Application of compound turtle shell liver-softening tablet in preparation of medicine for treating alveolar echinococcosis

Abstract

The invention discloses an application of a compound turtle shell liver-softening tablet in preparing a medicine for treating alveolar echinococcosis, after intervention of the compound turtle shell liver-softening tablet, the focus volume of an alveolar echinococcosis mouse model is reduced, the invasion ability of liver macrophages is reduced, the hepatic fibrosis degree is obviously reduced, and the action mechanism relates to activation of an STING-TBK1 signal channel, so that the liver-softening effect of the compound turtle shell liver-softening tablet is improved. The polarization of the macrophage M1 is induced, so that the fibrosis process is reversed. The dosage forms of the medicine comprise tablets, granules, capsules and oral liquid, and preferably 1g / kg of the medicine is taken for intragastric administration. The invention provides an innovative treatment strategy for the alveolar echinococcosis and the hepatic fibrosis caused by the alveolar echinococcosis, and has a clear molecular mechanism and a remarkable clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of Compound Biejia Ruangan Tablets in the preparation of drugs for treating alveolar echinococcosis. Background Art

[0002] Hydatidosis, also known as echinococcosis, is a zoonotic parasitic disease caused by the larvae of echinococcus tapeworms parasitizing in humans or animals, including cystic echinococcosis (CE) and alveolar echinococcosis (AE), which are infected by Echinococcus granulosus (E. granulosus) and Echinococcus multilocularis (E. multilocularis), respectively. Echinococcus multilocularis often grows invasively and almost always originates in the liver and gradually infiltrates into adjacent tissues and organs, similar to metastatic malignant tumors, so it is also known as "worm cancer". This disease is globally distributed and seriously threatens human health and life safety.

[0003] At present, patients often present at the late stage of Echinococcus multilocularis infection, often accompanied by complex clinical symptoms. With the increase in infiltration of various immune cells around the lesion, especially the infiltration of M2 macrophages, it will induce the body to produce an "immune suppression" microenvironment, which is conducive to the continuous infection of parasites and aggravates liver fibrosis. Currently, experts and scholars generally believe that radical surgical resection of the lesion tissue infected with Echinococcus multilocularis is the most ideal treatment method, but the recurrence rate after surgery is extremely high, and anthelmintic drugs are usually used for maintenance treatment after surgery; however, most patients miss the best treatment window period for radical resection when they seek medical treatment, and usually need lifelong conservative anthelmintic drug treatment, so drug treatment is particularly important for this disease.

[0004] Studies have confirmed that Compound Biejia Ruangan Tablets have good efficacy in the treatment of liver fibrosis. The results of a multi-center randomized controlled study showed that when patients with chronic hepatitis B naive received entecavir antiviral treatment, the cumulative incidence of liver cancer and liver-related mortality rates after 7 years of combined anti-liver fibrosis treatment with Compound Biejia Ruangan Tablets were 4.7% and 0.2%, respectively, lower than 9.3% and 2.2% of entecavir monotherapy; the liver biopsy pathological results showed that the liver fibrosis reversal rate of patients with chronic hepatitis B treated with Compound Biejia Ruangan Tablets increased from 59.2% to 76.7%. At the same time, it has been reported that for liver fibrosis caused by Clonorchis sinensis, Compound Biejia Ruangan Tablets can reduce the oxidative stress injury of the liver and delay the progression of liver fibrosis. However, there is currently no relevant report on the use of Compound Biejia Ruangan Tablets to treat liver fibrosis caused by alveolar echinococcosis. Summary of the Invention

[0005] To achieve the above object, the present invention provides an application of Compound Biejia Ruangan Tablets in the preparation of a drug for treating alveolar echinococcosis, thus solving the above technical problems.

[0006] The present invention provides an application of Compound Biejia Ruangan Tablets in the preparation of a drug for treating alveolar echinococcosis, which induces macrophage M1 polarization by activating the STING-TBK1 signaling pathway, thereby alleviating liver fibrosis caused by alveolar echinococcosis infection.

[0007] Further, the alveolar echinococcosis is caused by infection with Echinococcus multilocularis.

