System for evaluating spleen deficiency and phlegm stasis syndrome of diabetic nephropathy based on combined detection of basophilic granulocytes and total bilirubin and application

Abstract

The invention discloses a system for evaluating spleen deficiency and phlegm stasis syndromes of diabetic nephropathy based on combined detection of basophilic granulocytes and total bilirubin and application, and belongs to the technical field of biology. A ZDF (fa / fa) rat model is used, BASO% and TBIL levels are quantitatively detected, a modernization biology evaluation system capable of simultaneously reflecting DKD'spleen deficiency 'and'phlegm stasis' core pathogenesis is provided for the first time, and the technical problem that objective and quantitative traditional Chinese medicine syndrome evaluation indexes are lacked in the existing DKD model research field is solved. The method specifically comprises the following steps: associating two indexes belonging to an immune system and a metabolic system for the first time to form a brand-new collaborative evaluation combination, and overcoming the limitation of single index evaluation; a quantifiable and repeatable operation method is provided, and a key tool is provided for traditional Chinese medicine mechanism research and new drug research and development of DKD; traditional Chinese medicine syndromes and modern medical indexes are organically combined, and modernization and internationalization development of traditional Chinese medicine is promoted.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a system for evaluating diabetic kidney disease with spleen deficiency and phlegm stasis syndrome based on combined detection of basophilic granulocytes and total bilirubin and use thereof. BACKGROUND

[0002] Diabetic kidney disease (DKD) is one of the most severe and common microvascular complications of diabetes, and its pathogenesis is complex, which has become the main cause of end-stage renal disease. DKD research highly depends on animal models, among which the Zucker Diabetic Fatty (ZDF) rat is widely used in pathogenesis research and drug intervention evaluation as a spontaneous type 2 diabetes model, because it can simulate the core pathological characteristics of human DKD (such as obesity, insulin resistance, progressive proteinuria, and glomerular sclerosis).

[0003] However, although the ZDF model performs well in simulating disease pathologies in Western medicine, it has two significant technical bottlenecks in application, especially in the modernization of traditional Chinese medicine: (1) The lack of TCM syndrome evaluation system and the bottleneck of subjectivity. Currently, the evaluation of DKD animal models completely relies on Western medical pathophysiological indicators (such as blood glucose, urine protein, serum creatinine, and histological staining). However, the core essence of traditional Chinese medicine in preventing and treating DKD lies in "treatment based on syndrome differentiation", among which "spleen deficiency and phlegm stasis" is an important syndrome throughout its early and middle stages. Traditional TCM syndrome evaluation relies on macroscopic observation (such as listless spirit, dull and withered fur, dark purple tongue), but this method highly depends on the evaluator's subjective experience, lacks objective, quantifiable, and repeatable biological indicators, leading to ambiguous model syndrome attribute determination and difficulty in associating with modern molecular mechanism research. This "syndrome-index" disconnection seriously hinders the in-depth explanation of the mechanism of TCM in preventing and treating DKD and the standardized research and development of innovative traditional Chinese medicines.

[0004] (2) The existing biomarkers are disconnected with the overall concept of traditional Chinese medicine and the demand for efficacy evaluation. Although modern medicine has discovered many molecular markers related to DKD, there are two major problems: a) singleness and limitation: most studies focus on single pathway or single type of markers (such as only focusing on inflammatory factors IL-6, TNF-a, or only focusing on metabolic products TMAO). DKD is a complex disease, which is the result of the interweaving of multiple systems such as metabolism, immunity, inflammation, etc. A single indicator cannot fully reflect the overall state of the disease, and it is impossible to correspond to the complex biological connotation of the traditional Chinese medicine "spleen deficiency and phlegm stasis" syndrome. b) Weak correlation with the connotation of traditional Chinese medicine syndrome: the essence of traditional Chinese medicine "spleen deficiency" involves transportation disorder (corresponding to modern metabolic disorder) and external defense weakness (corresponding to modern immune disorder); "phlegm stasis" corresponds to lipid deposition (dyslipidemia) and sluggishness of collaterals (microcirculation disorder, inflammation). Although ZDF rats spontaneously exhibit some of the above characteristics, there has always been a lack of a combination of quantifiable biomarkers that can accurately reflect the two core pathogenesis of "spleen deficiency" (immunity) and "phlegm stasis" (metabolism) in DKD, which can be used to construct an objective evaluation system for syndrome.

[0005] Specifically, although some studies have reported that there is a decrease in total bilirubin (TBIL) levels in DKD (reflecting impaired liver metabolic function and decreased antioxidant capacity of the body, related to "phlegm turbidity" internalization), or speculated immune dysfunction, no study has revealed the change pattern of basophilic granulocytes (BASO%) in DKD, and no one has conducted correlation analysis and combined "TBIL" and "BASO%" as an integrated evaluation system.

