Application of ammonium glycyrrhizinate in preparation of medicine for preventing and treating renal fibrosis caused by urinary tract obstruction

Abstract

The invention relates to the technical field of medicines, discloses application of ammonium glycyrrhizinate in preparation of medicines for preventing and treating renal fibrosis caused by urinary tract obstruction, and discloses a method for treating renal fibrosis more safely and effectively. According to the method, the natural anti-inflammatory characteristic of ammonium glycyrrhizinate is utilized, the side effects of the medicine are reduced, and the treatment safety is improved. In addition, it is proved that ammonium glycyrrhizinate can significantly reduce mitochondrial autophagy, maintain normal functions of mitochondria and relieve renal fibrosis, a new therapeutic target is provided for treating renal fibrosis, meanwhile, ammonium glycyrrhizinate can be used as a supplement or replacement of an existing therapeutic method, a new therapeutic choice is provided for patients, and clinical application prospects are broad. The medicine is especially suitable for patients with poor response to traditional medicines. The results show that ammonium glycyrrhizinate has a significant clinical application value and a wide market prospect, and provides a new choice for treatment of related diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of ammonium glycyrrhizinate in preparing a medicine for preventing and treating renal fibrosis caused by urinary tract obstruction. Background Art

[0002] Renal fibrosis is a typical pathological change in chronic kidney disease (CKD), and epithelial-mesenchymal transition (EMT) is the main stage. Tubulointerstitial fibrosis is characterized by tubular atrophy and extracellular matrix (ECM) accumulation in renal tissue. Since the expression level of TGF-β1 (transforming growth factor, β1) in cells is closely related to the production of EMT and ECM, it is considered to be a strong mediator of renal fibrosis and CKD and a potential key driver of renal fibrosis. Mitochondrial autophagy is a selective autophagy that can selectively degrade damaged or dysfunctional mitochondria. Mitochondrial autophagy reduces renal fibrosis by reducing the production of reactive oxygen species (ROS). Therefore, inhibiting the occurrence of EMT and regulating mitochondrial autophagy are of great significance for further and better treatment of renal injury. Pathological changes in CKD renal fibrosis is very common. When the ureter is obstructed, urine retention compresses the renal tubules, causing progressive necrosis of renal tubular epithelial cells, interstitial acute and chronic inflammatory cell infiltration, and necrotic renal tubular tissue is gradually replaced by fibrous scars, eventually forming progressive renal fibrosis. Because fibrosis is considered to be irreversible, inhibiting the progress of fibrosis is considered to be a potential strategy to prevent the development of CKD. At present, renal fibrosis becomes a medical problem to be solved urgently, and the present invention proposes a new solution for this reason. Therefore, it is of great clinical significance to alleviate or delay the renal interstitial fibrosis process in the CKD process and find new drugs for preventing and treating renal fibrosis. Summary of the invention

[0003] Ammonium glycyrrhizinate (AG) is a compound extracted from licorice and belongs to the saponin class. The applications of ammonium glycyrrhizinate in the pharmaceutical industry include antitussive and expectorant, anti-inflammatory, anti-allergic, protecting cell membrane stability, immunomodulatory, promoting phagocyte activity and anti-fibrosis. In addition, according to the provisions of the current "Standards for the Use of Food Additives" (GB2760-2014) in my country, ammonium glycyrrhizinate can be used as a flavoring agent, flavor enhancer and surfactant. Studies in recent years have shown that ammonium glycyrrhizinate has certain safety and scientificity in clinical applications, and its application in the preparation of drugs for the prevention and treatment of various diseases has gradually attracted attention. In view of this, the present invention proposes the use of ammonium glycyrrhizinate in the preparation of drugs for the prevention and treatment of renal fibrosis caused by urinary tract obstruction, aiming to provide a new prevention and treatment strategy to improve the therapeutic effect of renal fibrosis.

