Application of glycoursodeoxycholic acid in treating cholestatic acute liver injury induced by podophyllotoxin

Abstract

The invention provides application of glycoursodeoxycholic acid in treating cholestasis type acute liver injury induced by podophyllotoxin, and relates to the field of chemical medicines. Glycine ursodeoxycholic acid as an HSPA9 micromolecule agonist can reduce synthesis of bile acid, promote excretion of the bile acid and improve the cholestasis condition by adjusting FXR / Cyp7a1 / BSEP bile acid metabolic pathway. Meanwhile, the glycoursodeoxycholic acid can improve oxidative stress injury, promote cell proliferation and reduce the cell apoptosis rate to a certain extent, so that pathological injury of the liver is improved, and a new choice is provided for treating related diseases accompanied by acute liver injury.

Description

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office on August 28, 2024, with application number 202411193810.3 and application name “Application of glycoursodeoxycholic acid in the treatment of acute liver injury”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of chemical medicine, and in particular to application of glycoursodeoxycholic acid in treating podophyllotoxin-induced cholestatic acute liver injury. Background Art

[0003] Acute liver injury is an acute and severe liver disease caused by a variety of factors such as drugs, toxins, and infection. Among them, cholestatic acute liver injury is characterized by rapid disease progression and poor clinical prognosis due to liver cell damage and inflammatory response caused by bile acid metabolism disorders and excretion disorders.

[0004] In-depth research has revealed that the core mechanisms of cholestatic acute liver injury involve abnormalities in two key pathways: imbalanced bile acid metabolism and exacerbated oxidative stress. Impaired function of the farnesoid X receptor (FXR), a core regulator of bile acid homeostasis, leads to decreased expression of downstream target genes (such as the bile salt export pump BSEP) and deregulation of key enzymes involved in bile acid synthesis, creating a vicious cycle of excessive synthesis and impaired excretion. Accumulation of reactive oxygen species (ROS) in hepatocytes inhibits the Keap1 / Nrf2 / NQO1 antioxidant pathway, leading to downregulation of antioxidant factor expression, further exacerbating lipid peroxidation and hepatocyte apoptosis. Manifestations include abnormally elevated serum direct bilirubin (DBIL) and total bile acid (TBA), hepatocyte edema, bile duct epithelial hyperplasia, and inflammatory cell infiltration. In severe cases, it can progress to liver failure, posing a significant challenge to existing treatment options.

[0005] While some drugs have been shown to enhance the antioxidant capacity of hepatocytes, their targets are unclear. Their ability to regulate excessive bile acid synthesis, a core pathological process (e.g., overactivation of cholesterol 7α-hydroxylase), is insufficient. This inability to simultaneously address the dual imbalance of synthesis and excretion and increased oxidative stress makes it difficult to precisely intervene in the complex pathological mechanisms. To address the complex pathological mechanisms of cholestatic acute liver injury, there is an urgent need to develop novel therapeutic approaches that target key regulatory targets and have clear mechanisms of action to enhance clinical efficacy. Summary of the Invention

[0006] In order to address the above-mentioned defects and deficiencies in the prior art, the present application aims to provide an application of glycoursodeoxycholic acid in the treatment of podophyllotoxin-induced cholestatic acute liver injury.

[0007] The present invention provides a use of glycoursodeoxycholic acid as an HSPA9 small molecule agonist in the preparation of a medicine for treating podophyllotoxin-induced cholestatic acute liver injury.

[0008] Preferably, the drug is used in vitro to improve cholestatic acute liver injury at a drug concentration of 50 to 120 nM.

[0009] Preferably, the drug is administered by intraperitoneal injection, oral administration or intravenous injection.

[0010] Preferably, the dosage of glycoursodeoxycholic acid during intravenous injection is 80-110 mg / kg.

[0011] Beneficial effects of the present invention: The present invention discloses the use of glycoursodeoxycholic acid (GUDCA) as a small molecule agonist of HSPA9 for the treatment of podophyllotoxin-induced cholestatic acute liver injury. GUDCA regulates the entire chain of bile acid metabolism, from synthesis to liver entry and excretion, by targeting and activating the FXR pathway. Following GUDCA action, HSPA9 undergoes nuclear translocation to form a functional complex with the farnesoid X receptor (FXR). After entering the nucleus, GUDCA-bound HSPA9 directly binds to the DNA binding domain of FXR, stabilizing FXR binding to target gene promoters, thereby inhibiting CYP7A1-mediated bile acid oversynthesis. Reducing NTCP expression alleviates the burden of bile acid entry into hepatocytes, while promoting BSEP enhances excretion. This approach addresses the source of bile stasis, prevents liver toxicity, and addresses the core pathological issues of cholestatic liver injury. While regulating metabolic pathways, GUDCA activates the Nrf2 antioxidant pathway, increasing the expression of antioxidant proteins such as HO-1 and NQO1, alleviating oxidative stress. It also inhibits pro-apoptotic proteins and promotes anti-apoptotic proteins, reducing hepatocyte apoptosis. Metabolic regulation synergizes with antioxidant and anti-apoptotic effects to protect hepatocytes from multiple perspectives, improve pathological liver damage, and enhance therapeutic efficacy and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention, in which: Figure 1 The results are the body weight of rats in each group; Figure 2 These are the liver organ index results of rats in each group; Figure 3 is the alkaline phosphatase (ALP) content in the serum of rats in each group; Figure 4 is the alanine aminotransferase (ALT) content in the serum of rats in each group; Figure 5 is the total bile acid (TBA) content in the serum of rats in each group; Figure 6 is the total bilirubin (TBIL) content in the serum of rats in each group; Figure 7 is the direct bilirubin (DBIL) content in the serum of rats in each group; Figure 8 The repair effect of GUDCA on Bsep gene after PPT damage in BRL cells; Figure 9 The figure shows the inhibitory effect of GUDCA on Cyp7a1 gene after PPT damage in BRL cells; Figure 10 The activation effect of GUDCA on Fxr gene after PPT injury in BRL cells; Figure 11 The down-regulation effect of GUDCA on Ntcp gene after PPT injury in BRL cells; Figure 12 The figure shows the inhibitory effect of multiple doses of GUDCA on Cyp7a1 protein in BRL cells damaged by PPT; Figure 13 The activation effect of multiple doses of GUDCA on Bsep protein in PPT-damaged BRL cells; Figure 14 This is the down-regulation effect of multiple doses of GUDCA on Ntcp protein in PPT-damaged BRL cells; Figure 15 The activation effect of multiple doses of GUDCA on Fxr protein in PPT-damaged BRL cells; Figure 16 The results of protein expression of FXR / Cyp7a1 / BSEP pathway in BRL cells are shown in Figure 2. Figure 17 The repair effect of GUDCA on Bsep excretion gene after PPT damage in liver tissue; Figure 18 The inhibitory effect of GUDCA on Cyp7a1 synthesis gene after PPT injury in liver tissue; Figure 19 The activation effect of GUDCA on Fxr core regulatory genes after PPT injury in liver tissue; Figure 20 The down-regulation effect of GUDCA on Ntcp translocation into the liver after PPT injury in liver tissue; Figure 21 This is the result of BSEP protein expression in rat liver tissue; Figure 22 The results of CYP7A1 protein expression in rat liver tissue are shown in Figure 2. Figure 23This is the result of FXR protein expression in rat liver tissue; Figure 24 The results of NTCP protein expression in rat liver tissue are shown in Figure 2. Figure 25 is the surface plasmon resonance (SPR) binding curve of GUDCA and HSPA9; Figure 26 is the microthermophoresis (MST) binding curve of GUDCA and HSPA9; Figure 27 The results of HSPA9 mRNA expression in BRL cells of different treatment groups; Figure 28 The results of HSPA9 protein expression in BRL cells of different treatment groups; Figure 29 The CCK8 detection results of BRL cell proliferation rate in different treatment groups; Figure 30 The results of PCR detection of Bsep gene expression in BRL cells of different treatment groups; Figure 31 The PCR detection results of Cyp7a1 gene expression in BRL cells of different treatment groups; Figure 32 The results of PCR detection of Fxr gene expression in BRL cells of different treatment groups; Figure 33 The results of PCR detection of Ntcp gene expression in BRL cells of different treatment groups; Figure 34 The results of PCR detection of HSPA9 gene expression in BRL cells of different treatment groups; Figure 35 The WB detection results of BSEP protein expression in BRL cells of different treatment groups; Figure 36 The WB detection results of CYP7A1 protein expression in BRL cells of different treatment groups; Figure 37 The WB detection results of FXR protein expression in BRL cells in different treatment groups; Figure 38 The WB detection results of HSPA9 protein expression in BRL cells in different treatment groups; Figure 39 The WB detection results of NTCP protein expression in BRL cells of different treatment groups; Figure 40 Effects of different doses of GUDCA on the proliferation of BRL cells damaged by PPT; Figure 41Effects of different doses of GUDCA on CAT activity in BRL cells injured by PPT; Figure 42 Effects of different doses of GUDCA on GSH content in PPT-damaged BRL cells; Figure 43 Effects of different doses of GUDCA on MDA content in BRL cells damaged by PPT; Figure 44 Effects of different doses of GUDCA on SOD activity in BRL cells damaged by PPT; Figure 45 Effects of different doses of GUDCA on the apoptosis rate of BRL cells damaged by PPT. DETAILED DESCRIPTION

