Use of a composition of paeonia and licorice in the preparation of a drug for preventing and treating organ fibrosis disease

Abstract

The application discloses application of a peony and licorice composition in preparation of a medicine for preventing and treating organ fibrosis diseases, belongs to the technical field of clinical application of the peony and licorice composition, and specifically relates to application of a traditional Chinese medicine classical prescription in preparation of a medicine for preventing and treating organ fibrosis diseases. The application provides an optimal weight ratio of the peony and licorice composition for generating the prevention and treatment effect on organ fibrosis. It is found that the peony and licorice composition with a weight ratio of 1:1 can not only obviously reduce the change of cell hardness in the process of organ fibrosis, reduce inflammatory cell infiltration of the organ, relieve organ damage and collagen deposition, but also improve the function of the fibrotic organ and reverse the morphology of the fibrotic organ, thereby delaying the development process of organ fibrosis. The application finds the medical use of the peony and licorice composition in preventing and treating organ fibrosis diseases, and innovatively finds the component ratio of the peony and licorice composition in preventing and treating organ fibrosis diseases.

Description

Technical Field

[0001] This invention relates to the field of clinical application technology of peony and licorice composition, specifically to the use of peony and licorice composition in the preparation of drugs for the prevention or treatment of organ fibrosis. Background Technology

[0002] Fibrosis is the end-stage pathological outcome of a chronic inflammatory disease characterized by excessive deposition of extracellular matrix components, tissue structural damage, and loss of function. Fibrosis can occur in various organs, including vital organs such as the kidneys, liver, heart, and lungs. During the development of fibrosis, various etiologies induce the overexpression of cytokines, leading to the activation and transformation of intrinsically functional cells and interstitial cells, and the massive accumulation of matrix proteins, thereby destroying tissue structure and resulting in organ dysfunction. Related studies indicate that fibrosis is mainly related to repeated organ cell damage, secretion of pro-fibrotic factors, inflammatory cell infiltration, epithelial-mesenchymal transition, and an imbalance between extracellular matrix production and degradation. Statistics show that organ fibrosis accounts for 45% of all disease-related deaths worldwide, exhibiting high insidiousness and mortality. Currently, clinical treatment for organ fibrosis primarily involves the combined use of anti-inflammatory and immunosuppressant drugs to slow the pathological progression. However, this approach is not widely used due to the uncertainty of its efficacy and the high incidence of adverse reactions. Organ transplantation remains the only effective treatment for end-stage organ fibrosis, but it also faces challenges such as organ donor shortages, difficult postoperative recovery, and high medical costs. Therefore, the development of safe, effective and economical anti-organ fibrosis drugs is of great significance.

[0003] As the birthplace of natural Chinese herbal medicines, my country boasts thousands of medicinal herbs. The combination of various herbs to form traditional Chinese medicine formulas embodies the accumulated medical experience of the Chinese people in their daily lives, representing the essence of traditional Chinese medicine culture and possessing significant research value. Due to its multi-component, multi-target, and holistic approach, traditional Chinese medicine plays an irreplaceable role in disease treatment compared to Western medicine. With technological advancements and in-depth research, its advantages in disease treatment are becoming increasingly apparent.

[0004] Peony and Licorice Decoction, composed of peony root and licorice root, is a classic ancient formula recorded by Zhang Zhongjing in *Treatise on Febrile and Miscellaneous Diseases*. Pharmacological studies have shown that Peony and Licorice Decoction has antispasmodic, analgesic, hepatoprotective, antitussive, antiasthmatic, and anti-inflammatory effects. Clinically, it can be used to treat inflammatory diseases, spastic diseases, painful diseases, bronchial asthma, and gynecological diseases. The main innovation of this invention lies in the first discovery that the optimal 1:1 weight ratio of peony root and licorice root can delay the pathological process of fibrosis in different organs, thereby producing a significant anti-organ fibrosis effect, which can be used to prepare drugs for the prevention and treatment of organ fibrosis. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the lack of effective drugs for the prevention and treatment of organ fibrosis in clinical practice, and to provide a new use for a classic Chinese medicine formula, namely, the application of a classic formula composed of peony and licorice in the preparation of drugs for the prevention or treatment of organ fibrosis.

[0006] The aforementioned classic Chinese medicine formula is prepared by decocting and extracting peony root and licorice root in a 1:1 weight ratio with water. The peony root is white peony root, and the licorice root is raw licorice root, prepared licorice root, or stir-fried licorice root.

[0007] The total raw weight of peony and licorice in the aforementioned classic Chinese medicine formula is 1-200g, preferably 2-100g, and most preferably 5-50g.