[0008] Further, the dosage forms of the drug include tablets, granules, capsules and oral liquids.

[0009] Further, the fibrosis caused by treating alveolar echinococcosis is manifested in inhibiting the growth of lesions in animals infected with alveolar echinococcosis, or alleviating liver injury and liver fibrosis in animals infected with alveolar echinococcosis.

[0010] Further, the administration concentration of Compound Biejia Ruangan Tablets for inhibiting mice infected with alveolar echinococcosis is 1 g / kg, the administration method is oral gavage, and continuous administration is for more than 4 weeks.

[0011] Further, the therapeutic effect of the drug is reflected by at least one of the reduction of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and the decrease of the content of four liver fibrosis markers (HA, LN, PCIII, CIV).

[0012] Further, the preparation method of the drug includes crushing Compound Biejia Ruangan Tablets and passing through a 120-mesh sieve, and dissolving with physiological saline to prepare a suspension with a concentration of 50 g / L.

[0013] Further, the effect of the drug in alleviating liver fibrosis is verified by at least one of the reduction of the positive expression area of αSMA around the lesions and the reduction of collagen fiber (Sirius red staining) deposition.

[0014] Further, the manifestations of the drug activating the STING-TBK1 signaling pathway include: up-regulation of the expression of STING and TBK1 proteins or mRNAs, and up-regulation of iNOS expression and down-regulation of Arg1 expression.

[0015] Further, the drug is used in combination with an auxiliary component, and the auxiliary component includes an antibiotic or an anti-inflammatory agent; the antibiotic includes penicillin and streptomycin; the drug is used to treat a mouse model infected with alveolar echinococcosis, and the model is constructed by injecting 2000 protoscolices through the portal vein.

[0016] The application of Compound Biejia Ruangan Tablets proposed by the present invention in the preparation of drugs for treating alveolar echinococcosis has the following beneficial effects:

[0017] 1. Compound Biejia Ruangan Tablets proposed by the present invention have a significant anti-parasitic effect in the preparation of drugs for treating alveolar echinococcosis. In the mouse model infected with alveolar echinococcosis, after intragastric administration at a dose of 1 g / kg for 4 weeks, the lesion volume was reduced by 42.5% compared with the model group ( Figure 3 ), confirming its direct inhibitory effect on parasite growth; Compound Biejia Ruangan Tablets can also reverse liver fibrosis. Sirius red staining of liver tissue in the treatment group showed that the collagen deposition area decreased by 58.3% ( Figure 3 ), and the protein expressions of αSMA and Collagen1 decreased by 62% and 57% respectively ( Figure 6 ), significantly superior to existing anti-fibrotic drugs; while the serum ALT and AST levels decreased by 51.8% and 47.3% respectively compared with the model group ( Figure 2 ), and the indexes of the four liver fibrosis markers (HA, LN, PCIII, CIV) decreased synchronously by 35 - 48% ( Figure 2 C-F), indicating its curative effect in reducing liver injury;

[0018] 2. By activating the STING-TBK1 pathway, the protein expressions of STING and TBK1 in liver tissue of the treatment group were up-regulated by 2.3 times and 1.8 times respectively ( Figure 6 ), the expression of the macrophage M1 polarization marker iNOS was increased by 2.1 times, and at the same time, the M2 marker Arg1 was inhibited ( Figure 6 H-M), breaking through the bottleneck of fibrosis treatment from the level of immune regulation. Description of the Drawings

[0019] Figure 1 It is an operation diagram of treating the mouse model of alveolar echinococcosis with Compound Biejia Ruangan Tablets;

[0020] Figure 2 It is for ELISA to detect the four liver fibrosis markers and liver function in the sera of mice in the Compound Biejia Ruangan Tablets (treatment group) and alveolar echinococcosis model (control group);

[0021] Figure 3 It is for the evaluation of the therapeutic effect of Compound Biejia Ruangan Tablets on the mouse model of alveolar echinococcosis. A: General view of the parasitic lesion; B: HE staining map of liver tissue; C: Sirius red staining map of liver tissue;

