Liver-intestine chip model and method for evaluating effect of oligosaccharide on liver using same

Abstract

The application belongs to the technical field of organ chip models, and specifically discloses a liver-intestine chip model and a method for evaluating the influence of oligosaccharides on the liver by using the same. The liver-intestine chip model comprises a multilayer microfluidic structure, the main body material of the multilayer microfluidic structure is modified polydimethylsiloxane, the multilayer microfluidic structure comprises an intestinal microcavity, a liver microcavity and a microchannel connecting the two, the intestinal microcavity is inoculated with intestinal epithelial cells, the liver microcavity is inoculated with liver cells, the bottom of the liver microcavity is provided with a window array, the corner of the liver microcavity is provided with a groove, the bottom of the intestinal microcavity is integrated with an elastic membrane, the elastic membrane is communicated with an external air pressure driving system, and the upper surface of the elastic membrane is etched with a microstructure similar to small intestinal villi. By using the above liver-intestine chip model and the method for evaluating the influence of oligosaccharides on the liver by using the same, the liver-intestine organ chip model can simulate the microenvironment of the intestinal-liver axis in vivo, and provides a more real and reliable platform for studying the influence of oligosaccharides on the liver.

Description

Technical Field

[0001] This invention relates to the field of organ-on-a-chip modeling technology, and in particular to liver-intestinal microarray models and methods for evaluating the effects of oligosaccharides on the liver. Background Technology

[0002] The liver is a vital metabolic organ in the human body, and the development of many diseases is closely related to liver function. Simultaneously, the gut and liver are closely linked through the gut-hepatic axis, and the gut microbiota and its metabolites can influence the physiological and pathological processes of the liver. Oligosaccharides, as a class of carbohydrates with potential prebiotic effects, have attracted increasing attention regarding their impact on the liver. Traditional research methods, such as cell culture and animal experiments, have certain limitations. Cell culture struggles to simulate the complex physiological microenvironment in vivo, while animal experiments suffer from species differences and are costly and time-consuming. The emergence of organ-on-a-chip technology offers a new approach to addressing these issues, enabling the construction of highly biomimetic organ microsystems in vitro to better simulate the structure and function of organs in vivo. However, currently, an effective liver-gut organ-on-a-chip model is lacking for evaluating the effects of oligosaccharides on the liver. Summary of the Invention

[0003] The purpose of this invention is to provide a liver-intestinal microarray model and a method for evaluating the effects of oligosaccharides on the liver. The liver-intestinal microarray model can simulate the microenvironment of the gut-liver axis in vivo, providing a more realistic and reliable platform for studying the effects of oligosaccharides on the liver.

[0004] To achieve the above objectives, this invention provides a hepatic-intestinal chip model, comprising a multilayer microfluidic structure. The main material of the multilayer microfluidic structure is modified polydimethylsiloxane. The multilayer microfluidic structure includes intestinal microchambers, liver microchambers, and microchannels connecting the two. The intestinal microchambers are connected to various types of intestinal cells, and the liver microchambers are connected to various types of liver cells. The bottom of the liver microchambers is provided with an array of windows, and the corners of the liver microchambers are provided with grooves. An elastic membrane is integrated at the bottom of the intestinal microchambers. The elastic membrane is connected to an external pneumatic drive system, and the upper surface of the elastic membrane is etched with microstructures similar to small intestinal villi.

[0005] Preferably, the microchannel is provided with a micro flow sensor and a regulating valve in the middle, and a selective permeable membrane with a pore size of 20-50 nm is provided on the side of the microchannel near the intestinal microchamber.

[0006] Preferably, the intestinal microcompartments mimic the crypt-villi structure of the small intestine, and the liver microcompartments mimic the sinusoidal structure of the liver.

[0007] Preferably, the intestinal-like cells include Caco-2 cells and HT-29 cells, with a Caco-2 cell:HT-29 cell ratio of 9:1.

[0008] Preferably, the liver-like cells include HepaRG cells, hepatic stellate cells, Kupffer cells, and endothelial cells, and a mixed cell system is constructed according to the ratio of HepaRG:hepatic stellate cells:Kupffer cells:endothelial cells = 6:2:1:1.

