Application of agrimonin in hepatic lung syndrome pulmonary pathological angiogenesis

Abstract

The invention relates to the technical field of medicine, in particular to application of agrimonin in hepatic lung syndrome pulmonary pathological angiogenesis, in the invention, agrimonin promotes mitochondrial biogenesis by repairing mtDNA damage, increasing mtDNA and regulating mitochondrial homeostasis so as to further improve the OXPHOS function of mitochondria, so that the agrimonin can be used for treating hepatic lung syndrome pulmonary pathological angiogenesis. The HPS liver is reduced, and the glycolysis flux of tissues is used for reducing pathological angiogenesis; according to the application of the PGC1-alpha as the target spot of the hairyvein agrimony for treating the liver cirrhosis, the hairyvein agrimony promotes expression of the PGC-1 alpha, promotes the mitochondrial biogenesis function, increases the OXPHOS capacity of PMVECs mitochondria, increases the OXPHOS / glycolysis ratio of tissues and reduces HPS angiogenesis. According to the invention, the active monomers of the traditional Chinese medicines are used for preparing the medicine for treating the pulmonary pathological angiogenesis of the liver-lung syndrome, so that the toxic and side effects of the kidney can be better avoided, the exact action target of the medicine can be analyzed, the living quality and the compliance of a patient are improved, and a new thought is provided for the prevention and treatment of the HPS by utilizing the'simultaneous treatment of the liver and the lung 'of the holism concept.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of agrimony in pathological pulmonary angiogenesis in hepatopulmonary syndrome. Background Art

[0002] Hepatopulmonary syndrome (HPS) is a liver-induced lung injury that occurs on the basis of chronic liver disease, with an incidence of approximately 5-30%. The three main pathological changes of HPS are systemic hypoxemia caused by liver disease, IPVD, and PPA. The 5-year survival rate of HPS patients is poor compared with that of patients with chronic liver disease (23% vs 63%, P = 0.003). HPS patients have an increased incidence of perioperative pulmonary complications and risk of death. HPS patients are significantly different from ordinary cirrhosis patients in the use of perianesthetic-related drugs, ventilation mode, and fluid volume management. The median survival of HPS cirrhosis patients with hypoxemia is shorter than that of non-HPS patients (10.6 months vs 40.8 months, P < 0.05).

[0003] Pulmonary PPA and IPVD changes in HPS play an important role in hypoxemia. IPVD causes the capillary diameter to expand from the normal 8-15μm to 15-500μm, increasing the oxygen diffusion distance and leading to ventilation dysfunction; PPA causes ventilation-perfusion ratio (V / Q) mismatch, leading to hypoxemia. Increased carbon monoxide (NO) plays a major role in IPVD. Patients with cirrhosis and intrapulmonary vasodilation have elevated plasma endothelin-1 (ET-1) levels, which induce NO-related vasodilation through activation of endothelin b receptor (ETBR) on endothelial cells. In addition, increased phagocytosis of bacterial endotoxins in the lungs promotes the activation of inducible nitric oxide synthase (iNOS), which also helps to increase the production of NO. Macrophages produce heme oxygenase-1 (HO-1) and metabolize heme through heme oxygenase to produce carbon monoxide (CO), which helps vasodilation.

[0004] Angiogenesis is the driving factor of extrahepatic target organ damage in HPS. In the process of chronic liver disease progressing to cirrhosis, the more obvious the pathological angiogenesis, the more severe the damage. Angiogenic factors from the diseased liver, including plasma von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), vascular endothelial growth factor receptor 2 (VEGFR-2), placental growth factor (PLGF) and soluble vascular endothelial growth factor 1 (sFlt-1), continue to increase in expression. Pathological extrahepatic angiogenesis is the pathological basis of extensive target organ damage in HPS.

[0005] In recent years, clinical observation studies using drugs such as pentoxifylline, methylene blue, and sorafenib have not found a definite effect on improving HPS hypoxemia, and serious side effects reduce the quality of life and compliance of patients. However, 85%-100% of patients after liver transplantation have improved oxygenation and returned to normal within 1 year. Therefore, we consider that the use of drugs that "treat both the liver and lungs, and treat multiple targets" can achieve a better therapeutic effect, and the side effects of the drug should be limited to increase the patient's compliance with long-term use.

[0006] Traditional Chinese medicine has a history of thousands of years in my country for treating various chronic liver diseases and is a natural synthetic drug source for treating liver diseases. Compared with chemical synthetic drugs, Chinese herbal natural synthetic drugs have long-term medicinal experience. The use of multiple dosage forms can increase patient compliance and can be used as a long-term adjuvant treatment for chronic liver disease. Modern pharmacology has also conducted extensive research on the role of Chinese medicine monomers. The use of Chinese medicine monomers can minimize the toxic side effects of Chinese medicine and is more conducive to clinical transformation.

[0007] To this end, the present application aims to provide the use of agrimony in pathological pulmonary angiogenesis in hepatopulmonary syndrome, so as to solve the above-mentioned problems in the prior art. Summary of the invention

[0008] The purpose of the present invention is to solve the technical problems raised in the above-mentioned background technology and to provide the application of agrimony in pathological angiogenesis of the lung in hepatopulmonary syndrome.

[0009] The above-mentioned purpose of the present invention is achieved like this:

[0010] On the one hand, the scheme of the present invention provides the use of agrimony in the preparation of a drug for treating pathological pulmonary angiogenesis in hepatopulmonary syndrome.

[0011] Furthermore, the agrimonycetin is used to regulate mitochondrial fusion and fission homeostasis, promote mitochondrial fusion, increase mtDNA levels, and improve OXPHOS function; the agrimonycetin is used to repair mtDNA damage and promote mitochondrial biogenesis, increase tissue OXPHOS / glycolysis ratio, restore normal energy metabolism of tissues, and reduce pathological angiogenesis in HPS liver and lung tissues.

[0012] Another aspect of the present invention provides the use of PGC1-α as a target for treating liver cirrhosis with Agrimoniae scabra.

[0013] Furthermore, agrimonycin was used to promote PGC-1α expression, promote mitochondrial biogenesis function, increase PMVECs mitochondrial OXPHOS capacity, increase tissue OXPHOS / glycolysis ratio, and reduce HPS angiogenesis.

[0014] The difficulty and significance of the technical problem solved by the present invention are:

[0015] Chinese herbal medicine has a history of thousands of years of application in the treatment of liver diseases, so patients have good compliance with Chinese medicine treatment. Modern medicine also often uses Chinese patent medicine as an auxiliary drug in the treatment of chronic liver diseases. In the scheme of the present invention, the active monomer of the drug that plays the main therapeutic role in the traditional Chinese medicine prescription is used, which can better avoid the side effects of nephrotoxicity and analyze the exact target of the drug, and by selecting Agrimoniin (C 82 H 54 O 52 ) were used to test its protective effect on lung injury in HPS rats and its improvement on pathological angiogenesis, confirming its specific mechanism of action.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The application of agrimony in the preparation of a drug for treating pathological angiogenesis in the lung of hepatopulmonary syndrome provided by the scheme of the present invention. On the one hand, agrimony in the present invention promotes mitochondrial biogenesis by repairing mtDNA damage, increasing mtDNA and regulating mitochondrial homeostasis, thereby improving mitochondrial OXPHOS function, reducing HPS liver and tissue glycolysis flux to reduce pathological angiogenesis; on the other hand, the present invention confirms the application of PGC1-α as a target of agrimony in the treatment of liver cirrhosis, and agrimony is used to promote the expression of PGC-1α, promote mitochondrial biogenesis function, increase the mitochondrial OXPHOS capacity of PMVECs, increase the tissue OXPHOS / glycolysis ratio, and reduce HPS angiogenesis.

