Application of corydalis saxicola in preparation of medicine for preventing and treating liver microcirculation disturbance or liver injury caused by lipopolysaccharide

Abstract

The invention discloses application of corydalis saxicola in preparation of a medicine for preventing and treating liver microcirculation disturbance or liver injury caused by lipopolysaccharide. When lipopolysaccharide induction is carried out or 6 hours after lipopolysaccharide induction is carried out; corydalis saxicola bunting injection with corydalis saxicola bunting alkali as active ingredient for intravenous or intramuscular injection, which can reduce the number of leukocytes rolling and adhering to the central vein region of the liver, the level of serum alanine transaminase (ALT) and aspartate transaminase (AST), the necrosis area of the liver tissue, the tumor necrosis factor-alpha (TNF-alpha) of the liver tissue and interferon-gamma (IFN-gamma) of the liver tissue, wherein the number of leukocytes rolling and adhering to the central vein region of the liver is increased due to lipopolysaccharide increase. And content.

Description

Technical Field

[0001] The present invention relates to liver prevention and treatment drugs, and in particular to the use of fusinine in the preparation of drugs for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide, belonging to the field of medical technology. Background Art

[0002] Acute liver injury (ALI) is a common disease that seriously threatens the health of patients. A large number of liver cells die in a short period of time, resulting in a sharp decline in liver function.

[0003] Lipopolysaccharide (LPS), also known as endotoxin, is a component of the cell wall of Gram-negative bacteria and can induce acute liver injury.

[0004] Corydalis saxicolabunting injection (CSBI) is a traditional Chinese medicine prepared from Corydalis saxicolabunting extract, which is mainly used in the clinic to treat acute and chronic hepatitis, cirrhosis, liver cancer, etc. However, it is still unclear whether Corydalis saxicolabunting injection can improve liver microcirculation disorders and liver damage caused by lipopolysaccharide (endotoxin) and its potential mechanism. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide the use of coptisine in the preparation of drugs for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] 1. Application of coptis chinensis alkaloids in the preparation of drugs for the prevention and treatment of liver microcirculation disorders or liver damage caused by lipopolysaccharide.

[0008] As one of the preferred technical solutions, fuscin reduces liver damage through the following mechanisms:

[0009] (A) Reduce the accumulation of ferrous ions in the liver caused by LPS;

[0010] (B) Reduce lipopolysaccharide-induced liver lipid peroxidation;

[0011] (C) Alleviate the liver redox imbalance caused by LPS;

[0012] (D) Reduced lipopolysaccharide-induced hepatic inflammatory cell infiltration.

[0013] As one of the preferred technical solutions, coptis chinensis reduces the number of leukocytes rolling and adhering in the central vein area of ​​the liver.

[0014] As one of the preferred technical solutions, coptisine reduces the area of ​​liver necrosis.

[0015] As one of the preferred technical solutions, coptisine reduces the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum.

[0016] As one of the preferred technical solutions, fuscin reduces the content of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in liver tissue.

[0017] As one of the preferred technical solutions, coptisine reduces the ferrous ion content in liver tissue, alleviates the level of liver lipid peroxidation, and thus alleviates liver damage.

[0018] 2. A pharmaceutical composition for preventing and treating hepatic microcirculation disorders or liver damage caused by lipopolysaccharide, comprising coptisine.

[0019] 3. Use of a pharmaceutical composition containing coptisine in the preparation of drugs for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide.

[0020] 4. A drug for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide, comprising the aforementioned pharmaceutical composition.

[0021] 5. A drug for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide, wherein the active ingredient is coptisine.

[0022] Beneficial effects of the present invention:

[0023] The invention discloses the use of coptis chinensis in the preparation of drugs for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide (endotoxin, LPS). Intravenous or intramuscular injection of coptis chinensis injection containing coptis chinensis as an effective ingredient at the same time as or 6 hours after lipopolysaccharide induction can reduce the number of leukocytes rolling and adhering in the central vein area of ​​the liver increased by lipopolysaccharide, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the area of ​​liver tissue necrosis, and the contents of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in liver tissue.

