WO2015120679A1 - 重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药物中的应用 - Google Patents

重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药物中的应用 Download PDF

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WO2015120679A1
WO2015120679A1 PCT/CN2014/079833 CN2014079833W WO2015120679A1 WO 2015120679 A1 WO2015120679 A1 WO 2015120679A1 CN 2014079833 W CN2014079833 W CN 2014079833W WO 2015120679 A1 WO2015120679 A1 WO 2015120679A1
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fibrosis
rlz
tissue fibrosis
mice
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张喜田
孙非
梁重阳
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张喜田
孙非
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A61K36/074—Ganoderma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

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  • the invention relates to a recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) for preparing a drug for treating tissue fibrosis use.
  • the mouse is used as an experimental object to establish a tissue fibrosis model, and a reasonable administration route and dosage are designed.
  • rLZ-8 Ganoderma lucidum immunoregulatory protein
  • Fibrosis can occur in a variety of organs, the main pathological changes are fibrous connective tissue in organ tissues. Increased, parenchymal cells, continuous progression can cause structural damage and dysfunction of organs, and even exhaustion, a serious threat to human health Kanghe life.
  • the body organ consists of two parts: the parenchyma and the interstitial.
  • Substance refers to the main structural and functional cells of the organ, the interstitial It is composed of interstitial cells and extracellular matrix, distributed between parenchymal cells, mainly for mechanical support and connection. Also fine The extracellular matrix constitutes a microenvironment that maintains the physiological activities of cells, is a bridge of signal transmission between cells, and participates in various physiological processes. Pathological processes play an important role in tissue wound repair and fibrosis.
  • pulmonary fibrosis As an example of pulmonary fibrosis, pulmonary fibrosis, as a disease with high lethality, has become more and more Received attention. Among them, idiopathic pulmonary fibrosis is the most common type of idiopathic interstitial pneumonia, accounting for approximately 43% to 68%. At present, we often say that pulmonary fibrosis diseases mainly include idiopathic pulmonary fibrosis, silicosis, and allergic alveoli. Inflammation, as well as pulmonary fibrosis caused by radiation and drugs, also include lung inflammation associated with collagen vascular disease For a wide range of chronic lung diseases.
  • interstitial lung diseases various types of chronic and acute lung diseases are usually accompanied by varying degrees Pulmonary inflammation and fibrosis, both of which are called interstitial lung diseases. Interstitial lung disease caused by various causes The common outcome is pulmonary interstitial fibrosis, which is also the primary cause of respiratory failure.
  • pulmonary fibrosis The pathogenesis of pulmonary fibrosis is mainly caused by a variety of inflammatory cells and related media involved in the damage process. Inflammatory reaction, excessive deposition of collagen fibers during fibroproliferative repair of lung tissue, destruction of lung tissue structure, loss of Normal gas exchange function. Pulmonary fibrosis is the accumulation of fibroblasts in the lung parenchyma, extracellular matrix deposition. The excess is accompanied by the destruction of tissue structure caused by inflammation and damage of lung tissue as a pathological feature.
  • the invention relates to the application of the recombinant Ganoderma lucidum immunoregulatory protein rLZ-8 in preparing a medicament for treating tissue fibrosis, After a series of experimental results, the recombinant Ganoderma lucidum immunomodulatory protein has a significant effect in the treatment of tissue fibrosis. Other positive control samples have the advantages of significant therapeutic effect and no side effects.
  • the specific invention is as follows:
  • the invention adopts experimental mice as research objects, and separately designs and establishes different tissue fibrosis models, wherein bleomycin (BLM) is used as a modeling drug for pulmonary fibrosis, and atomization method is used to establish mouse lung.
  • BLM bleomycin
  • Dox Doxorubicin
  • CDDP Cisplatin
  • the mouse model of renal fibrosis was established by using the method of 50% carbon tetrachloride (CCl 4 ) olive oil solution as the liver fibrosis model, and the rat liver fibrosis model was established by subcutaneous injection of the back.
  • the experimental method compares the role of recombinant Ganoderma lucidum immunomodulatory protein in the treatment of tissue fibrosis.
  • the changes of biochemical parameters of tissue fibrosis are taken as the main parameters.
  • the statistical results of the experimental results are analyzed by SPSS statistical software. The statistical results show that In the treatment of tissue fibrosis, rLZ-8 was significantly different from other positive drugs, and no obvious side effects were produced.
  • the invention is beneficial in that the recombinant Ganoderma lucidum immunoregulatory protein (rLZ-8) provided by the invention is treated Significant efficacy in tissue fibrosis, represented by four typical cases of tissue fibrosis, illustrating rLZ-8
  • tissue fibrotic disorders represented by four typical cases of tissue fibrosis, illustrating rLZ-8
  • the therapeutic effects of rLZ-8 and positive drugs have obvious differences and advantages, and no accompanying side effects occur, prolonging the disease In the life of sick mice, in the experiments of heart, kidney and liver, the therapeutic effects of rLZ-8 have obvious differences and advantages.
  • Tissue wound recovery and fibrosis content and serum ALT (U/L), AST (L/L), ALB (g/L) activity in rat models There was a positive effect on the recovery of force, and it was statistically significant compared with the positive control group.
  • the treatment group showed a significant reduction or even disappearance of fibrosis, which was unexpectedly invented by the inventors and was also achieved by rLZ-8. best effect.
  • Example 1 Establishment of a mouse lung tissue fibrosis model
  • Kunming mouse female weighing in the range of 18-22g, bleomycin (BLM), hydroxyproline kit, propylene dione kit, free Epidemiology staining kit, antibody, HE staining reagent, medical nebulizer, transparent glass container, conventional reagent (NaCl, poly Formaldehyde, PBS, etc., anatomical tools, upright fluorescence microscope, mouse litter and feed, Real time PCR kit, PCR Different primers, cryo slicer and paraffin slicer.