[0018] 2. The present invention uses the active monomers of traditional Chinese medicine to prepare drugs for treating pathological angiogenesis of the lung in hepatopulmonary syndrome, which can better avoid nephrotoxic side effects and analyze the exact target of the drug, thereby facilitating the improvement of the patient's quality of life and compliance. The present invention utilizes the holistic concept of "treating the liver and lungs together" and provides a new idea for the prevention and treatment of HPS. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a comparison of UCBDL and M-UCBDL surgical methods in the embodiments of the present invention (A: UCBDL (Upper common bileduct ligation) surgical method; B: M-UCBDL (Modified common bileduct ligation) surgical method);

[0020] Figure 2 is a comparison of liver tissue changes and liver injury markers between M-CBDL and UCBDL in the embodiment of the present invention (A: HE and Masson liver staining, CD31 fluorescence staining results (n=8); B: METAVIR score of liver fibrosis; C: comparison of changes in liver injury markers ALT, ALP, AST and TBA. Compared with the sham group, ns, P>0.05, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; scale bar=50μm);

[0021] Figure 3is a comparison of lung tissue changes and lung injury indicators between M-CBDL and UCBDL in the embodiment of the present invention (AB: HE lung staining, eNOS and CD31 fluorescence staining and statistical results (n=8); C: expression comparison of angiogenesis-related proteins VEGFR1 and VEGFR2 at 3 and 6 weeks after CBDL surgery; D: comparison of changes in P(Aa)O2 and PO2 between different groups. Compared with the sham group, ns, P>0.05, *, P<0.05, **, P<0.01, ****, P<0.0001; scale=50μm);

[0022] Figure 4 is a comparison of inflammatory factor-related indicators between M-CBDL and UCBDL in the embodiment of the present invention (A: changes in the expression of IL-6, IL-10, IL-1β and TNF-α in M-CBDL and UCBDL groups at 3 and 6 weeks; B: comparison of common bile duct epithelial cells between sham and UCBDL (3 weeks); C: bile accumulation in the common bile duct 3 weeks after CBDL. Compared with the common bile duct ligation group 3, ns, P>0.05; *, P<0.1; scale of electron microscope images = 50 μm);

[0023] Figure 5 is a comparison of the survival rates of M-UCBDL and UCBDL in the embodiment of the present invention (AB: comparison of the survival rates of M-CBDL and UCBDL. Compared with the UCBDL group, ****, P<0.0001);

[0024] Figure 6 The improvement effect of different Chinese medicine monomers on the liver and lung function of CBDL rats in the embodiment of the present invention (A: comparison of P(Aa)O2 and PO2 in different treatment groups; B: comparison of liver enzyme metabolism levels in different treatment groups; C: comparison of bilirubin metabolism levels in different treatment groups; D: comparison of renal function in different treatment groups. Compared with the sham group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001);

[0025] Figure 7 is the improvement effect of the drug treatment in the embodiment of the present invention on the pathological changes of liver and lung in the CBDL group (A: comparison of liver HE and Masson staining; B: comparison of liver fibrosis staining area; C: comparison of lung HE and Masson staining; D: lung injury score and liver fibrosis METAVIR score. Compared with the sham group, *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001; HE and Masson scale = 50 μm);

[0026] Figure 8is the improvement effect of the Chinese medicine treatment in the embodiment of the present invention on liver and lung fibrosis in the CBDL group (A: Fluorescence staining of collagen-Ⅰ and α-SMA in liver tissue; B: Fluorescence staining of collagen-Ⅰ and α-SMA in lung tissue. Compared with the sham group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; immunofluorescence scale = 50 μm);

[0027] Fig. 9 It is the effect of the treatment of the Chinese medicine monomer in the embodiment of the present invention on the pathological angiogenesis of the liver and lung of CBDL rats (A: CD31 fluorescence staining of liver and lung tissues; B: VEGF immunohistochemical staining of liver and lung tissues; C: WB experimental results of CD31 and VEGF in liver and lung tissues. Compared with the sham group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; immunohistochemical scale = 50 μm; immunofluorescence scale = 50 μm);

[0028] Fig.10 The effects of high and low doses of agrimony in the embodiments of the present invention on the ratio of pyruvate / lactate in liver and lung tissues (A: changes in the ratio of pyruvate / lactate and lactate dehydrogenase in liver and lung tissues; BC: changes in the expression of PFKFB3 determined by immunohistochemistry and Elisa; DE: changes in the spatial expression of PFKFB3 assessed by laser confocal microscopy. Compared with the control group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; immunohistochemistry scale = 50 μm; laser confocal microscopy scale = 10 μm);

[0029] Fig.11 It is that the agrimonycetin in the embodiment of the present invention improves the mitochondrial damage and mitochondrial function decline caused by HPS (A: changes in mtDNA levels and mtDNA damage in liver and lung tissues; BC: immunohistochemical evaluation of the expression changes of mitochondrial respiratory chain-related proteins CoxⅣ and PHB1. Compared with the control group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; immunohistochemical scale = 50μm);

[0030] Fig.12It is that the agrimony in the embodiment of the present invention enhances mitochondrial biogenesis by promoting mitochondrial fusion function (A: immunohistochemistry of liver and lung tissues to detect changes in PGC-1α expression; B: statistical graph of OD values ​​of immunohistochemistry of PGC-1α expression in liver and lung tissues; C: Elisa to evaluate changes in expression of PGC-1α in liver and lungs; DE: laser confocal immunofluorescence to evaluate changes in expression of DRP1; FG: laser confocal immunofluorescence to evaluate changes in expression of MFN1. Compared with the control group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; immunohistochemistry scale = 50 μm; laser confocal scale = 10 μm);

[0031] Fig.13 It is that the agrimonycin in the embodiment of the present invention promotes the mitochondrial biogenesis function by stimulating PGC-1α (A: PMVECs, PGC-1α expression changes after PGC-1α siRNA plasmid transfection; B: PMVECs tubule experiment; CD: PMVECs scratch experiment; E: Elisa evaluates the change of pyruvate / lactate ratio; F: Elisa evaluates the expression change of lactate dehydrogenase in PMVECs; G: Elisa evaluates the expression change of citrate synthase in PMVECs. Compared with the control group, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001; tubule formation picture scale = 200μm; scratch picture scale = 500μm). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] The implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0034] As shown in the drawings, a preferred embodiment of the present invention is provided.

[0035] The embodiments of the present invention provide the use of agrimony in the preparation of a drug for treating pathological angiogenesis in the lungs of hepatopulmonary syndrome, wherein agrimony promotes mitochondrial fusion and increases mtDNA levels by regulating mitochondrial fusion and fission homeostasis, thereby improving OXPHOS function; agrimony repairs mtDNA damage and promotes mitochondrial biogenesis, thereby increasing the tissue OXPHOS / glycolysis ratio, restoring normal energy metabolism of the tissue, and reducing pathological angiogenesis in the liver and lung tissues of HPS.

[0036] The expression of PGC-1α was reduced by siRNA transfection in PMVECs. After the reduction of PGC-1α protein, the inhibitory effect of agrimony on angiogenesis was significantly reduced. Therefore, agrimony reduces HPS angiogenesis by promoting PGC-1α expression, promoting mitochondrial biogenesis function, increasing PMVECs mitochondrial OXPHOS capacity, and increasing tissue OXPHOS / glycolysis ratio.

[0037] The following is an experiment of the implementation scheme of the present invention:

[0038] 1. Construction of the Improved M-UCBDL Rat Model

[0039] 1. Experimental Animals

[0040] 6-8 week old SPF male SD rats weighing 230-260 g were purchased from the Experimental Animal Center of the Army Medical University and raised in the Animal Room Center of the First Affiliated Hospital of the Army Medical University. A week of feeding was performed before modeling so that the experimental animals could reach a weight of more than 300 g during the adaptation process. The circadian rhythm of feeding was 12 hours, the ambient temperature was 22±1°C, the humidity was 55±6%, and the diet and water were free. All protocols complied with the ethical standards for experimental animals. The experimental protocol was approved by the Animal Research Committee of Chongqing Hospital of Traditional Chinese Medicine (approval number: 2021-DWSY-ZZY).

