Method for evaluating liver lipid regulation by using liver lipid regulating reagent and application of method

Abstract

The invention provides a method for evaluating liver lipid regulation by a liver lipid regulating reagent and application of the method. The method comprises the steps of organ chip processing, 3D non-alcoholic fatty liver organ formation and liver lipid regulating reagent intervention and evaluation. According to the invention, the principles of engineering and stem cell development are utilized, the response of the liver tissue and the liver lipid regulating reagent under the pathological state of the human liver is remodeled in vitro, and the method has great application value in the aspects of in-vitro organ reconstruction, drug metabolism, disease generation mechanism exploration, drug efficacy evaluation and innovative drug development.

Description

Technical Field

[0001] The invention relates to the fields of tissue engineering and biomedicine, and in particular to an evaluation method of a liver lipid regulating agent such as ginsenoside Rb1. Background Art

[0002] The liver is an important and complex organ that participates in protein and fat metabolism, bile production, detoxification, immune regulation and other functions. Ginseng is a natural product extracted from plants with important medicinal and food homology, and is widely used in health care worldwide. Ginsenoside Rb1 is the most abundant monomer extracted from ginseng. In recent years, more and more studies have focused on exploring the medicinal value of ginsenoside Rb1 in anti-hyperlipidemia and liver protection. At present, the application of ginsenosides in liver lipid regulation is mainly concentrated in animal experiments and two-dimensional monolayer cell experiments. Unfortunately, due to species differences, the existing animal models cannot fully represent human biology. In addition, most two-dimensional monolayer cell experiments use static culture, lack a dynamic environment similar to the body, and it is difficult to simulate key organ functions, and the drug prediction ability is poor. In other words, the traditional ginsenoside efficacy evaluation system has deficiencies in both technical means and methods. Therefore, exploring new evaluation systems and discovering innovative drugs are still hot topics in current research. In addition, high-throughput drug testing in a pathology-related manner can greatly improve the efficiency of drug development and guide clinical drug use.

[0003] Human liver in vitro models can accurately predict drug response and efficacy, effectively making up for the shortcomings of animal models. Human organoids are the mainstream human organoid equivalents in vitro. They refer to 3D cell aggregates formed by self-assembly of stem cells or progenitor cells, which summarize the key structures and functions of in vivo organ / tissue-like organs. In particular, organoids derived from human induced pluripotent stem cells have shown great potential in biomedicine, including disease modeling, efficacy evaluation, sensitivity testing and safety testing. Organ chips can accurately reshape physiological microenvironmental factors such as fluid shear force, information interaction, and drug gradients, partially overcoming the limitations of traditional models and have been widely used in liver physiological and pathological modeling. At present, organoid chips are a forward-looking technology that combines the advantages of organoids and organ chips. They have great application value in in vitro organ regeneration, drug metabolism, disease mechanism exploration, drug efficacy evaluation and innovative drug development. Therefore, organoid chip technology can make up for the shortcomings of animal models and monolayer cell culture models in liver lipid regulation reagents such as ginsenoside Rb1 in liver lipid regulation. Summary of the invention

[0004] The purpose of the present invention is to provide an evaluation method for liver lipid regulation and its application. The invention effectively realizes the processing of organ chips, the formation of 3D non-alcoholic fatty liver organoids, and the intervention and evaluation of ginsenoside Rb1 and obeticholic acid Ob. The dynamic perfusion system reshapes the key microenvironmental elements such as complex cellular components, physiological fluids, 3D tissue morphology, etc. in the body, not only realizing the construction of an in vitro pathological liver model, but also reproducing the response of the human liver to liver lipid regulating agents. The invention has great application value in in vitro organ regeneration, drug metabolism, disease mechanism exploration, drug efficacy evaluation and innovative drug development. The invention is applicable to the evaluation method of liver lipid regulation, which is not limited to ginsenoside Rb1 or obeticholic acid Ob, and can be extended to the lipid regulation evaluation of other formed drugs or lead drugs.