[0009] Preferably, before inoculation of the intestinal microcavities, a polymethacrylic acid-co-acrylic acid copolymer coating with a thickness of 5 μm is grafted onto the internal surface of the intestinal microcavities.

[0010] Preferably, the liver microcavity is provided with a liver microscaffold constructed from poly(N-isopropylacrylamide) hydrogel.

[0011] Preferably, the modification method of polydimethylsiloxane is as follows: the cured polydimethylsiloxane substrate is ultrasonically cleaned with anhydrous ethanol, then rinsed with deionized water, the treated polydimethylsiloxane substrate is placed in a plasma cleaner, oxygen is introduced, and it is treated for 30-60 seconds. After treatment, it is immediately removed, a 5% (v / v) 3-aminopropyltriethoxysilane ethanol solution is prepared, the plasma-treated polydimethylsiloxane substrate is completely immersed in the solution, and it is reacted on a shaker at 30°C in the dark for 2 hours. After the reaction is completed, the surface of polydimethylsiloxane is rinsed with anhydrous ethanol and then vacuum dried.

[0012] This invention also provides a method for evaluating the effects of oligosaccharides on the liver using a liver-gut microarray model, which is implemented using a liver-gut microarray model and includes the following steps:

[0013] Step 1: Dissolve different types and concentrations of oligosaccharides in culture medium and inject them into the liver-intestinal microarray model;

[0014] Step 2: At different time points after oligosaccharide treatment, the activity of cells in the intestinal and liver microcavities was detected;

[0015] Step 3: Collect the culture medium of the liver-intestinal microarray model and detect liver function indicators;

[0016] Step 4: Perform metabolomics analysis on cells within the liver microcavity chamber to detect changes in intracellular metabolites;

[0017] Step 5: Add fluorescently labeled gut microbiota into the intestinal microcavities and observe the changes in the composition and distribution of gut microbiota after oligosaccharide treatment.

[0018] The advantages and beneficial effects of this invention, which uses the above-mentioned hepato-gut microarray model and the method for evaluating the effects of oligosaccharides on the liver, are as follows:

[0019] 1. This invention introduces more cell types into the chip model to construct a cell community that more closely resembles that in vivo. Goblet cells are added to the intestinal microcompartment to simulate the secretory and barrier functions of the intestine. Hepatic stellate cells, Kupffer cells, and endothelial cells are added to the liver microcompartment to form a hybrid cell system, better simulating the physiological and pathological processes of liver metabolism, immunity, and fibrosis. Modified polydimethylsiloxane (PDMS) is used, and through the synergistic effect of plasma activation and chemical grafting, hydrophilic amino groups are introduced onto the PDMS surface. This reduces the adsorption of small molecules such as oligosaccharides while maintaining its good biocompatibility and mechanical properties.

[0020] 2. This invention simulates intestinal peristalsis by placing an elastic membrane at the bottom of the intestinal microcavities and driving the periodic vibration of the elastic membrane with external air pressure. The elastic membrane can achieve uniform and stable periodic vibration under the inflation and deflation of the air chambers, accurately simulating the mechanical stimulation of intestinal epithelial cells by human intestinal peristalsis, thus providing more realistic experimental conditions for studying the effects of oligosaccharides on the liver in a dynamic intestinal environment.

[0021] 3. Compared with traditional cell culture and animal experiments, the method of the present invention has the advantages of low cost, short cycle and high reproducibility, and reduces species differences in animal experiments, thereby improving the accuracy and reliability of experimental results.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the liver-intestinal chip model of the present invention;

[0024] Figure 2 This is a cell activity diagram from Example 2 of the present invention;

[0025] Figure 3 This is a graph showing the liver marker detection results in Example 2 of the present invention, where A is alanine aminotransferase (ALT), B is aspartate aminotransferase (AST), and C is triglycerides (TG).

[0026] Figure 4 This is a graph showing the results of liver microcompartment cell metabolism analysis in Embodiment 2 of the present invention;

[0027] Figure 5 This is a cell activity diagram from Example 3 of the present invention;

[0028] Figure 6 This is a graph showing the liver marker detection results in Example 3 of the present invention, where A is alanine aminotransferase (ALT), B is aspartate aminotransferase (AST), and C is triglycerides (TG).

