Application of Cm-CATH2 in preparation of medicine for treating silicosis

Abstract

The invention provides application of Cm-CATH2 in preparation of a medicine for treating silicosis, and belongs to the technical field of biological medicine. The invention provides an application of Cm-CATH2 and / or pharmaceutically acceptable salt of Cm-CATH2 in preparation of medicines for treating silicosis, a treatment effect of Cm-CATH2 on a silicosis animal model is verified by constructing the silicosis animal model, and a result shows that Cm-CATH2 can act on the silicosis in a multi-target manner after an early-stage silicosis animal model, so that the treatment effect of Cm-CATH2 on the silicosis is improved, and the treatment effect of Cm-CATH2 on the silicosis is improved. According to the present invention, lactic dehydrogenase (LDH) is down-regulated to reduce the oxidative stress level, down-regulate matrix metalloproteinase 2 (MMP2) and extracellular matrix glycosaminoglycan (GAG), and finally reduce the level of collagen III (COL-III) so as to achieve the anti-fibrosis effect, and the multi-target effect can effectively alleviate the fibrosis progress, such that the Cm-CATH2 can be used for treating silicosis, has the dose effect, and can provide the anti-fibrosis effect, and the high-dose effect is good.

Description

Technical Field

[0001] The present invention provides the field of biomedicine technology, and specifically relates to the use of Cm-CATH2 in preparing a drug for treating silicosis. Background Art

[0002] Silicosis is a lung disease caused by long-term inhalation of large amounts of free silicon dioxide (SiO 2 ) A systemic disease with pulmonary fibrosis as the main lesion caused by dust particles. Workers who have been engaged in mining, quarrying, tunneling, and production in quartz powder factories, glass factories, refractory materials factories, ceramic factories, and enamel factories for a long time are susceptible to this disease. Silicosis is the most serious occupational disease, characterized by slow development. Even after leaving the silica dust operation, the lesion continues to develop slowly. Most patients develop the disease 10 to 15 years after exposure to silica dust. Silicosis has lung function damage in the early stage, but because the lungs have a strong compensatory ability, patients are often asymptomatic. As the lesions develop, especially when combined with pulmonary tuberculosis and cor pulmonale, respiratory and cardiopulmonary dysfunction of varying degrees gradually appear. The basic lesions of silicosis are the formation of silicosis nodules in the lung tissue and diffuse interstitial fibrosis.

[0003] There is currently no effective treatment for silicosis, and all current treatments are inadequate. Although lung lavage is feasible, it is limited by indications and contraindications, and can aggravate the condition when the condition is severe or combined with other lung diseases. Drug therapy is the most convenient, economical and efficient treatment method, but its current efficacy is limited. Hanfangji A is a clinically recommended drug, but its effect still needs to be improved. Summary of the invention

[0004] The present invention provides use of Cm-CATH2 in preparing a drug for treating silicosis. The Cm-CATH2 acts on silicosis at multiple targets and can effectively alleviate the progression of fibrosis.

[0005] The present invention provides the potential application of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the preparation of a drug for treating silicosis.

[0006] In a preferred embodiment of the present invention, the pharmaceutically acceptable salts of Cm-CATH2 include sodium salt and phosphate salt.

[0007] In a preferred embodiment of the present invention, the therapeutic effect of the drug includes at least one of the following: reducing LDH to reduce the level of oxidative damage, down-regulating MMP2, down-regulating GAG, reducing the level of collagen III and alleviating the progression of fibrosis.

[0008] In a preferred embodiment of the present invention, the reducing the level of oxidative damage comprises reducing the level of oxidative damage caused by at least one of the following oxidants: 2 O2 OH - and O 2 - .

[0009] In a preferred embodiment of the present invention, the dosage form of the drug for treating silicosis includes an injection.

[0010] The present invention also provides a medicine for treating silicosis, wherein the active ingredient includes Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2, and also includes pharmaceutically acceptable excipients.

[0011] In a preferred embodiment of the present invention, the dosage form of the drug includes injection.

[0012] In a preferred embodiment of the present invention, the concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the injection is not less than 2 mg / mL.