[0013] The present application will be further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to explain the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the invention are shown in the accompanying drawings. The embodiments and features in the embodiments of the present application may be combined with each other unless there is a conflict. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the examples.

[0014] According to a first aspect of the present invention, there is provided a use of glycoursodeoxycholic acid as a small molecule agonist of HSPA9 in the preparation of a medicament for treating podophyllotoxin-induced cholestatic acute liver injury.

[0015] In this study, GUDCA specifically binds to HSPA9 and promotes its nuclear translocation, forming a functional complex with FXR. This inhibits CYP7A1-mediated bile acid oversynthesis, reducing bile acid accumulation at the source. It also enhances the expression of the efflux protein BSEP, accelerating bile acid excretion and breaking the vicious cycle of excessive synthesis and impaired excretion. Compared to traditional drugs that only regulate excretion or synthesis, this drug achieves precise regulation of bile acid metabolic homeostasis. Furthermore, by downregulating Keap1 expression and activating Nrf2 and its downstream antioxidant factors (HO-1 and NQO1), GUDCA enhances the ability of hepatocytes to scavenge reactive oxygen species (ROS), reduces the production of the lipid peroxidation product MDA, and enhances the activity of antioxidants such as superoxide dismutase (SOD), thus preventing secondary hepatocyte damage caused by oxidative stress, which is often unsatisfactory with bile acid-modulating drugs alone.

[0016] In a preferred embodiment of the present invention, the drug is used in vitro to improve cholestatic acute liver injury, and the drug concentration is 50 to 120 nM.

[0017] In a preferred embodiment of the present invention, the drug is administered by intraperitoneal injection, oral administration or intravenous injection.

[0018] In a preferred embodiment of the present invention, the dosage of glycoursodeoxycholic acid during intravenous injection is 80-110 mg / kg.

[0019] Unless otherwise specified, the raw materials in the examples of this application were purchased from commercial channels and used directly without any special treatment. Unless otherwise specified, the analytical methods in the examples were all based on conventional settings of instruments or equipment and conventional analytical methods.

[0020] Example 1 1.1 Evaluation of the ameliorative effect of GUDCA on podophyllotoxin-induced cholestatic acute liver injury based on in vivo experiments 1.1.1 Experimental animals Fifty SPF-grade SD male healthy rats weighing 210-230 g were purchased with the animal license number SCXK (Beijing) 2021-0006. They were housed in the Animal Center of Tianjin University of Traditional Chinese Medicine, with a 12-hour day and night cycle, an ambient temperature of 23±2°C, and an ambient humidity of 35±5%. They were fed adaptively for 3 days, and their status was recorded. All SD rats were then randomly divided into 5 groups, with 10 rats in each group; namely, Healthy Control (CON), PPT administration group (Podophyllotoxin dose group, PPT), PPT+GUDCA administration group (Podophyllotoxin dose group+Glycoursodeoxycholic acid, PPT+G), PPT+Silymarin positive drug silymarin administration group (Podophyllotoxin dose group +Silymarin, PPT+S), GUDCA administration group (Glycoursodeoxycholic acid The PPT group received an intraperitoneal injection of 45 mg / kg. The PPT+G group received intraperitoneal injections of PPT (45 mg / kg) and GUDCA solution (100 mg / kg). The PPT+S group received intraperitoneal injections of PPT (45 mg / kg) and the positive drug silymarin (100 mg / kg). The GUDCA group received intraperitoneal injections of GUDCA solution (100 mg / kg). The dosages for each group are shown in Table 1.

[0021] Table 1 Dosage regimen

[0022] 1.1.2 Preparation of Podophyllotoxin Solution Prepare a 4.50 mg / mL drug solution based on the dosage. Accurately weigh an appropriate amount of podophyllotoxin and dissolve it in a trace amount of 2% DMSO solution (low concentrations avoid toxicity). Then, add 0.50% CMC-Na solution to the desired volume. Ultrasonicate for 1 hour until dissolved and milky white. Store in a refrigerator at 4°C. For the healthy control group, prepare a 2% DMSO solution and dilute to the mark with 0.50% CMC-Na solution.

[0023] 1.1.3 Preparation of Glycine Ursodeoxycholic Acid Solution Prepare a 10 mg / mL drug solution based on the dosage. Accurately weigh an appropriate amount of GUDCA and dissolve it in a trace amount of 2% DMSO solution (low concentrations avoid toxicity). Then add 0.5% CMC-Na solution to the required volume. Ultrasonicate for 1 hour until it dissolves and turns milky white. Store in a refrigerator at 4°C.

[0024] 1.1.4 Sample collection and processing Except for the PPT+GUDCA group (PPT and GUDCA were intraperitoneally injected, respectively), rats in each group were given drugs by a single intraperitoneal injection, and samples were collected 24 hours after administration.

[0025] Whole blood was collected from the abdominal aorta and placed in a 5 mL heparinized test tube. After standing for 1 hour, the blood was centrifuged at 3000 rpm, 4°C, and 15 minutes to obtain the supernatant. The supernatant was centrifuged at 3000 rpm, 4°C, and 8 minutes, and then the supernatant was obtained again and divided into multiple portions for the detection of serum biochemistry, inflammatory factors, oxidative stress and other indicators.

[0026] The rat liver was removed, and connective tissue, including fat and tendons, was quickly stripped. The liver was washed several times with saline to remove residual blood and contaminants, then dried with absorbent paper. The whole liver was weighed. The liver was then separated into two parts using tissue scissors. One part was soaked in 4% paraformaldehyde and stored at room temperature for subsequent pathological sectioning. The other part was fixed and aliquoted with a scalpel and stored at -80°C.

[0027] 1.1.5 Animal behavior observation After each group was administered with the drug, the behavior of the rats in each group was observed at 3 h, 6 h, 8 h, 18 h, 21 h, and 24 h after administration, and the body weight of the rats in each group was recorded.

[0028] 1.1.6 Determination of liver organ index Each group of mice was weighed, and then the anesthetic avertin (10 mL / kg) was injected intraperitoneally according to the dose. After blood was collected from the posterior abdominal aorta, the mice were immediately killed. The cavity was opened, the liver and other organs were removed, and the fat, tendon and other connective tissues were quickly stripped off. The residual blood and contaminants were washed with saline several times, and the saline on the surface of the organs was dried with filter paper, and then weighed and recorded. The weight results are shown in the figure below. Figure 1 The organ index of each group of rats was calculated using the following formula: Organ index (100%) = organ weight (g) / rat body weight (g) × 100% The organ index results are as follows Figure 2 shown.

[0029] 1.1.7 Serum biochemical index detection Alanine aminotransferase (ALT) in serum was determined at a wavelength of 340 nm using a fully automatic biochemical analyzer, ALP was determined at a wavelength of 405 nm using the NPP substrate-AMP buffer method, and TBA was determined at a wavelength of 405 / 505 nm using the fifth-generation cyclic enzyme method.

[0030] The ratio of ALT to ALP levels can be used to categorize drug-induced liver injury as hepatocellular, cholestatic, or a combination of both. This is typically expressed as an R value, calculated as the ratio of the measured serum ALT value to the upper limit of the normal ALT range and the ratio of the measured serum ALP value to multiples of the upper limit of the normal ALT range. Hepatocellular injury is defined as R > 5, cholestatic injury as R < 2, and mixed injury as 2 < R < 5.

