| Purpose:To clarify the evolution of syndromes and medication rules of diabetic kidney disease(DKD)in ancient and modern times.To explore the mechanism of Yiqi Yangyin Huashihuoxue Fang(YYHHF)in treating and delaying the progression of early DKD.To provide theoretical and data evidence from ancient books and literature,clinical evidence and experimental evidence for the clinical application of YYHHF.Material and method:Paper one:Exploring the syndromes and medication rules of DKD in ancient literatures based on data miningThis study collected ancient literatures such as medical classics,diagnostic methods,comprehensive medical books,medical records,etc.by using search terms of "Shen Xiao","Xiao Shen","Xia Xiao","Shen Xiao" and "Nei Xiao".Ancient literatures were organized into a database that can be recognized by data mining software to explore the syndromes and medication rules of DKD deeply by the methods of association rule analysis,network relationship mining,knowledge graph visualization analysis and prescription drug cluster analysis.This study explores the theory and prescription rules of different dynasties for the diagnosis and treatment of DKD in the order of time,and present the evolution and development of the theory of diagnosis and treatment of DKD in ancient literatures objectively.Paper two:Construction of risk factors and predictive equations for the progression of DKDThis study collected the medical records of patients who were hospitalized and diagnosed with DKD in the Department of Endocrinology,Nephrology,and Endocrine Rehabilitation of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine from December 1,2018 to December 1,2021.And 834 medical records of patients were screened according to the inclusion and exclusion criteria.The medical records of the patients included in the study were randomly divided into 584 cases in the training set and 250 cases in the validation set at the ratio of 7:3.The training set was divided into a microalbuminuria group of 442 cases and a macroalbuminuria group of 142 cases according to the medical records of follow-up visits and with reference to the staging standard of DKD.The basic data,anthropometric indexes,course of type 2 diabetes mellitus,comorbidities,TCM standard and laboratory indexes of the patients in the microalbuminuria group and the macroalbuminuria group in the training set were analyzed and compared by software of SPSS 26.0.The independent risk factors for the progression of DKD were screened by multivariate Logistic regression analysis.The prediction equation was constructed according to the independent risk factors and the ROC curve was drawn,and the consistency of the prediction equation was verified by the external verification method.Paper three:Observation on the curative effect of YYHHF in the treatment of early DKDThis study observed the patients with Qi-yin deficiency and damp stasis syndrome in the microalbuminuria stage in the patients of the paper two,who returned to the outpatient clinic of Professor Ma Xiaoyan with the method of retrospective study.The basic treatment combined with irbesartan tablets was the control group,with a total of 40 cases,and the basic treatment combined with YYHHF was the observation group,with a total of 52 cases.The observation period is 12 weeks.The basic data,laboratory indexes and TCM syndrome scores before and after treatment were collected from the two groups.This study summarized Professor Ma Xiaoyan’s academic thoughts on the diagnosis and treatment of DKD based on the theory of "preventive treatment",and explored the intervention effect of YYHHF on the risk factors for the progression of DKD.The overall curative effect of YYHHF was evaluated in combination with syndrome curative effect and clinical curative effect standard.Paper four:Study on the mechanism of action of YYHHF on podocyte-related protein in DKD model rats.SPF SD rats were adaptively reared to a body weight of 200±20g.The left renal artery was isolated and ligated in the modeling group,and fed with high-sugar and high-fat diet 1 week after the operation,and intraperitoneally injected with 2%streptozotocin solution(40mg·kg-1)2 weeks later.The left renal artery in the sham operation group was directly sutured without separation and ligation,and was fed with normal chow 1 week after operation,and intraperitoneally injected with sodium citrate buffer(40mg·kg-1)2 weeks later.SD rats that met the modeling criteria started intervention therapy.The sham operation group was renamed the normal group,and the modeling group was randomly divided into a model group,an irbesartan control group,a YYHHF high-dose group(YYHHF-H),a