  • Modeling experiments 1) Modeling experiment grouping: divided into three groups, namely saline group (group A) and BLM low dose group (Group B 4 mg/mL), BLM high dose group (Group C 8 mg/mL), 12 mice per group. Divide 12 mice from each group into two groups 6 sets of each group were placed in transparent glass containers with a length, width and height of 20 cm each, and the corresponding marks were made. The saline and BLM are sprayed into the corresponding transparent glass in the form of a mist, respectively. 2) Modeling the administration procedure: Each time the amount of atomization was 1 ml, the glass cover was closed for 15 min, and the mouse was placed in the cage for 5 min. This operation was repeated 6 times.
  • mice were recorded at 9:00 and 17:00 every day and the activity was observed at any time. Feeding for 3 days, At 4 time points of 7 days, 14 days and 21 days, the mice were sacrificed and the lungs were observed to observe the changes in lung appearance, color and lung surface. Bronchoalveolar lavage was performed after taking the lungs, and then part of the lung tissue was taken for paraffin section, and histological examination of HE staining was performed. Another lung tissue was taken for RT-PCR to detect RNA levels. According to histological analysis and RNA levels, proteins Changes in levels and inflammatory cell mass were used to assess whether modelling of pulmonary fibrosis was available. The following table is by Szapiel et al. Methods The grading criteria for the severity of pathological changes in lung tissue were evaluated.
  • Szapiel lung tissue pathological changes grade divided into four levels: "0" is level 1; “I” is level 2; “II” is level 3; “III” For grade 4, the higher the number of grades, the more severe the degree of fibrosis.
  • BLM induced pathological grading and modeling dose of pulmonary fibrosis in mice and alveolar structure of mice in saline group on the 21st day Complete, thin alveolar wall, no extracellular matrix deposition in the periphery of alveolar wall, no inflammatory cell exudation;
  • BLM low dose group The alveolar structure of the mouse is partially destroyed, and a small amount of extracellular matrix deposition and inflammatory cell exudation around the alveolar wall;
  • BLM In the high-dose group the structural damage of the lung tissue was severe, and there were a lot of extracellular matrix deposition and inflammatory cell infiltration around the alveolar wall. Out, there is almost no complete alveolar structure as shown in Figure 1.
  • Paraffin section of lung tissue and HE staining according to Szapiel et al. Graded by standard, the degree of fibrosis of the tissues was counted, and it was found that the mouse slices of the saline group were almost free of fibrotic areas. In the BLM low-dose group, 66% of the lung sections were moderately fibrotic, while in the BLM high-dose group, 75% of the lung sections were severely fiberized. Chemical. No pulmonary fibrosis was found in the saline group of the saline group; most of the lung tissue of the BLM low-dose group was formed during BLM atomization modeling.
  • the lung tissue sections of the saline group were all normal; the degree of pulmonary fibrosis in the low dose group Mainly concentrated in moderate fibrosis; high-dose group pulmonary fibrosis in the level of severe fibrosis.
  • Experimental grouping 7 groups of experimental design, 12 mice in each group, respectively, saline group (group A), model group (group B), The low-dose group (group C), the middle-dose group (group D), the high-dose group (group E), the positive control group (group F), and one week after modeling
  • group A 7 groups of experimental design, 12 mice in each group, respectively, saline group (group A), model group (group B), The low-dose group (group C), the middle-dose group (group D), the high-dose group (group E), the positive control group (group F), and one week after modeling
  • the drug group (Group G) the molding process is the same as that described in Example 1.
  • Treatment courses Groups B, C, D, E, F, and G were all modeled with low-dose BLM. Then group A and B were injected with normal saline daily. As a control, groups C, D, and E were injected with rLZ-8 every day, and body weight was weighed every 3 days, and administered according to body weight. Group C agent The dose was 3.84375 ⁇ g/kg, the D dose was 7.6875 ⁇ g/kg, and the E dose was 19.21875 ⁇ g/kg. The positive drug was dexamethasone 4 mg/kg, and the G group was injected with rLZ-8 for 1 week, and the dose was 7.6875 ⁇ g/kg.
  • mice The physiological indexes and deaths of the mice were recorded at 9:00 and 17:00, respectively. On the 14th day and the 21st day, 6 rats were randomly selected. After the sacrifice, the lungs were observed to observe the color of the lungs, the appearance of the mice, and the changes in the surface of the lungs. Part of the lung tissue was taken for paraffin section and HE staining for histological examination; the other part was RT-PCR. Detection of RNA levels. Based on histological analysis and changes in RNA levels, protein levels and inflammatory cell mass The degree of fibrosis.
  • the experimental results show that the therapeutic effect of the rLZ-8 middle dose group (D group and G group) and the positive control drug group (F group) Comparison, the treatment effect is obvious.
  • the comparison of severe fibrosis shows that rLZ-8 can positively restore lung tissue.
  • group F the number of mice still at the level of severe fibrosis was significantly reduced, without fiber
  • the rLZ-8 medium dose group (group D and group G) has a significant advantage compared with the positive control group (group F). Partial cure occurred during the treatment cycle, but there was no cure in the positive control group (Group F).
  • mice The activity of the mice was observed at any time, and the death of the mice was recorded every day at 9 am and 5 pm. Select 35 days, 42 days and 49 days at three time points to take the material, after the death, take the heart and rinse the remaining blood with cold PBS, placed in a clean The filter paper is blotted dry and placed in a paraformaldehyde solution for fixation.