[0029] Figure 7 This is a cell activity diagram from Example 4 of the present invention;

[0030] Figure 8 This is a graph showing the liver marker detection results in Example 4 of the present invention, where A is alanine aminotransferase (ALT), B is aspartate aminotransferase (AST), and C is triglycerides (TG).

[0031] Figure 9 This is a cell activity diagram from Example 5 of the present invention;

[0032] Figure 10 This is a graph showing the liver marker detection results in Example 5 of the present invention, where A is alanine aminotransferase (ALT), B is aspartate aminotransferase (AST), and C is triglycerides (TG).

[0033] Figure 11 This is a graph showing the results of liver microcompartment cell metabolism analysis in Example 5 of the present invention;

[0034] Figure 12 This is a graph showing the results of gut microbiota analysis in Example 2 of the present invention;

[0035] Figure 13 This is a graph showing the results of gut microbiota analysis in Example 3 of the present invention;

[0036] Figure 14 This is a graph showing the results of gut microbiota analysis in Example 4 of the present invention;

[0037] Figure 15 This is a graph showing the results of gut microbiota analysis in Example 5 of the present invention.

[0038] Figure Labels

[0039] 1. Intestinal microchambers; 2. Flexible trachea; 3. Micropump; 4. Fenhole array; 5. Liver microchambers; 6. Microchannels; 7. Air cavities; 8. Elastic membrane; 9. Small intestinal villi microstructure; 10. Grooves. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0041] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning understood by one of ordinary skill in the art to which this invention pertains. The terms "first," "second," and similar terms used in this invention do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as "comprising" or "including" mean that the element or object preceding the word encompasses the elements or objects listed following the word and their equivalents, without excluding other elements or objects. Terms such as "connected" or "linked" are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as "upper," "lower," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0042] Unless otherwise defined, all reagents, equipment and other materials used in this invention are commercially available.

[0043] Example 1

[0044] like Figure 1 As shown, the liver-intestinal chip model includes a multilayer microfluidic structure. The main material of the multilayer microfluidic structure is modified polydimethylsiloxane. The multilayer microfluidic structure includes an intestinal microchamber 1, a liver microchamber 5, and a microchannel 6 connecting the two. The intestinal microchamber 1 simulates the crypt-villi structure of the small intestine, and the liver microchamber 5 simulates the sinusoidal structure of the liver.

[0045] The intestinal microcompartment 1 is connected to various intestinal cells, including Caco-2 cells and HT-29 cells, with a Caco-2 cell to HT-29 cell ratio of 9:1.

[0046] Five types of liver cells were connected to the liver microcompartment. These liver cell types included HepaRG cells, hepatic stellate cells, Kupffer cells, and endothelial cells. A mixed cell system was constructed according to the ratio of HepaRG:hepatic stellate cells:Kupffer cells:endothelial cells = 6:2:1:1.

[0047] A window array 4 is located at the bottom of the liver microchamber 5. The window array 4, with a diameter of 100-150 nm and a spacing of 200-300 nm, is fabricated using nanolithography on the endothelial cell seeding layer of the liver microchamber 5, with a distribution density of approximately [missing information]. Each microchamber has a per-mm² aperture. These apertures mimic the natural aperture structure of hepatic sinusoidal endothelial cells, allowing small molecule metabolites (such as glucose and short-chain fatty acids) and proteins to pass through, while restricting large molecules such as blood cells, thus replicating the liver's filtration function. The corners of the liver microchambers 5 are equipped with recesses 10 for collecting hepatocyte metabolites, facilitating subsequent detection and analysis, and evaluating liver metabolic function.

[0048] An elastic membrane 8 is integrated at the bottom of the intestinal microchamber 1. The elastic membrane 8 is connected to an external pneumatic drive system, and its upper surface is etched with microstructures 9 resembling small intestinal villi. In the epithelial cell culture area of ​​the intestinal microchamber 1, a columnar microstructure array with a height of 5-10 μm, a diameter of 0.5-1 μm, and a spacing of 2-3 μm is constructed using photolithography combined with electrospinning technology. After cell seeding, the microvilli array can induce the formation of functional microvilli on the cell surface, increasing the intestinal absorptive area and mimicking the absorptive function of the small intestinal mucosa.