[0041] 2. Methods

[0042] 2.1. Establishment of Modified upper commonbileduct ligation (UCBDL) and Upper common bileduct ligation (UCBDL) models in rats Rats were fasted from food and water for 8 hours before the establishment of the model. Anesthesia was induced with 5% sevoflurane and maintained with 2% sevoflurane. After the anesthetic took effect, a small incision of about 2.5 cm was made along the rectus abdominis incision. After the stomach was exposed, the duodenum was explored downwards, and the common bile duct was pulled outward and flipped free. UCBDL method: Try to make a single ligation as close to the confluence of the hepatobiliary duct as possible, make a second single ligation near the small intestine of the bile duct, and cut the common bile duct between the two ligature points ( Figure 1 A in the figure). Modified M-UCBDL method: double ligation is performed below the confluence of the hepatobiliary duct, and a second double ligation is performed near the small intestine of the bile duct. The entire common bile duct is carefully separated and removed between the two ligature points, minimizing the common bile duct stump ( Figure 1 B).

[0043] 2.2. Serum collection of CBDL model rats

[0044] Anesthetize the rat, use a 10mL syringe to draw a dilute heparin solution (diluted at 2500IU:100mL saline), evenly fill the empty needle and then drain to prepare for lubrication, connect the syringe to the scalp needle (to facilitate blood drawing operation) and draw blood through the rat's inferior vena cava puncture, shake the syringe gently during blood drawing to fully mix it with the dilute heparin solution, and then transfer it to a pre-cooled sterile centrifuge tube. Precool the centrifuge to 4℃, balance the centrifuge tube and place it in the centrifuge at 4℃, centrifuge at 3500 rpm for 20 minutes. Disinfect the centrifuge tube by ultraviolet irradiation for more than 30 minutes, draw the upper clear serum and place it in a -20℃ refrigerator for standby use.

[0045] 2.3. Collection of liver and lung tissues

[0046] Through right ventricular catheterization, perfusion is performed with a perfusion pump (paraformaldehyde is used for paraffin section specimens; saline is used for protein extraction specimens). Rats perfused with paraformaldehyde need to continue perfusion until the muscles are stiff; rats perfused with saline need to continue perfusion until the outflowing fluid contains almost no blood. Remove the lung and liver tissues in turn. Specimens that need to be used for biochemical experiments should be immediately stored in liquid nitrogen; lung tissues that need to be sliced ​​should be injected with a sufficient amount of paraformaldehyde solution from the trachea, and the trachea should be ligated to ensure that the filled paraformaldehyde maintains the shape of the lungs. Then, the lung tissues are placed in 4% paraformaldehyde for fixation and preservation. Liver tissues also need to be placed in 4% paraformaldehyde for tissue fixation. After 48 hours, paraffin embedding is performed, and paraffin tissue sections with a thickness of 4μm are selected according to experimental needs.

[0047] 2.4 HE staining of liver and lung tissue sections

[0048] (1) Paraffin sections were placed in a 60°C oven and baked at a constant temperature for 50 minutes to dry the sections, dehydrated with xylene and anhydrous ethanol, and washed three times;

[0049] (2) Add a small amount of hematoxylin stain to the tissue section, keep it for about 10 seconds, and then rinse it with distilled water (do not rinse the tissue section) to clean it; (3) Add differentiation solution to further process the section, keep it for about 1 minute, and then rinse it 3 times (tap water), each time for 5 minutes; (4) Add a small amount of eosin stain to the tissue section, keep it for about 1 minute, and then rinse it 3 times (distilled water), each time for 5 minutes; (5) Dehydrate and make the stained sections transparent according to the following steps: (75% ethanol - 85% ethanol - 95% ethanol) Each step of the above three operations takes about 3 seconds - keep it in anhydrous ethanol for 5 seconds - keep it in anhydrous ethanol for 1 minute - keep it in xylene for 3 minutes; (6) Place the processed sections in a ventilation room to dry for about 2 hours. After the xylene evaporates completely, seal the sections with neutral gum and dry them again at room temperature for 24 hours. 2.3.5 Masson staining of liver and lung tissue sections (1) Paraffin sections were routinely dewaxed in a gradient manner: anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and 75% ethanol for 5 minutes; washed with PBS three times, each time for 5 minutes; (2) A few drops of periodic acid aqueous solution were added to the tissue sections for 20 minutes, and then rinsed with tap water three times, each time for 5 minutes; (3) The sections were immersed in hexamine silver solution heated to 55-60°C, and the solution was changed once every 30 minutes, for a total of two changes, until the basement membrane of liver and lung tissue cells appeared black under the microscope, and then rinsed with tap water three times, each time for 5 minutes; (4) The color was adjusted with 0.2% gold chloride aqueous solution for about 2 minutes, and then rinsed with tap water three times, each time for 5 minutes; (5) The sections were immersed in 0.2% gold chloride aqueous solution. .25% sodium thiosulfate aqueous solution for 3 minutes, and rinse with tap water three times, 5 minutes each time; (6) Add modified Carazzis hematoxylin solution to the tissue section, stain for 20-30 minutes, and rinse with tap water three times, 5 minutes each time; (7) Add 1% hydrochloric acid aqueous solution to the tissue section for differentiation for a few seconds, and rinse with tap water three times, 5 minutes each time; (8) Add Masson solution to the tissue section and stain for 5 minutes, and rinse with tap water three times, 5 minutes each time; (9) Add 0.1% fast green aqueous solution to the tissue section and stain for 5 minutes, and rinse with tap water three times, 5 minutes each time; (10) After the xylene on the section is completely evaporated at room temperature, add a few drops of neutral gum to the tissue, cover with a coverslip, and dry at room temperature for 24 hours. 2.3.6 METAVIR score of liver fibrosis and pathological score of lung injury The sample size of each group of mice was 10, and METAVIR score was performed (F0: no fibrosis, F1: portal fibrosis without septa, F2: portal fibrosis with a small number of septa, F3: portal fibrosis with a large number of septa, F4: cirrhosis). Microscopic photos were taken with an optical microscope (Olympus BX51-PMS, Tokyo, Japan), and the images were analyzed with Image-ProPlus software (version 6.0, Media Cybernetics, Inc, USA).

[0050] 2.5 Immunohistochemistry

[0051] (1) Preparation of tissue sections is the same as in 2.4.

[0052] (2) Bake the paraffin sections in a 65°C constant temperature oven for 60 minutes.

[0053] (3) Dehydrate the sections by soaking them in gradient solutions: xylene solution (10 min) – xylene solution (10 min) – anhydrous ethanol (5 min) – anhydrous ethanol (5 min) – 95% ethanol (5 min) – 95% ethanol (5 min) – 90% ethanol (5 min) – 80% ethanol (5 min). Take them out and soak them in distilled water, and wash them on a shaker for 3 times.

[0054] (4) Dry the tissue sections at room temperature, draw a circle around each tissue with an immunohistochemistry pen, add 3% H2O2 solution within the circle and incubate for 30 minutes. Then soak in distilled water and shake on a shaker for 3 times, 5 minutes each time.

[0055] (5) Add antigen repair solution (Tris 1.21g, EDTA 0.33g, Tween-20 1mL) to 1L PBS solution) and perform antigen repair in a pressure cooker for 20 minutes (maintaining the heat with small bubbles is the best). Take it out and place it at room temperature until the antigen repair solution cools naturally to room temperature. Wash it with PBS 3 times, 5 minutes each time.

[0056] (6) Add 10% goat serum to the tissue and incubate for 30 minutes at room temperature. Pour off the goat serum.

[0057] (7) Dilute the primary antibody according to the instructions (check the official website to obtain the appropriate antibody dilution concentration), add the primary antibody to the circle drawn by the immunohistochemistry pen, and incubate overnight at 4°C. The next day, remove the slices and wash them three times in PBS with shaking for 5 minutes each time.

[0058] (8) Dilute the corresponding secondary antibody (HRP-labeled Goat Anti-Rabbit IgG H+L) according to the instructions, incubate on the tissue sections according to the primary antibody drop method, and keep at room temperature for 60 minutes. Wash again with PBS for 3 times with shaking, 5 minutes each time.

[0059] (9) Immediately prepare DAB colorimetric solution and add it to the slices for 1 minute. Rinse with tap water for 1 minute. Pipette an appropriate amount of hematoxylin stain solution and centrifuge at 3000 rpm for 1 minute. Take the upper layer of liquid and add it to the tissue slices for incubation for 10 seconds. Wash the stain solution 3 times with tap water, 5 minutes each time.

