Application of traditional Chinese medicine compound composition in preparation of anti-renal fibrosis medicine

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a traditional Chinese medicine compound composition in preparation of medicines for resisting acute and chronic renal injury and renal fibrosis. The traditional Chinese medicine compound composition has remarkable effects of resisting acute kidney injury and chronic kidney injury and improving the degree of renal fibrosis caused by the kidney injury, and can be used for improving and treating acute and chronic kidney injury and renal fibrosis diseases caused by the kidney injury. According to the traditional Chinese medicine compound composition, the acute and chronic renal injury is treated by reducing the oxidative stress level, regulating an inflammation signal channel and inhibiting kidney inflammation so as to regulate and control the immune microenvironment of the kidney, and the renal fibrosis caused by the renal injury is improved. The invention provides a new medicine with obvious curative effect and small adverse reaction for patients with acute and chronic renal injury and renal fibrosis caused by renal injury, so as to relieve the pain of the patients.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a new use of a Chinese medicine compound composition in preparing drugs for resisting acute and chronic kidney damage and renal fibrosis. Background Art

[0002] Tangcao Tablets is a pure Chinese medicine preparation, composed of 20 kinds of Chinese medicines, including Geranium, Honeysuckle, Bupleurum, Elsholtzia, Trichosanthes peel, Pomegranate peel, Astragalus, Licorice, Kapok, etc. It has the effects of clearing away heat and detoxifying, promoting blood circulation and invigorating Qi. It is currently used clinically to treat HIV-infected people and AIDS patients, and has the effect of increasing CD4 lymphocyte counts, and can improve symptoms such as fatigue, hair loss, loss of appetite and diarrhea.

[0003] The kidney is an important excretory organ in the human body and has the function of immunoregulation to maintain the stability of the body's internal environment. Acute kidney injury is a syndrome of reduced glomerular filtration rate and decreased urine volume, characterized by high morbidity and mortality. A large number of epidemiological evidence shows that there is a causal relationship between the clinical syndrome of acute kidney injury and the subsequent development of chronic kidney disease and renal fibrosis. Therefore, even if patients with acute kidney injury survive the acute critical period, they are at risk of developing chronic kidney disease in the future. The prevalence of chronic kidney disease is approximately 8-16% worldwide, and it is also one of the three major causes of early death.

[0004] Acute kidney injury to renal fibrosis is a complex pathological process involving changes in the renal immune microenvironment, and its balance is closely related to the stability of the body. At the same time, the renal immune microenvironment is also an important guarantee mechanism for the defense function of the kidney, including a variety of immune cells (such as neutrophils, dendritic cells, macrophages, B lymphocytes and T cells), resident renal cells, cell secretion of cytokines, chemokines, adhesion factors, etc., involving the activation of inflammatory pathways, the production of reactive oxygen species, tubular damage, apoptosis, and the balance of immune cells, playing an important role in the development of renal metabolism and injury. After the kidney is damaged, cytokines related to the proinflammatory signaling pathway are activated, thereby inhibiting the degradation of nuclear transcription factor (NF-κB), inducing phosphorylation of nuclear transcription factors and subsequent translocation to the nucleus. In the nucleus, activated NF-κB leads to the transcription of inflammatory mediators including tumor necrosis factor α (TNF-α). In turn, tumor necrosis factor α (TNF-α) induces the expression of other inflammatory cytokines and recruits inflammatory cells into renal tissue, thereby causing renal inflammation. At the same time, renal injury causes the generation of reactive oxygen species, affects the tumor suppressor protein p53, induces apoptosis through the interaction of death receptor-tumor necrosis factor and intrinsic pathway caspase, leads to mitochondrial dysfunction and oxidative stress. In addition, the Th1 / Th2 balance affects the degree of renal injury by regulating the ratio of immune cells. Overexpression of Th1 cytokines, such as T-bet and interferon-γ (IFN-γ), participates in the regulation of inflammatory response, enhances the chemotaxis and adhesion of inflammatory cells, leads to the aggregation and activation of inflammatory cells, thus causing inflammatory response in renal tissue, and inhibits the expression of Th2 cytokines, breaks the balance of Th1 / Th2, causes excessive inflammation and oxidative stress, further promotes the imbalance of the immune microenvironment of renal injury, and leads to acute renal injury. With the changes in the immune microenvironment of the kidney, renal tubular epithelial cells are stimulated to produce a series of inflammatory mediators and profibrotic factors, which promote the development of chronic kidney disease. When the growth of regenerating tubules that cannot be differentiated is atrophied, pathological signals and autocrine activity that disrupts the interaction between different renal cells and renal interstitial cells will be displayed. There is a clear cellular crosstalk between tubular epithelial-fibroblasts, epithelial-endothelial cells, and epithelial-inflammatory cells. One of the major changes is the interaction between pericytes and the peritubular capillary endothelium, which leads to the separation of pericytes from capillaries, causing capillary disintegration and microvascular rarefaction. Pericytes transform into myofibroblasts, leading to endothelial dysfunction, chronic inflammatory infiltration, oxidative stress, mitochondrial dysfunction, and epigenetic changes; in addition, renal parenchymal cells include endothelial cells, tubular epithelial cells, mesangial cells, and podocytes. These parenchymal cells work closely with immune cells to maintain renal homeostasis. Under pathological conditions, renal parenchymal cells express Toll-like receptors and induce innate immune responses.Immune cells present antigens to T cells, producing cytokines (such as TNF-α, interleukin, IFN-γ), chemokines, inducible nitric oxide synthase, hyaluronic acid and other substances that cause renal inflammation. The inflammatory response leads to glomerular cell proliferation and renal dysfunction, and immune cells invade the renal interstitium. All pathological changes eventually lead to dysregulation of the renal immune microenvironment and the formation of renal fibrosis. Therefore, the occurrence and development of renal injury is a complex pathological process involving multiple mechanisms, and a single treatment may not produce a good effect.

[0005] At present, there is a lack of effective methods for treating kidney injury in clinical practice. Existing therapeutic drugs are mainly diuretics, antioxidants, statins, immunosuppressants, etc., which have limited efficacy. Amifostine, a drug approved by the US Food and Drug Administration for the treatment of acute kidney injury, has not been widely used because of its multiple adverse reactions and high price. Therefore, there is a wide demand in clinical practice to develop drugs targeting the molecular mechanism of kidney injury and to achieve better therapeutic effects through multi-angle synergistic treatment.

[0006] The main ingredients of Tangcao Tablets are Geranium, Honeysuckle, Trichosanthes, Bupleurum, Elsholtzia, Astragalus, Licorice, etc. Geranium is a common Chinese medicine with the effects of clearing away heat and dampness, and stopping diarrhea. Studies have shown that the extract of Geranium can reduce the level of lipid peroxides and increase the oxygen free radical scavenging activity in the rat nephritis model induced by aminonucleoside puromycin (PA), thereby inhibiting nephritis; in the study of the effect of TNF-α on the expression of interleukin 1 (IL-1) and human monocyte chemotactic enzyme 1 (MCP-1) in mouse glomerular mesangial cells, it was found that honeysuckle water extract inhibited the proliferation of glomerular mesangial cells and downregulated the expression of proinflammatory factors such as interleukin 6 (IL-6) and MCP-1 to achieve the purpose of treating kidney disease; Bupleurum polysaccharide has an effect on the treatment of kidney diseases induced by lead. In the lead-induced liver and kidney injury model, it can significantly increase the levels of superoxide dismutase (SOD) and glutathione (GSH) in mice, improve the body's antioxidant capacity, and regulate the metabolism of free radicals in the body, thereby achieving a protective effect on the kidneys of lead-poisoned mice; the various active ingredients contained in licorice can reduce oxidative damage, reduce cell apoptosis, improve renal structure, and alleviate renal inflammatory response in mice with acute renal injury; therefore, it can be speculated that the various ingredients in Tangcao tablets synergistically regulate the renal immune microenvironment, and jointly exert anti-inflammatory, antioxidant and immunomodulatory effects, and may be a potential drug for the treatment of renal injury and renal fibrosis.

