Traditional Chinese medicine composition for treating pancreatic fibrosis and preparation method thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating pancreatic fibrosis and a preparation method thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition disclosed by the invention takes isoflavone as a main component, and the isoflavone not only can reverse the fibrosis degree of pancreatic fibrosis cells by retarding the proliferation and migration ability of fibrosis cells, influencing the cell activity and reducing the generation of various fibrosis proteins, but also can inhibit the illness state of mice with chronic pancreatic fibrosis; the disease progression is slow or blocked. According to the traditional Chinese medicine composition, the pancreatic fibrosis process is effectively reversed through retardation of isoflavone on pancreatic stellate cell fibrosis induced by TGF-beta1, the inhibition effect of the traditional Chinese medicine composition on pancreatic fibrosis can be improved in combination with other traditional Chinese medicine ingredients, and the ingredients are safe.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to a traditional Chinese medicine composition for treating pancreatic fibrosis and a preparation method thereof. Background Art

[0002] Pancreatic fibrosis is the main pathological feature of chronic pancreatitis. Chronic pancreatitis (CP) is a progressive, fibro-inflammatory disease that eventually leads to endocrine and exocrine dysfunction. The pathological characteristics of CP include acinar cell atrophy, fibrosis, inflammatory cell infiltration, and duct obstruction. Pancreatic fibrosis is one of the most important pathological features of CP and a sign of CP progression. Fibrosis is essential for tissue repair after injury, but the excessive deposition of extracellular matrix (ECM) caused by irreversible damage to the pancreas and subsequent pancreatic fibrosis in turn seriously impairs pancreatic function. There is currently a lack of drugs that can effectively alleviate or reverse pancreatic fibrosis and thus cure chronic pancreatitis.

[0003] Since pancreatic fibrosis is progressive and irreversible during the course of chronic pancreatitis, existing treatments are aimed at solving or alleviating the clinical symptoms of chronic pancreatitis, and cannot fundamentally reverse or delay the development of pancreatic fibrosis. Therefore, screening drugs that improve pancreatic fibrosis from natural or synthetic drugs is the key to the current research field of chronic pancreatitis.

[0004] Isoflavone (Calycison, Cal) is a secondary metabolite belonging to the flavonoids, which has strong antioxidant, anti-inflammatory and apoptosis regulating effects. The prior art uses isoflavones to have different degrees of preventive and therapeutic effects on other fibrotic diseases, such as liver fibrosis, pulmonary fibrosis, myocardial fibrosis, etc. However, the causes of different diseases are different, and the treatment methods are also different. The use of isoflavones to prepare a method for treating pancreatic fibrosis caused by chronic pancreatitis has important preventive and therapeutic significance. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a traditional Chinese medicine composition for treating pancreatic fibrosis, which effectively reverses the degree of pancreatic fibrosis through the effect of isoflavones on pancreatic stellate cells induced by TGF-β1.

[0006] The Chinese medicine composition for treating pancreatic fibrosis of the present invention comprises the following raw materials in parts by weight:

[0007] 20-30 parts of isoflavones; 0-5 parts of isoginkgo biloba; 0-5 parts of dansholtzia rhamnoides; 0-5 parts of rutin; 0-5 parts of naringin; 0-5 parts of bergenin; 0-5 parts of pomegranate peel tannin; 0-5 parts of sophora flavescens.

[0008] Preferably, the Chinese medicine composition for treating pancreatic fibrosis comprises the following raw materials in parts by weight:

[0009] 20-30 parts of isoflavones; 1-5 parts of isoginkgo biloba; 1-5 parts of dansholtzia rhamnoides; 1-5 parts of rutin; 1-5 parts of naringin; 1-5 parts of bergenin; 1-5 parts of punica granatum; 1-5 parts of sophora flavescens.

[0010] Another object of the present invention is to provide a method for preparing a Chinese medicine composition for treating pancreatic fibrosis, the preparation method comprising the following steps:

[0011] (1) Weigh the raw materials according to the proportion;

[0012] (2) fully grinding and mixing the above-mentioned Chinese medicinal components, filtering, and obtaining a mixed powder;

[0013] (3) adding the mixed powder into water for ultrasonic dissolution, collecting the filtrate after filtering, and spray drying or vacuum concentrating to obtain the traditional Chinese medicine composition for treating pancreatic fibrosis.

