Method for observing influence of icaritin on hepatic fibrosis by using laser fluorescence

Abstract

The invention discloses a method for observing the influence of icaritin on hepatic fibrosis by using laser fluorescence, and relates to the technical field of hepatic fibrosis research, laser fluorescence is used for recording and deducing the influence of icaritin on hepatic fibrosis, and deducing conditions comprise liver function detection, inflammation detection and hepatic fibrosis level evaluation. Compared with the prior art, the method has the advantages that the icaritin is mixed by using the fluorescent substance and injected into the rat body, so that the fluorescent substance can be dispersed into the hepatic fibrosis tissue through the icaritin, and then the fluorescent substance is measured through the optical fiber sensor; according to the method, the specific distribution and concentration change of the icaritin on the hepatic fibrosis and the interaction condition of the icaritin and related biomolecules can be accurately measured, meanwhile, the action change of the icaritin in the dynamic development process of the hepatic fibrosis can be timely captured, and the influence of the icaritin on hepatic fibrosis tissues can be reflected in real time.

Description

Technical Field

[0001] The present invention relates to the technical field of liver fibrosis research, and specifically provides a method for observing the effect of icariin on liver fibrosis using laser fluorescence. Background Art

[0002] Liver fibrosis plays a crucial role in the development of liver diseases. It is a progressive pathological process, and various etiologies such as chronic viral hepatitis (hepatitis B, hepatitis C, etc.), long-term alcohol abuse, non-alcoholic fatty liver disease, etc. can trigger liver fibrosis. During the process of liver fibrosis, hepatocytes are continuously damaged, hepatic stellate cells are activated and transformed into myofibroblasts, which begin to synthesize a large amount of extracellular matrix components such as collagen. With the continuous deposition of extracellular matrix, the structure of liver lobules is gradually destroyed, and the normal tissue structure and function of the liver are affected. If liver fibrosis cannot be effectively controlled, it will further develop into liver cirrhosis or even liver cancer, seriously threatening the life and health of patients. Currently, the clinical diagnosis of liver fibrosis mainly relies on invasive means such as liver biopsy, but this method has certain risks and cannot dynamically monitor the progression of liver fibrosis in real time. Therefore, there is an urgent need for more accurate and real-time observation methods to deeply study the occurrence and development mechanism of liver fibrosis in order to provide more effective strategies for the treatment and prevention of liver fibrosis.

[0003] As a natural product, icariin has attracted attention in recent years for its potential role in anti-liver fibrosis. Researchers initially believe that icariin has the effect of anti-liver fibrosis, but its specific mechanism of action and effects during the process of liver fibrosis have not been fully clarified. Existing observation methods have many deficiencies in studying the effect of icariin on liver fibrosis. On the one hand, the accuracy is poor, and it is difficult to accurately measure the specific distribution, concentration changes of icariin in liver fibrosis tissues or cells, and the interaction with related biomolecules. On the other hand, the real-time performance is insufficient, and it cannot timely capture the changes in the role of icariin during the dynamic development of liver fibrosis. Therefore, we propose a method for observing the effect of icariin on liver fibrosis using laser fluorescence. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for observing the effect of icariin on liver fibrosis using laser fluorescence.

[0005] To solve the problems raised in the above background art, the present invention provides the following technical solution: A method for observing the effect of icariin on liver fibrosis using laser fluorescence, which uses laser fluorescence to record and infer the effect of icariin on liver fibrosis. The inference conditions include liver function detection, inflammation detection, and liver fibrosis level assessment. The specific operation steps of the method for observing the effect of icariin on liver fibrosis using laser fluorescence are as follows:

[0006] Step 1: Mix the fluorescent substance and icariin-loaded serum. Take the fluorescent substance and add it to a centrifuge tube containing icariin-loaded serum. Use a sterile stirring rod to slowly stir to preliminarily mix the substances in the centrifuge tube to obtain a mixed solution. Place the centrifuge tube containing the mixed solution on a constant temperature oscillator for oscillating mixing. After mixing, transfer it to a centrifuge and centrifuge for 10 min. After completion, take the supernatant and pour it into a sterile centrifuge tube, and store it in a dark room for standby;