[0007] Cisplatin is a drug commonly used in the clinic to treat a variety of malignant tumors. Its high accumulation, high excretion, and high metabolism in the kidneys make the renal damage effect particularly obvious, and the renal toxicity is positively correlated with the dose. The results of cisplatin-induced renal damage are universal, can simulate complex clinical pathological processes, and are also recognized as a classic inducer of renal damage. After free filtration by the glomeruli, cisplatin accumulates in the renal tubular epithelial cells, with a concentration several times that of blood, causing tubular cell damage, dysfunction, edema, and gradually worsening renal damage. As the disease progresses, the body weight gradually decreases, and related immune organs atrophy. With the increase in the accumulation of cisplatin in renal tubular epithelial cells, renal function gradually weakens, and creatinine (Cr) and urea nitrogen (BUN) levels increase.

[0008] NF-κB is a multifunctional transcription factor that can regulate the expression and synthesis of a variety of inflammatory factors, adhesion factors, and growth factors, and therefore plays a key role in immune regulation and inflammatory response. In acute kidney injury, changes in the kidneys occur mainly through acute inflammation, so the expression of NF-κB is crucial in acute kidney injury.

[0009] In chronic renal injury, IL-6 can promote the increase of fibrinogen expression by activating STAT3 and participate in renal fibrosis. Transforming growth factor β (TGF-β) plays a key driving role in the development of renal fibrosis. Its mechanism mainly includes promoting the synthesis of extracellular matrix, inhibiting the degradation of matrix metalloproteinases and regulating the transformation of renal tubular epithelial cells to fibroblasts, thereby promoting the process of renal fibrosis. In addition, TGF-β promotes the expression of α-smooth muscle actin (α-SMA) through the induction of various cytokines, thereby promoting the development of myofibroblasts. Smad protein belongs to the downstream protein of the TGF-β family and is its key downstream regulatory factor. Its activation is mainly achieved through the regulation of TGF-β. Therefore, the TGF-β / Smad3 signaling pathway plays an important regulatory role in the development of renal fibrosis.

[0010] In the process of renal fibrosis, the imbalance of Th1 and Th2 cells may play an important role. Long-term chronic inflammation may lead to the activation of Th1 cells, release inflammatory factors, and cause tissue damage. At the same time, the activation of Th2 cells may lead to excessive humoral immune response and fibrosis. IFN-γ, IL-4, T-bet, and Gata3 are specific inflammatory factors secreted by Th1 and Th2 cells. The high and low expression levels of IFN-γ / IL-4 and T-bet / Gata3 reflect the number of Th1 and Th2 cells; the ratio of IFN-γ / IL-4 and T-bet / Gata3 is generally used to measure the direction of Th1 and Th2 in the body. Improper regulation of the immune system may lead to a vicious cycle of inflammation and fibrosis, ultimately leading to continuous damage to the structure and function of the kidney. Therefore, in the treatment of renal fibrosis, regulating the balance of the immune system may be an important therapeutic strategy. Summary of the invention

[0011] In order to better treat acute and chronic renal injury and renal fibrosis, and relieve the pain of patients. The present invention provides a new use of a Chinese medicine compound composition, specifically, a new use in the preparation of drugs for chronic kidney disease and renal fibrosis caused by acute renal injury. The Chinese medicine compound composition is produced by Shanghai Baisuixing Pharmaceutical Co., Ltd., and the product name is Tangcao Tablets, and the approval number is a pure Chinese medicine preparation of National Medicine Standard Z20050291. The present invention is a secondary research and development of the existing product, and it is unexpectedly found that Tangcao Tablets have a certain effect of resisting acute and chronic renal injury and renal fibrosis caused by renal injury.

[0012] The specific technical scheme of the present invention is:

[0013] The present invention provides a use of a Chinese medicine compound composition in preparing a drug for treating acute and chronic renal injury and renal fibrosis. The Chinese medicine compound composition is composed of the following components in parts by weight: 120-150 parts of geranium, 120-150 parts of astragalus, 120-200 parts of nightshade, 120-200 parts of honeysuckle, 60-100 parts of kapok, 40-60 parts of terminalia chebula, 80-100 parts of oldenlandia diffusa, 100-200 parts of scutellaria baicalensis ... 50 parts, pomegranate peel, 150-200 parts of glutinous rice roots, 80-150 parts of water chestnuts, 40-60 parts of trichosanthes peels, 60-100 parts of bupleurum, 40-60 parts of elsholtzia, 60-80 parts of liquorice, 120-150 parts of millettia reticulata, 40-60 parts of safflower, 40-60 parts of ginkgo leaves, 60-120 parts of purslane, 40-60 parts of picrophora, and 28-40 parts of scorpions.

[0014] Furthermore, the Chinese medicine compound composition is prepared into conventional oral dosage forms including tablets, capsules, powders and granules.

[0015] Furthermore, the Chinese medicine compound composition is made into film-coated tablets.

[0016] Furthermore, the dosage of the Chinese medicine compound composition is adjusted based on the severity of the disease and the presence or absence of previous medical history.

[0017] The tangerine tablets of the present invention have a significant effect of resisting acute kidney injury. By increasing body weight, reducing renal edema and renal tissue pathological damage, reducing creatinine and urea nitrogen levels, increasing glutathione content and superoxide dismutase content in renal tissue, reducing tumor necrosis factor α and interleukin 6 levels, and reducing the phosphorylation level of nuclear transcription factor p65, regulating the expression of interferon γ and interleukin 4 in renal tissue, correcting the deviation in the Th1 / Th2 balance system toward Th1, and improving the degree of acute kidney injury.

[0018] The tangerine tablets of the present invention have significant anti-chronic kidney injury effects and have good improvement and treatment effects on chronic kidney disease. By reducing creatinine and urea nitrogen levels, reducing renal tissue collagen deposition to varying degrees, reducing the expression of α-smooth muscle actin, regulating the interleukin 6 / STAT3 signaling pathway to reduce the expression of inflammatory factors, increasing the content of glutathione and superoxide dismutase, increasing the Th1-specific transcription factor T-bet, reducing the expression of Th2-specific transcription factor Gata3 and related cytokines, the deviation of the Th1 / Th2 balance system in renal tissue to the Th2 direction is corrected.

[0019] The tangerine tablets of the present invention have significant anti-renal fibrosis effects and have good improvement and treatment effects on renal fibrosis diseases. The renal function is improved by reducing the levels of creatinine and urea nitrogen in mice with renal fibrosis, the expression of α-smooth muscle actin is reduced, the renal collagen deposition is reduced, the transforming growth factor β / Smad3 signaling pathway is regulated to alleviate renal fibrosis, the expression of inflammatory factors is reduced, the content of glutathione and superoxide dismutase is increased, the ratio of anti-apoptotic protein BCL-2 / pro-apoptotic protein BAX is increased, cell apoptosis is reduced, and the deviation to the Th2 direction in the Th1 / Th2 balance system in renal tissue is continuously corrected. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a line graph of the survival rate of mice with acute kidney injury treated with Tangcao tablets.

