Spleen-invigorating, dampness-eliminating and turbidity-eliminating formula and application thereof

Abstract

The invention provides a spleen-invigorating, dampness-eliminating and turbidity-discharging formula and application thereof, and belongs to the field of traditional Chinese medicines. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 20-60 parts of rhizoma smilacis glabrae, 5-20 parts of pyrrosia lingua, 5-25 parts of yam rhizome, 10-50 parts of lysimachia christinae hance, 5-15 parts of achyranthes bidentata, 10-30 parts of pearl barley, 5-15 parts of endive, 1-10 parts of rhizoma atractylodis, 1-10 parts of golden cypress and 5-25 parts of The spleen-invigorating, dampness-eliminating and turbidity-eliminating prescription can reduce the activity of xanthine oxidase and adenosine deaminase in liver tissues, inhibit the generation of uric acid, regulate the expression of an intestinal and kidney urate transporter, reduce uric acid reabsorption and increase uric acid excretion, plays a role in protecting kidney organs while reducing uric acid, and does not cause damage to the liver structure. The spleen-invigorating, dampness-eliminating and turbidity-eliminating formula has a good application prospect in preparation of medicines for preventing and / or treating hyperuricemia.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a spleen-strengthening, dampness-removing and turbidity-eliminating prescription and an application thereof. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease characterized by increased uric acid production and / or decreased excretion, leading to elevated serum uric acid levels. HUA is diagnosed when fasting serum uric acid levels >420 μmol / L in men and postmenopausal women, or >360 μmol / L in non-menopausal women, measured on two separate days while on a normal purine diet. HUA not only underlies the pathogenesis of gout but is also an independent risk factor for diabetes, chronic kidney disease, and cardiovascular disease. Therefore, early identification and prevention of HUA are crucial.

[0003] Modern medicine primarily uses two types of drug interventions: xanthine oxidase inhibitors (such as allopurinol and febuxostat) that inhibit uric acid production, and urate transporter inhibitors (such as benzbromarone) that promote uric acid excretion. While these drugs can effectively lower blood uric acid levels, they also have limitations that should not be ignored: long-term use may cause adverse reactions such as abnormal liver function, severe skin reactions, and gastrointestinal discomfort. Some drugs (such as febuxostat) have controversial cardiovascular risks and are prone to relapse after discontinuation. Furthermore, they primarily focus on regulating blood uric acid levels and have relatively limited effects on improving accompanying metabolic disorders and inflammatory states.

[0004] Traditional Chinese Medicine (TCM) has accumulated extensive theoretical and practical experience in the prevention and treatment of hyperuricemia, demonstrating its unique advantages in holistic regulation and addressing both the symptoms and the root causes. Based on core pathogenesis principles such as turbidity and stasis, dampness and heat, and spleen and kidney deficiency, TCM treatment emphasizes syndrome differentiation and treatment, utilizing a comprehensive approach involving clearing heat and dampness, strengthening the spleen and kidneys, activating blood circulation and removing stasis, and unblocking the meridians and dispersing turbidity. Studies have linked the onset of HUA to factors such as pre-existing spleen deficiency and a preference for fatty, sweet, and greasy foods. Spleen deficiency leads to poor fluid distribution, resulting in fluid accumulation and dampness. This dampness and turbidity, retained in the body, exacerbates spleen deficiency, creating a vicious cycle of spleen deficiency and internal dampness, and is a key cause of elevated serum uric acid. While TCM holds great promise in the prevention and treatment of HUA, further research is needed in several key areas to achieve more precise and effective application. Therefore, it is necessary to develop a TCM compound formula that is more effective in addressing the internal dampness and turbidity caused by spleen deficiency and dysfunction in patients with hyperuricemia, thereby providing a new treatment option for these patients. Summary of the Invention

[0005] The present invention aims to provide a spleen-strengthening, dampness-removing and turbidity-eliminating prescription and its application.

[0006] The invention provides a spleen-strengthening, dampness-removing and turbidity-eliminating prescription for treating hyperuricemia of internal accumulation of dampness and turbidity due to spleen deficiency. The prescription is prepared from the following raw materials in parts by weight: 20-60 parts of Smilax glabra, 5-20 parts of Pyrola, 5-25 parts of Radix Dipterocarpa, 10-50 parts of Herba Lysimachiae, 5-15 parts of Radix Achyranthis Bidentatae, 10-30 parts of Coix seeds, 5-15 parts of Chicory, 1-10 parts of Atractylodes, 1-10 parts of Cortex Phellodendri and 5-25 parts of Radix Puerariae.

[0007] Furthermore, it is prepared from the following raw materials in parts by weight: 30-41 parts of Smilax glabra, 11-13 parts of Pyrola, 14-16 parts of Radix Dipterocarpa, 29-31 parts of Herba Lysimachiae, 9-11 parts of Rhizoma Cyperi, 19-21 parts of Coix seeds, 9-11 parts of Cichorium, 5-7 parts of Atractylodes, 5-7 parts of Phellodendron, and 14-16 parts of Radix Puerariae.

[0008] Furthermore, it is prepared from the following raw materials in parts by weight: 40 parts of Smilax glabra, 12 parts of Pyrola, 15 parts of Radix Dichroae, 30 parts of Herba Lysimachiae, 10 parts of Rhizoma Cyperi, 20 parts of Coix seeds, 10 parts of Chicory, 6 parts of Atractylodes, 6 parts of Phellodendron, and 15 parts of Radix Puerariae.

