Complex Network Analysis Of Professor Liu Ximing's Treatment Of DKD And Research On The Mechanism Of Regulating Vasculature-globe Crosstalk By Raising The Clear And Lowering The Turbi

Objective1 Based on multiple complex network analyses,to visualize and present the different stages of DKD treatment by Professor Ximing Liu and the rules of prescriptions and medication,to extract the core drug combinations of DKD prescriptions.Refining the core prescriptions and connotations of DKD treatment by Professor Ximing Liu ’s using sheng-qing-jiang-zhuo treatment method.2 Using db/db mice as the vehicle and the core prescriptions as the intervention drugs.Explored the regulatory mechanism of the HE4/PEX19/PEX3 tubulo-globular crosstalk signalling pathway.MethodsClinical study1.1 Collecting DKD patients attending Professor Ximing Liu ’s clinic from 1 January 2012 to 30 September 2022,cases were screened by nadir criteria,and data on basic patient information(gender,age,date of consultation),disease diagnosis(staging diagnosis,evidence diagnosis,evidence diagnosis),symptoms,tongue and pulse,and prescribed medication were extracted.1.2 Patients with DKD were included in their initial medical records and the clinical features of the different stages of DKD were analysed with reference to the joint UACR and cGFR staging criteria.1.3 The prescription data set of each visit of DKD patients was recorded,and the drugs were treated as separate item sets and subjected to second-and third-order association rule analysis and complex network topology analysis,and the drugs were treated as separate data clusters and subjected to cohesive hierarchical clustering analysis.The core drug combinations were extracted from the data sets to identify the drug use patterns and core drug combinations for the treatment of DKD by the tutors at different stages and evidence.Experimental study2.1 Grouping method:Astragalus sanguinarius ascending,clearing and descending formula(QSF),which embodies the core treatment method of DKD(ascending,clearing and descending method),was used as the formula vehicle.db/db mice were used as the study subjects and divided into 4 groups,normal group(db/m),model group(db/db+Veh),astragalus sanguinarius ascending,clearing and descending formula group(db/db+QSF)and Irbesartan group(db/db+Irb).The mice were administered by gavage for 12 weeks and their body weight and blood glucose were monitored dynamically.2.2 Evaluation of renal function:24h urine was collected at weeks 0,4,8 and 12,and urine volume was counted.uALB was detected by Elisa method,Ucr,Scr and Urea were detected by biochemical instrument,and UACR was calculated to evaluate glomerular filtration function,and urine β2-MG and KIM-1 were detected to evaluate renal tubular reabsorption function.2.3 Urinary differential metabolic profile:To determine mouse urinary metabolites by liquid chromatography-mass spectrometry,to identify and screen endogenous urinary differential metabolites in db/db mice involved in QSF backregulation,and to screen differential metabolic pathways by enrichment analysis.2.4 Evaluation of tubulointerstitial injury:HE staining to observe renal pathological changes,PASM and Masson staining to assess the extent of interstitial fibrosis,immunohistochemistry to detect the expression of α-SMA,a marker of renal fibrosis.Ecadherin,a marker of tubular epithelial cell injury,and CD2AP,a foot cytoskcletal protein,and immunofluorescence to detect the foot cytoskeletal protein α-actin-4 The expression of the renal pro-inflammatory regulators p53,NF-κB and endothelial injury marker ICAM-1 was measured by Western blot.qPCR was used to detect the expression levels of the above proteins at the mRNA level.2.5 Tubulo-globular crosstalk signalling pathway:Elisa assay was used to detect the concentration of HE4 in serum,and immunohistochemistry was used to observe the localisation of HE4 in the kidney,western blot and qPCR were used to detect the protein and mRNA expression levels of HE4 and peroxisome generating factors PEX19 and PEX3,respectively,Elisa assay was performed to determine the concentration of peroxisome Catalase protein in the kidney.Results1 Clinical study1.1 Clinical Characteristics(1)General Information:The study included 180 patients with diabetic kidney disease(DKD),with a male to female ratio of approximately 1.6:1 and a higch incidence age range of 51-60 years.Among them,there were 7 cases in the normal albuminuria period,90 cases in the microalbuminuria period,52 cases in the massive albuminuria period,22 cases in the severe renal dysfunction period,and 9 cases in the renal failure period.(2)Symptom and Tongue Pulse:From the normal albuminuria period to the renal failure period,the proportion of dry mouth and thirst gradually decreased,while the proportion of fatigue,limb edema,and pale tongue gradually increased.