[0014] Preferably, the filtration in step (2) is performed through a 100-110 mesh sieve.

[0015] Preferably, the weight ratio of the mixed powder to water in the ultrasonic dissolution in step (3) is 1:2-3.

[0016] Preferably, the temperature of the ultrasonic dissolution in step (3) is 50-60°C.

[0017] Preferably, the ultrasonic dissolution time in step (3) is 30-45°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a Chinese medicine composition for treating pancreatic fibrosis and a preparation method thereof. The Chinese medicine composition of the present invention has isoflavones as the main component. Isoflavones can not only block the proliferation and migration ability of fibrotic cells, affect cell activity, and reduce the production of various fibrotic proteins, thereby reversing the degree of fibrosis of pancreatic fibrotic cells, but also inhibit the condition of mice with chronic pancreatic fibrosis, slowing down or blocking the progression of the disease. The Chinese medicine composition of the present invention effectively reverses the process of pancreatic fibrosis by blocking pancreatic stellate cell fibrosis induced by TGF-β1 with isoflavones, and can be combined with other Chinese medicine ingredients to improve the inhibitory effect of the Chinese medicine composition on pancreatic fibrosis, and the ingredients are safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of ELISA test of relevant markers after TGF-β1 induced HPSC;

[0021] Figure 2 The results of WB detection of relevant markers after TGF-β1 induced HPSC;

[0022] Figure 3 To detect the ELISA results of related markers after Cal acted on HPSC induced by TGF-β1 for 48h;

[0023] Figure 4 To detect the WB results of related markers after Cal acted on HPSC induced by TGF-β1 for 48h;

[0024] Figure 5 To detect the results of Cal acting on HPSC induced by TGF-β1 for 48 hours;

[0025] Figure 6 The results of the cell scratch test after Cal acted on HPSC induced by TGF-β1 for 48 hours;

[0026] Figure 7 The results are the results of the detection of apoptosis level of HPSC induced by TGF-β1 after 48 hours of Cal action;

[0027] Figure 8 The results are the results of the cell cycle test after Cal acted on HPSC induced by TGF-β1 for 48 hours;

[0028] Fig. 9 The results are the results of the detection of cell clone formation after Cal acted on HPSC induced by TGF-β1 for 48 hours;

[0029] Fig.10 The results are for the detection of Lipase levels in the pancreatic fibrosis animal model after Cal treatment;

[0030] Fig.11 The results are for the detection of collagen I levels in pancreatic fibrosis animal models after Cal treatment;

[0031] Fig.12 The results are for the detection of α-SMA levels in the pancreatic fibrosis animal model after Cal treatment;

[0032] Fig.13 The results of HE staining of tissues in the pancreatic fibrosis animal model after Cal treatment;

[0033] Fig.14 The results are for the detection of tissue mRNA levels in the pancreatic fibrosis animal model after Cal treatment;

[0034] Fig.15 The results of tissue protein level detection in pancreatic fibrosis animal model after Cal treatment;

[0035] Fig.16 The results of tissue mRNA level detection after the pancreatic fibrosis animal model was treated with a combination of traditional Chinese medicines;

[0036] Fig.17 The results of tissue protein level test after the pancreatic fibrosis animal model was treated with a combination of traditional Chinese medicines;

[0037] Fig.18 These are the results of HE staining of tissues after the animal model of pancreatic fibrosis was treated with a combination of traditional Chinese medicines. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the examples. The raw materials used in the examples are all products commonly purchased on the market, and the proportions of the raw materials are all weight ratios unless otherwise specified.

[0039] Example 1

[0040] A Chinese medicine composition for treating pancreatic fibrosis, comprising the following raw materials:

[0041] 25 parts of isoflavones.

[0042] The isoflavone (Calycosin) for treating pancreatic fibrosis was purchased from MCE, item number: HY-N0519. It was dissolved in DMSO to prepare a drug stock solution with a concentration of 1 mM, and then the drug was further diluted with DMSO to 50 μM and 100 μM for later use.

[0043] Example 2

[0044] A Chinese medicine composition for treating pancreatic fibrosis, comprising the following raw materials:

[0045] 25 parts of isoflavones; 1 part of isoginkgetin; 1 part of danye-rhubarb; 1 part of rutin; 1 part of naringin; 1 part of bergenin; 1 part of pomegranate peel tannin; 1 part of sophora flavescens.

