 3,and cleaved PARP1 were significantly decreased(P<0.05).The variation trend of IHC and RT-PCR results was consistent with WB results.Part 4:Exploring the mechanism of Shenzhuo formula on microcirculation based on zebrafish model1.Compared with the normal control group,the diameter of the intersegmental blood vessels in the model group was obviously reduced(p<0.001).Compared with the model group,the diameter of the zebrafish intersegmental vessels in the middle-dose and high-dose groups of Shenzhuo formula increased obviously(p<0.05).2.Compared with the normal control group,the incidence of zebrafish thrombosis in the model group was significantly higher(p<0.001),and the incidence of zebrafish thrombosis in the low,medium and high dose groups of Shenzhuo formula was significantly lower(P<0.001).3.Compared with the normal control group,the blood flow velocity and cardiac output of the zebrafish in the model group were obviously decreased(p<0.001).Compared with the model group,the Shenzhuo formula significantly increased the blood flow of the zebrafish blood flow velocity(p<0.01),the middle and high dose groups of Shenzhuo formula significantly increased cardiac output(p<0.01)in a dose-dependent manner.Conclusion1.Clinical research(1)Based on the analysis of medical case data mining technology,it was found that the core drugs in the treatment of DKD by Xiaolin Tong were Hirudo,Radix Astragali seu Hedy sari,Radix et Rhizoma Rhei,and Radix Salviae Miltiorrhizae.To a certain extent,it reflects the supervisor’s understanding of the etiology and pathogenesis of DKD:Qi deficiency and blood stasis are the important core pathogenesis of DKD,and deficiency,blood stasis and turbidity are its pathological characteristics,and the treatment of nourishing qi,promoting blood circulation and clearing collasals runs throughout.(2)Based on the treatment of nourishing qi,activating blood and dredging collaterals,microalbuminuria takes into account clearing phlegm and dampness.The commonly used prescriptions are Gegen Qinlian decoction,Ganjiang Huanqqin Huanglian Renshen decoction,Dahuang Xiexin decoction and Xiaoxianxiong decoction,which aims to improve the pathological internal environment of the body and delay the development of DKD.macroalbuminuria takes into account both warming yang and diuresis.Commonly used prescriptions are Herba Leonuri,Herba Epimedii,and Poria,which aim to promote the metabolism of water retention in the body.kidney failure takes into account both warming yang and diuresis,dredging abdomen and relieving turbidity,and tonifying kidney and astringent essence.The commonly used prescriptions are Dahuang Fuzi decoction,Shuilu Erxiandan,Herba Epimedii,Herba Leonuri,Poria,and Semen Cuscutae,aiming to protect kidney function and improve the life of patients quality.It reflects the flexibility of the instructor in dispatching prescriptions and medication,good at using classical prescriptions to adjust the state and targeting,the medicines are few and precise,and the medicines are specific,and powerful.(3)The "four qi" of the medicine are mainly flat,warm,and cold,the combination of cold and warm reflects the treatment method of strengthening the righteousness and eliminating pathogenic factors,attacking and supplementing simultaneously.The"five flavors" are mainly bitter,acrid,and sweet,taking the meaning of acrid to open the bitterness and lowering the bitterness,and adjust them with sweet-flavored medicines,so as to prevent the acrid,acrid and lowering drugs from damaging the human body’s righteousness,so as to achieve the purpose of eliminating evil without harming righteousness.(4)Different stages of DKD,the same drug,the dosage is different,especially the four core drugs of Hirudo,Radix Astragali seu Hedysari,Radix et Rhizoma Rhei,and Radix Salviae Miltiorrhizae,with the progression of the disease,the drug dosage showed a gradual upward trend.2.Experimental research(1)Shenzhuo formula contains a variety of chemical components and multiple targets.Network pharmacology predicts that the mechanisms involved in the treatment of DKD are mainly apoptosis,angiogenesis,cell adhesion regulation,immune and inflammatory response,which reflects the characteristics of Shenzhuo formula’s synergistic intervention of DKD through "multi components,multi targets and multi pathways".(1)Shenzhuo formula can significantly reduce UACR,kidney function related indicators,and renal injury factors,improve glomerular basement membrane thickening,mesangial cell proliferation,mesangial matrix accumulation and collagen fiber deposition,etc.,and can significantly relieve foot process fusion and protect podocytes.In addition,Shenzhuo formula can improve microcirculation and microvascular dysfunction by improving hemodynamics and reducing the incidence of thrombosis.(3)Shenzhuo formula may reduce urinary albumin,protect renal function,and delaying the progression of DKD by up-regulating WT-1,Nephrin,Podocin,BCL-2,down-regulating Desmin,cleaved caspase 3,and cleaved PARP1,inhibiting the apoptosis of glomerular podocytes,maintaining the connection between podocytes,stabilizing the basement membrane,slowing podocyte damage,and protecting the function of the glomerular filtration barrier.