[0060] (10) Dehydrate the tissue sections in the following order: 75% ethanol (5 min) – 80% ethanol (5 min) – 95% ethanol (5 min) – 95% ethanol (5 min) – anhydrous ethanol (5 min) – anhydrous ethanol (5 min) – xylene (15 min) – xylene (15 min) soak.

[0061] (11) Place the slices in a fume hood to dry, then seal with neutral gum and dry again at room temperature for 24 hours.

[0062] 2.6 Immunofluorescence

[0063] (1) Preparation of tissue sections is the same as in 2.4.

[0064] (2) Bake the paraffin sections in a 65°C constant temperature oven for 60 minutes.

[0065] (3) Dehydrate the sections by soaking them in gradient solutions: xylene solution (10 min) – xylene solution (10 min) – anhydrous ethanol (5 min) – anhydrous ethanol (5 min) – 95% ethanol (5 min) – 95% ethanol (5 min) – 90% ethanol (5 min) – 80% ethanol (5 min). Take them out and soak them in distilled water, and wash them on a shaker for 3 times.

[0066] (4) Shake off the water on the tissue sections at room temperature, draw a circle around each tissue with an immunohistochemical pen, add 3% H2O2 solution within the circle and incubate for 30 minutes. Soak in distilled water and shake on a shaker to wash 3 times, 5 minutes each time.

[0067] (5) Add antigen repair solution (Tris 1.21g, EDTA 0.33g, Tween-201mL) in 1L PBS solution) and perform antigen repair in a pressure cooker for 20 minutes (maintaining the heat with small bubbles is the best). Take it out and place it at room temperature until the antigen repair solution cools naturally to room temperature. Wash it with PBS three times, 5 minutes each time.

[0068] (6) Dilute the primary antibody according to the instructions (check the official website to obtain the antibody dilution concentration), add the primary antibody to the circle drawn by the immunohistochemistry pen, and incubate overnight at 4°C. The next day, remove the slices and wash them three times in PBS with shaking for 5 minutes each time.

[0069] (7) Dilute the corresponding secondary antibody (HRP-labeled Goat Anti-Rabbit IgG H+L) according to the instructions, incubate on the tissue sections according to the primary antibody drop method, and keep at room temperature for 60 minutes. Wash again with PBS for 3 times with shaking, 5 minutes each time.

[0070] (8) The sections were counterstained with Cy3-labeled Goat Anti-Rabbit IgG (H+L) and FITC-labeled Goat Anti-Rabbit IgG (H+L) (Biyuntian, China) and washed again with PBS for 3 times with shaking for 5 minutes each time.

[0071] (9) Add DAPI solution (containing anti-tissue fluorescence) to the tissue section for staining and cover with a coverslip;

[0072] (10) The slides were sealed and air-dried overnight, and photographed under a microscope (A2 microscope, ZEISS, Germany).

[0073] 2.7 Enzyme-linked immunosorbent assay (ELISA)

[0074] (1) Preparation before the experiment:

[0075] 1) Take out the Elisa kit in advance and place it at room temperature for equilibrium according to the time required by the instructions.

[0076] 2) Preparation of standard gradient working solution (slightly different for each kit): add 1 mL PBS to the standard, let it stand for 15 minutes and then gently mix (concentration is 200 pg / mL), then use the standard as number 1 and prepare 8 centrifuge tubes in sequence to dilute at the following concentrations: 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, 6.25 pg / mL, 3.12 pg / mL, 0 pg / mL.

[0077] 3) Preparation of biotin working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 0.5 min (to collect the biotin at the bottom of the centrifuge tube), dilute the 100× concentrated biotinylated antibody to 1× with PBS or diluent, and the preparation and use time shall not exceed 3 hours.

[0078] 4) Preparation of enzyme conjugate working solution: Centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute, and dilute the 100× concentrated HRP enzyme conjugate to 1× using the diluent provided in the kit. The preparation and use time shall not exceed 3 hours.

[0079] 5) Preparation of washing solution: prepare washing solution according to the instructions of the kit (the washing solution can be prepared once to facilitate the operation of machine washing well plates).

[0080] (2) Experimental operation (the steps of the preliminary experiment are the same as those of the formal experiment):

[0081] 1) Take the slats out of the refrigerator 10 minutes in advance and allow to equilibrate to room temperature.

[0082] 2) Sample addition: Add 50 μl of sample and standard to the corresponding wells in sequence according to the kit instructions, and add 100 μl of PBS to the blank wells. Cover with a sealing film and incubate at 37°C for 60 minutes.

[0083] 3) Add biotinylated antibody: Take out the ELISA plate, pour out the liquid in the well plate, add 100 μL of biotinylated antibody working solution, cover with sealing film and incubate at 37°C for 60 minutes.

[0084] 4) Washing: Pre-experimental washing: discard the liquid, add 300μL of washing solution to each well, let it stand for 1 minute to spin off the washing solution, and repeat 3 times; for formal experiments, the plate can be washed by machine.

[0085] 5) Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes.

[0086] 6) Wash the plate again.

[0087] 7) Add substrate (reagent three): Add 90 μL substrate to each well, cover with sealing film and incubate at 37°C in the dark for 15-20 minutes (according to the time required by the instructions, until a gradient blue color appears in the well plate).

[0088] 8) Add stop solution: Take out the ELISA plate, add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm. (The pre-experiment needs to calculate the fitting formula based on the OD value to improve the protein dilution multiple in the formal experiment)

[0089] 2.8 Statistical Analysis

[0090] This experimental study used SPSS software for Windows, V.23.0 (IBM Corp., Armonk, NY, USA) and GraphPad PRISM (version 8.00; GraphPad Software, San Diego, California, USA). Data statistical analysis was performed. According to observation and statistical judgment, if the descriptive data obeyed the normal distribution, the mean ± standard deviation was used; if not, the median and quartile were used. If the numerical variables of the test data obeyed the normal distribution, one-way analysis of variance was used, and when the variances between the groups of the data to be processed were homogeneous, the least significant difference method (Least Significance Difference Method, LSD) was used for multiple comparisons, otherwise Dunnett's T3 was used for correction; if the numerical variables did not obey the normal distribution, the Kruskal-Wallis H test was used for inter-group comparison. In the present study, the observation endpoints of all groups were 5 weeks after M-UCBDL and UCBDL surgery, and the Log-rank method was used to compare the differences in survival rates of the three groups of rats. All statistical tests were two-sided, and P < 0.05 was considered statistically significant.

[0091] 3. Results

[0092] 3.1. Comparison of liver tissue changes and liver injury markers between M-CBDL and UCBDL. HE-stained liver tissue sections: In the sham group, the liver lobules and portal areas were normal in structure, with neat arrangement of the lobules and no fibrosis; in the UCBDL and M-UCBDL groups, the liver lobules were structurally destroyed, the pseudolobules formed by regeneration were arranged in disorder, and fibrosis and regenerative nodules were formed in the portal areas (see Figure 2 Compared with the sham group, the liver fibrosis score, CD31 positive cell count, total bilirubin, alanine aminotransferase, and aspartate aminotransferase in the UCBDL and M-UCBDL groups were increased, but there was no statistical difference between the two groups (ALT: UCBDL vs M-UCBDL: 138.6.3 IU / L vs 141.4 IU / LP>0.05; TBA: UCBDL vs M-UCBDL: 108.4 mg / dl vs 114.2 mg / dl, P>0.05) (see Figure 2 AC in).

