Use of lactobacillus acidophilus in the preparation of a product for the prevention or treatment of gallbladder cholesterol stones

Abstract

The application discloses application of lactobacillus acidophilus in preparation of products for preventing or treating gallbladder cholesterol stones, and relates to the technical field of probiotics, and the lactobacillus acidophilus is used in the products for preventing or treating cholesterol stones by regulating LXR-alpha channel and NPC1L1 protein expression; the product is a medicine or a functional food, the product contains an effective dose of lactobacillus acidophilus live bacteria and a pharmaceutically or food-acceptable carrier, the lactobacillus acidophilus is lactobacillus acidophilus ATCC4356; the product is used for reducing the ratio of cholesterol and total bile acid in bile, thereby improving the stone-forming property of bile; the product is used for down-regulating NPC1L1 protein expression, so as to reduce intestinal cholesterol absorption; it is clear that the lactobacillus acidophilus has a significant intervention effect on the formation of gallbladder cholesterol stones of mice induced by high cholesterol diet.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to the application of Lactobacillus acidophilus in the preparation of products for the prevention or treatment of gallbladder cholesterol stones. Background Technology

[0002] Gallstones are a common digestive system disease that seriously affects patients' quality of life and also imposes a heavy medical burden on society. Among various types of gallstones, cholesterol stones account for over 70%, making them the most common type.

[0003] The formation of cholesterol gallstones is a complex pathological process involving multiple factors. The core mechanism lies in an imbalance in cholesterol metabolism, leading to cholesterol supersaturation in bile. Under normal physiological conditions, cholesterol absorption in the intestines, transport in the liver, and bile secretion are in dynamic equilibrium. However, when the body's metabolism is disrupted, this balance is broken, and excess cholesterol is secreted into the bile, eventually crystallizing and forming gallstones.

[0004] In the molecular regulatory network of cholesterol transport and secretion, hepatic X receptor α (LXR-α) is a core transcription factor maintaining cholesterol metabolic homeostasis. ATP-binding cassette transporters G5 and G8 (ABCG5 / ABCG8), members of the adenosine triphosphate (ATP)-binding cassette transporter family, assemble into functional heterodimers in the endoplasmic reticulum and are located on the hepatocyte capillary bile duct lateral membrane and the intestinal epithelial cell apical membrane, serving as key proteins for cholesterol transport and excretion. Niemann-Pick C1-like protein 1 (NPC1L1), as a core transmembrane protein mediating intestinal cholesterol uptake, directly determines the efficiency of intestinal cholesterol absorption through its expression level.

[0005] In recent years, the close relationship between gut microbiota and cholesterol metabolism has received widespread attention. Probiotics, as a class of live microorganisms that, when ingested in sufficient quantities, can produce beneficial effects on host health, colonize and multiply in the gut, intervening in lipid metabolism through multiple pathways, including regulating gut microbiota structure, enhancing intestinal barrier function, and influencing the enterohepatic circulation of bile acids. *Lactobacillus acidophilus* (LA) is one of the most widely used probiotics in clinical practice, exhibiting good gastrointestinal tolerance and small intestinal colonization ability. However, the specific intervention effects and molecular mechanisms of *Lactobacillus acidophilus* in the formation of cholesterol gallstones remain unclear.

[0006] This study aims to investigate whether Lactobacillus acidophilus can influence cholesterol absorption and transport in the body by regulating the protein expression of the liver LXR-α-ABCG5 / ABCG8 signaling axis and the small intestine NPC1L1, thereby intervening in the formation of cholesterol gallstones. This research is expected to provide experimental evidence and theoretical basis for the clinical translation and application of probiotic preparations. Summary of the Invention

[0007] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an application of *Lactobacillus acidophilus* in the preparation of products for the prevention or treatment of gallbladder cholesterol stones. It clarifies that *Lactobacillus acidophilus* has a significant intervention effect on the formation of gallbladder cholesterol stones induced by a high-cholesterol diet in mice. It reveals the potential molecular mechanism by which *Lactobacillus acidophilus*, based on the LXR-α and NPC1L1 pathways, improves the imbalance of bile components and synergistically inhibits the formation of gallbladder cholesterol stones.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The use of Lactobacillus acidophilus in the preparation of products for the prevention or treatment of cholesterol gallstones, wherein the Lactobacillus acidophilus regulates the LXR-α pathway and NPC1L1 protein expression in the products for the prevention or treatment of cholesterol gallstones.