  • Establish a histological analysis to evaluate myocardial fibrosis A model of refinement, combined with mouse ventricular cavity changes, perivascular fibrosis, interstitial fibrosis, and vacuolization Determine if the model is available.
  • mice/group design 8 experimental groups, 10 mice/group, saline group (group A), model group (group B), rLZ-8 3 ⁇ g/kg (group C), rLZ-86 ⁇ g/kg (group D), rLZ-812 ⁇ g/kg (group E), rLZ-824 ⁇ g/kg (F) Group), rLZ-848 ⁇ g/kg (group G) and positive control group (group H).
  • Treatment procedure The modeling process was the same as in Example 3, and the B, C, D, E, F, G, and H groups used Dox 3 mg/kg twice a week. Modeling. Then, groups A and B were intraperitoneally injected with normal saline as a control, and groups C, D, E, G, and H were injected once every two days. rLZ-8 treatment. The body weight was weighed every 3 days and administered according to the body weight. The injection dose of group C was 3 ⁇ g/kg, and the D group was 6 ⁇ g/kg, group E was 12 ⁇ g/kg, group F was 24 ⁇ g/kg, group G was 48 ⁇ g/kg, group H was perfused for two days. Fusinopril sodium tablets 10 mg/kg were administered as a positive control group.
  • mice The death of the mice was recorded at 9 am and 5 pm every day and the activity was observed at any time.
  • the mice were sacrificed and the heart was washed with cold PBS, placed on a clean filter paper, blotted dry, and placed in a large amount. Fixation is carried out in a polyoxymethylene solution. Then the myocardial tissue was made into paraffin sections for HE staining and trichrome staining for tissue analysis. Academic test.
  • mice In order to detect myocardial fibrosis grade and myocardial fibrosis status in mice, according to the seventh week The mortality of the mice was sampled, and the number of mice from the negative control group to the positive control group was 10, 2, 1, 4, 6, 6, 8, 7 respectively. As shown in Table 6.
  • the sampling method was performed by injecting an appropriate amount of anesthetic, the heart was separated under aseptic conditions, and the cold PBS was used. Rinse the remaining blood and blot the remaining blood with filter paper. Cross-section was performed at the 1/2 segment of the ventricle, and the myocardial tissue was placed in 4% paraformaldehyde. Fix in the middle, then perform HE staining on paraffin sections, cut three sections of each mouse tissue and perform under the same conditions.
  • mice were subjected to Masson staining and photographed under 20-fold and 40-fold microscopes.
  • Got Results Only one of the 10 mice in the negative control group developed +-stage perivascular fibrosis; the model group appeared more severe Heavy interstitial fibrosis and vacuolization; rLZ-83ug/kg, rLZ-86ug/kg appeared in the low-dose treatment group Interstitial fibrosis and the formation of vacuoles, but with the increase of therapeutic dose, the indicators of myocardial fibrosis in mice gradually weakened.
  • mice When the therapeutic dose was rLZ-848ug/kg, only two mice developed +grade perivascular fibrosis and a +-grade interstitial Fibrosis; in the positive control group, 1 out of 7 mice showed + grade perivascular fibrosis and + grade interstitial fibrosis, One appeared ++ perivascular fibrosis and the formation of ++ vacuoles.
  • mice The experimental results show that rLZ-8 has a significant therapeutic effect on Dox-induced myocardial injury in mice.
  • the comparison between the treatment group G group and the positive drug group H group showed that the effect of rLZ-8 treatment was very significant.
  • CDDP cisplatin
  • the 5mg/kg dose treatment can cause the weight loss of the mice, and the concentration of cisplatin at the 7mg/kg dose concentration
  • the normal survival of the rat is not suitable as the model dose concentration for this experiment.
  • 5mg/kg dose concentration of cisplatin is guaranteed to be small
  • the weight loss of the mice is caused, and subsequent histological and molecular indicators are also detected.
  • the results of the report are consistent. Therefore, cisplatin at a dose of 5 mg/kg was selected as the model dose concentration for this experiment.
  • mice 30 18-22g female Kunming mice were randomly divided into 3 groups and treated as follows: blank The control group (group A) was intraperitoneally injected with PBS for 5 consecutive days from the first day. Negative control group (group B), starting from the first day, 5 mg/kg cisplatin was intraperitoneally injected for 5 consecutive days, and PBS was injected into the tail vein from the 7th day to the 28th day.
  • rLZ-8 treatment group Group C rLZ-8123 ⁇ g/Kg
  • 5mg/kg cisplatin was injected intraperitoneally for 5 consecutive days from the first day, from the 7th day to the first For 28 days, 123 ⁇ g/kg rLZ-8 was injected into the tail vein.
  • mice On days 14 and 28, 2 mice were taken from each group, and the kidneys were taken after sacrifice. 4% poly Formaldehyde fixation, paraffin-embedded sections, and assessment of tissue damage and extracellular matrix using Masson's trichrome staining (MTS) It is collagen) deposition.
  • MTS Masson's trichrome staining
  • the experimental results show that rLZ-8 is caused by renal fibrosis caused by cisplatin at a dose of 5 mg/kg.
  • the increase of matrix components such as collagen in the cytoplasm has a certain inhibitory effect, and the results of MTS staining on kidney tissue sections show that On day 14, the degree of tissue damage in group C was lower than that in CDDP group, and the collagen content in the matrix was also lower than that in CDDP group; kidney on day 28
  • the degree of renal damage in the group was inhibited or improved.
  • Experimental group model group, positive drug group, treatment group were given subcutaneous injection of 50% carbon tetrachloride olives in the back.
  • the oil solution twice a week, starts at 8:00 am on Monday and Thursday for 8 weeks and replicates the cirrhosis model.