[0009] Furthermore, it is a pharmaceutical preparation prepared with the powder of the raw material drug, the water extract of the raw material drug or the organic solvent extract of the raw material drug as the active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients.

[0010] The pharmaceutical preparation is an oral preparation.

[0011] Furthermore, the oral preparation is a decoction, oral liquid, granules, capsules, powders, pills or tablets.

[0012] Furthermore, the content of the raw material drug in the unit preparation of the pharmaceutical preparation is 87.5g to 91g.

[0013] The conversion method for the content of the Chinese herbal compound in the unit dose of the drug of the present invention is as follows: According to the preparation method of Example 1, the dosage of the Chinese herbal compound for mice is 11.36g / kg to 11.88g / kg, calculated based on the raw material. According to "Pharmacological Test Methodology" edited by Xu Shuyun, the dosage for mice = 9.1 × the dosage for humans (70kg body weight). Therefore, the dosage for humans (70kg body weight) converted by the Chinese herbal compound is 1.25g / kg to 1.3g / kg. Therefore, the content of the Chinese herbal compound in the unit dose of the drug is 1.25g / kg to 1.3g / kg × 70kg = 87.5g to 91g.

[0014] The unit dosage form of the present invention is calculated based on an average adult weight of 70 kg.

[0015] The present invention also provides a method for preparing the above-mentioned spleen-strengthening, dampness-removing and turbidity-eliminating prescription, which comprises the following steps: taking the raw material drug, directly powdering it, or adding water or an organic solvent for extraction, and then adding pharmaceutically acceptable excipients or auxiliary ingredients to obtain the spleen-strengthening, dampness-removing and turbidity-eliminating prescription.

[0016] The present invention also provides use of the spleen-strengthening, dampness-removing and turbidity-eliminating prescription in preparing a medicament for preventing and / or treating hyperuricemia.

[0017] Furthermore, the drug is a drug that reduces uric acid production and increases uric acid excretion.

[0018] Furthermore, the medicine is a medicine for treating hyperuricemia of spleen deficiency and internal accumulation of dampness and turbidity.

[0019] The present invention has been engaged in clinical and basic research on hyperuricemia (HUA) for a long time. Based on the characteristics of HUA, the author proposed a pathogenesis view that spleen deficiency leads to internal accumulation of dampness and turbidity, and based on this, proposed a clinical treatment principle of "strengthening the spleen to eliminate dampness and turbidity". It is believed that the pathogenesis of HUA is based on spleen deficiency, and the pathological products include dampness and turbidity, damp heat, phlegm dampness, phlegm heat, and heat toxins. These are related to factors such as congenital deficiency, spleen deficiency, and irregular diet of patients, which lead to spleen dysfunction, water loss, loss of body fluid distribution, and dampness and turbidity. Dampness and turbidity retained in the body exacerbate spleen deficiency and dysfunction, creating a vicious cycle of internalized dampness and turbidity, resulting in symptoms such as poor appetite, abdominal distension, heaviness in the head and body, and sticky stools. Clinically, this manifests as elevated blood uric acid. Endogenous dampness and turbidity, damaged by dampness, and persistently retained are the long-term pathological state of HUA. Once established, dampness and turbidity can obstruct the middle jiao and accumulate in the lower jiao, exacerbating the spleen's dysfunctional transport and transformation function, impacting the body's ability to separate clear and turbid fluids, raising the clear and lowering the turbid, leading to exacerbated HUA and the development of complications such as hyperuricemia and nephropathy. In the recipes of this invention, "parts" represent parts by weight, with 1 part being 1g.

[0020] The formula of the present invention is composed of: 40g of Smilax glabra, 12g of Pyrola, 15g of Radix Dichroae, 30g of Herba Lysimachiae, 10g of Rhizoma Cyathulae, 20g of Coix seeds (i.e., Coix lachryma-jobi), 10g of Chicory, 6g of Atractylodes lancea, 6g of Phellodendron chinense, and 15g of Radix Puerariae.

[0021] The Chinese medicinal compound of the present invention has the effects of strengthening the spleen, removing dampness and clearing turbidity; and is mainly used to treat hyperuricemia with spleen deficiency and internal accumulation of dampness and turbidity as the main pathogenesis.

[0022] Solution of the present invention:

[0023] Based on the principle of "prescriptions are derived from methods, and methods are established based on symptoms," and based on the internal pathogenesis of hyperuricemia caused by spleen deficiency, dampness, and turbidity, clinical treatment focuses on strengthening the spleen, removing dampness, and clearing turbidity. Atractylodes macrocephala, a bitter, pungent, and warm herb, enters the spleen and stomach meridians, excels at drying dampness and dispelling wind, and can also strengthen the spleen. Coix seed, a sweet, mild, and slightly cold herb, enters the spleen, stomach, and lung meridians, promoting diuresis and dampness removal, dispelling numbness, and also strengthening the spleen. These two herbs are the main herbs. They are supplemented by the bitter and cold Phellodendron chinense, which clears heat and dries dampness, and the sweet and mild Smilax glabra, which detoxifies, removes dampness, and promotes joint circulation. Modern research indicates that these herbs are essential for the clinical treatment of gout. The bitter and cold Radix Dichroae, known for its ability to clear turbidity and eliminate dampness, also serve as the auxiliary herbs. The bitter and cold chicory is used as a diuretic and detumescent, strengthening the spleen and stomach for digestion. The bitter and cold herb Jinqiancao dispels dampness, promotes diuresis, and relieves stranguria. The bitter and cold herb Ligusticum wallichii is also used as a diuretic and cooling blood. The pungent, sweet, and cool Pueraria root raises the clear and lowers the turbid, promoting fluid production and quenching thirst, and unblocking the meridians. Together, these herbs eliminate dampness, dissipate dampness, promote diuresis, and relieve stranguria without damaging yin essence. Finally, the herb Achyranthes bidentata is added directly to the affected area, guiding the herbs downward and taking advantage of the situation to further promote the elimination of dampness and turbidity. The entire formula strengthens the spleen qi and restores the spleen's function of transporting and transforming dampness to address the root cause. It dispels dampness and turbidity, promotes diuresis, and relieves stranguria, guiding dampness through urination to eliminate it. It addresses both the symptoms and the root cause, eliminating pathogenic factors without harming the vital essence, and strengthening the spleen and supporting the vital essence without clinging to it.

[0024] The present invention has achieved the following beneficial effects:

[0025] (1) The present invention proposes an understanding of the pathogenesis of internal dampness and turbidity caused by spleen deficiency and dysfunction and a treatment principle of strengthening the spleen, eliminating dampness and eliminating turbidity, and creates a spleen-strengthening, dampness-eliminating and turbidity-eliminating prescription with the effects of strengthening the spleen, eliminating dampness and eliminating turbidity. The present invention screens and optimizes the formula of the spleen-strengthening, dampness-eliminating and turbidity-eliminating prescription to obtain the formula shown in Example 1, which contains 40g of Smilax glabra, 12g of Pyrola, 15g of Radix Dipterocarpa, 30g of Lysimachia chinensis, 10g of Radix Achyranthis Bidentatae, 20g of Coix seeds, 10g of Cichorium indicum, 6g of Atractylodes macrocephala, 6g of Phellodendron chinense and 15g of Radix Puerariae.

[0026] (2) The present invention establishes a hyperuricemia rat model with increased uric acid production and decreased uric acid excretion, and studies the effects of the Jianpi Huashi Xiezhuo recipe on the blood uric acid level, uric acid production, and uric acid excretion-related indicators of the hyperuricemia rat model. This further explains the mechanism of action of the recipe in treating hyperuricemia and provides an experimental basis for its promotion and application.

[0027] (3) The spleen-strengthening, dampness-removing and turbidity-eliminating prescription of the present invention can reduce the activities of xanthine oxidase (XOD) and adenosine deaminase (ADA) in liver tissue, inhibit uric acid production, regulate the expression of intestinal and renal urate transporters, reduce uric acid reabsorption, and increase uric acid excretion. While lowering uric acid, it also protects the kidneys without causing damage to the liver structure. It is safe and can be taken for a long time.

[0028] (4) The Chinese herbal compound of the present invention has good application prospects in the preparation of drugs for preventing and / or treating hyperuricemia.

[0029] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0030] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The liver tissue changes of rats in each group (200×, yellow arrows: fatty vacuoles; green arrows: hepatocyte edema).

[0032] Figure 2 The diagram shows the changes in renal tissues of rats in each group (200×, yellow arrows: dilated renal tubules; green arrows: ruptured renal tubular epithelial cells).

[0033] Figure 3 The liver tissue changes of rats in each group (200×).

[0034] Figure 4 The diagram shows the changes in kidney tissue of each rat (200×, yellow arrows: renal tubular epithelial cell damage; green arrows: glomerular epithelial cell vacuoles).

[0035] Figure 5 These are the electrophoresis images of ABCG2 and GLUT9 proteins in the small intestine of rats in each group (A. blank group; B. model group; C. Chinese medicine group; D. benzbromarone group).

[0036] Figure 6 The electrophoresis images of URAT1 and OAT1 proteins in the kidney tissues of rats in each group (A. blank group; B. model group; C. Chinese medicine group; D. benzbromarone group).

[0037] Figure 7 The expression of ABCG2 protein in kidney tissue of rats in each group (200×). DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0039] The "room temperature" referred to in the present invention is 25±10°C, and the time of "overnight" is 24±5 hours.

[0040] Example 1: Preparation of the Chinese herbal compound for invigorating the spleen, eliminating dampness and eliminating turbidity

[0041] The ingredients of the prescription for invigorating the spleen, eliminating dampness and purging turbidity are: 40g of wild yam, 12g of stonecrop, 15g of radix polygoni multiflori, 30g of herba schizonepetae, 10g of radix achyranthis Bidentatae, 20g of coix seed, 10g of chicory, 6g of atractylodes, 6g of phellodendron, and 15g of kudzu root.

[0042] Preparation method: Weigh the above raw materials according to the ratio, add 8 to 10 times the amount of water and boil twice, each time for 20 to 30 minutes, filter, and combine the filtrate to obtain a 2 g / mL solution of the spleen-strengthening, dampness-removing and turbidity-removing prescription.