[0093] 3.2. Comparison of lung tissue changes and lung injury indicators between M-CBDL and UCBDL HE staining showed that compared with the sham group, the lung respiratory membrane thickened and inflammatory cell infiltration was obvious in the UCBDL and M-UCBDL groups (see Figure 3A in the figure). Compared with the sham group, the expression of eNOS and the expression of angiogenesis marker proteins VEGFR1 and VEGFR2 in the lung tissues of UCBDL and M-UCBDL were increased compared with those in the sham group, and the change trends were consistent (see Figure 3B-C). At the same time, compared with the sham group, the PaO2 of the UCBDL and M-UCBDL groups decreased, and the P(Aa)O2 increased, and the difference was statistically significant (PaO2: sham vs UCBDL vs M-UCBDL: 98.2 mmHg vs 72.3 mmHg vs 79.6 mmHg P < 0.01; P(Aa)O2: sham vs UCBDL vs M-UCBDL: 7.6 mmHg vs 18.8 mmHg vs 21.2 mmHg, P < 0.001), but there was no statistically significant difference between the two in the UCBDL and M-UCBDL groups (see Figure 3 D).

[0094] 3.3 Comparison of inflammatory factor-related indicators between M-CBDL and UCBDL

[0095] By comparing the expression of IL-6, IL-10, IL-1β and TNF-α in the rat serum between the M-CBDL and UCBDL groups at 3 and 6 weeks, no statistical difference was found (see Figure 4 A in the figure indicates that the inflammatory response of the two modeling methods is consistent. The proliferation of common bile duct epithelial cells in the sham group and the UCBDL group was compared under an electron microscope, and it was found that the common bile duct epithelial cells in the UCBDL group proliferated significantly (see Figure 4 B). By dissecting the common bile duct model between the ligature points, it was found that bile accumulated in the common bile duct and the common bile duct epithelial cells secreted bile in the M-UCBDL and UCBDL models (see Figure 4 C), indicating that bile continued to flow into the peritoneal cavity after common bile duct cutting.

[0096] 3.4 Comparison of survival rates between M-UCBDL and UCBDL

[0097] M-CBDL improved the survival rate of the UCBDL model (UCBDL vs M-UCBDL: 24.2% vs 73.4%, P < 0.001), laying the foundation for subsequent experiments (see Figure 5 AB in).

[0098] In summary, the present invention further improved the previous model into the M-CBDL model, reduced the high mortality rate of the SPF-level SD rat CBDL model, and constructed an HPS rat model that meets the diagnostic criteria for hepatopulmonary syndrome. Blood gas analysis, relevant indicators of liver function, and pathological sections of liver and lung tissues showed that there was no statistical difference between the various indicators of M-UCBDL and UCBDL. Both M-UCBDL and UCBDL can be used as effective models of liver injury and hepatopulmonary syndrome. Compared with UCBDL, the M-UCBDL constructed by the present invention has a lower mortality rate and is a more reliable and repeatable model of cirrhosis combined with HPS, which is particularly suitable for research requiring SPF-level rats.

[0099] 2. The therapeutic effect of traditional Chinese medicine on HPS pulmonary pathological angiogenesis

[0100] 1. Methods

[0101] 1.1. Traditional Chinese medicine intervention strategy

[0102] SPF grade SD male rats, weighing 230-300g, were purchased from the Experimental Animal Center of the Army Medical University. Baicalein (C15H10O5, HPLC>98%, CAS#49167-8, Yuanye Bio-Technology, China), catalpol (C15H22O10, HPLC>98%, CAS#2415-24-9, Yuanye Bio-Technology, China) and agrimony (C82H54O52, HPLC>98%, CAS#82203-01-8, Yuanye Bio-Technology, China) were diluted with normal saline. CBDL rats were randomly divided into 4 groups, CBDL positive control group, baicalein, catalpol and agrimony intervention group. The drug intervention group began intraperitoneal injection 2 weeks after CBDL surgery, and the dosage of baicalein (40mg·kg -1 ·d -1 ), catalpol (8 mg kg -1 ·d -1 ) and agrimonycin (5 mg kg -1 ·d -1 ), and after 3 weeks of continuous intervention (i.e., 5 weeks after CBDL surgery), the CBDL group and the control group were given an equal amount of normal saline. The abdominal cavity of the rats in the sham group was opened, and the common bile duct was separated, and then the abdominal cavity was closed.

[0103] 1.2. The experimental methods for arterial blood gas collection and serum preparation in the experimental group rats are detailed in the first part above.

[0104] 1.3. For details on the experimental methods for tissue sampling, please refer to the first part above.

[0105] 1.4. The experimental method of HE staining of liver and lung tissues is detailed in the first part above.

[0106] 1.5. The experimental method of Masson staining of liver and lung tissues is detailed in the first part above.

[0107] 1.6. The experimental methods for METAVIR scoring of liver fibrosis and pathological scoring of lung injury are detailed in the first part above.

[0108] 1.7 PAS staining of liver and lung tissue sections

[0109] PAS staining (Periodic Acid-Schiffstain) is mainly used in histology to detect glycogen or other polysaccharides in tissues. In the study, it was mainly used to observe the content and distribution of glycogen in liver tissue. The main steps are as follows: (1) The paraffin sections were placed in a constant temperature of 60°C for 45 minutes and immediately immersed in xylene for dewaxing after baking. The specific dewaxing sequence is as follows: 15 minutes in xylene - 15 minutes in xylene - 6 minutes in anhydrous ethanol - 6 minutes in anhydrous ethanol - 5 minutes in 95% ethanol - 5 minutes in 85% ethanol - 5 minutes in 75% ethanol. (1) Wash with ultrapure water for 5 minutes × 3 times; (2) Add peroxy acid solution, oxidize for 5 minutes, and wash with distilled water for 10 minutes; (3) Add Schiff reagent and stain for 10-20 minutes; (4) Pour off Schiff reagent and rinse with running water for 10 minutes; (5) Add hematoxylin staining solution and stain the nucleus for 1-2 minutes, and rinse with running water for 5 minutes; (6) Differentiate with acidic ethanol differentiation solution; (7) Wash with Scott anti-blue water for 3 minutes; (8) Dehydrate with 95% ethanol for 2-3 seconds, dehydrate with anhydrous ethanol 3 times for 5 seconds each, and transparentize with xylene 3 times for 1 minute each, and seal with neutral gum; (9) Take images under a microscope (Olympus BX51, Japan) at 200 times. Select 3 slices from each group, and randomly select 4 fields of view for each slice for subsequent statistical analysis.

[0110] 1.8 Immunohistochemistry of liver and lung tissues

[0111] The experimental method is detailed in the first part above.

[0112] 1.9 Immunofluorescence of liver and lung tissues

[0113] The experimental method is detailed in the first part above.

[0114] 1.10. Enzyme-linked immunosorbent assay (ELISA)

[0115] The experimental method is detailed in the first part above.

[0116] 1.11. Western Blot (Wb)

[0117] (1) Tissue lysis

[0118] 1) Place the tissue separation tools on ice for precooling. The liver and lung specimens for the experiment should also be placed on ice as soon as possible after being taken out of liquid nitrogen to prevent protease hydrolysis;

[0119] 2) Weigh the tissue according to the experimental requirements and put it into a centrifuge tube (add about 200 μl lysis buffer to about 10 mg of tissue), homogenize it with a homogenizer (note that the centrifuge tube should be sealed with sealing glue in advance to prevent the homogenate from bursting during centrifugation), and then centrifuge it at 12,000 rpm for 20 minutes at 4°C. After standing for 5 minutes, aspirate the supernatant with a pipette tip, transfer it to a pre-cooled microcentrifuge tube, and place it on ice again;

[0120] (2) Determination of protein concentration

[0121] 1) Add 10 μl of protein standard to a 96-well plate.

[0122] 2) Add 10 μl of sample to a 96-well plate.

[0123] 3) Add 300 μl of G250 staining solution (detergent compatible) to each well.

[0124] 4) Measure the absorbance at a wavelength of 595 nm using an enzyme-labeled instrument.

[0125] 5) Calculate the sample protein concentration based on the standard curve and sample volume.