[0046] The preparation method of the traditional Chinese medicine composition for treating pancreatic fibrosis comprises the following steps:

[0047] (1) Weigh the raw materials according to the proportion;

[0048] (2) Grinding and mixing the above Chinese medicinal components thoroughly, filtering through a 100-mesh sieve to obtain a mixed powder;

[0049] (3) The mixed powder is added into 2 times the weight of water, and dissolved at 60° C. for 45 min under ultrasound. The filtrate is collected after filtration, and solid powder is obtained after spray drying to prepare a granular preparation, which is the Chinese medicine composition for treating pancreatic fibrosis, and is recorded as drug Y1.

[0050] Example 3

[0051] A Chinese medicine composition for treating pancreatic fibrosis, comprising the following raw materials:

[0052] 20 parts of isoflavones; 1 part of isoginkgetin; 1 part of danye-rhubarb; 1 part of rutin; 1 part of naringin; 1 part of bergenin; 2 parts of pomegranate peel tannin; 1 part of sophora flavescens.

[0053] The preparation method of the traditional Chinese medicine composition for treating pancreatic fibrosis comprises the following steps:

[0054] (1) Weigh the raw materials according to the proportion;

[0055] (2) Grinding and mixing the above Chinese medicinal components thoroughly, filtering through a 100-mesh sieve to obtain a mixed powder;

[0056] (3) The mixed powder is added into 2 times the weight of water, and dissolved at 60° C. for 45 min under ultrasound. The filtrate is collected after filtration, and solid powder is obtained after spray drying to prepare a granular preparation, which is the Chinese medicine composition for treating pancreatic fibrosis, and is recorded as drug Y2.

[0057] Example 4

[0058] A Chinese medicine composition for treating pancreatic fibrosis, comprising the following raw materials:

[0059] 25 parts of isoflavones; 1 part of isoginkgetin; 1 part of danye-rhubarb; 1 part of rutin; 1 part of naringin; 1 part of bergenin; 1 part of pomegranate peel tannin; 1 part of sophora flavescens.

[0060] The preparation method of the traditional Chinese medicine composition for treating pancreatic fibrosis comprises the following steps:

[0061] (1) Weigh the raw materials according to the proportion;

[0062] (2) Grinding and mixing the above Chinese medicinal components thoroughly, filtering through a 100-mesh sieve to obtain a mixed powder;

[0063] (3) The mixed powder is added into 2 times the weight of water, and dissolved at 50° C. for 30 min under ultrasound. The filtrate is filtered and collected, and vacuum concentrated to the same weight volume as the mixed raw materials to prepare a spray preparation, which is the Chinese medicine composition for treating pancreatic fibrosis, denoted as drug Y3.

[0064] The following experiment was conducted on the Chinese medicinal composition (isoflavones) for treating pancreatic fibrosis prepared in Example 1:

[0065] Experiment 1: Construction of pancreatic fibrosis cell model

[0066] The cells selected for this part of the experiment are human pancreatic stellate cells (HPSC) (Pricella, CP-H024), and the specific processing process is as follows.

[0067] 1.1 Cell culture

[0068] (1) Human pancreatic stellate cells were cultured in DMEM (Bio-channel, BC-M-005) complete medium containing 10% fetal bovine serum and maintained in a 37° C., 5% CO 2 incubator.

[0069] (2) When the cells have grown to about 80%-90%, they can be digested and then passaged or plated.

[0070] 1.2 Establishment of pancreatic fibrosis cell model

[0071] HPSCs in the logarithmic growth phase were selected and treated with TGF-β1 (10 ng / mL) (MCE, HY-P70543) for 24 h;

[0072] 1.3 Cell treatment

[0073] The pancreatic fibrosis cell model established above was divided into three groups: TGF-β1 group, TGF-β1+Cal (50 μM) group and TGF-β1+Cal (100 μM) group. The Cal treatment time was 48 hours, and then the cell proteins were collected to detect fibrosis-related markers.