[0024] The specific mechanism of the present invention is that cyperine reduces the accumulation of ferrous ions and lipid peroxidation levels in liver tissue caused by lipopolysaccharide, and ultimately alleviates liver damage mediated by ferroptosis. Cell experiments further confirmed that cyperine inhibits hepatocyte ferroptosis induced by inflammatory cytokines TNF-α and IFN-γ. Therefore, cyperine inhibits hepatocyte ferroptosis promoted by inflammatory cytokines in the liver, thereby playing a therapeutic and preventive role in liver damage caused by lipopolysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The results show that after LPS modeling, 1.68 ml / kg of Rhizoma Coptidis injection was injected intramuscularly to observe its therapeutic effect on liver microcirculation disorders. Rhodamine 6G was used to label leukocytes and observe the rolling and adhesion of leukocytes in liver microvessels in vivo. *P<0.05 vs. control group; #P<0.05 vs. LPS alone treatment group; nsP>0.05 vs. control group; n=3.

[0026] Figure 2 The results of intramuscular injection of 1.68 ml / kg Coptis chinensis injection after lipopolysaccharide modeling; A shows the representative pictures of HE staining and TUNEL staining of the liver tissue of rats in each group after intramuscular injection of 1.68 ml / kg Coptis chinensis injection; B shows the statistical chart of HE inflammation score and the number of TUNEL positive cells; C shows the statistical chart of AST and ALT content in the serum of rats in each group; D shows the statistical chart of TNF-α and IFN-γ content in the liver tissue of rats in each group. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=3-12.

[0027] Figure 3 The results of intramuscular injection of 1.68 ml / kg Rhizoma Coptidis injection after lipopolysaccharide modeling; A shows the content of ferrous ions in the liver tissue of rats in each group; B shows the protein expression of TF and TFR, which mediate iron transfer, in the liver tissue of rats in each group. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=3 / 4.

[0028] Figure 4 The results of intramuscular injection of 1.68 ml / kg Rhizoma Coptidis injection after lipopolysaccharide modeling; A shows the content of lipid peroxidation product LPO in the liver tissue of rats in each group; B shows the content of lipid peroxidation product MDA in the liver tissue of rats in each group. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=4 / 5.

[0029] Figure 5 The results of intramuscular injection of 1.68 ml / kg Rhizoma Coptidis injection after lipopolysaccharide modeling; A shows the fluorescence expression of 8-OHdG, a marker of oxidative stress, in the liver tissue of rats in each group; B shows the redox state in the liver tissue of rats in each group, green is the oxidized state, and red is the reduced state. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=3.

[0030] Figure 6The results of intramuscular injection of 1.68 ml / kg Rhizoma Coptidis injection after lipopolysaccharide modeling; A shows the changes in the content of GSH / GSSG in the liver tissue of rats in each group; B shows the changes in the content of GSH in the liver tissue of rats in each group. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=3-5.

[0031] Figure 7 The results of intramuscular injection of 1.68 ml / kg Rhizoma Coptidis injection after lipopolysaccharide modeling; A shows the infiltration of MPO-positive cells and CD68-positive cells in the liver tissue of rats in each group; B shows the protein expression of ICAM1, a molecule that mediates leukocyte adhesion, in the liver tissue of rats in each group. *P<0.05 vs. control group; #P<0.05 vs. lipopolysaccharide alone treatment group; nsP>0.05 vs. control group; n=3.

[0032] Figure 8 The results of the observation of the preventive effect of 0.84 ml / kg Rhizoma Corydalis injection on liver microcirculation disorder and liver injury by tail vein injection of lipopolysaccharide modeling at the same time; A shows the rolling and adhesion of leukocytes in liver microvessels; B shows the representative HE staining of liver tissues of rats in each group after tail vein injection of Rhizoma Corydalis injection and the corresponding inflammatory scores. *P<0.05vs. control group; #P<0.05vs. lipopolysaccharide alone treatment group; nsP>0.05vs. control group; n=3 / 4.

[0033] Fig. 9 It shows that cyperine can reduce the ferroptosis of Hepg2 cells caused by combined treatment with cytokines; A shows the immunofluorescence co-staining of ferroptosis marker protein TF and hepatocyte marker protein Alb; B shows the analysis of blood components of cyperine injection; C shows the cell death of each group after cyperine and cytokine treatment of Hepg2 cells; D shows the accumulation of ferrous ions in each group of cells; E shows the lipid peroxidation of each group of cells. *P<0.05vs. control group; #P<0.05vs.TNF-α+IFN-γ combined treatment group; nsP>0.05vs. control group; n=3. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0035] The present invention uses the marketed product Coptis chinensis injection whose active ingredient is Coptis chinensis alkaloids to carry out relevant experiments.

