Peritoneal dialysis fluid containing baicalein and preparation method and use thereof

Abstract

The present invention belongs to the field of medical technology, and in particular to a peritoneal dialysis fluid containing baicalein and its preparation method and use. The preparation method of the peritoneal dialysis fluid containing baicalein, comprises the following steps: step 1: baicalein is dissolved in DMSO and configured to baicalein-DMSO storage solution; step 2: baicalein-DMSO storage solution is added to the peritoneal dialysis fluid, and then supplemented with ultrasound, and the ultrasound is completed to obtain the peritoneal dialysis fluid containing baicalein. By the above-mentioned preparation method, baicalein is successfully dissolved in the peritoneal dialysis fluid and can be stably stored at room temperature, which provides a new strategy for developing novel peritoneal dialysis (PD) drugs to prevent and treat peritoneal fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and in particular relates to a peritoneal dialysis fluid containing baicalein, and a preparation method and application thereof. Background Art

[0002] Peritoneal dialysis (PD) is an important alternative treatment for end-stage renal disease (ESRD). Effective PD relies on the structural and functional integrity of the peritoneum. Progressive peritoneal fibrosis is a common complication of long-term PD, ultimately leading to embedded peritoneal sclerosis (EPS), ultrafiltration failure, and increased mortality. EPS is characterized by chronic inflammation, significant peritoneal thickening, systemic nutritional failure, and associated intestinal adhesions and obstruction. Although the precise mechanisms remain to be determined, accumulating evidence suggests that peritoneal fibrosis responds to a cascade of insults, including persistent exposure to bioincompatible dialysate, uremic toxins, and peritonitis. Under these stimuli, inflammation, neoangiogenesis, and mesothelial-to-mesenchymal transition (MMT) are considered key pathological changes that ultimately lead to discontinuation of PD treatment. Various treatment options for PD-related peritoneal fibrosis have been attempted, but most are derived from animal studies and lack further clinical validation. Therefore, we sought to identify a safe antifibrotic compound targeting EPS. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a peritoneal dialysis fluid containing baicalein and its preparation method and use. The preparation method provided by the present invention successfully dissolves baicalein in peritoneal dialysis fluid and can be stably stored at room temperature, providing conditions for the development of new peritoneal dialysis (PD) drugs to prevent and treat peritoneal fibrosis. In addition, the present invention explores the therapeutic effect of peritoneal dialysis fluid containing baicalein on peritoneal dialysis (PD)-related peritoneal fibrosis through human cell and mouse animal experiments, and reports for the first time the anti-fibrotic effect of baicalein dissolved in peritoneal dialysis fluid as an intraperitoneal injection drug on PD-related peritoneal fibrosis, providing a new strategy for the development of new PD drugs to prevent and treat peritoneal fibrosis.

[0004] The technical solution of the present invention is:

[0005] A method for preparing a peritoneal dialysis fluid containing baicalein comprises the following steps:

[0006] Step 1: dissolving baicalein in DMSO to prepare baicalein-DMSO stock solution;

[0007] Step 2: adding the baicalein-DMSO storage solution to the peritoneal dialysis fluid, and then performing ultrasound to obtain the peritoneal dialysis fluid containing baicalein.

[0008] During the research and development process, the inventors discovered that baicalein was difficult to dissolve when added to peritoneal dialysis fluid (either at room temperature or heated), resulting in the precipitation of a pale yellow flocculent precipitate, making it unsuitable for the prevention and treatment of peritoneal fibrosis. The preparation method provided by the present invention successfully dissolves baicalein in peritoneal dialysis fluid and allows for stable storage at room temperature, paving the way for the development of new PD drugs to prevent and treat peritoneal fibrosis.

[0009] Furthermore, in step 2, the ultrasonic frequency is 40-60 Hz; and the total time of the ultrasonic process is 8-13 minutes.

[0010] Furthermore, during the ultrasonic process in step 2, the interval is 9-11 seconds per minute.

[0011] Intermittent sonication of the baicalein-DMSO storage solution and peritoneal dialysis fluid under these ultrasound conditions effectively ensured the stable and complete dissolution of baicalein in the peritoneal dialysis fluid. The 9-11 second intervals per minute during sonication also effectively prevented heat accumulation. Without intermittent sonication, heat would accumulate and scorch the solute. Furthermore, if the fluid heats up during sonication, the resulting solute (after centrifugation) would appear charred.

