Common bile duct flow limiting tube for constructing chronic cholestasis rat model and construction method

Abstract

The invention relates to the technical field of experimental animal model construction in biomedical research, and discloses a common bile duct flow limiting tube for constructing a chronic cholestasis rat model and a construction method, the flow limiting tube comprises a hard tubular body and a soft protective fixing wing wrapping the tubular body, the inner diameter of the tubular body is 0.12-0.15 mm, and the outer diameter of the tubular body is 0.12-0.15 mm. The length of the tubular body is 0.5 to 0.8 cm; protruding fixing rings are arranged at the two ends of the protection fixing wing, and the total outer diameter of the flow limiting pipe is smaller than or equal to 0.7 mm. The common bile duct flow limiting tube is implanted into the specified position of the common bile duct of an experimental animal through a precise operation, precise control over bile flow is achieved, and therefore a standardized animal model capable of highly simulating the human chronic cholestatic liver disease in the aspects of the pathological process, physiological compensation and systematic expression is established. The model and the construction method thereof can be directly applied to multiple directions of liver pathophysiology mechanism research, drug target verification, new drug curative effect and safety evaluation and the like.

Description

Technical Field

[0001] This invention relates to the field of experimental animal model construction technology in biomedical research, specifically to a common bile duct flow restrictor and its construction method for constructing a rat model of chronic cholestasis. Background Technology

[0002] Cholestatic liver disease is a chronic, progressive disease caused by impaired bile secretion or excretion. It is represented by primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Its core pathological features include intrahepatic bile duct damage, portal venous inflammation, liver fibrosis, and even cirrhosis, accompanied by systemic symptoms and a prolonged course. In-depth investigation of its pathological mechanisms and the development of therapies requires animal models that can reproduce the chronic, progressive, and compensatory pathological process. However, current mainstream modeling methods have limitations due to their disconnect from clinical pathology, making it difficult to meet the needs of translational medicine research.

[0003] To better suit research on chronic cholestasis, existing animal models have developed three main directions for improvement: targeting the genetic level, developing... Mdr2 ( Abcb4 Genetic engineering models such as gene knockout are used to simulate diseases by addressing genetic mechanisms; chemical induction models such as α-naphthyl isothiocyanate (ANIT) gavage are used to damage bile duct epithelial cells through drugs; surgical models such as complete common bile duct ligation (CBDL) and partial bile duct ligation (PBDL) are optimized to construct pathological states by regulating bile duct patency.

[0004] However, all three existing models have key shortcomings: genetic engineering models are costly, have large individual variations, pre-set pathological processes, and their compensatory regulation differs greatly from clinical practice, making them unable to simulate diseases driven by non-genetic factors; chemically induced models are acute toxicity models, causing severe liver damage in a short period of time and failing to reproduce the dynamic evolution of chronic lesions; in surgical models, CBDL completely blocks enterohepatic circulation, leading to rapid disease progression and death of animals within a short time, while PBDL only causes focal cholestasis, which is inconsistent with the clinical diffuse pathological basis. All of these models are insufficient to meet the needs of chronic cholestasis mechanism research and long-term efficacy evaluation, and there is an urgent need for new modeling technologies and devices that can achieve "precise flow restriction". Summary of the Invention

[0005] The present invention aims to provide a common bile duct flow-limiting tube and a method for constructing a rat model of chronic cholestasis, in order to solve the technical problem that existing animal models of cholestasis cannot simulate the progressive and compensatory pathological features of chronic cholestasis.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a common bile duct restrictor for constructing a rat model of chronic cholestasis, comprising a rigid tubular body and a soft protective and fixing wing covering the tubular body, wherein the inner diameter of the tubular body is 0.12~0.15mm. The inventors have confirmed through extensive experiments that an inner diameter below this range is equivalent to complete ligation of the common bile duct, resulting in an acute severe illness model; while an inner diameter greater than this range fails to form effective cholestasis.

[0007] Preferably, as an improvement, the tubular body is made of one of medical-grade stainless steel, alloy, or ceramic. This ensures excellent mechanical stability and chemical inertness in the in vivo environment, maintains a constant inner diameter parameter over a long period, and avoids affecting the flow-limiting effect due to deformation or corrosion.

[0008] Preferably, as an improvement, the length of the tubular body is 0.5~0.8cm. This length can minimize irritation to the bile duct tissue while ensuring surgical operability and fixation stability.

[0009] Preferably, as an improvement, the inner diameter of the tubular body is 0.12 mm, and the length of the tubular body is 0.5 cm.

[0010] Preferably, as an improvement, the outer diameter of the tubular body is 0.32~0.37 mm.

[0011] Preferably, as an improvement, both ends of the protective fixing wing are provided with protruding fixing rings, and the total outer diameter of the flow-limiting tube is less than or equal to 0.7 mm. Limiting its total outer diameter to less than the diameter of the common bile duct in the hepatic hilum of the rat facilitates operation during model construction.

[0012] Preferably, as an improvement, the protective fixing wing is made of medical-grade silicone or medical-grade polyurethane. This design utilizes its softness as a buffer to prevent the rigid inner tube from abrading or puncturing the bile duct wall; furthermore, its structure provides a reliable anchor point for intraoperative ligation and fixation, and can disperse ligation force, effectively preventing tissue cutting.