[0007] Step 2: Establish an in vitro model simulating the physiological environment in rats. Use a buffer solution with an ion concentration and pH value similar to that of rat blood, and add substances simulating the internal metabolic enzymes of the liver. Add the mixed solution and growth hormone to the in vitro model, place it in a constant temperature incubator at 37 °C, and take out the mixed solution at 1 h, 3 h, 6 h, and 12 h respectively to detect the content changes of icariin and the fluorescent substance therein, and observe the physical state changes of the mixed solution in the in vitro model. After passing the evaluation, store it for standby;

[0008] Step 3: Anesthetize 8-week-old rats with liver fibrosis. Use a syringe to inject the mixed solution through the tail vein. After completion, install a non-invasive physiological monitoring device on the rat's abdomen, paste electrode patches on its chest to connect to a heart rate monitor, observe the behavioral changes of the rats, adaptively raise the rats for 7 d, and after normal culture for 35 d, gavage-feed the rats with the mixed solution at a dose of 1 mg / kg / d - 16 mg / kg / d;

[0009] Step 4: After 36 d of administering the mixed solution to the rats, insert the optical fiber sensor probe near the liver, detect and record the changes in the optical properties of the fluorescent substance, and infer the biochemical changes inside the liver fibrosis based on the changes in the optical properties;

[0010] Step 5: Take blood from the rats' orbital caudal veins once on the 42nd and 57th days of the experiment. Sacrifice all the rats on the 63rd day, and collect their liver, spleen, and blood specimens. Immediately weigh the liver and spleen tissues, and divide the liver into three parts to compare the results.

[0011] As a further scheme of the present invention: in the above Step 1, surface-modified carbon dots in the second near-infrared region are selected as the fluorescent substance mixed into icariin. The carbon dots in the second near-infrared region will emit fluorescence with a specific wavelength under the excitation of near-infrared light. In liver fibrosis tissues, the fluorescence characteristics of the carbon dots will change according to the increase in collagen components and the change in tissue structure.

[0012] As a further scheme of the present invention: in the above Step 1, before the mixing experiment, the icariin-loaded serum needs to be precooled at 4 °C for 30 min, and at the same time, all the instruments to be used in the experiment are placed in a super clean bench for ultraviolet disinfection for 30 min.

[0013] As a further solution of the present invention: in the first step, when oscillating the mixed solution, the ambient temperature is controlled at 37 °C, the oscillation frequency is 2 Hz, and the oscillation time lasts for 1 h. Among them, a sterile glass rod is used for stirring every 15 min. After mixing is completed, the mixed solution needs to be centrifuged at a rate of 3000 r / min for 10 min - 15 min at a temperature of 4 °C.

[0014] As a further solution of the present invention: in the second step, before simulating the in vivo environment of the mixed solution, it is necessary to observe the color and transparency of the mixed solution, and use a viscosimeter to measure the viscosity of the mixed solution. The measurement temperature is 25 °C, the growth hormone is insulin-like growth factor-1, and the excludability of the growth hormone in the simulated rat in vivo environment is detected. When preparing the growth hormone, a glycosylation modification enzyme is added thereto, and the mass ratio of the glycosylation modification enzyme to insulin-like growth factor-1 is 1:10 - 20.

[0015] As a further solution of the present invention: in the second step, the content of icariin in the mixed solution is detected by HPLC method. The mixed solution of methanol and water is used as the mobile phase in the HPLC method, and the mass ratio of methanol to water is 75:50. A fluorescence spectrometer is used to detect the fluorescent substances in the mixed solution. The mixed solution is pretreated with a microporous filter membrane with a pore size of 0.22 μm - 0.24 μm. The wavelength range scanning parameters of the initial excitation light source are 800 nm - 1200 nm, the scanning step is 5 nm - 10 nm, the excitation wavelength is 900 nm - 1050 nm, and the emission wavelength is 1100 nm - 1300 nm.