(3)Distribution of Syndrome Patterns:The top six syndrome patterns of DKD were phlegm-heat stagnation(20%),qi-yin deficiency(18.33%),kidney yin deficiency(16.67%),yin deficiency and blood stasis(12.22%),deficiency of the original qi(10%),and dampness turbidity(8.89%).Among them,the highest syndrome pattern in the normal albuminuria period was phlegm-heat stagnation(57.14%),in the microalbuminuria period was also phlegm-heat stagnation(27.78%),in the massive albuminuria period was qi-yin deficiency(36.54%),in the severe renal dysfunction period was deficiency of the original qi(54.55%).and in the renal failure period was also deficiency of the original qi(55.56%).(4)Distribution of Herbal Ingredients:The highest herbal ingredient in the normal albuminuria period was heat(60.71%),in the microalbuminuria period was yin deficiency(53.28%),in the massive albuminuria period was also yin deficiency(65.93%),in the severe renal dysfunction period was qi deficiency(81.11%),and in the renal failure period was also qi deficiency(92.68%).1.2 Complex Network Analysis of Medication(1)Complex Network Analysis:A total of 780 prescription data were collected from 180 DKD patients.It involved 222 medicinal ingredients,with a cumulative frequency of traditional Chinese medicine usage of 5,071 times.On average,each prescription consisted of 6.5 medicinal ingredients.The most frequently used medications,in descending order,were Danshen,Maidong,Huangqi,Shan Yao,Beisha Shen,and Shihu.Among them,the frequency of Huangqi,a medication for invigorating qi and promoting clearing,increased from 7.14%to 80.49%from the normal stage of urinary albumin to the stage of renal failure,with the average dosage increasing from 45±21 g to 114±27 g.(2)Agglomerative hierarchical clustering of prescriptions in different stages of DKD:There were a total of 7 prescriptions for the stage of normal urinary albumin(including Banxia Xiexin Tang with modifications,Sanmiao Pill with modifications,Shashen Maidong Tang with modifications,etc.),6 prescriptions for the stage of microalbuminuria(including Zhibai Dihuang Tang with modifications,Huangqi Jianzhong Tang,Zisheng Pill with moditications,etc.),7 prescriptions for the stage of macroalbuminuria(including Zishen Pill with modifications,Jisheng Shenqi Pill with modifications,etc.),6 prescriptions for the stage of severe renal function decline(including Erchen Tang with modifications,Qishen Shengqing Jiangzhuo Fang with modifications,etc.),and 10 prescriptions for the stage of renal failure(including Renshen Lingli Pill with modifications,Linggui Zhugan Tang with modifications,Qishen Shengqing Jiangzhuo Fang with modifications,etc.).(3)Network topology of medication combinations for different patterns of DKD:Phlegmheat obstruction pattern(Danshen+Huanglian+Banxia+Huangqin+Zhishi),Qi-Yin deficiency pattern(Huangqi+Shanyao+Maidong+Yuzhu),Kidney Yin deficiency pattern(Shudihuang+Shanzhuyu+Shanyao+Duzhong),Yin deficiency and blood stasis pattern(Danshen+Maidong+Shihu),Yuan Qi deficiency pattern(Huangqi+Baizhu+Taizishen+Danshen),Dampness accumulation pattern(Huangqi+Fuling+Baizhu+Chenpi).(4)The core medication combination of Huangqi+Danshen+Maidong+Chuanshanlong co-occurs in multiple complex medication networks of DKD across different stages and patterns.Experimental Research2.1 Effect of the sheng-qing-jiang-zhuo treatment method on renal function in db/db mice(1)General observations:As db/db mice aged,their blood glucose levels continued to rise.QSF had an improvement effect on blood glucose levels and significantly lowered the blood glucose levels of 12-week-old mice(P<0.01).Irb had no significant effect on blood glucose levels in mice.(2)Serum renal function:Compared with db/m mice,the urea level of model group mice increased slightly at week 12(P<0.05).and the Scr level showed no difference.Compared with the model group,there was no statistically significant difference in Urea and Scr levels in the QSF and Irb groups.(3)Urine renal function evaluation:Compared with db/m mice,the urine volume of model group mice increased,and the difference was significant at week 12(P<0.01).Compared with the model group,QSF reduced the urine volume of mice at week 12(P<0.05),while Irb had no significant effect on urine volume in mice.As db/db mice aged,their UACR levels continued to rise.Compared with the model group,the UACR levels of the Irb group at weeks 8 and 12 were significantly lower(P<0.01),and the UACR levels of the QSF group at week 12 were significantly lower(P<0.01).As db/db mice aged,the β2-MG levels in their urine continued to rise.Compared with the model group,the β2-MG levels in the urine of the QSF and Irb groups at week 12 were significantly lower(P<0.01).As db/db mice aged,the KIM-1 levels in their urine also continued to rise.Compared with the model group,the urine KIM-1 levels in the QSF and Irb groups at week 12 were significantly lower(P<0.01).2.2 Effect of the ascending clearing and descending turbidity method on endogenous urinary metabolites in db/db mice.