[0005] The present invention provides an application of ginsenoside Rb1 in liver lipid regulation, characterized in that the main steps of the model construction method are: processing of organ chips, formation of 3D non-alcoholic fatty liver organoids, and intervention and evaluation of ginsenoside Rb1 and obeticholic acid Ob.

[0006] The organ chip is mainly composed of several microarray units, wherein each array unit is composed of a cell and culture medium inlet (1), a perfusion channel (2), a microarray structure (3), and a culture medium outlet (4). After entering through the inlet (1), the cells are retained in the microarray structure, assembled in situ and induced to form organoids. After the culture medium enters through the inlet (1) at a certain flow rate, it passes through the perfusion channel containing the microarray structure (3) and then flows out through the outlet (4).

[0007] The organ chip is composed of an upper and lower layer, and the material is polydimethylsiloxane (PDMS). The number of array units is 1-8, and the micro-array structure (3) can be pit-shaped or column-shaped, and the diameter of the micro-pit / column ranges from 200μm to 1mm, the depth ranges from 200μm to 800μm, and the spacing ranges from 50-500μm.

[0008] The flow rate of the culture medium in the organ chip is 30-100 μL / h.

[0009] The processing of the organ chip specifically comprises the following steps: preparing the chip by using soft lithography technology, pouring PDMS prepolymer on the formed template to obtain the upper and lower layers, sealing after oxygen plasma treatment for 10-90s, and then hydrophobically modifying the chip with a certain concentration of PF127 for use.

[0010] The concentration range of the PF127 modified chip is 0.1%-1%, and the action time is 1-24h.

[0011] The formation of 3D non-alcoholic fatty liver organoids provided by the present invention comprises the following specific steps:

[0012] (1) Differentiation of human pluripotent stem cells into liver-specific endoderm: The culture medium was treated with RPMI-1640, GlutaMAX, KSR, B27, and Activin A for 5 days, then replaced with a culture medium containing GlutaMAX, KSR, B27, BMP4, bFGF, and RPMI-1640 for 3 days.

[0013] The concentration ranges of GlutaMAX, KSR and B27 are all 0.1-5%, the concentration range of ActivinA is 50-100 ng / mL, the concentration range of BMP4 is 10-40 ng / mL, and the concentration range of bFGF is 5-20 ng / mL.

[0014] (2) Differentiation into hepatic progenitor cells: The culture medium components are RPMI-1640, GlutaMAX, KSR, B27, and HGF for 5 days. The concentrations of GlutaMAX, KSR, and B27 are all in the range of 0.1-5%, and the concentration of HGF is in the range of 10-40 ng / mL.

[0015] (3) Hepatocyte differentiation and organoid formation: The culture medium consisted of HCM, OSM, and Dex for 5 days.

[0016] The OSM concentration range is 5-20 ng / mL, and the Dex concentration range is 10 -8 -10 -7 M.

[0017] (4) Long-term culture of liver organoids: The culture medium consists of HCM and Dex treatment for 2 days.

[0018] The Dex concentration range is 10 -8 -10 -7 M.

[0019] (5) Free fatty acid exposure: The culture medium components are HCM and Dex. This is the formation of pathological 3D non-alcoholic fatty liver organoids.

[0020] The free fatty acid concentration range is 100-1000 μM, and the Dex concentration range is 10 -8 -10 -7 M.

[0021] The liver lipid regulating reagent provided by the present invention, such as ginsenoside Rb1 or obeticholic acid Ob intervention, specifically comprises the following steps: preparing a certain concentration of ginsenoside working solution or obeticholic acid Ob working solution to intervene in 3D non-alcoholic fatty liver organoids, wherein the culture medium components are HCM and Dex.

[0022] The concentration range of the liver lipid regulating agent, such as ginsenoside Rb1, is 0.01 μM-1 mM, and the action time of ginsenoside is 5 days. The concentration range of obeticholic acid Ob is 1-50 μM, the action time is 5 days, and the concentration range of Dex is 10 -8 -10 -7 M.