[0013] Preferably, as an improvement, this scheme also provides a method for constructing a rat model of chronic cholestasis, including implanting and fixing the above-mentioned flow-limiting tube in the common bile duct of the rat's porta hepatis, including the following steps: Step 1: Selection of implantation site: Select the common bile duct within 0.5~0.8cm from the opening of the duodenum as the implantation site for the flow restriction tube; Step 2, Surgical Procedure: After anesthetizing the animal, make an abdominal incision to expose the common bile duct in the porta hepatis; perform a preliminary ligation on the side of the common bile duct near the duodenum; make a small longitudinal incision on the anterior wall of the common bile duct; gently insert the flow-limiting tube into the common bile duct along the physiological flow direction of bile; then, use the protective wings at both ends of the flow-limiting tube as fixation points to ligate and fix it, with the ligation force being such that the flow-limiting tube does not move and the bile duct wall only slightly deforms; finally, close the abdomen layer by layer.

[0014] Technical benefits: The selection of the ductal insertion site in this approach has three major advantages: First, it effectively avoids the complex vascular network and pancreatic tissue in the porta hepatis, significantly reducing the risk of intraoperative bleeding and postoperative acute pancreatitis; second, this segment of the common bile duct has sufficient freedom, providing ample space for microsurgical operations; third, it reserves a safe space for the adaptive dilation of the bile duct due to postoperative stasis, avoiding bile duct rupture.

[0015] Preferably, as an improvement, the ligation is performed using 7-0, 8-0, or 9-0 non-absorbable sutures. The use of non-absorbable sutures in this procedure is for the site where the common bile duct is cut and the hollow tube is inserted during the ligation surgery. If absorbable sutures were used, bile within the common bile duct would leak from the ligation site later, leading to model failure and animal death due to bile leakage. Therefore, to maintain the ligation, absorbable sutures cannot be used.

[0016] Preferably, as an improvement, the entire surgical procedure follows the principle of minimally invasive surgery and uses microsurgical instruments for blunt dissection to minimize tissue damage.

[0017] The principle and advantages of this scheme are: This invention provides a common bile duct flow restrictor with specific structural parameters. Through precise surgery, it is implanted at a designated location in the common bile duct of experimental animals, enabling precise control of bile flow. This establishes a standardized animal model that highly simulates human chronic cholestatic liver disease in terms of pathological progression, physiological compensation, and systemic manifestations. This model and its construction method can be directly applied to various fields, including research on liver pathophysiology mechanisms, drug target validation, and evaluation of the efficacy and safety of new drugs. Specific advantages are as follows: 1. Achieved artificial control over the pathological process: This scheme, through the innovative idea of ​​physical flow restriction, transforms the process of cholestasis from "acute outbreak" to "chronic evolution", successfully simulating the slow and gradual process of clinical onset of cholestasis. It is the only animal model of cholestasis that can realize the early events and compensatory mechanisms of cholestasis caused by bile duct injury.

[0018] 2. The model's pathological features are more closely aligned with clinical practice: Because this approach preserves a limited amount of bile enterohepatic circulation, the model can not only stably reproduce core pathological changes such as intrahepatic bile duct hyperplasia, portal inflammation, and fibrosis, but also effectively simulate the enterohepatic axis disorder and its systemic metabolic effects caused by reduced intestinal bile acids. This is something that complete blockage models such as bile duct ligation cannot achieve. For example, when intestinal bile is reduced to a limited extent, the composition and function of the intestinal flora differ significantly from those in complete absence, and their roles in the pathological process also differ.

[0019] 3. High surgical success rate and good model stability: The "hard inside and soft outside" flow restriction tube design of this scheme, combined with the optimized implantation site, fundamentally solves the problem that traditional surgery is prone to excessive swelling of the bile duct and easy rupture, thereby significantly improving the success rate of model establishment and the long-term survival rate of animals, making chronic intervention studies lasting for several weeks possible.

[0020] 4. Significant Application Value: The model construction method of this scheme is the first to realize the study of early pathological changes and compensatory regulation caused by cholestasis in animal models. Furthermore, it provides a highly standardized and clinically consistent ideal experimental platform for in-depth research on the pathogenesis of chronic cholestatic diseases, validation of novel therapeutic targets, and screening and efficacy evaluation of long-acting drugs. Attached Figure Description

[0021] Figure 1 This is a three-dimensional view of the common bile duct flow restrictor in an embodiment of the present invention.

[0022] Figure 2 This is a cross-sectional schematic diagram of the common bile duct flow restrictor in an embodiment of the present invention.

[0023] Figure 3 This is a top view of the common bile duct flow restrictor in an embodiment of the present invention.

[0024] Figure 4 This is a schematic diagram of the surgical steps for common bile duct restrictor implantation in an embodiment of the present invention ((A) schematic diagram of common bile duct restrictor implantation; (B) schematic diagram of common bile duct restrictor ligation and fixation).

[0025] Figure 5 This is a schematic diagram illustrating the mechanism and effects of the chronic cholestasis model of the present invention.

[0026] Figure 6Serum biochemical indicators and liver histopathology of rats in the sham-operated group, common bile duct ligation group, and common bile duct ligation model of this invention (comparison of results at different time points after surgery in different groups: (A) serum alkaline phosphatase (ALP), (B) serum total bilirubin (TBIL), (C) serum alanine aminotransferase (ALT), (D) serum aspartate aminotransferase (AST), and (E) liver H&E staining; **P<0.01: compared with the sham-operated group; #P<0.05, ##P<0.01: compared with the common bile duct ligation group).