YYHHT medium-dose group(YYHHF-M),and a Chinese medicine low-dose group(YYHHF-L).Normal group and model group were given normal saline by gavage.Irbesartan control group was given irbesartan solution by gavage.Each dose group of traditional Chinese medicine was given the YYHHF(crude medicinal amount 11.16g·ml-1,5.58g·ml-1,2.79g·ml-1)by gavage.Rats were weighed weekly.24-hour urine protein quantification and blood biochemical indexes were collected at the 4th and 8th weeks after treatment.Rats were sacrificed at week 8 to collect kidney tissue.Kidney tissue was observed morphologically under light microscope with HE staining.The contents of inflammatory markers basic fibroblast growth factor(bFGF),insulin-like growth factor(IGF)and monocyte chemoattractant protein-1(MCP-1)in renal tissue were detected by enzyme-linked immunosorbent assay(ELISA).The expression of podocyte marker protein(PCX)and hole septum protein molecule(Nephrin)in renal podocytes was detected by western blotting(WB).Results:Paper one:Exploring the syndromes and medication rules of DKD in ancient literatures based on data mining1 A total of 167 articles related to disease location elements and pathogenic elements of DKD were retrieved in this study,which includes 9 kinds of disease location elements,mainly kidney,heart and lung,and 16 pathogenic elements,mainly fire(heat),followed by yin deficiency,body(liquid)injury,dampness,blood stasis,etc.Association rule analysis shows that stomach-fire(heat),stomach+kidney-fire(heat)are relatively closely related.Network relationship mining shows that the relationship between kidney-fire(heat),kidney-yin deficiency,kidney-jin(liquid)injury,kidney-qi deficiency,etc.is relatively close.Knowledge spectrum map visualization shows that the elements of disease location are mostly expressed in kidney,heart,lung,spleen,stomach and liver;the pathogenic elements are most expressed in terms of fire(heat),yin deficiency,qi deficiency,and body(liquid)injury;yang deficiency,dampness,blood stasis,etc.form a group of elements with close co-occurrence levels.The disease location element contains the regular characteristics of the restraint of the five elements.The pathogenic element of fire(heat)is mostly deficient heat,with dampness as the core and blood stasis throughout.The pathogenesis of DKD shows the evolution law of "heat-deficiency-stasis" in general.Compound syndromes include spleen and kidney qi deficiency,lung and kidney qi deficiency,heart and kidney yin deficiency,liver and kidney yin deficiency,spleen and kidney yang deficiency,heart and kidney yang deficiency,etc.Concurrent syndromes include damp-heat syndrome,phlegm-damp syndrome,phlegm-drinking syndrome,qi deficiency and blood stasis syndrome,qi stagnation and blood stasis syndrome,yin deficiency and blood stasis syndrome,yang deficiency and blood stasis syndrome,etc.Dampness,damp-heat,blood stasis and other pathogenic elements are seen in all syndromes,and are common in all stages of the disease.2 A total of 416 prescriptions related to DKD were retrieved in this study,with a total of 224 prescription drugs,of which 46 are commonly used drugs(more than 5%of usage frequency).Commonly used medicines are mainly tonic,heat-clearing,and dampness-dampening medicines,among which the tonic medicines are headed by qitonifying medicines.The medicinal properties of commonly used drugs in DKD prescriptions are more cold,warm,and flat;the medicinal tastes are more sweet,bitter,and acrid;the main purpose is to return to the kidney,heart,and lung meridians.Drug association rules and network relationship analysis show that Liuwei Dihuang Pill and Shengmai Yin are implied.Knowledge graph visualization and drug cluster analysis show that the core prescription contains Baifuling Pill,Sangqizhu Powder,Shugan Dihuang Powder,Jinkui Shenqi Pill,Jisheng Shenqi Pill,Siwu Decoction,etc.The principles of treatment include nourishing qi and nourishing yin,clearing heat and moistening dryness and promoting body fluid,strengthening the spleen and kidney,dispelling dampness and reducing swelling,promoting blood circulation and removing blood stasis.3 From sorting out the development context of the syndrome and treatment of DKD in ancient literatures:DKD in Han and Tang Dynasties has not yet been distinguished from diabetes mellitus.Zhongjing pioneered the treatment of XiaXiao and clarified the status of kidney deficiency in diabetes."Outside Taiwan Secrets" has laid the foundation for the SanXiao theory of governance.Fire(heat)was mostly used for treatment during the Han and Tang dynasties,and the treatment focused on clearing away heat and benefiting qi,while taking