[0013] In a preferred embodiment of the present invention, the solvent of the injection solution includes sterile physiological saline.

[0014] In a preferred embodiment of the present invention, based on rats, the administration method of the injection solution includes 400 μg intravenous injection / each time.

[0015] Beneficial effects: The present invention constructs a silicosis animal model and uses Cm-CATH2 to treat the silicosis animal model. The results show that after the Cm-CATH2 acts on the silicosis animal model, it can act on silicosis at multiple targets, significantly reduce the level of oxidative stress, down-regulate metalloproteinase MMP2 and extracellular matrix GAG, and ultimately reduce the level of collagen III, thereby achieving an anti-fibrosis effect. The multi-target effect can effectively alleviate the progression of fibrosis. Therefore, the Cm-CATH2 can be used to treat silicosis, and there is a dose effect, and a high dose has a good effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The photographs of the lungs of silicosis rats under different treatments are shown in Figure A: Gamble solution control group; B: silicosis group; C: Cm-CATH2 low-dose group; D: Cm-CATH2 high-dose group; E: Cm-CATH2 control group; F: tetrandrine group;

[0017] Figure 2 The HE staining results of lung tissues under different treatments are shown in Figure A: Gamble solution control group; B: silicosis group; C: low-dose Cm-CATH2 group; D: high-dose Cm-CATH2 group; E: Cm-CATH2 control group; F: tetrandrine group;

[0018] Figure 3These are the results of Masson staining of lung tissue under different treatments, A: Gamble solution control group; B: silicosis group; C: low-dose Cm-CATH2 group; D: high-dose Cm-CATH2 group; E: Cm-CATH2 control group; F: tetrandrine group. DETAILED DESCRIPTION

[0019] The present invention provides the use of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the preparation of a medicine for treating silicosis.

[0020] The Cm-CATH2 described in the present invention is a molecule of the Cathelicidins family, has good antibacterial and anti-inflammatory activity, can recruit immune cells, and exert immunomodulatory function. The Cm-CATH2 described in the present invention is a linear polypeptide containing 33 amino acid residues, a theoretical isoelectric point of 12.96, a molecular weight of 4089.97Da, and a net charge of +12. Its source, preparation method and toxicity can refer to the contents disclosed in Chinese patent CN106188265A. The Cm-CATH2 used in the embodiment of the present invention is powdered, and the purity of Gill Biochemical synthesis is 99.7%.

[0021] In a preferred embodiment of the present invention, the pharmaceutically acceptable salts of Cm-CATH2 include sodium salt and phosphate salt.

[0022] In the present invention, after silica dust enters the lung tissue, it first reacts with alveolar macrophages, which continuously stimulate and activate macrophages and neutrophils to produce reactive oxidative metabolites (H 2 O 2 OH - , O 2 - After the alveolar macrophages engulf dust, a respiratory burst occurs, stimulating the intracellular electron transfer system and secreting various active mediators, which eventually trigger lipid peroxidation reactions. Under normal conditions, LDH is present in the cytoplasm and lysosomes of alveolar macrophages with intact membrane structures. The content in the alveolar cavity is very small. After the alveolar macrophage cell membrane is severely damaged, it will appear in large quantities in the alveoli; therefore, increased LDH enzyme activity in bronchoalveolar lavage fluid is an important indicator of lung macrophage damage, dissolution and necrosis. When dust acts on tissue cells and changes the permeability of their membranes, LDH in the cells overflows to the outside of the cells. Therefore, changes in serum LDH in patients with pneumoconiosis are one of the auxiliary indicators for the diagnosis of pneumoconiosis. The LDH level in patients with silicosis was significantly higher than that in the control group.