[0027] Figure 7 This study investigated the changes in the content and composition of bile acids in rats subjected to sham surgery, common bile duct ligation, and the common bile duct restriction model of this invention (comparison of results at different time points after surgery in different groups: (A) serum levels of conjugated bile acids, unconjugated bile acids, and total bile acids, as well as the levels of representative bile acids ursodeoxycholic acid (UDCA), β-mouse cholic acid (β-MCA), and taurine cholic acid / mouse cholic acid (T-α / β-MCA); (B) levels of conjugated bile acids, unconjugated bile acids, and total bile acids in the liver, (C) small intestine, and (D) feces; **P<0.01: compared with the sham surgery group, ##P<0.01: compared with the common bile duct ligation group).

[0028] Figure 8 The results of the sham surgery, common bile duct ligation, and common bile duct closure model rats of the present invention were compared 14 days after surgery, including (A) the main biosynthetic enzymes of bile acids in the liver, the mRNA of uptake and efflux transporters, (B) the expression level of MRP4 protein, and (C) the activity of CYP8B1 enzyme in the liver (*P<0.05, **P<0.01: compared with the sham surgery group, #P<0.05, ##P<0.01: compared with the common bile duct ligation group).

[0029] Figure 9 This is a community bar graph of the intestinal flora at the phylum taxonomic level in rats subjected to sham surgery, common bile duct ligation, and the model of this invention (common bile duct dysregulation).

[0030] Figure 10 The pathological damage and biochemical indicators of the α-naphthyl isothiocyanate (ANIT)-induced acute cholestasis mouse model of the present invention are as follows: (A) serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP) and serum total bilirubin (TBIL) levels at 24 h and 48 h after administration of ANIT to mice; (B) serum and liver levels of conjugated bile acids, unconjugated bile acids and total bile acids; (C) liver H&E staining; *P<0.05, **P<0.01: compared with blank control group).

[0031] Figure 11 For the present invention Mdr2 Pathological damage and biochemical indicators in gene knockout mouse models Mdr2 (A) Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), and serum total bilirubin (TBIL) levels in gene knockout mice; (B) Serum and liver levels of conjugated bile acids, unconjugated bile acids, and total bile acids; (C) Liver H&E and Masson staining; **P<0.01: compared with wild-type mice.

[0032] Figure 12 The effects of different inner diameter flow-limiting tubes of the present invention on bile flow rate, bile duct swelling, and liver injury are as follows: (A) Effect of different inner diameter flow-limiting hollow tubes on bile flow rate in rats; (B) Schematic diagram of common bile duct swelling in rats with common bile duct ligation and common bile duct flow restriction models 7 and 14 days after surgery (white dashed lines represent the outline of the common bile duct); (C) Quantitative results of common bile duct swelling 14 days after surgery (expressed as the amount of fluid accumulation in the common bile duct); (D) Liver histopathology (H&E staining) and serum ALT activity 14 days after implantation of hollow flow-limiting tubes with different inner diameters; (E) The ratio of secondary bile acids to total bile acids in feces 10 days after implantation of hollow flow-limiting tubes with different inner diameters (%); *P<0.05, **P<0.01 There is a significant difference between the two groups).

[0033] Figure 13 This invention relates to the effect of the length of the flow-limiting tube on surgical safety and model stability.

[0034] Figure 14 The effect of the presence or absence of a soft protective fixation wing on the construction of a rat model of chronic cholestasis is investigated in this invention.

[0035] Figure 15 This invention relates to the impact of implantation location on surgical safety.

[0036] The reference numerals in the accompanying drawings include: 1. flow restrictor tube; 2. tubular body; 3. protective fixing wing; 4. fixing ring. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0038] Overview of the Plan This protocol provides a common bile duct restrictor for constructing a rat model of chronic cholestasis. (See attached document.) Figures 1-3 The common bile duct restrictor tube 1 of this invention adopts a unique "hard inside and soft outside" composite structure design, mainly composed of a rigid tubular body 2 and a soft protective fixing wing 3.

[0039] The rigid tubular body 2 is the core component of the flow-limiting function, made of medical-grade stainless steel or other rigid materials (such as specific alloys, ceramics, etc.) with excellent mechanical strength, chemical inertness, and biocompatibility. Its core function is to provide a bile flow channel that maintains a constant shape and size in the body environment over a long period, effectively preventing luminal collapse or deformation caused by surgical ligation or long-term compression from surrounding tissues, thereby ensuring the accuracy and repeatability of bile flow restriction. The inner diameter (id) of the tubular body 2 is 0.12~0.15mm; this parameter range is crucial for achieving accurate flow restriction and establishing a chronic model. The outer diameter (OD1) of the tubular body 2 is approximately 0.32~0.37mm. The length (L1) of the tubular body 2 is 0.5~0.8cm (i.e., 5~8mm).

[0040] The soft protective fixation wing 3 tightly covers the outside of the tubular body 2 and is made of soft biocompatible materials such as medical-grade silicone and medical-grade polyurethane. Both ends of the protective fixation wing 3 have protruding fixation rings 4, the total outer diameter (OD2) of which preferably does not exceed 0.7 mm. This design has a dual advantage: firstly, the soft fixation wing (201) provides an ideal anchoring point for firmly fixing the flow-limiting tube to the common bile duct and surrounding tissue using sutures (see...). Figure 2 Secondly, its flexible nature can effectively buffer and disperse the ligation force, significantly reducing the friction, pressure and irritation of the bile duct wall by the rigid inner tube, thereby reducing the risk of postoperative tissue inflammation, adhesion and bile duct perforation, and greatly improving the success rate of animal models and animal welfare.