[0036] Embodiment 1:

[0037] 1.68ml / kg of Coptis chinensis injection was injected intramuscularly 6 hours after lipopolysaccharide induction. Liver microcirculation was observed and samples were taken 24 hours after lipopolysaccharide induction to evaluate the therapeutic effect of Coptis chinensis injection on liver microcirculation disorders and liver damage caused by lipopolysaccharide (endotoxin). 0.84ml / kg of Coptis chinensis injection was injected intravenously at the same time as lipopolysaccharide induction. Liver microcirculation was observed and samples were taken 24 hours after lipopolysaccharide induction to evaluate the preventive effect of Coptis chinensis injection on liver microcirculation disorders and liver damage caused by lipopolysaccharide (endotoxin).

[0038] Experimental data show that Coptis chinensis injection can reduce the number of white blood cells rolling and adhering in the central vein area of ​​the liver. Coptis chinensis injection can reduce the area of ​​liver necrosis, reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and reduce the levels of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in liver tissue. Coptis chinensis injection can reduce the content of ferrous ions in liver tissue and reduce the level of liver lipid peroxidation, thereby reducing liver damage. This study shows that Coptis chinensis injection can play a therapeutic and preventive role in liver damage caused by lipopolysaccharide.

[0039] 1 Materials and Methods

[0040] 1.1 Main Reagents

[0041] Yanhuanglian injection was provided by Guangxi Hefeng Pharmaceutical Co., Ltd.; tissue lysis buffer RIPA: CST, USA; PBS buffer powder: Zhongshan Jinqiao, China; anti-rabbit TF, TFR: Abmart, China; anti-rabbit GPX4 antibody: Abcam, USA; anti-rabbit 8-OHdG antibody: Beijing Biosen Biotechnology Co., Ltd., China; BODIPY TM 581 / 591C11 probe: Thermo Fisher Scientific, USA; horseradish enzyme-labeled goat anti-rabbit IgG (H+L): Zhongshan Jinqiao, China. BCA protein quantification kit: Pierce, USA; LPO, MDA, GSH, GSSG content detection kit: Beijing Solaibao Technology Co., Ltd., China; primers: Beijing Qingke Biotechnology Co., Ltd.

[0042] 1.2 Animal and model establishment

[0043] Clean grade SD male rats, weighing 200±20g, were ordered from the Experimental Animal Center of Peking University Health Science Center. The animal was kept at a temperature of 23±2℃, a relative humidity of 45±5%, 12-hour light / dark alternation, and free access to food and water. The animals were fasted for 12 hours before the experiment and had free access to water. The experimental operation procedures were carried out in accordance with the guidelines of the Peking University Animal Research Committee.

[0044] Post-drug administration (treatment): Induce anesthesia in rats with isoflurane, then inject 10 mg / kg lipopolysaccharide into the tail vein (the control group rats were injected with an equal amount of normal saline into the tail vein), and 6 hours after modeling, inject 1.68 ml / kg of Coptis chinensis injection into the lateral thigh muscles of the rats for treatment (the control group rats were injected with an equal amount of normal saline into the lateral thigh muscles). 24 hours after modeling (i.e. 18 hours of treatment), observe the microcirculation and collect samples, including rat serum and liver tissue.

[0045] Simultaneous administration (prevention): Rats were anesthetized by isoflurane, and then 10 mg / kg lipopolysaccharide was injected into the tail vein, and 0.84 ml / kg of Coptis chinensis injection was injected into the tail vein for treatment (the control group rats were injected with an equal amount of normal saline into the tail vein). Microcirculation was observed and samples were collected 24 hours after modeling, including rat serum and liver tissue.

[0046] 1.3 Analysis of blood components of Coptis chinensis injection

[0047] 1.68ml / kg of Rhizoma Coptis injection was injected into the lateral thigh muscles of rats. Blood was collected from the inner canthus of the rats at 0h, 0.5h, 1h, 2h, and 4h after intramuscular injection. Serum was collected by centrifugation and sent for testing. Mass spectrometry data was searched in the Thermo Compound Discovery database. Mass spectrometry identification was performed using the MetFrag reference retention time and intensity. Blood components were screened according to the standard of error / ppm<5.

[0048] 1.4 Paraffin sections and HE staining

[0049] Tissue fixation: Fresh liver tissue was washed with PBS (10 mM, pH = 7.4), cut into pieces and immediately placed in 4% paraformaldehyde and fixed overnight in a 4°C refrigerator;

[0050] Dehydration and transparency: After the tissue blocks were trimmed again, they were placed in 70%, 80%, 90%, 95%, 100%, and 100% ethanol aqueous solutions for gradient dehydration, respectively, for 2 hours, 2 hours, 1 hour, 1 hour, 30 minutes, and 30 minutes, and then transparentized in xylene twice, each time for 15 minutes;

[0051] Wax immersion: Place the tissue block in pre-melted wax for 3 hours;

[0052] After embedding the wax blocks, the sections were sliced ​​on a microtome and placed in an incubator at 37°C overnight.