[0006] Therefore, there is a long-standing technical problem in the prior art: the lack of a combination of biomarkers that can objectively, quantitatively, and systematically reflect the core pathogenesis of DKD "spleen deficiency and phlegm stasis" syndrome (immune-metabolic disorder) and its application method, resulting in a series of problems such as inaccurate evaluation of DKD traditional Chinese medicine syndrome model and unclear explanation of drug efficacy mechanism. SUMMARY

[0007] The present application is proposed to directly overcome the above technical bottlenecks. That is, based on the above-mentioned deficiencies existing in the prior art, the present application first provides a system and use for evaluating diabetic kidney disease (DKD) spleen deficiency and phlegm stasis syndrome based on combined detection of basophilic granulocytes and total bilirubin. Specifically, using a ZDF (fa / fa) rat model, by quantitatively detecting BASO% and TBIL levels, the present application first provides a modern biological evaluation system that can simultaneously reflect the core pathogenesis of DKD "spleen deficiency" and "phlegm stasis", solving the technical problem of lack of objective and quantitative TCM syndrome evaluation indicators in the existing DKD model research field. Innovation: For the first time, two indicators belonging to the immune system and the metabolic system are associated to form a new synergistic evaluation combination, overcoming the limitations of single indicator evaluation. Practicality: A quantifiable and repeatable operation method is provided, which provides a key tool for TCM mechanism research and new drug research and development of DKD. Prospective: The combination of TCM syndrome and modern medical indicators promotes the modernization and internationalization development of traditional Chinese medicine.

[0008] In order to achieve the above technical purposes, the present application adopts the following technical solutions: One of the purposes of the present application is to provide a biomarker combination comprising basophilic granulocyte percentage (BASO%) and total bilirubin (TBIL).

[0009] The second purpose of the present application is to provide an application of a product for detecting the biomarker combination in screening drugs for preventing and treating diabetic kidney disease (DKD).

[0010] The third purpose of the present application is to provide an application of a product for detecting the biomarker combination in evaluating the efficacy of DKD prevention and treatment drugs.

[0011] The fourth purpose of the present application is to provide an application of a product for detecting the biomarker combination in studying DKD "spleen deficiency and phlegm stasis" syndrome.

[0012] Further, the product for detecting the biomarker combination includes detection instruments and / or detection kits.

[0013] Further, the screening, evaluation and / or research method includes using a DKD animal model.

[0014] Further, the animal model includes a ZDF (fa / fa) rat model.

[0015] Further, the screening, evaluation and / or research method is realized by quantitatively detecting BASO% and TBIL levels in the DKD animal model.

[0016] Further, the BASO% is BASO% in peripheral blood.

[0017] Still further, the TBIL level is a TBIL level in serum.

[0018] Compared with the prior art, the present application has the following beneficial effects: (1) Advantage one: realizing objective and quantitative evaluation of TCM syndromes, overcoming subjective drawbacks At present, TCM differentiation of DKD mainly relies on "observation, smell, inquiry and palpation" of physicians, and the diagnosis result is easily affected by personal experience and subjective judgment of physicians, lacking unified and quantifiable standards. This seriously hinders the clinical popularization, research repeatability and new drug development of traditional Chinese medicine. The present application firstly establishes specific correlation between BASO and TBIL, two blood indexes which can be accurately measured by full-automatic blood routine and biochemical analyzers, and "spleen deficiency and phlegm stasis" syndrome. The originally abstract TCM syndromes have specific and repeatable laboratory data support, realizing the leap from subjective experience judgment to objective data measurement. The present application has the following advantages: (1) index quantification: BASO and TBIL are routine items of clinical examination, the detection method is standardized, and the result is presented in digital form, eliminating human subjective interference. (2) deep agreement with pathogenesis: the present application is not a simple list of indexes, but a deep modern interpretation of "spleen deficiency and phlegm stasis" pathogenesis (BASO consumption corresponds to "spleen deficiency and weak camp, immune disorder", TBIL consumption corresponds to "turbidity toxin consuming essence"), which closely combines objective indexes with TCM theory.

[0019] (2) Advantage two: excellent diagnostic specificity and sensitivity, high evaluation accuracy Some existing objective researches on TCM syndromes often use single index or index combination with weak correlation, resulting in low diagnostic efficiency (AUC) or difficulty in balancing sensitivity and specificity, and easy misjudgment. As shown in the examples of the present application, the area under the curve (AUC) of BASO and TBIL combined detection is equal to 1, which is significantly higher than that of single index or other conventional index combination (such as blood lipids, conventional inflammation indexes). This indicates that the combination can extremely accurately distinguish "spleen deficiency and phlegm stasis" syndrome from non-syndrome state. Therefore, the present application can: (1) multi-target point cooperation: the combination simultaneously captures two core pathological links of "spleen deficiency" (immune disorder, BASO↓) and "phlegm stasis and turbidity toxin" (oxidative stress / bilirubin metabolism imbalance, TBIL↓), providing more comprehensive syndrome information. (2) internal biological correlation: immune disorder caused by spleen deficiency and oxidative stress produced by phlegm stasis and turbidity toxin interact with each other in DKD process, forming a vicious cycle. The combination of BASO and TBIL appropriately reflects this internal relationship, thus producing a "1+1>2" synergistic diagnostic effect.