[0022] Figure 4 It is for immunohistochemical staining to detect the expressions of STING, TBK1, α-SMA, and Collagen1 in liver tissue;

[0023] Figure 5 It is for immunofluorescence co-localization staining to observe the expressions of STING and TBK1 on hepatocytes at different infection times;

[0024] Figure 6 qRT-PCR and Western Blot were used to detect the expression levels of genes related to the STING-TBK1 pathway. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0026] Unless otherwise specified, the examples and comparative examples are parallel tests with the same components, component contents, preparation steps and preparation parameters. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0027] Unless otherwise stated, all reagents were used as received without further purification.

[0028] In the preparation examples and embodiments of the present invention, the “parts” are parts by mass unless otherwise specified, and the concentration percentages are mass concentrations unless otherwise specified.

[0029] Example 1 Construction of Alveolar Echinococcus Mouse Model

[0030] 1.1 Preparation of protoscolex

[0031] Take a gerbil with intraperitoneal infection of alveolar larvae for more than 6 months, anesthetize it with 4% chloral hydrate, kill it by cervical dislocation, and use 75% ethanol to spray the mouse body surface for sterilization. Separate the lesion tissue of intraperitoneal infection under sterile conditions, select the vesicles with acceptable activity, and aseptically extract the cystic fluid with a 50ml syringe. After the remaining infected liver tissue is fully cut into pieces, put it into a sterile mortar for grinding, and use the prepared physiological saline containing 500U / ml penicillin and 100U / ml streptomycin to sieve the ground tissue through a 200-mesh nylon mesh. After multiple sieving, collect the filtrate in a 50ml centrifuge tube and let it stand to precipitate the protoscolecus. After most of the protoscolecus is precipitated, discard the supernatant, and then rinse it repeatedly with physiological saline containing double antibodies until the supernatant shows a clear color. After the last washing, the protoscolecus precipitate is resuspended with physiological saline containing antibiotics to prepare a suspension for use.

[0032] 1.2 Establishment of an animal model of mouse liver infection by portal vein route

[0033] Adjust the concentration of the protoscolex suspension to make the total system 200 - 300 μL containing 2,000 protoscoleces. Select female BALB / c mice at 6 - 8 weeks old (from the Animal Experiment Center of Xinjiang Medical University), puncture and inject the protoscoleces of Echinococcus multilocularis through the hepatic portal vein, and press the puncture point with a dry cotton swab for about 2 minutes until hemostasis. Place the postoperative mice on a heating table at 30 °C for resuscitation until they wake up, and then put them back into the cage for normal feeding.

[0034] Example 2 Treatment with Compound Biejia Ruangan Tablets to construct a treatment group model

[0035] 2.1 The mice successfully inoculated with Echinococcus multilocularis in Example 1 above were sacrificed at 8 days, 30 days, 60 days, and 90 days after infection respectively, and liver tissues were collected. After 60 days of Echinococcus multilocularis infection, mice with uniform infection degree were selected for the therapeutics experiment. Compound Biejia Ruangan Tablets were administered by gavage (the tablets of Compound Biejia Ruangan Tablets were repeatedly pulverized, passed through a 120 - mesh sieve, and dissolved in normal saline), and the doses were divided into 0.5 g / (kg·d) and 1 g / (kg·d), and administered continuously for 4 weeks (the drug was prepared into a 50 g / L concentration with normal saline, the administration volume was 0.1 mL / 10 g body weight, once a day). After continuous administration for 4 weeks, the mice were fasted but allowed to drink water for 12 h, then weighed, anesthetized by intraperitoneal injection of 4% chloral hydrate, blood was collected from the orbital cavity, the abdomen of the mice was disinfected with 75% ethanol, and the liver tissues were collected by laparotomy. The specific operation is shown in Figure 1 .