[0021] Figure 2 The figure shows the effect of Tangcao tablets on the body weight and organ index of mice with acute renal injury. A is the body weight of each group, B is the thymus index of each group, C is the spleen index of each group, and D is the kidney coefficient of each group.

[0022] Figure 3 The figure shows the effect of Tangcao tablets on renal function in mice with acute renal injury. A is the Cr level graph of each group, and B is the BUN level graph of each group.

[0023] Figure 4The figure shows the effect of Tangcao tablets on the renal tissue pathology of mice with acute kidney injury, where A is the HE staining image of the renal tissue pathology section of mice in the blank group, B is the HE staining image of the renal tissue pathology section of mice in the model group, C is the HE staining image of the renal tissue pathology section of mice in the low-dose group of Tangcao tablets, D is the HE staining image of the renal tissue pathology section of mice in the medium-dose group of Tangcao tablets, E is the HE staining image of the renal tissue pathology section of mice in the high-dose group of Tangcao tablets, and F is the renal tubular injury score.

[0024] Figure 5 The effect of Tangcao tablets on the oxidative stress level in renal tissue of mice with acute renal injury. A is the GSH level of each group, and B is the SOD level of each group.

[0025] Figure 6 The figure shows the effect of Tangcao tablets on serum inflammation levels in mice with acute kidney injury. A is the TNF-α level graph, and B is the IL-6 level graph.

[0026] Figure 7 The figure shows the effect of Tangcao tablets on the NF-κB signaling pathway in renal tissue of mice with acute renal injury. A is an immunoblot of inflammatory signaling pathway proteins, B is an expression level of NF-κB-p65 in each group, C is an expression level of heme oxygenase 1 (HO-1) in each group, D is an expression level of interleukin 10 (IL-10) in each group, and E is an expression level of TGF-β in each group.

[0027] Figure 8 The figure shows the effect of Tangcao tablets on the balance of Th1 / Th2 inflammatory factors in the kidney tissue of mice with acute renal injury. A is the immunoblotting of inflammatory factor proteins, B is the expression level of IFN-γ in each group, C is the expression level of IL-4 in each group, and E is the ratio of IFN-γ to IL-4 in each group.

[0028] Fig. 9 The figure shows the effect of Tangcao tablets on the body weight and organ index of mice with chronic renal injury. A is the body weight of each group, B is the thymus index of each group, C is the spleen index of each group, and D is the kidney coefficient of each group.

[0029] Fig.10 The figure shows the effect of Tangcao tablets on renal function in mice with chronic renal injury. A shows the Cr level in each group, and B shows the BUN level in each group.

[0030] Fig.11 The figure shows the effect of Tangcao tablets on the degree of renal injury in mice with chronic renal injury. A is the HE staining of the renal tissue pathology section of mice in the blank group, B is the HE staining of the renal tissue pathology section of mice in the model group, C is the HE staining of the renal tissue pathology section of mice in the low-dose group of Tangcao tablets, D is the HE staining of the renal tissue pathology section of mice in the medium-dose group of Tangcao tablets, and E is the HE staining of the renal tissue pathology section of mice in the high-dose group of Tangcao tablets.

[0031] Fig.12 The figure shows the effect of Tangcao tablets on collagen deposition in mice with chronic renal injury. A is the MASSON staining of the renal tissue pathology section of mice in the blank group, B is the MASSON staining of the renal tissue pathology section of mice in the model group, C is the MASSON staining of the renal tissue pathology section of mice in the low-dose group of Tangcao tablets, D is the MASSON staining of the renal tissue pathology section of mice in the medium-dose group of Tangcao tablets, E is the MASSON staining of the renal tissue pathology section of mice in the high-dose group of Tangcao tablets, and F is the percentage level of collagen-positive areas in each group.

[0032] Fig.13 The figure shows the effect of Tangcao tablets on the oxidative stress level in renal tissue of mice with chronic renal injury. A shows the GSH level of each group, B shows the SOD level of each group, and C shows the myeloperoxidase level (MPO).

[0033] Fig.14 The figure shows the effect of Tangcao tablets on the level of inflammation in renal tissue of mice with chronic renal injury. A is the immunoblotting of inflammatory factors in each group, B is the expression level of IFN-γ in each group, C is the expression level of IL-4 in each group, D is the expression level of IL-10 in each group, E is the expression level of TNF-α in each group, and F is the expression level of IL-6 in each group.

[0034] Fig.15 The figure shows the effect of Tangcao tablets on the IL-6 / STAT3 and NF-κB signaling pathways in the kidney tissue of mice with chronic renal injury. A is an immunoblot of proteins related to the IL-6 / STAT3 and NF-κB signaling pathways, B is an expression level of IL-6 in each group, C is an expression level of phosphorylated STAT3 protein (p-STAT3) in each group, D is an expression level of phosphorylated nuclear transcription factor p65 (p-NF-κB-p65), and E is an expression level of α-SMA.

[0035] Fig.16 The figure shows the effect of Tangcao tablets on the Th1 / Th2 balance in the renal tissue of mice with chronic renal injury. A is the immunoblotting of immune cytokine proteins in each group, B is the IFN-γ expression level in each group, C is the IL-4 expression level in each group, D is the ratio of IFN-γ to IL-4 expression levels in each group, and E is the ratio of GATA3 to T-bet expression levels in each group.

[0036] Fig.17 The figure shows the effect of Tangcao tablets on the organ index of mice with renal fibrosis. A is the thymus index of each group, B is the spleen index of each group, and C is the kidney coefficient of each group.

[0037] Fig.18 The figure shows the effect of Tangcao tablets on renal function in mice with renal fibrosis. A shows the Cr level in each group, and B shows the BUN level in each group.

[0038] Fig.19 The figure shows the effect of Tangcao tablets on the degree of renal injury in mice with renal fibrosis. A is the HE staining of the renal tissue pathology section of mice in the blank group, B is the HE staining of the renal tissue pathology section of mice in the model group, C is the HE staining of the renal tissue pathology section of mice in the low-dose group of Tangcao tablets, D is the HE staining of the renal tissue pathology section of mice in the medium-dose group of Tangcao tablets, and E is the HE staining of the renal tissue pathology section of mice in the high-dose group of Tangcao tablets.

[0039] Fig. 20 The figure shows the effect of Tangcao tablets on collagen deposition in mice with renal fibrosis. A is the MASSON staining of the renal tissue pathology section of mice in the blank group, B is the MASSON staining of the renal tissue pathology section of mice in the model group, C is the MASSON staining of the renal tissue pathology section of mice in the low-dose group of Tangcao tablets, D is the MASSON staining of the renal tissue pathology section of mice in the medium-dose group of Tangcao tablets, E is the MASSON staining of the renal tissue pathology section of mice in the high-dose group of Tangcao tablets, and F is the percentage level of collagen-positive areas in each group.

[0040] Fig.21 The figure is the effect of Tangcao tablets on the oxidative stress level of renal tissue in mice with renal fibrosis. A is the GSH level of each group, B is the SOD level of each group, and C is the MPO level of each group.

[0041] Fig. 22 The figure shows the effect of Tangcao tablets on the level of inflammation in renal tissue of mice with renal fibrosis. A is the immunoblotting of inflammatory factors in each group, B is the expression level of IL-10 in each group, C is the expression level of IFN-γ in each group, D is the expression level of IL-4 in each group, E is the expression level of TGF-β in each group, and F is the expression level of interleukin 1β (IL-1β) in each group.