[0012] Furthermore, in step 1, the ratio of baicalein to DMSO is 1.0 g:3.7 mL.

[0013] Furthermore, in step 2, the baicalein-DMSO stock solution is added to the peritoneal dialysis fluid heated to 36.5-38° C. Under this condition, the precipitation of baicalein is reduced compared with room temperature (25° C.).

[0014] More preferably, the baicalein-DMSO stock solution is added to the peritoneal dialysis fluid heated to 37° C. At this temperature, the solubility of baicalein is higher than that at room temperature.

[0015] Furthermore, in step 2, the peritoneal dialysis fluid contains 35-45g of glucose per 1000mL. Currently, the peritoneal dialysis fluids used in clinical practice have sugar contents of 1.5%, 2.5%, and 4.25%, so the peritoneal dialysis fluid with a sugar content of 4.25% is the peritoneal dialysis fluid with the highest glucose concentration. Studies have shown that high-glucose peritoneal dialysis fluid directly stimulates human peritoneal mesothelial cells to induce changes in the phenotype of peritoneal mesothelial cells and express transforming growth factor-β (Transforming Growth Factor-β, TGF-β). TGF-β mediates mesothelial cell MMT and extracellular matrix synthesis by activating the SMAD signaling pathway. At the same time, high-glucose peritoneal dialysis fluid will produce a large amount of glucose degradation products (Glucose Degradation Products, GDPs) during high-temperature sterilization, which directly damage peritoneal mesothelial cells, upregulate mesothelial cell TGF-β and vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF), promote endothelial cell MMT, and the formation of new small blood vessels. After a series of glycation reactions, GDPs can also be converted into advanced glycation end products (AGEs), accelerating the process of peritoneal fibrosis.

[0016] Furthermore, the peritoneal dialysis fluid in step 2 is a compound preparation: each 1000 mL contains 42.5 g of glucose, 5.67 g of sodium chloride, 0.257 g of calcium chloride, 0.152 g of magnesium chloride, and 5 g of sodium lactate.

[0017] Furthermore, after the baicalein-DMSO storage solution in step 1 is prepared, it is packaged and refrigerated for storage.

[0018] Furthermore, the present invention also provides a peritoneal dialysis fluid containing baicalein prepared by the above-mentioned preparation method.

[0019] The present invention also provides a use of the baicalein-containing peritoneal dialysis fluid in the preparation of drugs for preventing and treating peritoneal fibrosis and drugs for treating peritoneal fibrosis.

[0020] Baicalein (5,6,7-trihydroxyflavone, BAI) is a flavonoid compound extracted from the root of Scutellaria baicalensis, with a long history of use. Baicalein has been shown in multiple studies to have anti-inflammatory, antioxidant, and anti-cancer effects. Furthermore, previous studies have shown that baicalein improves fibrosis in multiple organs, including the heart, lungs, liver, and kidneys, by regulating different inflammatory signaling pathways. These studies also showed that baicalein is safe and well tolerated in animal experiments. However, it has not yet been found whether baicalein can be used in the treatment of embedded peritoneal sclerosis (EPS), whether baicalein can act on the peritoneum, and whether it has a therapeutic effect on peritoneal fibrosis.

[0021] During the research and development of the present invention, the inventors have discovered the mechanism of peritoneal fibrosis: long-term exposure of peritoneal tissue to dialysate leads to peritoneal mesothelial cell MMT, which is a key link in the process of peritoneal fibrosis. Traditional peritoneal dialysis fluid uses glucose as a solvent to provide osmotic pressure, and has the characteristics of high sugar, hypertonicity, low pH value and other poor biocompatibility. The present invention explores the preventive and therapeutic effects of peritoneal dialysis fluid containing baicalein on peritoneal dialysis (PD)-related peritoneal fibrosis through human cell and mouse animal experiments; and when preparing drugs for the prevention and treatment of peritoneal fibrosis, it is preferred that the effective concentration of the flavonoid compound baicalein in the peritoneal dialysis fluid is 10-30μM. At this concentration, the peritoneal dialysis fluid containing baicalein of the present invention has a better effect in preventing and treating peritoneal fibrosis; more preferably, the effective concentration of baicalein is 10μM. This is the first report of the anti-fibrotic effect of baicalein dissolved in peritoneal dialysis fluid as an intraperitoneal injection drug on PD-related peritoneal fibrosis, providing a new strategy for the development of new PD drugs to prevent and treat peritoneal fibrosis.