[0043] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0044] Experimental Example 1: Effect of the Chinese herbal compound of the present invention - Jianpi Huashi Xiezhuo recipe on uric acid production

[0045] 1. Experimental methods

[0046] 1.1 Animal grouping, modeling, and drug administration

[0047] Forty SPF male SD rats were taken and adaptively fed for 1 week, and randomly divided into a blank group (10 rats) and a modeling group (30 rats). The blank group was fed a normal diet every day and gavaged with an equal dose of normal saline. A hyperuricemia (HUA) rat model was constructed by gavage with 10% fructose combined with 600 mg / kg potassium oxonate once a day for 3 weeks. After successful modeling, the rats were randomly divided into: a model group of 10 rats, a Chinese medicine group of 10 rats, and an allopurinol group (a first-line drug for reducing uric acid production, a positive control group) of 10 rats, and the modeling was maintained; based on the conversion of human and animal body surface areas, the corresponding drugs were gavaged for 3 weeks: the Chinese medicine group was gavaged with 11.36 g / kg of the Jianpi Huashi Xiezhuo prescription liquid (prepared in Example 1), the allopurinol group was gavaged with 13.85 mg / kg of allopurinol suspension, and the blank group and the model group were gavaged with an equal dose of normal saline.

[0048] 1.2 Sampling and sample collection

[0049] After 3 weeks of intervention, rats in each group were fasted but not watered, and tissues were collected and packaged the next morning: ① Part of the rat liver tissue was quickly removed on an ice surface, placed in a 50 mL centrifuge tube, quickly placed in dry ice for storage, and promptly transferred to a -80°C freezer for storage; ② Part of the rat liver tissue, approximately 1.5 cm × 1.5 cm × 0.3 cm in size, was cut and placed in 4-10 times the volume of 4% paraformaldehyde for fixation for 24-48 hours. The paraformaldehyde solution was then discarded, and the rats were rinsed with running water for 30 minutes to one hour and then stored in 75% alcohol. The alcohol was replaced once a week.

[0050] 1.3 Observation indicators

[0051] 1.3.1 Enzyme colorimetric method for detecting blood uric acid content

[0052] At the end of the first, second, and third week of the intervention, blood uric acid levels in each group were measured by sampling blood from the orbital venous plexus. The blood uric acid levels in each group were measured using an automatic microplate reader and an enzymatic colorimetric method according to the kit instructions.

[0053] 1.3.2 Enzyme colorimetric assay for detection of liver XOD and ADA activity

[0054] Quickly harvest 0.1 g of liver tissue on ice and add it to 0.9 g of pre-chilled saline. Add grinding beads and homogenize in an ice-water bath until no unground tissue fragments are visible. Centrifuge at 12,000 rpm for 20 minutes at 4°C. Remove the supernatant and transfer it to a pre-chilled 1.5 mL centrifuge tube. Strictly follow the kit instructions.

[0055] 1.3.3 Observation of liver and kidney tissue pathological morphology by HE staining

[0056] Hematoxylin and eosin staining was used to observe fixed, dehydrated, transparent, wax-impregnated, and embedded rat liver and kidney tissue sections. The sections were dewaxed in xylene, hydrated with graded alcohols, and stained with hematoxylin for 5 minutes. After washing, the sections were differentiated with 1% hydrochloric acid and alcohol for 3-5 seconds, and then stained with eosin for 2 minutes. After dehydration, transparentization, and mounting, the pathomorphological changes of the liver and kidney tissues were observed under a microscope.

[0057] 2. Experimental results

[0058] 2.1 Comparison of blood uric acid levels among groups

[0059] On day 21 of intervention, compared with the blank group, the model group had an elevated serum uric acid level (P < 0.05). There was no statistically significant difference in serum uric acid between the TCM group and the blank group (P > 0.05). Compared with the model group, the TCM group and the allopurinol group showed statistically significant differences (P < 0.05). The results (Table 1) showed that the serum uric acid level in HUA model rats was elevated, and the Jianpi Huashi Xiezhuo recipe was able to reduce the serum uric acid level in HUA model rats.

[0060] Table 1 Effects of Jianpi Huashi Xiezhuo Decoction on blood uric acid levels in rats (n=10, μmol / L)

[0061]

[0062] Note: Compared with the blank group, 1) P < 0.05; compared with the model group, 2) P < 0.05.

[0063] 2.2 Comparison of liver XOD and ADA activities in each group

[0064] The results (Table 2) showed that compared with the blank group, the liver XOD activity of rats in the model group and allopurinol group increased, with statistical significance (P < 0.05); compared with the model group, the liver XOD activity of rats in the TCM group and allopurinol group decreased, with statistical significance (P < 0.05); compared with the TCM group, the liver XOD activity of rats in the allopurinol group increased, with statistical significance (P < 0.05). Compared with the blank group, the liver ADA activity of rats in the model group increased, with statistical significance (P < 0.05). Compared with the model group, the ADA activity of rats in the TCM group and allopurinol group decreased, with statistical significance (P < 0.05).

[0065] Table 2 Effects of Jianpi Huashi Xiezhuo Decoction on XOD and ADA activities in rat liver (n=10, (U / gprot))

[0066]

[0067]

[0068] Note: Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05; compared with the Chinese medicine group 3)

[0069] P<0.05.