[0053] Sectioning: Slice the embedded lung tissue into 5 μm thick slices on a paraffin slicer, adhere it to a glass slide, and store it at room temperature;

[0054] Wax melting: put the slices into a 60℃ oven for 1 hour;

[0055] Xylene I 8 min, xylene II 8 min, anhydrous ethanol I 4 min, anhydrous ethanol II 4 min, 95% ethanol aqueous solution 2 min, 80% ethanol aqueous solution 2 min;

[0056] Stain with hematoxylin for 2-3 min, wash with tap water and observe the nuclei under a microscope;

[0057] 1% hydrochloric acid alcohol differentiates for a few seconds;

[0058] Washing with tap water turns blue;

[0059] Eosin staining for 3-10 seconds;

[0060] Rinse the back with tap water;

[0061] 80% ethanol aqueous solution for a few seconds, 90% ethanol aqueous solution for 2 minutes, 95% ethanol aqueous solution for 2 minutes, 100% ethanol I for 4 minutes, 100% ethanol II for 4 minutes, xylene I for 8 minutes, xylene II for 8 minutes;

[0062] Seal the slides with neutral gum.

[0063] 1.5 Immunohistochemical staining

[0064] Waxing: Place the slices in a 60°C oven for 1 hour;

[0065] Xylene I 8 min, xylene II 8 min, anhydrous ethanol I 4 min, anhydrous ethanol II 4 min, 95% ethanol 2 min, 80% ethanol 2 min;

[0066] Sodium citrate antigen repair for 10 minutes, then cool at room temperature;

[0067] Wash with PBS three times, 3 min each time;

[0068] The membrane was permeabilized with 0.3% Triton and incubated at 37°C for 30 min;

[0069] Wash with PBS three times, 3 min each time;

[0070] Incubate with 3% hydrogen peroxide aqueous solution at room temperature for 10 min to eliminate endogenous peroxidase activity;

[0071] Wash with distilled water and soak in PBS for 5 min;

[0072] Block with serum consistent with the source of the secondary antibody and incubate at room temperature for 30 min;

[0073] Pour off the serum, do not wash, add primary antibody and keep in 4℃ refrigerator overnight, take out the slide the next day;

[0074] Wash with PBS three times, 3 min each time;

[0075] Add reaction enhancement solution and incubate at 37°C for 20 min;

[0076] Wash with PBS three times, 3 min each time;

[0077] Add enhanced enzyme-labeled goat anti-rabbit / mouse IgG polymer dropwise and incubate at 37°C for 20 min;

[0078] Wash with PBS three times, 3 min each time;

[0079] DAB was used for color development and incubated at room temperature for 5 to 8 minutes.

[0080] Rinse with tap water, incubate in hematoxylin staining solution for 20 seconds; differentiate, rinse and turn blue.

[0081] The steps of dehydration, transparency and sealing are the same as above.

[0082] 1.6 Immunofluorescence staining

[0083] Wax melting: put the slices into a 60℃ oven for 1 hour;

[0084] Xylene I 8 min, xylene II 8 min, anhydrous ethanol I 4 min, anhydrous ethanol II 4 min, 95% ethanol 2 min, 80% ethanol 2 min;

[0085] Sodium citrate antigen repair for 10 minutes, then cool at room temperature;

[0086] Wash with PBS three times, 3 min each time;

[0087] The membrane was permeabilized with 0.3% Triton and incubated at 37°C for 30 min;

[0088] Wash with PBS three times, 3 min each time;

[0089] Block with serum consistent with the source of the secondary antibody and incubate at room temperature for 30 min;

[0090] Pour off the serum, do not wash, add primary antibody and keep in 4℃ refrigerator overnight, take out the slide the next day;

[0091] Wash with PBS three times, 3 min each time;

[0092] Add secondary antibody and incubate at room temperature for 2 hours;

[0093] Wash with PBS three times, 3 min each time;

[0094] Seal the sections with anti-fade mounting medium containing DAPI;

[0095] Photography: Take pictures under SP8 or STED fluorescence confocal microscope.