  • First time 0.5ml/100g Body weight, thereafter, was injected 0.3 ml/100 g body weight every 4 days. Free drinking and eating. At the same time, monitor the body of the rat every Monday. Heavy changes, preliminary estimates of the possibility of liver failure to reduce the mortality caused by replication of cirrhosis model.
  • Subcutaneous injections were given at doses of 15 ug/kg, 30 ug/kg, and 60 ug/kg.
  • liver wet weight Due to portal hypertension in liver cirrhosis, liver wet weight has increased.
  • Statistics show that the model group has the highest liver wet weight. In the normal group, the liver wet weight of the treatment group decreased compared to the model group. The differences between the groups were statistically significant. P ⁇ 0.05. It can be seen from Table 7 that the liver wet weight of the control group has the lowest mean value and the amplitude change is small; the mean value of the liver wet weight of the model group The highest, the magnitude of change; the liver wet weight of the treatment group decreased compared with the mean of the model group. The trend of liver index changes The same was true for the control group, which was the lowest, the model group was the highest, and the treatment group was lower than the model group. Among them, rLZ-8 high dose group The effect is most obvious.
  • the rat liver fibrosis model was replicated by CCl 4 : olive oil (1:1) for 8 weeks, and the serum AST and ALT activities in the rats were significantly higher than those in the normal control group (P ⁇ 0.05). The difference was statistically significant. The ALB content was significantly lower than that of the normal control group (P ⁇ 0.05). The difference was statistically significant.
  • the rats were injected with LZ-8 drug at different doses every day. The AST, ALT enzyme activity, and ALB content and amount in the serum of the rats were measured at 10 days, 20 days, and 30 days. Results The serum levels of AST and ALT in the rLZ-8 treatment group decreased gradually, and the ALB content increased gradually. The difference was significant (P ⁇ 0.05). The rLZ-8 medium dose has the best effect.

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Abstract

本发明涉及重组灵芝免疫调节蛋白（rLZ-8）在制备治疗组织纤维化药物中的应用。

Description

重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药 物中的应用 技术领域