[0020] (3) Advantage three: convenient detection, low cost, easy to popularize and transform clinically Some new omics technologies (such as genomics, proteomics) can also find biomarkers, but the detection cost is high, the cycle is long, the technology is complex, and it is difficult to be widely used in routine clinical. BASO% and TBIL are routine detection items that can be carried out in the clinical laboratory of hospitals at all levels without additional equipment or expensive reagents. This makes the transformation threshold of the application very low, easy to quickly promote in clinical practice, and used for TCM auxiliary diagnosis and efficacy monitoring of DKD. The application is based on the existing and mature detection platform in the clinic, and ingeniously excavates new diagnostic values with TCM characteristics from it, which embodies strong practicality and transformation potential.

[0021] In summary, the application creatively solves the key technical problem of objective evaluation of DKD "spleen deficiency and phlegm stasis" syndrome by combining objective indicators of modern medicine with traditional wisdom of TCM. The advantages are reflected in objectivity, accuracy, practicality and other aspects, which not only has important theoretical significance, but also has broad clinical transformation and industrialization prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 For the body weight (A), blood glucose (B), food intake (C), water intake (D), urine volume (E) of ZDF-DKD model rats in Example 1 of the application, BUN (F), TT (G), FIB (H), TG (I), TC (J) comparison (X̄± s, n=10). Compared with the control group, # p<0.05, ## p<0.01, ### p<0.001.

[0023] Figure 2 For the 24-hour urine albumin (24h-uALB) content (A), urine albumin / urine creatinine ratio (UACR) (B), kidney weight / body mass (C) and kidney tissue pathological score (D) of ZDF-DKD model rats in Example 1 of the application (X̄± s, n=10). Compared with the control group, #p<0.05, ##p<0.01, ###p<0.001.

[0024] Figure 3 For the pathological morphological changes of kidney tissue of ZDF-DKD model rats in Example 1 of the application (HE, PAS, Masson x400; TEM x10000). A. ZDF rat control group; B. ZDF rat DKD model group.

[0025] Figure 4 For the peripheral blood BASO% (A), BASO (B) of ZDF-DKD model rats in Example 1 of the application (X̄± s, n=10). Compared with the control group, #p<0.05, ## p<0.01, ### p<0.001.

[0026] Figure 5 The serum AST (A), ALT (B), ALB (C), GLB (D), ALP (E), TBiL-1 (F) of the ZDF-DKD model rats in Example 1 of the application were compared (X±s, n=10). Compared with the control group, # p<0.05, ## p<0.01, ### p<0.001.

[0027] Figure 6 The peripheral blood BASO% (A), serum TBiL-1 (B), UACR (C) and kidney tissue pathological score (D) of the ZDF-DKD rats after MD treatment in Example 2 of the application were compared (X±s, n=10). Compared with the model group, p<0.05, p<0.01, p<0.001.

[0028] Figure 7 The changes in the pathological morphology of the kidney tissue of the ZDF-DKD model rats in Example 2 of the application (HE×400, PAS×600, Masson×200) were A. ZDF rat DKD model group, B. ZDF rat MD group, C. ZDF rat EN group.

[0029] Figure 8 The body weight (A), blood glucose (B), FIB (C), TT (D), TC (E), TG (F), BUN (G), CRE (H), UACR (I) and kidney tissue pathological score (J) of the ZDF and STZ-SD DKD model rats in Example 3 of the application were compared (X±s, n=10). Compared with the ZDF-C control group, # p<0.05, ## p<0.01, ### p<0.001; compared with the SD-C control group, p<0.05, p<0.01, p<0.001.

[0030] Figure 9 The peripheral blood BASO% (A), serum TBiL-1 (B) of the ZDF and STZ-DKD model rats in Example 3 of the application were compared (X±s, n=10). Compared with the ZDF-C control group,# p<0.05, ## p<0.01, ### p<0.001; compared with SD-C control group, p<0.05, p<0.01, p<0.001.

[0031] Figure 10 AUC area of BASO% alone, TBIL alone and BASO%+TBIL combined detection in the present application comparative example 1.

[0032] Figure 11 AUC area of TT alone, FIB alone and TT+FIB combined detection in the present application comparative example 2.

[0033] Figure 12 AUC area of HDLC alone, LDLC alone and HDLC+LDLC combined detection in the present application comparative example 2.