[0023] The evaluation after intervention with liver lipid regulating agents such as ginsenoside Rb1 or obeticholic acid Ob is as follows: liver organoids without free fatty acids, with free fatty acids, with free fatty acids and liver lipid regulating agents such as ginsenoside Rb1, and with free fatty acids and liver lipid regulating agents such as obeticholic acid Ob are collected, cholesterol and triglyceride levels are detected by the kit microplate method, the production and accumulation of lipid droplets in liver organoids are characterized by oil red staining, and frozen sections are subjected to lipid immunofluorescence staining. At a certain concentration, ginsenoside Rb1 and obeticholic acid Ob can effectively reduce triglyceride and cholesterol levels and reduce lipid accumulation.

[0024] The present invention also provides the application of the method described in the evaluation of identifying liver lipid regulating reagents, or the authenticity or purity of the drug to be tested, specifically for whether an unknown drug has the function of regulating liver lipids. For example, it reduces the levels of triglycerides and cholesterol under free fatty acid exposure, improves lipid metabolism ability, and reduces oxidative stress. Specifically, liver lipid accumulation can be measured by triglycerides and cholesterol. If it increases, it means lipid accumulation occurs. Immunofluorescence staining or oil red staining is used to observe lipids, and lipid accumulation can be clearly seen. Lipid metabolism is mainly regulated by real-time fluorescence quantitative PCR to detect the expression of lipid decomposition and lipid synthesis genes. If the unknown drug can regulate lipid metabolism, it will reduce the expression of lipid synthesis genes and increase the expression of lipid decomposition genes, thereby reducing lipid accumulation. It is used to develop new drugs; or specifically, the liver lipid regulating reagent to be tested is evaluated according to the above method to obtain the evaluation result, and the standard liver lipid regulating reagent is compared with the evaluation result obtained by the above method to judge.

[0025] The present invention utilizes the principles of engineering and stem cell development to reshape the response of liver tissue and liver lipid regulating agents under human liver pathological conditions in vitro. The invention has great application value in in vitro organ regeneration, drug metabolism, disease mechanism exploration, drug efficacy evaluation and innovative drug development. Specifically, the present invention can eliminate the interference of matrix materials and the resulting uncertainty in the organ development process, and can overcome the heterogeneity of conventional organoid culture, such as the difference in subsequent functions caused by different sizes. First of all, the matrix-free system with clear components used in the present invention is more conducive to analyzing key events in the organ development process. In addition, the chip used in the present invention can be designed as a high-throughput bit array form, and microfluidic technology realizes the manipulation and monitoring of fluids, forming hundreds of organoids in a unit body system, while avoiding their mutual fusion, and expanding the demand for biological samples. At the same time, the microarray chip of the present invention can also be combined with a commercial well plate, so as to have greater compatibility and higher efficiency. Furthermore, the present invention uses a microfluidic chip with excellent biocompatibility, which is easy to manipulate fluids and monitor organoid behavior. Finally, the species differences between animals and humans make it difficult to directly extrapolate the results obtained in animal models to humans to guide human medication and drug development. The microfluidic chip of the present invention is expected to replace animal models for the development and evaluation of lead drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the organ chip structure. Figure A is a high-throughput array chip; Figure B is a single array unit of the organ chip; 1 is the cell and culture medium inlet, 2 is the perfusion channel, 3 is the micro-array structure (columnar), and 4 is the culture medium outlet.

[0027] Figure 2 This is a diagram of the formation and identification results of 3D non-alcoholic fatty liver organoids. Figure A shows the accumulation of lipid droplets; Figure B shows the levels of triglycerides and cholesterol under the action of 0.5μM, 1μM, and 10μM concentrations of Rb1.

[0028] Figure 3 The results of the regulation of liver lipid metabolism in 3D non-alcoholic fatty liver organoids under the intervention of 50μM and 100μM concentrations of Rb1. Figure A shows the levels of triglycerides and cholesterol under the action of 50μM and 100μM concentrations of Rb1, and Figure B shows the expression of lipid metabolism-related genes under the action of 100μM concentration of Rb1.

