Method for improving cholestatic hepatic fibrosis by using gastrodin

Abstract

The invention relates to the technical field of biological medicines, in particular to a method for improving cholestatic hepatic fibrosis by using gastrodin. Comprising the following steps that a therapeutically effective dose of gastrodin is applied to mammals suffering from cholestatic hepatic fibrosis, the administration dosage of the gastrodin is 12.5-150 mg / kg / day, and the administration period is 2-4 weeks. The invention provides a method for improving cholestatic hepatic fibrosis by using gastrodin, which realizes multi-target regulation and control of the fibrosis process through a specific dosage and period administration scheme.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for improving cholestatic liver fibrosis by utilizing gastrodin. Background Art

[0002] Cholestatic fibrosis is a progressive liver disease caused by disorders in bile production, secretion, or excretion. The core pathological manifestation is the abnormal accumulation of bile acids in the liver, which activates hepatic stellate cells, bile duct epithelial cells, and immune cells, forming a profibrotic microenvironment. This process, through multi-dimensional mechanisms such as bile acid toxicity, inflammatory cascades, and oxidative stress, leads to excessive deposition of extracellular matrix and ultimately progresses to liver fibrosis. As a common terminal pathological outcome of diseases such as primary biliary cholangitis and biliary atresia, cholestatic fibrosis currently lacks effective reversal methods and is a major challenge in the clinical treatment of liver disease.

[0003] Current clinical interventions primarily rely on bile acid regulators (such as ursodeoxycholic acid and its derivatives), which primarily promote bile excretion but have limited regulatory capabilities for core drivers of fibrosis, such as collagen deposition and myofibroblast activation. Particularly in advanced patients, existing drugs struggle to block the abnormally high expression of profibrotic genes (such as COL1A1 and ACTA2) and pathological collagen deposition, and are unable to significantly improve imaging and histological signs of fibrosis (such as enhanced ultrasound echogenicity and expanded Masson stain-positive areas). Furthermore, conventional treatments are ineffective for cholestatic fibrosis induced by surgical or chemical injury models, necessitating the development of new strategies that can inhibit the progression of fibrosis through multiple targets. Summary of the Invention

[0004] The present invention provides a method for improving cholestatic liver fibrosis by utilizing gastrodin, and realizes multi-target regulation of the fibrosis process through a specific dosage and cycle administration scheme.

[0005] The technical solution adopted by the present invention is: a method for improving cholestatic liver fibrosis using gastrodin, comprising the following steps: administering a therapeutically effective dose of gastrodin to a mammal suffering from cholestatic liver fibrosis, wherein the dosage of the gastrodin is 12.5-150 mg / kg / day, and the administration cycle is 2-4 weeks.

[0006] As a further improvement of the present invention, the dosage of the gastrodin is 50-150 mg / kg / day, preferably administered by intraperitoneal injection or oral gavage.

[0007] As a further improvement of the present invention, the cholestatic liver fibrosis is induced by the following model:

[0008] Chemical induction model: Feeding a diet containing 1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) for 2 weeks to establish;

[0009] The surgically induced model was established by double ligation of the common bile duct (BDL), and drug administration was continued for 4 weeks after ligation.

[0010] As a further improvement of the present invention, the gastrodin improves liver fibrosis through the following mechanisms:

[0011] Downregulation of profibrotic gene expression: reduction of mRNA levels of COL1A1, ACTA2 (α-SMA encoding gene), and TIMP1 in the liver;

[0012] Inhibit collagen deposition: Reduce the protein expression of collagen 1A1, α-SMA and fibronectin (FN) in the liver;

[0013] Improve cholestasis markers: reduce serum total bilirubin (TBIL) and total bile acid (TBA) levels.

[0014] As a further improvement of the present invention, the evaluation of the therapeutic effect includes at least one of the following indicators:

[0015] Indicator 1, ultrasound imaging detection: decreased echo intensity around the liver portal;

[0016] Indicator 2, histological analysis: Masson staining showed reduced collagen deposition, and immunohistochemistry showed decreased α-SMA expression;

[0017] Indicator three, improvement in gallbladder function: the volume of bile in the gallbladder is significantly reduced.

[0018] As a further improvement of the present invention, the mammal is a mouse model, preferably a C57 strain male mouse.