[0041] This protocol also provides a method for constructing a rat model of chronic cholestasis, see reference. Figure 4 First, it is necessary to determine the critical implantation site. This site should ideally be located within 0.5–0.8 cm of the common bile duct opening into the duodenum. This precise site selection is based on three main advantages, ensuring the safety of the surgery and the effectiveness of the model: 1. Avoidance of critical structures: Effectively avoids the complex vascular network of the porta hepatis, pancreatic tissue, and duodenal wall, fundamentally reducing the risk of intraoperative bleeding, postoperative acute pancreatitis, and duodenal injury. 2. Optimized operating space: This segment of the common bile duct has sufficient free length and a wide surgical field, facilitating microsurgical operations. 3. Reserved expansion space: Sufficient space is reserved for the compensatory dilation of the bile duct that inevitably occurs postoperatively due to bile stasis, effectively avoiding the risk of rupture caused by excessive tension on the bile duct wall.

[0042] like Figure 4 As shown, the construction method specifically includes the following surgical steps: 1. Preoperative preparation and exposure: After anesthetizing the experimental animals (such as rats), a midline abdominal incision is made to gently expose the common bile duct in the porta hepatis.

[0043] 2. Initial ligation and incision: At the distal end of the selected implantation site (on the side closer to the duodenum), the common bile duct is initially ligated with sutures to temporarily block bile flow. Subsequently, a small longitudinal incision is made on the anterior wall of the common bile duct at the implantation site, with the incision length approximately 90% of the outer diameter of the flow restrictor.

[0044] 3. Implantation and Fixation: Following the physiological flow direction of bile (towards the duodenum), gently insert the flow-limiting tube 1 into the lumen of the common bile duct, ensuring the tube is centered. The incision in the common bile duct is located in the middle of the flow-limiting tube. Subsequently, using the fixing rings 4 at both ends of the flow-limiting tube as fixation points, ligate and fix it using 7-0, 8-0, or 9-0 non-absorbable sutures. The ligation force must be strictly controlled, aiming for no movement of the flow-limiting tube and only slight deformation of the bile duct wall (see [link to procedure]). Figure 4 This step is crucial to ensuring the stability of the flow-limiting tube and preventing damage to the bile duct.

[0045] 4. Abdominal Closure and Postoperative Care: After confirming there is no active bleeding, suture the abdominal cavity layer by layer. Postoperatively, place the animal on a 37°C constant-temperature mat until it awakens, and provide soft food and glucose saline to promote recovery.

[0046] The mechanism and effects of this chronic cholestasis model are as follows: Figure 5 As shown in the diagram. This protocol, based on precise measurements of the effect of the inner diameter of the flow-limiting tube on bile flow rate, restricts the enterohepatic circulation of bile by implanting a 0.12-0.15 cm hollow flow-limiting tube into the common bile duct of rats at a distance of 0.5-0.8 cm from the duodenal opening, thus creating a slowly developing animal model of cholestatic liver injury. The pathological damage in this model is clear but relatively mild: moderate liver damage, changes in intestinal flora, abnormal accumulation of bile acids in the liver, and mild regulation of bile acid transporters and synthases are observed. Importantly, the serum transaminases and alkaline phosphatase levels in this model animals show a gradual rather than explosive increase, demonstrating an early compensatory decrease in conjugated bile acids and a compensatory increase in unconjugated bile acids—a pathological process not observed in any other existing cholestasis models. 。

[0047] Example 1: A mild chronic cholestasis model was constructed using a 0.12 mm inner diameter flow-limiting tube. 1. Preparation of flow limiting tube The flow-limiting tube used in this embodiment is made of medical-grade stainless steel precision tubing with a rigid hollow core. Its inner diameter is precisely 0.12 mm, outer diameter 0.3 mm, and length 5 mm. These materials and parameters ensure that the extremely fine flow channel can resist protein deposition or pressure from surrounding tissues during long-term implantation, thus preventing blockage and maintaining the long-term stability of the flow-limiting effect. The outer protective fixation wing is made of highly biocompatible medical-grade silicone, precisely molded onto the inner tube to form a soft fixation wing structure with an outer diameter of 0.6 mm, facilitating surgical manipulation and effectively protecting bile duct tissue.

[0048] 2. Animal model surgery Healthy adult SD rats (weighing 220-250g) were anesthetized, and a midline abdominal incision was made to expose the common bile duct. A small incision was made in the middle of the common bile duct (approximately 5mm from the duodenal opening) using a microsurgery. Then, the flow-limiting tube of this invention was held with forceps, and its two ends were inserted sequentially into the lumen of the common bile duct, adjusting its position so that the incision in the common bile duct was located in the middle of the flow-limiting tube. Finally, the flow-limiting tube was ligated and fixed at the fixation wing protrusions at both ends using 8-0 non-absorbable sutures. After confirming secure fixation and no bile leakage, the abdominal cavity was sutured layer by layer, and standard postoperative care was performed.