[0004] The present invention provides an application of ammonium glycyrrhizinate or its preparation, and the application includes any one of the following: A1) Application of ammonium glycyrrhizinate in the preparation of drugs for preventing and treating renal fibrosis caused by urinary tract obstruction; A2) Application of ammonium glycyrrhizinate in the preparation of drugs for inhibiting the epithelial-mesenchymal transition process induced by TGF-β1 in human renal tubular epithelial cells HK-2; A3) Application of ammonium glycyrrhizinate in increasing the expression of E-cadherin protein in human renal tubular epithelial cells HK-2; A4) Application of ammonium glycyrrhizinate in the preparation of drugs for increasing the expression of E-cadherin protein in human renal tubular epithelial cells HK-2; A5) Application of ammonium glycyrrhizinate in reducing the expression of N-cadherin protein in human renal tubular epithelial cells HK-2; A6) Application of ammonium glycyrrhizinate in the preparation of drugs for reducing the expression of N-cadherin protein in human renal tubular epithelial cells HK-2; A7) Application of ammonium glycyrrhizinate in reducing the expression of Vimentin protein in human renal tubular epithelial cells; A8) Application of ammonium glycyrrhizinate in the preparation of drugs for reducing the expression of Vimentin protein in human renal tubular epithelial cells; A9) Application of ammonium glycyrrhizinate in the preparation of drugs for preventing and / or treating diseases related to renal fibrosis.

[0005] A10) Application of ammonium glycyrrhizinate in reducing the expression of Fn protein in human glomerular mesangial cells HRMC; A11) Application of ammonium glycyrrhizinate in the preparation of drugs for reducing the expression of Fn protein in human glomerular mesangial cells HRMC; A12) Application of ammonium glycyrrhizinate in reducing the expression of COL protein in human glomerular mesangial cells HRMC; A13) Application of ammonium glycyrrhizinate in the preparation of drugs for reducing the expression of COL protein in human glomerular mesangial cells HRMC; Preferably, the disease related to renal fibrosis is chronic kidney disease or renal sclerosis caused by urinary tract obstruction.

[0006] The present invention also provides a drug for treating kidney disease, and the active ingredient of the drug is ammonium glycyrrhizinate.

[0007] Preferably, the kidney disease is renal fibrosis, chronic kidney disease or renal sclerosis caused by urinary tract obstruction.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Provide a safer and more effective method for treating renal fibrosis. By utilizing the natural anti-inflammatory properties of ammonium glycyrrhizinate, the side effects of drugs are reduced, and the safety of treatment is improved; 2. The ammonium glycyrrhizinate of the present invention can be used as a supplement or alternative to existing treatment methods, providing new treatment options for patients, especially for those who do not respond well to traditional drugs. 3. The preparation process of the ammonium glycyrrhizinate of the present invention is relatively mature, with high extraction efficiency and purity, easy for large-scale production and application, helping to reduce the treatment cost and making it affordable for more patients. 4. The experimental results of the present invention show that ammonium glycyrrhizinate has significant effects in reversing oxidative stress and improving mitochondrial function, providing new ideas and methods for the development of new drugs for treating renal fibrosis.

[0009] 5. The experiments of the present invention also confirm that ammonium glycyrrhizinate can significantly reduce the occurrence of mitophagy. This regulatory mechanism helps to protect cells from damage caused by excessive autophagy, while maintaining the normal function of mitochondria and alleviating the occurrence of renal fibrosis, providing a new therapeutic target for the treatment of renal fibrosis.

[0010] In summary, through the various pharmacological activities of ammonium glycyrrhizinate, the present invention provides a new treatment strategy for the prevention and treatment of renal fibrosis, with significant clinical application value and broad market prospects, providing new options for the treatment of related diseases.

[0011] By reading the detailed description of the preferred embodiments below, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. And throughout the drawings, the same reference numerals are used to represent the same components. In the drawings: Figure 1 Shows the effect of ammonium glycyrrhizinate on renal fibrosis in UUO rats in Example 1 of the present invention.

[0012] Figure 2 Shows the effect of ammonium glycyrrhizinate on the viability of HK-2 cells in Example 2 of the present invention.

[0013] Figure 3 Shows the effect of ammonium glycyrrhizinate on the expression of E-cadherin, N-cadherin and Vimentin in TGF-β1-induced HK-2 cells in Example 3 of the present invention.

[0014] Figure 4 Shows the effect of ammonium glycyrrhizinate on the opening degree of mitochondrial permeability transition pores in TGF-β1-induced HK-2 cells in Example 4 of the present invention.