[0034] Figure 13 AUC area of NEUT alone, LYMPH alone and NEUT+LYMPH combined detection in the present application comparative example 2. DETAILED DESCRIPTION

[0035] The following examples are intended to illustrate the present application but not to limit the scope of the present application. Modifications or replacements of the methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application. The reagents, kits and instruments used in the following examples can be obtained from the market, and the methods used in the examples are consistent with the commonly used methods unless otherwise specified.

[0036] (1) Core invention content of the present application The present application initiates a biomarker combination based on the combined detection of basophil percentage (BASO%) and total bilirubin (TBIL), which is used for objectively evaluating the animal model of diabetic kidney disease (DKD), the TCM syndrome attribute of "spleen deficiency and phlegm stasis", and for drug screening and efficacy evaluation.

[0037] (2) Technical solution points of the present application Evaluation method: by quantitatively detecting the BASO% in the peripheral blood and the TBIL level in the serum of the DKD animal model, when both levels are significantly reduced, it indicates that the model successfully simulates DKD and its "spleen deficiency and phlegm stasis" syndrome.

[0038] Model application: the animal model is preferably ZDF (fa / fa) rats, and this model can spontaneously present the characteristic changes of the above-mentioned biomarkers.

[0039] Application expansion: Expand the application of the biomarker combination to: screening drugs for preventing and / or treating DKD; evaluating the efficacy of drugs on DKD and the syndrome of "spleen deficiency with phlegm and blood stasis".

[0040] (3) Key problems solved by the present invention The present invention solves the technical problem of the lack of objective and quantitative TCM syndrome evaluation indexes in the existing research field of DKD models, and for the first time provides a modern chemical biology evaluation system that can simultaneously reflect the core pathogenesis of "spleen deficiency" (immune disorder - BASO%↓) and "phlegm and blood stasis" (metabolic disorder - TBIL↓) in DKD.

[0041] (4) Advantages and innovation of the present invention Innovation: For the first time, two indexes belonging to the immune system and the metabolic system are associated to form a new synergistic evaluation combination, overcoming the limitations of single-index evaluation.

[0042] Practicality: It provides a quantifiable and reproducible operation method, providing a key tool for the research on the mechanism of traditional Chinese medicine and the development of new drugs for DKD.

[0043] Prospectiveness: It organically combines TCM syndromes with modern medical indexes, promoting the modernization and internationalization of traditional Chinese medicine.

[0044] (5) Method scheme of the present invention: Animal model: 6-week-old SPF-grade ZDF(fa / fa) and ZDF(fa / +) rats were used as (controls), male, with body weight (200±20) g, certificate number SCXK(E)2022-0030, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental conditions maintained the temperature at (23±2) °C and the humidity at 50%±10%. The animals were raised under a 12 h / 12 h light-dark cycle. The animals freely drank pure water, and all rats freely ate K5008 feed (purchased from Keao Xieli (Tianjin) Feed Co., Ltd., batch number 2024072904). After 7 weeks, if the fasting blood glucose of ZDF rats was ≥11.1 mmol / L twice, the 24-hour urine volume increased, and the urine protein increased, it was determined that the early DKD rat model was successfully established, and the rats were continued to be fed for 14 weeks. The general state of the rats was observed daily, the body weight was weighed weekly and the food intake was recorded, the fasting blood glucose was detected every two weeks, and the urine albumin / creatinine ratio (UACR) was detected by a urine analyzer by collecting urine monthly.

[0045] Sample collection: At the end of the experiment, rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (5 mL / kg), and blood was collected from the abdominal aorta. A portion of the whole blood was anticoagulated with EDTA-K2 for routine blood tests; another portion of the whole blood was anticoagulated with sodium citrate, and the plasma was obtained after centrifugation for coagulation parameters such as PT (prothrombin time), APTT (activated partial thromboplastin time), TT (thrombin time), and FIB (fibrinogen). A third portion of the whole blood was anticoagulated with heparin for hemodynamic parameters such as blood viscosity and plasma viscosity; the remaining blood was centrifuged at 3500 rpm for 15 min using a Thermo centrifuge to obtain serum for biochemical tests. Longitudinal sections of kidney tissue from each rat were fixed in 10% formalin, embedded, sectioned, and stained with hematoxylin and eosin (HE), phyll-aldosterone (PAS), and Msasson staining. Each section was scanned and observed using a digital tissue scanner and optical microscope. HE staining of kidney tissue in each group was used to score damage (glomerular hypertrophy, mesangial cell proliferation with basement membrane thickening, basophilic tubules, protein casts, and interstitial inflammatory cell infiltration) (0 points: normal; 1 point: lesion area <1 / 4; 2 points: 1 / 4 < lesion area <1 / 2; 3 points: lesion area ≥1 / 2; 4 points: lesion area >1 / 2, with tuberous sclerosis). The percentage of glomerular mesangial proliferation area and the percentage of renal fibrosis area in each group were calculated using Fiji Image J software. In addition, 1 mm³ of renal cortex from 3 kidneys in each group was used to prepare electron microscopy samples in 2.5% glutaraldehyde osmium tetroxide fixative (to observe ultrastructural changes in glomeruli and renal tubules), and the thickness of the glomerular basement membrane and the fusion rate of podocyte foot processes were calculated using ImageJ software.