[0031] In this experiment, we judged the form of liver damage based on the R value, such as Figure 3 and Figure 4 As shown, the liver function indexes ALP (P < 0.0001) and ALT (P < 0.0001) in the PPT group were significantly increased compared with the CON group, and ALP (P < 0.0001) and ALT (P < 0.001) in the GUDCA + PPT group were significantly decreased compared with the PPT group.

[0032] According to the data of the PPT group, R = (168.73 / 40) / (877.50 / 110) = 0.53 < 2. Therefore, we judged the acute liver injury caused by PPT as cholestatic liver injury.

[0033] The serum biochemical index TBA was used to investigate the improvement effect of GUDCA on PPT-induced cholestatic acute liver injury. Figure 5As shown in the figure, the TBA level in the PPT group (mean value 32.272 μmol / L) was significantly higher than that in the healthy control group (CON, mean value 13.173 μmol / L), indicating that bile acid metabolism and excretion were severely obstructed, and bile acid accumulated in the liver and blood. Figure 6 As shown in Figure 2, the TBIL level in the PPT group (mean 87.164 μmol / L) was significantly higher than that in the healthy control group (CON, mean 6.218 μmol / L), indicating increased bilirubin production and / or excretion disorder. Figure 7 As shown in the data, the DBIL level in the PPT group (mean value 112.791 μmol / L) was significantly higher than that in the healthy control group (CON, mean value 4.297 μmol / L), which is one of the most direct signs of cholestasis and clearly indicates that damage to hepatocytes or bile duct system leads to the inability of conjugated bilirubin to be normally excreted into bile.

[0034] The PPT group showed significant increases in all three key cholestatic markers (TBA, TBIL, and DBIL), far exceeding those in the healthy control group. This strongly demonstrates that podophyllotoxin successfully induced a typical cholestatic acute liver injury model, the main characteristics of which are the abnormal accumulation of bile acids and bilirubin (especially conjugated bilirubin) in the body.

[0035] like Figure 5 As shown in the figure, the TBA level in the PPT+G group (GUDCA treatment group) (mean 21.569 μmol / L) was significantly lower than that in the PPT injury group (32.272 μmol / L), indicating that GUDCA effectively promoted the excretion of bile acids and / or alleviated liver damage, reducing bile acid accumulation. Although still higher than the CON group, the improvement was significant. Figure 6 As shown in the figure, the TBIL level in the PPT+G group (GUDCA treatment group) (mean 57.855 μmol / L) was significantly lower than that in the PPT injury group (87.164 μmol / L), indicating that GUDCA improved the metabolism and excretion of bilirubin. Figure 7 As shown, the DBIL level in the PPT+G group (GUDCA treatment group) (mean, 30.341 μmol / L) was significantly lower than that in the PPT injury group (112.791 μmol / L). This is the most prominent indicator of GUDCA's therapeutic effect, indicating that it has a significant effect on ameliorating hepatocyte or bile duct damage and promoting conjugated bilirubin excretion.

[0036] After GUDCA treatment (PPT+G group), all three cholestasis markers (TBA, TBIL, and DBIL) were significantly lower than those in the untreated PPT injury group, with DBIL showing the greatest decrease. This clearly demonstrates that GUDCA can effectively alleviate podophyllotoxin-induced cholestasis and improve liver function.

[0037] like Figure 5 、 Figure 6 and Figure 7 As shown, the TBA (mean, 19.351 μmol / L), TBIL (mean, 7.564 μmol / L), and DBIL (mean, 7.560 μmol / L) levels in the GUDCA control group (administered only GUDCA, without PPT injury) were very close to those in the healthy control group (CON) and significantly lower than those in the PPT injury group. This result demonstrates that, under these experimental conditions, GUDCA alone did not cause cholestasis or liver injury and exhibited a favorable safety profile.

[0038] The present invention confirms through biochemical indicators that podophyllotoxin can cause typical cholestatic acute liver injury. More importantly, the research results strongly demonstrate that GUDCA can significantly improve this injury, effectively alleviate the cholestatic state, and reduce serum bile acid and bilirubin levels, suggesting that GUDCA is an effective drug for treating cholestatic liver injury, especially cholestatic liver injury caused by toxins such as podophyllotoxin.

[0039] Example 2 Mechanism verification experiment based on HSPA9 target analysis of GUDCA regulating bile acid metabolism and antioxidant pathway to improve podophyllotoxin-induced cholestatic acute liver injury This example focuses on the core mechanism by which GUDCA synergistically regulates the FXR / Cyp7a1 / BSEP bile acid metabolism pathway and the Nrf2 antioxidant pathway by targeting HSPA9. Using cell models (BRL cells) and animal models (rat liver tissue), we validated the key role of HSPA9 in GUDCA's improvement of podophyllotoxin-induced cholestatic acute liver injury from multiple perspectives, including gene expression (RT-PCR), protein regulation (Western Blot), and cell morphology.

[0040] 2.1 Verify the regulatory effect of different doses of GUDCA on the FXR / Cyp7a1 / BSEP bile acid metabolism axis in PPT-induced cholestatic acute liver injury.

[0041] FXR is the master switch of bile acid metabolism. Activated FXR inhibits bile acid synthase Cyp7a1, reduces bile acid synthesis, promotes bile salt export pump BSEP to accelerate bile acid excretion, and thus improves cholestasis. If FXR function is abnormal, Cyp7a1 is overexpressed, bile acid synthesis is excessive, BSEP expression is reduced, and excretion is impaired, then cholestasis is aggravated.

[0042] 2.1.1 Cell experiments demonstrated the regulatory effect of GUDCA on hepatocyte pathways; This experiment used a basic cell model to detect the mRNA expression of key genes in the FXR / Cyp7a1 / BSEP pathway, verified the regulatory effect of GUDCA on the FXR pathway in PPT-damaged BRL cells, and confirmed that GUDCA can restore the expression of Bsep and Fxr and inhibit the overactivation of Cyp7a1 and Ntcp.

[0043] 2.1.1.1 Experimental methods (1) BRL cell culture: After rapid thawing at 37°C, add the corresponding complete medium (89% DMEM medium + 10% FBS + 1% double antibody) and resuspend, centrifuge at 1000 r / min for 5 minutes to obtain the precipitate, resuspend with the corresponding complete medium and culture in a 37°C, 5% CO2 incubator; (2) Observe the cell growth and subculture when the cells grow to about 80%; (3) Pour out the original culture medium in the culture dish, rinse twice with PBS, add 0.25% trypsin to digest the cells, observe the cell digestion, and after the cells become round, remove the trypsin. Add complete culture medium to blow the cells into a single cell suspension, mix the cells, divide the cells into plates according to a certain ratio, add appropriate amount of culture medium and culture them in a 37℃, 5% CO2 incubator until the cells are in a stable state for subsequent experiments.

[0044] (4) The cells were inoculated at a volume of 5*10 5 Cells were seeded into 6-well plates in 3 mL of culture medium at 37°C with 5% CO 2 Culture overnight in an incubator. After cells adhered, treat with PPT / GUDCA for 48 h according to the above groups. Then collect samples according to the groups for RT-PCR detection. ①. Total RNA extraction from cells Total RNA was extracted using the Trizol extraction kit. The specific steps are as follows: 1) Collect 1*10 6 Transfer the cell samples to a 1.5 ml centrifuge tube.

[0045] 2) Add 1 ml of Trizol and homogenize thoroughly, then let it stand at room temperature for 5 minutes.

[0046] 3) Add 0.2 ml of chloroform, shake vigorously for 15 seconds, and let it stand for 3 minutes.

[0047] 4) Centrifuge at 12000 rpm for 10 min at 4°C and collect the supernatant.

[0048] 5) Add 0.5 ml of isopropanol, mix well, and let stand on ice for 20-30 minutes.

[0049] 6) Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant.

[0050] 7) Add 1 ml of 75% ethanol to wash the precipitate. Centrifuge at 7500 g for 5 minutes at 4°C and discard the supernatant.

[0051] 8) Place at room temperature to dry or blow dry in a clean bench for about 5 minutes, then add an appropriate amount of RNase-free H2O to dissolve.