[0015] Figure 5 Shows the effect of ammonium glycyrrhizinate on mitochondrial membrane potential and mitochondrial reactive oxygen species in TGF-β1-induced HK-2 cells in Example 5 of the present invention.

[0016] Figure 6 Effect of ammonium glycyrrhizinate on mitochondrial autophagy of TGF-β1-induced HK-2 cells in Example 6 of the present invention.

[0017] Figure 7 Effect of ammonium glycyrrhizinate on fibronectin and collagen of TGF-β1-induced HRMC cells in Example 7 of the present invention. Detailed implementation manners Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be fully conveyed to those skilled in the art. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the drawings and in conjunction with the embodiments.

[0019] Example 1 Improvement of renal fibrosis in UUO rats by ammonium glycyrrhizinate I. Experimental materials SD rats provided by Spf (Beijing) Biotechnology Co., Ltd. (Animal Certificate Number: NO.110324231106182758); ammonium glycyrrhizinate (Product Number: T2891) was purchased from Shanghai TargetMol Co., Ltd.

[0020] II. Experimental methods Thirty-six SPF-grade male SD rats weighing 150±10 g were selected, placed in a standard SPF-level environment for breeding, and provided with standard feed. The rats could drink water freely. After one week of normal feed adaptation breeding, the animals were randomly divided into six groups: normal control group, UUO model group, UUO model group + AG (2 mg / kg, 10 mg / kg, 50 mg / kg) groups, and UUO model group + pirfenidone group, with 6 rats in each group. The mice were dieted one day before modeling. After the fasting ended, the CON, UUO group, UUO + AG group, and UUO + pirfenidone group were modeled. When the rats were anesthetized, atropine sulfate was subcutaneously injected 15 minutes in advance, and then Zoletil 50 was intramuscularly injected for anesthesia. The rats were fixed in the lateral position on the operating board, and a longitudinal incision was made along the kidney position to expose the kidney. The normal group was not treated, while the model group and the drug administration groups were subjected to ureteral ligation. After the operation, the inner and outer skins were quickly sutured and disinfected. After the rats woke up, they were routinely bred and their states were observed. After the modeling was successful, the drugs were administered. The breeding methods of each group were as follows: Control group (sham operation group, also known as normal group or Con group): Normal water and feed were provided daily; UUO group (urinary tract obstruction renal fibrosis model group): Normal water and feed were provided daily; UUO + AG group (ammonium glycyrrhizinate group): Starting from the second day after surgery, ammonium glycyrrhizinate was administered by gavage daily at doses of 2 mg / kg, 10 mg / kg, and 50 mg / kg, and normal water and feed were provided daily for 14 days; UUO + PFD group (pirfenidone group): Starting from the second day after surgery, pirfenidone was administered by gavage daily at a dose of 500 mg / kg, and normal water and feed were provided daily.

[0021] During the model establishment and the above-mentioned treatment process, the rats were weighed weekly. On the 14th day after drug administration, the rats were anesthetized. An ultrasound workstation MyLab™ X7 with an abdominal probe L 4 - 15 and a working frequency range of 4 - 15 MHz was used. All rats were in the supine position. The gray scale and depth were adjusted to the optimal state to observe the kidney morphology, size, cortical thickness, parenchymal echo, and blood flow. After the B-ultrasound examination, the anesthetized rats were sacrificed, and the kidney cortical tissues were collected. The collected kidney cortical tissues were fixed in 4% paraformaldehyde and subjected to dehydration, embedding, and sectioning to prepare paraffin sections. The sections were stained with hematoxylin-eosin staining (HE), Masson staining, and Periodic Acid-Schiff stain (PAS) respectively.

[0022] III. Experimental Results Under ultrasonic detection ( Figure 1 A), it can be seen that compared with normal rats, the kidney morphology of UUO rats has changed, and the kidney volume has increased significantly. This is because the ureter of the surgical side kidney is obstructed, resulting in the obstruction of urine excretion, the increase of intrapelvic pressure, the dilation of renal pelvis and calyces, and then the formation of hydronephrosis, ultimately leading to weight gain. Under the detection of color Doppler mode, red usually indicates blood flow towards the probe, and blue indicates blood flow away from the probe. Compared with normal rats, the blood flow distribution in the kidneys of UUO rats is less, and there may be a situation of vascular stenosis or occlusion caused by urine retention. Ammonium glycyrrhizinate and pirfenidone can improve the increase of intrapelvic pressure and the dilation of renal pelvis and calyces, and partially restore the renal blood flow of UUO rats, and the improvement effect of ammonium glycyrrhizinate is better than that of pirfenidone.