本发明涉及重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药物中的应 用。具体涉及以小鼠作为实验对象，建立组织纤维化模型，设计合理的给药途径和剂量，定 期检测小鼠组织纤维化症状，以改善小鼠的生活质量和延长小鼠寿命为治疗目标，评价重 组灵芝免疫调节蛋白治疗组织纤维化过程中的意义。
背景技术
纤维化(Fibrosis)能发生于多种器官，主要病理改变为器官组织内纤维结缔组织 增多，实质细胞减少，持续进展可致器官结构破坏和功能减退，乃至衰竭，严重威胁人类健 康和生命。
机体器官由实质和间质两部分构成。实质是指器官的主要结构和功能细胞，间质 由间质细胞和细胞外基质构成，分布在实质细胞之间，主要起机械支撑和连接作用。此外细 胞外基质构成维持细胞生理活动的微环境，是细胞之间的信号传导的桥梁，参与多种生理 病理过程，在组织创伤修复和纤维化过程中起重要作用。
以肺纤维化举例说明，肺纤维化作为一种高致死率的疾病，在医学界已经越米越 受到重视。其中，特发性肺纤维化是特发性间质性肺炎中最为常见的一种，发病比例大约占 43％～68％。目前，我们经常说的肺纤维化疾病主要包括特发性肺纤维化、矽肺、过敏性肺泡 炎以及放射线和药物所导致的肺纤维化，还包括与胶原血管病相关联的肺部炎症等范围较 为广泛的一类慢性肺部疾病。此外，各类的慢性以及急性的肺部疾病通常会伴有不同程度 上的肺部炎症和纤维化，它们均称为肺间质性疾病。各种不同病因引发的肺间质性疾病的 共同结局是肺间质纤维化，也是引发呼吸衰竭的首要因素。
肺纤维化发病机理主要是多种炎性细胞以及相关的介质参与的损伤过程引发的 炎症反应、肺组织的纤维增生修复过程中胶原纤维的过量沉积，肺组织结构被破坏，丧失了 正常的气体交换功能。肺纤维化是以肺实质组织成纤维细胞的大量聚集、细胞外基质沉积 过量同时伴随有肺组织的炎症和损伤所致组织结构破坏为病理特征。
因肺纤维化的发病机理并不十分清楚，故在治疗方面还存在着一些困难。以前医 生只是用一些非特异性抗炎药物减少炎症反应来减轻纤维化程度；或是用糖皮质激素及免 疫抑制剂等药物来缓解病人的病症，但并没有对纤维化的根源进行根治，故治疗效果并不 是很理想。通常在治疗过程中伴随有其他的并发症发生或是治疗药物的副作用过大而导致 损伤其他的器官，同时也不能很好的改善病人的生活质量。因此，不断的研究新的药物成为 一种迫切需求。目前，深入的研究肺纤维化发病机制后，人们猜想通过阻断发病的早期的炎 症反应或是减轻修复过程中胶原的过量沉积的治疗方法是可以有效的治疗肺纤维化，由此 建立了多种新治疗方法。现在一般认为，加强对肺纤维化的临床诊断和早期识别，对治疗是 非常有必要的。了解肺纤维化的一些临床表现能更早的进行确诊，为治疗争取宝贵的时间； 对肺纤维化发病机制的研究可以找出最佳的治疗方法。
针对目前的治疗组织纤维化药物都有很严重的副作用，而且并不能非常有效地改 善小鼠的生活质量或是延长寿命，寻找一种能解决上述问题的药物显得十分重要。本研究 主要探讨重组灵芝免疫调节蛋白rLZ-8用于预防和治疗药物诱导的组织纤维化。
发明内容
本发明涉及重组灵芝免疫调节蛋白rLZ-8在制备治疗组织纤维化药物中的应用， 经过一系列实验结果表明，重组灵芝免疫调节蛋白在治疗组织纤维化过程中，药效显著，与 其他阳性对照样比较具有治疗效果显著，无副作用等优势。具体发明内容如下：
本发明采用实验鼠作为研究对象，分别设计并建立了不同组织纤维化模型，其中，以博 来霉素(BLM)作为肺纤维化的造模用药，雾化法造模技术，建立小鼠肺组织纤维化模型；以 多柔比星(Dox)作为心肌纤维化造模用药，采用尾静脉注射法建立小鼠心肌纤维化模型； 采用顺铂(CDDP)作为肾脏纤维化造模用药，腹腔注射法建立小鼠肾脏纤维化模型；以体积 分数为50％四氯化碳(CCl4)橄榄油溶液作为肝脏纤维化造模用药，采用背部皮下注射法建 立大鼠肝脏纤维化模型；通过设置对照实验法对比说明重组灵芝免疫调节蛋白在治疗组织 纤维化过程中的作用，以组织纤维化的生化参数改变作为主要考察参数，利用SPSS统计学 软件对实验结果进行了统计分析，统计结果表明，在治疗组织纤维化方面rLZ-8与其它阳 性药比较具有显著差异，无明显的毒副作用产生。
本发明的有益之处在于：本发明所提供的重组灵芝免疫调节蛋白(rLZ-8)在治疗 组织纤维化方面具有显著疗效，采用了4种典型的组织纤维化病例为代表，说明了rLZ-8 在治疗组织纤维化病症中的应用是显而易见的，在肺组织纤维化实施例中我们可以看到， rLZ-8与阳性药的治疗效果具有明显的差异和优势，并且没有伴随的副反应发生，延长了患 病小鼠的生命，在心脏、肾脏、肝脏的实验中，rLZ-8的治疗效果具有明显的差异和优势，在 组织创伤恢复和纤维化含量以及大鼠模型血清中ALT(U/L)、AST(L/L)、ALB(g/L)的活 力恢复方面具有积极的影响，同时与阳性对照组相比较具有统计学意义，部分实验中期在 治疗组出现纤维化大幅减少甚至消失，是发明人始料未及的，同时也是rLZ-8所能达到的 最佳效果。
附图说明：
图1rLZ-8对肾纤维化的组织损伤对比的影响
图2rLZ-8对肾纤维化间质中胶原成分含量的影响
具体实施方式
实施例1：小鼠肺组织纤维化模型的建立
实验材料
昆明鼠雌性，体重范围在18-22g，博来霉素(BLM)，羟脯氨酸试剂盒，丙二酮试剂盒，免 疫组化染色试剂盒，抗体，HE染色试剂，医用雾化器，透明玻璃容器，常规试剂(NaCl、多聚 甲醛、PBS等)，解剖刀具，正置荧光显微镜，小鼠垫料及饲料，Real time PCR试剂盒，PCR特 异引物，冰冻切片机及石蜡切片机。
实验方法与操作
雾化法造模、肺泡灌洗技术、冰冻及石蜡切片技术、HE染色及Masson氏三色染色技 术、ELISA法检测灌洗液中蛋白的变化、流式法检测灌洗液中的炎性细胞的量。
