Application of Danshensu in preparation of products for preventing and treating peritoneal dialysis related peritoneal fibrosis

Abstract

The application discloses application of Danshensu in preparation of a product for preventing and treating peritoneal dialysis related peritoneal fibrosis, and belongs to the field of medicines. The application proves that direct use of Danshensu can improve peritoneal dysfunction induced by peritoneal dialysis, reduce peritoneal thickening and collagen deposition, inhibit epithelial-mesenchymal transition of peritoneal mesothelial cells, reduce expression of alpha-SMA and VEGFA, increase expression of E-cadherin, and resist peritoneal fibrosis through inhibition of a STAT3 signal pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of tanshinone in the preparation of products for the prevention and treatment of peritoneal dialysis-related peritoneal fibrosis (PD-PF). Background Technology

[0002] Peritoneal dialysis (PD) is an important alternative treatment for end-stage renal disease, offering advantages such as home-based convenience and hemodynamic stability. However, long-term use of high-glucose dialysate can lead to peritoneal fibrosis (PF), characterized by peritoneal thickening, collagen deposition, and decreased ultrafiltration function, potentially resulting in dialysis failure in severe cases. One of the core mechanisms of PF is the epithelial-mesenchymal transition (EMT) in peritoneal mesothelial cells, which leads to a decrease in E-cadherin, an increase in α-SMA, and promotes extracellular matrix deposition.

[0003] Currently, there are no specific drugs for pulmonary fibrosis (PF) associated with liver disease (PD). Existing research on tanshinone (DSS) mainly focuses on liver and lung fibrosis, and its application in PD-related PF has not yet been observed. Although there are studies on the anti-fibrotic effect of "tanshinone pretreatment of MSC exosomes," this is an exosome engineered system, which is not compatible with the direct application of small molecules in terms of technology and mechanism of action. Therefore, the development of small molecule drugs that can inhibit the progression of PF has significant clinical implications. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides for the first time the use of tanshinone in the prevention and treatment of PD-PF through direct application.

[0005] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: This invention mainly provides the application of tanshinone in the preparation of products for the prevention or treatment of PD-PF, wherein tanshinone is the only active pharmaceutical ingredient.

[0006] The molecular formula of tanshinone is C9H. 10 O5, with a molecular weight of 198.17 g / mol, has the following structural formula: Furthermore, the product can improve peritoneal dysfunction associated with peritoneal dialysis, including at least one of the following: decreased ultrafiltration function, increased protein leakage of dialysate, abnormal glucose transport, and abnormal urea nitrogen clearance.

[0007] Furthermore, the product can reduce peritoneal thickening and / or collagen fiber deposition.

[0008] Furthermore, the product can inhibit the epithelial-mesenchymal transition of peritoneal mesothelial cells.

[0009] Furthermore, the product can reduce the expression of α-SMA and / or VEGFA, and / or increase the expression of E-cadherin.

[0010] Furthermore, the product exerts its anti-peritoneal fibrosis effect by inhibiting the STAT3 and / or phosphorylating STAT3 signaling pathways.

[0011] Furthermore, the product includes pharmaceutical preparations.

[0012] Furthermore, the pharmaceutical preparation also includes a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the dosage form of the pharmaceutical preparation is an oral preparation or an injectable preparation. The oral preparation includes any one of tablets, capsules, granules, and solutions, and the injectable preparation includes any one of solutions, emulsions, or suspensions.

[0014] Furthermore, the product can be used in combination with peritoneal dialysis fluid.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to demonstrate that tanshinone small molecules can be directly used for the treatment of PD-PF; (2) This invention demonstrates, through a mouse peritoneal fibrosis model induced by peritoneal dialysis fluid, that tanshinone can significantly reduce peritoneal thickening, inflammatory cell infiltration and collagen fiber deposition. (3) This invention confirms that tanshinone can improve peritoneal ultrafiltration function, reduce protein leakage of dialysate, and improve glucose and urea nitrogen clearance function; (4) This invention confirms that tanshinone can reduce the expression of α-SMA and VEGFA and increase the expression of E-cadherin, thereby inhibiting EMT and angiogenesis; (5) This invention relates to the direct drug use of tanshinone small molecules, which is significantly different from existing exosome-engineered therapeutic systems; (6) This invention provides a theoretical basis and application value for the subsequent development of tanshinone into a drug for the prevention and treatment of PD-PF. Attached Figure Description

[0016] Figure 1 Tanshinone improves PD-induced peritoneal structural and functional damage in mice. A: HE staining; B: Masson staining; C: Peritoneal thickness; D: Ultrafiltration volume; E: Drainage fluid protein concentration; F: Blood urea nitrogen clearance rate; G: Glucose clearance rate.