[0049] 3. Evaluation of the effectiveness of animal models 3.1 Pathological progression and degree of damage Postoperative dynamic monitoring of serum biochemical indicators was conducted. Results showed significant increases in serum total bilirubin (TBIL), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), but the upward trend was gradual: the increase in serum biochemical indicators was mild on postoperative days 1 and 3; until day 14 postoperatively, no obvious liver tissue damage was observed in the rats of this invention group; only on day 28 were mild hepatocellular necrosis and inflammatory cell infiltration observed. During the 28-day observation period, all 12 rats in the invention group survived, and the pathological damage among individuals was basically consistent, demonstrating the excellent stability, reproducibility, and animal survival rate of the model. Figure 6 ).

[0050] 3.2 Changes in bile acid metabolism A comprehensive analysis was conducted on bile acids in the liver, serum, intestines, and feces.

[0051] Serum bile acids: Conjugated bile acids showed a slight increase followed by a decrease 6 hours post-surgery, indicating early compensatory regulation, and peaked on the 5th day. Unconjugated bile acids exhibited a slow fluctuation process of first decreasing, then increasing, and then decreasing again. This important compensatory change was observed for the first time in an animal model of cholestasis, and this pattern was distinctly different from that in the acute model. Figure 7 ).

[0052] Liver bile acids: On postoperative days 7, 14 and 28, the levels of total bile acids, conjugated and unconjugated bile acids in the liver were significantly increased, confirming the successful induction of intrahepatic cholestasis.

[0053] Intestinal and fecal bile acids: Levels of various bile acids were significantly reduced in the small intestine, while conjugated bile acids were significantly increased in feces, and unconjugated bile acids were decreased. This pattern demonstrates that "the enterohepatic circulation of bile is weakened but not completely interrupted," which is highly consistent with the characteristic of partial bile inflow into the intestine in clinical patients with chronic cholestasis.

[0054] 3.3 Changes in gut microbiota Changes in major gut microbiota were detected using 16S rRNA gene sequencing. At the phylum level, the relative abundance of Firmicutes was downregulated, while the relative abundance of Bacteroidetes and Proteobacteria was increased. This indicates that partial restriction of the enterohepatic circulation of bile acids can mildly alter the composition of the gut microbiota. Figure 8 ).

[0055] 3.4 Effects on the expression of key liver synthases and transporter genes To further explore the model mechanism, we examined the mRNA and protein expression levels of key genes involved in bile acid synthesis and transport in the liver, as well as the enzyme activity of synthases. Figure 9 ).

[0056] Regarding synthases: the mRNA expression and enzyme activity of CYP8B1 were mildly inhibited, indicating that the liver is attempting to adapt to cholestasis by reducing the synthesis of more toxic hydrophobic bile acids.

[0057] Transporters: The mRNA expression of the basolateral uptake transporter Ntcp was mildly downregulated to reduce the accumulation of bile acids in the liver, while the expression level of the basolateral outgoing transporter MRP4 protein was mildly upregulated to divert intrahepatic bile acids to the kidneys for excretion. This demonstrates the body's physiological compensatory mechanism under cholestasis.

[0058] Conclusion: The pathological changes induced by the model of this invention are gradual, coordinated and mild, and realistically simulate the compensatory regulatory process carried out by the body to maintain homeostasis in human chronic cholestasis.

[0059] In summary, the model constructed in this embodiment has four major advantages: 1. The slow onset and progression of the pathology provides a sufficient time window for studying early disease events and compensatory regulation.

[0060] 2. The liver tissue damage level is mild, and the animal survival rate is high, making it suitable for long-term intervention studies.

[0061] 3. The bile acid metabolism pattern is more consistent with clinical characteristics, especially in that it preserves part of the enterohepatic bile circulation.

[0062] 4. For the first time, we observed compensatory regulation at the molecular level in an animal model of cholestasis, which is something that cannot be achieved in other existing animal models of cholestasis.

[0063] Comparative Example 1: Sham Surgery Group (Negative Control) 1. Experimental Methods This comparative example served as a negative control. The surgical procedure was identical to that of Example 1, including anesthesia, laparotomy, exposure and dissection of the common bile duct, etc. The key difference was the absence of common bile duct incision and drainage tube implantation. This was intended to eliminate the potential influence of the surgical procedure itself on the experimental results and to establish a baseline for all observed indicators.

[0064] 2. Model Performance Evaluation Throughout the 28-day observation period, the sham-operated rats showed normal behavior and no abnormal clinical manifestations.

[0065] Serum biochemical indicators: On postoperative days 1, 3, 7, 14, and 28, serum total bilirubin (TBIL), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels remained within the normal range without significant fluctuations. Liver histopathology: H&E staining of liver tissue on postoperative days 14 and 28 showed intact liver lobule structure, neatly arranged hepatocytes, and no pathological changes such as necrosis, inflammatory cell infiltration, edema, degeneration, or bile duct hyperplasia. Bile acid metabolism: The levels of total bile acids, conjugated and unconjugated bile acids in serum, liver, small intestinal contents, and feces showed no statistically significant difference from preoperative baseline levels at all testing time points (postoperative days 7, 14, and 28), and their composition ratios remained stable. Figure 6 and Figure 7 ).

[0066] Conclusion: The sham surgery group confirmed that all the pathological and biochemical changes observed in Example 1 of this invention originated from the implantation effect of the flow-limiting tube, rather than the surgical procedure itself.

[0067] Comparative Example 2: Complete Common Bile Duct Ligation (CBDL) Model 1. Experimental Methods The experimental method was basically the same as in Example 1, except that instead of implanting a flow-limiting tube, a complete ligation was performed at the same location in the common bile duct using a ligation suture. The mechanism and effects of this complete common bile duct ligation (CBDL) model are as follows: Figure 5 As shown.