[0008] The organ fibrosis diseases mentioned include fibrosis diseases or diseases induced by fibrosis, such as renal fibrosis, pulmonary fibrosis, cardiac fibrosis, liver fibrosis, pancreatic fibrosis, ocular fibrosis, splenic fibrosis, endometrial fibrosis, bone marrow fibrosis, and skin fibrosis.

[0009] The renal fibrosis mentioned refers to renal fibrosis caused by hypertension, scleroderma, glomerulonephritis, kidney stones, hyperlipidemia, diabetes, pyelonephritis, kidney transplant rejection, hyperuricemia, hypercalciuria, systemic lupus erythematosus, and other causes of renal fibrosis, as well as other renal fibrosis of unknown etiology and diseases induced by renal fibrosis.

[0010] The pulmonary fibrosis diseases mentioned include drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, pneumoconiosis, sarcoidosis, allergic pneumonia, and radiation-induced pulmonary fibrosis, as well as other pulmonary fibrosis of unknown etiology and diseases induced by pulmonary fibrosis.

[0011] The aforementioned cardiac fibrosis refers to cardiac fibrosis, cardiac remodeling, and myocardial hypertrophy caused by ischemic heart disease, hypertension, viral myocarditis, metabolic cardiomyopathy, Keshan disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmia, as well as cardiac fibrosis of other unknown causes and diseases induced by cardiac fibrosis.

[0012] The liver fibrosis mentioned refers to liver fibrosis caused by various reasons such as fatty liver, viral hepatitis, alcoholic hepatitis, autoimmune diseases, malnutrition, chronic congestive heart failure, and drugs, as well as liver fibrosis of unknown etiology and diseases induced by liver fibrosis.

[0013] The pancreatic fibrosis mentioned refers to acute pancreatitis, pancreatic duct obstruction, chronic alcoholism, Oddi sphincter dysfunction, pancreatic ischemia, and diseases induced by pancreatic fibrosis.

[0014] The ocular fibrosis mentioned refers to retinal fibrosis caused by eye trauma, eye surgery, and diabetes, as well as diseases induced by ocular fibrosis.

[0015] The term "endometrial fibrosis" refers to endometrial fibrosis caused by various factors, such as endometriosis, as well as diseases induced by endometrial fibrosis.

[0016] The aforementioned myelofibrosis includes idiopathic and drug-induced myelofibrosis, polycythemia vera, chronic myeloid leukemia, Hodgkin's disease, and diseases induced by myelofibrosis.

[0017] This invention investigates the preventive and therapeutic effects of a classic traditional Chinese medicine formula composed of peony and licorice on organ fibrosis by constructing in vivo and in vitro organ fibrosis models. In vitro experimental results show that, compared with peony alone, licorice alone, a 2:1 weight ratio of peony to licorice, or a 1:2 weight ratio of peony to licorice, the 1:1 weight ratio of peony to licorice significantly reversed the pathological changes of kidney, lung, heart, and liver fibrosis. In vivo anti-organ fibrosis experiments show that, in a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO), the 1:1 weight ratio of peony to licorice dose-dependently improved the changes in kidney morphology and function caused by renal fibrosis, improved kidney damage and collagen deposition, and significantly inhibited the pathological changes of renal fibrosis. Attached Figure Description

[0018] Figure 1 This study demonstrates how the use of peony and licorice alone, as well as peony and licorice combinations in different weight ratios, can block changes in cell stiffness in the in vitro TGF-β1-stimulated renal, pulmonary, cardiac, and hepatic fibrosis processes.

[0019] Figure 2 The study demonstrated that a 1:1 weight ratio composition of peony and licorice improved 24-hour urine volume, 24-hour urine protein, creatinine, and blood urea nitrogen in UUO mice at different doses.

[0020] Figure 3 This study demonstrated that a 1:1 weight ratio composition of peony and licorice improved the appearance, volume, weight, and renal coefficient of the kidneys in UUO mice at different doses.

[0021] Figure 4 This study demonstrated that a 1:1 weight ratio composition of peony and licorice improved renal pathology in UUO mice at different doses. Detailed Implementation

[0022] Example 1

[0023] Preparation of concentrated aqueous extracts of peony, licorice and their combination

[0024] The Chinese medicinal herbs, white peony root, stir-fried licorice root, or a combination of white peony root and stir-fried licorice root in different weight ratios, were pulverized in a herb cutter and soaked for 12 hours. The herbs were then decocted twice with water every other day. For the first decoction, the water mass was 10 times the total weight of the herbs. The decocted liquid was filtered and reserved for later use. The remaining dregs were decocted again with water. For the second decoction, the water mass was 8 times the total weight of the herbs. When the liquid began to boil, it was simmered over low heat for 1 hour. The two decoctions were combined to obtain a concentrated aqueous extract of peony root, licorice root, and the combination.