[0049] The periodic vibration of the elastic membrane 8, driven by external air pressure, simulates intestinal peristalsis, promoting the maintenance of intestinal cell function and the transport of substances. When constructing the intestinal microchamber 1, the elastic membrane 8 is laid flat across the entire bottom area of ​​the microchamber. Its material can be a modified polydimethylsiloxane (PDMS) material with mechanical properties similar to the intestinal lamina propria, with a thickness controlled at 50-100 μm to ensure good flexibility and appropriate rigidity. Microfabrication techniques such as photolithography are used to etch microstructures similar to the base of the small intestinal villi onto the surface of the elastic membrane 8 to enhance cell adhesion and substance exchange efficiency. Simultaneously, the elastic membrane 8 is seamlessly bonded to the sidewalls of the microchamber, ensuring that vibrational energy is evenly distributed to the entire bottom of the microchamber under external air pressure, avoiding localized stress concentration.

[0050] A micro-pump 3 interface is installed on one side of the microchamber, connected to the air chamber 7 inside the elastic membrane via a flexible air tube 2 with an inner diameter of 100-200 μm. The air chamber 7 is designed as a distributed structure, evenly distributed below the elastic membrane 8, and consists of multiple small air chambers with a diameter of 500 μm and a depth of 100 μm, interconnected by microchannels 6 with a diameter of 50 μm. Thus, when the micro-pump 3 outputs air pressure at a frequency of 0.1-0.5 Hz and an amplitude of 5-10 μm, the elastic membrane 8 can achieve uniform and stable periodic vibration under the inflation and deflation of the air chamber 7, accurately simulating the mechanical stimulation of intestinal peristalsis on intestinal epithelial cells, thereby providing more realistic experimental conditions for studying the effects of oligosaccharides on the liver in a dynamic intestinal environment.

[0051] The microchannel 6 is equipped with a miniature flow sensor and a regulating valve in the middle. A selectively permeable membrane with a pore size of 20-50 nm is located on the side of the microchannel near the intestinal microchamber 1. This allows small molecule metabolites and cytokines to pass through while blocking bacteria and undigested macromolecules, mimicking the intestinal barrier function and preventing harmful substances from entering the liver microchamber 5.

[0052] Before inoculation, the inner surface of intestinal microchamber 1 was grafted with a polymethacrylic acid-co-acrylic acid copolymer coating with a thickness of 5 μm. The coating was functionally modified to address the dynamic changes in the intestinal microenvironment, and its core function was to achieve biomimetic regulation of intestinal barrier function through the pH responsiveness of the material.

[0053] The liver microchamber 5 is equipped with a liver microscaffold constructed from poly(N-isopropylacrylamide) hydrogel. The swelling and shrinkage of the gel (volume change rate of 20-50%) is achieved by temperature control at 32-37℃, simulating the volume change of liver tissue under metabolically active conditions.

[0054] The modification method of polydimethylsiloxane (PDMS) is as follows: the cured PDMS substrate is ultrasonically cleaned with anhydrous ethanol and then rinsed with deionized water. The treated PDMS substrate is then placed in a plasma cleaner, oxygen is introduced, and the treatment is carried out for 30-60 seconds. After treatment, it is immediately removed. A 5% (v / v) 3-aminopropyltriethoxysilane ethanol solution is prepared, and the plasma-treated PDMS substrate is completely immersed in the solution. The reaction is carried out on a shaker at 30°C in the dark for 2 hours. After the reaction is completed, the surface of the PDMS is rinsed with anhydrous ethanol and then vacuum dried.

[0055] This modification method introduces hydrophilic amino groups onto the PDMS surface through the synergistic effect of plasma activation and chemical grafting. This reduces the adsorption of small molecules such as oligosaccharides by the material while maintaining its good biocompatibility and mechanical properties.