[0046] Detection methods: The number and percentage of basophils (BASO) were detected using a Sysmex XN-350 fully automated blood analyzer; the serum total bilirubin (TBIL-1) content was detected using the diazo method with a Sysmex BX3010 fully automated biochemical analyzer. The TBIL-1 diazo reagent kit was purchased from Dipo / Guangzhou Huaxin Technology Co., Ltd.

[0047] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0048] Example 1: Basic validation (syndrome attribute association) in the ZDF rat DKD model (1) Objective: To verify the correlation between changes in BASO% and TBIL levels and “spleen deficiency and phlegm stasis” syndrome in the spontaneously formed DKD model of ZDF rats.

[0049] (2) Experimental animals and grouping: Twenty SPF-grade male ZDF (Zucker Diabetic Fatty) rats, 7 weeks old, were used as the model group.

[0050] Ten SPF-grade male age-matched, non-diabetic ZL (Zucker Lean) mice were used as a normal control group.

[0051] (3) Modeling and feeding: All rats were fed a high-fat Purina #5008 diet with free access to food and water.

[0052] Record weight, food intake, and water intake weekly. Starting from 12 weeks of age, test random blood glucose weekly. When two consecutive random blood glucose levels are ≥16.7 mmol / L, the patient is considered to have developed diabetes.

[0053] Continue feeding until 26 weeks of age, at which point the model group is expected to show significant proteinuria and renal function impairment.

[0054] (4) Sample collection and testing: At 28 weeks of age, after fasting for 12 hours but with free access to water, all rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital.

[0055] Abdominal aortic blood sampling: A portion of the blood is injected into an EDTA-K2 anticoagulant tube, and a complete blood cell count is immediately performed using a fully automated blood analyzer (such as the Sysmex XN series), recording the percentage of basophils (BASO%).

[0056] Another portion of the blood was centrifuged and the serum was separated. The serum was then tested using a fully automated biochemical analyzer (such as Sysmex BX series) for total bilirubin (TBIL), total cholesterol (TCHO), triglycerides (TG), creatinine (Scr), blood urea nitrogen (BUN), etc.

[0057] 24-hour urine samples were collected to detect urinary albumin and urinary creatinine, and the urinary albumin-creatinine ratio (UACR) was calculated.

[0058] The rats were euthanized, and the bilateral kidneys and part of the liver were quickly removed, fixed in formalin, embedded in paraffin, sectioned, and stained with HE, PAS, and Masson. The kidneys were then observed and scored for histopathological examination.

[0059] (5) Results and Conclusions: ① General condition, metabolism, and characteristics of kidney damage like Figures 1-3 As shown, compared with the normal control group (ZL rats), the model group (ZDF rats) exhibited typical characteristics of obesity-related type 2 diabetes mellitus (T2DM): significant early weight gain ( P <0.01 indicates spleen deficiency with damp-heat and internal accumulation of fat; weight loss occurs in the later stages ( P <0.05%, indicating prolonged illness and depletion of kidney essence and vital energy. Simultaneously presenting with hyperglycemia, polyphagia, polydipsia, and polyuria (all... P<0.01). Blood lipid (total cholesterol TC, total triglyceride TG) and blood coagulation indicators (prothrombin time TT, fibrinogen FIB) were significantly increased ( P <0.01), and the renal function index (urea nitrogen BUN) was also significantly increased ( P <0.01), and 24h urine albumin (24h-ALB) and the reaction of kidney filtration function (urine albumin / urine creatinine ratio UACR) were significantly increased ( P <0.001). In addition, the kidney mass / body mass was significantly increased ( P <0.01), and under light microscope and electron microscope, glomerular hypertrophy, mesangial matrix proliferation, basement membrane thickening, foot process fusion and collagen fiber deposition were observed, which were consistent with the typical DKD pathological changes of "deficiency of collateral vessels and phlegm and blood stasis", suggesting that this ZDF rat well simulated the characteristics of "spleen deficiency and phlegm and blood stasis" DKD rat model.

[0060] ②Changes in blood routine indicators As Figure 4 shown, the main significant changes in blood routine of ZDF rat DKD model group were concentrated in basophilic granulocytes, and the proportion BASO% and BASO# were significantly reduced ( P <0.01), which reflected the model rat's spleen deficiency leading to imbalance of Ying and Wei, and immune cell differentiation disorder, which was consistent with the essence of phlegm and dampness of lipid metabolism disorder and chronic inflammation.