[0070] 2.3 Comparison of liver tissues among groups

[0071] Compared with the blank group, hepatocyte edema was observed in the model group, and round, tense vacuoles of varying sizes were observed in the cytoplasm. Compared with the model group, hepatocyte edema and fat-like vacuoles were reduced in the TCM group, and hepatocyte edema was observed in the allopurinol group ( Figure 1 ).

[0072] 2.4 Changes in kidney tissue of rats in each group

[0073] Compared with the blank group, the model group rats showed less tubular dilatation and tubular epithelial cell rupture; compared with the model group, the Chinese medicine group rats showed improved tubular dilatation and no obvious tubular epithelial cell rupture, while the allopurinol group showed tubular dilatation and tubular epithelial cell rupture ( Figure 2 ).

[0074] Experimental Example 2: Effect of the Chinese herbal compound of the present invention - Jianpi Huashi Xiezhuo recipe on uric acid excretion

[0075] 1. Experimental methods

[0076] 1.1 Animal grouping, modeling, and drug administration

[0077] 40 SPF male SD rats were taken and adapted to feeding for 1 week. They were randomly divided into a blank group (10 rats) and a modeling group (30 rats). The blank group rats were gavaged with an equal amount of normal saline daily. The rat modeling method was: 200mg / kg ethambutol suspension + 600mg / kg potassium oxonate suspension gavage, to construct a hyperuricemia (HUA) rat model. The gavage dose was determined based on body weight once a day, and the modeling was carried out for 3 weeks. The rats successfully modeled were randomly divided into: 10 model groups, 10 Chinese medicine groups, and 10 benzbromarone groups (a first-line drug for promoting uric acid excretion, a positive control group); and the modeling was maintained. The corresponding drugs were gavaged for 3 weeks: the Chinese medicine group was gavaged with 11.88g / kg of the Jianpi Huashi Xiezhuo prescription liquid (prepared in Example 1), the benzbromarone group was gavaged with 7.24mg / kg of benzbromarone suspension, and the blank group and the model group were gavaged with equal volumes of normal saline.

[0078] 1.2 Sampling and sample collection

[0079] After the third week of intervention, serum was collected using 3% isoflurane inhalation anesthesia, followed by blood sampling from the orbital venous plexus. Serum was separated promptly, and blood biochemical analysis was completed within 4 hours. The following morning, tissue samples were collected: partial kidney and small intestinal tissues were quickly removed on dry ice, placed in 50 mL centrifuge tubes, and promptly transferred to a -80°C freezer for storage. Portions of liver and kidney tissues, approximately 1.5 cm × 1.5 cm × 0.3 cm in size, were excised and fixed in 10 volumes of 4% paraformaldehyde for 24 hours, rinsed with running water for 30 minutes, and preserved in 75% alcohol.

[0080] 1.3 Observation indicators

[0081] 1.3.1 Enzyme colorimetric method for detecting blood uric acid content

[0082] The blood uric acid content of rats in each group was detected by enzyme colorimetry using a fully automatic microplate reader. The specific operations were carried out according to the instructions of the kit.

[0083] 1.3.2 Observation of morphological changes in rat liver and kidney tissues by HE staining

[0084] After routine tissue fixation, dehydration, embedding, and sectioning, HE staining, dewaxing, hematoxylin staining, hydrochloric acid alcohol differentiation, eosin staining, and neutral plastic sealing were performed according to the kit instructions; tissue morphological changes were observed under an upright microscope.

[0085] 1.3.3 Real-time fluorescence quantitative PCR (qPCR) detection of mRNA expression of ATP-binding cassette subfamily G member 2 (ABCG2), glucose transporter 9 (GLUT9) in small intestine, urate transporter 1 (URAT1), and organic anion transporter 1 (OAT1) in kidney

[0086] Total RNA was extracted according to the instructions of the RNA extraction kit. RNA samples of each group were thawed on ice, shaken and mixed, and heated at 4°C at 12000 r / min. -1 After centrifugation for 5 minutes (centrifuge radius 6.5 cm), the tubes were added to each set of centrifuge tubes, the volume was adjusted to 18 μL with ddH2O, 6 μL of 4× gDNA wiper mix was added, the mixture was placed in a real-time PCR instrument, and the tubes were incubated at 42°C for 2 minutes. The tubes were removed and 6 μL of 5× RT Master Mix II was added to each tube, the mixture was mixed, and the tubes were placed in a gradient PCR instrument with the following settings: 50°C for 15 minutes, 85°C for 2 minutes for RNA reverse transcription. The amplification reaction system was prepared according to the reagent instructions and placed in a qPCR instrument with the following reaction conditions: 94°C for 5 minutes, 95°C for 10 seconds, 60°C for 30 seconds, 72°C for 20 seconds, and 72°C for 10 minutes, for 40 cycles. The primer sequences for the qPCR experiment are shown in Table 3.