[0096] 1.7 Observation of liver microcirculation

[0097] The rolling and adhesion of leukocytes in liver microvessels were analyzed using an in vivo microcirculation device. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (3 mg / ml, 30 mg / kg, SigmaAldrich, MO, USA) according to their body weight. After anesthesia, the rats were placed on an observation board in the lateral position. The liver was placed in a heat-controlled (37°C) observation box and carefully handled to minimize the influence of respiratory movement. The left lateral lobe of the liver was observed with the assistance of an inverted intravital microscope (TE2000-E, Nikon, Tokyo, Japan). Small veins with a diameter of 20-45 μm, a length of 200 μm, and no adherent leukocytes were selected for observation. A DVD recorder (DVR-R25, Malata, Xiamen, China) was used to record the dynamic microcirculation of the liver. To evaluate the rolling and adhesion of leukocytes, 0.4 mL of the fluorescent tracer rhodamine 6G (0.5 mg / mL, Sigma-Aldrich, St. Louis, MO, USA, cat. no.: 83697) was administered via the left femoral vein to label leukocytes in vivo. Venules were observed under irradiation at a wavelength of 543 nm. Leukocytes adhering to the vessel wall for more than 10 seconds were considered stable adherent leukocytes. Leukocytes that stayed in the same position for less than 10 seconds were considered rolling leukocytes.

[0098] 1.8 Western Blot

[0099] (1) Sampling: After the rats were killed, the liver tissues were quickly removed and placed in cryotubes. The cryotubes were placed in a liquid nitrogen tank and then transferred to a -80°C refrigerator for storage.

[0100] (2) Protein quantification: Take out the tissue from the -80℃ freezer, thaw it on ice, cut the tissue into small pieces and weigh it (preferably 100 mg), and put it into a centrifuge tube. Add RIPA lysis buffer (1 mL / 100 mg) according to the weight of the tissue, and add protease inhibitors to RIPA. Ultrasonic homogenize the tissue in the centrifuge tube until the protein solution becomes transparent. Centrifuge the protein lysate at 4℃ for 55 min, 13,000 rpm, and take the supernatant. Dilute the sample with pure water and take 10 μl for quantification. Dilute the protein standard in multiples to prepare a standard curve. Detect the protein concentration of the sample to be tested according to the instructions of the BCA protein quantification kit (Pulile, Beijing, China). After the quantification, add the loading buffer to the protein lysate and boil the centrifuge tube at 100℃ for 10 min. Divide the boiled protein sample into aliquots and store it in a -80℃ refrigerator for testing.

[0101] (3) Gel preparation: The preparation method of the 10% one-step PAGE gel preparation kit is as follows:

[0102] i. Lower gel: add 4 ml of solution and buffer respectively, and add 80 μl of improved coagulant;

[0103] ii. Upper gel: add 1 ml of solution and buffer solution respectively, and add 20 μl of improved coagulant;

[0104] After injecting the prepared lower layer of glue into the glass plate, you can add the upper layer of glue without waiting for it to solidify. After waiting for about 15 minutes, it can be solidified and used.

[0105] (4) Electrophoresis: Fix the gel plate on the electrophoresis stand and fill the electrophoresis tank with electrophoresis solution. Pull out the comb evenly with force. The sample loading amount is 20 μg. Set the initial voltage to 80 V. After the Maker is layered, switch the voltage to 120 V until the blue band reaches the bottom of the gel plate, which takes about 90 minutes.

[0106] (5) Transfer: Cut the polyvinylidene fluoride (PVDF) membrane into 8.5×5.5 cm size. Soak the PVDF membrane in methanol before electrotransfer, take out the gel and soak it in electrotransfer solution. Make a transfer "sandwich": the bottom layer is the black plate, followed by sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and close the clamp. Fill the electrotransfer tank with pre-cooled electrotransfer solution in advance and place the "sandwich" in the electrotransfer tank. Set the transfer current to 220 mA, which takes about 120 min. After the transfer, take out the PVDF membrane and rinse it in TBST (150 mM, pH = 7.4) for 10 min. Put the PVDF membrane in 5% milk powder and block it on a shaker at room temperature for 60 min. Wash with TBST for 5 min × 3 times.

[0107] (6) Incubation with antibodies: Dilute the primary antibody to the desired concentration with TBST, place the PVDF membrane in the antibody diluent, and shake at 4°C overnight. Remove the PVDF membrane and wash the membrane with TBST for 5 min × 3 times. Dilute the secondary antibody with a 5% skim milk powder aqueous solution to a concentration of 1:5000 (volume). Place the PVDF membrane in the secondary antibody diluent and incubate on a shaker at room temperature for 60 min. Remove the PVDF membrane and wash the membrane with TBST for 5 min × 3 times.