[0017] Figure 2 Tanshinone improves abnormal expression of peritoneal fibrosis-related EMT and angiogenesis induced by PD. AB: α-SMA; CD: E-cadherin; EF: VEGFA. All are immunohistochemical.

[0018] Figure 3 Tanshinone improves abnormal EMT-related expression in HMrSV5 cells induced by PD dialysis fluid. A: Cell viability; B: Western blot; C: α-SMA protein expression; D: VEGFA protein expression; E: E-cadherin protein expression.

[0019] Figure 4 Effects of tanshinone on the abnormal expression of related proteins. A: Expression of STAT3 and p-STAT3 proteins; B: Quantitative results; C: Static intervention; D: Colivelin intervention.

[0020] Figure 5 Tanshinone interacts with STAT3 protein. A: Molecular docking; B: SPR detection. Detailed Implementation

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0022] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0023] Example 1 The purpose of this embodiment is to establish a peritoneal fibrosis model, compare the conditions of different treatment groups, and thus verify the role of tanshinone in alleviating PD-PF.

[0024] 1. Drug Information The raw material, tanshinone (DSS), was purchased from Selleck, with an HPLC purity >98%, CAS number 76822-21-4, and molecular weight of 198.17.

[0025] 2. Establishment of a peritoneal fibrosis model Male C57BL / 6J mice aged 10-12 weeks were selected and provided by Guangzhou Ruige Biotechnology Co., Ltd. All mice were housed in an SPF-grade animal facility with free access to food and water. The experimental environment was maintained at a temperature of 22-25℃, a relative humidity of 50%-60%, and a 12-hour day-night cycle.

[0026] A peritoneal fibrosis model related to peritoneal dialysis was established by continuous intraperitoneal injection of 4.25% glucose peritoneal dialysis solution. Except for the control group, mice in the other groups were injected intraperitoneally with 3 mL of 4.25% glucose peritoneal dialysis solution daily for 4 weeks to induce peritoneal thickening, collagen deposition, and peritoneal dysfunction.

[0027] 3. Mouse grouping All mice were randomly divided into four groups of six each: (1) Control group: The same volume of normal saline was injected into the peritoneum daily; (2) Model group (PD group): Daily intraperitoneal injection of 4.25% glucose peritoneal dialysis solution; (3) Low-dose tanshinone treatment group (29 mg / kg DSS group): 4.25% glucose peritoneal dialysis solution was injected into the peritoneum daily, and 29 mg / kg tanshinone was administered at the same time.

[0028] (4) High-dose tanshinone treatment group (58 mg / kg DSS group): 4.25% glucose peritoneal dialysis solution was injected into the peritoneum daily, and 58 mg / kg tanshinone was administered at the same time.

[0029] 4. Administration method and dosage in mice Tanshinone was prepared in a suitable solvent before use and administered via intraperitoneal injection once daily for 4 consecutive weeks. The model group received an equal volume of 4.25% glucose peritoneal dialysis solution, while the control group received an equal volume of physiological saline.

[0030] Throughout the experiment, the mice's mental state, diet, and activity level were observed regularly, and weight changes were recorded.

[0031] 5. Anesthesia and tissue sampling After the experiment, peritoneal function was tested on mice in each group.

[0032] Each mouse was intraperitoneally injected with 3 mL of 4.25% glucose peritoneal dialysis fluid. After retention for 120 min, the drainage volume of the peritoneal dialysis fluid was recorded. Subsequently, the peritoneal cavity was cut along the midline, and residual fluid in the peritoneal cavity was absorbed using sterile gauze and weighed. The peritoneal ultrafiltration volume (UF) was calculated by subtracting the weight of the dry gauze. Peritoneal dialysis drainage fluid and peripheral blood samples were collected simultaneously to detect dialysate protein concentration, blood urea nitrogen, and glucose levels. After peritoneal function testing, the mice were euthanized under isoflurane anesthesia, and the peritoneal cavity was opened along the midline to expose the peritoneal tissue. After separating the parietal peritoneal tissue, it was gently rinsed with physiological saline, the surface fluid was blotted dry with filter paper, and then fixed with 4% paraformaldehyde for subsequent HE staining, Masson staining, and immunohistochemical detection.