建模实验：1)建模实验分组：分设3个组，分别为生理盐水组(A组)、BLM低剂量组 (B组4mg/mL)、BLM高剂量组(C组8mg/mL)，每组12只小鼠。将各组12只鼠分为两个小组 每组6只，分别放入长宽高各为20cm的透明玻璃容器里，做好相应的标记，用医用超声雾化 器分别将生理盐水和BLM以雾状的形式喷到相应的透明玻璃器里，2)建模给药操作程序： 每次雾化量为1ml，关闭玻璃罩封闭15min，鼠放同笼内休息5min，此操作重复6次。3)实 验结果观察：每天9时和17时记录小鼠的死亡情况并随时观察活动情况。分别于饲养3天、 7天、14天和21天的4个时间点处死小鼠取肺观察小鼠肺外观、颜色、肺表面的变化情况； 取肺后先进行支气管肺泡灌洗，然后取肺组织一部分做石蜡切片，进行HE染色的组织学检 测；另取一部分肺组织做RT-PCR，进行RNA水平的检测。根据组织学分析和RNA水平，蛋白 水平和炎性细胞量的变化来评价肺纤维化的模型建立是否可用。下表是按Szapiel等的 方法评价肺组织病理变化严重程度的分级标准。
[根据细则26改正08.08.2014]　
表1 Szapiel肺组织病理变化等级
Figure WO-DOC-FIGURE-3
Szapiel肺组织病理变化等级，分为四级：“0”为1级；“I”为2级；“II”为3级；“III” 为4级，级数越高纤维化程度越严重。
实验结论：
BLM诱导小鼠肺纤维化病理分级与造模剂量摸索，第21天时生理盐水组小鼠肺泡结构 完整，肺泡壁较薄，肺泡壁外周无细胞外基质沉积，也没有炎性细胞的渗出；BLM低剂量组 小鼠肺泡结构部分被破坏，肺泡壁的周围有少量细胞外基质的沉积和炎性细胞的渗出；BLM 高剂量组肺组织结构破坏比较严重，肺泡壁周围有大量细胞外基质沉积和炎性细胞的渗 出，几乎没有完整的肺泡结构如图1所示。对肺组织石蜡切片并进行HE染色，按Szapiel等 级标准分级，统计组织的纤维化程度，发现生理盐水组的小鼠切片统计几乎无纤维化区域， BLM低剂量组小鼠肺切片有66％为中级纤维化，而BLM高剂量组小鼠肺切片75％为重度纤维 化。生理盐水组肺切片未发现纤维化病变；BLM雾化造模时，BLM低剂量组小鼠肺组织大部 分发生了中度纤维化，部分肺组织正常，具备继续存活条件；BLM高剂量组小鼠肺组织绝大 部分发生重度纤维化，大部分小鼠因呼吸衰竭而死亡，无法进行后续治疗实验。综上所述， 在药物治疗时选用了BLM低剂量进行造模。
[根据细则26改正08.08.2014]　
表2 造模实验结果及其Szapicl等级标准分级
实验组 n 0 I II III
生理盐水 12 12 0 0 0
BLM低剂量组 12 0 3 8 1
BLM高剂量组 12 0 1 2 9
按Szapiel的等级标准分级，生理盐水组肺组织切片全正常；低剂量组肺纤维化程度 主要集中在中度纤维化；高剂量组肺纤维化在重度纤维化水平。
实施例2 rLZ-8对小鼠肺组织纤维化治疗作用
实验分组：实验设计7个组，每组12只小鼠，分别为生理盐水组(A组)、模型组(B组)、 治疗低剂量组(C组)、中剂量组(D组)、高剂量组(E组)、阳性对照组(F组)和造模一周后给 药组(G组)，造模过程同实施例1所述。
治疗程疗：B、C、D、E、F、G组都用低剂量BLM造模。然后A、B组每天注射生理盐水 作为对照，C、D、E组每天注射rLZ-8治疗，每隔3天称一次体重，根据体重进行给药。C组剂 量为3.84375μg/kg，D组剂量为7.6875μg/kg，E剂量为19.21875μg/kg，F组每天注 射阳性药地塞米松4mg/kg，G组造模1周后注射rLZ-8治疗，给药剂量为7.6875μg/kg。
实验记录：每天9时和17时记录小鼠的各项生理指标和死亡情况，每组分别于第 14天和第21天随机取鼠6只，处死后取肺观察小鼠肺颜色、外观完整与否、肺表面的变化情 况；取肺组织一部分做石蜡切片和HE染色，进行组织学上的检测；另一部分则做RT-PCR进 行RNA水平的检测。根据组织学分析和RNA水平，蛋白水平和炎性细胞量的变化来进行判 断纤维化的程度。
实验结果：肺组织石蜡切片并进行HE染色，统计组织的纤维化程度。A组的小鼠 肺组织都没有纤维化；B纤维化程度主要是中度肺纤维化；D组的纤维化等级大部分恢复到 了轻微纤维化的水平，但C组的肺组织纤维化程度主要集中在中度纤维化水平，部分为轻 度的纤维化，而E组的肺纤维化各个等级的差不多，但大部分还是在轻度和中度等级；阳性 对照组(F组)的肺组织纤维化程度主要集中在中度纤维化，部分是轻度或重度。具体数值 详见表3。
[根据细则26改正08.08.2014]　
表3rlz-8抑制BLM引起的肺纤维化
Figure WO-DOC-FIGURE-5
实验结果可以看出，rLZ-8中剂量组(D组与G组)的治疗效果，与阳性对照药组(F组) 比较，治疗效果明显，首先，在重度纤维化方面进行比较说明rLZ-8能够使肺组织积极恢复 到中度纤维化水平，与F组比较看出仍处于重度纤维化水平的小鼠数量明显减少，在无纤 维化方面，rLZ-8中剂量组(D组与G组)与阳性对照药组(F组)比较，具有明显的优势，在 治疗周期内出现了部分治愈的情况，而在阳性对照药组(F组)没有治愈的情况出现。
实施例3 小鼠心肌纤维化模型的建立
选取100只18-22g的昆明雌鼠，设5个组，分别设为生理盐水组，模型1组(DOX1mg/ kg)，模型2组(Dox2mg/kg)，模型3组(Dox3mg/kg)，模型4组(Dox4mg/kg)组，每组 小鼠数量分别为15，15，25，30，15。分别每周两次，注射量为200μl/次/只，其中溶有1 mg/kg Dox，2mg/kg Dox，3mg/kg Dox或4mg/kg Dox。