[0061] ③ Changes in biochemical indicators As Figure 5 shown in Table 1, ZDF rat DKD model showed metabolic hepatitis (serum glutathione pyruvate transaminase (AST), glutathione alanine transaminase (ALT) increased significantly ( P <0.01), albumin / globulin ratio decreased significantly (ALB / GLB) ( P <0.01)), cholestasis (alkaline phosphatase ALP↑ / total bilirubin TBIL↓) and overall blood lipid disorder. TBIL as the depletion of endogenous antioxidant substances is the key biological marker of "turbidity resistance", thus on the basis of "spleen deficiency and liver stagnation, phlegm and blood stasis", it completely simulates the pathogenesis evolution chain of "spleen deficiency-phlegm and blood stasis-turbidity" of diabetes.

[0062] Table 1 Changes in main pathological changes of ZDF-DKD model group compared with control group (X̄±s, n=10)

[0063] In summary, the ZDF rat model group is characterized by the "spleen and kidney qi deficiency with phlegm and blood stasis" syndrome of chronic progression: ① Metabolic dynamics: weight first increased (phlegm retention) and then decreased (kidney essence loss) is a typical sign of spleen deficiency and kidney; ② Blood characteristics: basophil depletion reflects spleen deficiency and weak immunity; ③ Coagulation disorder: FIB significantly increased as the material basis for phlegm and blood stasis; ④ Liver lesions: TG / TCHO significantly increased and TBIL-1 significantly decreased reflect the formation of phlegm from cream and the depletion of essence by turbidity toxin; ⑤ Kidney damage: kidney hypertrophy, increased urinary albumin, and UNCR rising suggest that the spleen and kidney are failing to capture, showing spleen deficiency and kidney deficiency, leading to the excretion of nutrient essence (protein) and the formation of proteinuria; and the mutual accumulation of phlegm, blood stasis, and obstruction of kidney collaterals can further promote the destruction of kidney unit structure and loss of function (Table 1), which is consistent with the typical DKD pathological changes of "yin deficiency and collateral damage" and "phlegm and blood stasis blocking collaterals". Its pathogenesis follows the progressive rule of "spleen deficiency → phlegm → stasis → essence depletion".

[0064] The experimental results show that in the successfully constructed'spleen deficiency with phlegm and blood stasis' DKD animal model, the percentage of basophils (BASO%) and total bilirubin (TBIL) levels are significantly lower than the normal control group, and this change is consistent with the classic pathological indicators of diabetic nephropathy and the macroscopic manifestations of traditional Chinese medicine syndromes. The combined decrease of BASO% and TBIL is highly related to the severity of DKD and the syndrome manifestations of "spleen deficiency and phlegm and blood stasis" (such as phlegm from cream, turbidity toxin depleting essence), which can be used for objective evaluation of the animal model of this syndrome.

[0065] Example 2 Traditional Chinese medicine intervention counter-evidence experiment ("test the syndrome with the prescription" to verify the specificity of the marker) (1) Purpose: By using traditional Chinese medicine compound for invigorating the spleen, resolving phlegm, and promoting blood circulation, it is verified that the changes in the levels of BASO% and TBIL can be reversed with the improvement of the syndrome, thereby counter-evidence the specificity of the syndrome biomarker.

[0066] (2) Experimental animals and grouping: SPF male ZDF rats, 30, 8 weeks old. After modeling (about 14 weeks old), they were randomly divided into 3 groups: Model group (n=10): given the same volume of pure water by gavage.

[0067] Traditional Chinese medicine treatment group (n=10): given traditional Chinese medicine MD (main components: Huangqi, Yansu (vinegar), Sanqi, Chishao, Danshen, Chuanxiong, etc.) by gavage, the dosage is 2g / kg / d (equivalent to the clinical equivalent dose).

[0068] Positive drug control group (n=10): given Enalapril (EN) 1mg / kg / d (equivalent to the clinical equivalent dose) solution by gavage.

[0069] SPF level male same-age ZL rats, 10, as normal control group, given the same volume of pure water.

[0070] (3) Experimental procedure: Each group was given 12 weeks of continuous medication (to 26 weeks of age).

[0071] After 12 weeks of intervention, all blood, urine and tissue samples were collected for endpoint analysis according to the method of Example 1.

[0072] (4) Results and conclusions: As shown in Figure 6 and Figure 7 , compared with the model group, the MD treatment group and the enalapril group: ① Renal function (UACR, 24h-uALB) and pathological damage were significantly improved. ② BASO% and TBIL levels will be significantly rebounded (but still lower than the normal control group).

[0073] Conclusion: Effective intervention of traditional Chinese medicine can specifically reverse the abnormal changes of BASO% and TBIL, and the changes are synchronized with the improvement of the disease. This strongly proves that the combination of this marker is specifically related to "spleen deficiency and phlegm stasis" syndrome, and can be used as a specific biomarker of this syndrome, and for evaluating the efficacy of drugs.