[0016] As a further solution of the present invention: in the third step, the rats suffering from liver fibrosis are divided into three groups: a low-dose group, a medium-dose group, and a high-dose group according to the gavage feeding dose. The dose of the mixed solution given to the low-dose group is 1 mg / kg / d, the dose of the mixed solution given to the medium-dose group is 4 mg / kg / d, and the dose of the mixed solution given to the high-dose group is 16 mg / kg / d. Insulin-like growth factor-1 is co-injected into the rats with the mixed solution at an injection frequency of 2 mg / kg / d.

[0017] As a further solution of the present invention: in the fourth step: the fiber optic sensor is a tapered fiber optic sensor based on a micro-nano structure, the tip diameter is 100 nm - 200 nm, the sensor body is a high-purity quartz optical fiber, and the biochemical changes in the liver tissue include the degree of inflammation, the change in collagen content, and the liver fibrosis index.

[0018] As a further solution of the present invention: in step five, the first part of the liver is fixed in 4% formalin for 24 hours to prepare immunohistochemical sections, the second part of the liver is quickly frozen in liquid nitrogen and then stored in a refrigerator at -80 °C, and the third part of the liver is cut into pieces with scissors and made into a tissue suspension with trypsin.

[0019] Adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. By mixing icariin with a fluorescent substance and injecting it into rats, the present invention enables the fluorescent substance to be dispersed into the liver fibrosis tissue through icariin, and then measures the fluorescent substance through an optical fiber sensor. Through this method, the specific distribution, concentration change of icariin on liver fibrosis, and the interaction with related biomolecules can be accurately measured. At the same time, it can timely capture the change of the role of icariin in the dynamic development process of liver fibrosis and reflect the impact of icariin on liver fibrosis tissue in real time. By injecting growth hormone into rats, the time for the experiment can be reduced, which plays a major role in inferring the impact of icariin on liver fibrosis and improves the efficiency of the experiment.

[0021] 2. By grouping and subsequent detection operations on experimental rats, including preparing icariin-containing drug serum, mixing fluorescent substances, simulating the in vivo environment detection, and gavage feeding with different doses, the present invention can comprehensively and systematically study the impact of icariin on liver fibrosis. At the same time, by using a variety of detection means to detect the content changes of icariin and fluorescent substances in the mixed solution and different treatment methods for rat livers, data on the impact and role of icariin on liver fibrosis can be obtained from three perspectives, which helps to more deeply and accurately infer the anti-liver fibrosis mechanism of icariin and provides rich experimental basis and data support for the further development of effective anti-liver fibrosis treatment strategies. Brief Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the process for observing the impact of liver fibrosis in the embodiment of the present invention. Detailed Embodiments

[0023] The following further describes the specific embodiments of the present invention with reference to the drawings. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0024] A method for observing the effect of icariin on liver fibrosis using laser fluorescence. The method records and infers the effect of icariin on liver fibrosis using laser fluorescence. The inference conditions include liver function detection, inflammation detection, and liver fibrosis level assessment. The specific operation steps of the method for observing the effect of icariin on liver fibrosis using laser fluorescence are as follows:

[0025] Step 1: Mix the fluorescent substance and icariin-containing serum. Take the fluorescent substance and add it to a centrifuge tube containing icariin-containing serum. Slowly stir it with a sterile stirring rod to preliminarily mix the substances in the centrifuge tube to obtain a mixed solution. Place the centrifuge tube containing the mixed solution in a thermostatic oscillator for oscillating mixing. After mixing, transfer it to a centrifuge and centrifuge for 10 min. After completion, take the supernatant and pour it into a sterile centrifuge tube, and store it in a dark room for later use;

[0026] Step 2: An in vitro model simulating the physiological environment in rats. Use a buffer solution with an ion concentration and pH value similar to that of rat blood, and add substances simulating the internal metabolic enzymes of the liver. Add the mixed solution and growth hormone to the in vitro model, and place it in a thermostatic incubator at 37°C. Take out the mixed solution at 1 h, 3 h, 6 h, and 12 h respectively, detect the content changes of icariin and the fluorescent substance therein, and observe the physical state changes of the mixed solution in the in vitro model. After passing the evaluation, store it for later use;