into account the gastrointestinal fluids.The Song and Yuan Dynasties began to distinguish the SanXiao theory.The "Cold and Cool School" prevails.The important position of the spleen and stomach was emphasized during the Song and Yuan Dynasties.While treating fire(heat),it also took into account yang deficiency and dampness.The Ming Dynasty advocated the theory of MingMen.The "Warm and Tonic School" is gradually established on the basis of the "Cold and Cool School".Both cold and warm can be treated.It was believed that while nourishing yin and clearing away heat,it emphasized syndrome differentiation to warm the lower yuan,replenish the essence and transform the qi during the Ming Dynasty.Exploring the pathogenesis of DKD in terms of fire(heat),deficiency and cold,and pay attention to the status of pathological products such as phlegm-dampness and blood stasis during the Qing Dynasty.The main treatment is to invigorate Qi and blood,promote blood circulation and remove blood stasis,and dispel dampness and dispel turbidity.The academic thoughts of the physicians of the past dynasties were unified during the period of the Republic of China.It was emphasized that the deficiency of righteousness was the foundation During the period of the Republic of China.In the treatment,more qi and yin were invigorated,while promoting blood circulation and removing blood stasis,diuresis and turbidity,and taking into account deficiency and excess.Paper two:Construction of risk factors and predictive equations for the progression of DKD1 Comparison of microalbuminuria group and macroalbuminuria group in DKD:The difference of groups in the gender,age,systolic blood pressure(SBP),history of hypertension,history of diabetic retinopathy,wet syndrome,phlegm syndrome,dampheat syndrome,serum total protein(TP),serum albumin(ALB),total cholesterol(TC),glycerol Triesters(TG),low density lipoprotein cholesterol(LDL-C),glycosylated hemoglobin(HbA1c),fasting plasma glucose(FPG),blood uric acid(UA),blood urea nitrogen(BUN),serum creatinine(Scr),cystatin(CysC),serum calcium(Ca2+),thyroid-stimulating hormone(TSH)and free triiodothyronine(FT3)were statistically significant(P<0.05).2 Multivariate Logistic regression analysis showed that gender(male),history of hypertension,history of diabetic retinopathy,wet syndrome,damp-heat syndrome,HbA1c(≥7%),LDL-C(≥2.6(without coronary heart disease)/1.8(with coronary heart disease)Heart disease)mmol·L-1),ALB(<35g·L-1),Ca2+,FT3,CysC were independent risk factors for the progression from microalbuminuria to macroalbuminuria in DKD.3 Based on independent risk factors,the prediction equation for DKD progression was established as follows:P=(exp(6.150+0.936SEX+0.678HH+0.627HDR+1.852DS+2.023DHS-2.477ALB+2.923LDL-C+1.452HbA1c-0.444FT3-4.251Ca2++1.207CysC))/(1+exp(6.150+0.936SEX+0.678HH+0.627HDR+1.852DS+2.023DHS-2.477ALB+2.923LDL-C+1.452HbA1c-0.444FT3-4.251Ca2++1.207CysC))The area under the ROC curve of the prediction equation was 0.920(95%CI:0.8950.946),the sensitivity was 80.99%,the specificity was 90.05%,and the accuracy was 88.87%.External validation suggests that the prediction equations are consistent.Paper three:Observation on the curative effect of YYHHF in the treatment of early DKD1 Professor Ma Xiaoyan believes that the pathogenesis of the essence of DKD is virtual and the surface of DKD is real.In the early and middle stages,both qi and yin are mainly deficient,and in the later stage,both qi and blood are deficient in yin and yang.Damp heat is an important pathological element for kidney collateral damage,and blood stasis runs through the course of the disease.Damp heat and blood stasis are both pathological products and pathogenic factors that affect the progression and outcome of the disease.Professor Ma Xiaoyan differentiates and treats early DKD based on the theory of "preventing disease and preventing changes in preventive treatment".DKD is based on the principles of nourishing qi and nourishing yin,clearing heat and removing dampness,promoting blood circulation and removing blood stasis,supplemented by the methods of soothing,nourishing,softening and calming the liver.Professor Ma Xiaoyan created YYHHF in order to achieve the purpose of nourishing qi and yin,eliminating dampness and clearing away heat,and opening the kidneys and collaterals.2 There were no significant differences between the control group and the observation group in gender,age,comorbidities,smoking history,drinking history,and course of type 2 diabetes(P>0.05).3 Compared before and after treatment,the control group and the observation group had better therapeutic effects in terms of FPG,HbAlc,high-density lipoprotein