[0126] (3) Electrophoresis

[0127] 1) Absorb the lower layer of glue and inject it into the middle of the glass plate to the upper edge of the green plate, and flatten the liquid surface with 75% ethanol;

[0128] 2) Place at room temperature for 20 minutes until a clear straight line is formed between the water and the glue surface, pour out the ethanol, and add the upper layer of glue;

[0129] 3) Insert the comb, place at room temperature for 20 minutes, remove the comb, and place the glass plate into the electrophoresis device;

[0130] 4) Add samples and protein molecular weight markers;

[0131] 5) Keep constant voltage at 80V for about 20min (after the bromophenol blue marker band can be observed to have run out of the upper gel color); adjust the electrophoresis condition to 120V for about 90min;

[0132] 6) Stop electrophoresis when the bromophenol blue marker strip runs to the bottom of the glass plate and the marker strips are separated enough;

[0133] (4) Transfer

[0134] 1) Take the PVDF membrane and soak it in methanol for about 5 minutes to activate it;

[0135] 2) Pry open the glass plate, keep the gel containing the target protein, and immerse it in the transfer solution;

[0136] 3) Place the gel and PVDF membrane in a sandwich pattern and place them in the transfer tank;

[0137] 4) Ice bath, constant current 400mA 50min (adjust the transfer time according to the different molecular weights of the detected proteins)

[0138] (5) Blocking: Place the PVDF membrane in the blocking solution at room temperature for 2 h;

[0139] (6) Primary antibody incubation

[0140] 1) Discard the blocking solution;

[0141] 2) Add primary antibody diluted in TBST and incubate at 4°C overnight;

[0142] 3) Wash the membrane three times with TBST, 10 min each time;

[0143] (7) Secondary antibody incubation:

[0144] 1) Add secondary antibody diluted in blocking solution and incubate at room temperature for 60 minutes;

[0145] 2) Wash with TBST 5 times × 5 min or 3 times × 10 min;

[0146] (8) Color development: Add an appropriate amount of developer solution onto the protein surface of the PVDF membrane for development.

[0147] 2. Statistical analysis

[0148] The present invention uses SPSS software for Windows, V.23.0 (IBM Corp., Armonk, NY, USA) and GraphPad PRISM (version 8.00; GraphPad Software, San Diego, California, USA). Data statistical analysis was performed. According to observation and statistical judgment, if the descriptive data obeys normal distribution, the mean ± standard deviation is used to express; if not, the median and quartile are used to express. If the numerical variables of the test data obey normal distribution, one-way analysis of variance is used, and when the variances between the groups of the data to be processed are homogeneous, the least significant difference method (Least Significance Difference Method, LSD) is required for multiple comparisons, otherwise Dunnett's T3 is used for correction; if the numerical variables do not obey normal distribution, the Kruskal-Wallis H test is used for inter-group comparison. All statistical tests are bilateral, and P<0.05 indicates statistical significance.

[0149] 3. Results

[0150] 3.1. Effects of different Chinese medicine monomers on the improvement of liver and lung function in CBDL rats

[0151] Hypoxemia in the CBDL group was improved after drug treatment. Compared with the sham group, the P(Aa)O2 difference in the CBDL group was increased and PO2 was decreased, and drug treatment improved hypoxemia. (P(Aa)O2: sham vs CBDL: 7.9 mmHg vs 19.1 mmHg, P < 0.001; CBDL vs Baicalein vs Agrimoniin vs Catalpol: 19.1 mmHg vs 11.7 mmHg vs 9.2 mmHg vs 10.4 mmHg, P < 0.001) (see Figure 1A); at the same time, after drug intervention, the liver function of rats improved to varying degrees (ALT: sham vs CBDL: 52 μmol / L vs 350.2 μmol / L, P < 0.001; CBDL vs Baicalein vs Agrimoniin vs Catalpol: 350.2 μmol / L vs 69.8 μmol / L vs 70.5 μmol / L vs 103.5 μmol / L, P < 0.001; GGT: sham vs CBDL: 26.6 μmol / L vs 93.2μmol / L, P<0.001; CBDL vs Baicalein vs Agrimoniin vs Catalpol: 93.2μmol / Lvs 58.7μmol / L vs 46.9μmol / Lvs 27.9μmol / L, P<0.001; TBiL:sham vs CBDL: 3.73μmol / L vs111μmol / L, P<0.001; CBDLvs BaicaleinvsAgrimoniin vs Catalpol: 111μmol / L vs 89.6μmol / L vs48μmol / L vs46.33μmol / L, P<0.001) (see Figure 6 No renal impairment was observed after drug treatment (see Figure 6 D).

[0152] 3.2. Effect of drug therapy on the improvement of liver and lung pathological changes in the CBDL group

[0153] Histological observations of liver and lung showed that compared with the sham group, the liver and lung tissues of CBDL were severely damaged, with obvious liver lobule damage and fibrosis, inflammatory cell infiltration in lung tissue, and edema of alveolar epithelial cells. Masson staining showed that the METAVIR score of liver fibrosis and the area of ​​pulmonary fibrosis in the CBDL group increased (see Figure 6 Compared with the CBDL group, the area of ​​liver fibrosis and METAVIR score were reduced after TCM treatment, and the lung injury score was alleviated (see Figure 6 B, D).

[0154] 3.3 Effect of Chinese medicine treatment on liver and lung fibrosis in the CBDL group

[0155] The expression of fibrotic proteins Collagen-Ⅰ and α-SMA in liver and lung tissues were labeled with fluorescent antibodies to further detect the degree of fibrosis in liver and lung tissues in the CBDL group. Among them, agrimonin could significantly reduce the fibrosis of liver and lung tissues (Fibrosis of liver collagen-Ⅰ: sham vs CBDL: 9.7 vs 120.9, P < 0.0001; CBDL vs Baicalein vs Agrimoniin vs Catalpol: 120.9 vs 35.6 vs 8.5 vs 28.4, P < 0.0001; Fibrosis of lung α-SMA: sham vs CBDL: 8.9 vs 89, P < 0.0001; CBDL vs Baicalein vs Agrimoniin vs Catalpol: 89 vs 14.3 vs 8.8 vs 17.2, P < 0.0001) (see Figure 8 In terms of changing the pathological changes of liver and lung tissues, the improvement effect of agrimony was the most obvious.

[0156] 3.4 Effects of Chinese medicine monomer intervention on pathological angiogenesis in the liver and lungs of CBDL rats

[0157] Compared with the CBDL group, Chinese medicine treatment can reduce the number of CD31 positive cells in liver and lung tissues, and reduce the expression of VEGF in liver and lung tissues. Among them, the expression of VEGF in the agrimonycetin group was the lowest (see Fig. 9 Western blot experiments also showed that compared with the CBDL group, the expression of CD31 and VEGF in the liver and lung tissues of the agrimony-treated group was the lowest (see Fig. 9 C). Therefore, compared with the improvement of CBDL liver and lung function, tissue pathological changes and reduction of pathological angiogenesis, among the three Chinese medicine monomers, Herba Agrimoniae has greater potential in the treatment of HPS.

[0158] In summary, agrimony has an inhibitory effect on angiogenesis pathway-related proteins and can reduce NO release by inhibiting iNOS activity. Its drug target is highly consistent with the therapeutic target of HPS; and agrimony has the effect of regulating glucose metabolism and repairing mitochondrial damage. The therapeutic effects of baicalin, catalpol and agrimony on the liver and kidney function, blood gas analysis, improvement of liver and lung tissue pathological structure, fibrosis and reduction of angiogenesis in HPS animal models were compared, and it was found that agrimony had a better therapeutic effect than baicalin and catalpol; and baicalin, agrimony and catalpol could significantly reduce P(Aa)O2 and increase PO2 in HPS animal models after treatment, thereby improving HPS animal hypoxemia; by comparing the effects of baicalin, agrimony and catalpol on the liver and kidney function, blood gas analysis, improvement of liver and lung tissue pathological structure, fibrosis and reduction of angiogenesis in HPS animal models, agrimony had a better therapeutic effect than baicalin and catalpol.

[0159] 3. Study on the effect and mechanism of Agrimoniae in reducing lung pathological angiogenesis in HPS rats

[0160] 1. Methods

[0161] 1.1. Rats grouping and agrimony extract intervention regimen

[0162] The high-dose (8 mg / kg / d, AH) and low-dose (3 mg / kg / d, AL) groups of agrimony were treated with intraperitoneal injection of agrimony for 3 weeks starting 2 weeks after M-UCBDL. PMVECs were set up in 3 observation groups: agrimony group (concentrations were 100 Um, 200 Um, and 300 Um, respectively), the most suitable concentration was selected according to the experimental results, and siRNA was further used to reduce PGC-1α expression.