[0023] The effects of ammonium glycyrrhizinate on histological changes and mesangial matrix deposition in the kidneys were evaluated by HE, PAS, and Masson staining. HE staining showed that compared with normal rats, UUO rats had hydronephrosis, progressive dilation of the renal pelvis, and thinning of the renal cortex. Ammonium glycyrrhizinate and pirfenidone could improve the damage of renal tubules and glomeruli, and the improvement effect of ammonium glycyrrhizinate was better than that of pirfenidone ( Figure 1B). Masson staining showed that the collagen deposition in the glomeruli of the kidneys of UUO rats increased ( Figure 1 B), ammonium glycyrrhizinate and pirfenidone could reduce the collagen deposition in the glomeruli of UUO rats, and the effect of ammonium glycyrrhizinate was better than that of pirfenidone ( Figure 1 B). PAS staining showed that compared with normal rats, the PAS-positive mesangial matrix in the glomeruli of UUO rats increased ( Figure 1 B), ammonium glycyrrhizinate and pirfenidone could reduce the PAS-positive mesangial matrix in the glomeruli of UUO rats, and the effect of ammonium glycyrrhizinate was better than that of pirfenidone ( Figure 1 B). Thus, it can be seen that ammonium glycyrrhizinate has the potential to improve renal fibrosis in UUO rats, providing a theoretical basis and experimental evidence for the development of new anti-fibrotic drugs.

[0024] Example 2 Effect of ammonium glycyrrhizinate on the viability of HK2 cells I. Experimental materials Including human renal tubular epithelial cells HK-2 (provided by Shanghai Meiwan Biotechnology Co., Ltd.), human glomerular mesangial cells HRMC (provided by Shanghai Meiwan Biotechnology Co., Ltd.) and DMEM / F-12 (1:1) medium (purchased from Thermo Fisher Scientific).

[0025] II. Experimental methods To determine the appropriate drug concentration, a CCK8 cell proliferation and toxicity detection kit was used to evaluate cell viability. Briefly, HK-2 cells were seeded in 96-well plates at a density of 1×10 4 cells per well, cultured for 24 h, and after sufficient attachment, were treated with different doses of ammonium glycyrrhizinate (0, 0.5, 2.5, 5, 10, 20, 40, 80, 160 μM) added to the medium and cultured for 48 h. After washing with PBS, a basic medium containing CCK8 solution was added and incubated in the dark for 2 h. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay reader. To more intuitively observe the cell morphology, HK-2 cells were seeded in 6-well plates at a density of 8×10 5 cells per well, cultured for 24 h, and after sufficient attachment, were treated with different doses of ammonium glycyrrhizinate (0, 0.5, 2.5, 5, 10, 20, 40, 80, 160 μM) added to the medium and cultured for 48 h. After washing twice with PBS, the 6-well plates were placed on the stage of a microscope and light microscope images of the cells were taken.

[0026] III. Experimental results From the light microscope images of the cells and the results of CCK8 ( Figure 2 ), it can be seen that ammonium glycyrrhizinate had little effect on the viability of human renal tubular epithelial cells HK-2, and the cell morphology and viability basically remained normal.

[0027] Example 3. Expression of E-cadherin, N-cadherin and Vimentin proteins in HK-2 human renal tubular epithelial cells induced by ammonium glycyrrhizinate and improved by TGF-β1 I. Experimental materials The experimental materials follow the same materials as in Example 2.