[0042] Fig.23 The figure shows the effect of Tangcao tablets on apoptotic proteins in the kidney tissue of mice with renal fibrosis. A is the immunoblot of anti-apoptotic protein BCL-2 and pro-apoptotic protein BAX in the kidney tissue of mice, and B is the ratio of the expression levels of anti-apoptotic protein BCL-2 and pro-apoptotic protein BAX in each group.

[0043] Fig.24 The figure shows the effect of Tangcao tablets on the TGF-β / Smad3 signaling pathway in renal tissue of mice with renal fibrosis. A is an immunoblot of proteins related to the TGF-β / Smad3 signaling pathway, B is a graph showing the expression level of TGF-β in each group, C is a graph showing the expression level of phosphorylated Smad3 protein, and D is a graph showing the expression level of α-SMA.

[0044] Fig.25The figure shows the effect of Tangcao tablets on the Th1 / Th2 balance in the renal tissue of mice with renal fibrosis. A is the immunoblot of cytokine proteins in each group, B is the ratio of T-bet and GATA3 expression levels in each group, C is the ratio of IFN-γ and IL-4 expression levels in each group, D is the IFN-γ expression level in each group, and E is the IL-4 expression level in each group. DETAILED DESCRIPTION

[0045] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0046] Example 1: The preventive and therapeutic effects of Tangcao tablets on cisplatin-induced acute renal injury in mice

[0047] 1 Main experimental materials

[0048] 1.1 Experimental animals

[0049] SPF grade mice, male BALB / c strain, were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (animal certificate number: No. 210726220101395215), aged about 6 to 8 weeks, weighing 20 ± 2 g. They were kept in the IVC animal laboratory, which maintained a constant temperature (21 to 25 ° C), humidity 40% to 60%) and 12 hours of light / dark alternation. The bedding of the cages was sterilized by high pressure, and the cages and drinking bottles were disinfected regularly. The mice were kept in separate cages and had free access to food and water, with 8 to 10 mice in each cage. The experiment began after the mice were adapted to feeding for one week.

[0050] 1.2 Experimental drugs

[0051] Tangcao tablets, batch number: 190308, approval number: National Medicine Standard Z20050291, Shanghai Baisuixing Pharmaceutical Co., Ltd.

[0052] 1.3 Main reagents and consumables

[0053] Sodium carboxymethyl cellulose, Shanghai Linen Technology Development Co., Ltd.; No. 12 mouse gavage needle, Henan Zhike Hongrun Environmental Protection Technology Co., Ltd.; methanol, Komiou Chemical Reagent Co., Ltd. (Tianjin); 1× PBS buffer, Beijing Solebow Technology Co., Ltd.; 0.5% Evans blue dye, Beijing Solebow Technology Co., Ltd.; formamide, Tianjin Damao Chemical Reagent Factory; GSH assay kit, Nanjing Jiancheng Bioengineering Institute; 5× protein loading buffer, Shanghai Yazyme Biomedical Technology Co., Ltd.; protein phosphatase inhibitor cocktail (100×), Beijing Solebow Technology Co., Ltd.; BCA protein concentration assay kit, GLPBIO; PVDF membrane, Millipore; MPO assay kit, Nanjing Jiancheng Bioengineering Institute; skim milk powder, Beijing Solebow Technology Co., Ltd.; 10× electrotransfer solution, Beijing Solebow Technology Co., Ltd.; PVDF membrane (pore size 0.45 μm), Merck, Germany Millipore Corporation; prestained protein molecular weight standard (marker), CISTRO; SDS-PAGE gel preparation kit, Beijing Solebao Technology Co., Ltd.; ultrasensitive ECL chemiluminescence detection kit, Shanghai Yazyme Biotechnology Co., Ltd.; Tris, Beijing Solebao Technology Co., Ltd.; glycine, Beijing Solebao Technology Co., Ltd.; SDS, Beijing Solebao Technology Co., Ltd.; GAPDH antibody, Wuhan Sewell Biotechnology Co., Ltd.; β-actin antibody, Wuhan Sewell Biotechnology Co., Ltd.; goat anti-rabbit IgG-HRP, SAB Shanghai Sabo Biotechnology Co., Ltd.; goat anti-mouse IgG-HRP SAB, Shanghai Sabo Biotechnology Co., Ltd.; IL-6 antibody, Signal way Antibody, USA; TGF-β antibody, Protein tech, USA; IL-1β antibody, SAB Shanghai Sabo Biotechnology Co., Ltd.; TNF-α antibody, SAB Shanghai Sabo Biotechnology Co., Ltd.; IL-4 antibody, Protein tech, USA; IL-10 antibody, Protein tech, USA; TGF-β antibody, Protein tech, USA tech; T-bet antibody, Beijing Bioson Biotechnology Co., Ltd.; Gata3 antibody, Wuhan Fine Biotechnology Co., Ltd.; IFN-γ antibody, Beijing Bioson Biotechnology Co., Ltd.; HO-1 antibody, Beijing Bioson Biotechnology Co., Ltd.; NF-κB-p65 antibody, Wuhan Fine Biotechnology Co., Ltd.; STAT3 antibody, Signal way Antibody, USA; P-STAT3 antibody, Signal way Antibody, USA.

[0054] 1.4 Main instruments

[0055] Animal room IVC feeding system, Beijing Zhongshi Technology Co., Ltd.; -80℃ low temperature refrigerator, Suzhou Antai Air Technology Co., Ltd.; -20℃ low temperature refrigerator, Sanyo Co., Ltd. (Japan); ice maker, Panasonic Electric Industrial Co., Ltd. (Japan); pipette, Eppendof, Germany; horizontal centrifuge, Thermo Fisher Scientific; electric blast drying oven, Shanghai Senxin Co., Ltd. (DGG-9140); Milli-Q ultrapure water manufacturing system, Millipore (USA); analytical balance Quintix type, Sartorius Group (Germany); pipette, Eppendorf Life Sciences, Germany; water bath, Shanghai Jinghong Experimental Equipment Co., Ltd.; low temperature high-speed centrifuge, Eppendorf, Germany; vortex oscillator, Vortex-Genie2; full wavelength microplate reader Multiskan GO type, Thermo Fisher Scientific; horizontal shaker, Sartorius Group (Germany); high-throughput tissue grinder, Ningbo Xinzhi Biotechnology Co., Ltd.; Western Blot device, Bio-Rad, USA; WB chemiluminescence imager, Shanghai Peiqing Technology Co., Ltd.; analytical balance, Sartorious BP121S.

[0056] 2 Experimental methods

[0057] 2.1 Drug preparation: Tangcao tablets are divided into three dosage groups: 0.25g / kg, 0.5g / kg, and 1g / kg, which are Tangcao tablet low-dose group, Tangcao tablet medium-dose group, and Tangcao tablet high-dose group. 0.5% sodium carboxymethyl cellulose solution is used to prepare Tangcao tablet suspensions with concentrations of 0.025g / mL, 0.05g / mL, and 0.1g / mL, respectively. 6mL of cisplatin (specification 5mg / mL) is absorbed and dissolved in physiological saline, and the volume is adjusted to 20mL to prepare a cisplatin solution with a concentration of 1.5mg / mL. All drugs are prepared on demand.