[0052] ②. RNA concentration determination Table 2 RNA concentration determination

[0053] ③.cDNA synthesis cDNA was synthesized using a reverse transcription synthesis kit. The specific steps are as follows: For the reverse transcription reaction system, add the following reagents to a 0.2 ml PCR tube, see Table 3: Table 3 Reverse transcription reaction system

[0054] PCR conditions: incubate at 42°C for 40 min, heat at 85°C for 5 min, and store at 4°C. Store cDNA at -20°C.

[0055] ④. Polymerase chain reaction (PCR) 1) Primer synthesis Primers used for real-time PCR were designed and synthesized using Primer Premier 5.0 software from Shanghai Bioengineering Corporation, using the housekeeping gene Gapdh as an internal reference. Primer sequences are shown in Table 4.

[0056] Table 4 Real-time PCR primer sequences

[0057] 2) Real-time PCR reaction system Prepare the reaction mixture according to the real-time PCR system. Add ddH2O, SybrGreen qPCR Master Mix, forward primer, reverse primer, and cDNA template to a PCR reaction tube and mix thoroughly. Prepare the reaction system according to Table 5.

[0058] Table 5 Real time-PCR reaction system

[0059] 3) PCR amplification conditions Table 6 Real time-PCR reaction conditions

[0060] ⑤. Real-Time PCR Data Processing After PCR amplification, the real-time fluorescence quantitative PCR instrument automatically analyzes the results, adjusts the threshold and baseline according to the negative control to determine the Ct value of each sample, and determines whether the Ct value is valid according to the melting curve. -△△CT The expression difference of target gene between the control group and each experimental group was analyzed by the following calculation formula: △Ct = Ct target gene - Ct internal reference, recorded as △Ct control, the average value of △Ct control in the control group was obtained, and the average value of △Ct control was subtracted from the △Ct of each group to obtain the △△Ct value, and then the 2 -△△CT The value is the relative expression level of the gene in each group.

[0061] 2.1.1.2 Experimental Results Real-time PCR results showed that compared with the BRL group, after PPT treatment, the mRNA levels of CYP7A1 and NTCP increased significantly, and the expression levels of FXR and BSEP decreased significantly. This was reversed after GUDCA treatment. No significant changes were observed after GUDCA treatment.

[0062] like Figure 8 、 Figure 9 、 Figure 10 and Figure 11 Shown are the repair effect of GUDCA on Bsep gene after PPT-damaged BRL cells, the inhibitory effect of GUDCA on Cyp7a1 gene after PPT-damaged BRL cells, the activation effect of GUDCA on Fxr gene after PPT-damaged BRL cells, and the down-regulation effect of GUDCA on Ntcp gene after PPT-damaged BRL cells.

[0063] like Figure 8 As shown in the results, there was no significant difference in Bsep expression between the CON / GUDCA groups, and GUDCA alone did not affect Bsep expression in normal cells; Bsep expression in the PPT group was significantly reduced, and PPT damage led to downregulation of bile acid excretion genes; Bsep expression in the PPT+GUDCA group was significantly increased, indicating that GUDCA can restore Bsep expression in PPT-damaged cells and promote bile acid excretion. Figure 9As shown in the results, there was no significant difference in Cyp7a1 expression between the CON / GUDCA group, and GUDCA alone did not affect the synthesis of Cyp7a1 in normal cells; Cyp7a1 expression was significantly increased in the PPT group, and PPT damage led to upregulation of bile acid synthesis genes; Cyp7a1 expression was significantly decreased in the PPT+GUDCA group, and GUDCA could inhibit the excessive synthesis of Cyp7a1 in PPT-damaged cells and reduce bile acid production. Figure 10 As shown in the results, there was no significant difference in Fxr expression between the CON / GUDCA groups, and GUDCA alone did not affect the Fxr regulatory function of normal cells; Fxr expression was significantly reduced in the PPT group. Fxr expression in the PPT+GUDCA group was significantly increased, indicating that GUDCA could restore Fxr expression in PPT-damaged cells and restart bile acid metabolism regulation. Figure 11 As shown, there was no significant difference in Ntcp expression in the CON / GUDCA group, and GUDCA alone did not affect the Ntcp entry into the liver function of normal cells; Ntcp expression was significantly increased in the PPT group, and PPT damage led to upregulation of bile acid entry genes into the liver, increasing the burden on hepatocytes; Ntcp expression was significantly decreased in the PPT+GUDCA group, and GUDCA could inhibit the overexpression of Ntcp in PPT-damaged cells and reduce the burden on hepatocytes.

[0064] In summary, when PPT induces liver damage, the FXR pathway is disordered, Cyp7a1 is oversynthesized, bile acids are increased, BSEP excretion is insufficient, bile acid accumulates, NTCP enters the liver overloaded, and hepatocytes are overburdened, thereby exacerbating cholestasis; GUDCA can dose-dependently activate the FXR pathway, inhibit Cyp7a1 and NTCP, reduce synthesis and entry into the liver, promote enhanced excretion of BSEP, and improve cholestasis from synthesis to entry into the liver to excretion; GUDCA has no adverse effects on the bile acid metabolic pathway of normal hepatocytes and is highly safe.

[0065] 2.1.1.3 FXR / Cyp7a1 / BSEP pathway protein expression experiment in cell model This experiment tested protein expression to verify whether GUDCA can regulate the entire pathway from gene transcription to protein translation. The experimental method is as follows: Collect cells (1) BRL cell culture: After rapid thawing at 37°C, add the corresponding complete medium (89% DMEM medium + 10% FBS + 1% double antibody) and resuspend, centrifuge at 1000 r / min for 5 minutes to obtain the precipitate, resuspend with the corresponding complete medium and culture in a 37°C, 5% CO2 incubator; (2) Observe the cell growth and subculture when the cells grow to about 80%; (3) Pour out the original culture medium in the culture dish, rinse twice with PBS, add 0.25% trypsin to digest the cells, observe the cell digestion, and after the cells become round, remove the trypsin. Add complete culture medium to blow the cells into a single cell suspension, mix the cells, divide the cells into plates according to a certain ratio, add appropriate amount of culture medium and culture them in a 37℃, 5% CO2 incubator until the cells are in a stable state for subsequent experiments.

[0066] (4) The cells were inoculated into 6-well plates at a density of 5*10^5 cells, cultured in 3 mL of culture medium, and cultured overnight in a 37°C, 5% CO2 incubator. After the cells adhered to the wall, podophyllotoxin was added according to the above groups. GUDCA stock solution was dissolved in sterile water and treated with different concentrations (27.75 nM, 55.5 nM, and 111 nM). After 48 hours of induction, samples were collected according to the groups for WB detection. Protein extraction and quantification 1) Sample Preparation: After 6 hours of culture, discard the supernatant from the plate, wash the cells twice with 3 ml of PBS, add 0.25% trypsin to digest the cells, observe the cell digestion process, and after the cells become rounded, remove the trypsin and add 1 mL of complete medium. Centrifuge at 1200 rpm for 3 minutes to collect the cell pellet (approximately 1 x 10^6 cells). Add 200 μL of pre-chilled RIPA lysis buffer containing PMSF (1:100 ratio) to the collected cell sample, mix well, and after thorough lysis, centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant, perform protein quantification, and store in a -80°C freezer.

[0067] 2) Protein quantification: 3) Draw the standard curve: Take a piece of ELISA plate and add reagents according to Table 7 below: Table 7 ELISA plate reagent addition amount

[0068] 4) Prepare an appropriate amount of BCA working solution based on the number of samples, mixing BCA Reagent A and B at a volume ratio of 50:1 and mix thoroughly. 5) Add 200 μl of BCA working solution to each well; 6) Oscillate the plate on a shaker for 30 seconds, then incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm. Plot a standard curve with absorbance as the y-axis and protein concentration (mg / ml) as the x-axis (see the attached table for protein quantification). 7) Add 2.5 μl of the test protein and 17.5 μl of PBS (diluted 8-fold) to the ELISA plate. Add 200 μl of BCA working solution. Oscillate the plate on a shaker for 30 seconds and incubate at 37°C for 30 minutes. Measure the absorbance at 562 nm. 8) Based on the absorbance value of the measured sample, the corresponding protein concentration (mg / ml) can be found on the standard curve, and the sample loading amount can be determined based on the sample concentration.