[0023] In the early stage of pulmonary fibrosis, i.e. the alveolitis stage, the MMP-2 / TIMP-1 ratio increases, and its degradation effect is enhanced, causing damage to lung tissue and triggering pulmonary fibrosis. In the later stage, i.e. the fibrosis stage, the MMP-2 / TIMP-1 ratio decreases, its degradation effect weakens, matrix deposition increases, and stable pulmonary fibrosis is formed. One of the basic components of the extracellular matrix is ​​GAG. Studies have found that the total amount of GAG in silicosis tissue increases, and the higher the degree of fibrosis, the denser its distribution, and it exists with collagen fibers. Collagen III is one of the typical markers of fibrosis. Therefore, in an embodiment of the present invention, the above-mentioned markers are detected.

[0024] In one embodiment of the present invention, a silicosis rat model was treated with Cm-CATH2. In terms of gross changes in the lungs, high doses of Cm-CATH2 had similar effects to tetrandrine, with normal morphology, light red color, uniform color, soft texture, smooth surface of the lung lobes, and basically disappeared nodules and hemorrhages. After HE staining of lung tissue, high doses of Cm-CATH2 can significantly reduce the degree of fibrosis in lung tissue. It has been verified that the Cm-CATH2 can act on silicosis at multiple targets, by reducing the level of oxidative stress, down-regulating metalloproteinases MMP2 and GAG, and ultimately reducing the level of collagen III, thereby achieving an anti-fibrotic effect, and thus can effectively alleviate the progression of fibrosis. Therefore, in the present invention, the therapeutic effects of the drug include at least one of the following: down-regulating LDH to reduce the level of oxidative damage, down-regulating MMP2, down-regulating extracellular matrix GAG, reducing the level of collagen III (COL-III), and alleviating the progression of fibrosis. The reduction of oxidative damage levels in the present invention includes reducing the level of oxidative damage caused by at least one of the following oxidants: H 2 O 2 OH - and O 2 - .

[0025] The present invention also provides a medicine for treating silicosis, wherein the active ingredient includes Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2, and also includes pharmaceutically acceptable excipients.

[0026] In a preferred embodiment of the present invention, the dosage form of the drug includes an injection, and the concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the injection is not less than 2 mg / mL. The solvent of the injection includes sterile physiological saline. In terms of rats, the administration method of the injection includes 400 μg intravenous injection / each time.

[0027] In order to further illustrate the present invention, the use of Cm-CATH2 provided by the present invention in the preparation of a drug for treating silicosis is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0028] The materials used in the embodiments of the present invention are conventional materials in the art unless otherwise specified, and the experimental methods used are also conventional methods in the art unless otherwise specified.

[0029] 1. Construction of silicosis animal model:

[0030] Male SD rats were purchased from Changsha Tianqin Biotechnology Co., Ltd., license number SCXK (Xiang) 2024-0021. They were raised in the SPF animal room of Guizhou University of Traditional Chinese Medicine, with a total of 48 rats, each weighing 250 g. The quality certificate number is SCXK (Guizhou) 2021-0003; the experimental facility certificate number is SYXK (Guizhou) 2021-0005. The temperature is about 25°C and the humidity is about 45%. After one week of adaptive feeding, animal modeling was performed. Prepare Gamble solution with a 0.22 μm filter membrane for sterilization and set aside. Weigh 2500 mg SiO 2 After high pressure sterilization, dry for 3 hours for later use. Use 50mL Gamble solution to fully resuspend SiO 2 , making the concentration 50mg / mL, and using a sterile 1mL syringe to infuse 1mL into each rat. The infusion of Gamble solution was used as the control. The tetrandrine injection was used as the positive control (TGS), and the high-dose injection of Cm-CATH2 after infusion of Gamble solution was used as the negative control group (Cm-CATH2 control group). The drug was administered in the early stage of silicosis (i.e., 21 days after dust stimulation).

[0031] The composition of Gamble solution: MgCl 2 6H 2 O 0.212g / L, NaCl 6.415g / L, Na 2 HPO 4 0.148g / L, Na 2 SO 4 ·2H 2 O 0.179g / L, CaCl 2 ·4H 2 O 0.319 g / L, NaHCO 3 2.703g / L, sodium tartrate 0.180g / L, sodium citrate 0.186g / L, sodium lactate 0.175g / L, sodium pyruvate 0.172g / L and glycine 0.118g / L, pH = 7-7.4.