[0074] Example 3 Specificity comparison of different syndrome models (excluding universality) (1) Purpose: To verify that the combined changes of BASO% and TBIL are specific manifestations of DKD "spleen deficiency and phlegm stasis" syndrome, rather than a universal phenomenon of other kidney disease models, highlighting its specificity.

[0075] (2) Experimental animals and grouping: Group A (spleen deficiency and phlegm stasis syndrome DKD model, ZDF-M): ZDF rats (same as Example 1), n=10.

[0076] Group B (homologous non-diabetic ZL (Zucker Lean) rats, ZDF-C): ZDF control rats (same as Example 1), n=10.

[0077] Group C (kidney yin deficiency and blood deficiency DKD model, SD-M): 6-week-old male SD rats, after 1 week of adaptive feeding, given high-sugar high-fat feed for 4 weeks, intraperitoneal injection of 1% STZ (35 mg / kg, dissolved in ph=4.2-4.5 citric acid), n=10. One week after injection, measure fasting blood glucose once a week for three consecutive weeks. If the fasting blood glucose is ≥11.1 mmol / L for 3 times, the body weight of the rat after modeling is lower than that before modeling, and the urine volume is significantly increased, it is determined that the diabetic rat modeling is successful, and the high-sugar high-fat feed is continued to be given for 12 weeks until the end of the experiment.

[0078] Group D (SD normal control group, SD-C): Healthy SD rats, n=10.

[0079] (3) Experimental procedure: When each model was successfully established (confirmed by Scr, 24h urine protein quantification, etc.), blood samples of all animals were collected simultaneously with the method in Example 1 to detect BASO% and TBIL, and specific indicators of each model were also detected.

[0080] (4) Results and Conclusions: like Figure 8 As shown, compared with their respective homologous rat control groups, the body weight of the SD-M group was also significantly reduced ( P< 0.001), blood sugar rose significantly ( P< 0.001), prothrombin time (TT) increased significantly ( P< 0.05), BUN, UACR and renal tissue pathology score were also significantly increased ( P< 0.01). These data indicate that the model successfully induced the typical biochemical and pathological features of diabetic nephropathy. However, its lipid profile did not change significantly, suggesting that the model did not exhibit the key elements of the "kidney deficiency and phlegm stasis" syndrome. Furthermore, as... Figure 9 As shown, only the ZDF-M group showed a significant combined decrease in BASO% and TBIL; the SD-M group showed no significant change in either TBIL or BASO%.

[0081] Conclusion: The combined decrease of BASO% and TBIL is a relatively specific manifestation of DKD with "spleen deficiency and phlegm stasis syndrome," rather than a general pattern in all DKD cases. This further strengthens its persuasiveness as a specific biomarker for this syndrome.

[0082] Comparative Example 1: AUC Comparison between Combined Detection and Individual Detection Data from the normal control group (n=10) and the model group (n=10) in Example 1 were used to calculate the AUC of the ROC curves for BASO% alone, TBIL alone, and BASO%+TBIL combined detection (new variables were generated through logistic regression).

[0083] Table 2. Area under the AUC curve for BASO% alone, TBIL alone, and BASO%+TBIL combined detection.

[0084]

[0085] The results are as follows Figure 10and Table 2: BASO% alone AUC is 0.083 (95% CI: 0.00-0.241), TBIL alone AUC is 0.00 (95% CI: 0.00-1.00), and joint detection AUC is 1.00 (95% CI: 1.00-1.00). DeLong test shows that joint detection is significantly better than BASO% alone (p=0.08) and TBIL alone (p=0.02), indicating that joint detection is significantly better than single indicators.

[0086] Conclusion: This comparative example demonstrates the necessity of using BASO% in combination with TBIL, producing a "1+1>2" synergistic effect, with a discriminant performance far superior to that of a single indicator. This indicates that the combination is an indivisible whole technical solution.

[0087] Comparative Example 2: Comparison with other conventional or easily thought-of biomarker combinations The samples and methods of Example 1 were also used. Several groups of indicator combinations commonly used in the art or logically deduced were selected for comparison: Combination A (conventional coagulation indicators): prothrombin time (TT) + fibrinogen (FIB) Combination B (conventional lipid indicators): high-density lipoprotein (HDLC) + low-density lipoprotein (LDLC) Combination C (inflammatory indicators): neutrophil percentage (NEUT%) + lymphocyte percentage (LYMPH%) The ROC curve AUC values, sensitivity, and specificity of these comparative combinations were calculated respectively.

[0088] Table 3: AUC curve area of TT alone, FIB alone, and TT+FIB joint detection

[0089] Table 4: AUC curve area of HDLC alone, LDLC alone, and HDLC+LDLC joint detection

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[0091] Table 5: AUC curve area of NEUT alone, LYMPH alone, and NEUT+LYMPH joint detection

[0092] The results are shown in Figures 11-13 and Tables 3-5: Combination A (TT+FIB) may only change when kidney function is severely impaired, with poor sensitivity and cannot be used for early syndrome evaluation.