(1)Differential urinary metabolites in DKD:There were a total of 387 different metabolites,with the main differential metabolic pathways being central carbon metabolism,pentose phosphate pathway,prolactin signaling pathway,and FoxO signaling pathway.(2)Regulation of differential metabolite profiles by QSF:QSF was found to modulate 32 endogenous differential metabolites,upregulating 18 and downregulating 14.The primarily affected metabolites included cAMP,steroid hormones secreted by the adrenal cortex,and their intermediate metabolites such as dehydroepiandrosterone,corticosterone,18hydroxycorticosterone,and acylphosphates.(3)Metabolic pathways regulated by QSF:The main pathways influenced were aldosterone synthesis and secretion,aldosterone-regulated sodium reabsorption,and ovarian steroidogenesis.2.3 Protective effects of the sheng-qing-jiang-zhuo treatment method on glomerular injury in db/db mice.(1)Renal interstitial fibrosis:Compared to the model group,HE staining showed that QSF and Irb reduced glomerular hypertrophy and mesangial expansion to varying degrees.PASM and Masson staining demonstrated decreased positive staining substances and improved renal interstitial fibrosis in the QSF and Irb groups.QSF reduced the gene expression of the fibrosi s marker α-SMA(P<0.05),while QSF and Irb had no significant effect on the protein expression of α-SMA(P>0.05).(2)Tubular injury:Compared to the model group.QSF and Irb increased the protein and gene expression of the tubular injury marker E-cadhcrin to varying degrees(P<0.05 or P<0.01).(3)Glomerular injury:Both QSF and Irb upregulated the protein and gene expression levels of the endothelial injury factor ICAM-1.QSF and Irb also upregulated the protein and gene expression of the podocyte cytoskeleton proteins CD2AP and α-actin-4 to varying degrees(P<0.05 or P<0.01).(4)Glomerular injury-related pro-inflammatory regulatory factors:Compared to the model group,both QSF and Irb downregulated the protein and gene expr ession levels of p53 and NFκB(P<0.01).2.4 Effect of the sheng-qing-jiang-zhuo treatment method on the HE4/PEX19/PEX3 Tubule-Glomerular Crosstalk Pathway in db/db Mice.(1)Key protein in the tubule-glomerular crosstalk pathway,HE4:Compared to db/m mice.the model group exhibited significantly increased levels of HE4 in both serum and kidneys(P<0.01).The QSF and Irb groups showed varying degrees of reduction in HE4 protein expression in serum and kidneys,and HE4 gene expression was significantly decreased(P<0.01).(2)Peroxisome biogenesis factors PEX19 and PEX3:Compared to the model group,the QSF and Irb groups showed increased protein expression of PEX19 and PEX3 in the kidneys to varying degrees(P<0.01 or P<0.05).QSF upregulated the gene expression of PEX19 and PEX3 to different extents,while the differences in the Irb group were not statistically significant(P>0.05).(3)Peroxisome content:Compared to the model group,both QSF and Irb were able to reduce the protein concentration of Catalase in the kidneys to varying degrees(P<0.01 or P<0.05).However,there were no significant differences in Catalase gene expression between the two groups and the model group(P>0.05).Conclusion(1)Clinical characteristics of DKD:Through complex network analysis,as DKD progresses with disease staging,the proportion of Phlegm-Heat obstruction pattern and FireHeat pattern gradually decreases,while the proportion of Qi Deficiency pattern,DampnessInternal Excess pattern,and Qi Deficiency pattern gradually increases.This reflects the trend of the pathogenesis of "inefficient clearing of qi and failure to eliminate turbid toxins"worsening with disease progression.(2)Regularities of medication and characteristics of treatment methods:The core drug combination of Huangqi,Danshen,Maidong,Chuanshanlong in multiple complex drug networks under different stages and patterns of DKD,demonstrating the sheng-qing-jiang-zhuo core treatment method.(3)The sheng-qing-jiang-zhuo treatment method can inhibit renal inflammation and fibrosis,improve structural damage to renal tubular epithelial cells,glomerular endothelial cells,and podocytes,regulate overall renal function in DKD model mice,and delay disease progression.(4)The beneficial effects of the ascending clearing and descending turbidity method on renal tubular reabsorption function and glomerular filtration function are related to the metabolic pathways associated with steroid hormones.The deep mediation of the tubuleglomerular crosstalk signal pathway involving HE4/PEX19/PEX3 may be a key mechanism underlying its therapeutic effect in DKD.