[0036] 2.2 After 4 weeks of treatment with Compound Biejia Ruangan Tablets, the treatment effect was evaluated: the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected. Blood was collected from the orbital cavity of the mice into a 1.5 mL centrifuge tube, left to stand at 4 °C for 2 h, centrifuged at 3,000 rpm for 15 min, the serum was separated and transferred to a clean centrifuge tube. 150 μL of serum was mixed with 150 μL of sterile pure water, and detected using an automatic biochemical analyzer BS - 120 (the Fifth Affiliated Hospital of Xinjiang Medical University). The experimental results are shown in Figure 2 (A, B). Compared with the Em group, the levels of ALT and AST in the treatment group of Compound Biejia Ruangan Tablets were significantly reduced. The results indicate that Compound Biejia Ruangan Tablets can reduce liver injury by gavage administration. At the same time, the contents of four serum liver fibrosis markers were detected, and the ELISA detection steps refer to the kit instructions (Saipai Biology), and the results are as shown in Figure 2 (C - F). Compared with the Em group, the contents of four serum liver fibrosis markers in the treatment group of Compound Biejia Ruangan Tablets were significantly decreased.

[0037] Example 3 Observation of lesion size, HE staining and Sirius red staining

[0038] 3.1 Observe and compare the lesion sizes of the Em group and the treatment group. It can be seen that Figure 3 in the Em group, the lesions grew progressively with the number of days of infection, and the lesion size in the treatment group was significantly smaller than that in the Em group.

[0039] 3.2 Take the liver tissue obtained in Example 2. After fixing with 4% paraformaldehyde for 72 hours, dehydrate with gradient ethanol, clear with xylene, and then embed in paraffin and section. After cutting the paraffin block of liver tissue into 4 μm sections, place them in an oven at 60 °C for baking overnight. The next day, dewax with xylene, hydrate with gradient ethanol, and then stain with hematoxylin for 6 minutes, wash with tap water, differentiate with hydrochloric acid ethanol for 2 seconds, blue with PBS for 3 minutes, then stain with eosin for 3 minutes, wash with tap water, dehydrate with gradient ethanol, clear with xylene, and mount with neutral balsam. The results of HE staining are as Figure 3 shown. There was a large amount of inflammatory cell infiltration around the lesions in the alveolar echinococcosis-infected mouse model, indicating successful mouse modeling.

[0040] 3.3 Take the paraffin sections obtained by the treatment method in Example 3.2. After dewaxing with xylene and hydrating with gradient ethanol, directly put them into the tap water connected in a beaker. Preheat the wet box in an incubator at 37 °C for 30 min. Prepare hematoxylin staining solution in a 1:1 ratio, add the staining solution and incubate for 10 min (operate in the incubator), wash with distilled water 3 times, 5 min each time. Stain with Sirius red staining solution for 15 min (operate in the incubator), wash with distilled water 3 times, 5 min each time. Dehydrate and clear, wait to dry, and mount with neutral resin. The Sirius red staining is as Figure 3 shown. The collagen fibers are stained red. Compared with the control group of mice, the degree of tissue fibrosis around the lesions in the compound turtle shell and malan head herb tablet treatment group of mice was reduced.

[0041] Example 4 Compound turtle shell and malan head herb tablet promotes macrophage M1 polarization by activating the STING-TBK1 signaling pathway and alleviates liver fibrosis

[0042] 4.1 Take the paraffin sections obtained by the treatment method in Example 3.2. After dewaxing with xylene and hydrating with gradient ethanol, thaw with Tris-EDTA buffer in a microwave oven for 10 min, let the sections cool naturally to room temperature in the repair solution for 45 min, block with 10% goat serum at room temperature for 1 hour, add α-SMA, STING, and TBK1 primary antibodies and incubate overnight at 4 °C in a wet box. The next day, rewarm in an incubator at 37 °C for 40 minutes, soak and wash with PBS 3 times, 5 minutes each time. Then add the secondary antibody and incubate at room temperature for 2 hours, soak and wash with PBS 3 times, 5 minutes each time. After adding the DAB chromogenic solution, observe under the microscope that the target area turns brown and other background areas are lightly stained. Immediately put it into water to terminate, stain with hematoxylin for 3 minutes, differentiate with hydrochloric acid ethanol for 2 s, blue in tap water for 3 minutes, then dehydrate with gradient ethanol, clear with xylene, and mount with neutral balsam. The α-SMA immunohistochemical staining is as Figure 4As shown, a large number of α-SMA positive cells (brown in the figure) were found around the lesions of the Echinococcus multilocularis-infected mouse model, and the positive expression in the treatment group with Compound Biejiaruangan Tablets was significantly reduced. α-SMA is a marker of hepatic stellate cell activation. When extensive positivity appears, fibrosis can be considered to exist in this area. Thus, it is indicated that Compound Biejiaruangan Tablets can reduce the degree of fibrosis around the lesions of the Echinococcus multilocularis-infected mouse model. At the same time, the expressions of STING and TBK1 around the lesions in the treatment group were significantly increased, and the significant upregulation of the STING-TBK1 pathway further alleviated liver fibrosis.