[0093] Combination B (HDLC+LDLC) will increase significantly, but the specificity is poor, because any hyperlipidemia will cause changes, can not be specifically directed to "spleen deficiency and phlegm stasis" syndrome.

[0094] Combination C (NEUT%+LYMPH%) reflects non-specific inflammation, and the correlation with DKD syndrome is not strong, and the effect is far inferior to BASO%.

[0095] The discriminant performance (AUC) of all these easily thought combinations will be significantly lower than the "BASO%+TBIL" combination.

[0096] Conclusion: This example proves that the present application is not routine or easily thought of in the art, and its effect is unexpected. It solves the problems of low sensitivity, poor specificity or weak correlation with TCM syndromes of other marker combinations, and embodies significant technical progress.

[0097] Although relevant staff may think of the following alternatives based on different research directions: (1) "easily thought of" combinations based on the same pathological mechanism: Combination A (inflammation-oriented): Neutrophil percentage (NEUT%) + Lymphocyte percentage (LYMPH%). Reason: inflammation is the core link of DKD and "phlegm stasis" syndrome, and NEUT% and LYMPH% are more conventional inflammation indicators.

[0098] Combination B (lipid-oriented): Total cholesterol (TCHO) + Triglyceride (TG). Reason: lipid metabolism disorder is the material basis of "phlegm turbidity", and these two are the most classic blood lipid indicators.

[0099] Combination C (liver function-oriented): Total bilirubin (TBIL) + Alanine aminotransferase (ALT). Reason: TBIL is one of the indicators, and ALT is a more sensitive and conventional liver cell damage indicator.

[0100] (2) combinations based on other TCM syndrome theories: Combination D: Platelet count (PLT) + Fibrinogen (FIB). Reason: This is a more direct blood coagulation indicator for "blood stasis" pathogenesis.

[0101] (3) use more complex statistical models: Combination E: Put BASO% and TBIL together with other multiple indicators (such as age, TCHO, FIB, etc.) into a machine learning model (such as random forest, support vector machine) for modeling. But even if a complex model is used, its effect may not be better than this simple linear combination we carefully discovered.

[0102] But the above comparative examples fully show that, although there are other possible biomarker combinations, their diagnostic efficiency is significantly lower than the present application. The present application first discovered that the combination of BASO and TBIL has a high specific correlation with the "spleen deficiency and phlegm stasis" syndrome of DKD, which is not obvious and achieves unexpected technical effects.

[0103] In the present application, BASO + TBIL is not a simple superposition of two indicators, but a deep revelation of the modern biological connotation of the "spleen deficiency and phlegm stasis" syndrome. Among them: BASO: related to "spleen deficiency". Traditional Chinese medicine believes that "spleen is the source of blood and qi", "spleen is responsible for transportation and transformation", and is closely related to modern immune and metabolic functions. The depletion of BASO accurately reflects the state of "spleen deficiency and immune disorder".

[0104] TBIL: related to "phlegm and stasis" and "consumption of essence". As an endogenous antioxidant, its depletion means "internal turbidity and consumption of essence", which is highly consistent with the oxidative stress mechanism of DKD organ damage in the late stage.

[0105] Synergistic effect: the combination of the two, one pointing to "spleen deficiency" (the subject), and one pointing to "turbidity and essence consumption" (the marker), completely covers the core pathogenesis of the "spleen deficiency and phlegm stasis" syndrome, which is unmatched by any other combination based on surface phenomena (such as inflammation, blood lipids).

[0106] The above-described embodiments are only descriptions of preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A biomarker combination, characterized in that, The biomarker combination comprises basophil percentage (BASO%) and total bilirubin (TBIL).

2. Use of a product for detecting the biomarker combination in claim 1 in screening drugs for preventing and treating diabetic kidney disease (DKD).

3. Use of a product for detecting the biomarker combination in claim 1 in evaluating the efficacy of drugs for preventing and treating DKD.

4. Use of a product for detecting the biomarker combination in claim 1 in studying the "spleen deficiency and phlegm stasis" syndrome of DKD.

5. Use according to any one of claims 2 to 4, characterized in that, The product for detecting the biomarker combination in claim 1, wherein the product form comprises a detection instrument and / or a detection kit.

6. Use according to claim 5, characterized in that, The screening, evaluation and / or study method comprises using an animal model of DKD.

7. Use according to claim 6, characterized in that, The animal model comprises a ZDF (fa / fa) rat model.

8. Use according to claim 7, characterized in that, The screening, evaluation and / or study method is achieved by quantitatively detecting the BASO% and TBIL levels in the animal model of DKD.

9. Use according to claim 8, characterized in that, The BASO% is the BASO% in peripheral blood.

10. Use according to claim 9, characterized in that, The TBIL level is the TBIL level in serum.

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