[0033] 6. Peritoneal function test Peritoneal function in mice of different groups was evaluated using a peritoneal dialysis equilibrium test. The main indicators measured included peritoneal ultrafiltration volume, protein concentration in the dialysis drainage fluid, urea nitrogen clearance rate, and glucose clearance rate. Peritoneal permeability was evaluated by measuring the ratio of urea nitrogen concentration in the dialysate to that in the blood (D / P) and the change in glucose concentration in the dialysate (D / D0).

[0034] The results showed that long-term treatment with 4.25% glucose peritoneal dialysis fluid resulted in significant peritoneal thickening, decreased peritoneal ultrafiltration function, and increased protein concentration in the drainage fluid in the model group mice, along with abnormal urea nitrogen and glucose clearance. However, treatment with tanshinone resulted in reduced peritoneal thickness, significantly increased ultrafiltration volume, significantly decreased protein concentration in the drainage fluid, and significantly improved urea nitrogen and glucose clearance. These results indicate that tanshinone can improve peritoneal dysfunction induced by peritoneal dialysis. Figure 1 C, D, E, F, G).

[0035] 7. HE and Masson staining After fixation, the peritoneal tissue was dehydrated with graded ethanol, embedded in paraffin, and sectioned. It was then stained with hematoxylin and eosin (HE) and Masson's stain, respectively. HE staining was performed using hematoxylin and eosin. Masson's staining was performed using hematoxylin, brilliant green, and aniline blue. After staining, the tissue was dehydrated, cleared, and mounted. Images were then observed and acquired under a microscope. Ten high-power fields were randomly selected from each section to measure the peritoneal thickness, and the average value was taken.

[0036] HE staining results showed that the peritoneum of mice in the blank group was intact, thin, and had neatly arranged cells; the peritoneum of mice in the model group was significantly thickened, with increased inflammatory cell infiltration and disordered tissue structure; after treatment with tanshinone, the peritoneal tissue was more densely arranged, inflammatory cell infiltration was significantly reduced, and peritoneal thickness was significantly decreased. Masson staining results showed that the peritoneal tissue of the model group had significantly increased blue-stained collagen fibers, indicating increased extracellular matrix deposition; after treatment with tanshinone, collagen fiber deposition in the peritoneal tissue was significantly reduced. These results indicate that tanshinone can alleviate peritoneal thickening, inflammatory infiltration, and collagen deposition induced by peritoneal dialysis. Figure 1 A, B).

[0037] 8. Immunohistochemical detection To further evaluate the effects of tanshinone on peritoneal fibrosis and EMT, immunohistochemical (IHC) analysis was performed on peritoneal tissues from mice in each group. Paraffin sections were dewaxed with xylene and then hydrated using a gradient of ethanol. They were subsequently heated with citrate antigen retrieval solution, cooled naturally, and washed with PBS buffer. Sections were treated with 3% hydrogen peroxide to block endogenous peroxidase activity, followed by blocking with goat serum at room temperature for 30 min. After blocking, α-SMA antibody, E-cadherin antibody, and VEGFA antibody were added, and the sections were incubated overnight at 4°C. The next day, the sections were washed three times with PBS buffer for 5 min each time, followed by incubation with HRP-labeled secondary antibody at room temperature for 30 min. After a second wash, DAB chromogenic solution was used for development, with the development time controlled under a microscope. Finally, hematoxylin counterstaining was performed on the cell nuclei, followed by dehydration, clearing, and mounting. Images were then observed and acquired under a microscope.

[0038] Immunohistochemical results showed that E-cadherin expression was high and α-SMA and VEGFA expression was low in the peritoneal tissue of mice in the blank control group. In the model group, α-SMA expression was significantly increased and E-cadherin expression was significantly decreased in the peritoneal tissue, suggesting epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells; simultaneously, VEGFA expression was significantly increased, suggesting enhanced peritoneal angiogenesis. After treatment with tanshinone, the expression of α-SMA and VEGFA in the peritoneal tissue was significantly decreased, while E-cadherin expression was significantly increased. These results indicate that tanshinone can inhibit peritoneal dialysis-induced EMT and angiogenesis, thereby alleviating peritoneal fibrosis. Figure 2 ).

[0039] Example 2 The purpose of this embodiment is to further evaluate the effects of tanshinone on peritoneal mesothelial cell damage and EMT induced by peritoneal dialysis fluid by selecting human peritoneal mesothelial cells HMrSV5 for in vitro experiments.

[0040] 1. Cell Culture Human peritoneal mesothelial cells (HMrSV5) were purchased from Guangzhou Genio Biotechnology Co., Ltd. Cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were incubated at 37°C in a 5% CO2 incubator. Cells were used for subsequent experiments after reaching 70%-80% confluence.