[0058] 2.2 Establishment and treatment of acute kidney injury model: After one week of adaptive feeding, mice were randomly divided into two batches, each batch was divided into 5 groups, namely, blank group (control), model group (model), Tangcao tablet low-dose group (Tang-0.25g / kg), Tangcao tablet medium-dose group (Tang-0.5g / kg), Tangcao tablet high-dose group (Tang-1g / kg). The first batch of animals in the three Tangcao tablet groups were gavaged with different doses of Tangcao tablets according to body weight at a fixed time every morning. One hour after administration on the fifth day, the model group and the Tangcao tablet group were intraperitoneally injected with 15mg / kg cisplatin according to body weight, and the blank group was injected with normal saline. The weight and general condition of the mice were recorded daily during the modeling period, and the Tangcao tablet group continued to be administered. The survival period of each group was observed and recorded. The operation of the second batch of animals was the same as the first batch. After modeling on the eighth day, the mice were anesthetized and killed, and specimens were collected for subsequent testing. See 2.3 for details.

[0059] 2.3 Specimen collection: The animals were fasted for 12 hours after the experiment and had free access to water. The mice were anesthetized with sodium pentobarbital and blood was collected from the eye sockets. After the blood was allowed to stand for 2 to 4 hours, the supernatant was collected by ultra-low temperature centrifuge and frozen at -80°C for later use. The thymus, spleen, and kidney tissues were collected and the organ weights were weighed. The right kidney was fixed in 4% paraformaldehyde solution, and the remaining tissues were quickly frozen in liquid nitrogen and then transferred to -80°C for storage.

[0060] 2.4 Calculation of mouse organ index: Organ index = organ mass (mg) / body weight (g)

[0061] 2.5 HE staining to observe the pathological changes of renal tissue: The renal tissue retained in 2.3 was fixed, dehydrated, waxed, embedded, paraffin sliced ​​(4-5μm), dewaxed to water, HE stained, dehydrated and sealed, and microscopic examination and image acquisition and analysis were performed. The pathological sections were observed under an optical microscope for the morphology of renal tissue and inflammatory infiltration. Histological scoring was also performed.

[0062] 2.6 Determination of Cr and BUN in serum: Add the serum retained in 2.3 to a 96-well plate, add relevant colorimetric reagents in sequence according to the instructions of the kit, determine the absorbance value, and calculate the Cr and BUN contents in serum respectively.

[0063] 2.7 Determination of GSH and SOD levels in renal tissue: Take the renal tissue retained in 2.3, accurately weigh the renal tissue of each group of mice, add the homogenate medium in proportion, and grind mechanically while placing in an ice water bath to prepare a renal tissue homogenate. Centrifuge it in an ultra-low temperature centrifuge, take the supernatant and add it to a 96-well plate. Prepare and add the required reagents in sequence according to the requirements of the kit instructions, determine the absorbance value after color development, and calculate the content of GSH and SOD.

[0064] 2.8 Determination of serum inflammatory factors TNF-α and IL-6 levels: Add the serum retained in 2.3 to a 96-well plate, and add relevant reagents in sequence according to the instructions of the enzyme-linked immunosorbent assay kit. After color development, measure the absorbance value and calculate the TNF-α and IL-6 content.

[0065] 2.9 Protein immunoblotting analysis was used to detect the expression levels of NF-κB-p65, HO-1, IL-10, TGF-β, IFN-γ, and IL-4 in renal tissue: the renal tissue retained in 2.3 was cut into pieces for protein sample preparation, protein quantification and denaturation, and SDS-PAGE gel electrophoresis was used to quantitatively analyze the protein expression of inflammatory signaling pathway proteins NF-κB-p65, HO-1, IL-10, TGF-β and cytokines IFN-γ and IL-4.

[0066] 2.10 Statistical analysis: Data were expressed as mean ± standard deviation (Mean ± SD). Graphpad Prism 9.1.0 software was used to plot the data, and SPSS 27.0 software was used for statistical analysis. For multiple groups of data that followed a normal distribution, one-way analysis of variance was used to compare the mean differences between groups. If the variances of the data groups were equal, the LSD method was used for pairwise comparisons; the Games-Howell method was used for pairwise comparisons of data with unequal variances. For multiple groups of data that did not follow a normal distribution, the Kruskal-Wallis method was used for mean comparisons, and the Bonferroni method was used for pairwise comparisons between groups. The T test was used to compare the means of two groups of data that followed a normal distribution. The Mann-Whitney U method was used to compare the means of two groups of data that did not follow a normal distribution. The Person method was used for correlation analysis of two groups of data that followed a normal distribution. The Spearman method was used for correlation analysis of two groups of data that did not follow a normal distribution. If the calculated P < 0.05, it was considered to be statistically significant.

[0067] 3 Experimental results

[0068] 3.1 Effect of Tangcao Tablets on the survival of mice with acute renal injury: There was no death in the blank group, while mice in the model group developed diarrhea and severe weight loss, and died at 48h. The survival rate at 120h was only 18%, while the survival rates of the low-, medium-, and high-dose Tangcao Tablets groups at 120h were 45%, 36%, and 64%, respectively. Among them, the high-dose Tangcao Tablets group was the most significant, with statistically significant differences compared with the model group (P<0.05). Figure 1 .

[0069] 3.2 Effect of Tangcao Tablets on Body Weight and Organ Index of Mice with Acute Kidney Injury: Compared with the blank group, the body weight of mice in the model group decreased (P<0.05), the thymus tissue and spleen tissue were significantly atrophied (P<0.001), and the kidneys were enlarged (P<0.01), indicating that the model was seriously damaged at this time. Compared with the model, different doses of Tangcao Tablets can significantly reduce the kidney coefficient (P<0.01), and have no significant effect on body weight, thymus coefficient, and spleen coefficient. See for details. Figure 2 .

[0070] 3.3 Effect of Tangcao Tablets on Renal Function of Mice with Acute Kidney Injury: Compared with the blank group, the Cr and BUN levels in the model group were significantly increased and statistically significant (P<0.001). Compared with the model, the Cr and BUN levels in each dose group of Tangcao Tablets were reduced to varying degrees and statistically significant (P<0.01, P<0.001). This suggests that Tangcao Tablets can reduce the Cr and BUN levels in mice with acute kidney injury and protect renal function. Figure 3 .

[0071] 3.4 Effect of Tangcao tablets on renal tissue pathology in mice with acute renal injury: In the blank group, the morphology and size of renal tubules in renal tissue were normal, the glomerular structure was clear and not deformed, and there was no proliferation of mesangial matrix. The renal pathology of the model group showed obvious deformation and expansion of the renal tubular lumen, degeneration and shedding of epithelial cells, edema and degeneration, local diffuse bleeding, and renal interstitial edema accompanied by inflammatory cell infiltration. Compared with the model group, the Tangcao tablets in each group alleviated renal tubular injury to varying degrees, the renal tubular lumen did not show deformation and expansion, the epithelial cell morphology was normal, and the inflammatory cell infiltration and congestion were not obvious. The renal tubular injury scores were all lower than those of the model group (P<0.01). See for details. Figure 4 .

[0072] 3.5 Effect of Tangcao Tablets on the oxidative stress level in renal tissue of mice with acute renal injury: Compared with the blank group, the GSH and SOD contents in the model group were significantly reduced and statistically significant (P<0.05, P<0.01). Compared with the model group, each group of Tangcao Tablets was able to increase the GSH content, and the high-dose group of Tangcao Tablets was statistically significant (P<0.01); each group of Tangcao Tablets was able to increase the SOD content and there were statistical differences (P<0.001). This suggests that Tangcao Tablets can enhance antioxidant capacity and reduce the oxidative stress level in mice with acute renal injury. See for details. Figure 5 .