[0009] As a preferred embodiment, the product is a pharmaceutical or functional food, the product contains an effective dose of live Lactobacillus acidophilus and a pharmaceutically or food-acceptable carrier, the Lactobacillus acidophilus having the preservation number ATCC4356.

[0010] As a preferred embodiment, the product is used to reduce the ratio of cholesterol to total bile acids in bile, thereby improving bile litholytic properties.

[0011] As a preferred option, the product is used to downregulate NPC1L1 protein expression to reduce intestinal cholesterol absorption.

[0012] As a preferred embodiment, the product is used to inhibit the expression of LXR-α and downregulate the protein expression of its downstream ATP-binding cassette transporter ABCG5 / ABCG8, thereby reducing the secretion of cholesterol from hepatocytes into bile.

[0013] As a preferred embodiment, the viable bacterial concentration of the *Lactobacillus acidophilus* is at least 1 × 10⁻⁶. 9 CFU / mL.

[0014] As a preferred embodiment, the method for preparing the live Lactobacillus acidophilus preparation includes the following steps: First, activation culture: After thawing the strain preserved in glycerol at -80℃, take a small amount of bacterial solution and add it to MRS liquid medium. Then, place it in an anaerobic incubator at 37℃ and culture for 24 hours to obtain activated bacterial solution. Second, expand culture: Take the bacterial culture from the cultured solution after 24 hours each day, inoculate it into fresh MRS liquid medium for expanded culture, mix well and continue to culture at 37°C for 24 hours. Third, collect the bacterial cells: After 24 hours of culture, collect the bacterial culture, centrifuge to collect the bacterial cell precipitate, resuspend it in physiological saline, and adjust the viable cell concentration to 1×10⁻⁶. 9 CFU / mL yields a live Lactobacillus acidophilus preparation.

[0015] A pharmaceutical composition or functional food composition for the prevention or treatment of gallbladder cholesterol stones, comprising Lactobacillus acidophilus in the application described herein, and a pharmaceutically or food-grade acceptable carrier, wherein Lactobacillus acidophilus is the active ingredient.

[0016] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, as can be seen from the above technical solution: First, this application demonstrates that Lactobacillus acidophilus has the effect of inhibiting lipid metabolism disorders. Compared with the high-cholesterol diet-induced lithotripsy model group (LD group), the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the Lactobacillus acidophilus intervention group (LA group) were reduced, while the level of high-density lipoprotein cholesterol (HDL-C) increased. This provides a new idea and method for the prevention and treatment of related diseases caused by dyslipidemia.

[0017] Secondly, the balance between cholesterol and bile acids in bile is crucial for maintaining the soluble state of cholesterol. An elevated TC / TBA ratio leads to cholesterol supersaturation in gallbladder bile, which is an important indicator of bile lithogenesis. In this application, the TC / TBA ratio in the bile of mice in the LD group was significantly higher than that in the normal control group (NC group). However, after intervention with Lactobacillus acidophilus, the TC / TBA ratio in the LA group was significantly lower than that in the LD group, suggesting that Lactobacillus acidophilus can effectively improve bile lithogenesis and reduce the risk of cholesterol gallstone formation.

[0018] Third, this study reveals the regulatory role of Lactobacillus acidophilus in the expression of key proteins. Lactobacillus acidophilus can inhibit the activation of the LXR-α-ABCG5 / ABCG8 signaling pathway in the liver and reduce the active secretion of cholesterol from hepatocytes into bile.

[0019] Fourth, intervention with Lactobacillus acidophilus can significantly reduce the expression of NPC1L1 in the intestine of mice fed a high-fat diet, thereby leading to a decrease in serum cholesterol levels and a reduction in cholesterol gallstone formation.