[0029] Figure 4 Figure 3. Oxidative stress and lipid peroxidation levels in 3D non-alcoholic fatty liver organoids treated with 5 μM obeticholic acid and 100 μM Rb1. DETAILED DESCRIPTION

[0030] The present invention will be further described in the following examples, but the present invention is not limited thereto.

[0031] Example 1

[0032] Lipid regulation by ginsenoside Rb1 in the liver.

[0033] The present invention provides an application of ginsenoside Rb1 in liver lipid regulation, characterized in that the invention includes main steps: processing of organ chips, formation of 3D non-alcoholic fatty liver organoids and intervention and evaluation of ginsenoside Rb1.

[0034] The organ chip is mainly composed of several microarray units, such as Figure 1 The figure shows an organoid chip composed of four microarray units, wherein each array unit is composed of a cell and culture medium inlet (1), a perfusion channel (2), a microarray structure (3), and a culture medium outlet (4). After entering through the inlet (1), the cells are retained in the microarray structure, assembled in situ and induced to form organoids. After the culture medium enters through the inlet (1) at a certain flow rate, it passes through the perfusion channel containing the microarray structure (3) and then flows out through the outlet (4).

[0035] The organ chip is composed of upper and lower layers, and the material is polydimethylsiloxane (PDMS). The number of array units is 4, and the micro-array structure is columnar, with a diameter of 500 μm, a depth of 600 μm, and a spacing of 50 μm.

[0036] The flow rate of the culture medium in the organ chip was 40 μL / h.

[0037] The specific steps of constructing the organ chip are as follows: preparing the chip by conventional soft lithography technology, pouring PDMS prepolymer on the formed template to obtain upper and lower layers, sealing after oxygen plasma treatment for 15 seconds, and then hydrophobically modifying the chip with 0.2% PF127 for 4 hours for use.

[0038] The formation of 3D non-alcoholic fatty liver organoids, the specific steps are:

[0039] (1) Differentiation of human pluripotent stem cells into liver-specific endoderm: The culture medium was treated with RPMI-1640, GlutaMAX, KSR, B27, and Activin A for 5 days, then replaced with a culture medium containing GlutaMAX, KSR, B27, BMP4, bFGF, and RPMI-1640 for 3 days.

[0040] The concentrations of GlutaMAX, KSR and B27 were all 1%, the concentration of ActivinA was 100 ng / mL, the concentration of BMP4 was 20 ng / mL, and the concentration of bFGF was 10 ng / mL.

[0041] (2) Differentiation into hepatic progenitor cells: The culture medium components are RPMI-1640, GlutaMAX, KSR, B27, and HGF for 5 days. The GlutaMAX, KSR, and B27 are all 1%, and the HGF concentration is 20 ng / mL.

[0042] (3) Hepatocyte differentiation and organoid formation: The culture medium consisted of HCM, OSM, and Dex for 5 days.

[0043] The OSM concentration was 10 ng / mL and the Dex concentration was 10 -7 M.

[0044] (4) Long-term culture of liver organoids: The culture medium consists of HCM and Dex for 2 days. This is physiological 3D liver organoid formation. The Dex concentration is 10 -7 M.

[0045] (5) Free fatty acid exposure: After 2 days of treatment in step (4), free fatty acids were added for 5 days. The culture medium components were HCM and Dex. This is the formation of pathological non-alcoholic fatty liver organoids, such as Figure 2 The non-alcoholic fatty liver organoid shown in center A has a large amount of lipid droplet accumulation.

[0046] The free fatty acid concentration was 600 μM and the Dex concentration was 10 -7 M.

[0047] The intervention and evaluation of ginsenoside Rb1 provided by the present invention specifically comprises the following steps: preparing a ginsenoside working solution of a certain concentration to intervene in 3D non-alcoholic fatty liver organoids, wherein the culture medium components are HCM and Dex.

[0048] The ginsenoside type is Rb1 with a concentration of 0.5 μM, 1 μM, and 10 μM. The ginsenoside action time is 5 days. The Dex concentration is 10 -7 M. It is well known that the typical feature of non-alcoholic fatty liver disease is dyslipidemia, which is manifested by high levels of triglycerides and cholesterol in serum. Figure 2 As shown in B, ginsenoside Rb1 can effectively reduce triglyceride and cholesterol levels at different concentration gradients.