[0073] 3.6 Effect of Tangcao Tablets on Inflammation Levels in Serum of Mice with Acute Kidney Injury: Compared with the blank group, the levels of TNF-α and IL-6 in the model group were significantly increased and statistically significant (P<0.05, P<0.01). Compared with the model group, the levels of TNF-α and IL-6 in each group of Tangcao Tablets were significantly reduced and statistically significant (P<0.05, P<0.01, P<0.001). This suggests that Tangcao Tablets can reduce the level of inflammation in mice with acute kidney injury by reducing the expression of TNF-α and IL-6. See for details. Figure 6 .

[0074] 3.7 Effect of Tangcao Tablets on NF-κB signaling pathway in renal tissue of mice with acute kidney injury: Compared with the blank group, the phosphorylated NF-κB-p65 in the model group was significantly increased (P<0.05), and HO-1, IL-10, and TGF-β were significantly decreased (P<0.05, P<0.01); Compared with the model group, the Tangcao Tablets group significantly reduced the phosphorylation level of NF-κB-p65 and increased the levels of HO-1, IL-10, and TGF-β, all of which were statistically significant (P<0.05, P<0.01). This suggests that Tangcao Tablets can inhibit the activation of NF-κB-p65 in the renal tissue of mice with acute kidney injury and alleviate the level of renal tissue inflammation. See for details. Figure 7 .

[0075] 3.8 Effect of Tangcao Tablets on the balance of Th1 / Th2 inflammatory factors in renal tissue of mice with acute kidney injury: Compared with the blank group, the expression of pro-inflammatory factor IFN-γ in the model group was significantly increased, the expression of anti-inflammatory factor IL-4 was significantly decreased, and the ratio of IFN-γ / IL-4 increased (P<0.01), indicating that the renal inflammatory environment in the model group tended to develop in the direction of Th1. Compared with the model group, the Tangcao Tablets group significantly reduced the ratio of IFN-γ / IL-4 and the differences were statistically significant (P<0.05, P<0.01). This suggests that Tangcao Tablets can correct the imbalance of Th1 / Th2 inflammatory factors in renal tissue of mice with acute kidney injury. See details. Figure 8 .

[0076] Example 2: The preventive and therapeutic effects of Tangcao tablets on cisplatin-induced chronic renal injury in mice

[0077] 1 Main experimental materials

[0078] α-SMA antibody, Abcam (Shanghai) Trading Co., Ltd.; MASSON staining kit, Shanghai ELISA; MPO assay kit, Nanjing Jiancheng Bioengineering Institute. The rest of the main experimental materials are the same as those in Example 1.

[0079] 2 Experimental methods

[0080] 2.1 Drug preparation: Take 1.5 mL of cisplatin (specification 5 mg / mL) and dissolve it in physiological saline, dilute to 15 mL, and prepare 0.5 mg / mL cisplatin solution, which is prepared before use. The preparation method of Tangcao tablets is the same as that of Example 1.

[0081] 2.2 Establishment of chronic renal injury model and drug treatment: After adaptive feeding for one week, 48 male BALB / c mice were randomly divided into 5 groups. They were: blank group (Control), model group (Model), Tangcao tablet low-dose group (Tang-0.25g / kg), Tangcao tablet medium-dose group (Tang-0.5g / kg), Tangcao tablet high-dose group (Tang-1g / kg), 10 mice each. The blank group had normal diet and water. The model group and the drug group were intraperitoneally injected with cisplatin 5mg / kg on the 1st, 7th, 14th and 21st days to establish chronic renal injury, and cisplatin was injected four times in total. The drug group was gavaged with Tangcao tablets on the second day after the first injection of cisplatin. The drug was administered continuously for 6 weeks, and the model group and the blank group were given an equal amount of sodium carboxymethyl cellulose solution. The entire experimental process lasted 43 days.

[0082] 2.3 Specimen collection: Same as specimen collection in Example 1.

[0083] 2.4 Calculation of mouse organ index: Same as 2.4 of Example 1.

[0084] 2.5 HE staining to observe the pathological changes of renal tissue: Same as 2.5 of Example 1.

[0085] 2.6 Observation of renal fibrosis in renal tissue by MASSON staining: The renal tissue of 2.3 was fixed with wax, cooled and sliced ​​into 4 μm slices using a slicer, dewaxed, hydrated, stained, sealed and examined under a microscope. Five fields of view under a 200× optical microscope were randomly collected in the cortical area of ​​each slice. The absolute area of ​​positive staining was measured using Image J image analysis software, and the ratio of different staining areas to the overall area of ​​the test area (Area%) was calculated.

[0086] 2.7 Determination of Cr and BUN in serum: Same as 2.6 in Example 1.

[0087] 2.8 Determination of GSH, SOD and MPO content in renal tissue: Accurately weigh the sample tissue in 2.3, homogenize the tissue and add relevant reagents in sequence according to the instructions of the MPO assay kit, measure the absorbance value after color development, and calculate the MPO content. The rest of the operation is the same as 2.7 in Example 1.

[0088] 2.9 Protein immunoblotting analysis was used to detect the expression levels of NF-κB-p65, HO-1, IL-10, TGF-β, IFN-γ, and IL-4 in renal tissue: the same as 2.9 in Example 1.

[0089] 2.10 Statistical analysis: Same as 2.10 in Example 1.

[0090] 3 Experimental results

[0091] 3.1 Effects of Tangcao tablets on body weight and organ indexes of mice with chronic renal injury: Compared with the body weight of mice in the blank group, the body weight of mice in the model group began to decrease in the first week, and the body weight dropped to the lowest on the fifth day, and the body weight showed an upward trend on the sixth day. The body weight changed similarly in the four cycles. The low-dose group and the medium-dose group of Tangcao tablets showed a trend of recovering body weight. At the same time, the thymus coefficient, spleen coefficient, and kidney coefficient of the model group were significantly reduced (all P<0.001). This indicates that a chronic renal injury model has been established. Among them, the low- and medium-dose groups of Tangcao tablets significantly increased the thymus coefficient, which was statistically different from the model. There was no significant difference in the spleen coefficient and kidney coefficient compared with the model. See for details. Fig. 9 .

[0092] 3.2 Effect of Tangcao Tablets on Renal Function of Mice with Chronic Renal Injury: Compared with the blank group, the Cr and BUN levels in the model group were significantly increased and statistically significant (P<0.05, P<0.01). Compared with the model group, the Cr levels in the low, medium and high dose groups of Tangcao Tablets were significantly reduced, and there were statistical differences between the medium and high dose groups (P<0.001); the BUN levels in the low, medium and high dose groups of Tangcao Tablets were significantly reduced, and there were statistical differences (P<0.05, P<0.001). This suggests that Tangcao Tablets can reduce the Cr and BUN levels in mice with chronic renal injury and protect renal function. See for details. Fig.10 .

[0093] 3.3 Effect of Tangcao Tablets on the degree of renal injury in mice with chronic renal injury: There was no obvious abnormality in the renal tissue of mice in the blank group, the structure was clear, and no inflammatory cells and interstitial infiltration were observed. Compared with the blank group, the renal tubular epithelial cells in the model group were edematous, bulging into the tubular lumen, the cell volume increased, the density of the cytoplasmic matrix decreased, and a large number of vacuoles were observed, and a small number of fibrotic masses accompanied by inflammatory cell infiltration appeared. This indicates that the renal tissue in the model group was inflamed and fibrosis had been initiated. Compared with the model group, the above conditions in the Tangcao Tablets group were alleviated, the renal tubular epithelial cell structure was relatively complete, the amount of inflammatory cell infiltration was small, the cell volume did not increase significantly, and the renal tubular lumen morphology was normal. This suggests that Tangcao Tablets can significantly improve the degree of renal injury in mice with chronic renal injury. See for details. Fig.11 .