cholesterol(HDL-C),LDL-C,BUN,CysC,urinary albumin/creatinine ratio(UACR),and 24h urine protein quantification(P<0.05).The observation group also had control and regulation effects on TC and Ca2+(P<0.05),and the syndrome score was significantly reduced(P<0.01).Both groups had good clinical efficacy(P<0.05).Compared with the control group after treatment,the FPG,HbAlc,TC,LDL-C,HDLC,BUN and UA of the observation group were well controlled(P<0.05).The syndrome scores of the observation group were significantly decreased,and the syndrome efficacy was better(P<0.01).The clinical efficacy of the two groups was comparable(P>0.05).There were no obvious adverse reactions such as skin rash,itching,dizziness,headache,nausea and vomiting,or impaired liver and kidney function in both groups.Paper four:Study on the mechanism of action of YYHHF on podocyte-related protein in DKD model rats1 General status,body mass and renal histomorphology:Following the method of unilateral renal artery ligation+STZ induction+high-sugar and high-fat diet feeding,the model of early DKD model was successfully established.The rats in the normal group were generally in good condition.The rats in the model group developed symptoms such as lethargy,obvious polydipsia,polyphagia,polyuria,and weight loss.Although the general state of each treatment group has different degrees of abnormal performance,it is generally better than the model group.The body weight of the rats in the normal group was consistent with the age of the week.The model group decreased significantly.Compared with the model group,there was no significant change in the body weight of the rats in the irbesartan control group(P>0.05)and the body weight of each dose group of YYHHF after treatment was higher than that of the model group(P<0.05).The histomorphology of the kidneys of the rats in the normal group was normal.The glomerular structure,shape and size of the rats in the type group were abnormal,the glomerulus was atrophied,the glomerular cavity became larger,the proportion was unbalanced,the wall thickness of the proximal tubule and the distal tubule were uneven,the lumen size was different,and the swelling,a large number of inflammatory cells infiltrated.Compared with the rats in the model group,the glomerular size of the rats in each treatment group recovered to varying degrees,and the infiltration and damage of inflammatory cells were also alleviated to varying degrees.Among them,the renal tissue morphology of the rats in the YYHHG-H recovered the best.2 Biochemical Indicators:Compared with the normal group,the blood glucose,TG,TC,LDL-C,HDL-C,alanine aminotransferase(ALT),aspartate aminotransferase(AST),BUN,Scr,24h urine protein quantification of the rats in the model group and each treatment group all were abnormal(P<0.05).Compared with the model group,the blood glucose,TG,TC,HDL-C,LDL-C,ALT,AST,BUN,Scr,and 24h urinary protein quantification of each treatment group in the 4th and 8th weeks were significantly higher than those of the model group(P<0.05),except the blood glucose of the western medicine control group and the TG and TC of the low-dose traditional Chinese medicine group in the 4th week(P>0.05).Compared with the irbesartan control group,the quantitative control levels of blood glucose,TG,TC,HDL-C,LDL-C,ALT,AST,BUN,Scr and 24h urine protein in the YYHHF-H group were significantly higher in the 4th and 8th weeks(P<0.05).The control levels of blood glucose,TG,TC,HDL-C,LDL-C,ALT,AST,BUN and Scr in the 4th and 8th weeks of the YYHHF-M and YYHHF-L groups were comparable to those of the irbesartan control group(P>0.05),or worse than the irbesartan control group(P<0.05).3 Inflammatory factors:The contents of bFGF,IGF and MCP-1 in the tissues of normal rats were within the normal range.Compared with the normal group,the contents of bFGF,IGF and MCP-1 in the renal tissue of early DKD rats in each group were significantly increased(P<0.01).Compared with the model group,the contents of bFGF,IGF and MCP-1 in the kidney tissue of the irbesartan control group and the groups of each dose of YYHHF were significantly decreased.