[0041] 2. Establishment of a peritoneal dialysis fluid-induced cell damage model Previous studies have shown that stimulation with 4.25% glucose peritoneal dialysis fluid for 48 h can induce fibrosis-like changes in HMrSV5 cells. Therefore, in this embodiment, HMrSV5 cells were stimulated with 4.25% glucose peritoneal dialysis fluid for 48 h to establish an in vitro peritoneal mesothelial cell injury and EMT model.

[0042] 3. Tanshinone treatment method and concentration After culturing HMrSV5 cells for 24 h, a culture system containing 4.25% glucose peritoneal dialysis fluid was added, along with different concentrations of tanshinone. The final concentrations of tanshinone were 0 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM, with at least 3 replicates per group, and the cells were cultured for a total of 48 h.

[0043] 4. Cell viability detection After co-culturing for 48 h, 10 μL of CCK8 solution was added to each well, and the cells were incubated at 37℃ for 2 h. The absorbance at 450 nm was then measured using a microplate reader to evaluate the effect of different concentrations of tanshinone on the viability of HMrSV5 cells.

[0044] The results showed that tanshinone did not exhibit significant toxicity to HMrSV5 cells within the concentration range of 0-200 μM, and could improve PD-induced cell viability decline. Figure 3 A).

[0045] 5. Western blot detection HMrSV5 cells were stimulated with 4.25% glucose peritoneal dialysis solution and then treated with 20 μM, 100 μM, and 200 μM tanshinone for 48 h. After the experiment, the culture medium was discarded, and the cells were washed twice with PBS buffer, then lysed with RIPA lysis buffer and on ice for 30 min. The cells were then centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected for protein extraction. After determining the protein concentration using the BCA method, an equal volume of protein sample was added to loading buffer and denatured in a 100 °C metal bath for 10 min. Proteins were then separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 h, then α-SMA, VEGFA, and E-cadherin primary antibodies were added, and the membrane was incubated overnight at 4 °C. The next day, the membrane was washed three times with TBST buffer for 10 min each time, followed by incubation with the corresponding HRP-labeled secondary antibody at room temperature for 1 h. After washing the membrane again, it was developed using ECL chemiluminescent reagent, and images were acquired using a gel imaging system.

[0046] Western blot results showed that stimulation with 4.25% glucose peritoneal dialysis fluid significantly increased the expression of α-SMA and VEGFA proteins in HMrSV5 cells, while decreasing the expression of E-cadherin, suggesting EMT and fibrosis-related changes in the cells. Tanshinone treatment significantly decreased the expression of α-SMA and VEGFA proteins, while significantly increasing the expression of E-cadherin. Furthermore, the ameliorative effect gradually increased with increasing tanshinone concentration, with the 200 μM group showing the most significant effect. These results indicate that tanshinone can improve peritoneal dialysis fluid-induced EMT and fibrosis-related phenotypic changes in HMrSV5 cells, thereby exerting an anti-peritoneal fibrosis effect. Figure 3 B, C, D, E).

[0047] Example 3 The purpose of this embodiment is to further evaluate the effect of tanshinone on improving the abnormal expression of related proteins induced by peritoneal dialysis fluid, and to observe the changes in STAT3-related proteins.

[0048] 1. Cell culture and model establishment Human peritoneal mesothelial cells (HMrSV5) were selected for the experiment. Cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin, and streptomycin in a 37°C, 5% CO2 incubator. Once the cells reached 70%-80% confluence, they were stimulated with 4.25% glucose peritoneal dialysis solution for 48 h to establish an in vitro peritoneal mesothelial cell EMT injury model.

[0049] 2. Tanshinone treatment After culturing HMrSV5 cells for 24 h, a culture system containing 4.25% glucose peritoneal dialysis fluid was added, and different concentrations of tanshinone were administered simultaneously. The final concentration of tanshinone was 200 μM. The cells were cultured for a total of 48 h.

[0050] 3. STAT3 inhibitor and agonist intervention experiments To further verify the involvement of STAT3-related pathways in the action of tanshinone, intervention experiments were conducted using the STAT3 inhibitor Static (HY-13818, MCE) and the STAT3 agonist Colivelin (S9664, Selleck), respectively. The final concentration of Static was 10 μM, and the final concentration of Colivelin was 1 μM. Cells in each group were treated for 48 h before subsequent assays.