[0020] Fifth, a molecular model was proposed to demonstrate the synergistic mechanism of Lactobacillus acidophilus in preventing cholesterol gallstones through intestinal absorption inhibition and hepatic secretion downregulation. Lactobacillus acidophilus downregulates the expression of NPC1L1 protein in the small intestine, reducing intestinal cholesterol uptake; on the other hand, it inhibits the hepatic LXR-α-ABCG5 / ABCG8 signaling pathway, reducing cholesterol excretion from hepatocytes, synergistically correcting bile component imbalance and reducing bile stone formation, providing a theoretical basis for the clinical application of Lactobacillus acidophilus in the prevention and treatment of cholesterol gallstones.

[0021] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the trend of mouse weight change and the final weight gain of the mouse according to the present invention. Figure 2 This is a schematic diagram showing the comparison of protein expression levels in different groups of mice according to the present invention; Figure 3 This is a schematic diagram illustrating the correlation between protein expression levels and bile lithogenesis in this invention. Detailed Implementation

[0023] The present invention is as follows Figure 1 As shown in Figure 3, a type of Lactobacillus acidophilus is used in the preparation of products for the prevention or treatment of cholesterol gallstones. This Lactobacillus acidophilus regulates the LXR-α pathway and NPC1L1 protein expression in products for the prevention or treatment of cholesterol gallstones.

[0024] This product is a pharmaceutical or functional food containing an effective dose of live Lactobacillus acidophilus and a pharmaceutically or food-acceptable carrier, with the Lactobacillus acidophilus having the accession number ATCC4356.

[0025] This product is used to reduce the ratio of cholesterol to total bile acids in bile, thereby improving bile stone formation.

[0026] This product is used to downregulate NPC1L1 protein expression to reduce intestinal cholesterol absorption.

[0027] This product is used to inhibit the expression of LXR-α and downregulate the protein expression of its downstream ATP-binding cassette transporters ABCG5 / ABCG8, thereby reducing the secretion of cholesterol from hepatocytes into bile.

[0028] The viable concentration of this Lactobacillus acidophilus is at least 1 × 10⁻⁶. 9 CFU / mL.

[0029] The method for preparing the live Lactobacillus acidophilus preparation includes the following steps: First, activation culture: After thawing the strain preserved in glycerol at -80℃, take a small amount of bacterial solution and add it to MRS liquid medium. Then, place it in an anaerobic incubator at 37℃ and culture for 24 hours to obtain activated bacterial solution. Second, expand culture: Take the bacterial culture from the cultured solution after 24 hours each day, inoculate it into fresh MRS liquid medium for expanded culture, mix well and continue to culture at 37°C for 24 hours. Third, collect the bacterial cells: After 24 hours of culture, collect the bacterial culture, centrifuge to collect the bacterial cell precipitate, resuspend it in physiological saline, and adjust the viable cell concentration to 1×10⁻⁶. 9 CFU / mL yields a live Lactobacillus acidophilus preparation.

[0030] A pharmaceutical composition or functional food composition for the prevention or treatment of gallbladder cholesterol stones, comprising Lactobacillus acidophilus in the application thereto, and a pharmaceutically or food-acceptable carrier, wherein Lactobacillus acidophilus is the active ingredient.

[0031] Example: Application of Lactobacillus acidophilus in the preparation of products for the prevention or treatment of gallbladder cholesterol stones Materials and Methods: Animals: Eight-week-old adult male C57BL / 6 mice were purchased from Henan Skobes Biotechnology Co., Ltd., and housed in the SPF-grade animal facility of the Experimental Animal Center of Shihezi University. The indoor temperature was 22±2℃, and the humidity was 40%~60%, with a 12-hour light-dark cycle. After one week of acclimatization feeding, they were included in the experiment. This experiment was reviewed and approved by the Bioethics Committee of Shihezi University (Approval No.: A2024-382).