(P<0.01),except for the YYHHFL group,there was no significant difference in the content of MCP-1(P>0.05).Compared with the irbesartan control group,the contents of bFGF,IGF and MCP-1 in the kidney tissue of the rats in the YYHHF-L group were significantly higher(P<0.01),and the contents of bFGF and IGF in the kidney tissue of the rats in the YYHHF-M group were significantly higher(P<0.01),but there was no significant difference in the content of MCP-1(P>0.05),and there was no significant difference in the content of bFGF and IGF in the kidney tissue of YYHHF-H group(P>0.05),while the content of MCP-1 was significantly lower(P<0.01).4 Podocytes:The expression of PCX and Nephrin in the kidney tissue of normal rats was normal.Compared with the normal group,except that there was no significant difference in the expression of Nephrin in the renal tissue of the rats in the irbesartan control group(P>0.05),and the expression of Nephrin in the renal tissue of the rats in the YYHHF-H group was lower(P<0.05),the rest of the groups were significantly higher than those in the normal group.The expressions of PCX and Nephrin in rat kidney tissue were significantly decreased(P<0.01).Compared with the model group,the expressions of PCX and Nephrin in the renal tissue of the rats in the irbesartan control group were significantly higher(P<0.01),and the expression of Nephrin in the renal tissue of the rats in the YYHHF-H group was higher(P<0.05).There was no significant difference in the expressions of PCX and Nephrin among the other groups(P>0.05).Compared with the irbesartan control group,there was no significant difference in the expression of PCX and Nephrin in the YYHHF-H group(P>0.05);the expression of PCX and Nephrin in the YYHHF-M and YYHHF-L groups decreased(P<0.05).Conclusion:1 The pathogenesis of DKD in ancient literatures generally shows the evolution law of"heat-deficiency-stasis".Heat is mostly deficient heat,with dampness as the core and blood stasis throughout.The syndrome types are spleen-kidney qi deficiency syndrome,lung-kidney qi deficiency syndrome,heart-kidney yin-deficiency syndrome,liverkidney yin-deficiency syndrome,spleen-kidney-yang deficiency syndrome,heartkidney-yang deficiency syndrome,etc.Concurrent syndromes include damp-heat syndrome,phlegm-damp syndrome,phlegm-drinking syndrome,and blood stasis syndrome.The herbs used in prescriptions are mainly tonic,heat-clearing,and dampness-dampening herbs,among which the tonic herbs are mainly qi-invigorating herbs.The prescription contains Liuwei Dihuang Pill,Shengmai Yin,Baifuling Pill,Sangqizhu Powder,Shugan Dihuang Powder,Jinkui Shenqi Pill,Jisheng Shenqi Pill,Siwu Decoction,etc.The treatment principles include nourishing qi and nourishing yin,clearing heat and moistening dryness and promoting body fluid,invigorating the spleen and invigorating the kidney,dispelling dampness and reducing swelling,promoting blood circulation and removing blood stasis.2 Regarding its development context,in the Han and Tang Dynasties diabetes was mostly used to treat thirst.Zhongjing created the theory of XiaXiao.In the Song and Yuan Dynasties,the SanXiao were clearly distinguished,and the XiaXiao had a clear argument.From the "cold and cool school" to the "warm tonic school",the theory of diagnosis and treatment of DKD is gradually enriched.By the time of the Qing Dynasty and the Republic of China,dampness and blood stasis were given full attention.The treatment focuses on nourishing qi and nourishing yin,as well as promoting blood circulation and removing blood stasis,diverting dampness and eliminating turbidity and deficiency,which lays the foundation for the modern diagnosis and treatment of DKD.3 Gender(male),history of hypertension,history of diabetic retinopathy,dampness syndrome,dampness-heat syndrome,HbA1c(≥7%),LDL-C(≥2.6(without coronary heart disease)/1.8(with coronary heart disease)mmol·L-1),ALB(<35g·L-1),Ca2+,FT3 and CysC were independent risk factors for the progression from microalbuminuria to macroalbuminuria in DKD.The DKD progress prediction equation can predict the progression of DKD.4 Professor Ma Xiaoyan believes that the pathogenesis of the essence of DKD is essential deficiency and superficial wealth.In the early and middle stages,both qi and yin are mainly deficient,and in the later stage,both qi and blood are deficient in yin and yang.Damp heat is an important pathological element for kidney collateral damage,and blood stasis runs through the course of the disease.The treatment principle is to nourish qi and nourish yin,clear dampness and heat,promote blood circulation and remove blood stasis,supplemented by the methods of soothing,nourishing,softening and calming liver.YYHHF can achieve good clinical efficacy and effectively delay the progression of DKD by intervening in the independent risk factors of dampness syndrome,dampness-heat syndrome,LDL-C,HbAlc,Ca2+,and CysC.5 YYHHF can treat and delay the progression of early DKD by adjusting glucose and lipid metabolism,improving inflammatory response,repairing kidney tissue,and protecting podocytes.Its efficacy is positively correlated with concentration in a certain range. |
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