[0043] 4.2 Embed the liver tissue obtained in Example 2 in OCT. After freezing and sectioning the liver tissue, fix it with 4% paraformaldehyde for 15 min. After fixation, wash it 3 times with PBS for 5 min each time. Permeabilize it with 0.1% TritonX-100, block it with 2% BSA for 30 min, incubate it with the primary antibody overnight, incubate it with the secondary antibody in the dark for 2 h, add DAPI for 10 min, wash it 3 times with PBS for 5 min each time, and mount the slides. The results are as Figure 5 shown. When the Echinococcus multilocularis-infected mice were at 8 days, the STING-TBK1 pathway began to be activated as Figure 5 (B), and reached the peak at 30 days Figure 5 (C). At this time, it was consistent with the time node of enhanced inflammatory immunity in Echinococcus multilocularis infection. At this time, the inflammatory response in the Echinococcus multilocularis-infected mice was enhanced. As the infection time progressed, at 60 days, the co-localization fluorescence expression of STING-TBK1 decreased as Figure 5 (D), and the co-localization fluorescence expression was the lowest at 90 days as Figure 5 (E). Compared with the Echinococcus multilocularis-infected mice at 90 days, the Echinococcus multilocularis-infected mice intragastrically administered with Compound Biejiaruangan Tablets could reactivate the STING-TBK1 pathway as Figure 5 (F-I).

[0044] 4.3 Western Blot was used to detect the protein expression of STING, TBK1, α-SMA and Collagen1 in the liver. The liver tissue was lysed with RIPA lysis buffer, ground with a grinder, and the cells were disrupted by ultrasound. After centrifugation at 13,000 r / min for 15 min at 4°C, the supernatant was taken, and the protein was quantified by BCA. The protein extract was denatured in a metal bath at 100°C for 10 min. 60 μg of the protein extract was taken, and the proteins were separated by SDS-PAGE at room temperature. First, electrophoresis was carried out at a constant voltage of 80 V for 30 min. After the sample passed through the stacking gel, electrophoresis was then carried out at a constant voltage of 110 V for 90 min. The proteins were transferred to a PVDF membrane by wet transfer method at a constant voltage of 80 V for 3 h. The membrane was blocked in 5% non-fat milk blocking solution for 2 h, washed with 0.1% PBST 5 times, 5 min each time. The primary antibody was incubated overnight at 4°C. The membrane was washed with PBST 5 times, 5 min each time. The secondary antibody was incubated at room temperature for 2 h, and the membrane was washed as above. After incubation with ECL luminescent solution, the PVDF membrane was taken out and exposed and analyzed on a gel imaging analyzer. GAPDH was used as an internal reference, and Image Lab was used to analyze the protein expression by calculating the ratio of the gray value of the target protein to that of GAPDH. The results were as follows Figure 6 (A-G) showed that compared with the Em group, the protein expression levels of STING and TBK1 in the Compound Biejiaruanganpian treatment group were significantly up-regulated, while the protein expression levels of α-SMA and Collagen1 were significantly down-regulated. At the same time, it was found that the protein expression level of iNOS was up-regulated and the protein expression level of Arg1 was down-regulated. It was indicated that Compound Biejiaruanganpian treatment activated the STING-TBK1 signaling pathway, promoted M1 polarization, and alleviated liver fibrosis.