[0074] Experiment 2: Detection of protein levels of related markers in pancreatic fibrosis cell models and animal models

[0075] 2.1 Cell model sample extraction

[0076] (The following operations are performed on ice) Discard the supernatant of the culture plate, wash with pre-cooled PBS, add 0.5 mL of complete extraction buffer to each 100 mm well plate, collect the scraped cells in a pre-cooled EP tube, vortex briefly and incubate on ice for 30 min, centrifuge at 13000 rpm for 10 min at 4°C, take the supernatant and place it on ice to clean the cryotube, and store the sample at -80°C.

[0077] 2.2 Tissue model sample extraction

[0078] Take mouse pancreatic fibrosis tissue and place it on ice for immediate homogenization (or immerse it in liquid nitrogen for "quick freezing" and store the sample at -80°C), add about 300 μL of complete extraction buffer to about 5 mg of tissue block and homogenize, rinse the blade twice with 300 μL of complete extraction buffer each time, then stir continuously for 2 hours at 4°C, centrifuge at 13000 rpm for 20 minutes at 4°C. Place on ice, transfer the supernatant (i.e., soluble protein extract) to a new centrifuge tube, and store the sample at -80°C.

[0079] 2.3ELISA detection method

[0080] Coat the plate with 100 μL of coating solution per well, cover the plate and incubate at room temperature for 1 hour. Discard the supernatant and wash once with 300 μL of wash buffer per well, tap on absorbent paper to remove residual liquid. Block with 300 μL of blocking buffer per well for 1 hour at room temperature. Prepare standard and sample dilutions in blocking buffer, add 100 μL of standards (in duplicate) and samples to the designated wells and incubate at room temperature for 2 hours. Prepare detection antibody solution by diluting detection antibody with blocking buffer. Add 100 μL of detection antibody solution to each well and react gently at room temperature for 2 hours. Prepare streptavidin-HRP working solution by diluting 1:5000 with blocking buffer. (For example, to prepare a working solution that meets the needs of 1 well plate, mix 2 μL of streptavidin-HRP with 9.998 mL of blocking solution to prepare 10 mL of working solution.) Add 100 μL of streptavidin-HRP working solution to each well and react at room temperature for 1 hour. Add 100 μL of TMB substrate solution to each well and incubate at room temperature for 30 minutes. Finally, add 100 μL of stop solution to each well. After 30 minutes, use an enzyme reader (450 nm) to measure the absorbance of the solution in each well, and calculate the results using double logarithmic straight line fitting or four-parameter curve fitting. After collecting the supernatant, use ELISA to detect the expression levels of fibrosis-related markers α-SMA (the ELISA kit used was purchased from abcam, the product number is ab240678), Collagen I (the ELISA kit used was purchased from abcam, the product number is ab285250) and Fibronectin (the ELISA kit used was purchased from abcam, the product number is ab219046). The test results for the pancreatic fibrosis cell model are as follows. Figure 1 As shown in the figure, the expression of these indicators in human pancreatic stellate cells induced by TGF-β1 increased compared with the control group, indicating that the cell model of pancreatic fibrosis was successfully constructed. The test results of the pancreatic fibrosis cell model after isoflavone treatment are shown in the figure. Figure 3 As shown, the results showed that Cal drug can reduce the protein levels of fibrosis markers, so Cal can alleviate the degree of pancreatic cell fibrosis.

[0081] Experiment 3: Western Blot assay to detect marker levels of pancreatic fibrosis cells

[0082] The pancreatic fibrosis cells induced by TGF-β1 were evenly plated in a six-well plate. After the cells were treated with Cal at concentrations of 0, 50, and 100 μM for 48 hours, they were digested with trypsin and protein lysis buffer was prepared. The RIPA lysis buffer and the protease phosphatase mixture were prepared at a ratio of 100:1. The cells were lysed on ice for 30 minutes and centrifuged in a 4°C centrifuge for 30 minutes. The supernatant was taken and the protein loading buffer was added at a ratio of 4:1 between the protein supernatant and 5× protein loading buffer. After heating in a metal bath for 10 minutes, it can be used for protein molecule detection. After collecting the cell samples, the expression levels of fibrosis-related markers α-SMA (the antibodies used were purchased from proteintech, with the catalog number 14395-1-AP), Collagen I (the antibodies used were purchased from proteintech, with the catalog number 67288-1-Ig) and Fibronectin (the antibodies used were purchased from proteintech, with the catalog number 15613-1-AP) were detected by WB experiments. The detection results of the pancreatic fibrosis cell model are shown in the following figure. Figure 2 As shown in the figure, the protein expression levels of α-SMA, Collagen I and Fibronectin in HPSC induced by TGF-β1 were significantly increased. Figure 4 As shown, the results showed that the levels of related fibrosis markers (α-SMA, Collagen I and Fibronectin) were decreased after Cal treatment.