[0022] Compared with the prior art, the peritoneal dialysis fluid containing baicalein provided by the present invention, and its preparation method and use, have the following advantages:

[0023] (1) During the research and development process, the inventors found that baicalein was difficult to dissolve when added to peritoneal dialysis fluid (normal temperature or heated), and was manifested as the precipitation of light yellow flocculent precipitates, making it impossible to use it in the prevention and treatment of peritoneal fibrosis. In order to solve the problem of baicalein being difficult to dissolve in peritoneal dialysis fluid, the inventors tried a variety of methods to increase its solubility. Finally, they adopted the method of dissolving baicalein in dimethyl sulfoxide to prepare a baicalein-DMSO storage solution (BAI storage solution); then, the baicalein-DMSO storage solution was added to the peritoneal dialysis fluid heated to 36.5-38°C, and then assisted by ultrasound, which successfully made baicalein dissolve in the peritoneal dialysis fluid, forming a light yellow solution of varying depths depending on the concentration, which can be stably stored at room temperature. The preparation method provided by the present invention successfully makes baicalein dissolve in peritoneal dialysis fluid and can be stably stored at room temperature, providing conditions for the development of new PD drugs to prevent and treat peritoneal fibrosis.

[0024] (2) Baicalein is a flavonoid compound that is soluble in organic solvents such as methanol, ethanol, and dimethyl sulfoxide (DMSO). However, the inventors found that when baicalein was dissolved in methanol or ethanol and then mixed with peritoneal dialysis fluid, baicalein precipitated, failing to achieve the experimental concentration of baicalein in the peritoneal dialysis fluid or failing to maintain stable storage. However, the peritoneal dialysis fluid containing baicalein obtained in the present invention can be stably stored at room temperature.

[0025] (3) Peritoneal dialysis (PD) is one of the important alternative therapies for the treatment of end-stage renal disease (ESRD). However, the complication caused by long-term peritoneal dialysis - progressive peritoneal fibrosis, not only makes peritoneal dialysis, which depends on the integrity of peritoneal structure and function, impossible to complete, but also increases the mortality rate. Studies have shown that the high-glucose peritoneal dialysis fluid used in the existing peritoneal dialysis process is very likely to cause peritoneal fibrosis, and then pathological changes such as inflammation, new angiogenesis and mesothelial to mesenchymal transition (MMT) occur, which ultimately leads to the interruption of PD treatment. The present invention explores the therapeutic effect of peritoneal dialysis fluid containing baicalein on peritoneal dialysis (PD)-related peritoneal fibrosis through human cell and mouse animal experiments. It is the first time to report the anti-fibrotic effect of baicalein stably dissolved in peritoneal dialysis fluid as an intraperitoneal injection drug on PD-related peritoneal fibrosis, which provides a new strategy for the development of new PD to prevent and treat peritoneal fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the results of a study on the effect of baicalein in the peritoneal dialysis fluid containing baicalein on improving peritoneal fibrosis in mice induced by peritoneal dialysis fluid obtained in Example 1 of the present application, wherein:

[0027] (A) Sirius red staining and Masson staining of paraffin sections of mouse parietal peritoneal tissue (representative images);

[0028] (B) Semi-quantitative analysis of Sirius red staining, collagen area corrected for peritoneal length; #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (PDs group);

[0029] (C) Semi-quantitative analysis of Masson staining, collagen area corrected for peritoneal length; #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (PDs group);

[0030] (D) Immunohistochemical staining of α-SMA and Collagen Ⅰ in paraffin sections of mouse parietal peritoneal tissue (representative images); #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (PDs group);

[0031] (E) Semi-quantitative analysis of α-SMA immunohistochemistry, α-SMA area corrected for peritoneal length; #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (PDs group);

[0032] (F) Semi-quantitative analysis of Collagen I immunohistochemistry, Collagen I area corrected for peritoneal length; #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (PDs group);

[0033] (G) Western blot was used to detect the protein expression level of Fibronectin, a fibrosis marker in the visceral peritoneal tissue of mice.