[0068] 2. Model effect comparison 2.1 Acute pathological injury and high mortality rate Serum ALT, AST, ALP, and TBIL levels in CBDL model rats increased sharply after surgery. ALT and AST peaked on day 7, approximately twice that of the experimental group; ALP and TBIL continued to rise, significantly higher than in the experimental group. Significant liver tissue damage appeared on day 14 post-surgery, progressing to severe and extensive liver necrosis by day 28. Figure 6 The animals were in dire living conditions; of the 12 rats, 3 died within 14 days and 9 died within 28 days, resulting in a mortality rate as high as 75%.

[0069] 2.2 Severe Imbalance in Bile Acid Metabolism Serum bile acids: Total bile acids and conjugated bile acids surged sharply from 6 hours post-surgery, reaching their peak on day 3, with significantly higher levels than in the present invention group. Unconjugated bile acids, however, declined rapidly without any rebound or fluctuation. Figure 7 ).

[0070] Bile acids in the liver, intestines and feces: Bile acids are extremely stagnant in the liver, while the levels of various bile acids in the intestines and feces drop sharply to extremely low levels, proving that the enterohepatic bile circulation is completely blocked, which is inconsistent with most clinical cases.

[0071] 2.3 Significant changes in gut microbiota composition Because ligation of the common bile duct completely interrupts the bile acid cycle, it significantly alters the composition of the gut microbiota. The relative abundance of Firmicutes decreased significantly by about 50%, while the relative abundance of Proteobacteria increased significantly by about 4 times, reflecting a severe dysbiosis of the gut microbiota.

[0072] 2.4 The changes in synthases and transporters are dramatic. Detection of the same genes showed that the common bile duct ligation group (CBDL model) triggered very dramatic molecular compensatory changes, such as... Figure 9 As shown: (1) Synthesizer: The expression of CYP8B1 is strongly inhibited, which is a passive response to acute severe injury rather than an active adaptive regulation.

[0073] (2) Transporters: The expression of basal uptake transporters Ntcp, Oatp1a1, and Oatp1b2 was significantly downregulated, while the expression level of basal outtake transporter MRP4 protein was significantly upregulated. This change was more dramatic than that of the present invention group.

[0074] Conclusion: The gene expression profile of the comparative model shows a pathological compensatory regulation that is completely different from that of the present invention group. The regulation of related synthases and transporters is very intense and cannot simulate the real molecular pathophysiological process of human chronic diseases.

[0075] Comparative Example 3: Acute Cholestasis Model Induced by α-Naphthalene Isothiocyanate (ANIT) 1. Experimental Methods The most commonly used C57 / BL6 mouse model was established. The model was induced by a single administration of ANIT (50 mg / kg body weight) via gavage. Samples were collected at 0, 6, 12, 24 and 48 hours after ANIT administration for analysis to compare the pathological state of Example 1 of this invention at similar time points (e.g., day 3 and day 5).

[0076] 2. Model effect comparison Disease progression and injury pattern: The ANIT model rapidly developed acute severe cholestasis within 48 hours of administration. Serum ALT, AST, ALP, and TBIL levels peaked at 48 hours, significantly higher than the levels observed on days 3 and even 5 in Example 1. Liver pathology revealed acute bile duct epithelial cell necrosis, extensive neutrophil infiltration, and severe hepatolytic necrosis, in stark contrast to the slow, mild injury pattern observed in Example 1. Figure 10 ).

[0077] Bile acid metabolism: Serum total bile acids in the ANIT model increased sharply after 48 hours, but this change was a result of acute toxic injury, and the early compensatory regulatory phenomena observed in Example 1 (such as the initial increase followed by a decrease in conjugated bile acids) could not be observed. Due to acute bile duct injury and hepatocellular dysfunction, its bile acid metabolism profile was characterized by dramatic and chaotic features.

[0078] Applicability: This model cannot be used to study the core pathological processes of chronic cholestasis, such as progressive bile duct hyperplasia, fibrosis, and long-term compensatory metabolic regulation of the body. Its application is limited to the study of acute hepatotoxicity mechanisms.

[0079] Conclusion: Comparative Example 3 demonstrates the fundamental limitations of chemically induced models in simulating chronic, progressive cholestasis, highlighting the outstanding advantage of the model of this invention in terms of the controllability of pathological progression.

[0080] Comparative Example 4: Mdr2 Gene knockout mouse model 1. Experimental Methods This comparative example adopts Mdr2 Gene knockout mice are a hereditary cholestasis model. Samples are collected from animals at 6 weeks of age (corresponding to the early stage of disease development, this age is generally suitable for research).

[0081] 2. Model effect comparison The uncontrollability of pathological progression: Mdr2 - / -Mice spontaneously exhibited significant cholestasis, bile duct hyperplasia, and early fibrosis at 6 weeks of age. The disease progression was predetermined by a genetic defect, making it impossible to precisely control the severity and rate of progression by adjusting flow-limiting tube parameters (such as inner diameter) as is possible with this invention. Figure 11 ).

[0082] Differences in pathological features: This model is characterized by progressive, inflammatory-driven biliary response and portal hypertension. Its liver injury pattern is similar to that of human PSC, but it lacks the core pathophysiological basis of physical obstruction of bile flow that is common to various types of chronic cholestasis in humans.