[0027] Step 3: Anesthetize 8-week-old rats with liver fibrosis. Use a syringe to inject the mixed solution through the tail vein. After completion, install a non-invasive physiological monitoring device on the abdomen of the rats, paste electrode patches on their chests to connect to a heart rate monitor, observe the behavioral changes of the rats, adaptively raise the rats for 7 d, and after normal culture for 35 d, gavage-feed the rats with the mixed solution at a dose of 1 mg / kg / d - 16 mg / kg / d;

[0028] Step 4: After 36 d of administering the mixed solution to the rats, insert the optical fiber sensor probe near the liver, detect and record the changes in the optical properties of the fluorescent substance, and infer the biochemical changes inside the liver fibrosis based on the changes in the optical properties;

[0029] Step 5: At the 42nd and 57th days of the experiment, take blood from the rats' caudal vein once. At the 63rd day, sacrifice all the rats, and collect their liver, spleen, and blood specimens. Immediately weigh the liver and spleen tissues, and divide the liver into three parts to compare the results.

[0030] In one embodiment of the present invention: In step one, surface-modified carbon dots in the second near-infrared region are selected as the fluorescent substance to be mixed into icariin. The carbon dots in the second near-infrared region will emit fluorescence with a specific wavelength under the excitation of near-infrared light. In liver fibrosis tissues, the fluorescence characteristics of the carbon dots will change according to the increase in collagen components and the change in tissue structure.

[0031] In one embodiment of the present invention: In step one, before conducting the mixing experiment, the icariin-containing serum needs to be precooled at 4°C for 30 minutes, and at the same time, all the instruments to be used in the experiment are placed in a laminar flow hood for ultraviolet disinfection for 30 minutes.

[0032] In one embodiment of the present invention: In step one, when the mixed solution is oscillated, the ambient temperature is controlled at 37°C, the oscillation frequency is 2 Hz, and the oscillation time lasts for 1 hour. Among them, a sterile glass rod is used for stirring every 15 minutes. After mixing is completed, the mixed solution needs to be centrifuged at a rate of 3000 r / min at 4°C for 10 - 15 minutes.

[0033] In one embodiment of the present invention: In step two, before simulating the in-vivo environment of the mixed solution, it is necessary to observe the color and transparency of the mixed solution, and use a viscosimeter to measure the viscosity of the mixed solution. The measurement temperature is 25°C, and the growth hormone is insulin-like growth factor-1. The excludability of the growth hormone in the simulated rat in-vivo environment is detected. When preparing the growth hormone, a glycosylation modification enzyme is added to it. The mass ratio of the glycosylation modification enzyme to insulin-like growth factor-1 is 1:10 - 20. Insulin-like growth factor-1 is co-injected into the rats at an injection frequency of 2 mg / kg / d together with the mixed solution.

[0034] In one embodiment of the present invention: In step two, the content of icariin in the mixed solution is detected by HPLC method. The mixed solution of methanol and water is used as the mobile phase in the HPLC method. The mass ratio of methanol to water is 75:50. A fluorescence spectrometer is used to detect the fluorescent substance in the mixed solution. The mixed solution is pretreated with a microporous filter membrane with a pore size of 0.22 μm - 0.24 μm. The wavelength range scanning parameters of the initial excitation light source are 800 nm - 1200 nm, the scanning step is 5 nm - 10 nm, the excitation wavelength is 900 nm - 1050 nm, and the emission wavelength is 1100 nm - 1300 nm.

[0035] In one embodiment of the present invention: In step three, the rats with liver fibrosis are divided into three groups, namely, a low-dose group, a medium-dose group, and a high-dose group, according to the gavage feeding dose. The dose of the mixed solution given to the low-dose group is 1 mg / kg / d, the dose of the mixed solution given to the medium-dose group is 4 mg / kg / d, and the dose of the mixed solution given to the high-dose group is 16 mg / kg / d.