[0028] II. Experimental methods (1) Cell culture and grouping: Place sterile cover slips that have been passed through fire in a six-well plate. After the cover slips have cooled, inoculate HK-2 cells into the six-well plate at an appropriate density. When the cell density reaches 50%, replace the medium with serum-free DMEM / F12 medium and place it for 12 h. Subsequently, divide the cells into six groups for different treatments. One group serves as the blank control group with normal medium, and another group is replaced with 10% FBS medium containing 10 ng / mL TGF-β1 as the model group. The remaining three groups are respectively added with different concentrations of ammonium glycyrrhizinate (2.5 μM, 10 μM, 40 μM) on the basis of the model group. The last group is cultured with pirfenidone (0.5 mg / mL) on the basis of the model group for 24 hours and then subsequent experiments are carried out.

[0029] (2) Immunofluorescence detection: Detection of the expression of E-cadherin, N-cadherin, and Vimentin proteins in HK-2 cells was performed by immunofluorescence method (ImmunoFluroscence, IF). The specific steps of cell immunofluorescence detection are as follows: First, wash the collected cell samples twice with PBS, then fix them with 4% paraformaldehyde at room temperature for 15 minutes, and then permeabilize them with 0.1% Triton X-100 in PBS for 10 minutes; After that, block the cells with 10% goat serum for 30 minutes, and then incubate them overnight at 4°C with the primary antibody containing 10% goat serum; Subsequently, wash three times with PBS, and then incubate in the dark box with DyLight 488-conjugated AffiniPure goat anti-mouse IgG (H+L) or CY3-conjugated AffiniPure goat anti-rabbit IgG (H+L) for 1.5 hours. The cell nuclei were stained with DAPI staining reagent for 30 minutes. Finally, place the cover slips with cells on the glass slides, and there is an anti-fluorescence quenching mounting medium on the glass slides. Apply a circle of nail polish around the edge of the cover slip to fix the coverslip. After the coverslip is fixed, use a single-photon confocal microscope to collect images.

[0030] III. Experimental results E-cadherin, an inter-epithelial cell adhesion protein, has weak fluorescence intensity in the control group. After treatment with TGF-β1, its fluorescence intensity indicates increased cell-cell adhesion and aggravated fibrosis. After intervention with ammonium glycyrrhizinate, the fluorescence intensity shows a decreasing trend (Figure 3 ). N-cadherin is a calcium-dependent transmembrane glycoprotein mainly expressed in mesenchymal cells, with a relatively high fluorescence intensity in the control group (Con group). After treatment with TGF-β1, the fluorescence intensity decreased significantly. After intervention with ammonium glycyrrhizinate, the fluorescence intensity increased ( Figure 3 ). Vimentin, as an intermediate filament protein, had a relatively high fluorescence intensity in the control group (Con group), with significant expression in the cell-cell contact area, showing a concentrated specific fluorescence localization. After treatment with TGF-β1, the fluorescence intensity decreased significantly. After intervention with ammonium glycyrrhizinate, the fluorescence intensity increased ( Figure 3 ).

[0031] In summary, the effect of ammonium glycyrrhizinate on HK-2 cell fibrosis was manifested as inhibiting abnormal cell adhesion and migration ability, enhancing the structural stability of cells by regulating the expression of E-Cadherin, N-Cadherin and Vimentin, thereby reducing the degree of fibrosis, and its effect was better than that of PFD.

[0032] Example 4 Ammonium glycyrrhizinate reduces the opening of mitochondrial permeability transition pores in HK-2 cells I. Experimental materials The experimental materials followed the same materials as in Example 2 and the mitochondrial permeability transition pore (MPTP) detection kit.

[0033] II. Experimental methods The experimental methods were as follows: (1) Cell culture and grouping followed the same procedure as in Example 3.

[0034] (2) Detection of mitochondrial permeability transition pores: The MPTP detection kit was used to measure the opening degree of mitochondrial permeability transition pores. The fluorescence quenching solution was composed of Calcein AM staining solution and CoCl2. At 37 °C, the above mixed working solution was applied to the cells for 60 minutes, followed by incubation with fresh medium for 30 minutes, washing with PBS, and then staining with Hoechst 33342 for 45 minutes, and image acquisition was performed using a single-photon confocal microscope.

[0035] III. Experimental results The experimental results showed that in TGF-β1-induced HK-2 cells, an increase in the opening degree of MPTP was observed, indicating impaired mitochondrial function. In HK-2 cells treated with ammonium glycyrrhizinate, the opening degree of MPTP decreased ( Figure 4 ). The experimental results revealed that ammonium glycyrrhizinate could improve mitochondrial function by alleviating the opening degree of MPTP in HK2 cells.