[0032] Feed: The regular feed was provided by the Experimental Animal Center of Shihezi University, with a formula of 60% carbohydrates, 21% protein, 5% fat, and 14% other ingredients; the high-cholesterol litholytic feed was purchased from Sinobiotech Co., Ltd., with a formula of 1.25% cholesterol, 15% lard, 0.5% bile acid, and 83.25% basal feed.

[0033] The strain, *Lactobacillus acidophilus*, has the accession number ATCC 4356; its deposit address is 10801 University Boulevard, Manassas, VA20110, USA; it is deposited at the American Type Culture Collection (ATCC); this strain was provided by Shandong Lu Microbial Technology Co., Ltd. (its original source is ATCC). After thawing the strain from its glycerol-preserved state at -80℃, a small amount of the bacterial culture was inoculated into MRS medium and incubated at 37℃ for 24 hours. Subsequently, it was diluted 9 times with sterile physiological saline and inoculated onto MRS medium for plate colony counting. The strain concentration was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0034] Reagents: Total cholesterol (TC, catalog number: A111-1-1), triglycerides (TG, catalog number: A110-1-1), low-density lipoprotein cholesterol (LDL-C, catalog number: A113-1-1), high-density lipoprotein cholesterol (HDL-C, catalog number: A112-1-1), and total bile acids (TBA, catalog number: E003-2-1) ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute; LXR-α primary antibody (catalog number: 14351-1-AP) and ABCG5 primary antibody (catalog number: 27722-1-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; ABCG8 primary antibody (Bio-Sens Biotechnology Co., Ltd., catalog number: bs-10149R) was purchased from Wuhan Sanying Biotechnology Co., Ltd. ); NPC1L1 primary antibody (Thermo Fisher Scientific, catalog number: PA1-16800); ACTIN primary antibody (catalog number: GB15003), GAPDH primary antibody (catalog number: GB15004), and rabbit secondary antibody (catalog number: GB23303) were all purchased from Savill Biotechnology Co., Ltd.

[0035] Preparation of bacterial culture for gavage: In this experiment, bacterial culture was prepared daily using a continuous subculturing method for gavage in mice. Specific steps: Each day, 1 ml of the bacterial culture cultured at 37°C for 24 hours was aspirated and inoculated into 9 ml of fresh MRS culture medium. After mixing, the culture was placed in a 37°C incubator for another 24 hours. Simultaneously, the remaining old bacterial culture (i.e., the culture cultured for 24 hours the previous day) was used for the gavage experiment on the same day. These steps were repeated daily to ensure a relatively stable bacterial concentration.

[0036] Animal grouping and treatment: Mice were randomly divided into three groups: a normal control group (NC group), a litholytic model group (LD group), and a Lactobacillus acidophilus intervention group (LA group), with 6 mice in each group. The NC group was fed a normal diet, while the LD and LA groups were fed a high-cholesterol litholytic diet for a total of 8 weeks. Simultaneously, the LA group was administered 0.2 ml of Lactobacillus acidophilus bacterial solution (1×10⁻⁶) by gavage.9 The CFU / mL concentration was measured in CFU, and the NC and LD groups were administered equal volumes of normal saline by gavage for a total of 8 weeks.

[0037] Sample collection: After the last intervention, mice were fasted for 12 hours but allowed free access to water. Blood samples were collected from the mice, and the gallbladder, liver, and small intestine were preserved and frozen in EP tubes at -80 ℃ for subsequent protein expression detection.

[0038] Serum lipid and bile composition determination: According to the kit instructions, the levels of TC, TG, LDL-C, HDL-C in mouse serum and TC and TBA in bile were detected by enzyme colorimetric method.

[0039] Western blotting was used to detect the expression levels of LXR-α, ABCG5, ABCG8 in the liver and NPC1L1 in the small intestine. Total protein was extracted from mouse liver and small intestine tissues, and the protein concentration was determined by the BCA method. After electrophoresis, the total protein was transferred to a PVDF membrane and then incubated overnight at 4°C with primary antibodies against LXR-α, ABCG5, ABCG8, NPC1L1, ACTIN, and GAPDH (1:2000, 1:500, 1:1000, 1:5000, 1:1000, and 1:10000). After washing, rabbit secondary antibodies (NPC1L1 and ACTIN at 1:3000, the others at 1:5000) were added and incubated for 2 hours. The protein bands were then visualized using ECL chemiluminescence immunoassay, and the gray values ​​were calculated using ImageJ software.