[0036] In one embodiment of the present invention: In step four: The fiber optic sensor is a tapered fiber optic sensor based on a micro-nano structure, with a tip diameter of 100 nm - 200 nm. The sensor body is made of high-purity quartz fiber. The biochemical changes in the liver tissue include the degree of inflammation, changes in collagen content, and liver fibrosis indicators.

[0037] In one embodiment of the present invention: In step five, the first part of the liver is fixed in 4% formalin for 24 h to prepare immunohistochemical sections. The second part of the liver is quickly frozen in liquid nitrogen and then stored in a refrigerator at -80 °C. The third part of the liver is minced with scissors and made into a tissue suspension using trypsin.

[0038] In one embodiment of the present invention: For the degree of inflammation, when the mixed solution enters the liver fibrosis tissue, the reactive oxygen species released by inflammatory cells will change the fluorescence intensity and wavelength of the fluorescent substance. These changes are detected by the fiber optic sensor, and the degree of inflammation is recorded.

[0039] In one embodiment of the present invention: For the change in collagen content, the fluorescent substance in the mixed solution has a binding effect with collagen. During the process of liver fibrosis, collagen accumulates and degrades, resulting in changes in the chemical properties of the fluorescent substance. The fiber optic sensor is used to detect and record the changes, and the dynamic changes in collagen content are inferred based on the changes.

[0040] In one embodiment of the present invention: For liver fibrosis indicators, liver fibrosis at different stages will cause changes in the structure and composition of liver tissue. At the same time, the changes in liver fibrosis at different stages will affect the optical properties of the fluorescent substance. The fiber optic sensor is used to capture and record the changes in different fluorescent substances, and the problems of liver fibrosis indicators are inferred based on the recorded problems.

[0041] Example 1

[0042] First, prepare icariin-loaded serum. Divide healthy experimental rats into a drug administration group and a control group. Weigh icariin and put it into a stirring container, add normal saline to the container, and use the icariin solution to intragastrically administer to the rats in the drug administration group for seven consecutive days. Administer normal saline with the same volume as the icariin solution to the rats in the control group by gavage. Collect blood from the abdominal aorta of the rats within 1 h after the last administration. After centrifugation, inactivation, and sterilization of the blood, prepare icariin-loaded serum. Then, pre-cool the icariin-loaded serum at 4°C for 30 min. At the same time, put all the instruments to be used in the experiment into the ultra-clean workbench for ultraviolet disinfection for 30 min. Then, add the surface-modified carbon dots in the second near-infrared region to the centrifuge tube containing the icariin-loaded serum, and slowly stir with a sterile stirring rod to preliminarily mix the substances in the centrifuge tube. During the operation, control the ambient temperature at 37°C, the oscillation frequency at 2 Hz, and the oscillation time for 1 h. Stir with a sterile glass rod every 15 min. After preparing the mixed solution, place the centrifuge tube containing the mixed solution in a constant temperature oscillator for oscillating and mixing, centrifuge at a rate of 3000 r / min at 4°C for 10 min. After completion, take the supernatant and pour it into a sterile centrifuge tube, and store it in the dark for later use;

[0043] An in vitro model simulating the physiological environment in rats. Take a buffer solution with an ion concentration and pH value similar to that of rat blood, and add substances simulating the internal metabolic enzymes in the liver. Add the mixed solution and insulin-like growth factor-1 to the in vitro model. The mass ratio of the glycosylation-modifying enzyme to insulin-like growth factor-1 is 1:15. Place it in a constant temperature incubator at 37°C. Take out the mixed solution at 1 h, 3 h, 6 h, and 12 h respectively, and use HPLC method to detect the content of icariin in the mixed solution. Use the mixed solution of methanol and water as the mobile phase in the HPLC method, and the mass ratio of methanol to water is 75:50. Use a fluorescence spectrometer to detect the fluorescent substances in the mixed solution. Pretreat the mixed solution with a 0.22 μm microporous filter membrane. The wavelength range scanning parameters of the initial excitation light source are 900 nm, the scanning step is 6 nm, the excitation wavelength is 1000 nm, and the emission wavelength is 1300 nm. Observe the physical state changes of the mixed solution in the in vitro model. After passing the evaluation, store it for later use;