[0163] 1.2 Rat tissue collection

[0164] The experimental methods are detailed above.

[0165] 1.3. HE staining and Masson staining of rat tissue

[0166] The experimental methods are detailed above.

[0167] 1.4. Immunohistochemistry and immunofluorescence of rat tissues

[0168] The experimental methods are detailed in the above content.

[0169] 1.5. Enzyme-linked immunosorbent assay (ELISA)

[0170] The experimental methods are detailed above.

[0171] 1.6. PAS staining of liver and lung tissue sections

[0172] The experimental methods are detailed above.

[0173] 1.7. HE staining and Masson staining of rat tissue

[0174] The experimental methods are detailed above.

[0175] 1.8. Immunohistochemistry and immunofluorescence of rat tissues

[0176] The experimental methods are detailed above.

[0177] 1.9. Enzyme-linked immunosorbent assay (ELISA)

[0178] The experimental methods are detailed above.

[0179] 1.10 PAS staining of liver and lung tissue sections

[0180] The experimental methods are detailed above.

[0181] 1.11. Cell recovery (1) Store in liquid nitrogen 1 mL (1×10 5 ) of SPD rat PMVECs. Take them out of liquid nitrogen and prepare for the recovery experiment. Place the taken PMVECs in a 37°C water bath as soon as possible to wait for thawing; (2) Gently shake the cryovial during the thawing process to allow it to be evenly heated and thawed. After observing that it is completely thawed, wipe the cryovial with 75% alcohol and place it in a sterile operating table in a sterile manner; (3) Gently blow the cells in the cryovial with a pipette, aspirate the cell suspension and add it to a 10mL EP tube containing 5mL endothelial cell complete culture medium. Gently blow again to mix, and then centrifuge at room temperature at 1000rpm for 5 minutes; (4) After centrifugation, carefully pour out the supernatant, add 3mL endothelial cell complete culture medium to the EP tube again, gently blow with a pipette to resuspend the cells, and inoculate them in 25cm 2 The culture flask was placed in a cell culture flask, and after the culture flask was marked, it was placed in a cell culture incubator for static culture (culture conditions: 37°C, 5% CO2); (5) After 8 hours, the cells were observed, and the non-adherent cells were carefully aspirated with a pipette, and 3 mL of fresh endothelial cell complete culture medium was added again and the static culture was continued.

[0182] 1.12 Cell culture and passaging

[0183] PMVECs were placed under a microscope to observe their adherent growth. The culture medium of PMVECs needs to be changed every two days. When the cells grow to about 70% to 80% of the culture medium, the cells need to be subcultured (1) Use a clean bench according to the principle of sterility, suck out the old culture medium liquid with a pipette, wash the cells twice with sterile PBS, and gently shake the culture medium after each wash; (2) After washing, add 1mL of 0.25% trypsin to the culture medium and let it act on the cells in the culture medium for about 1.5-2min (observe that the cells have been digested as much as possible), then add 2ml of culture medium to terminate digestion; (3) Centrifuge the cells again at room temperature, 1000rpm, 5min; (4) After the cells are centrifuged, pour out the supernatant, subculture the cells at a ratio of 1:2, and then place them in a cell culture incubator for static culture.

[0184] 1.13. siRNA transfection of PMVECs

[0185] (1) Observe the cells grow to 70–80% of the inoculated area before starting the transfection experiment; (2) Use Opti-MEM TM Dilute Lipofectamine in culture medium TM 3000 reagent - shake gently to mix thoroughly; (3) use Opti-MEM TM medium to prepare the siRNA premix, then add P3000 TM Reagents: Mix thoroughly; (4) Incubate for 10-15 minutes; (5) Replace the culture medium without antibiotics and add siRNA-lipid complexes to the cells; After 24 hours, check the RNA level to see if the transfection is successful, and select three candidate sequences.

[0186] 1.14 Cell scratch test

[0187] (1) Marking the culture plate: Use a marker to draw lines on the back of the 6-well plate (note that the lines should not be too thick), and the spacing between the drawn parallel marking lines is 0.5-1cm; (2) Cell plating: Digest the cells with trypsin and prepare a cell suspension. After counting the cells, plate them to ensure that the density of each group of cells is consistent. Generally, about 5-10×105 cells are inoculated in each well; (3) Make scratches: After the cells are fully spread, use a 20ul pipette tip to make cell scratches so that the scratches intersect with the marking lines; (4) Wash the cells twice with PBS, and use the pipette tip to remove the scratched cells according to the markings. After the pipette tip scratches, replace the culture medium and continue to culture the cells in a 37℃, 5% CO2 incubator; (5) Cell culture and timed photography observation.

[0188] 1.15. Cell Tubule Formation Assay

[0189] (1) Preparation of endothelial cells

[0190] 1) Two days before the test, cells were passaged and cultured for 24 hours. 2) One day before the experiment, the medium was replaced with serum-free medium to starve the cells. 3) The day before use, the matrix gel was thawed overnight in ice at 4°C refrigerator, added to a 24-well plate after thawing, and stored at 4°C after centrifugation. 4) All tools, consumables and solutions in contact with the matrix gel were pre-cooled in advance, and the matrix gel was kept on ice throughout the experiment.

[0191] (2) Experimental operation

[0192] 1) Wash the cells with PBS, add 0.25% trypsin digestion solution to digest the cells, and count the cells. 2) Incubate the 24-well plate at 37°C and 5% CO2 for 30 minutes to solidify the matrix gel. 3) Add the cells to the 24-well plate according to the predetermined concentration and add them to the 48-well plate, and use an inverted microscope with a bright field of 100 times to take images of the tubular network.

[0193] 1.16. q-PCR

[0194] (1) After taking out the PMVEC from the incubator, aspirate the culture medium in the well plate, slowly add 2 ml of sterile PBS along the side wall of each well to wash the cells, and wash twice in total. (2) Add 1 ml of Trizol reagent to each well, and blow the cells to make the cells fall off the bottom of the well plate. Transfer the cell suspension to an enzyme-free 1.5 ml centrifuge tube, add 200 ul of chloroform to each well, vortex at the highest speed for 15 s, and let it stand for 3 min. (3) Place the 1.5 ml centrifuge tube in a 4°C precooled centrifuge and centrifuge at 13000g for 10 min. At this time, the RNA is located in the colorless liquid on the top. (4) Pipette 480 ul of supernatant from each well into a new 1.5 ml centrifuge tube, add 500 ul of isopropanol to each well, shake up and down, and let it stand at room temperature for 10 min. (5) Place the 1.5 ml centrifuge tube in a 4°C precooled centrifuge and centrifuge at 13000g for 10 min. At this time, the RNA is located in the precipitate. (6) Discard the supernatant and add 1 ml Wash the RNA precipitate by inverting the tube with 75% ethanol diluted with DEPC water (7) Place the 1.5 ml centrifuge tube in a centrifuge precooled at 4°C, centrifuge at 7500 rpm for 5 min, and discard the supernatant (8) Place the 1.5 ml centrifuge tube in a centrifuge precooled at 4°C, centrifuge at 7500 rpm for 5 min again, and discard the supernatant (9) Let the RNA precipitate dry at room temperature until it becomes translucent, and add an appropriate amount of DEPC water to dissolve the RNA (10) After NanoDrop3000 is calibrated, take 1 ul from each sample and place it in the measuring well to measure the RNA concentration, and uniformly dilute the RNA concentration of each sample to 500 ng / ul (11) Reverse transcription reaction: Thaw the RNA on ice and thaw the reverse transcription kit at room temperature (about 23°C); (12) Prepare the reaction mixture on ice according to the following ingredients: 5×gDNA Eraser Buffer 2ul, gDNA Eraser 1ul, Total RNA 2ul, RNaseFree dH2O 5ul, incubate at 42℃ for 2min, 4℃ for 3min on a PCR instrument. (13) Prepare the reaction solution on ice: 10ul of the reaction solution in step (12), 1ul of PrimeScript RT Enzyme Mix I, 1ul of RT Primer Mix*4, 4ul of 5×PrimeScript Buffer2 (for Real Time), 4ul of RNase Free dH2O, incubate at 37℃ for 15min, 85℃ for 5s, and 4℃ for 3min on a PCR instrument; (14) Set up the internal reference group and target gene group: add 5ul of TB Green, 0.2ul of internal reference / target gene primer F / R, 1ul of cDNA, and 3.6ul of ddH2O to each well. Set up 3 wells for internal reference and 5 wells for target gene. Set the PCR instrument at 95℃ for 5min, 95℃ for 20s, 55℃ for 20s, and 72℃ for 20s. Cycle 10 times.