[0019] A pharmaceutical composition is used in the preparation of a drug for improving cholestatic liver fibrosis. The pharmaceutical composition contains gastrodin as an active ingredient and is configured in a dosage form suitable for intraperitoneal injection or oral administration. The concentration of gastrodin in the dosage form is 1-10 mg / mL.

[0020] As a further improvement of the present invention, the pharmaceutical composition is used in combination with obeticholic acid (OCA), wherein the dosage of gastrodin is 12.5-50 mg / kg / day, and the dosage of OCA is 5 mg / kg / day.

[0021] Beneficial effects of the present invention: By limiting the administration of gastrodin to a specific dosage range (12.5–150 mg / kg / day) and administration cycle (2–4 weeks), the present invention achieves a multi-dimensional synergistic intervention in cholestatic liver fibrosis: (1) Targeted regulation of core fibrosis pathways: Gastrodin can simultaneously downregulate the mRNA expression of pro-fibrotic genes such as COL1A1, ACTA2, and TIMP1, and inhibit the pathological deposition of their encoded proteins (collagen 1A1, α-SMA, and FN), blocking the fibrosis process at both the transcriptional and translational levels, overcoming the defect of existing therapies in insufficient regulation of myofibroblast activation. (2) Significant improvement of cholestasis and liver injury: In both DDC chemical-induced and BDL surgical-induced models, it can significantly reduce serum total bilirubin (TBIL) and total bile acid (TBA) levels, and simultaneously reduce the volume of gallbladder cholestasis, confirming its universal efficacy in treating cholestasis caused by different etiologies. (3) Provide a quantifiable evaluation system: The therapeutic response can be dynamically monitored through intuitive indicators such as ultrasound imaging (reduced liver portal echo intensity) and histology (reduced Masson staining collagen area and decreased α-SMA immunohistochemical expression), providing an objective evaluation basis for clinical translation. (4) Expand the application scenarios of treatment: Through the design of intraperitoneal injection / or gavage dosage form (1–10 mg / mL concentration) and the combination with obeticholic acid (OCA), it takes into account the convenience of administration and the potential for synergistic enhancement, which is particularly suitable for combined intervention in patients with advanced fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of echo intensity around the liver portal in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0023] Figure 2 This is a bar graph of total bilirubin and total bile acid levels in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0024] Figure 3 Schematic diagram of Masson staining and immunohistochemistry detection in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0025] Figure 4 This is a Masson staining and immunohistochemistry histogram in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0026] Figure 5 This is a Western blot band diagram in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0027] Figure 63. A histogram of mRNA levels of pro-fibrotic and pro-inflammatory factors (COL1A1, ACTA2, and TIMP1) in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0028] Figure 7 This is a schematic diagram of a liver image in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0029] Figure 8 This is a histogram of the total bile volume in the gallbladder in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0030] Figure 9 This is a bar graph of serum total bilirubin (TBIL) and total bile acid (TBA) levels in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0031] Figure 10 Schematic diagram of liver collagen deposition and collagen 1A1 expression in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0032] Figure 11 This is a bar graph of the mRNA levels of COL1A1, ACTA2, and TIMP1 in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0033] Figure 12 In one embodiment of the present invention, a method for improving cholestatic liver fibrosis by using gastrodin is Figure 4 DDC immunohistochemistry data;

[0034] Figure 13 In one embodiment of the present invention, a method for improving cholestatic liver fibrosis by using gastrodin is Figure 6 and 11 PCR data;

[0035] Figure 14 In one embodiment of the present invention, a method for improving cholestatic liver fibrosis by using gastrodin is Figure 8 DBL gallbladder volume data;

[0036] Figure 15 In one embodiment of the present invention, a method for improving cholestatic liver fibrosis by using gastrodin is Figure 9 BDL serum data;

[0037] Figure 16 This is an experimental flow chart of snRNA-seq analysis of fresh liver tissue in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0038] Figure 17 This is a cell number statistics and UMAP visualization diagram in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0039] Figure 18 This is a diagram illustrating cell type identification in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0040] Figure 19 This is a cell type-specific gene expression verification diagram in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0041] Figure 20 This is a cell type ratio analysis diagram in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention;

[0042] Figure 21 This is a statistical analysis chart of the changes in the proportion of Kupffer cells in an embodiment of a method for improving cholestatic liver fibrosis using gastrodin of the present invention. DETAILED DESCRIPTION

[0043] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, this application is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the embodiments described herein are only used to explain this application and are not intended to limit this application.