[0044] Rats at 8 weeks of age with liver fibrosis were anesthetized and then fixed on an animal fixator. A syringe was used to inject the mixed solution through the caudal vein. After completion, a non-invasive physiological monitoring device was installed on the abdomen of the rats, and electrode patches were pasted on their chests to connect to a heart rate monitor. After the rats woke up, their behavioral changes were observed. The rats were adaptively fed for 7 days. After normal culturing for 35 days, the rats with liver fibrosis were divided into three groups: a low-dose group, a medium-dose group, and a high-dose group, according to the gavage feeding dose. The low-dose group was given a dose of 1 mg / kg / d of the mixed solution, the medium-dose group was given a dose of 4 mg / kg / d of the mixed solution, and the high-dose group was given a dose of 16 mg / kg / d of the mixed solution. The other two groups were injected with a blank CMC-Na solution. After 36 days of giving the rats the mixed solution, the fiber optic sensor probe was inserted near the livers of the five groups of rats with liver fibrosis. The fiber optic sensor was a tapered fiber optic sensor based on a micro-nano structure, with a tip diameter of 150 nm. The sensor body was made of high-purity quartz fiber. The optical property changes of the fluorescent substance were detected and recorded. Based on the optical property changes, the biochemical changes inside the liver fibrosis were inferred, including the degree of inflammation, changes in collagen content, and liver fibrosis indicators. At the 42nd and 57th days of the experiment, blood was taken once from the caudal vein of the rats' eyes. At the 63rd day, all the rats were sacrificed, and their liver, spleen, and blood specimens were collected. The liver and spleen tissues were immediately weighed, and the liver was divided into three parts to compare the results. The first part of the liver was fixed in 4% formalin for 24 h to make immunohistochemical sections. The second part of the liver was quickly frozen in liquid nitrogen and then stored in a -80 °C refrigerator. The third part of the liver was cut into pieces with scissors and made into a tissue suspension with trypsin.

[0045] The safety of icariin was evaluated by observing and detecting the changes in the food intake and body weight of the rats:

[0046] The optical characteristic changes of surface-modified carbon dots in the second near-infrared region near the liver were detected by a fiber optic sensor. Based on the fluorescence intensity changes of the surface-modified carbon dots in the second near-infrared region, the effects of icariin on inflammation-related molecules were inferred, and information about the internal biochemical state of liver fibrosis was obtained by detecting the wavelength changes of the fluorescence;

[0047] After the experiment started, the food intake of the rats was monitored every 2 days, and the body weight of the rats was monitored every 3 days. The changes in serum liver function (ALT, AST) and liver fibrosis indicators (HA, LN, PC III, HYP, Col IV) of the rats on the 42nd, 57th, and 63rd days were detected by ELISA;

[0048] The serum inflammatory factor levels (IL-6, IL-10, TNF-α, IFN-γ) of the rats on the 63rd day were detected by ELISA;

[0049] After sacrificing all the rats, the collected livers were finally recorded, including serum liver function, liver fibrosis indicators, serum inflammatory factor levels, and collagen content.

[0050] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations of these embodiments still fall within the protection scope of the present invention.

[0051] In the description of the specification, the description referring to terms such as "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0052] The above content is only an example and illustration of the present invention. Those skilled in the art of this technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution, as long as they do not deviate from the invention or exceed the scope defined by the claims of the present invention, they should fall within the protection scope of the present invention.