[0087] Table 3 Primer sequences for qPCR experiments

[0088]

[0089] 1.3.4 Western blot analysis of the expression of ABCG2, GLUT9, URAT1, and OAT1 proteins in the small intestine of rats

[0090] Rat small intestine and kidney tissues were collected and total protein was extracted using RIPA lysis buffer and protease inhibitors. Protein concentration was determined using a total protein quantification kit. 5× SDS-PAGE loading buffer was added at a ratio of 1:4, mixed, and heated in a metal bath for 20 minutes to denature the protein. After SDS-PAGE gel electrophoresis and transfer to a PVDF membrane, the membrane was blocked with rapid blocking buffer for 5 minutes at room temperature. Primary antibodies (URAT1 1:1000, OAT1 1:1000, ABCG2 1:1000, GLUT9 1:1000, GADPH 1:5000) were added and incubated at room temperature for 4 hours. Secondary antibodies (HRP goat anti-rabbit IgG 1:5000, HRP goat anti-mouse IgG 1:5000) were added and incubated at room temperature for 90 minutes. The PVDF membrane was developed with ECL developer, and the grayscale value of each band was analyzed using Image J and other software.

[0091] 1.3.5 Immunofluorescence detection of ABCG2 protein expression in rat kidney

[0092] Routine tissue embedding and sectioning were performed. After dewaxing, the sections were heated in EDTA antigen retrieval solution for 20 minutes. An endogenous peroxidase blocker was added and incubated at room temperature for 10 minutes. A sufficient amount of 5% BSA blocking solution was added and incubated for 30 minutes. After shaking off, the primary antibody working solution (ABCG2 1:100) was added and incubated at 4°C overnight. The secondary antibody working solution (FITC-labeled goat anti-rabbit IgG, 1:50) was added and incubated at room temperature in the dark for 1 hour. DAPI staining solution was added and incubated at room temperature in the dark for 10 minutes. An autofluorescence quencher was added and incubated at room temperature in the dark for 5 minutes. After rinsing, the sections were mounted with anti-fluorescence quenching mounting medium. Pictures were taken using an inverted fluorescence microscope, and fluorescence intensity was analyzed with Image J software.

[0093] 1.4 Statistical methods

[0094] Experimental data were collected and organized in Excel. SPSS 24.0 software was used to analyze the data. When measurement data conformed to a normal distribution, they were expressed as (x ± s). Within-group comparisons were performed with a paired t-test, and between-group comparisons were performed with a one-way ANOVA. When measurement data did not conform to a normal distribution, the median and interquartile range (IQR) were used, and between-group comparisons were performed with a rank-sum test. The test level was set at 0.05, and differences were considered statistically significant when P < 0.05.

[0095] 2. Experimental results

[0096] 2.1 Comparison of blood uric acid levels in rats of each group after the third week of intervention

[0097] On day 21 of intervention, compared with the blank group, the model group had an elevated serum uric acid level (P < 0.05). There was no statistically significant difference in serum uric acid between the TCM group and the blank group (P > 0.05). Compared with the model group, the TCM group showed a statistically significant difference (P < 0.05), and the Jianpi Huashi Xiezhuo formula group was superior to the benzbromarone group (P < 0.05). The results (Table 4) showed that the serum uric acid level in HUA model rats was elevated, and the Jianpi Huashi Xiezhuo formula could reduce the serum uric acid level in HUA model rats.

[0098] Table 4 Comparison of blood uric acid levels in rats in each group

[0099]

[0100] Note: Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05; compared with the Chinese medicine group 3)

[0101] P<0.05.

[0102] 2.2 Changes in liver tissue of rats in each group

[0103] Compared with the blank group, no obvious hepatocyte swelling, eosinophilia or necrosis was observed in the liver tissues of rats in the model group, Jianpi Huashi Xiezhuo group and benzbromarone group under HE staining. The results showed that Jianpi Huashi Xiezhuo did not damage the liver structure of HUA model rats ( Figure 3 ).

[0104] 2.3 Changes in kidney tissue of rats in each group

[0105] Compared with the blank group, the model group showed vacuolar changes in glomerular epithelial cells, rupture and necrosis of renal tubular epithelial cells, and more serious damage. After drug intervention, the renal pathology of rats showed that the pathological damage of glomeruli and renal tubules was alleviated compared with the model group. The results showed that HUA model rats had renal structural damage, and Jianpi Huashi Xiezhuo recipe could alleviate the renal structural damage of HUA model rats ( Figure 4 ).

[0106] 2.4 Comparison of ABCG2 and GLUT9 mRNA expression in small intestine tissue of rats in each group

[0107] The results (Table 5) showed that compared with the blank group, the mRNA expression level of ABCG2 in the model group was decreased (P < 0.05), and the mRNA expression level of GLUT9 was increased (P < 0.05); compared with the model group, the mRNA expression level of ABCG2 in the TCM group was increased (P < 0.05), and the mRNA expression level of GLUT9 was decreased (P < 0.05); the mRNA expression level of ABCG2 in the benzbromarone group was increased (P < 0.05), and the mRNA expression level of GLUT9 was decreased (P < 0.05); compared with the TCM group, the mRNA expression level of GLUT9 in the benzbromarone group was increased (P < 0.05).

[0108] Table 5 Comparison of ABCG2 and GLUT9 mRNA expression levels in small intestine tissue of rats in each group (n=3)

[0109]

[0110] Note: Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05; compared with the Chinese medicine group 3)

[0111] P<0.05.