[0108] (7) Exposure and analysis: Drop the prepared luminescent liquid on the front of the PVDF membrane and incubate at room temperature for 2 min. Place the PVDF membrane in a transparent plastic film and put it into the machine for exposure. The relative intensity of each band is normalized by its corresponding internal reference protein, and semi-quantitative analysis is performed using the image analysis software Image J.

[0109] 1.9 Statistical methods

[0110] All experimental results are expressed as mean ± standard error (mean ± SEM) and analyzed and plotted using GraphPad Prism software. One-way ANOVA was used for inter-group comparisons, and Tukey's honestly was used for further pairwise comparisons between groups. p<0.05 was considered statistically significant.

[0111] 2. Experimental results

[0112] 2.1 Coptis chinensis injection can improve liver microcirculation disorders caused by lipopolysaccharide

[0113] Figure 1 This is the result of intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Rhodamine 6G was used to label leukocytes and observe the adhesion of leukocytes in liver microvessels in vivo. No rolling and adhering leukocytes were found in the control group. Compared with the control group, Coptis chinensis injection alone did not affect the rolling and adhesion of leukocytes in liver tissue. A large number of rolling and adhering leukocytes were seen in liver tissue after lipopolysaccharide modeling alone. Compared with the lipopolysaccharide alone treatment group, intramuscular injection of Coptis chinensis injection 6 hours later can partially reduce the rolling and adhering leukocytes in liver tissue. The number of hepatic sinusoids perfused in the central venous area of ​​the liver was counted. Compared with the control group, Coptis chinensis injection alone did not affect the perfusion of hepatic sinusoids in the central venous area of ​​the liver. After lipopolysaccharide alone modeling, the number of hepatic sinusoids perfused in the central venous area of ​​the liver decreased. Compared with the lipopolysaccharide alone treatment group, simultaneous intramuscular injection of Coptis chinensis injection can partially restore the perfusion of hepatic sinusoids in the central venous area of ​​the liver. The results suggest that intramuscular injection of Coptis chinensis injection can improve liver microcirculation disorders caused by lipopolysaccharide.

[0114] 2.2 Coptis chinensis injection can alleviate liver damage caused by lipopolysaccharide

[0115] Figure 2 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 2 Results of tissue staining (HE) and necrosis staining (TUNEL) in Figure A Figure 2 The statistical analysis corresponding to B in the middle showed that the liver morphology after treatment with the single injection of Coptis chinensis was no different from that of the control group, and necrotic foci and TUNEL-positive cells appeared in the liver tissue after lipopolysaccharide alone modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could reduce the area of ​​necrotic foci and the number of TUNEL-positive cells. Figure 2C analyzed the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of rats in each group. Compared with the control group, the single injection of Coptis chinensis did not affect the levels of AST and ALT in the serum of rats, and the levels of AST and ALT in the serum of rats increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could reduce the levels of AST and ALT in the serum of rats. Figure 2 D analyzed the contents of TNF-α and IFN-γ in the liver tissue of rats in each group. Compared with the control group, the single injection of Coptis chinensis did not affect the contents of TNF-α and IFN-γ in the liver tissue of rats, and the contents of TNF-α and IFN-γ in the liver tissue of rats increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later can reduce the contents of TNF-α and IFN-γ in the liver tissue of rats. The results suggest that intramuscular injection of Coptis chinensis injection can alleviate liver damage caused by lipopolysaccharide.

[0116] 2.3 Coptis chinensis injection can reduce the accumulation of ferrous ions in the liver caused by lipopolysaccharide

[0117] Figure 3 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 3 Figure A detected the ferrous ion content in rat liver tissue. Compared with the control group, the single injection of Coptis chinensis did not affect the ferrous ion content in liver tissue. The ferrous ion content in liver tissue increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could reduce the ferrous ion content in liver tissue. Figure 3 Figure B shows the expression of proteins mediating iron ion transfer and the corresponding statistical analysis. Compared with the control group, the single injection of Coptis chinensis did not affect the protein expression of transferrin (TF) and transferrin receptor (TFR) in rat liver. The protein expression of TF and TFR in rat liver increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later can reduce the protein expression of TF and TFR in rat liver. The results suggest that intramuscular injection of Coptis chinensis injection can reduce the accumulation of ferrous ions in the liver caused by lipopolysaccharide.

[0118] 2.4 Coptis chinensis injection can reduce liver lipid peroxidation caused by lipopolysaccharide

[0119] Figure 4 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 4In A and B, the contents of lipid peroxide (LPO) and malondialdehyde (MDA) in rat liver tissue were detected, respectively. Compared with the control group, the single injection of Coptis chinensis did not affect the contents of LPO and MDA in liver tissue. The contents of LPO and MDA in liver tissue increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could reduce the contents of LPO and MDA in liver tissue. The results suggest that intramuscular injection of Coptis chinensis injection can reduce liver lipid peroxidation caused by lipopolysaccharide.