[0094] 3.4 Effect of Tangcao Tablets on Collagen Deposition in Mice with Chronic Renal Injury: MASSON staining was used to examine the degree of renal fibrosis in mice with chronic renal injury. No obvious collagen deposition was observed in the renal tissue of mice in the blank group, and the morphological structure of the glomeruli and renal tubules was normal, with only a small amount of blue collagen fibers visible. In the model group, the glomerular basement membrane was significantly thickened, fibroblasts proliferated in large numbers, and flocculent bright blue collagen fiber deposition and aggregation were observed near the renal tubules, and collagen deposition was significantly increased (P<0.001). Compared with the model group, the Tangcao Tablets group reduced the degree of renal fibrosis to varying degrees and all showed statistical differences. A significant decrease in blue collagen fibers, slight proliferation of the glomerular mesangial basement, no obvious collagen deposition near the renal tubules, a significant decrease in the percentage of collagen-positive areas, a milder degree of fibrosis, and a significantly reduced area of ​​renal fibrosis (P<0.001, P<0.01). This suggests that Tangcao Tablets can reduce the degree of renal fibrosis in mice with chronic renal injury. See for details. Fig.12 .

[0095] 3.5 Effect of Tangcao Tablets on the oxidative stress level in renal tissue of mice with chronic renal injury: Compared with the empty group, the GSH and SOD levels in the model group were significantly decreased, and the MPO level was significantly increased, which was statistically significant (P<0.001, P<0.05). The GSH and SOD levels in the low-, medium-, and high-dose groups of Tangcao Tablets were significantly increased compared with the model, and the GSH and SOD levels in the high-dose group of Tangcao Tablets were statistically different from those in the model group (P<0.05, P<0.001). The MPO level in each group of Tangcao Tablets was significantly lower than that in the model and was statistically different (P<0.05, P<0.001). See for details. Fig.13 .

[0096] 3.6 Effect of Tangcao Tablets on the Inflammation Level of Renal Tissue in Mice with Chronic Kidney Injury: Compared with the blank group, IFN-γ in the model group was significantly reduced (P<0.05), and IL-4, IL-10, TNF-α, and IL-6 were significantly increased and statistically significant (P<0.01, P<0.05). Compared with the model, IFN-γ in the medium and high dose groups of Tangcao Tablets was significantly increased (P<0.05), and IL-4, IL-10, TNF-α, and IL-6 in the low, medium, and high dose groups of Tangcao Tablets were significantly reduced, and statistically significant (P<0.05). This suggests that Tangcao Tablets can regulate various inflammatory factors and thus reduce the inflammation level in mice with chronic kidney injury. See for details. Fig.14 .

[0097] 3.7 Effect of Tangcao Tablets on IL-6 / STAT3 and NF-κB signaling pathways in renal tissue of mice with chronic renal injury: Compared with the blank group, IL-6, STAT3, p-NF-κB-p65, and α-SMA in the model group were significantly increased, and there were statistical differences (P<0.05, P<0.01). The Tangcao Tablets groups reduced the levels of IL-6, STAT3, p-NF-κB-p65, and α-SMA to varying degrees, and there were statistical differences compared with the model (P<0.05, P<0.01). This suggests that Tangcao Tablets can alleviate chronic renal injury by regulating the IL-6 / STAT3 and NF-κB-p65 signaling pathways. See for details. Fig.15 .

[0098] 3.8 Effect of Tangcao Tablets on the Th1 / Th2 balance in renal tissue of mice with chronic renal injury: Compared with the blank group, the expression of Gata3 in the model group was significantly increased, the expression of T-bet was decreased, the Gata3 / T-bet ratio was increased, and the model was biased towards Th2. The Gata3 / T-bet ratio of the low, medium and high dose groups of Tangcao Tablets was significantly lower than that of the model and there was a statistical difference (P<0.05, P<0.01). This suggests that Tangcao Tablets can correct the imbalance of Th1 / Th2 in mice with chronic renal injury. See for details. Fig.16 .

[0099] Example 3: The preventive and therapeutic effects of Tangcao tablets on cisplatin-induced renal fibrosis in mice

[0100] 1 Main experimental materials: TGF-β antibody, Shanghai SAB Biotechnology Co., Ltd.; α-SMA antibody, p-Smad3 antibody, (Shanghai) Abcam Trading Co., Ltd. The rest are the same as the main experimental materials in Example 1.

[0101] 2 Experimental methods

[0102] 2.1 Drug preparation: The preparation method of Tangcao tablets is the same as that of Example 1. Cisplatin uses a drug stock solution (5 mg / mL).

[0103] 2.2 Establishment of cisplatin-induced renal fibrosis mouse model and drug treatment: After adaptive feeding for one week, 48 male BALB / c mice were randomly divided into 5 groups. They were: 8 in the blank group (Control), 10 in the model group (Model), 10 in the low-dose group (Tang-0.25g / kg) of Tangcao tablets, 10 in the medium-dose group (Tang-0.5g / kg) of Tangcao tablets, and 10 in the high-dose group (Tang-1g / kg) of Tangcao tablets. The mice in the blank group ate and drank water normally. The model group and the drug group were injected with cisplatin at 5mg / kg body weight on the 1st, 7th, 14th and 21st days, respectively, for a total of four injections to establish a renal fibrosis model. After the model was successful, different doses of Tangcao tablets were gavaged and administered continuously for 8 weeks, and the model group was given an equal amount of sodium carboxymethyl cellulose solution. The entire modeling process lasted 89 days.

[0104] 2.3 Specimen collection: Same as specimen collection in Example 1.

[0105] 2.4 Calculation of mouse organ index: Same as 2.4 of Example 1.

[0106] 2.5 HE staining to observe the pathological changes of renal tissue: Same as 2.5 of Example 1.

[0107] 2.6 Observation of renal fibrosis in renal tissue by MASSON staining: Same as 2.6 of Example 2.

[0108] 2.7 Determination of Cr and BUN in serum: Same as 2.6 of Example 1.

[0109] 2.8 Determination of GSH and SOD levels in kidney tissue: Same as 2.7 of Example 1.

[0110] 2.9 Protein immunoblotting analysis was used to detect the expression levels of IL-10, IFN-γ, IL-4, TGF-β, IL-1β, anti-apoptotic protein BCL-2, pro-apoptotic protein BAX, p-Smad3, α-SMA, and transcription factor T-bet in renal tissue: the renal tissue retained in 2.3 was cut into pieces for protein sample preparation, protein quantification and denaturation, and SDS-PAGE gel electrophoresis was used to quantitatively analyze the protein expression of IL-10, IFN-γ, IL-4, TGF-β, IL-1β, anti-apoptotic protein BCL-2, pro-apoptotic protein BAX, p-Smad3, α-SMA, and transcription factor T-bet.

[0111] 2.10 is the same as 2.10 in Example 1.

[0112] 3 Experimental results

[0113] 3.1 Effect of Tangcao Tablets on Organ Indexes of Mice with Renal Fibrosis: Compared with the blank group, the thymus index and spleen index of mice in the model group did not change significantly, and the kidney index decreased significantly (P<0.001). This indicates that the kidneys of mice atrophy. No significant difference was observed between the drug group and the model group. Fig.17 .

[0114] 3.2 Effect of Tangcao Tablets on Renal Function of Mice with Renal Fibrosis: Compared with the blank group, the Cr and BUN levels in the model group were significantly increased and statistically significant (P<0.001, P<0.01). Compared with the model group, the Cr and BUN levels in the low, medium and high dose groups of Tangcao Tablets were significantly decreased and statistically significant (P<0.05, P<0.01, P<0.001). This suggests that Tangcao Tablets can reduce the Cr and BUN levels in mice with renal fibrosis and protect renal function. See for details. Fig.18 .