[0083] Experiment 4: CCK8 detection of the effect of Cal on the cell activity of pancreatic fibrosis cells

[0084] Pancreatic fibrotic cells induced by TGF-β1 were evenly plated in a 96-well plate at a number of 800 cells / well. Cal was applied to the cells at concentrations of 0, 50, and 100 μM for 48 h, and then CCK8 reagent (Vazyme, A311-01) was added to detect cell viability at a time gradient of 0, 24, 48, and 72 h. The results are shown in FIG. Figure 5 As shown, the results showed that the cell viability level decreased with the increase of Cal concentration, and the cell viability level was time-dependent, so Cal significantly affected the cell viability of pancreatic fibrosis cells.

[0085] Experiment 5: Cell scratch assay to detect the effect of Cal on the migration ability of pancreatic fibrosis cells

[0086] Pancreatic stellate cells (HPSC) were induced with TGF-β1. When the cells grew to about 70%, Cal (50 μM) and Cal (100 μM) were used to treat the cells for 48 hours. A 10 μL pipette tip was used to make a scratch in the middle and left and right of the well. The well was then treated with 2% FBS DMEM complete medium. Photos were taken and photos were taken again 24 hours later to observe the effect of the drug on the migration ability of pancreatic fibrosis cells. The results of the cell scratch level test after Cal acted on TGF-β1-induced HPSC for 48 hours are shown in Figure 2. Figure 6 The cell scratch test showed that the cell migration speed of the pancreatic fibrosis cell model was the fastest. As the concentration of Cal drug increased, the cell migration rate slowed down, indicating that Cal could inhibit the migration ability of HPSC induced by TGF-β1.

[0087] Experiment 6: Flow cytometry to detect the effect of Cal on apoptosis of pancreatic fibrosis cells

[0088] Pancreatic fibrotic cells induced by TGF-β1 were evenly plated in a six-well plate. Cal was applied to the cells at concentrations of 0, 50, and 100 μM for 48 h, and then digested with trypsin. After washing the cells with PBS, the cells were stained with 7-AAD / Annexin V-PE reagent (Vamyze, A213-01) and then tested on a microscope. The results of the detection of cell apoptosis levels in HPSCs induced by TGF-β1 for 48 h were as follows: Figure 7 Flow cytometry was used to detect the effect of Cal on apoptosis of pancreatic fibrosis cells. It can be seen that with the increase of drug concentration, the apoptosis rate of cells increased, indicating that Cal can induce apoptosis of pancreatic fibrosis cells.

[0089] Experiment 7: Flow cytometry to detect the effect of Cal on the cell cycle of pancreatic fibrosis cells

[0090] Pancreatic fibrotic cells induced by TGF-β1 were evenly plated in a six-well plate. Cal was applied to the cells at concentrations of 0, 50, and 100 μM for 48 h, followed by trypsin digestion. After washing the cells with PBS, the cells were fixed with 95% ethanol at -20°C for 2 h, and then dyed with a staining solution. The samples were incubated at 37°C for 30 min and then tested on a machine. The results of the cell cycle level test after Cal acted on the HPSC induced by TGF-β1 for 48 h were as follows: Figure 8 .from Figure 8 It can be seen that Cal can affect the cycle of pancreatic fibrosis cells, causing them to be blocked in the G2 / M phase, and the blocking effect of Cal is concentration-dependent.

[0091] Experiment 8: Clone formation assay to detect the effect of Cal on cell proliferation

[0092] After digestion of pancreatic fibrosis cells induced by TGF-β1, the cells were counted and plated in a six-well plate at a number of 500 cells / well. After adding Cal for 48 hours, the drug was removed and the culture medium was replaced with fresh medium every 48 hours. After the cell colonies visible to the naked eye appeared, the culture was stopped. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 10 minutes and then stained with crystal violet for 15 minutes. After washing, the cells were photographed to obtain the detection results of cell clone formation after Cal acted on TGF-β1-induced HPSC for 48 hours. Fig. 9 .from Fig. 9 It can be seen that with the increase of Cal drug concentration, the number of cell colonies decreased significantly, indicating that Cal can inhibit the proliferation ability of pancreatic fibrosis cells.