[0120] 2.5 Coptis chinensis injection can alleviate liver redox imbalance caused by lipopolysaccharide

[0121] Figure 5 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 5 Figure A detected the content of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative stress, in rat liver tissue. Compared with the control group, the injection of Rhizoma Corydalis alone did not affect the content of 8-OHdG in liver tissue. The content of 8-OHdG in liver tissue increased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Rhizoma Corydalis injection 6 hours later reduced the content of 8-OHdG in liver tissue. Figure 5 In Figure B, C11-BODIPY was used to detect the redox state in rat liver tissue. Compared with the control group, the single injection of Coptis chinensis did not affect the redox state in the liver tissue, and the redox imbalance in the liver tissue was observed after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could partially restore the unbalanced redox state in the liver tissue. This result suggests that intramuscular injection of Coptis chinensis injection can alleviate the liver redox imbalance caused by lipopolysaccharide.

[0122] 2.6 Coptis chinensis injection can improve liver glutathione metabolism disorder caused by lipopolysaccharide

[0123] Figure 6 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 6 In Figure A, the contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) in rat liver tissue were detected, and the ratio of reduced glutathione / oxidized glutathione was further analyzed. Compared with the control group, the injection of Coptis chinensis alone did not affect the ratio of reduced glutathione / oxidized glutathione in liver tissue. The ratio of reduced glutathione / oxidized glutathione in liver tissue decreased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection at 6 hours can partially restore the ratio of reduced glutathione / oxidized glutathione in liver tissue. Figure 6Figure B is the content of reduced glutathione (GSH) in rat liver tissue. Compared with the control group, the single injection of Coptis chinensis did not affect the content of reduced glutathione in liver tissue. The content of reduced glutathione in liver tissue decreased after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection at 6 hours can partially restore the content of reduced glutathione in liver tissue. The results suggest that intramuscular injection of Coptis chinensis injection can improve the liver glutathione metabolism disorder caused by lipopolysaccharide.

[0124] 2.7 Coptis chinensis injection can reduce the infiltration of hepatic inflammatory cells induced by lipopolysaccharide

[0125] Figure 7 This is the result of treatment with intramuscular injection of 1.68ml / kg Coptis chinensis injection after lipopolysaccharide modeling. Figure 7 Figure A detected the infiltration of granulocytes (MPO) and macrophages (CD68) in rat liver tissue. Compared with the control group, the single injection of Coptis chinensis did not affect the infiltration of granulocytes (MPO) and macrophages (CD68) in liver tissue. A large number of granulocytes (MPO) and macrophages (CD68) were infiltrated in liver tissue after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, intramuscular injection of Coptis chinensis injection 6 hours later could partially reduce the number of granulocytes (MPO) and macrophages (CD68) infiltrating in liver tissue. Figure 7 Figure B detected the protein expression of intercellular adhesion molecule (ICAM1) in rat liver tissue, which is a protein that induces leukocyte adhesion. Compared with the control group, the single injection of Coptis chinensis did not affect the protein expression of ICAM1 in liver tissue. The protein expression of ICAM1 in liver tissue increased after LPS modeling. Compared with the LPS-only treatment group, intramuscular injection of Coptis chinensis injection 6 hours later could partially downregulate the protein expression of ICAM1 in liver tissue. This result suggests that intramuscular injection of Coptis chinensis injection can reduce the infiltration of liver inflammatory cells caused by LPS.

[0126] 2.8 Coptis chinensis injection can prevent liver microcirculation disorders and liver damage caused by lipopolysaccharide

[0127] The above experiments show that Coptis chinensis injection can treat liver microcirculation disorders and liver damage caused by lipopolysaccharide. In addition, we also injected 0.84ml / kg Coptis chinensis injection into the tail vein while lipopolysaccharide modeling to observe its preventive effect on liver microcirculation disorders and liver damage. Figure 8A in the middle shows the adhesion of leukocytes in liver microvessels. Compared with the control group, intravenous injection of Coptis chinensis injection did not affect the rolling and adhesion of leukocytes in liver tissue. A large number of rolling and adhering leukocytes were observed in liver tissue after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, simultaneous intravenous injection of Coptis chinensis injection can partially reduce the rolling and adhering leukocytes in liver tissue. Figure 8 The results of tissue staining (HE) and the corresponding statistical analysis in Figure B showed that there was no difference in liver morphology between the control group and the intravenous injection of Coptis chinensis injection, and necrotic foci appeared in the liver tissue after lipopolysaccharide modeling. Compared with the group treated with lipopolysaccharide alone, the intravenous injection of Coptis chinensis injection can reduce the area of ​​necrotic foci. Therefore, intravenous injection of Coptis chinensis injection can prevent liver microcirculation disorders and liver damage caused by lipopolysaccharide. Compared with the dosage of Coptis chinensis injection during treatment, half the dose of Coptis chinensis injection can play a role in preventing liver damage.