[0069] Western blot assay Preparation of protein samples: Add 5× SDS loading buffer to the extracted protein, mix well, place in a boiling water bath for 5 minutes to denature the protein, centrifuge at 12000 rpm for 5 minutes, and the obtained protein sample can be directly loaded for detection or stored at -80°C.

[0070] Prepare polyacrylamide gel: Determine the concentration of the separating gel based on the amount of protein to be analyzed. Clean the glass plate, dry it, align it, and insert it into the clamps to tighten it. Slowly inject the separating gel along the glass plate, then inject an appropriate amount of water onto the surface. After the gel solidifies, pour out the water and absorb the remaining water with filter paper. Inject the concentrating gel along the glass plate to fill the remaining space. Insert a comb and cool the gel and mold in a 4°C refrigerator. After the gel solidifies, remove the comb. The preparation of the separating gel and concentrating gel is shown in Table 8: Table 8. Preparation of separating gel and stacking gel

[0071] Sample loading: Assemble the electrophoresis apparatus, pour in the electrophoresis buffer, and use a pipette to inject the marker and 30 μg of protein into the sample wells respectively.

[0072] Electrophoresis: Keep the voltage of the stacking gel constant at 80V for 30min; keep the voltage of the separation gel constant at 120V for 60min. Stop the electrophoresis when the bromophenol blue approaches the bottom of the gel and proceed to membrane transfer.

[0073] Gel cutting: Gently pry open the glass plate, take out the gel, and cut the gel using the marker as a control.

[0074] Transfer: Cut filter paper and PVDF membrane to the same size as the gel. Soak the PVDF membrane in methanol for 15 seconds. Once the membrane turns translucent from white, place it in ultrapure water and let it sit for 2 minutes. Equilibrate the treated PVDF membrane in transfer buffer for 15 minutes. Stack the layers in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad. Use a glass rod to remove any air bubbles between the layers. Place the membrane in a transfer tank and add transfer buffer.

[0075] Blocking: The membrane was rinsed three times with TBST for 5 min each time, and then washed with 5% skim milk powder at 37°C with slow shaking for 2 h.

[0076] Incubate with primary antibody: Determine the primary antibody dilution ratio according to the instructions, dilute the antibody to the appropriate concentration with blocking buffer, and incubate overnight at 4°C.

[0077] Secondary antibody incubation: After the primary antibody incubation is complete, rinse with TBST three times for 1 min each. Place the PVDF membrane in a 1:5000 dilution of HRP-labeled secondary antibody solution and incubate at 37°C with gentle shaking for 1 h. Rinse with TBST three times for 5 min each.

[0078] Color development: Add an appropriate amount of ECL luminescent liquid to the membrane and take pictures using an integrated chemiluminescence instrument.

[0079] Western blot test results showed that compared with the PPT model group, the expressions of BSEP, UGT1A1, and FXR were significantly increased, while the expressions of CYP7A1 and NTCP were significantly decreased after administration, and they were dose-dependent.

[0080] like Figure 12 、 Figure 13 、 Figure 14 and Figure 15 As shown, the inhibitory effect of multiple doses of GUDCA on Cyp7a1 protein in PPT-injured BRL cells, the activating effect of multiple doses of GUDCA on Bsep protein in PPT-injured BRL cells, the down-regulating effect of multiple doses of GUDCA on Ntcp protein in PPT-injured BRL cells, and the activating effect of multiple doses of GUDCA on Fxr protein in PPT-injured BRL cells.

[0081] like Figure 12 As shown in the data, the expression of Cyp7a1 was significantly increased in the PPT group, and PPT damage led to overactivation of bile acid synthase. Compared with the PPT group, the expression of Cyp7a1 in the GUDCA intervention group (L / M / H) gradually decreased. GUDCA can inhibit the excessive synthesis of Cyp7a1 in a dose-dependent manner and reduce bile acid production.

[0082] like Figure 13 As shown in the data, BSEP expression was significantly decreased in the PPT group, and PPT damage led to insufficient expression of bile acid excretion protein. Compared with the PPT group, BSEP expression in the GUDCA intervention group (L / M / H) gradually increased. GUDCA can promote BSEP expression in a dose-dependent manner and enhance bile acid excretion capacity.

[0083] like Figure 14 As shown in the data, NTCP expression was significantly increased in the PPT group. PPT damage may lead to excessive bile acid entry into the liver, increasing the burden on hepatocytes. Compared with the PPT group, NTCP expression in the GUDCA intervention group (L / M / H) gradually decreased. GUDCA can dose-dependently inhibit NTCP overexpression and reduce bile acid overload entering the liver.

[0084] like Figure 15As shown in the data, FXR expression was significantly decreased in the PPT group, and PPT damage led to the inhibition of FXR function, the master switch of bile acid metabolism; compared with the PPT group, FXR expression in the GUDCA intervention group (L / M / H) gradually increased, and GUDCA can activate FXR in a dose-dependent manner, restoring the core regulation of bile acid metabolism.

[0085] In summary, when PPT induces liver damage, the FXR pathway is disordered, Cyp7a1 is oversynthesized, bile acids are increased, BSEP excretion is insufficient, bile acid accumulates, NTCP enters the liver overloaded, and liver cells are burdened, thereby exacerbating cholestasis; GUDCA can dose-dependently activate the FXR pathway, inhibit Cyp7a1 and NTCP, reduce synthesis and entry into the liver, promote enhanced excretion of BSEP, and improve cholestasis from synthesis-entry into the liver-excretion; and within the safe dose range, the higher the GUDCA dose, the stronger the regulation of the FXR pathway, proving that GUDCA can accurately target the core pathway of bile acid metabolism.

[0086] Figure 16 The following table shows protein expression in the FXR / Cyp7a1 / BSEP pathway in BRL cells. CYP7A1 is a key enzyme in bile acid synthesis; higher protein expression indicates greater bile acid synthesis. The CON group showed a shallow band, indicating low CYP7A1 protein expression. The PPT group showed a deep band, indicating a significant increase in CYP7A1 protein expression, as PPT damage leads to overactivation of bile acid synthase. The bands in the GUDCA groups (L / M / H) became lighter, and CYP7A1 protein expression decreased, indicating that higher GUDCA doses resulted in a stronger inhibitory effect on CYP7A1 synthesis. Therefore, GUDCA can dose-dependently inhibit CYP7A1 protein expression and reduce bile acid synthesis.

[0087] BSEP protein is a bile salt export pump; higher protein expression indicates enhanced bile acid excretion. The CON group showed darker bands and higher BSEP protein expression, indicating strong excretion function in a normal liver. The PPT group showed lighter bands and significantly decreased BSEP protein expression, indicating that excretion function is impaired after PPT damage. The GUDCA group (L / M / H) showed darker bands and increased BSEP protein expression, indicating that higher GUDCA doses promote BSEP excretion. Therefore, GUDCA can dose-dependently promote BSEP protein expression and enhance bile acid excretion.

[0088] Higher NTCP sodium-taurocholic acid cotransporter expression indicates greater bile acid influx into the liver. In the CON group, NTCP protein bands were shallow, with low NTCP protein expression, indicating moderate bile acid influx in normal livers. In the PPT group, bands were deep, with a significant increase in NTCP protein expression, suggesting an overload of bile acid influx into the liver following PPT injury. In the GUDCA group (L / M / H), bands became lighter in each group, and NTCP protein expression decreased, indicating that higher GUDCA doses resulted in a stronger inhibitory effect on NTCP influx into the liver. Therefore, GUDCA can dose-dependently inhibit NTCP protein expression and reduce the burden of bile acid influx into the liver.

[0089] The farnesoid X receptor (FXR) is a core regulatory protein in bile acid metabolism. Higher expression indicates stronger pathway activation. The CON group showed darker FXR protein bands and higher FXR protein expression, indicating moderate pathway activation in normal livers. The PPT group showed lighter bands and significantly decreased FXR protein expression. Following PPT injury, the function of core regulatory factors is suppressed. In the GUDCA groups (L / M / H), the bands became darker and FXR protein expression increased in each group, indicating that higher GUDCA doses resulted in stronger activation of the FXR pathway. Therefore, GUDCA can dose-dependently activate FXR protein expression and restore core pathway regulation.