[0051] 4. Western blot detection After the experiment, the culture medium was discarded, and the cells were washed twice with PBS buffer. RIPA lysis buffer was added, and the cells were lysed on ice for 30 min. Subsequently, the cells were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected for protein extraction. After determining the protein concentration using the BCA method, an equal volume of protein sample was added to loading buffer and denatured in a 100°C metal bath for 10 min. SDS-PAGE gel electrophoresis was then performed, and the sample was transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder at room temperature for 1 h, and then primary antibodies against STAT3, p-STAT3, VEGFA, and E-cadherin were added, respectively, and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST buffer for 10 min each time, followed by incubation with the corresponding HRP-labeled secondary antibody at room temperature for 1 h. After washing again, the membrane was developed using ECL chemiluminescence reagent, and images were acquired using a gel imaging system.

[0052] The results showed that stimulation with 4.25% glucose peritoneal dialysis fluid increased the expression of STAT3 and p-STAT3 in HMrSV5 cells, while VEGFA expression increased and E-cadherin expression decreased. Tanshinone treatment decreased the expression of STAT3 and p-STAT3, decreased VEGFA expression, and increased E-cadherin expression, with the ameliorative effect gradually increasing with increasing tanshinone concentration. Furthermore, the STAT3 inhibitor Stattic enhanced the ameliorative effect of tanshinone on the expression of related abnormal proteins, while the STAT3 agonist Colivelin partially attenuated the ameliorative effect of tanshinone. These results indicate that tanshinone can improve peritoneal dialysis fluid-induced EMT-related abnormal expression (…). Figure 4 ).

[0053] 5. Detection of the binding activity of tanshinone to STAT3 protein The binding between tanshinone and STAT3 protein was detected using surface plasmon resonance (SPR) technology.

[0054] The experiment was conducted using a Biacore T200 surface plasmon resonance (SPR) instrument. A CM5 sensor chip was selected, with the Fc1 channel used as the reference channel and the Fc4 channel as the detection channel. After activating the chip surface using EDC / NHS, recombinant human STAT3 protein was immobilized in the Fc4 channel via amine coupling at a concentration of 60 μg / mL, with an immobilization level of approximately 8800 RU. Unreacted active groups were then blocked using ethanolamine. Tanshinone was prepared into gradient solutions of different concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, and 50 μM), and injected sequentially in ascending order of concentration. The injection flow rate was 30 μL / min, the binding time was 120 s, and the dissociation time was 300 s. After detection, the signals from the reference channel and the blank control were subtracted, and the data were analyzed using Biacore T200 Evaluation Software.

[0055] The results showed that tanshinone and STAT3 protein have a certain interaction ability. Figure 5 A) As the concentration of tanshinone increases, the binding response signal gradually strengthens. Kinetic fitting analysis shows that the equilibrium dissociation constant (KD) between the two is approximately 2.53 μM, indicating a certain binding activity between tanshinone and STAT3 protein. Figure 5 B).

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of tanshinone in the preparation of products for the prevention or treatment of peritoneal fibrosis associated with peritoneal dialysis, characterized in that, Tanshinone is the only active pharmaceutical ingredient in the product.

2. The application according to claim 1, characterized in that, The product can improve peritoneal dysfunction associated with peritoneal dialysis, including at least one of the following: decreased ultrafiltration function, increased protein leakage of dialysate, abnormal glucose transport, and abnormal urea nitrogen clearance.

3. The application according to claim 1, characterized in that, The product can reduce peritoneal thickening and / or collagen fiber deposition.

4. The application according to claim 1, characterized in that, The product can inhibit the epithelial-mesenchymal transition of peritoneal mesothelial cells.

5. The application according to claim 4, characterized in that, The product can reduce the expression of α-SMA and / or VEGFA, and / or increase the expression of E-cadherin.

6. The application according to claim 1, characterized in that, The product exerts its anti-peritoneal fibrosis effect by inhibiting the STAT3 and / or phosphorylated STAT3 signaling pathways.

7. The application according to claim 1, characterized in that, The products include pharmaceutical preparations.

8. The application according to claim 7, characterized in that, The pharmaceutical preparation also includes a pharmaceutically acceptable carrier or excipient.

9. The application according to claim 7, characterized in that, The dosage form of the pharmaceutical preparation is an oral preparation or an injectable preparation. The oral preparation includes any one of tablets, capsules, granules, and solutions, and the injectable preparation includes any one of solutions, emulsions, or suspensions.

10. The application according to claim 1, characterized in that, The product can be used in combination with peritoneal dialysis fluid.

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