[0036] Example 5 Ammonium Glycyrrhizinate Increases the Mitochondrial Membrane Potential of HK-2 Cells and Decreases the Mitochondrial Superoxide Level I. Experimental Materials The experimental materials follow the same materials as in Example 2, a mitochondrial membrane potential detection kit (JC-1) and a mitochondrial superoxide red fluorescent probe (Mito SOX).

[0037] II. Experimental Methods Apply a mitochondrial membrane potential assay kit containing JC-1 to measure the mitochondrial membrane potential (MMP) level. Dilute the probe to a 1× concentration with JC-1 buffer and continue to incubate the cells in each group at 37°C for 90 minutes. Counterstain the cell nuclei with Hoechst 33342 staining solution at room temperature for 60 minutes, and perform image acquisition using a single-photon confocal microscope. Detect mitochondrial superoxide using a mitochondrial superoxide red fluorescence (MitoSOX Red) probe. Dilute the probe to 5 µM with Hank's balanced salt solution (HBSS) and incubate the cells in each group at 37°C for 30 minutes in the dark. After washing twice with PBS, counterstain the cell nuclei with Hoechst 33342 solution diluted with HBSS for 45 minutes, wash four times with PBS for 5 minutes each, and then perform image acquisition using a single-photon confocal microscope.

[0038] III. Experimental Results The experimental results showed that in TGF-β1-induced HK-2 cells, a decrease in MMP was observed, indicating impaired mitochondrial function. However, after treatment with ammonium glycyrrhizinate, the decrease in MMP was significantly reversed ( Figure 5 ). At the same time, TGF-β1 induced a large amount of ROS in the mitochondria of HK-2 cells, while after treatment with ammonium glycyrrhizinate, the ROS level in the mitochondria was significantly reduced. Based on the above results, ammonium glycyrrhizinate can improve the reduction of MMP and the increase in mitochondrial ROS level induced by TGF-β1 in HK-2 cells, thereby improving mitochondrial function, and its effect is better than that of PFD.

[0039] Example 6 Ammonium Glycyrrhizinate Significantly Reduces the Level of Mitophagy in TGF-β1-Induced HK-2 Cells I. Experimental Materials The experimental materials follow the same materials as in Example 2. A mitophagy fluorescent probe (Mtphagy Dye), a lysosome fluorescent dye (Lyso Dye), and a MitoTracker® Deep Red FM mitochondrial deep red fluorescent probe.

[0040] II. Experimental Methods The culture and grouping of cells followed the same procedure as in Example 3. HK-2 cells were seeded in glass-bottom dishes at an appropriate density and cultured overnight. The culture medium was removed, and the cells were washed twice with serum-free medium. Working solution 1 was prepared at a ratio of Mtphagy Dye stock solution: serum-free medium = 1:1000. 1 mL of working solution 1 was added to each glass-bottom dish, and the cells in each group were incubated at 37 °C for 30 minutes. After incubation, the cells were washed twice with serum-free medium. The corresponding concentration of the modeling drug or therapeutic drug was added to the glass-bottom dishes according to the grouping, and the cells were incubated at 37 °C for 48 hours. After the culture was completed, the cells were washed twice with serum-free medium. Working solution 2 was prepared at a ratio of Lyso Dye stock solution: serum-free medium = 1:1000. 1 mL of working solution 2 was added to each glass-bottom dish, and the cells in each group were incubated at 37 °C for 30 minutes. After the culture was completed, the cells were washed twice with serum-free medium. Working solution 3 was prepared at a ratio of MitoTracker® Deep Red FM stock solution: serum-free medium = 1:4000. 1 mL of working solution 3 was added to each glass-bottom dish, and the cells in each group were incubated at 37 °C for 45 minutes. After the culture was completed, the cells were washed twice with serum-free medium. Finally, the cell nuclei were counterstained with Hoechst 33342 solution diluted with HBSS for 45 minutes, washed three times with serum-free medium, and then images were collected using a single-photon confocal microscope.