随时观察小鼠的活动情况，每天上午9点和下午5点记录小鼠的死亡情况。选取 35天，42天和49天三个时间点进行取材，处死后取心脏用遇冷PBS冲洗余血，放置于干净 的滤纸上吸干，并放入多聚甲醛溶液中进行固定处理。建立通过组织学分析来评价心肌纤 维化的模型，结合小鼠的小鼠心室腔变化、血管周纤维化、间质纤维化以及空泡的形成等变 化确定模型是否可用。
[根据细则26改正08.08.2014]　
表4 根据J.P.Bertinchant等人的心肌纤维化分级标准：
Figure WO-DOC-FIGURE-6
实验结果：如表5所示，从表中可以看到随着造模时间的延长，小鼠心肌组织中血管周 维纤维化，间质纤维化以及组织之间空泡的形成逐渐呈现上升趋势，并且在造模累积剂量 为42mg时，即3mg/kgDox每周两次腹腔注射，在第七周时小鼠出现较为明显的心肌纤维化 的现象，并且在该造模方法下小鼠的死亡率相对较低，有利于利用Dox建立稳定的小鼠心 肌纤维化稳定模型。以上结果说明Dox导致小鼠心肌损伤加重，随着造模时间的延长严重 程度加重，最适宜造模剂量为42mg即3mg/kgDox每周两次腹腔注射共注射七周。
[根据细则26改正08.08.2014]　
表5 多柔比星或生理盐水处理后组织学变化
Figure WO-DOC-FIGURE-7
实施例4rLZ-8对Dox诱导的小鼠心肌损伤的治疗作用
实验分组：设计8组实验组，10只小鼠/每组，生理盐水组(A组)、模型组(B组)、rLZ-8 3μg/kg(C组)、rLZ-86μg/kg(D组)、rLZ-812μg/kg(E组)、rLZ-824μg/kg(F 组)、rLZ-848μg/kg(G组)和阳性对照组(H组)。
治疗程序：造模过程同实施例3，B、C、D、E、F、G、H组都用每周两次3mg/kg的Dox 造模。然后A、B组每天腹腔注射生理盐水作为对照，C、D、E、G、H组每两天尾静脉注射一次 rLZ-8治疗。每隔3天称一次体重，根据体重进行给药。C组注射剂量为3μg/kg，D组为 6μg/kg，E组为12μg/kg，F组为24μg/kg，G组为48μg/kg，H组为每两天小鼠灌胃 给药福辛普利钠片10mg/kg作为阳性对照组。
实验记录：每天上午9点和下午5点记录小鼠的死亡情况并随时观察活动情况。 于第7周时，处死小鼠后取心脏用遇冷PBS冲洗余血，放置于干净的滤纸上吸干，并放入多 聚甲醛溶液中进行固定处理。然后心肌组织做石蜡切片进行HE染色和三色染色进行组织 学上的检测。
实验结果：为了检测小鼠心肌纤维化等级以及心肌纤维化状况，在第七周时根据 小鼠死亡率进行取样，从阴性对照组至阳性对照组各组小鼠只数分别为10，2，1，4，6，6，8，7 如表6所示。取样方法通过注射适量麻醉剂进行处死，无菌条件下分离心脏，并用遇冷PBS 冲洗余血并用滤纸吸干余血。选取心室1/2段处进行横切，并将心肌组织置于4％多聚甲醛 中进行固定，然后进行石蜡切片HE染色，每个小鼠组织均切三张切片并在相同条件下进行 染色。对小鼠心肌组织切片进行Masson染色并在20倍与40倍显微镜下进行拍照统计。得 到结果：阴性对照组10只小鼠中只有一只出现+级血管周纤维化；模型组中出现了较为严 重的间质纤维化以及空泡的形成；在低剂量治疗组中rLZ-83ug/kg，rLZ-86ug/kg均出现 间质纤维化以及空泡的形成，但随着治疗剂量的上升小鼠心肌纤维化各项指标逐渐减弱， 当治疗剂量为rLZ-848ug/kg时，仅有两只小鼠出现+级的血管周纤维化以及+级的间质 纤维化；阳性对照组中，7只小鼠中有1只出现+级血管周纤维化和+级间质纤维化，另有 一只出现++血管周纤维化和++空泡的形成。
[根据细则26改正08.08.2014]　
表6 rLZ-8对Dox诱导的小鼠心肌损伤的治疗作用
Figure WO-DOC-FIGURE-8
实验结论：实验结果表明，rLZ-8对对Dox诱导的小鼠心肌损伤的治疗作用十分显著， 治疗组G组与阳性药组H组比较说明，rLZ-8治疗效果十分显著。
实施例5 小鼠肾脏纤维化模型的建立
选取36只18-22g的昆明雌鼠，设3个组，分别设为对照组(PBS)，模型1组(CDDP5mg/ kg)，模型2组(CDDP7mg/kg)，每组小鼠数量分别为12，12，12。连续腹腔注射5天，注射量 为200μl/次/只，含有相应剂量顺铂(CDDP)。第1、3、5、7、14、28天时分别称量小鼠体重， 并统计各组小鼠生存率。
实验结果：我们发现顺铂(CDDP)注射剂量为7mg/kg时，小鼠死亡率较高，无法正 常存活。从第3天开始，小鼠陆续死亡，第7天时，存活率为60％，到第28天时，只有30％的 小鼠存活。5mg/kg剂量处理组，小鼠可正常存活，从第14天开始，有小鼠死亡，到第28天 时，小鼠存活率为90％。
在实验中我们发现，在保征小鼠正常存活的前提下，由于顺铂的肾毒性及对其它 器官的毒副作用，5mg/kg剂量处理可以导致小鼠体重下降，7mg/kg剂量浓度的顺铂严重影 响小鼠的正常存活，不适宜作为本实验的造模剂量浓度。5mg/kg剂量浓度的顺铂在保证小 鼠存活率的情况下，造成小鼠体重下降，且后续的组织学和分子指标检测，也与已知的相关 报道结果一致。因此，选用5mg/kg剂量浓度的顺铂作为本实验的造模剂量浓度。
实施例6 rLZ-8对顺铂(CDDP)所导致的小鼠肾纤维化的治疗作用
实验分组与治疗程序：随机将30只18-22g雌性昆明鼠分为3组，并作如下处理：空白 对照组(A组)，从第1天开始，连续5天腹腔注射PBS。阴性对照组(B组)，从第1天开始， 连续5天腹腔注射5mg/kg顺铂，从第7天开始到第28天，尾静脉注射PBS。rLZ-8治疗组 (C组rLZ-8123μg/Kg)从第1天开始连续5天腹腔注射5mg/kg顺铂，从第7天开始到第 28天，尾静脉注射123μg/kg rLZ-8。