[0056] Example 2

[0057] The method for evaluating the effects of oligosaccharides on the liver using a liver-gut microarray model is implemented using the liver-gut microarray model mentioned in Example 1, and includes the following steps:

[0058] Step 1: Dissolve different types and concentrations of oligosaccharides in culture medium and inject them into the liver-intestinal microarray model;

[0059] Step 2: At different time points after oligosaccharide treatment, the activity of cells in the intestinal and liver microcavities was detected;

[0060] Step 3: Collect the culture medium of the liver-intestinal microarray model and detect liver function indicators;

[0061] Step 4: Perform metabolomics analysis on cells within the liver microcavity chamber to detect changes in intracellular metabolites;

[0062] Step 5: Add fluorescently labeled gut microbiota into the intestinal microcavities and observe the changes in the composition and distribution of gut microbiota after oligosaccharide treatment.

[0063] Fructooligosaccharides were dissolved in the culture medium to prepare solutions with concentrations of 0.1 mM, 1 mM, and 10 mM. These solutions of different concentrations were then injected into the chip, and the treatment time was 7 days. A control group was also included, receiving the same amount of culture medium.

[0064] On days 1, 3, 5, and 7 after treatment with fructooligosaccharides, CCK-8 reagent was added to the chip. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. Figure 2 As shown, the results indicated that, compared with the control group, the cell activity of the fructooligosaccharide-treated group was slightly increased in the concentration range of 0.1 mM-1 mM, while the cell activity of the 10 mM fructooligosaccharide-treated group showed no significant change.

[0065] On day 7 of treatment, the chip culture medium was collected, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglycerides (TG) were measured using a biochemical reagent kit. Figure 3 As shown, the results indicated that, compared with the control group, the ALT and AST levels in the 0.1 mM fructooligosaccharide treatment group were slightly lower, while the TG level showed no significant change; the ALT, AST, and TG levels in the 1 mM fructooligosaccharide treatment group were significantly lower; and the ALT and AST levels in the 10 mM fructooligosaccharide treatment group were lower, but the TG level was higher.

[0066] Cells were collected from liver microchambers on day 7 after treatment and metabolomics analysis was performed using LC-MS. Figure 4 As shown, data analysis revealed that the levels of metabolites related to energy metabolism and lipid metabolism in cells of the fructooligosaccharide-treated group changed significantly. The levels of citric acid and α-ketoglutarate, intermediate products of the tricarboxylic acid cycle, increased, while the levels of palmitic acid, a fatty acid synthesizer, decreased.

[0067] Fluorescently labeled gut microbiota (including Bifidobacterium, Lactobacillus, and Escherichia coli) were pre-added to the intestinal microcavities. After 7 days of treatment with fructooligosaccharides, the composition and distribution changes of the gut microbiota were observed using a fluorescence microscope. Quantitative data were used to present the differences in the quantity and distribution of Bifidobacterium, Lactobacillus, and Escherichia coli. The horizontal axis represents the fructooligosaccharide treatment concentration (control group, 0.1 mM, 1 mM, 10 mM), and the vertical axis represents the relative fluorescence intensity (mean ± standard deviation), highlighting the promoting effect of fructooligosaccharides on beneficial bacteria and the inhibitory effect on harmful bacteria. Results are shown in [Figure number missing]. Figure 12 The results showed that the number of Bifidobacteria (p value control group / treatment group <0.001) and Lactobacillus (p value <0.001) in the fructooligosaccharide treatment group was significantly increased, while the number of Escherichia coli (p value <0.005) was decreased.

[0068] Example 3

[0069] Xylooligosaccharides were dissolved in a mixed culture medium to prepare solutions with concentrations of 0.05 mM, 0.5 mM, and 5 mM. The xylooligosaccharide solutions of different concentrations were injected into the chip, and the treatment time was 5 days. A control group was set up, which was injected with the same amount of culture medium. The detection method was the same as in Example 2.

[0070] Cell viability assay: such as Figure 5 As shown, the results indicated that the cell viability of the 0.05 mM and 0.5 mM xylooligosaccharide treatment groups was higher than that of the control group on both day 3 and day 5 of treatment, while the cell viability of the 5 mM xylooligosaccharide treatment group was slightly reduced on day 5.