[0040] Statistical methods: Experimental data were statistically analyzed using SPSS 26.0 software. Normally distributed continuous data were expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons between groups; the LSD method was used for homogeneous variances, and the Tamhane T² method was used for unequal variances. P < 0.05 was considered statistically significant.

[0041] Discussion of Results: Mouse body weight changes: The initial average body weight of mice in each group was 23.28 ± 0.57 g (P > 0.05). During continuous gavage, the body weight of mice showed a consistent and gradual increasing trend (see...). Figure 1 A). After 8 weeks of experimentation, compared with the NC group, the LD group mice showed a significant increase in body weight, while the body weight gain of the LA group mice receiving Lactobacillus acidophilus via gavage was controlled (P<0.05) (see [link to experiment]). Figure 1 B). This indicates that feeding mice with a high-cholesterol diet leads to a significant increase in weight, while Lactobacillus acidophilus can effectively inhibit this growth.

[0042] Gallstone formation in mice: In this experiment, the bile of mice in the NC group was observed to be crystal clear and clearly visible through the gallbladder wall, with no obvious crystals or stones inside. In the LD group, many large stones were visible in the gallbladder, and the bile appeared more turbid compared to other groups, with a stone formation rate of 100%. In the LA group, only muddy or flocculent stones were visible in the gallbladder, with no stones observed in the gallbladders of 4 mice, and the bile remained clear and transparent, with a stone formation rate of 33.3%. This indicates that a high-cholesterol diet can increase the risk of gallstone formation in mice, while Lactobacillus acidophilus can alleviate gallstone formation (see Table 1).

[0043] Table 1: Stone formation in mice of each group

[0044] Serum lipid and bile component levels in mice: Compared with the NC group, the serum TC, TG, and LDL-C levels in the LD group were significantly increased (P<0.05), while the HDL-C level was significantly decreased (P<0.001). Compared with the LD group, the serum TC, TG, and LDL-C level ratio in the LA group was significantly decreased (P<0.05), while the HDL-C level was significantly increased (P<0.001). Compared with the NC group, the TC level in the bile of mice in the LD group was significantly increased, while the TBA level was significantly decreased, leading to a significant increase in the TC / TBA ratio (P<0.001). Compared with the LD group, the TC level in the bile of mice in the LA group was significantly decreased, while the TBA level was significantly increased, leading to a significant decrease in the TC / TBA ratio (P<0.001) (see Tables 2 and 3).

[0045] Table 2: Serum biochemical levels in each group (x±s, n=6)

[0046] Table 3: Levels of bile biochemical indicators in each group (x±s, n=6)

[0047] Expression of proteins affecting cholesterol gallstone formation in mouse liver and small intestine: Compared with the NC group, the expression of LXR-α, ABCG5, and ABCG8 proteins in the liver of mice in the LD group was significantly upregulated (P<0.01); the expression of NPC1L1 protein in the small intestine also showed an upregulated trend (P<0.001). Compared with the LD group, the expression of LXR-α, ABCG5, and ABCG8 proteins in the liver of mice in the LA group was significantly downregulated (P<0.05); the expression of NPC1L1 protein in the small intestine also showed a downregulated trend (P<0.01) (see...). Figure 2 ).

[0048] Correlation analysis of key protein expression levels and bile lithogenesis: Pearson correlation analysis showed that the relative expression level of liver LXR-α protein was significantly positively correlated with the bile TC / TBA ratio (R2=0.8365, P=0.0006); the relative expression level of liver ABCG5 protein was extremely significantly positively correlated with the bile TC / TBA ratio (R2=0.9000, P<0.0001); the relative expression level of liver ABCG8 protein was significantly positively correlated with the bile TC / TBA ratio (R2=0.7243, P=0.0036); and the relative expression level of intestinal NPC1L1 protein was also significantly positively correlated with the bile TC / TBA ratio (R2=0.8791, P=0.0002). The above results suggest that high-fat diet-induced upregulation of intestinal NPC1L1 expression and activation of the hepatic LXR-α-ABCG5 / 8 pathway can jointly increase bile lithogenesis by increasing intestinal cholesterol absorption and hepatic bile cholesterol excretion (see...). Figure 3 ).