[0053] As mentioned above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A method for observing the effect of icariin on liver fibrosis using laser fluorescence, characterized in that: Laser fluorescence is used to record and infer the effect of icariin on liver fibrosis. The inference conditions include liver function test, inflammation test and liver fibrosis level assessment. The specific operation steps of the method for observing the effect of icariin on liver fibrosis using laser fluorescence are as follows: Step 1, mixing the fluorescent substance and the icariin-containing serum, taking the fluorescent substance and adding it to the centrifuge tube containing the icariin-containing serum, mixing and storing in a dark room for later use; Step 2: creating an in vitro model that simulates the physiological environment of a rat, adding the mixed solution and growth hormone into the in vitro model, detecting changes in the contents of icariin and fluorescent substances therein, and observing changes in the physical state of the mixed solution in the in vitro model; Step 3, anesthetize 8-week-old rats with liver fibrosis, inject the mixed solution through the tail vein, observe the behavioral changes of the rats, and feed the rats with the mixed solution by gavage at a dose of 1 mg / kg / d-16 mg / kg / d; Step 4: 36 days after the rats were given the mixed solution, the fiber optic sensor probe was inserted near the liver to detect and record the changes in the optical properties of the fluorescent substance, and to infer the biochemical changes inside the liver fibrosis; Step 5: Blood was collected once through the caudal vein of the rats on the 42nd and 57th days of the experiment. All rats were killed on the 63rd day, and their livers, spleens and blood samples were collected. The livers were divided into three parts to compare the results.

2. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In the step 1, surface-modified near-infrared zone II carbon dots are selected as the fluorescent substance to be mixed into icariin. The near-infrared zone II carbon dots will emit fluorescence of a specific wavelength under the excitation of near-infrared light. In liver fibrosis tissue, the fluorescence properties of the carbon dots will change according to the increase of collagen components and changes in tissue structure.

3. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In step 1, before the mixing experiment, the icariin-containing serum needs to be precooled at 4° C. for 30 minutes, and all the instruments required for the experiment are placed in a clean bench for ultraviolet disinfection for 30 minutes.

4. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In the step 1, when the mixed solution is oscillated, the ambient temperature is controlled at 37°C, the oscillation frequency is 2 Hz, and the oscillation time lasts for 1 hour, wherein a sterile glass rod is used for stirring every 15 minutes. After the mixing is completed, the mixed solution needs to be centrifuged at a temperature of 4°C and a rate of 3000 r / min for 10 minutes to 15 minutes.

5. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In the step 2, before simulating the in vivo environment of the mixed solution, the color and transparency of the mixed solution need to be observed, and the viscosity of the mixed solution is measured using a viscosity tester at a measurement temperature of 25°C.

6. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In the step 2, the icariin content in the mixed solution is detected by HPLC, a mixture of methanol and water is used as the mobile phase in the HPLC method, the mass ratio of methanol to water is 75:50, a fluorescence spectrometer is used to detect the fluorescent substance in the mixed solution, a 0.22 μm-0.24 μm microporous filter membrane is used to pretreat the mixed solution, the wavelength range scanning parameter of the initial excitation light source is 800nm-1200nm, the scanning step is 5nm-10nm, the excitation wavelength is 900nm-1050nm, and the emission wavelength is 1100nm-1300nm.

7. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In the step three, rats with liver fibrosis are divided into three groups according to the oral feeding dose, namely, a low-dose group, a medium-dose group and a high-dose group. The dose of the mixed solution administered to the low-dose group is 1 mg / kg / d, the dose of the mixed solution administered to the medium-dose group is 4 mg / kg / d, and the dose of the mixed solution administered to the high-dose group is 16 mg / kg / d.

8. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In step 4: the optical fiber sensor is a tapered optical fiber sensor based on a micro-nano structure, the tip diameter is 100nm-200nm, the sensor body is a high-purity quartz optical fiber, and the biochemical changes of liver tissue include the degree of inflammation, changes in collagen content, and liver fibrosis indicators.

9. The method of observing the effect of icariin on liver fibrosis using laser fluorescence according to claim 1, characterized in that: In step 5, the first part of the liver is fixed in 4% formalin for 24 hours to prepare immunohistochemical sections, the second part of the liver is quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator, and the third part of the liver is chopped with scissors and prepared into a tissue suspension using pancreatic enzymes.

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