[0112] 2.5 Comparison of OAT1 and URAT1 mRNA expression levels in kidney tissue of rats in each group

[0113] The results (Table 6) showed that compared with the blank group, the mRNA expression of OAT1 in the model group was decreased (P < 0.05), and the mRNA expression of URAT1 was increased (P < 0.05); compared with the model group, the mRNA expression of URAT1 in the Chinese medicine group was decreased (P < 0.05), and the mRNA expression of OAT1 was increased (P < 0.05); compared with the model group, the mRNA expression of URAT1 in the benzbromarone group was decreased (P < 0.05), and the mRNA expression of OAT1 was increased (P < 0.05).

[0114] Table 6 Comparison of OAT1 and URAT1 mRNA expression levels in kidney tissue of rats in each group (n=3)

[0115]

[0116] Note: Compared with the blank group, 1) P < 0.05; compared with the model group, 2) P < 0.05.

[0117] 2.6 Comparison of ABCG2 and GLUT9 protein expression in small intestine tissue of rats in each group

[0118] result( Figure 5 Table 7 shows that compared with the blank group, the model group had decreased ABCG2 protein expression (P < 0.05) and increased GLUT9 protein expression (P < 0.05); compared with the model group, the TCM group had increased ABCG2 protein expression (P < 0.05) and decreased GLUT9 protein expression (P < 0.05); the benzbromarone group had increased ABCG2 protein expression (P < 0.05) and decreased GLUT9 protein expression (P < 0.05); and compared with the TCM group, the benzbromarone group had increased GLUT9 protein expression (P < 0.05). These results suggest that uric acid reabsorption is increased and excretion is decreased in the small intestine of HUA model rats. The Jianpi Huashi Xiezhuo recipe can reduce uric acid reabsorption and promote uric acid excretion in the small intestine of HUA model rats.

[0119] Table 7 Western blot results of small intestine tissue (n=3)

[0120]

[0121] Note: Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05; compared with the Chinese medicine group 3)

[0122] P<0.05.

[0123] 2.7 Comparison of OAT1 and URAT1 protein expression in kidney tissue of rats in each group

[0124] result( Figure 6Table 8 shows that compared with the blank group, URAT1 protein expression increased in the model group (P < 0.05) and OAT1 protein expression decreased (P < 0.05). Compared with the model group, URAT1 protein expression decreased in the TCM group (P < 0.05) and OAT1 protein expression increased (P < 0.05), while URAT1 protein expression decreased in the benzbromarone group (P < 0.05) and OAT1 protein expression increased (P < 0.05). These results suggest that uric acid excretion is decreased and reabsorption is increased in the kidneys of HUA model rats. The Jianpi Huashi Xiezhuo formula can reduce uric acid reabsorption and promote uric acid excretion in the kidneys of HUA model rats.

[0125] Table 8 Western blot test results of kidney tissue (n=3)

[0126]

[0127] Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05.

[0128] 2.8 Comparison of ABCG2 protein expression in the kidneys of rats in each group

[0129] result( Figure 7 Table 9 shows that compared with the blank group, ABCG2 protein expression was decreased in the model group (P < 0.05); compared with the model group, ABCG2 protein expression was increased in the TCM group (P < 0.05), and in the benzbromarone group (P < 0.05); compared with the TCM group, ABCG2 protein expression was decreased in the benzbromarone group (P < 0.05). These results suggest that uric acid excretion in the kidneys of HUA model rats is reduced, and the Jianpi Huashi Xiezhuo formula can promote uric acid excretion.

[0130] Table 9 Average fluorescence intensity analysis (n=3)

[0131]

[0132] Note: Compared with the blank group 1) P < 0.05; compared with the model group 2) P < 0.05; compared with the Chinese medicine group 3)

[0133] P<0.05.

[0134] Spleen deficiency and dysfunction, along with internal accumulation of dampness and turbidity, are the core pathogenesis of elevated blood uric acid levels. A prescription for invigorating the spleen, transforming dampness, and eliminating turbidity, developed around this pathogenesis, incorporates Atractylodes lancea and Coix seed as the main ingredients, invigorating the spleen and eliminating dampness; Chicory, Smilax glabra, and Phellodendron chinense serve as the secondary ingredients, eliminating turbidity and dispelling dampness; Radix Dianthus, Herba Lysimachiae, Herba Glechomae, and Radix Puerariae serve as the envoys, distinguishing clear and turbid properties, promoting diuresis and relieving stranguria, and channeling dampness through urination; and Radix Achyranthis Bidentatae guides the downward movement of these herbs. Together, these herbs achieve the desired effect of invigorating the spleen, transforming dampness, and eliminating turbidity. This study found that treatment with the prescription significantly decreased blood uric acid levels, reduced XOD and ADA activity, and reduced hepatocyte edema and fatty vacuoles, demonstrating that it can improve the essential characteristics of spleen deficiency and dysfunction and lower blood uric acid levels. Allopurinol is a first-line medication for reducing uric acid production in clinical practice. It is effective in inhibiting uricosuric enzyme activity, lowering uric acid levels, inhibiting inflammasome activation, and reducing hepatocyte damage. Recent studies have also confirmed that allopurinol can correct intestinal flora disturbances. Using allopurinol as a positive control, the efficacy of the Jianpi Huashi Xiezhuo formula in reducing uricosuric enzyme activity and lowering serum uric acid levels was further validated, demonstrating that the Jianpi Huashi Xiezhuo formula improves the pathological changes associated with spleen deficiency and internal accumulation of dampness and turbidity. The present study also found that traditional Chinese medicine intervention reduced serum uric acid levels in rats with HUA. Reduced mRNA and protein expression of GLUT9 in the small intestine and URAT1 in the kidneys was observed, while increased mRNA and protein expression of ABCG2 in the small intestine and OAT1 in the kidneys was observed, as was increased protein expression of ABCG2 in the kidneys. This suggests that the formula can ameliorate the essential characteristics of spleen deficiency and turbidity, restore normal expression of urate transporter proteins, lower serum uric acid levels, and improve the pathological state of internal accumulation of dampness and turbidity, consistent with the results of other current studies on the pharmacodynamics of traditional Chinese medicine. Benzbromarone is the first-line drug used in clinical practice to promote uric acid excretion. It has a definite effect in regulating the expression of urate transporter proteins and lowering uric acid. Using benzbromarone as a positive control group, we further verified the efficacy of the Jianpi Huashi Xiezhuo formula in restoring the normal expression of urate transporter proteins and lowering blood uric acid levels, demonstrating the definite efficacy of the Jianpi Huashi Xiezhuo formula in improving core pathological changes such as spleen deficiency and internal accumulation of dampness and turbidity.