实验检测方法：第14和28天时，每组分别取2只小鼠，处死后取肾脏。采4％多聚 甲醛固定，石蜡包埋切片，利用马松三色染色(MTS)评估组织损伤程度及细胞外基质(主要 是胶原)沉积。
实验结果：实验结果表明，rLZ-8对于5mg/kg剂量浓度的顺铂造成的肾纤维化间 质中胶原等基质成分的的增加具有一定的抑制效果，对肾脏组织切片MTS染色结果显示， 第14天时，C组组织损伤程度低于CDDP组，基质中胶原含量也低于CDDP组；第28天时肾 脏结构的损毁程度以及萎缩程度加剧，同时，基质中肌纤维的含量增加，与CDDP组相比，C 组肾脏受损程度有抑制或改善的效果。通过Image-Pro Plus6.0分析(图1)，第14天时，与 CDDP组相比C组对于间质中胶原的沉积具有显著地抑制作用；第28天时，基质中肌纤维的 含量对比，rLZ-8组与CDDP组有显著差异(图2)。表明，rLZ-8具有促进抑制胶原的合成或 者促进胶原的降解作用。
实施例7 rLZ-8对CCl4所导致的小鼠肾纤维化的治疗作用
实验分组：模型组、阳性药组、治疗组予以背部皮下注射体积分数为50％四氯化碳橄榄 油溶液，每周2次，周一、周四早8：00开始，持续8周，复制肝硬化模型。首次0.5ml/100g 体重，其后，每4日注射0.3ml/100g体重。自由饮水与进食。同时每周周一监测大鼠的体 重变化，初步估计肝脏功能衰竭的可能性，以降低复制肝硬化模型所造成的死亡率。
治疗程序：对照组(A)模型组(B组)、阳性药组(甘利欣C组)、治疗低剂量组(D组)、 治疗中剂量组(E组)、治疗高剂量组(F组)的肝硬化模型建模开始至8周后，将大鼠用乙醚 麻醉，眼底静脉丛取血，进行血清肝功能生化指标的检测；阳性药组背部皮下注射甘利欣溶 液，注射量为12.5mg/kg。每天注射1次，持续4周；rLZ-8低、中、高剂量组按浓度分别为 15ug/kg、30ug/kg、60ug/kg的剂量背部皮下注射。每天注射1次，持续4周；实验至12周 末，禁食、不禁水18个小时，乙醚麻醉，采用大鼠摘眼球取血法，留取血的样本约10ml，离心 取血清后放入-20℃的低温冰箱，用来进行血清生化指标等的检测。
实验结果：复制动物肝纤维化模型，随着大鼠肝硬化程度的加重，大鼠体质量明显 减轻，大鼠的组间体质量比较，正常组与模型组之间的差异有统计学意义，P＜0.05。模型组 与灵芝免疫调节蛋白低剂量组之间的差异有统计学意义，P＜0.05。模型组与灵芝免疫调节 蛋白中剂量组之间的差异有统计学意义P＜0.05。模型组与灵芝免疫调节蛋白高剂量组之 间的差异有统计学意义，P＜0.05。对照组的体质量增高较为明显，模型组的体质量最低，治 疗组相对于模型组，体质量有所上升。预防高、中、低剂量组相对于模型组比较差异不显著， P＞0.05。
由于肝硬化存在门静脉高压，肝湿重有所升高。统计显示，模型组的肝湿重最高， 正常组的较低，治疗组的肝湿重相对于模型组有所下降。各组之间的差异有统计学意义， P＜0.05。通过表7可看出，对照组的肝湿重均值最低，幅度变化较小；模型组的肝湿重均值 最高，变化幅度较大；治疗组的肝湿重相对于模型组均值有所下降。肝指数的变化趋势大体 也是如此，对照组最低，模型组最高，治疗组相对于模型组有所下降。其中，rLZ-8高剂量组 的效果最为明显。
[根据细则26改正08.08.2014]　
表7  rLZ-8对肝脏纤维化大鼠模型的体质量、肝湿重、肝指数的影响
Figure WO-DOC-FIGURE-9
与对照组比较，*P＜0.05；与模型组相比，#P＜0.05。
在本实验中，经CCl4：橄榄油(1∶1)复制大鼠肝纤维化模型8周，其大鼠血清中 AST、ALT酶活性与正常对照组比较均显著性升高(P＜0.05)，差异具有统计学意义，ALB含量 与正常对照组比较显著性降低(P＜0.05)，差异具有统计学意义，复制模型成功后按不同剂 量每天给大鼠注射LZ-8药物，在给药10天、20天、30天时，对大鼠血清中的AST、ALT酶活 性、ALB含与量测定。结果rLZ-8治疗组随着给药时间增加，大鼠血清中AST、ALT酶活性逐渐 降低，ALB含量逐渐升高，与模型组比较差异显著(P＜0.05)，具有统计学意义。rLZ-8中剂量 效果最好。
[根据细则26改正08.08.2014]　
表8 rLZ-8对肝脏纤维化大鼠模型血清中ALT(U/L)、A5T(L/L)、ALB(g/L)的活力影响
Figure WO-DOC-FIGURE-10
注：*▲#分别表示ALT、AST、ALB与模型组对比p＜0.05。

Claims (7)

  1. 重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药物中的应用。
  2. 如权利1所述组织纤维化，其特征在于包括肺组织纤维化。
  3. 如权利1所述组织纤维化，其特征在于包括心脏组织纤维化。
  4. 如权利1所述组织纤维化，其特征在于包括肾脏组织纤维化。
  5. 如权利1所述组织纤维化，其特征在于包括肝脏组织纤维化。
  6. 如权利1所述药物，其特征在于该药物制剂核心成分是由权利要求1所述的重组灵 芝免疫调节蛋白(rLZ-8)和任选的药学可接受的辅剂组成。
  7. 如权利要求1所述的药物制剂给药途径为口服和非肠道给药，其中，口服包括口服 液，片剂，丸剂和胶囊；非肠道给药包括外用药和注射剂。
PCT/CN2014/079833 2014-02-12 2014-06-13 重组灵芝免疫调节蛋白(rLZ-8)在制备治疗组织纤维化药物中的应用 WO2015120679A1 (zh)

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