[0025] Example 2

[0026] In vitro anti-organ fibrosis effects of peony, licorice and their combination

[0027] Our previous work has confirmed that cell stiffness can serve as a biomarker for fibrosis (Biochem Biophys Res Commun, 2015, 468(1-2):214-220, CN201410776356.4). Kidney tubular epithelial cells (HK-2), lung epithelial cells (A549), primary cardiac fibroblasts (CFs), and hepatic stellate cells (LX-2) were pretreated with a total crude drug concentration of 20 mg / mL of peony water extract (ShaoYao), licorice water extract (GanCao), a peony and licorice water extract at a weight ratio of 1:1 (SGD(1:1)), a peony and licorice water extract at a weight ratio of 1:2 (SGD(1:2)), and a peony and licorice water extract at a weight ratio of 2:1 (SGD(2:1)). Then, these four cell types were stimulated with 10 ng / mL TGF-β1 to transform into myofibroblasts, thus constructing kidney, lung, heart, and liver fibrosis models. Atomic force microscopy was used to detect changes in cell stiffness before and after TGF-β1 stimulation, and cell stiffness was used as a marker to assess the degree of fibrosis. The results showed that ( Figure 1 In the aforementioned fibrosis model, concentrated aqueous extracts of peony root, concentrated aqueous extracts of licorice root, and concentrated aqueous extracts of peony root and licorice root at different weight ratios all showed a trend towards anti-fibrotic effects. Statistical analysis of the hardness results between groups indicated that only the concentrated aqueous extract of peony root and licorice root at a weight ratio of 1:1 had a significant effect on improving fibrosis, suggesting that the traditional Chinese medicine composition of peony root and licorice root at a 1:1 weight ratio has significant anti-organ fibrosis activity.

[0028] Example 3

[0029] In vivo anti-renal fibrosis effect of peony and licorice combination

[0030] 1. Model preparation and drug administration:

[0031] A peony and licorice combination prepared by decocting white peony and stir-fried licorice in a 1:1 weight ratio was used as the test sample. Adult male C57BL / 6 mice weighing 20-25g were randomly divided into 5 groups of 10 mice each. (1) Sham operation group (Sham), (2) Model group (UUO), (3) Pirfenidone group (PFD), (4) Low-dose peony and licorice combination group (SGD-L), (5) High-dose peony and licorice combination group (SGD-H). The UUO mice were fasted the night before modeling and anesthetized on the day of modeling. After cutting open the skin and muscle layers, the left kidney and ureter were ligated with 6-0 sutures. The sham operation group was the same as the model group except that no ligation was performed. On the second day after modeling, the Sham group and UUO group were administered physiological saline by gavage once a day, the PFD group was administered pirfenidone by gavage once a day at a dose of 100 mg / kg, the SGD-L group was administered a peony and licorice combination by gavage once a day at a dose of 8.34 g total crude drug / kg, which is a combination of 4.17 g peony and 4.17 g licorice per kg of mice, and the SGD-H group was administered a peony and licorice combination by gavage once a day at a dose of 16.68 g total crude drug / kg, which is a combination of 8.34 g peony and 8.34 g licorice per kg of mice. After 14 days of continuous administration, subsequent indicators were measured.

[0032] 2. Methods for detecting 24-hour urine volume, 24-hour urine protein, creatinine (Scr), and blood urea nitrogen (BUN)

[0033] After 14 days of continuous drug administration, urine samples were collected from mice in each group using metabolic cages. The 24-hour urine volume was measured, and the supernatant was used to detect 24-hour urinary protein according to the kit instructions. Whole blood was collected by enucleation and centrifuged twice at 3000 rpm and 4°C for 10 minutes each time. The supernatant was then collected to obtain a pale yellow serum sample. Scr and BUN levels were detected using a biochemical kit.

[0034] 3. Methods for tissue sampling, kidney appearance, and pathological examination

[0035] After 14 days of continuous administration, mice were euthanized by cervical dislocation and cardiac perfusion was performed. Physiological saline was slowly injected into the heart and perfused through the systemic circulation until the kidneys turned white. The left kidney was removed, and the long diameter (L), short diameter (W), and height (H) of the left kidney of each mouse were measured with calipers. The kidney volume was calculated using the formula: V = π / 6 × L (long diameter) × W (short diameter) × H (height). The weight of the left kidney of the mouse was weighed using an analytical balance, and the kidney coefficient of the mouse was calculated using the formula: kidney coefficient = weight of left kidney / weight of mouse body.