[0071] Liver function tests: On day 5 of treatment, the culture medium was collected to measure ALT, AST, and TG levels. Figure 6 As shown, the results indicated that the ALT and AST levels in the 0.05 mM xylooligosaccharide treatment group decreased slightly on day 5, while the TG level showed no significant change; the ALT, AST, and TG levels in the 0.5 mM xylooligosaccharide treatment group all decreased significantly on day 5; and the ALT and AST levels in the 5 mM xylooligosaccharide treatment group increased on day 5, as did the TG level.

[0072] Metabolomics analysis: On day 5 after treatment, liver microchamber cells were collected for LC-MS analysis. The results showed that the levels of amino acid metabolism and purine metabolism-related metabolites in the cells of the xylooligosaccharide-treated group were altered. The differences were quantified as "relative content (treatment group / control group, mean ± standard deviation)", and the results are shown in Table 1.

[0073] Table 1 Test Results

[0074]

[0075] Gut microbiota analysis: Changes in fluorescently labeled gut microbiota were observed. It was found that the number of Bifidobacteria (p<0.001) and Lactobacillus (p<0.001) increased in the xylooligosaccharide-treated group, while the number of Enterococci (p<0.001) decreased. Results are shown below. Figure 13 .

[0076] Example 4

[0077] Galacto-oligosaccharides were dissolved in a mixed culture medium to prepare solutions with concentrations of 0.2 mM, 2 mM, and 20 mM. These solutions of different concentrations were injected into the chip, and the treatment time was 6 days. A control group was set up, receiving the same amount of culture medium. The detection method was the same as in Example 2.

[0078] Cell viability assay: Cell viability was measured using the CCK-8 assay on days 2, 4, and 6 after galactooligosaccharide treatment. Figure 7 As shown, the results indicated that the cell viability of the 0.2 mM and 2 mM galactooligosaccharide treatment groups was higher than that of the control group at all time points, while the cell viability of the 20 mM galactooligosaccharide treatment group was lower than that of the control group on day 6.

[0079] Liver function tests: Culture medium was collected and tested on day 6 of treatment. Figure 8As shown, the ALT and AST levels in the 0.2 mM galactooligosaccharide treatment group decreased slightly, while the TG level remained basically unchanged; the ALT, AST, and TG levels in the 2 mM galactooligosaccharide treatment group decreased significantly; and the ALT, AST, and TG levels in the 20 mM galactooligosaccharide treatment group increased.

[0080] Metabolomics analysis: On day 6 after treatment, LC-MS analysis of liver cells revealed significant changes in glucose metabolism-related metabolites in the galactooligosaccharide-treated group, with increased glucose-6-phosphate content and elevated levels of glycogen synthesis-related metabolites. Changes in core glucose metabolism metabolites were quantified using "relative content (treatment group / control group, mean ± standard deviation)," highlighting the increasing trends of glucose-6-phosphate and glycogen synthesis-related metabolites. The results are shown in Table 2.

[0081] Table 2 Test Results

[0082]

[0083] Gut microbiota analysis: After 6 days of treatment with galactooligosaccharides, the number of Bifidobacteria (p < 0.001) and Clostridium (p < 0.005) decreased significantly in the fluorescently labeled gut microbiota. See results below. Figure 14 .

[0084] Example 5

[0085] Isomaltooligosaccharide was dissolved in a mixed culture medium to prepare solutions with concentrations of 0.5 mM, 5 mM, and 50 mM. These solutions were injected into the chip via a microfluidic system, and the treatment time was 4 days. A control group was included, receiving the same amount of culture medium. The detection method was the same as in Example 2.

[0086] Cell viability assay: Detected on days 1, 2, and 4 of treatment, such as... Figure 9 As shown, the cell viability of the 0.5 mM and 5 mM isomaltooligosaccharide treatment groups was higher than that of the control group, while the cell viability of the 50 mM treatment group decreased significantly on day 4.

[0087] Liver function test: Tested on day 4. Figure 10 As shown, ALT and AST were slightly decreased in the 0.5mM group, while TG showed no significant change; ALT, AST, and TG were all significantly decreased in the 5mM group; and all three were increased in the 50mM group.

[0088] Metabolomics analysis: Analysis on day 4 showed that cells collected from liver microchambers were analyzed by LC-MS, such as... Figure 11 As shown, the levels of glutathione, a detoxification-related metabolite, increased in the 5 mM treatment group and decreased in the 50 mM treatment group.