[0049] In summary, this study aimed to investigate the intervention effect of *Lactobacillus acidophilus* on cholesterol gallstone formation induced by a high-cholesterol diet in mice, and to elucidate its potential molecular mechanism based on the LXR-α-ABCG5 / ABCG8 and NPC1L1 pathways, in order to provide experimental evidence for the clinical application of probiotic preparations in the prevention and treatment of cholesterol gallstones. Eighteen 8-week-old male C57BL / 6 mice were randomly divided into a control group (NC group), a gallstone model group (LD group), and an *Lactobacillus acidophilus* intervention group (LA group), with six mice in each group. The NC group was fed a standard diet; the LD and LA groups were fed a high-cholesterol gallstone-inducing diet. The LA group was also administered *Lactobacillus acidophilus* bacterial solution (1×10⁻⁶) daily by gavage. 9The mice in the NC and LD groups were administered the same volume of saline by gavage for 8 consecutive weeks (CFU / mL). After the intervention, changes in body weight and gallstone formation were recorded in each group. Serum lipid levels and bile biochemical components were detected by enzyme colorimetry. The protein expression levels of liver LXR-α, ABCG5 / ABCG8, and small intestinal NPC1L1 were detected by Western blotting. Pearson correlation analysis was used to assess the correlation between the protein expression levels and the bile TC / TBA ratio. Compared with the NC group, mice in the LD group showed significantly increased body weight, a gallstone formation rate of 100%, and significantly elevated serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels, while significantly decreased high-density lipoprotein cholesterol (HDL-C) levels (all P < 0.05). Bile TC levels were significantly increased, TBA levels were significantly decreased, and the TC / TBA ratio was significantly increased (all P < 0.05). The expression of liver LXR-α, ABCG5, ABCG8 proteins, and small intestinal NPC1L1 protein was significantly upregulated (all P < 0.05). Compared with the LD group, mice in the LA group showed effectively inhibited body weight gain, a significantly reduced gallstone formation rate to 33.3%, and significantly improved serum lipids and bile biochemical indicators, with a significantly decreased bile TC / TBA ratio. The expression of liver LXR-α, ABCG5, ABCG8 proteins, and small intestinal NPC1L1 protein was significantly downregulated (all P < 0.05). Pearson correlation analysis showed that the relative expression levels of hepatic LXR-α, ABCG5, ABCG8, and small intestinal NPC1L1 proteins were all significantly positively correlated with the bile TC / TBA ratio (R² = 0.8365, 0.9000, 0.7243, and 0.8791, respectively; P < 0.01). Lactobacillus acidophilus reduces intestinal cholesterol absorption by downregulating small intestinal NPC1L1 expression, while simultaneously inhibiting the hepatic LXR-α-ABCG5 / ABCG8 signaling pathway to improve bile component imbalance, thereby synergistically inhibiting the formation of cholesterol gallstones in mice, demonstrating potential clinical translational value.

[0050] The key design focus of this invention is: First, this application demonstrates that Lactobacillus acidophilus has the effect of inhibiting lipid metabolism disorders. Compared with the high-cholesterol diet-induced lithotripsy model group (LD group), the serum levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the Lactobacillus acidophilus intervention group (LA group) were reduced, while the level of high-density lipoprotein cholesterol (HDL-C) increased. This provides a new idea and method for products that prevent and treat related diseases caused by dyslipidemia.