[0044] The present invention provides a method for improving cholestatic liver fibrosis by utilizing gastrodin, comprising the following steps: administering a therapeutically effective dose of gastrodin to a mammal suffering from cholestatic liver fibrosis, wherein the dosage of the gastrodin is 12.5-150 mg / kg / day and the administration cycle is 2-4 weeks.

[0045] The dosage of the gastrodin in the present invention is 50-150 mg / kg / day, preferably administered by intraperitoneal injection or oral gavage.

[0046] The cholestatic liver fibrosis described in the present invention is induced by the following models: (1) chemical induction model: feeding a feed containing 1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) for 2 weeks; (2) surgical induction model: established by double ligation of the common bile duct (BDL), and drug administration is continued for 4 weeks after ligation.

[0047] The gastrodin described in the present invention improves liver fibrosis through the following mechanisms: (1) downregulating the expression of profibrotic factor genes: reducing the mRNA levels of COL1A1, ACTA2 (α-SMA encoding gene) and TIMP1 in the liver; (2) inhibiting collagen deposition: reducing the protein expression of collagen 1A1, α-SMA and fibronectin (FN) in the liver; and (3) improving cholestasis markers: reducing serum total bilirubin (TBIL) and total bile acid (TBA) levels.

[0048] The evaluation of the therapeutic effect in the present invention includes at least one of the following indicators: (1) Indicator 1, ultrasound imaging detection: decreased echo intensity around the liver portal; (2) Indicator 2, histological analysis: Masson staining shows reduced collagen deposition, and immunohistochemistry shows decreased α-SMA expression; (3) Indicator 3, improvement of gallbladder function: a significant decrease in the volume of bile in the gallbladder.

[0049] The mammal described in the present invention is a mouse model, preferably a C57 strain male mouse.

[0050] A pharmaceutical composition for the preparation of a drug for ameliorating cholestatic liver fibrosis, comprising gastrodin as an active ingredient and formulated in a dosage form suitable for intraperitoneal injection or oral administration, wherein the concentration of gastrodin in the dosage form is 1–10 mg / mL. The pharmaceutical composition is used in combination with obeticholic acid (OCA), wherein the gastrodin dosage is 12.5–50 mg / kg / day and the OCA dosage is 5 mg / kg / day.

[0051] Example:

[0052] (1) DDC chemical induction model experiment

[0053] Model establishment and dosing regimen:

[0054] (1) Animal grouping: 30 male C57 mice (8 weeks old) were randomly divided into 5 groups (n=6): ① blank control group (Control): ordinary feed + intraperitoneal injection of normal saline; ② model group (Model): 1% DDC feed + intraperitoneal injection of normal saline; ③ positive drug group (OCA): 1% DDC feed + obeticholic acid (5 mg / kg / day by gavage); ④ low-dose gastrodin group (GAS-L): 1% DDC feed + gastrodin (12.5 mg / kg / day by intraperitoneal injection); ⑤ high-dose gastrodin group (GAS-H): 1% DDC feed + gastrodin (50 mg / kg / day by intraperitoneal injection).

[0055] (2) Dosage cycle: Administer once daily for 14 consecutive days (dosage volume 0.2 mL / 20 g).

[0056] Test results: ① Ultrasound imaging: The echo intensity around the liver portal in the GAS treatment group was significantly lower than that in the model group ( Figure 1 );② Serum indicators: The levels of serum total bilirubin (TBIL) and total bile acid (TBA) in the GAS-H group were significantly lower than those in the model group (P<0.05), and the decrease in TBIL was dose-dependent ( Figure 2 ); ③ Histopathology: Masson staining showed that collagen deposition (blue area) in the GAS group was reduced ( Figure 3 ); Immunohistochemistry showed decreased expression of α-SMA ( Figure 4 );④Protein and gene expression: Western blot: GAS down-regulated the expression of collagen 1A1, α-SMA and FN in a dose-dependent manner ( Figure 5 ); qPCR detection: The mRNA levels of profibrotic factors (COL1A1, ACTA2, TIMP1) in the GAS group were significantly decreased ( Figure 6 ).

[0057] (3) To further evaluate the cellular effects of GAS on DDC liver fibrosis, single-nuclear RNA sequencing (snRNA-seq) analysis was performed.

[0058] SnRNA-seq analysis was performed on fresh liver tissue (three groups, n = 6 each). Figure 16 After strict quality control, a total of 71,047 high-quality cell nuclei were obtained (Control: 24238; DDC: 24118; DDC-GAS: 22691), which were reduced in dimension by principal component analysis (PCA) and visualized in the UMAP map ( Figure 17 ).