[0034] (H) The grayscale values of the bands were analyzed semi-quantitatively, and α-Tubulin was used as an internal reference for correction. *P<0.05, compared with the model group (PDs group).

[0035] Figure 2 This figure shows the results of a study on the toxic and side effects of baicalin at therapeutic concentrations on the heart, liver, and kidneys of mice.

[0036] Figure 3 This is the result of screening the optimal experimental concentration of baicalein.

[0037] Figure 4 This is a graph showing the results of a study on the inhibition of MMT in human mesothelial cells Met-5a.

[0038] (A) Schematic diagram of cell experiment.

[0039] (B) Western blot analysis of the expression levels of fibrosis marker proteins Fibronectin and α-SMA in Met-5a cells treated with different concentrations of baicalein under TGF-β1 stimulation.

[0040] (C) Figure b shows semi-quantitative analysis of the grayscale value of Fibronectin bands, with α-Tubulin used as an internal reference for correction. #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (TGF-β1 group).

[0041] (D) Semi-quantitative analysis of grayscale values of α-SMA bands in Figure b, with α-Tubulin used as internal reference for correction, #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (TGF-β1 group).

[0042] (E) Immunofluorescence detection of α-SMA expression in cells. The green fluorescence in the figure represents α-SMA, and the blue fluorescence represents DAPI.

[0043] (F) Figure 4 (E) Semi-quantitative analysis of α-SMA fluorescence intensity; DAPI fluorescence intensity was used as an internal reference for correction, #P < 0.05, compared with the control group (Control group); *P < 0.05, compared with the model group (TGF-β1 group).

[0044] (G) RT-qPCR analysis of RNA expression levels of fibrosis markers fibronectin and α-SMA. CT values were corrected using 18s rRNA as an internal reference, and RNA expression was calculated using the formula 2^(-ΔΔt). #P < 0.05, compared with the control group; *P < 0.05, compared with the model group (TGF-β1 group). DETAILED DESCRIPTION

[0045] The present invention is further illustrated below through the description of specific implementation methods, 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 protection of the present invention.

[0046] In the following examples and comparative examples, reagents not otherwise specified are conventional reagents and can be purchased from conventional reagent production and sales companies. The manufacturer information of the peritoneal dialysis fluid is as follows:

[0047] [Generic name]: Peritoneal Dialysis Solution (Lactate-G 4.25%); English name: Peritoneal Dialysis Solution (Lactate-G 4.25%).

[0048] [Ingredients] This product is a compound preparation. Each 1000ml contains 42.5g of glucose, 5.67g of sodium chloride, 0.257g of calcium chloride, 0.152g of magnesium chloride, and 5g of sodium lactate.

[0049]

Specifications

[0050]

Approval number

[0051]

Manufacturer

[0052] The abbreviations involved in the present invention are shown in the following table:

[0053] Table 1: Glossary of abbreviations

[0054]

[0055]

[0056] Example 1 Preparation of peritoneal dialysis fluid containing baicalein

[0057] Step 1: Weigh 1.0 g of baicalein and dissolve it in 3.7 ml of DMSO to prepare baicalein-DMSO storage solution (BAI storage solution), and store it at -80°C after aliquoting.

[0058] Step 2: Add the baicalein-DMSO stock solution to peritoneal dialysis fluid heated to 37°C, and perform ultrasound at 60 Hz for 10 minutes (with a 10-second pause every minute to prevent heat accumulation) to completely dissolve the baicalein in the peritoneal dialysis fluid, thereby obtaining a peritoneal dialysis fluid containing baicalein. The peritoneal dialysis fluid containing baicalein is a light yellow solution that is stable at room temperature.

[0059] Comparative Example 1

[0060] At room temperature (25° C.), baicalein was added to peritoneal dialysis fluid, and the amounts of baicalein and peritoneal dialysis fluid used were the same as those in Example 1. Results: Baicalein could not be completely dissolved in the peritoneal dialysis fluid, and a large amount of light yellow flocs appeared.