[0115] 3.3 Effect of Tangcao Tablets on the degree of renal damage in mice with renal fibrosis: There was no obvious abnormality in the renal tissue of mice in the blank group, the renal structure was intact, and no inflammatory cell infiltration was observed. In contrast, the renal tubular epithelial cells in the model group were edematous, bulged into the tubular lumen, the cell volume increased, the renal tubular epithelial cells were edematous, degenerated and vacuolated, the renal tubules atrophied, the fibroblasts proliferated massively, and a large number of monocytes and lymphocytes infiltrated in the renal interstitium. This indicates that the inflammation of the renal tissue in the model group is continuing to develop at this time. Compared with the model group, the above conditions in the Tangcao Tablets group were alleviated, the amount of inflammatory cell infiltration was small, the cell volume did not increase significantly, the fibroblasts proliferated slightly, and the morphology of the renal tubular lumen was relatively normal. This indicates that Tangcao Tablets can significantly improve the degree of renal damage in mice with renal fibrosis. See for details. Fig.19 .

[0116] 3.4 Effect of Tangcao Tablets on Collagen Deposition in Mice with Renal Fibrosis: MASSON staining was used to examine the degree of renal fibrosis in mice with renal fibrosis. The morphological structures of the glomeruli and renal tubules in the normal group of mice were normal, and a small amount of blue collagen fibers were visible in the renal interstitium, without fibrosis. Compared with the blank group, the glomerular basement membrane in the model group was significantly thickened, fibroblasts proliferated in large numbers, and collagen deposition increased significantly. Compared with the early stage of renal fibrosis, fibrosis was aggravated, and a large number of flocculent bright blue collagen fibers were deposited and aggregated in the glomeruli and renal interstitium, the mesangial matrix was severely proliferated, the glomeruli were atrophied, and the capillary walls were significantly thickened. There were fewer bright blue collagen fiber clusters in the renal interstitium in the low, medium, and high dose groups of Tangcao Tablets, showing mild to moderate fibrosis, mild hyperplasia of the glomerular mesangial matrix, mild thickening of the basement membrane, and no obvious thickening of the glomerular wall. The degree of fibrosis was lighter than that of the model. Among them, the low-dose group and the medium-dose group were statistically different from the model, and the area of ​​renal fibrosis was significantly reduced (P<0.01). This indicates that Tangcao tablets can reduce collagen deposition caused by renal fibrosis in mice. Fig. 20 .

[0117] 3.5 Effect of Tangcao Tablets on the level of oxidative stress in renal tissue of mice with renal fibrosis: The GSH and SOD contents in the model group were significantly lower than those in the blank group and were statistically different (P<0.05, P<0.01); the low, medium and high dose groups of Tangcao Tablets increased the GSH and SOD contents, which were statistically different from the model (P<0.05, P<0.01, P<0.001). This suggests that Tangcao Tablets can reduce the level of oxidative stress in mice with renal fibrosis. There was no significant change in the expression of MPO among the groups. See for details. Fig.21 .

[0118] 3.6 Effect of Tangcao Tablets on the Inflammation Level of Renal Tissue in Mice with Renal Fibrosis: In the model group, the levels of IL-10 and IFN-γ in the renal tissue of mice were significantly reduced, while the levels of IL-4, TGF-β, and IL-1β were significantly increased, and there were significant statistical differences compared with the blank group (P<0.05); in the different dose groups of Tangcao Tablets, the levels of IL-10 and IFN-γ were significantly increased, and the levels of IL-4 and IL-1β in the high-dose group of Tangcao Tablets were significantly reduced, and there were significant statistical differences compared with the model group (P<0.05). This indicates that Tangcao Tablets may alleviate cisplatin-induced renal fibrosis in mice by regulating the expression levels of inflammatory factors. See details. Fig. 22 .

[0119] 3.7 Effect of Tangcao Tablets on apoptotic proteins in renal tissue of mice with renal fibrosis: Compared with the blank group, the anti-apoptotic protein BCL-2 in the model group decreased, and the pro-apoptotic protein BAX increased, resulting in a significant decrease in the ratio of the two, which was statistically different from the blank group (P<0.05). This suggests that the apoptosis of related cells in the renal tissue of the model group was more obvious. Compared with the model, the different doses of Tangcao Tablets increased the ratio of BCL-2 to BAX, and were statistically different from the model group (P<0.05, P<0.01). This suggests that Tangcao Tablets can reduce the apoptosis of related cells in the renal tissue of mice with renal fibrosis. See for details. Fig.23 .

[0120] 3.8 Effect of Tangcao Tablets on TGF-β / Smad3 Signaling Pathway in Renal Tissue of Mice with Renal Fibrosis: TGF-β, p-Smad3, and α-SMA in the model group were significantly increased, and there were statistical differences compared with the blank group (P<0.05, P<0.01). This shows that the renal tissue of the model group is in a severe state of fibrosis. In the different dose groups of Tangcao Tablets, it was observed that the expression of the above cytokines was reduced, especially in the low dose group, where the effect was most significant, and there was a significant statistical difference compared with the model group (P<0.05, P<0.01). This suggests that Tangcao Tablets can reduce the degree of renal fibrosis by regulating the TGF-β / Smad3 signaling pathway. See for details. Fig.24 .

[0121] 3.9 Effect of Tangcao Tablets on Th1 / Th2 Balance in Renal Tissue of Mice with Renal Fibrosis: Compared with the blank group, the Th1 transcription factor T-bet and cytokine IFN-γ in the model group were significantly decreased, while the Th2 transcription factor Gata3 and cytokine IL-4 were significantly increased, resulting in a decrease in T-bet / Gata3 and IFN-γ / IL-4, and there were statistical differences (P<0.01, P<0.05). This indicates that the renal tissue microenvironment in the model group shifted toward Th2 at this time. The low-dose group of Tangcao Tablets significantly increased the ratios of T-bet / Gata3 and IFN-γ / IL-4, and there were statistical differences compared with the model group (P<0.05). This shows that Tangcao Tablets can correct the imbalance of Th1 / Th2 in mice with renal fibrosis. See for details. Fig.25 .

Claims

1. Use of a Chinese medicine compound composition in the preparation of a medicine for treating acute and chronic renal injury and renal fibrosis, the Chinese medicine compound composition comprising the following components in parts by weight: 120-150 parts of geranium, 120-150 parts of astragalus, 120-200 parts of nightshade, 120-200 parts of honeysuckle, 60-100 parts of kapok, 40-60 parts of terminalia chebula, 80-150 parts of oldenlandia diffusa, 40-60 parts of pomegranate peel, 150-200 parts of glutinous rice roots, 80-150 parts of water chestnuts, 40-60 parts of trichosanthes peel, 60-100 parts of bupleurum, 40-60 parts of elsholtzia, 60-80 parts of liquorice, 120-150 parts of millettia, 40-60 parts of safflower, 40-60 parts of ginkgo leaves, 60-120 parts of purslane, 40-60 parts of picrorhizome, and 28-40 parts of scorpion.

2. The use according to claim 1, characterized in that The Chinese medicine compound composition is prepared into conventional oral dosage forms including tablets, capsules, powders and granules.

3. The use according to claim 1, characterized in that The Chinese medicinal compound composition is made into film-coated tablets.

4. The use according to claim 1, characterized in that The dosage of the Chinese medicine compound composition is adjusted based on the severity of the disease and whether or not there is a previous medical history.

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