[0090] GUDCA activates FXR protein in a dose-dependent manner, reversely regulates the downstream pathway, inhibits CYP7A1 and NTCP, and promotes the enhanced excretion of BSEP; the higher the dose of GUDCA, the stronger the regulation of the FXR pathway, that is, the lower the CYP7A1 / NTCP decreases, the higher the BSEP / FXR increase, and the lower the FXR decreases. Figure 12-15 The genetic data completely matched.

[0091] This experiment verified the mechanism of GUDCA regulating the FXR / Cyp7a1 / BSEP pathway at the protein level, proving that GUDCA can simultaneously regulate gene transcription and protein translation.

[0092] 2.1.2 Animal experiments demonstrate the regulatory effect of GUDCA on the overall liver pathway This experiment is an RT-PCR experiment of rat liver tissue, focusing on the regulatory effect of GUDCA on the FXR / Cyp7a1 / BSEP bile acid metabolic pathway in PPT-induced liver injury, and verifying the core mechanism of GUDCA in improving cholestasis at the whole animal level.

[0093] 2.1.2.1 FXR / Cyp7a1 / BSEP pathway PCR experiments in animal models Experimental methods Basic process: RNA extraction → reverse transcription into cDNA → primer design → Q-PCR amplification The experiment was divided into healthy control group (Healthy Control, CON), PPT administration group (Podophyllotoxin dose group, PPT), PPT + GUDCA administration group (Podophyllotoxin dose group + Glycoursodeoxycholic acid, PPT + GUDCA), GUDCA administration group (Glycoursodeoxycholic acid, GUDCA) ①. Total RNA extraction from tissues Total RNA from tissues was extracted using the Trizol extraction kit. The specific steps are as follows: 1) Collect an appropriate amount of sample into a 1.5 ml centrifuge tube.

[0094] 2) Add 1 ml of Trizol and homogenize thoroughly, then let it stand at room temperature for 5 minutes.

[0095] 3) Add 0.2 ml of chloroform, shake vigorously for 15 seconds, and let it stand for 3 minutes.

[0096] 4) Centrifuge at 12000 rpm for 10 min at 4°C and collect the supernatant.

[0097] 5) Add 0.5 ml of isopropanol, mix well, and let stand on ice for 20-30 minutes.

[0098] 6) Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant.

[0099] 7) Add 1 ml of 75% ethanol to wash the precipitate. Centrifuge at 7500 g for 5 minutes at 4°C and discard the supernatant.

[0100] 8) Place at room temperature to dry or blow dry in a clean bench for about 5 minutes, then add an appropriate amount of RNase-free H2O to dissolve.

[0101] ②. RNA concentration determination Table 9 RNA concentration determination

[0102] ③.cDNA synthesis cDNA was synthesized using a reverse transcription synthesis kit. The specific steps are as follows: For the reverse transcription reaction system, add the following reagents to a 0.2 ml PCR tube, see Table 10: Table 10 Reverse transcription reaction system

[0103] PCR conditions: incubate at 42°C for 40 min, heat at 85°C for 5 min, and store at 4°C. Store cDNA at -20°C.

[0104] ④. Polymerase chain reaction (PCR) 1) Primer synthesis Primers used for real-time PCR were designed and synthesized using Primer Premier 5.0 software from Shanghai Bioengineering Corporation, using the housekeeping gene Gapdh as an internal reference. Primer sequences are shown in Table 11.

[0105] Table 11 Real-time PCR primer sequences

[0106] 2) Real-time PCR reaction system Prepare the reaction mixture according to the real-time PCR system. Add ddH2O, SybrGreen qPCR Master Mix, forward primer, reverse primer, and cDNA template to a PCR reaction tube and mix thoroughly. Prepare the reaction system according to Table 12.

[0107] Table 12 Real time-PCR reaction system

[0108] 3) PCR amplification conditions Table 13 Real time-PCR reaction conditions

[0109] ⑤. Real-Time PCR Data Processing After PCR amplification, the real-time fluorescence quantitative PCR instrument automatically analyzes the results, adjusts the threshold and baseline according to the negative control to determine the Ct value of each sample, and determines whether the Ct value is valid according to the melting curve. -△△CT The expression difference of target gene between the control group and each experimental group was analyzed by the following calculation formula: △Ct = Ct target gene - Ct internal reference, recorded as △Ct control, the average value of △Ct control in the control group was obtained, and the average value of △Ct control was subtracted from the △Ct of each group to obtain the △△Ct value, and then the 2 -△△CT The value is the relative expression level of the gene in each group.

[0110] Experimental results RT-PCR results showed that compared with the CON group, the expressions of Fxr1, Bsep and Ugt1a1 were significantly decreased, while the expressions of Cp7a1 and Ntcp were significantly increased after PPT treatment, which could be effectively reversed after GUDCA treatment.

[0111] like Figure 17 、 Figure 18 、 Figure 19 and Figure 20 As shown, GUDCA in liver tissue repairs Bsep excretion genes after PPT injury, inhibits Cyp7a1 synthesis genes after PPT injury, activates Fxr core regulatory genes after PPT injury, and downregulates Ntcp liver entry genes after PPT injury.

[0112] like Figure 17 As shown, there was no significant difference in Bsep expression in the CON / GUDCA groups, and GUDCA alone did not affect Bsep expression in normal liver tissue; Bsep expression was significantly decreased in the PPT group, and PPT damage led to downregulation of bile acid excretion genes in liver tissue; Bsep expression was significantly increased in the PPT+GUDCA group, and GUDCA could restore Bsep expression in PPT-damaged liver tissue and promote bile acid excretion.

[0113] like Figure 18 As shown, there was no significant difference in Cyp7a1 expression in the CON / GUDCA group, and GUDCA alone did not affect Cyp7a1 synthesis in normal liver tissue; Cyp7a1 expression was significantly increased in the PPT group, and PPT damage led to upregulation of bile acid synthesis genes in liver tissue; Cyp7a1 expression was significantly decreased in the PPT+GUDCA group, and GUDCA could inhibit the excessive synthesis of Cyp7a1 in PPT-damaged liver tissue and reduce bile acid production.

[0114] like Figure 19 As shown, there was no significant difference in Fxr expression in the CON / GUDCA group, and GUDCA alone did not affect the Fxr regulatory function of normal liver tissue; Fxr expression was significantly decreased in the PPT group, and PPT damage led to downregulation of core bile acid regulatory genes in liver tissue; Fxr expression was significantly rebounded in the PPT+GUDCA group, and GUDCA could restore Fxr expression in PPT-damaged liver tissue and restart bile acid metabolism regulation.

[0115] like Figure 20 As shown, there was no significant difference in Ntcp expression in the CON / GUDCA group, and GUDCA alone did not affect the Ntcp entry function of normal liver tissue; Ntcp expression was significantly increased in the PPT group, and PPT damage led to upregulation of bile acid entry genes in liver tissue, increasing the burden on hepatocytes; Ntcp expression was significantly decreased in the PPT+GUDCA group, and GUDCA could inhibit the overexpression of Ntcp in PPT-damaged liver tissue and reduce the burden on hepatocytes.

[0116] like Figures 17-20As shown in the results, when PPT induced liver injury in rats, the FXR pathway was significantly disturbed, Cyp7a1 and Ntcp genes were upregulated, Bsep and Fxr genes were downregulated, bile acid synthesis increased, bile acid entered the liver more, excretion decreased, and congestion was aggravated, which was consistent with the cell experiment. Figures 8-11 The results were consistent. GUDCA can improve cholestasis from synthesis to liver entry to excretion by restoring Fxr expression, inhibiting Cyp7a1, inhibiting Ntcp, and promoting Bsep.

[0117] Figures 17-20 and Figures 8-11 The results were highly consistent, proving that the mechanism by which GUDCA regulates the FXR pathway is universal both in vivo and in vitro.

[0118] 2.1.2.2 FXR / Cyp7a1 / BSEP pathway protein expression experiment in animal models Experimental methods (1) Extraction of tissue protein Use pre-cooled scissors to separate the target tissue and keep it on ice as much as possible to prevent protease hydrolysis; Place the tissue block in a round-bottom EP tube, add liquid nitrogen to freeze the tissue, homogenize and grind on ice, and store it at -80°C for a long time; add about 200 μl of pre-chilled lysis buffer (add protease inhibitors before use: 1:100) for every approximately 10 mg of tissue, homogenize in an ice bath, and shake at 4°C for 2 hours.