[0059] Thirteen major cell types were identified using classic marker genes ( Figure 18): Hepatocytes (Hnf4a, Cyp2e1, Fah and Cyp2f), cholangiocytes (Krt19, Epcam and Krt7), sinusoidal endothelial cells (LSECs, Clec4g, Dnase13 and Aqp1), hepatic stellate cells (HSCs, Lrat, Vipr1, Colec11, Reln, Lhx2 and Hgf), erythrocytes (Ery, Hba-a1, Hab-a2 and Hbb-bt), Kupffer cells (Kupffer, Clec4f, Timd4, Cxcl13, Fabp7, Vsig4, Cd163 and Folr2), dendritic cells (Dendritic cells, H2-Ab, H2-Eb1, Flt3, CD7, Clec10a, Runx2 and Siglech), MoMph (Vcan, Cd177, Chil3 andS100a4), plasma cells (Plasma, lgkc, Sdc1, Xbp1, Eaf2 and Jchain), B cells (B cells, Cd79a, Cd79b, CD19, Pax5 and Ebf1), and T cells (T cells, Cd3d, Trac, Nkg7 and Cd3g). Furthermore, projecting gene expression onto UMAP revealed cell lineage specificity, which was consistent with the unique transcriptional profile of each cell type ( Figure 19 ), indicating that the correct assignment of cell types is strongly supported by evidence.

[0060] Next, the percentage of each cell type in the three groups was analyzed. Compared with the model group, some cell types changed significantly. The proportion of hepatocytes was only 15.92% in the model group, but increased to 28.25% in the GAS group, indicating that GAS treatment improved the proportion of hepatocytes. It is worth noting that Kupffer cells, T cells, B cells, and plasma cells increased by 26.16%, 9.79%, 4.81%, and 0.88% in the model group, respectively, while they decreased to 15.34%, 5.04%, 2.65%, and 0.40% in the GAS group, respectively, indicating that GAS treatment may have suppressed these immune cell populations. Other cell types such as red blood cells, LAM, LVEC, etc. showed smaller changes between the two groups ( Figure 20 In addition, among the above cell types, we found that the proportion of Kupffer cells in the GAS group was significantly decreased (p = 0.0021), while the difference between the model group and the GAS group was not significant (p = 0.49), indicating that GAS has a significant inhibitory effect on Kupffer cells (KC) ( Figure 21 ).

[0061] The results showed that GAS-induced changes in cell distribution included an increase in the proportion of hepatocytes and a decrease in certain immune cells, and these changes were partially restored after GAS treatment.

[0062] (II) BDL surgical induction model experiment

[0063] Model establishment and dosing regimen:

[0064] (1) Surgical procedure: 30 male C57 mice underwent double bile duct ligation (BDL).

[0065] (2) Group adjustment: ①GAS-L group: 50 mg / kg / day intraperitoneal injection of gastrodin; ②GAS-H group: 150 mg / kg / day intraperitoneal injection of gastrodin. (Other groups were treated with the same DDC model)

[0066] (3) Dosage cycle: Administer the drug once every other day starting from 3 days after surgery, for a total of 7 times (total cycle 4 weeks).

[0067] Test results: ① Improvement of cholestasis: Cholestasis in the liver of GAS group was reduced by macroscopic observation ( Figure 7 ); The bile volume in the gallbladder of the GAS-H group was significantly lower than that of the model group ( Figure 8 );② Serum indicators: The serum TBIL and TBA levels in the GAS group decreased significantly ( Figure 9 ); ③ Histological and molecular detection: Masson staining and collagen 1A1 immunohistochemistry showed decreased liver collagen deposition ( Figure 10 ); COL1A1, ACTA2, and TIMP1 mRNA expressions were significantly downregulated ( Figure 11 ).

[0068] Experimental Conclusion

[0069] In the DDC model, gastrodin (12.5-50 mg / kg / day for 14 days) significantly reduced serum TBIL / TBA levels, decreased collagen deposition, and downregulated the expression of pro-fibrotic genes. In the BDL model, gastrodin (50-150 mg / kg / day for 4 weeks) effectively reduced the volume of cholestasis and improved tissue fibrosis. By inhibiting COL1A1 / ACTA2 / TIMP1 expression, it blocked the pathological deposition of collagen 1A1, α-SMA, and FN.