[0089] Gut microbiota analysis: After treatment, the number of Lactobacillus (p<0.001) increased significantly in the 0.5mM and 5mM groups, while the change was not significant in the 50mM group. The number of Escherichia coli (p<0.001) decreased to varying degrees in all treatment groups. Results are shown below. Figure 15 .

[0090] Therefore, this invention employs the above-mentioned hepato-gut microarray model and the method for evaluating the effects of oligosaccharides on the liver. The hepato-gut microarray model can simulate the microenvironment of the gut-liver axis in vivo, providing a more realistic and reliable platform for studying the effects of oligosaccharides on the liver.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A liver-intestinal microarray model, characterized by: The invention includes a multilayer microfluidic structure, the main material of which is modified polydimethylsiloxane. The multilayer microfluidic structure includes an intestinal microchamber, a liver microchamber, and microchannels connecting the two. The intestinal microchamber is connected to a type of intestinal cell, and the liver microchamber is connected to a type of liver cell. The bottom of the liver microchamber is provided with an array of windows, and the corners of the liver microchamber are provided with grooves. An elastic membrane is integrated at the bottom of the intestinal microchamber. The elastic membrane is connected to an external pneumatic drive system, and the upper surface of the elastic membrane is etched with microstructures similar to small intestinal villi. Before inoculation of the intestinal microcavities, a polymethacrylic acid-co-acrylic acid copolymer coating with a thickness of 5 μm is grafted onto the internal surface of the intestinal microcavities. The liver microcavity is provided with a liver microscaffold constructed from poly(N-isopropylacrylamide) hydrogel. The modification method of polydimethylsiloxane is as follows: the cured polydimethylsiloxane substrate is ultrasonically cleaned with anhydrous ethanol, then rinsed with deionized water. The treated polydimethylsiloxane substrate is then placed in a plasma cleaner, oxygen is introduced, and the treatment lasts for 30-60 seconds. After treatment, it is immediately removed. A 5% (v / v) 3-aminopropyltriethoxysilane ethanol solution is prepared, and the plasma-treated polydimethylsiloxane substrate is completely immersed in the solution. The reaction is carried out on a shaker at 30°C in the dark for 2 hours. After the reaction is completed, the surface of the polydimethylsiloxane is rinsed with anhydrous ethanol and then vacuum dried.

2. The liver-intestine chip model according to claim 1, characterized in that: The microchannel is equipped with a micro flow sensor and a regulating valve in the middle, and a selective permeable membrane with a pore size of 20-50 nm is provided on the side of the microchannel near the intestinal microchamber.

3. The liver-intestinal microarray model according to claim 1, characterized in that: The intestinal microcompartments mimic the crypt-villi structure of the small intestine, and the liver microcompartments mimic the sinusoidal structure of the liver.

4. The liver-intestinal chip model according to claim 1, characterized in that: The intestinal-like cells include Caco-2 cells and HT-29 cells, with a Caco-2 cell to HT-29 cell ratio of 9:

1.

5. The liver-intestinal microarray model according to claim 1, characterized in that: The liver-like cells include HepaRG cells, hepatic stellate cells, Kupffer cells, and endothelial cells, and a mixed cell system is constructed according to the ratio of HepaRG:hepatic stellate cells:Kupffer cells:endothelial cells = 6:2:1:

1.

6. A method for evaluating the effects of oligosaccharides on the liver using a liver-gut microarray model, characterized in that, It is implemented using the hepatobiliary chip model as described in any one of claims 1-5, and includes the following steps: Step 1: Dissolve different types and concentrations of oligosaccharides in culture medium and inject them into the liver-intestinal microarray model; Step 2: At different time points after oligosaccharide treatment, the activity of cells in the intestinal and liver microcavities was detected; Step 3: Collect the culture medium of the liver-intestinal microarray model and detect liver function indicators; Step 4: Perform metabolomics analysis on cells within the liver microcavity chamber to detect changes in intracellular metabolites; Step 5: Add fluorescently labeled gut microbiota into the intestinal microcavities and observe the changes in the composition and distribution of gut microbiota after oligosaccharide treatment.

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