[0119] Centrifuge at 12,000 rpm for 20 minutes at 4°C. Gently aspirate the supernatant and transfer it to a freshly chilled microcentrifuge tube on ice. This is the protein sample and discard the precipitate. If the protein sample is not being processed, it can be stored at -80°C.

[0120] (2) Protein extraction and quantification Sample preparation: Add 200 μL of pre-cooled RIPA lysis buffer containing PMSF (1:100 ratio) to the collected cell samples, mix well, and centrifuge at 12,000 rpm for 10 minutes after sufficient lysis. Collect the supernatant, perform protein quantification, and store in a -80°C refrigerator.

[0121] Protein quantification: To create a standard curve: Prepare a microplate and add reagents according to Table 7. Based on the number of samples, prepare an appropriate amount of BCA working solution using a 50:1 volume ratio of BCA Reagent A to Reagent B. Mix thoroughly. Add 200 μl of BCA working solution to each well. Oscillate the microplate on a shaker for 30 seconds, incubate at 37°C for 30 minutes, and then measure absorbance at 562 nm. Plot a standard curve with absorbance as the y-axis and protein concentration (mg / ml) as the x-axis. Add 2.5 μl of the test protein and 17.5 μl of PBS (diluted 8-fold) to the microplate, then add 200 μl of BCA working solution. Oscillate the microplate on a shaker for 30 seconds, incubate at 37°C for 30 minutes, and then measure absorbance at 562 nm. Based on the absorbance of the sample being tested, the corresponding protein concentration (mg / ml) can be found on the standard curve. Determine the sample loading amount based on the sample concentration.

[0122] (3) Western blot detection Preparation of protein samples: Add 5× SDS loading buffer to the extracted protein, mix well, place in a boiling water bath for 5 minutes to denature the protein, centrifuge at 12000 rpm for 5 minutes, and the obtained protein sample can be directly loaded for detection or stored at -80°C.

[0123] Prepare polyacrylamide gel: Determine the concentration of the separating gel based on the amount of protein to be analyzed. Clean the glass plate, dry it, align it, and insert it into the clamps to secure it. Slowly inject the separating gel along the glass plate, then inject an appropriate amount of water onto the surface. After the gel solidifies, pour out the water and absorb the remaining water with filter paper. Inject the stacking gel along the glass plate to fill the remaining space. Insert a comb and cool the gel and mold in a 4°C refrigerator. After the gel solidifies, remove the comb. The preparation of the separating gel and stacking gel is shown in Table 8.

[0124] Sample loading: Assemble the electrophoresis apparatus, pour in the electrophoresis buffer, and use a pipette to inject the marker and 30 μg of protein into the sample wells respectively.

[0125] Electrophoresis: Keep the voltage of the stacking gel constant at 80V for 30min; keep the voltage of the separation gel constant at 120V for 60min. Stop the electrophoresis when the bromophenol blue approaches the bottom of the gel and proceed to membrane transfer.

[0126] Gel cutting: Gently pry open the glass plate, take out the gel, and cut the gel using the marker as a control.

[0127] Transfer: Cut filter paper and PVDF membrane to the same size as the gel. Soak the PVDF membrane in methanol for 15 seconds. Once the membrane turns translucent from white, place it in ultrapure water and let it sit for 2 minutes. Equilibrate the treated PVDF membrane in transfer buffer for 15 minutes. Stack the layers in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad. Use a glass rod to remove any air bubbles between the layers. Place the membrane in a transfer tank and add transfer buffer.

[0128] Blocking: The membrane was rinsed three times with TBST for 5 min each time, and then washed with 5% BSA at 37°C with slow shaking for 2 h.

[0129] Incubate with primary antibody: Determine the primary antibody dilution ratio according to the instructions (see the attached table for antibody information), dilute the antibody to the appropriate concentration with blocking buffer, and incubate overnight at 4°C.

[0130] Secondary antibody incubation: After the primary antibody incubation is complete, rinse with TBST three times for 1 min each. Place the PVDF membrane in a 1:5000 dilution of HRP-labeled secondary antibody solution and incubate at 37°C with gentle shaking for 1 h. Rinse with TBST three times for 5 min each.

[0131] Color development: Add an appropriate amount of ECL luminescent liquid to the membrane and take pictures using an integrated chemiluminescence instrument.

[0132] Table 14. Antibody dilution ratio and basic information

[0133] The experimental results are as follows Figure 21 As shown in the results, there was no significant difference in BSEP protein expression in the CON / GUDCA groups, and GUDCA alone did not affect BSEP protein in normal liver tissue; BSEP protein in the PPT group was significantly reduced, and PPT damage led to a downregulation of bile acid excretion protein in liver tissue; BSEP protein in the PPT+GUDCA group was significantly increased, and GUDCA could restore BSEP protein in PPT-damaged liver tissue and enhance bile acid excretion. Figure 22 As shown in the results, there was no significant difference in CYP7A1 protein expression in the CON / GUDCA groups. GUDCA alone did not affect CYP7A1 synthesis in normal liver tissue. CYP7A1 protein was significantly increased in the PPT group. PPT damage led to upregulation of bile acid synthesis protein in liver tissue. CYP7A1 protein was significantly decreased in the PPT+GUDCA group. GUDCA can inhibit CYP7A1 protein in PPT-damaged liver tissue and reduce bile acid synthesis. Figure 23As shown in the results, there was no significant difference in FXR protein expression in the CON / GUDCA groups, and GUDCA alone did not affect FXR regulatory proteins in normal liver tissue; FXR protein in the PPT group was significantly reduced, and PPT damage led to downregulation of bile acid core regulatory proteins in liver tissue; FXR protein in the PPT+GUDCA group was significantly increased, and GUDCA could restore FXR protein in PPT-damaged liver tissue and restart bile acid metabolism regulation. Figure 24 As shown, there was no significant difference in NTCP protein expression in the CON / GUDCA groups, and GUDCA alone did not affect the upregulation of NTCP protein entry into normal liver tissue; NTCP protein was significantly increased in the PPT group, and PPT damage led to upregulation of bile acid protein entry into liver tissue; NTCP protein was significantly decreased in the PPT+GUDCA group, and GUDCA could inhibit the NTCP protein in liver tissue damaged by PPT and reduce the burden on hepatocytes.

[0134] In this study, GUDCA was used to intervene in the PPT-induced acute liver injury model in rats to detect the expression of key proteins in the FXR / Cyp7a1 / BSEP pathway and to clarify the in vivo protein mechanism of GUDCA in regulating bile acid metabolism.

[0135] Example 3: 3.1 Direct interaction between GUDCA and its target protein HSPA9 The affinity of GUDCA to HSPA9 was measured by surface plasmon resonance (SPR) technology, and the KD was 8.434×10 -7 M, such as Figure 25 As shown in Figure 2, the binding is concentration-dependent. Figure 26 As shown, the experiment further confirmed by detecting the changes in the hydration layer caused by changes in molecular structure / conformation during molecular interactions that GUDCA induced the HSPA9 swimming rate to present an "S"-shaped dose-effect curve in the concentration range of 0.01-100μM, with a calculated KD=0.78μM, obvious upper and lower plateaus and strong concentration dependence, which is consistent with the high-affinity binding characteristics.

[0136] GUDCA targets Hspa9 to activate the FXR / Cyp7a1 / BSEP signaling pathway and improve PPT-induced ALI To verify that GUDCA improves PPT-induced acute liver injury (ALI) by targeting HSPA9 and activating the FXR / CYP7A1 / BSEP pathway, a BRL cell model with HSPA9 gene silencing was constructed. HSPA9 activity was modulated by the inhibitor MKT077 (10 μM) and the agonist tunicamycin (5 μg / mL). It was found that HSPA9 mRNA and protein levels were significantly reduced in the si-HSPA9 group, while co-treatment with tunicamycin partially restored expression. Figure 27 and Figure 28As shown in the data, overexpression experiments showed that HSPA9 mRNA and protein levels were significantly increased in the OE-HSPA9 group, while MKT077 treatment significantly inhibited their expression.