[0041] III. Experimental Results The experimental results showed that under normal conditions, the level of mitophagy was relatively low because the mitochondrial function in the cells was normal and there was no need to frequently remove damaged mitochondria ( Figure 6 ). When the cells were stimulated with TGF-β1, mitophagy increased significantly, indicating that TGF-β1 induced mitochondrial damage ( Figure 6 ). When ammonium glycyrrhizinate was added, the level of mitophagy decreased, inhibiting the signal of mitochondrial damage and thus reducing the occurrence of mitophagy ( Figure 6 ). This regulatory mechanism helps protect cells from damage caused by excessive autophagy, maintain normal mitochondrial function, alleviate the occurrence of renal fibrosis, and its effect is better than that of PFD.

[0042] Example 7 Ammonium Glycyrrhizinate Significantly Reduces the Expression of Fibronectin FN and Collagen COL in HRMC Cells I. Experimental Materials The experimental materials followed the same materials as in Example 2.

[0043] II. Experimental Methods The culture and grouping of cells followed the same procedure as in Example 3. Immunofluorescence detection followed the same procedure as in Example 3.

[0044] III. Experimental Results Both FN and COL play crucial roles in renal fibrosis, and their overexpression promotes the progression of renal interstitial fibrosis. The experimental results show that the contents of FN and COL proteins in TGF-β1-induced HRMC cells increase significantly. Ammonium glycyrrhizinate plays a positive role in improving renal interstitial fibrosis by reducing the expression of FN and COL proteins, and its effect is better than that of PFD ( Figure 7 ).

[0045] In summary, these results indicate that ammonium glycyrrhizinate can reduce renal fibrosis by decreasing extracellular matrix (ECM) deposition and improving renal cortex injury, and its effect is better than that of pirfenidone.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: the specific implementation manners of the present invention can still be modified or equivalently replaced, and any modification or equivalent replacement without departing from the spirit and scope of the present invention should be covered by the protection scope of the claims of the present invention.

Claims

1. An application of ammonium glycyrrhizinate or its preparation, characterized in that: The application includes any of the following: A1) Use of ammonium glycyrrhizinate in the preparation of a drug for preventing and treating renal fibrosis caused by urinary tract obstruction; A2) Use of ammonium glycyrrhizinate in a drug for inhibiting the process of epithelial-mesenchymal transition induced by TGF-β1 in human renal tubular epithelial cells; A3) Application of ammonium glycyrrhizinate in increasing the expression of E-cadherin protein in human renal tubular epithelial cells; A4) Use of ammonium glycyrrhizinate in the preparation of a drug for increasing the expression of E-cadherin protein in human renal tubular epithelial cells; A5) Use of ammonium glycyrrhizinate in reducing the expression of N-cadherin protein in human renal tubular epithelial cells; A6) Use of ammonium glycyrrhizinate in the preparation of a drug for reducing the expression of N-cadherin protein in human renal tubular epithelial cells; A7) Application of ammonium glycyrrhizinate in reducing the expression of Vimentin protein; A8) Use of ammonium glycyrrhizinate in the preparation of a drug for reducing the expression of Vimentin protein; A9) Use of ammonium glycyrrhizinate in the preparation of a medicament for preventing and / or treating diseases associated with renal fibrosis. A10) Use of ammonium glycyrrhizinate in reducing the expression of Fn protein in human glomerular mesangial cells; A11) Use of ammonium glycyrrhizinate in the preparation of a drug for reducing Fn protein expression; A12) Use of ammonium glycyrrhizinate in reducing COL protein expression in human glomerular mesangial cells; A13) Use of ammonium glycyrrhizinate in the preparation of a drug for reducing COL protein expression.

2. The use according to claim 1, characterized in that , the renal fibrosis-related improvements include increased intra-renal pelvic pressure, dilation of the renal pelvis and calyces, and restoration of renal blood flow.

3. The use according to claim 1, characterized in that: The disease associated with renal fibrosis is chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

4. A drug for treating kidney disease, characterized in that: The active ingredient of the medicine is ammonium glycyrrhizinate.

5. The drug according to claim 4, characterized in that The renal disease is renal fibrosis, chronic kidney disease or nephrosclerosis caused by urinary tract obstruction.

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