[0128] 2.9 Coptis chinensis can alleviate the ferroptosis of Hepg2 cells caused by combined cytokine treatment

[0129] Fig. 9 In Figure A, the ferroptosis marker protein transferrin (TF) was co-stained with the hepatocyte marker protein Alb. The results showed that TF and Alb were co-localized, indicating that ferroptosis occurred in hepatocytes. Fig. 9 The results of the analysis of the blood components of B showed that cyperine is the active ingredient of cyperine injection. The levels of TNF-α and IFN-γ in liver tissue increased significantly after lipopolysaccharide modeling, so we used these two cytokines and cyperine to treat Hepg2 cells in vitro. Fig. 9 As shown in C, compared with the control group, coptisine alone did not affect the viability of Hepg2 cells, combined treatment with cytokines induced Hepg2 cell death, and coptisine treatment could reduce the number of dead Hepg2 cells. Fig. 9 D in the figure shows the accumulation of ferrous ions in Hepg2 cells. Compared with the control group, cytokines combined with ferrous ions in Hepg2 cells increased the ferrous ion content in Hepg2 cells, and cytokines combined with ferrous ions in Hepg2 cells partially reduced the ferrous ion content in Hepg2 cells. Fig. 9 Figure E reflects the lipid peroxidation level of Hepg2 cells. Compared with the control group, cytokines combined with ferruginous pine did not affect the lipid peroxidation level of Hepg2 cells, and cytokines combined with ferruginous pine treatment could increase the lipid peroxidation level in Hepg2 cells. Treatment with ferruginous pine could partially reduce the lipid peroxidation level in Hepg2 cells. Therefore, ferruginous pine could reduce the ferroptosis of Hepg2 cells caused by cytokines combined with ferruginous pine.

[0130] 3. Conclusion

[0131] Coptis chinensis injection reduces inflammatory cell infiltration in liver tissue, reduces the release of inflammatory factors, and alleviates liver microcirculation disorders. Its mechanism of action is mainly mediated by Coptis chinensis injection reducing the accumulation of ferrous ions and lipid peroxidation levels in liver tissue caused by lipopolysaccharide, ultimately alleviating acute liver damage caused by hepatocyte iron death.

[0132] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. Application of coptis chinensis alkaloids in the preparation of drugs for the prevention and treatment of liver microcirculation disorders or liver damage caused by lipopolysaccharide.

2. The use according to claim 1, characterized in that: Fushenamine reduces liver damage through the following mechanisms: (A) Reduce the accumulation of ferrous ions in the liver caused by LPS; (B) Reduce lipopolysaccharide-induced liver lipid peroxidation; (C) Alleviate the liver redox imbalance caused by LPS; (D) Reduced lipopolysaccharide-induced hepatic inflammatory cell infiltration.

3. The use according to claim 1, characterized in that: Coptis chinensis reduces the number of rolling and adhering leukocytes in the central venous area of ​​the liver.

4. The use according to claim 1, characterized in that: Coptis chinensis reduces the area of ​​liver necrosis.

5. The use according to claim 1, characterized in that: Coptis chinensis reduces the levels of alanine aminotransferase and aspartate aminotransferase in serum.

6. The use according to claim 1, characterized in that: Fushenamine reduces the levels of tumor necrosis factor-α and interferon-γ in liver tissue; Coptis chinensis reduces the ferrous ion content in liver tissue and alleviates the level of liver lipid peroxidation, thereby reducing liver damage.

7. A pharmaceutical composition for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide, characterized in that: Contains fuscitine.

8. Use of a pharmaceutical composition containing coptisine in the preparation of drugs for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide.

9. A drug for preventing and treating liver microcirculation disorders or liver damage caused by lipopolysaccharide, characterized in that: A pharmaceutical composition comprising the pharmaceutical composition of claim 7.

10. A drug for preventing and treating liver microcirculation disorder or liver damage caused by lipopolysaccharide, characterized in that: Its active ingredient is coptisine.

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