[0083] Model stability and cost: Mdr2 - / - The severity of phenotypic changes in these models is greatly influenced by genetic background and rearing environment, with significant individual differences. Furthermore, their construction and maintenance are costly and time-consuming, making them unsuitable for rapid, economical, and standardized large-scale replication in common wild-type animals, unlike the model described in this invention.

[0084] Conclusion: Comparative Example 4 demonstrates that genetic models have inherent limitations in terms of disease course controllability, modeling cost, standardization, and breadth of pathological mechanisms. This invention provides a novel modeling scheme that is independent of specific genetic backgrounds, can be widely applied to any wild-type animal, and exhibits phenotypic uniformity and stability, thus addressing a long-standing pain point in this field.

[0085] Comparative Example 5: The Effect of Different Inner Diameters of Flow Restricting Tubes on Bile Flow Rate and Pathological Progress In order to systematically evaluate the quantitative impact of the core parameter of the flow-limiting tube inner diameter on bile flow rate, liver damage and changes in bile duct adaptability, and thus determine the optimal and only effective parameter range for establishing a chronic mild model.

[0086] 1. Experimental Methods Using the same SD rats and surgical methods as in Example 1, flow-limiting tubes with a length of 0.5 cm and inner diameters of 0.10 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, 0.15 mm, 0.16 mm, 0.20 mm, and 0.28 mm were implanted, with a complete common bile duct ligation (CBDL) group serving as a positive control. At specific postoperative time points, bile flow rate was precisely measured via bile duct drainage, and liver damage was assessed by serum biochemistry and histopathology. Simultaneously, the degree of common bile duct swelling was measured to assess bile duct pressure.

[0087] 2. Experimental Results and Comparative Analysis 2.1 Relationship between bile flow rate and inner diameter The experimental results generally conformed to Poiseuille's law of fluid dynamics. Within the inner diameter range of 0.10–0.12 mm, the bile flow velocity increased sharply with increasing inner diameter, indicating that even small changes in inner diameter within this range had a significant impact on fluid resistance. Notably, the bile flow velocity in the 0.11 mm inner diameter group exhibited extremely high inter-individual variability, indicating that this value represents a critical physical threshold where even small physiological differences in the rat common bile duct lead to significant variations in flow velocity, compromising the model's reproducibility. When the inner diameter exceeded 0.12 mm, the rate of increase in flow velocity with increasing inner diameter tended to moderate. Figure 12 The flow rate increases significantly starting from an inner diameter of 0.16 mm.

[0088] 2.2 Relationship between the degree of liver damage and its internal diameter Postoperative assessment at 14 days showed that the degree of liver damage was closely related to the liver diameter, and a clear threshold effect existed: For patients with an inner diameter ≤0.11mm: there was no statistically significant difference in liver injury (serum ALT / AST levels, necrosis area) between the 0.10mm group and the CBDL group, indicating that functional complete obstruction was achieved at this inner diameter, leading to an acute severe model.

[0089] Inner diameter = 0.11 mm: The degree of liver damage varied greatly, with some animals showing severe damage approaching that of the CBDL group, while others showed mild damage, making the model highly unstable. Inner diameter 0.12~0.15 mm: Flow-limiting tubes within this range induced significant and consistent liver damage, but the degree of damage was significantly lower (p<0.01) than that of the CBDL group and the 0.10 mm group, successfully achieving the technical effect of "flow restriction" rather than "blockage".

[0090] Inner diameter ≥0.16mm: No significant liver damage or cholestasis was induced. Serum biochemical indicators and histopathology were not different from those of the sham-operated group, indicating that bile flow was not effectively restricted.

[0091] 2.3 Relationship between the degree of bile duct swelling and its internal diameter The amount of fluid accumulation in the common bile duct 14 days post-surgery (reflecting intraductal pressure) further confirmed the above findings: The CBDL group and the 0.10mm group had extremely high fluid volumes (6.8±2.4mL and 6.5±2.9mL, respectively), indicating high intraductal pressure and a high risk. The 0.12mm and 0.15mm groups had significantly lower fluid volumes (2.7±0.7mL and 2.1±0.5mL, respectively), indicating that the bile ducts were in a state of controllable and moderate compensatory dilation. There were no significant changes in the 0.16mm and 0.20mm groups.

[0092] 2.4 Relationship between bile acid composition and inner diameter in the common bile duct Ten days after insertion of a 0.10 mm flow-limiting tube or bile duct ligation in rats, the proportion of secondary bile acids in feces significantly decreased, while 0.16 mm and 0.20 mm flow-limiting tubes did not change this proportion. The changes in the proportion of secondary bile acids in the 0.12 mm and 0.15 mm groups were less significant than those in the 0.10 mm group, but more significant than those in the sham-operated group.

[0093] Conclusion: Through extensive creative work and experimental verification, this comparative example unexpectedly discovered that 0.12 mm to 0.15 mm is the only effective critical window for stably establishing a chronic mild cholestasis model. Below this range (e.g., 0.10 mm), the effect is equivalent to complete ligation; above this range (e.g., 0.20 mm), no effective pathological changes are produced; and at the boundary value (0.11 mm), the model is unreliable due to individual differences. This proves that the selection of 0.12~0.15 mm as the core parameter in this invention is not a conventional choice in the field, but rather achieved unexpected technical results.