[0093] Experiments 1 to 8 show that Cal can inhibit the activity of pancreatic fibrosis cells by inhibiting cell migration and proliferation activity and inducing apoptosis, ultimately achieving the goal of alleviating pancreatic fibrosis.

[0094] Experiment 9: Construction of an animal model of pancreatic fibrosis

[0095] The present invention selects cerulet-induced C57BL / 6 female mice as an animal model for constructing chronic pancreatic fibrosis. The specific processing process is as follows: a total of 20 6-week-old C57BL / 6 female mice are selected and raised in a barrier, and the mice are divided into a control group and a model group. The model group is induced by repeated intraperitoneal injection of cerulet (at a dose of 50 μg / kg) (Ceruletide, MCE, HY-A0190), three times a week, for a total of 4 weeks, and the mice are euthanized 3 days after the last injection. The model group is divided into a model group and a Cal treatment group (10 mg / kg and 30 mg / kg). Starting from the second week after modeling, the Cal group treats the mice by gavage at the above dose every day until the mice are euthanized, and the mice in the model group are gavaged with the same dose of normal saline. After the mice are euthanized, the pancreatic tissues of the mice are collected and analyzed to obtain the test results of Lipase levels after Cal treatment of the pancreatic fibrosis animal model as shown below: Fig.10 ; The results of collagen I level detection after Cal treatment of pancreatic fibrosis animal model are as follows Fig.11 ; The results of α-SMA level detection after Cal treatment of pancreatic fibrosis animal model are as follows Fig.12 .from Fig.10It can be seen that compared with normal mice (control group), in pancreatic fibrosis mice (model group), the level of serum lipase detected by ELISA (LipaseAssay Kit III (Fluorometric) purchased from abcam, catalog number ab118969) was significantly reduced, indicating that the pancreas of the mice was damaged and the pancreatic fibrosis model was successfully established; in the pancreatic fibrosis model, different concentrations of Cal drugs were used for treatment (10 mg / kg and 30 mg / kg), and the results showed that the serum lipase level of the treated mice was restored, indicating that Cal has an inhibitory effect on chronic pancreatic fibrosis; compared with the model group, after the Cal drug treatment, protein samples were collected and the protein levels of collagen and α-SMA were verified by WB. Fig.11 and 12 The results showed that the levels of collagen I and α-SMA proteins decreased significantly with the increase of Cal drug concentration.

[0096] Experiment 10: Detection of related markers in mouse pancreatic tissue

[0097] The mouse pancreatic tissue was fixed with 4% paraformaldehyde for 24 hours and rinsed with running water to remove excess fixative and decomposition products produced by the tissue. The tissue was then dehydrated using ethanol from low to high concentrations, in order of 70% ethanol, 80% ethanol, 95% ethanol, and 100% ethanol. After dehydration, the tissue was made transparent, then waxed, embedded, and sliced. The sections were then stained with hematoxylin and eosin dye (HE staining) (Beyotime, C0105S). The results of tissue HE staining after Cal treated the pancreatic fibrosis animal model are shown below. Fig.13 .from Fig.13 It can be seen that the degree of fibrosis in the Cal-treated group was significantly lower than that in the model group.

[0098] Experiment 11: RT-PCR detection of mRNA levels of related markers in the chronic pancreatitis mouse model

[0099] The pancreatic tissue of pancreatic fibrosis mice was obtained, ground into fine powder using a tissue homogenizer, and Trizol (Vazyme, R401-01) was added, and the cells were lysed for 5 minutes. Then, 200 μL of chloroform was added, mixed by inversion, and kept on ice for 5 minutes. Centrifuged at 12000 rpm in a 4°C centrifuge for 15 minutes, 500 μL of the upper colorless liquid was aspirated, an equal volume of isopropanol was added, and the mixture was mixed by inversion. After being placed at -20°C for 2 hours, the mixture was centrifuged at 12000 rpm in a 4°C centrifuge for 15 minutes, the upper liquid was discarded, and the mixture was washed twice with 75% ethanol, each centrifuged for 5 minutes, dried in a fume hood, 25 μL of DEPC water was added, and heated at 55°C for 10 minutes. After the RNA concentration was detected, it was transcribed into cDNA using a reverse transcription kit (HisScriptⅡQ RT SuperMix, Vamyze, R222-01), and then its gene transcription level was detected using a qPCR kit (AceQ Qpcr SYBR Green MasterMix, Vamyze, Q111-02). The results of tissue mRNA level detection in the pancreatic fibrosis animal model after Cal treatment are shown in Fig.14 ; The results of tissue protein level detection in pancreatic fibrosis animal model after Cal treatment are as follows Fig.15 .from Fig.14 It can be seen that compared with the model group, the mRNA levels of the above markers were reduced in the Cal treatment group; Fig.15 It can be seen that Cal can significantly inhibit the protein levels of collagen I, α-SMA and TGF-β1.