[0061] Comparative Example 2

[0062] Baicalein was added to the peritoneal dialysis fluid heated to 37°C, and the amount of baicalein and peritoneal dialysis fluid was the same as in Example 1. Results: Baicalein could not be completely dissolved in the peritoneal dialysis fluid, and light yellow flocculent precipitated, the degree of which was less than that at room temperature (Comparative Example 1).

[0063] Comparative Example 3

[0064] Comparative Example 3 is basically the same as Example 1, except that: in step 1, DMSO is replaced by a co-solvent 0.5% sodium carboxymethyl cellulose / physiological saline.

[0065] In step 1, baicalein was completely dissolved by replacing DMSO with a cosolvent of 0.5% sodium carboxymethylcellulose / normal saline. However, in step 2, baicalein precipitated after dissolving it in peritoneal dialysis fluid. Furthermore, considering that the resulting peritoneal dialysis fluid containing baicalein needs to be administered to the human body, the toxic and side effects of adding 0.5% sodium carboxymethylcellulose / normal saline to the peritoneal dialysis fluid in comparative example 3 were not clearly established, so this experiment was not conducted.

[0066] Comparative Example 4

[0067] At room temperature (25°C), baicalein was added to peritoneal dialysis fluid and supplemented with ultrasound. The ultrasound frequency was 60 Hz and the ultrasound duration was 10 minutes (with a 10-second interval every 1 minute). This transformed the pale yellow precipitate of baicalein into extremely fine particles, which appeared to be almost completely dissolved by naked eye. However, the solution was essentially a suspension, and precipitation was observed after standing for 2 hours, indicating poor stability.

[0068] Comparative Example 5

[0069] Comparative Example 5 is basically the same as Example 1, except that the "10-second pause every 1 minute" operation was not performed during the ultrasonic operation in step 2. After the ultrasonic operation, the solution became hot, and after centrifugation, the solute was charred.

[0070] Comparative Example 6

[0071] Comparative Example 6 is basically the same as Example 1, except that DMSO is replaced by ethanol as a cosolvent, and then baicalein is precipitated after the heated peritoneal dialysis fluid is mixed with the baicalein-ethanol solution. Test Example: The therapeutic effect of baicalein-peritoneal dialysis fluid on peritoneal fibrosis is verified by human cell and mouse animal experiments

[0072] Experimental Example 1: Verification of the therapeutic effect of peritoneal dialysis fluid containing baicalein on peritoneal fibrosis through mouse animal experiments

[0073] The present invention explores the effect of baicalein on peritoneal dialysis-related peritoneal fibrosis by establishing a mouse peritoneal fibrosis model induced by peritoneal dialysis fluid (high glucose, 4.25% peritoneal dialysis fluid).

[0074] Experimental research process:

[0075] Mice were randomly divided into 5 groups:

[0076] There were three intervention groups. Mice were intraperitoneally injected with 10 mg / kg, 20 mg / kg, or 50 mg / kg of the product obtained in Example 1 daily for 6 weeks. Afterwards, the mice were killed and samples were collected. These included the peritoneal dialysis fluid + baicalein 10 mg / kg (PDs+BAI 10 mg / kg) group, the peritoneal dialysis fluid + baicalein 20 mg / kg (PDs+BAI 20 mg / kg) group, and the peritoneal dialysis fluid + baicalein 50 mg / kg (PDs+BAI 50 mg / kg) group.

[0077] The mice in the model group were intraperitoneally injected with the same volume (10 / 20 / 30) of peritoneal dialysis fluid every day for 6 weeks, and then the mice were killed and the samples were collected to form the peritoneal dialysis fluid (PDs) group.

[0078] The mice in the control group were intraperitoneally injected with the same volume of normal saline for 6 weeks before being killed and samples were collected, which is the control group. The specific operation is as follows:

[0079] Each group consisted of 5 mice, and each group received the following treatments:

[0080] Control group: 0.1 ml / g normal saline was injected intraperitoneally every day.

[0081] Peritoneal dialysates (PDs) group: 0.1 ml / g of 4.25% peritoneal dialysates (PDs) were injected into the abdominal cavity every day.