[0032] 2. Preparation of Cm-CATH2 injection

[0033] Cm-CATH2 powder (purity 99.7%, Gill Biochemical) was weighed and dissolved in sterile saline. The concentrations were 1 mg / mL (low-dose group) and 2 mg / mL (high-dose group).

[0034] 3. Intravenous administration

[0035] Each rat was injected with 0.2 mL of the drug into the tail vein every 3 days for a total of 5 times.

[0036] 4. Detection of efficacy evaluation indicators

[0037] After 36 days of administration, the rats were killed and their lung tissues were collected. The lungs were observed and HE (hematoxylin-eosin) and Masson (trichrome) stained. Sterile PBS was added for grinding. Centrifuged at 4°C, 3000rpm for 25min, the supernatant was taken, and ELISA was performed according to the instructions of the kit. The detection indicators were LDH, MMP2, GAG and collagen III.

[0038] Example 1

[0039] 1. Gross changes of rat lungs

[0040] The gross findings of the rat lungs are as follows Figure 1 As shown, the lungs of rats in the Gamble solution control group were bright red, smooth, soft, and without focal hemorrhages, which was the same as the Cm-CATH2 control group; the lungs of rats in the silicosis group were swollen, gray, hard, and solid foci were formed, and scattered, raised chestnut-shaped gray-white nodules and old hemorrhages were visible on the surface; the lung tissues in the Cm-CATH2 low-dose group were gray, swollen, and rough, with surface nodules and focal hemorrhages. The Cm-CATH2 high-dose group had normal morphology, light red, uniform color, soft texture, smooth lung lobe surface, and nodules and hemorrhages basically disappeared; the rats in the tetrandrine group were similar to the Cm-CATH2 high-dose group.

[0041] 2. HE staining of lung tissue

[0042] The staining results of rat lung tissue are as follows Figure 2As shown. Compared with the Gamble control group, the lung tissue of rats in the silicosis group was significantly different. Some alveoli shrank and became smaller, the alveolar septa widened, the alveolar structure was severely disordered, and the interstitial blood vessels were dilated and congested, and the fibrous tissue was significantly proliferated. The degree of lung tissue damage in the low-dose Cm-CATH2 group was not much different from that in the silicosis group. Compared with the silicosis group, the high-dose Cm-CATH2 group had a reduced degree of lung tissue fibrosis, less alveolar collapse, less widening of alveolar septa, less fibrous tissue in the interstitium, and a reduced number of fibroblasts. Compared with the Gamble control group, the integrity of the lung tissue in the Cm-CATH2 control group was similar, with basically no fibrous tissue hyperplasia.

[0043] 3. Masson staining of lung tissue

[0044] Masson staining results Figure 3 As shown, the lung tissue structure of rats in the Gamble solution control group was clear, the alveolar structure was intact, and there was no obvious blue collagen fiber deposition, which was the same as the Cm-CATH2 control group; the blue-stained area of ​​the lung tissue of rats in the silicosis group increased, the alveolar wall was thickened, the collagen fiber deposition was significantly increased, and the pulmonary fibrosis was significantly aggravated; compared with the silicosis group, there was no significant difference in the blue collagen fiber deposition in the low-dose Cm-CATH2 group, and the lung tissue structure of the high-dose Cm-CATH2 group and the tetrandrine group was slightly damaged, and the blue collagen fiber deposition was significantly reduced.

[0045] 4. ELISA detection of LDH, GAG, MMP2, COL-III

[0046] The results of ELISA tests are shown in Tables 1 to 4. In the early stage of silicosis, the levels of LDH, MMP2, GAG, and COL-III in the silicosis group increased significantly compared with the control group, which is consistent with the characteristics of silicosis. Compared with the silicosis group, low-dose Cm-CATH2 was ineffective, while high-dose significantly reduced the levels of LDH, MMP2, and COL-III; although TGS significantly reduced LDH and MMP2, COL-III was only slightly lower than that in the silicosis group and could not significantly affect its level. Compared with TGS, low / high-dose Cm-CATH2 significantly reduced LDH, and high-dose Cm-CATH2 significantly reduced COL-III. At the same time, high-dose Cm-CATH2 and TGS significantly reduced MMP2 and GAG, and the effects of the two were comparable, but low-dose was ineffective. Therefore, in general, high-dose Cm-CATH2 has better effects on LDH and COL-III than TGS, especially in the early stage of silicosis, TGS has poor effect on reducing collagen III production: while the effects on MMP2 and GAG are not much different, so high-dose Cm-CATH2 is better than TGS in the early stage of silicosis.