[0070] The present invention provides a method for improving cholestatic liver fibrosis using gastrodin, which has been experimentally verified to exhibit significant and multi-dimensional advantages in improving cholestatic liver fibrosis. Compared with traditional treatments, the therapeutic effect of gastrodin is more comprehensive and in-depth. Traditional drugs often find it difficult to achieve effective multi-target intervention in the face of the increasingly complex pathological mechanisms of cholestatic liver fibrosis. The gastrodin treatment method of the present invention can not only downregulate the expression of pro-fibrotic factors at the genetic level, but also inhibit the deposition of collagen at the protein level, while improving relevant markers of cholestasis.

[0071] In clinical application scenarios, this method has good operability and safety. Treatment within a specific dosage range and administration cycle can provide clinicians with a clear and feasible treatment plan. Gastrodin can exert an effective therapeutic effect on cholestatic liver fibrosis induced by different etiologies, whether in chemically induced or surgically induced models, demonstrating its wide applicability.

[0072] Furthermore, the combination with obeticholic acid further expands the effectiveness and flexibility of treatment. For patients with advanced fibrosis, combined therapy can synergistically improve treatment efficacy without excessively increasing risks, offering new hope for this difficult-to-treat patient population. Furthermore, the quantitative assessment system provided by this invention enables physicians to understand treatment outcomes promptly and accurately, allowing them to tailor treatment plans to their individual circumstances.

[0073] In summary, the method of the present invention using gastrodin to improve cholestatic liver fibrosis provides a safe, effective, multi-target innovative strategy for the treatment of cholestatic liver fibrosis, which has broad clinical application prospects and important medical value.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving cholestatic liver fibrosis using gastrodin, characterized in that: The following steps are involved: A therapeutically effective dose of gastrodin is administered to a mammal suffering from cholestatic liver fibrosis, wherein the gastrodin is administered at a dose of 12.5-150 mg / kg / day for a period of 2-4 weeks.

2. The method for improving cholestatic liver fibrosis using gastrodin according to claim 1, characterized in that: The dosage of the gastrodin is 50-150 mg / kg / day, preferably administered by intraperitoneal injection or oral gavage.

3. The method for improving cholestatic liver fibrosis using gastrodin according to claim 1, characterized in that: The cholestatic liver fibrosis was induced by the following model: Chemical induction model: Feeding a diet containing 1% DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) for 2 weeks to establish; The surgically induced model was established by double ligation of the common bile duct (BDL), and drug administration was continued for 4 weeks after ligation.

4. The method for improving cholestatic liver fibrosis using gastrodin according to claim 1, characterized in that: The gastrodin improves liver fibrosis through the following mechanisms: Downregulation of profibrotic gene expression: reduction of mRNA levels of COL1A1, ACTA2 (α-SMA encoding gene), and TIMP1 in the liver; Inhibit collagen deposition: Reduce the protein expression of collagen 1A1, α-SMA and fibronectin (FN) in the liver; Improve cholestasis markers: reduce serum total bilirubin (TBIL) and total bile acid (TBA) levels.

5. The method for improving cholestatic liver fibrosis using gastrodin according to claim 1, characterized in that: Evaluation of treatment efficacy includes at least one of the following indicators: Indicator 1, ultrasound imaging detection: decreased echo intensity around the liver portal; Indicator 2, histological analysis: Masson staining showed reduced collagen deposition, and immunohistochemistry showed decreased α-SMA expression; Indicator three, improvement in gallbladder function: the volume of bile in the gallbladder is significantly reduced.

6. The method for improving cholestatic liver fibrosis using gastrodin according to claim 1, characterized in that: The mammal is a mouse model, preferably a C57 strain male mouse.

7. Use of a pharmaceutical composition in the preparation of a medicament for improving cholestatic liver fibrosis, characterized in that: The pharmaceutical composition contains gastrodin as an active ingredient and is configured into a dosage form suitable for intraperitoneal injection or oral administration, wherein the concentration of gastrodin in the dosage form is 1-10 mg / mL.

8. Use of a pharmaceutical composition according to claim 7 in the preparation of a medicament for improving cholestatic liver fibrosis, characterized in that: The pharmaceutical composition is used in combination with obeticholic acid (OCA), wherein the dosage of gastrodin is 12.5-50 mg / kg / day, and the dosage of OCA is 5 mg / kg / day.

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