[0082] Peritoneal dialysis solution + baicalein 10 mg / kg (PDs + BAI 10 mg / kg) group: 0.1 ml / g of 4.25% peritoneal dialysis solution + 10 mg / kg baicalein was intraperitoneally injected daily.

[0083] Peritoneal dialysis solution + baicalein 20 mg / kg (PDs + BAI 20 mg / kg) group: 0.1 ml / g of 4.25% peritoneal dialysis solution + 20 mg / kg baicalein was intraperitoneally injected daily.

[0084] Peritoneal dialysis solution + baicalein 50 mg / kg (PDs + BAI 50 mg / kg) group: 0.1 ml / g of 4.25% peritoneal dialysis solution + 50 mg / kg baicalein was intraperitoneally injected daily.

[0085] Normal saline and 4.25% peritoneal dialysis fluid were incubated in a 37°C oven for 1 hour to preheat before injection. The mice were intraperitoneally injected daily for 6 weeks and then killed and their tissues were collected.

[0086] Experimental design and results Figure 1 shown.

[0087] Experimental research results: Experimental design as Figure 1 As shown in A; Sirius red and Masson staining ( Figure 1 B) showed that the parietal peritoneum of mice in the control group (Control group) was covered with a layer of mesothelial cells, and the peritoneum was thin and had no collagen accumulation. However, after 6 weeks of intraperitoneal injection of peritoneal dialysis fluid (PDs group), the subcutaneous mesothelial area of the parietal peritoneum of mice was significantly thickened, and the intercellular collagen deposition increased. Compared with the PDs model group, administration of different doses (10 / 20 / 50 mg / kg) of baicalein can significantly reduce the thickening of the parietal peritoneum of mice induced by high-glucose peritoneal dialysis fluid. Among them, although there was no statistical difference in the peritoneal collagen deposition of mice in the 10 mg / kg baicalein treatment group, it still showed a significant improvement trend. The results of peritoneal immunohistochemistry showed that ( Figure 1 C). Compared with the PDs group, baicalein supplementation reduced the expression of α-SMA and Collagen I in parietal peritoneal cells induced by high-glucose peritoneal dialysis fluid. Western blot results were also consistent with other experiments, that is, the protein levels of Fibronectin and α-SMA in the visceral peritoneal tissue of mice in the baicalein treatment group were significantly lower than those in the PDs group ( Figure 1 D) The above results suggest that baicalein has a protective effect on high glucose-induced peritoneal fibrosis in mice, but no significant difference was observed in the improvement effect of different doses of baicalein.

[0088] Moreover, HE staining was used to evaluate the effects of baicalein intervention on the whole body organs of mice, such as Figure 2 The results showed that the therapeutic concentration of baicalein had no toxic side effects on the heart, liver and kidneys of mice.

[0089] Experimental Example 2: Verification of the therapeutic effect of baicalein on peritoneal fibrosis through human cell experiments

[0090] During peritoneal dialysis, under the stimulation of multiple factors such as high-glucose peritoneal dialysis fluid, mechanical tension, and uremic toxins, some peritoneal mesothelial cells transform into fibroblasts, secreting extracellular matrix and participating in the disease process of peritoneal fibrosis. Therefore, the inventors cultured the Met-5a cell line in vitro to further explore the protective effect of baicalein treatment on mesothelial cells.

[0091] Experimental research process:

[0092] The specific grouping and experimental methods are as follows:

[0093] Cells: Human mesenchymal cells (Met-5a cells)

[0094] TGF-β1 stimulation model grouping and drug administration:

[0095] Preparation of baicalein: Prepare baicalein in DMSO. Weigh 100 mg of baicalein and dissolve it in 3.7 ml of DMSO to prepare a 100 μM stock solution. Aliquot and store in a -80°C freezer. Dilute to the experimental concentration in culture medium based on experimental needs.

[0096] (1) Blank control group (Control group): During the modeling process, cells were cultured only in M199 medium without adding stimulation or drugs.

[0097] (2) Baicalein group: The modeling process was to culture cells in M199 medium and add baicalein at experimental concentrations (0, 2, 5, 10 μM) and incubate for 24 hours.