[0051] Secondly, the balance between cholesterol and bile acids in bile is crucial for maintaining the soluble state of cholesterol. An elevated TC / TBA ratio leads to cholesterol supersaturation in gallbladder bile, which is an important indicator of bile lithogenesis. In this application, the TC / TBA ratio in the bile of mice in the LD group was significantly higher than that in the normal control group (NC group). However, after intervention with Lactobacillus acidophilus, the TC / TBA ratio in the LA group was significantly lower than that in the LD group, suggesting that Lactobacillus acidophilus can effectively improve bile lithogenesis and reduce the risk of cholesterol gallstone formation.

[0052] Third, this study reveals the regulatory role of Lactobacillus acidophilus in the expression of key proteins. Lactobacillus acidophilus can inhibit the activation of the LXR-α-ABCG5 / ABCG8 signaling pathway in the liver and reduce the active secretion of cholesterol from hepatocytes into bile.

[0053] Fourth, intervention with Lactobacillus acidophilus can significantly reduce the expression of NPC1L1 in the intestine of mice fed a high-fat diet, thereby leading to a decrease in serum cholesterol levels and a reduction in cholesterol gallstone formation.

[0054] Fifth, a molecular model was proposed to demonstrate the synergistic mechanism of Lactobacillus acidophilus in preventing cholesterol gallstones through intestinal absorption inhibition and hepatic secretion downregulation. Lactobacillus acidophilus downregulates the expression of NPC1L1 protein in the small intestine, reducing intestinal cholesterol uptake; on the other hand, it inhibits the hepatic LXR-α-ABCG5 / ABCG8 signaling pathway, reducing cholesterol excretion from hepatocytes, synergistically correcting bile component imbalance and reducing bile stone formation, providing a theoretical basis for the clinical application of Lactobacillus acidophilus in the prevention and treatment of cholesterol gallstones.

[0055] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of Lactobacillus acidophilus in the preparation of products for the prevention or treatment of gallbladder cholesterol stones, characterized in that: The Lactobacillus acidophilus is used in products for the prevention or treatment of cholesterol gallstones by regulating the LXR-α pathway and NPC1L1 protein expression.

2. The application according to claim 1, characterized in that: The product is a drug or functional food, and contains an effective dose of live Lactobacillus acidophilus and a pharmaceutically or food-acceptable carrier, wherein the Lactobacillus acidophilus has the accession number ATCC4356.

3. The application according to claim 1, characterized in that: The product is used to reduce the ratio of cholesterol to total bile acids in bile, thereby improving bile stone formation.

4. The application according to claim 1, characterized in that: The product is used to downregulate NPC1L1 protein expression to reduce intestinal cholesterol absorption.

5. The application according to claim 1, characterized in that: The product is used to inhibit the expression of LXR-α and downregulate the protein expression of its downstream ATP-binding cassette transporters ABCG5 / ABCG8, thereby reducing the secretion of cholesterol from hepatocytes into bile.

6. The application according to claim 1, characterized in that: The viable concentration of the Lactobacillus acidophilus is at least 1 × 10⁻⁶. 9 CFU / mL.

7. The application according to claim 1, characterized in that: The method for preparing the live Lactobacillus acidophilus preparation includes the following steps: First, activation culture: After thawing the strain preserved in glycerol at -80℃, take a small amount of bacterial solution and add it to MRS liquid medium. Then, place it in an anaerobic incubator at 37℃ and culture for 24 hours to obtain activated bacterial solution. Second, expand culture: Take the bacterial culture from the cultured solution after 24 hours each day, inoculate it into fresh MRS liquid medium for expanded culture, mix well and continue to culture at 37°C for 24 hours. Third, collect the bacterial cells: After 24 hours of culture, collect the bacterial culture, centrifuge to collect the bacterial cell precipitate, resuspend it in physiological saline, and adjust the viable cell concentration to 1×10⁻⁶. 9 CFU / mL yields a live Lactobacillus acidophilus preparation.

8. A pharmaceutical composition or functional food composition for the prevention or treatment of cholesterol gallstones, characterized in that: The application includes Lactobacillus acidophilus as described in any one of claims 1-7, and a pharmaceutically or food-grade acceptable carrier, wherein the Lactobacillus acidophilus is the active ingredient.

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