[0036] 4. Statistical methods

[0037] Statistical analysis was performed using GraphPad Prism 8.0 software. Data are expressed as mean ± standard deviation. This indicates that the distribution conforms to a normal distribution. One-way ANOVA was used for comparisons among multiple groups, where #P < 0.05, ##P < 0.01, and ###P < 0.001 were compared with the sham surgery group; and *P < 0.05, **P < 0.01, and ***P < 0.001 were compared with the model group.

[0038] 5. In vivo evaluation results of the anti-renal fibrosis effect of the combination of peony and licorice.

[0039] 5.1 Evaluation results of 24-hour urine volume, 24-hour urine protein, serum creatinine (Scr), and urine protein (BUN)

[0040] After UUO modeling, the total urine output of mice was significantly reduced within 24 hours. Administration of low-dose peony and licorice combination showed a tendency for urine output to recover, while high-dose administration significantly restored urine output, indicating that the peony and licorice combination can restore urine output reduced by UUO. Figure 2 A). Furthermore, 24-hour urinary protein, serum creatinine (Scr), and blood urea nitrogen (BUN) levels were significantly elevated after UUO modeling, indicating glomerular filtration dysfunction and kidney damage. The groups treated with the peony and licorice combination showed a dose-dependent improvement in these indicators, and both high and low doses of the peony and licorice combination were more effective than the positive control drug pirfenidone in improving 24-hour urinary protein. The high-dose peony and licorice combination also showed better improvement in creatinine and BUN than pirfenidone. Figure 2 (BD). The above indicates that a 1:1 weight ratio of peony and licorice can, in a dose-dependent manner, reverse the changes in urine output and renal biochemical parameters induced by UUO in mice, and improve glomerular filtration impairment and renal dysfunction in a UUO-induced mouse model of renal fibrosis.

[0041] 5.2 Evaluation results of kidney appearance, kidney volume, kidney weight and kidney coefficient

[0042] Following UUO modeling, the kidneys became enlarged, with a reduction in the renal parenchyma area. In some areas, only the cortex remained, accompanied by urine accumulation. Figure 3 A), and after treatment with a combination of high and low doses of peony and licorice, the degree of kidney enlargement was significantly improved, the renal parenchyma area increased, and the fluid accumulation decreased. Furthermore, the kidney volume after UUO modeling ( Figure 3 B) Kidney weight ( Figure 3 C) and kidney coefficient ( Figure 3 D) The levels were significantly increased, but these indicators were reversed in a dose-dependent manner after administration of the peony and licorice combination. This indicates that the peony and licorice combination can significantly improve urinary retention, kidney enlargement, and increased kidney weight caused by UUO.

[0043] 5.3 Evaluation results of kidney pathological section staining

[0044] H&E staining results showed that the model group mice exhibited renal tubular structural damage, extensive infiltration of interstitial inflammatory cells, and glomerular atrophy. The high- and low-dose peony and licorice combination groups showed relatively intact glomerular and tubular structures compared to the model group, and significantly improved interstitial inflammatory cell infiltration. Figure 4 A). Masson results showed that, compared with the sham-operated group, the model group had a large number of collagen-stained areas in the glomerular mesangium and basement membrane regions, with tubules disappearing in some areas and dilating in others. Treatment with the peony and licorice combination significantly reduced collagen fiber deposition in a dose-dependent manner, and in high-dose peony and licorice combination treatments, the number of disappeared tubules decreased, and tubular morphology was largely restored. Figure 4 B). The above pathological tissue section results suggest that a 1:1 weight ratio of peony and licorice can reduce the infiltration of inflammatory cells to alleviate kidney inflammation, improve renal tubular loss and dilation to restore renal tubular structure, and reduce collagen fiber deposition, thereby delaying the progression of renal fibrosis.

Claims

1. The application of a peony and licorice composition in the preparation of a drug for preventing and treating renal fibrosis, characterized in that, The peony and licorice composition is prepared by decocting peony and licorice in a 1:1 weight ratio with water, and is the only active ingredient of the drug.

2. The application according to claim 1, characterized in that, The peony is white peony root, and the licorice is raw licorice, roasted licorice, or stir-fried licorice.

3. The application according to claim 1, characterized in that, The total raw drug weight of the peony and licorice composition is 1-200 g.

4. The application according to claim 3, characterized in that, The total raw drug weight of the peony and licorice composition is 2-100 g.

5. The application according to claim 4, characterized in that, The total raw drug weight of the peony and licorice composition is 5-50 g.

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