[0049] Example 2

[0050] Lipid regulation by ginsenoside Rb1 in the liver.

[0051] The steps are the same as in case 1, except that the concentration of the ginsenoside Rb1 is 50 μM and 100 μM. Figure 3 As shown in Figure A, ginsenoside Rb1 at different concentrations can effectively reduce the levels of triglycerides and cholesterol in non-alcoholic fatty liver organoids. Figure 3As shown in Figure B, 100 μM ginsenoside Rb1 can alleviate the abnormal lipid metabolism caused by free fatty acid exposure. Specifically, in the development of non-alcoholic fatty liver disease, the gene of carbamoyl phosphate synthetase (CPS1) and the key enzyme of lipolysis, phosphodiesterase 3B (PDE3B), were significantly downregulated, and the expression of these genes was significantly upregulated after Rb1 intervention. Low-density lipoprotein-related protein 2 (LRP2) is expressed in absorptive epithelial tissue and is involved in the occurrence and progression of metabolic diseases. Figure 3 As shown in Figure B, the expression level of LRP2 gene in the Rb1-treated group was significantly higher than that in the pathological group, showing an inhibitory effect on lipid formation. Low expression of insulin-induced gene 1 (INSIG1) can promote hepatic lipid remodeling, protect against oxidative stress, reduce inflammation and fibrosis, and inhibit the progression of non-alcoholic fatty liver disease. Figure 3 As shown in B, Rb1 intervention can significantly upregulate INSIG1 gene expression induced by free fatty acid exposure.

[0052] Example 3

[0053] Lipid regulation by obeticholic acid Ob in the liver.

[0054] The steps are the same as those in Case 1, except that, as described, the concentration of obeticholic acid Ob is 5 μM. It is well known that reactive oxygen species (ROS), as intermediates in lipid oxidation reactions, can trigger oxidative stress responses in hepatocytes and promote the development of non-alcoholic fatty liver disease. In this study, abnormal lipid metabolism in fatty acid-induced non-alcoholic fatty liver disease may be partly responsible for the occurrence of oxidative stress. An increase in ROS was observed in free fatty acid-induced liver organoids, which is consistent with the results in human non-alcoholic fatty liver disease patients. The results showed that Ob intervention can reduce this level ( Figure 4 A). CYP2E1, as a key enzyme, is involved in the formation of oxidative stress during the development of nonalcoholic fatty lipoprotein. After free fatty acid treatment, the mRNA expression of CYP2E1 increased significantly. Ob treatment downregulated its expression to the control level ( Figure 4 Malondialdehyde (MDA) is a key marker of lipid peroxidation ( Figure 4 C), fatty acid treatment also upregulated MDA levels. In addition, the relative mRNA level of glutathione peroxidase GPx1 was restored by the addition of Ob ( Figure 4 (D), indicating the favorable role of Ob in regulating the anti-lipid oxidation response.

Claims

1. A method for evaluating the regulation of liver lipids by a liver lipid regulating agent, characterized in that: The steps include: S1. Processing of organ chips; S2, formation of 3D non-alcoholic fatty liver organoids; S3, intervention of liver lipid regulating agents on organoids; S4. Evaluation of liver lipid regulation by liver lipid regulating agents.

2. The method according to claim 1, characterized in that: The organ chip is mainly composed of several microarray units, wherein each array unit is composed of a cell and culture medium inlet (1), a perfusion channel (2), a microarray structure (3), and a culture medium outlet (4). After the cells enter through the inlet (1), they are retained in the microarray structure, assembled in situ and induced to form organoids. After the culture medium enters through the inlet (1) at a flow rate of 30-100 μL / h, it passes through the perfusion channel containing the microarray structure (3) and then flows out through the outlet (4).

3. The method according to claim 2, characterized in that: The organ chip is composed of an upper and lower layer, and the material is polydimethylsiloxane; preferably, the number of array units is 1-8, and the micro-array structure (3) is pit-shaped or column-shaped, and the diameter of the micro-pit / column ranges from 200 μm to 1 mm, the depth ranges from 200 μm to 800 μm, and the spacing ranges from 50 to 500 μm.