[0135] Prolonged retention of dampness and turbidity, which accumulates in the lower abdomen, can cause damage to organs such as the kidneys. In the present invention, model rats showed rupture and necrosis of glomerular and tubular epithelial cells in their kidney tissue. After three weeks of traditional Chinese medicine intervention, renal tissue damage was alleviated. This demonstrates that the Jianpi Huashi Xiezhuo formula can simultaneously protect the kidneys while lowering uric acid, alleviating damage to the body caused by pathogenic factors such as dampness and turbidity, and improving the course of HUA. The present invention validates the safety of the Jianpi Huashi Xiezhuo formula. Liver tissue sections showed no significant damage in the Jianpi Huashi Xiezhuo group compared to the blank group, demonstrating the safety and long-term suitability of the Jianpi Huashi Xiezhuo formula.

[0136] In summary, the spleen-strengthening, dampness-removing, and turbidity-clearing prescription of the present invention can reduce the activity of xanthine oxidase and adenosine deaminase in liver tissue, inhibit uric acid production, regulate the expression of intestinal and renal urate transporters, reduce uric acid reabsorption, and increase uric acid excretion. While lowering uric acid, it also protects the kidneys without causing damage to the liver structure. The spleen-strengthening, dampness-removing, and turbidity-clearing prescription of the present invention has good application prospects in the preparation of medicaments for the prevention and / or treatment of hyperuricemia.

Claims

1. A spleen-strengthening, dampness-removing and turbidity-eliminating prescription for treating hyperuricemia of spleen deficiency and dampness-turbidity type, characterized by: The invention is prepared from the following raw materials in parts by weight: 20-60 parts of rhizoma smilacis gracile, 5-20 parts of pyrrosiae, 5-25 parts of radix polygoni multiflori, 10-50 parts of herba schizonepetae, 5-15 parts of radix achyranthis Bidentatae, 10-30 parts of coix seeds, 5-15 parts of chicory, 1-10 parts of smilax china, 1-10 parts of cortex phellodendri and 5-25 parts of kudzu root.

2. The spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to claim 1, characterized in that: The invention is prepared from the following raw materials in parts by weight: 30-41 parts of Smilax glabra, 11-13 parts of Pyrola, 14-16 parts of Radix Dipterocarpa, 29-31 parts of Herba Lysimachiae, 9-11 parts of Radix Achyranthis Bidentatae, 19-21 parts of Coix seeds, 9-11 parts of Chicory, 5-7 parts of Atractylodes, 5-7 parts of Cortex Phellodendri and 14-16 parts of Radix Puerariae.

3. The spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to claim 2, characterized in that: The drug is prepared from the following raw materials in parts by weight: 40 parts of rhizoma smilacis gracile, 12 parts of pyrrosiae, 15 parts of radix polygoni multiflori, 30 parts of herba schizonepetae, 10 parts of radix achyranthis Bidentatae, 20 parts of coix seeds, 10 parts of chicory, 6 parts of atractylodes, 6 parts of phellodendron amurense and 15 parts of kudzu root.

4. The spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to any one of claims 1 to 3, characterized in that: It is a pharmaceutical preparation prepared with the powder of the raw material drug, the water extract of the raw material drug or the organic solvent extract of the raw material drug as the active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients.

5. The spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to claim 4, characterized in that: The pharmaceutical preparation is an oral preparation.

6. The spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to claim 5, characterized in that: The oral preparation is a decoction, oral liquid, granule, capsule, powder, pill or tablet.

7. A method for preparing the spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: taking the raw material medicine, directly grinding it into powder, or adding water or organic solvent to extract it, and then adding pharmaceutically acceptable excipients or auxiliary ingredients to obtain the spleen-strengthening, dampness-removing and turbidity-eliminating prescription.

8. Use of the spleen-strengthening, dampness-removing and turbidity-eliminating prescription according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating hyperuricemia.

9. The use according to claim 8, characterized in that The medicine is a medicine that reduces uric acid production and increases uric acid excretion.

10. The use according to claim 8, characterized in that The medicine is a medicine for treating hyperuricemia of spleen deficiency and internal accumulation of dampness and turbidity.

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