[0045] 4.4 Real-time fluorescence quantitative PCR was used to detect the gene expression of STING, TBK1, iNOS, Arg1, α-SMA and Collagen1 in liver tissue: Total RNA of liver tissue was extracted by the TRIzol method, and RNA was reverse transcribed into cDNA according to the RNA reverse transcription reagent. The gene fragments were amplified using a real-time fluorescence quantitative PCR detection kit. The reaction conditions were: 95°C for 2 min; 95°C for 5 s, 60°C for 10 s, for 40 cycles. 18S was used as an internal reference, and the 2^-ΔΔCt method was used for calculation. The results were as follows Figure 6As shown in (H-M), compared with the model group, the mRNA expression levels of STING and TBK1 were significantly up-regulated, the mRNA expression levels of the fibrosis indicators α-SMA and Collagen1 were down-regulated, the mRNA expression level of the M1 macrophage marker iNOS was up-regulated, and the mRNA expression level of the M2 macrophage marker Arg1 was down-regulated. Further suggesting that the activation of the STING-TBK1 signaling pathway, M1 macrophage polarization, inhibits the progression of liver fibrosis. In summary, the present invention for the first time discloses the application of Compound Biejia Ruangan Tablets in the preparation of drugs for the treatment of alveolar echinococcosis, reduces the lesion volume of mice infected with alveolar hydatid cysts. Compound Biejia Ruangan Tablets induce M1 polarization of macrophages by activating the STING-TBK1 signaling pathway to alleviate liver fibrosis caused by alveolar echinococcosis, providing a new way for the treatment of alveolar echinococcosis.

[0046] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should all be covered within the scope of the present invention.

Claims

1. A use of a compound Biejiaruangan tablet in the treatment of alveolar echinococcosis, characterized in that: It induces macrophage M1 polarization by activating the STING-TBK1 signaling pathway, thereby alleviating liver fibrosis caused by alveolar Echinococcus infection.

2. The use according to claim 1, characterized in that: The alveolar echinococcosis is caused by infection with Echinococcus multilocularis.

3. The use according to claim 1, characterized in that: The dosage forms of the drug include tablets, granules, capsules and oral liquids.

4. The use according to claim 1, characterized in that: The method for treating fibrosis caused by alveolar echinococcosis is to inhibit the growth of lesions in animals infected with alveolar echinococcosis, or to alleviate liver damage and liver fibrosis in animals infected with alveolar echinococcosis.

5. The use according to claim 1, characterized in that: The compound Biejiaruangan tablet has an administration concentration of 1 g / kg for inhibiting mice infected with alveolar echinococcosis, and is administered orally orally for more than 4 weeks.

6. The use according to claim 1, characterized in that: The therapeutic effect of the drug is manifested by at least one of the following indicators: a decrease in serum alanine aminotransferase and aspartate aminotransferase levels, and a decrease in four liver fibrosis levels.

7. The use according to claim 1, characterized in that: The preparation method of the medicine comprises the following steps: crushing the compound Biejiaruangan tablets, passing the tablets through a 120-mesh sieve, and dissolving the tablets with physiological saline to prepare a suspension with a concentration of 50 g / L.

8. The use according to claim 1, characterized in that: The effect of the drug in reducing liver fibrosis is confirmed by at least one of the following tests: Sirius red staining shows a reduction in collagen fiber deposition; immunohistochemistry detection shows a reduction in the positive expression area of ​​αSMA.

9. The use according to claim 1, characterized in that: The manifestations of the drug activating the STING-TBK1 signaling pathway include: up-regulation of STING and TBK1 protein or mRNA expression, up-regulation of iNOS expression, and down-regulation of Arg1 expression.

10. The use according to claim 1, characterized in that: The drug is used in combination with auxiliary components, wherein the auxiliary components include antibiotics or anti-inflammatory agents; the antibiotics include penicillin and streptomycin; the drug is used to treat a mouse model infected with alveolar echinococcosis, wherein the model is established by injecting 2,000 protoscolex into the portal vein.

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