[0137] like Figure 29 The results of CCK8 showed that GUDCA could reverse the cell proliferation inhibition of PPT, and this effect was weakened by HSPA9 knockdown and enhanced by overexpression. Figure 30-39 The results of gene rescue experiments confirmed that HSPA9 mediates the protective effect of GUDCA against PPT-induced liver injury. Tunicamycin (HSPA9 agonist) restored the proliferation capacity of the knockdown group, while MKT077 (HSPA9 inhibitor) abolished the protective effect of the overexpression group. Figure 30-34 For PCR results, Figure 35-Figure 39 Western blot test results.

[0138] Example 4 4.1 GUDCA improves PPT-induced acute injury in BRL cells 4.1.1 Experimental Grouping The cells were divided into a healthy control group (CON group), a model group (PPT group), a low-dose GUDCA treatment group (L-GUDCA), a medium-dose GUDCA treatment group (M-GUDCA), and a high-dose GUDCA treatment group (H-GUDCA). BRL cells in the CON group were cultured normally and added with 0.1% DMSO; the PPT group was exposed to 3.765 nM podophyllotoxin for 24 hours; the low-dose GUDCA treatment group was exposed to 3.765 nM podophyllotoxin and 27.75 nM GUDCA for 24 hours; the medium-dose GUDCA treatment group was exposed to 3.765 nM podophyllotoxin and 55.5 nM GUDCA for 24 hours; and the high-dose GUDCA treatment group was exposed to 3.765 nM podophyllotoxin and 111 nM GUDCA for 24 hours.

[0139] 4.1.2 Culture conditions BRL cells: 89% DMEM medium + 10% FBS + 1% P / S 4.1.3 Drug preparation Weigh 10 mg of podophyllotoxin and dissolve it in 100 μL of 0.1% DMSO to a stock solution concentration of 241.31 nM. Dilute it with culture medium to the desired working concentrations (0.941 nM, 1.882 nM, 3.765 nM, 7.531 nM, 15.062 nM, 30.125 nM, 60.25 nM, 120.5 nM, 241.3 nM). Weigh 10 mg of GUDCA and dissolve it in 100 μL of 0.1% DMSO to a stock solution concentration of 222.41 mM. Dilute it with culture medium to the desired working concentrations (6.937 nM, 13.875 nM, 27.75 nM, 55.5 nM, 111 nM, 222 nM, 444 nM, 888 nM).

[0140] 4.1.4 Cell culture and treatment BRL cells were revived. After thawing at 37°C, 2 mL of complete culture medium was added, and the cells were centrifuged at 1200 rpm for 3 min. The supernatant was discarded. 3 mL of the corresponding complete culture medium was added and cultured at 37°C in 5% CO2. Growth was observed after 24 h of recovery. When the cell density reached more than 80%, the cells were passaged. The cell concentration was adjusted to 0.8 × 10 4 Cells were seeded into 96-well cell culture plates at 100 μL per well in triplicate and cultured in a 37°C, 5% CO2 incubator for 24 hours before drug administration according to grouping. After 24 hours of culture, 20 μL of CCK-8 was added to each well and incubated at 37°C for 2 hours. The absorbance of each well was measured using a microplate reader.

[0141] 4.1.5 Detection of oxidative stress indicators Sample processing: Collect cells (cell number about 1×10 6 After washing with PBS, the cells were divided into After resuspending the cells, ultrasonically disrupt the cells and centrifuge them. The supernatant is collected as the sample to be tested. GSH value is measured at 412nm, MDA activity is measured by absorbance at 532nm and 600nm, SOD activity is measured at 560nm, and CAT activity is measured at 240nm.

[0142] 4.1.6 Flow cytometry for cell apoptosis First, the cells were digested and collected with EDTA-free trypsin, and then centrifuged at 1500 rpm for 5 minutes at room temperature to collect the cells. Secondly, the cells were washed and labeled with Annexin V-FITC and PI. After mixing, the cells were incubated at room temperature for 10 minutes in the dark. Finally, the cells were detected by flow cytometry and analyzed using FlowJo7.6 software.

[0143] 4.1.7 Experimental Results 4.1.7.1 CCK8 Experimental Results Depend on Figure 40 It can be seen that compared with the CON group, cell proliferation in the PPT group was significantly inhibited (P<0.0001); compared with the PPT group, the addition of low, medium and high concentrations of GUDCA in the PPT group can significantly promote cell proliferation (P<0.01, P<0.0001, P<0.0001), and it was dose-dependent.

[0144] 4.1.7.2 Oxidative stress index test results Depend on Figures 41-44 It can be seen that compared with the CON group, the CAT (P<0.0001), GSH (P<0.0001) and SOD (P<0.0001) contents were significantly decreased, and the MDA (P<0.0001) content was significantly increased after PPT treatment. The low, medium and high concentrations of GUDCA treatment significantly increased the CAT (P<0.05, P<0.001, P<0.001) and SOD (P<0.05, P<0.001, P<0.00001) contents, and decreased the MDA (P<0.05, P<0.01, P<0.00001) content in a dose-dependent manner. The medium and high concentrations of GUDCA treatment significantly increased the GSH (P<0.05, P<0.00001) content.

[0145] 4.1.7.3 Flow cytometry detection of cell apoptosis results Depend on Figure 45 The results showed that compared with the CON group, the apoptosis rate was significantly increased after treatment with PPT (P<0.0001), and low, medium and high concentrations of GUDCA treatment could significantly reduce the cell apoptosis rate (P<0.0001, P<0.0001, P<0.0001), and it was dose-dependent. As the concentration increased, the apoptosis rate gradually decreased.

[0146] All statistical analyses and graphs in the present invention were performed using GraphPad Prism 10.0 software. Experimental data were expressed as mean ± standard deviation (x̅±s), and the analysis process included three key steps: first, the data distribution characteristics were evaluated by normality test, followed by homogeneity of variance verification. For multiple groups of data that conformed to normal distribution and had homogeneous variance, one-way analysis of variance (One-Way ANOVA) was used for inter-group comparisons; if the variances were unequal, a non-parametric test was used instead, and the t-test was used uniformly for comparisons between two groups of data. The significance annotation system is specifically: NS indicates no statistical difference (P ≥ 0.05), *P < 0.05, **P < 0.01, ***P < 0.001.

[0147] In summary, GUDCA-induced HSPA9 can form a functional complex with the farnesoid X receptor (FXR) through nuclear translocation. GUDCA-bound HSPA9 enters the nucleus and directly binds to the DNA binding domain of FXR, stabilizing FXR binding to target gene promoters. This in turn inhibits CYP7A1-mediated bile acid overproduction, enhances expression of the efflux protein BSEP, and inhibits NTCP function, reducing portal bile acid reabsorption and thus significantly reducing the intracellular bile acid load in hepatocytes. This finding, by reversing the quadruple mechanism of the FXR / CYP7A1 / BSEP axis—synthesis overload, transport inhibition, impaired excretion, and toxic accumulation—ameliorates the progression of cholestatic ALI, laying an important theoretical foundation for the development of liver-protective strategies targeting this regulatory network.

[0148] This study systematically demonstrates the mechanism of action of GUDCA in treating acute liver injury through multi-dimensional experiments in cell and animal models. From genes to proteins, from cellular function to tissue pathology, it verifies its precise regulation of the FXR bile acid metabolic pathway, as well as its synergistic antioxidant and anti-apoptotic effects. This comprehensive body of experimental data not only clarifies GUDCA's targets and pathways, but also demonstrates its multi-faceted protective advantages. This provides a solid scientific basis for clinical application and is expected to promote innovation in the treatment of acute liver injury, helping advance this field towards more precise and effective treatments.

Claims

1. Use of glycoursodeoxycholic acid as a small molecule agonist of HSPA9 in the preparation of a drug for the treatment of podophyllotoxin-induced cholestatic acute liver injury.

2. The use according to claim 1, characterized in that The drug is used in vitro to improve cholestatic acute liver injury, and the drug concentration is 50 to 120 nM.

3. The use according to claim 1, characterized in that The drug is administered by intraperitoneal injection, oral administration or intravenous injection.

4. The use according to claim 3, characterized in that The dosage of glycoursodeoxycholic acid during intravenous injection is 80-110 mg / kg.

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