[0098] (3) TGF-β1 group: The modeling process was to culture cells in M199 medium and add 5 ng / ml TGF-β1 for incubation for 24 h.

[0099] (4) TGF-β1+Baicalein group: The modeling process was to culture cells in M199 medium, and 5 ng / ml TGF-β1 and baicalein at experimental concentrations (0, 2, 5, 10 μM) were added and incubated for 24 hours.

[0100] Experimental research results:

[0101] First, the Baicalein group treated Met-5a cells with different concentrations of Baicalein for 24 hours, and observed the cell state to explore the toxicity of Baicalein and the maximum loading dose of cells. Figure 3 A) and microscopic morphology ( Figure 3 Results from B) showed that Met-5a cells exhibited mild proliferation at baicalein concentrations of 5-10 μM; baicalein concentrations of 20-30 μM had no effect on cell viability. However, at concentrations of 50 μM or higher, a significant number of Met-5a cells died. These results suggest that baicalein has no significant toxic effects on cells at concentrations of 0-30 μM and can be used in subsequent experiments.

[0102] To clarify the effect of baicalein on TGF-β1-induced MMT, we stimulated Met-5a cells with 5 ng / ml TGF-β1 and then treated them with different concentrations of baicalein. Figure 4 A; Met-5a cells were stimulated with TGF-β1 for 24 hours, and the baicalein intervention group was co-incubated with baicalein at concentrations of (0 / 2 / 5 / 10 μM). Cells were detected 24 hours later. Western blot was used to detect the protein levels of fibrosis markers Fibronectin and α-SMA. Compared with the control group, TGF-β1 stimulation significantly increased the expression levels of Fibronectin and α-SMA. Baicalein inhibited the above-mentioned effects mediated by TGF-β1 in a concentration-dependent manner. In addition, 10 μM baicalein restored the expression levels of Fibronectin and α-SMA to baseline ( Figure 4 BD). The above results suggest that baicalein can reduce TGF-β1-induced MMT in Met-5a cells in a concentration-dependent manner. Therefore, we selected 10 μM baicalein for subsequent studies. Cell immunofluorescence results showed that compared with the control group, TGF-β1 stimulation induced a significant increase in α-SMA expression in Met-5a cells, while 10 μM baicalein treatment effectively inhibited this change ( Figure 4 EF). In addition, we used RT-qPCR to detect the expression level of mRNA, as shown in Figure 4 G shows that 10 μM baicalein can significantly inhibit the mRNA expression of Fibronectin and α-SMA mediated by TGF-β1.

[0103] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a peritoneal dialysis fluid containing baicalein, comprising the following steps: Step 1: dissolve baicalein in DMSO to prepare baicalein-DMSO stock solution; Step 2: adding the baicalein-DMSO stock solution to the peritoneal dialysis fluid, and then performing ultrasound to obtain the peritoneal dialysis fluid containing baicalein; The ultrasonic frequency in step 2 is 40-60 Hz; the total time of the ultrasonic process is 8-13 minutes; During the ultrasonic process in step 2, the interval is 9-11 seconds per minute; In the step 2, the baicalein-DMSO stock solution is added to the peritoneal dialysis fluid heated to 36.5-38°C.

2. The use of the baicalein-containing peritoneal dialysis fluid according to claim 1 in the preparation of a drug for preventing and treating peritoneal fibrosis, wherein: The ratio of baicalein to DMSO in step 1 is 1.0 g:3.7 mL.

3. Use of the peritoneal dialysis fluid containing baicalein according to claim 1 in the preparation of a drug for preventing and treating peritoneal fibrosis, characterized in that: In step 2, the peritoneal dialysis fluid is heated to 37°C.

4. The use of the baicalein-containing peritoneal dialysis fluid according to claim 1 in the preparation of a drug for preventing and treating peritoneal fibrosis, wherein: In step 2, the peritoneal dialysis fluid contains 35-45 g of glucose per 1000 mL.

5. Use of the peritoneal dialysis fluid containing baicalein according to claim 1 in preparing a drug for preventing and treating peritoneal fibrosis, characterized in that: Step 1: After the baicalein-DMSO storage solution is prepared, it is packaged and refrigerated for storage.

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