[0047] In terms of drug toxicity and side effects, compared with the Gamble solution control group, although Cm-CATH2 downregulated the levels of LDH, MMP2, and GAG in the control group, it did not change the collagen III content, indicating that the injection of Cm-CATH2 can complete lung tissue repair faster and therefore has no toxic side effects.

[0048] In Tables 1 to 4, a relative to the Gamble solution control group, the difference was significant, P<0.05; b relative to the silicosis group, the difference was significant, P<0.05; c relative to the TGS group, the difference was significant, P<0.05; d relative to the Cm-CATH2 high-dose group, P<0.05.

[0049] Table 1 Effect of Cm-CATH2 on LDH in silicosis rats

[0050] Group n LDH (pg / mg.pro) Gamble solution control group 8 5555.3275±551.25371 Silicosis Group 8 <![CDATA[7556.5293±1084.24995 a ]]> Cm-CATH2 low dose group 8 <![CDATA[4334.3747±277.04159 abc ]]> Cm-CATH2 high dose group 8 <![CDATA[4333.1505±212.25233 abc <!-- 4 -->]]> Cm-CATH2 control group 8 <![CDATA[4707.4494±191.35429 a ]]> Tetrandrine group (TGS) 8 <![CDATA[5273.4076±498.53223 b ]]>

[0051] Table 2 Effects of Cm-CATH2 on MMP2 in silicotic rats

[0052]

[0053]

[0054] Table 3 Effects of Cm-CATH2 on GAG in silicotic rats

[0055] Group n GAG (μg / g.Pro) Gamble solution control group 8 2.3643±0.29903 Silicosis Group 8 <![CDATA[2.8583±0.26026 a ]]> Cm-CATH2 low dose group 8 <![CDATA[2.5725±0.59739 cd ]]> Cm-CATH2 high dose group 8 <![CDATA[2.1990±0.28914 b ]]> Cm-CATH2 control group 8 <![CDATA[2.0684±0.10693 a ]]> Tetrandrine group (TGS) 8 <![CDATA[2.0641±0.21880 b ]]>

[0056] Table 4 Effect of Cm-CATH2 on collagen III in silicotic rats

[0057]

[0058]

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the preparation of a medicament for treating silicosis.

2. The application according to claim 1, characterized in that: The pharmaceutically acceptable salts of Cm-CATH2 include sodium salt and phosphate salt.

3. The application according to claim 1, characterized in that: The therapeutic effects of the drug include at least one of the following: reducing the level of oxidative damage, down-regulating metalloproteinase MMP2, down-regulating extracellular matrix GAG, reducing the level of collagen III and effectively alleviating the progression of fibrosis.

4. The use according to claim 3, characterized in that: The reducing of the level of cellular oxidative damage comprises reducing the level of oxidative damage caused by at least one of the following oxidants: H2O2, OH - and O2 - .

5. The use according to claim 1, characterized in that: The dosage form of the drug for treating silicosis includes injection.

6. A drug for treating silicosis, characterized in that: The active ingredient includes Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2, and also includes a pharmaceutically acceptable excipient.

7. The drug according to claim 6, characterized in that: The dosage form of the drug includes injection.

8. The drug according to claim 7, characterized in that: The concentration of Cm-CATH2 and / or a pharmaceutically acceptable salt of Cm-CATH2 in the injection is not less than 2 mg / mL.

9. The drug according to claim 7 or 8, characterized in that The solvent of the injection includes sterile physiological saline.

10. The drug according to claim 9, characterized in that: In terms of rats, the administration method of the injection solution includes 400 μg intravenous injection / each time.

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