[0094] Comparative Example 6: The effect of flow-limiting tube length on surgical safety and model stability This comparative example investigated the effect of different flow-limiting tube lengths (0.5cm, 0.8cm, and 1.2cm) under a fixed inner diameter of 0.12mm. The results show that ( Figure 13 In the group with a 1.2cm flow-limiting tube (n=12), two animals died within 28 days post-surgery. Autopsy confirmed the cause of death as bile duct rupture. Furthermore, the serum alkaline phosphatase and conjugated bile acid levels, as well as the coefficient of variation, were increased in the surviving animals, indicating that excessively long flow-limiting tubes can cause blockage. In contrast, no animals died in the 0.5cm and 0.8cm groups, and the model parameters remained stable. This demonstrates that excessively long flow-limiting tubes, due to their increased rigidity, are prone to damaging the bile duct, and the 0.5-0.8cm length range selected in this invention is crucial for achieving surgical safety and model stability.

[0095] Comparative Example 7: An Examination of the Necessity of Soft Protective Fixed Wings This comparative example directly compares the flow-limiting tube of this invention with medical-grade silicone protective wings with a pure stainless steel tube without protective wings (all other parameters are the same). Results at 14 days post-surgery showed ( Figure 14 Of the 12 models without protective wings, 7 died, a mortality rate of 58.3%, primarily due to bile leakage caused by displacement of the flow-limiting tube and bile duct puncture. In contrast, all models of the 12 models with protective wings survived. This comparison strongly demonstrates that the protective wings are not merely auxiliary components, but rather crucial structures that prevent displacement, avoid tissue damage, and thus ensure successful model construction.

[0096] Comparative Example 8: The impact of implantation location on surgical safety This comparative example uses the optimal flow-limiting tube of this invention to compare the effects of different implantation sites (0.5cm, 0.8cm, and 1.2cm from the duodenal opening). The results show that ( Figure 15 In the 1.2cm implantation site group (n=12), three deaths occurred, and the serum alkaline phosphatase and conjugated bile acid levels in the surviving animals showed large variations, indicating a high operative risk due to the site's proximity to the hepatic hilum. In contrast, the 0.5cm and 0.8cm implantation sites showed no deaths, successful surgeries, and good model consistency. This confirms that the 0.5-0.8cm implantation site selected in this invention is crucial for avoiding critical anatomical structures, ensuring surgical safety, and guaranteeing model reproducibility.

[0097] In summary, the aforementioned comparative examples collectively demonstrate that this invention is not simply a patchwork of components, but rather, through extensive experimental verification, the optimal combination of a series of core technical features has been determined, including an inner diameter (0.12~0.15mm), length (0.5~0.8cm), a soft protective wing structure, and an implantation site (0.5~0.8cm from the duodenum). This specific combination solves the long-standing technical problems in the field of model pathological progression being too rapid and excessive, resulting in low animal survival rates and poor reproducibility. The chronic, mild, controllable, and stable modeling effect achieved is something that those skilled in the art could not have predicted beforehand, thus possessing outstanding substantive characteristics and significant progress.

[0098] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A common bile duct restrictor for constructing a rat model of chronic cholestasis, characterized in that: It includes a rigid tubular body and a soft protective fixing wing covering the tubular body, wherein the inner diameter of the tubular body is 0.12 ~ 0.15 mm.

2. The common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 1, characterized in that: The tubular body is made of one of the following: medical-grade stainless steel, alloy, or ceramic.

3. The common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 1, characterized in that: The length of the tubular body is 0.5~0.8 cm.

4. A common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 3, characterized in that: The tubular body has an inner diameter of 0.12 mm and a length of 0.5 cm.

5. A common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 4, characterized in that: The outer diameter of the tubular body is 0.32~0.37 mm.

6. A common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 5, characterized in that: Both ends of the protective fixed wing are provided with protruding fixing rings, and the total outer diameter of the flow limiting tube is less than or equal to 0.7 mm.

7. The common bile duct restrictor tube for constructing a rat model of chronic cholestasis according to claim 6, characterized in that: The protective fixed wing is made of medical-grade silicone or medical-grade polyurethane.

8. A method for constructing a rat model of chronic cholestasis, characterized in that: The procedure includes implanting and fixing the flow-limiting tube according to any one of claims 1 to 7 into the common bile duct in the porta hepatis of a rat, comprising the following steps: Step 1: Selection of implantation site: Select the common bile duct within 0.5-0.8 cm from the opening of the duodenum as the implantation site for the flow restriction tube; Step 2, Surgical Procedure: After anesthetizing the animal, make an abdominal incision to expose the common bile duct in the porta hepatis; perform a preliminary ligation on the side of the common bile duct near the duodenum; make a small longitudinal incision on the anterior wall of the common bile duct; gently insert the flow-limiting tube into the common bile duct along the physiological flow direction of bile; then, use the protective wings at both ends of the flow-limiting tube as fixation points to ligate and fix it, with the ligation force being such that the flow-limiting tube does not move and the bile duct wall only slightly deforms; finally, close the abdomen layer by layer.

9. The method for constructing a rat model of chronic cholestasis according to claim 8, characterized in that: The ligation fixation is performed using 7-0, 8-0, or 9-0 non-absorbable sutures.

10. A method for constructing a rat model of chronic cholestasis according to claim 8 or 9, characterized in that: The entire surgical procedure follows the principle of minimally invasive surgery and uses microsurgical instruments for blunt dissection to minimize tissue damage.

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