[0195] result:

[0196] 1. Effects of different doses of agrimony on the ratio of pyruvate / lactate in liver and lung tissues

[0197] The increased expression of pyruvate, a metabolite of tissue glycolysis, proves that the glycolytic flux of the tissue has increased. Lactate dehydrogenase (LDH) is the key rate-limiting enzyme of glycolysis. Therefore, we measured the glycolytic flux of liver and lung tissues by evaluating the pyruvate / lactate ratio and LDH content in liver and lung tissues. The study found that compared with the control, M-UCBDL led to an increase in the pyruvate / lactate ratio in liver and lung tissues and an increase in the expression of LDH; the pyruvate / lactate ratio was significantly reduced after treatment with scutellaria baicalensis, and the reduction effect of the AH group was more significant (see Fig.10 A). Immunohistochemistry and ELISA were used to evaluate the expression of glycolytic marker protein PFKFB3 (see Fig.10 BC in the figure), and the expression of PFKFB3 was localized by laser confocal microscopy (see Fig.10 The experimental results showed that the ectopic expression of PFKFB3 in the liver and lung of CBDL increased, while the expression of PFKFB3 in the cytoplasm decreased after treatment with agrimony, and the reduction effect was more significant in the AH group.

[0198] 2. Agrimonia extract improves mitochondrial damage and decreased mitochondrial function caused by HPS

[0199] Metabolic changes caused by mitochondrial dysfunction are one of the initiating factors for the occurrence and development of HPS. mtDNA levels, mtDNA damage, and the transmembrane potential generated by the mitochondrial electron transport chain are key to maintaining mitochondrial function. The ND1 / β2M ratio of cellular DNA in liver and lung mitochondrial mtDNA was measured by q-PCR to evaluate changes in the expression of mtDNA levels. The ratio of long-chain mitochondrial DNA / short-chain mitochondrial DNA (D-loop) in liver and lung tissues was measured by q-PCR to evaluate changes in mitochondrial damage. The study found that compared with the control group, the mtDNA levels in the liver and lung tissues of the M-UCBDL group decreased, and the increased long-chain DNA damage of mtDNA led to a decrease in the mtDNA long / short ratio. The mtDNA level increased after treatment with centaury extract, and the increase in the mtDNA long / short ratio improved the damage of mtDNA, and the improvement effect of the AH group was better than that of the AL group (see Fig.11A in the figure). At the same time, the expression changes of mitochondrial respiratory chain complex IV (Mitochondriacomplex-IV, Cox-Ⅳ) and mitochondrial membrane anti-proliferation protein (Prohibitin1, PHB1) were evaluated by immunohistochemistry. Compared with the control group, the expression of Cox-Ⅳ and PHB1 in the CBDL group was reduced. The expression of respiratory chain PHB1 and Cox-Ⅳ can be enhanced after treatment with agrimony. The AH group has a better effect on promoting the expression of Cox-Ⅳ and PHB1 than the AL group. The study showed that agrimony increased the function of the mitochondrial respiratory chain (see Fig.11 BC in.

[0200] 3. Agrimonia enhances mitochondrial biogenesis by promoting mitochondrial fusion function

[0201] Mitochondrial functional homeostasis and mtDNA levels are two key factors that promote mitochondrial biogenesis. Immunohistochemistry and Elisa were used to measure the expression changes of PGC-1α, a marker of mitochondrial biogenesis. Laser confocal microscopy was used to evaluate the expression changes of mitochondrial fission protein DRP1 and fusion protein MFN1. The study showed that compared with the control group, the expression of PGC-1α in M-UCBDL decreased. After intervention with different doses of AL and AH, the expression of PGC-1α in liver and lung tissues increased, and the increase effect of AH was better than that of AL (see Fig.12 AC in ); M-UCBDL reduced the expression of PGC-1α and MFN1, and enhanced the expression of DRP1, leading to increased mitochondrial fission function (see Fig.12 FG in the figure). Analysis of the agrimonycetin treatment group found that agrimonycetin could promote the increase of MFN1 and PGC-1α levels and inhibit the expression of DRP1, and the enhancement effect of the AH group was better than that of the AL group. The study confirmed that agrimonycetin promotes mitochondrial biogenesis by increasing mitochondrial fusion function and PGC-1α levels.

[0202] 4. Agrimonia promotes mitochondrial biogenesis by stimulating PGC-1α

[0203] Under conditions where OXPHOS energy supply is decreased due to mitochondrial damage and dysfunction, the glycolysis pathway outside the mitochondria becomes the main source of ATP for cells and tissues, and this shift in energy metabolism promotes pathological angiogenesis. Through the above studies, we have confirmed that agrimonycetin can reduce HPS angiogenesis, increase mitochondrial biogenesis capacity and restore mitochondrial function by increasing mtDNA levels and improving mtDNA damage, promoting PGC-1α expression. To further verify our research, we constructed siRNA to transfect PMVECs to reduce the expression of PGC-1α. Western blotting experiments verified that after successful transfection of PGC-1α siRNA, the PGC-1α protein of PMVECs was inhibited (see Fig.13 BA in it). The experiment showed that compared with the PGC-1α expression inhibition group (CBDL+siRNA; CBDL+AGR+siRNA), the angiogenesis inhibition effect of the CBDL+AGR group was significant. Tubule formation experimental analysis: Compared with the control group, the CBDL+siRNA; CBDL+AGR+siRNA groups had a significant increase in the number of nodes formed, vascular branches and total vascular length, while the number of nodes, vascular branches and total vascular length of the CBDL+AGR group were significantly reduced. This shows that agrimony inhibits angiogenesis by regulating PGC-1α (see Fig.13 B). The scratch test showed that the migration area of ​​PMVECs cells in the CBDL+siRNA and CBDL+AGR+siRNA groups was significantly larger than that in the CBDL+AGR group at 24 h, indicating that agrimony has a significant inhibitory effect on cell migration (see Fig.13 The pyruvate / lactate ratio, glycolysis marker protein LDH and protein expression of TCA key rate-limiting enzyme citrate synthase (CS) of PMVECs were measured by Elisa: CBDL+siRNA and CBDL+AGR+siRNA showed an increase in pyruvate / lactate ratio and LDH expression, and a decrease in CS expression, while the CBDL+AGR group showed a decrease in pyruvate / lactate ratio and LDH expression, and a decrease in CS expression (see Fig.13 Agrimoniae can promote the expression of PGC-1α and promote mitochondrial biogenesis, thereby reducing HPS angiogenesis.

[0204] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of agrimonycin in the preparation of drugs for treating pathological pulmonary angiogenesis in hepatopulmonary syndrome.

2. The use according to claim 1, characterized in that: The agrimonycetin is used to regulate mitochondrial fusion and fission homeostasis, promote mitochondrial fusion to increase mtDNA levels, and improve OXPHOS function; the agrimonycetin is used to repair mtDNA damage and promote mitochondrial biogenesis, increase tissue OXPHOS / glycolysis ratio, restore normal energy metabolism of tissues, and reduce pathological angiogenesis in HPS liver and lung tissues.

3. Application of PGC1-α as a target for the treatment of liver cirrhosis with Herba Agrimoniae.

4. The use according to claim 3, characterized in that: Agrimoniae is used to promote PGC-1α expression, promote mitochondrial biogenesis function, increase PMVECs mitochondrial OXPHOS capacity, increase tissue OXPHOS / glycolysis ratio, and reduce HPS angiogenesis.

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