4. The method according to claim 2, characterized in that: Step S1: Processing of organ chips, specifically, the following steps: preparing the chip by soft lithography technology, casting the formed template with PDMS prepolymer to obtain the upper and lower layers, sealing after oxygen plasma treatment for 10-90s, and hydrophobically modifying the chip with a certain concentration of PF127 for use.

5. The method according to claim 1, characterized in that: The formation of 3D non-alcoholic fatty liver organoids in step S2, specifically the following steps: (1) Differentiation of human pluripotent stem cells into liver-specific endoderm: The culture medium was treated with RPMI-1640, GlutaMAX, KSR, B27, and Activin A for 5 days, then replaced with a culture medium containing GlutaMAX, KSR, B27, BMP4, bFGF, and RPMI-1640 for 3 days; (2) Differentiation into hepatic progenitor cells: The culture medium consists of RPMI-1640, GlutaMAX, KSR, B27, and HGF for 5 days; (3) Hepatocyte differentiation and organoid formation: The culture medium consists of HCM, OSM, and Dex for 5 days; (4) Long-term culture of liver organoids: the culture medium consists of HCM and Dex treatment for 2 days; (5) Free fatty acid exposure: Free fatty acid treatment for 5 days, the culture medium components are HCM and Dex, that is, 3D non-alcoholic fatty liver organoids are formed.

6. The method according to claim 5, characterized in that: In step (1) and step (2), the concentrations of GlutaMAX, KSR, and B27 were all in the range of 0.1-5%. In step (1), the concentrations of ActivinA were in the range of 50-100 ng / mL, BMP4 were in the range of 10-40 ng / mL, and bFGF were in the range of 5-20 ng / mL. In step (2), the concentrations of HGF were in the range of 10-40 ng / mL. In step (3), the concentrations of OSM were in the range of 5-20 ng / mL, and the concentrations of Dex were in the range of 10 -8 -10 -7 M; the Dex concentration range in step (4) is 10 -8 -10 -7 M, the treatment time is 2-50 days; the free fatty acid concentration range in step (5) is 100-1000 μM, and the Dex concentration range is 10 -8 -10 -7 M.

7. The method according to claim 1, characterized in that: The specific steps of step S3 intervention and evaluation are as follows: preparing a liver lipid regulation reagent working solution to intervene in 3D non-alcoholic fatty liver organoids, and the culture medium components are HCM and Dex; The concentration of the liver lipid regulation reagent working solution is 0.01 μM-1 mM, the action time is 5 days, and the Dex concentration range is 10 -8 -10 -7 M.

8. The method according to claim 1, characterized in that: The evaluation of step S4 is specifically: The cholesterol level, triglyceride level, lipid immunofluorescence staining, oil red staining, lipid metabolism regulation, and oxidative stress level were evaluated to confirm whether the liver lipid regulation reagent working solution could regulate liver lipids; If the drug truly has the effect of regulating liver lipids, then the above evaluation can prove it in this system, that is, if the drug can regulate liver lipids, it will reduce triglyceride and cholesterol levels under pathological conditions, and improve lipid metabolism capacity and oxidative stress levels. Specifically, lipid accumulation and reduction can be observed through immunofluorescence and oil red staining.

9. The method according to any one of claims 1 to 8, characterized in that The liver lipid regulating agent is ginsenoside Rb1, obeticholic acid Ob or a combination thereof.

10. The use of the method according to any one of claims 1 to 9 in the evaluation of identifying liver lipid regulating reagents, or the authenticity or purity of a drug to be tested, specifically for determining whether an unknown drug has liver lipid regulating function (for example, using the following evaluation criteria: whether the unknown drug can reduce lipid accumulation, regulate lipid metabolism capacity, and improve oxidative stress levels) or specifically for determining whether the liver lipid regulating reagent to be tested is evaluated by the above method and compared with the evaluation result obtained by the above method for a standard liver lipid regulating reagent.

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