[0100] The following experiments were performed on the Chinese medicine compositions for treating pancreatic fibrosis prepared in Examples 2 to 4:

[0101] In this experiment, the pancreatic fibrosis animal model constructed in Example 9 was used to treat the model with a Chinese medicine combination (Y1, Y2, Y3) with Cal as the main ingredient. The dose was 20 mg / kg and the method of gavage was adopted for a total of two weeks. After the mice were euthanized, the pancreatic tissue of the mice was taken to extract mRNA and protein, and the levels of related markers were detected. The pathological conditions of the pancreatic tissue of the mice after Chinese medicine treatment were detected by HE staining.

[0102] The mRNA level of tissues in the pancreatic fibrosis animal model was detected after the combination of Chinese medicines was treated. Fig.16 ; Detection of tissue protein levels in pancreatic fibrosis animal models after treatment with Chinese medicine combination drugs Fig.17 ; HE staining of tissues in pancreatic fibrosis animal models treated with Chinese medicine combination drugs Fig.18 .

[0103] Fig.16The results of RT-PCR experiments showed that the combination of Chinese medicines mainly composed of Cal can also inhibit the progression of pancreatic fibrosis; Fig.17 ELISA experiments also further showed that the Chinese herbal medicine combination could significantly inhibit the protein levels of collagen I, α-SMA, and TGF-β1; Fig.18 HE staining results showed that the pathology of pancreatic tissue in animals with pancreatic fibrosis was restored after treatment with the Chinese medicine combination.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating pancreatic fibrosis, characterized in that: The invention comprises the following raw materials in parts by weight: 20-30 parts of isoflavones; 0-5 parts of isoginkgo biloba; 0-5 parts of dansholtzia rhamnoides; 0-5 parts of rutin; 0-5 parts of naringin; 0-5 parts of bergenin; 0-5 parts of pomegranate peel tannin; 0-5 parts of berberine.

2. The Chinese medicine composition for treating pancreatic fibrosis according to claim 1, characterized in that: The invention comprises the following raw materials in parts by weight: 20-30 parts of isoflavones; 1-5 parts of isoginkgetin; 1-5 parts of dansholtzia rutin; 1-5 parts of rutin; 1-5 parts of naringin; 1-5 parts of bergenin; 1-5 parts of pomegranate peel tannin; 1 to 5 parts of berberine.

3. The method for preparing the Chinese medicine composition for treating pancreatic fibrosis according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) Weigh the raw materials according to the proportion; (2) fully grinding and mixing the above-mentioned Chinese medicinal components, filtering, and obtaining a mixed powder; (3) adding the mixed powder into water for ultrasonic dissolution, collecting the filtrate after filtering, and spray drying or vacuum concentrating to obtain the traditional Chinese medicine composition for treating pancreatic fibrosis.

4. The method for preparing the Chinese medicine composition for treating pancreatic fibrosis according to claim 3, characterized in that: The filtration in step (2) is performed through a 100-110 mesh screen.

5. The method for preparing the Chinese medicine composition for treating pancreatic fibrosis according to claim 3, characterized in that: The weight ratio of the mixed powder to water in the ultrasonic dissolution in step (3) is 1:2-3.

6. The method for preparing the Chinese medicine composition for treating pancreatic fibrosis according to claim 3, characterized in that: The temperature of the ultrasonic dissolution in step (3) is 50-60°C.

7. The method for preparing the Chinese medicine composition for treating pancreatic fibrosis according to claim 3, characterized in that: The ultrasonic dissolution time in step (3) is 30-45°C.

Images