Application of haematococcus pluvialis in preparation of medicine for preventing and treating pulmonary fibrosis
By using Radix Chronicus or its preparations, inflammatory cell infiltration and collagen cell deposition are reduced, and the existing drugs have not been ideal for treating pulmonary fibrosis and obvious side effects are solved, providing a safer and more effective treatment plan.
Patent Information
- Application Number
- CN202510103588.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-04-08
AI Technical Summary
The existing drugs used to treat pulmonary fibrosis are not effective, and the side effects are obvious, resulting in serious impact on the quality of life of patients.
Using Radix Chronicus or its preparations, it is used as a new drug to prevent and treat pulmonary fibrosis by reducing inflammatory cell infiltration and collagen cell deposition.
It provides a safer and more effective treatment method for pulmonary fibrosis, reducing drug side effects, improving the safety of treatment, and providing new treatment options as a supplement or alternative to existing treatment methods.
Smart Images
Figure CN119770532A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medicines, and in particular to the use of Haematococcus pluvialis in the preparation of medicines for preventing and treating pulmonary fibrosis. Background Art
[0002] Pulmonary fibrosis (PF) is a chronic progressive, interstitial fibrotic lung tissue damage disease. Its pathogenesis is still unclear and it is the terminal manifestation of various diffuse interstitial lung diseases. This type of disease is characterized by progressive exertional dyspnea and deterioration of lung function. Its pathological mechanism is not fully understood. In recent years, many studies have considered that it is due to pathogens, autoimmunity, chemical factors and other factors that damage the lungs, causing abnormal inflammation and damage repair reactions in the body. The degree and type of this reaction are affected by age, genetic factors, and environmental factors. Abnormal inflammation and damage repair reactions can cause pathological alveolar microenvironment, leading to pathological processes such as alveolar epithelial cell damage, epithelial-mesenchymal transition and abnormal activation of fibroblasts, and a large amount of extracellular matrix deposition, which ultimately causes progressive destruction of lung tissue structure and irreversible loss of function. At present, the pathogenesis of PF is not fully understood. The two drugs used in the clinic to treat PF, pirfenidone and nintedanib, are not ideal, with obvious side effects, which seriously affect the quality of life of patients. Therefore, it is of great clinical significance to find new drugs for the prevention and treatment of pulmonary fibrosis. Summary of the invention
[0003] Haematococcus pluvialis Haematococcus Pluvialis , HP) is a freshwater green algae that can accumulate a large amount of astaxanthin under stress conditions such as nitrogen deficiency, high light and high salt, which can reach 5% of the cell dry weight. It is the best source of natural astaxanthin in nature. Astaxanthin is a red, fat-soluble carotenoid. It is the substance with the strongest antioxidant capacity discovered so far. It also has the effects of inhibiting tumors, anti-inflammatory, and improving immunity. It is widely used in food, medicine, cosmetics, aquaculture and other fields. Based on these characteristics, the use of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of various diseases has gradually attracted attention. In view of this, the present invention proposes the use of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of pulmonary fibrosis, aiming to provide a new strategy for the prevention and treatment of pulmonary fibrosis to improve the therapeutic effect of pulmonary fibrosis.
[0004] The present invention provides an application of Haematococcus pluvialis or a preparation thereof, wherein the application comprises any one of the following: A1) Application of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of pulmonary fibrosis; A2) Use of Haematococcus pluvialis as a medicament for inhibiting the process of fibroblast-to-myofibroblast transformation induced by TGF-β1 in human embryonic lung fibroblasts; A3) Use of Haematococcus pluvialis to reduce Fibronectin protein expression in human embryonic lung fibroblasts; A4) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of Fibronectin protein in human embryonic lung fibroblasts; A5) Use of Haematococcus pluvialis to reduce the expression of a-smooth muscle actin protein in human embryonic lung fibroblasts; A6) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of a-smooth muscle actin protein in human embryonic lung fibroblasts; A7) Use of Haematococcus pluvialis in the preparation of drugs for preventing and / or treating diseases related to pulmonary fibrosis.
[0005] The invention also provides a medicine for treating pulmonary fibrosis, wherein the active ingredient of the medicine is Haematococcus pluvialis.
[0006] Compared with the prior art, the present invention has the following beneficial effects: 1. Provide a safer and more effective method for treating pulmonary fibrosis, by utilizing the anti-inflammatory properties of Haematococcus pluvialis to reduce inflammatory cell infiltration, collagen cell deposition, etc., reduce drug side effects, and improve the safety of treatment; 2. The Haematococcus pluvialis of the present invention can be used as a supplement or substitute for existing treatment methods to provide patients with new treatment options, especially for patients who have poor response to or severe side effects of current clinical drugs for the treatment of pulmonary fibrosis (pirfenidone and nintedanib).
[0007] 3. The Haematococcus pluvialis of the present invention is of natural and safe origin, has a simple preparation and production process, is low in cost, has a large yield, and has a low treatment cost, which is affordable for more patients.
[0008] 4. The Haematococcus pluvialis of the present invention can not only treat pulmonary fibrosis, but also prevent its occurrence, slow down the progression of the disease to a certain extent, and improve the quality of life of patients.
[0009] In summary, the present invention provides a new treatment strategy for preventing pulmonary fibrosis through the multiple biological activities of Haematococcus pluvialis, and has significant clinical application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Various other advantages will become apparent to those skilled in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, throughout the accompanying drawings, the same reference symbols are used to represent the same components. In the accompanying drawings: Figure 1The effect of Haematococcus pluvialis on pulmonary respiratory function in a mouse pulmonary fibrosis model induced by bleomycin.
[0011] Figure 2 HE and Masson staining show the effects of Haematococcus pluvialis on lung tissue morphology and collagen deposition in a mouse pulmonary fibrosis model.
[0012] Figure 3 qRT-PCR was used to detect the effect of Haematococcus pluvialis on the expression level of fibrotic proteins in the HFL1 cell model induced by TGF-β1.
[0013] Figure 4 Immunofluorescence was used to detect the effect of Haematococcus pluvialis on the expression level of fibrotic proteins in the HFL1 cell model induced by TGF-β1. DETAILED DESCRIPTION
[0014] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0015] Example 1 Effect of Haematococcus pluvialis on improving lung function in bleomycin-induced pulmonary fibrosis model 1. Experimental Materials Hunan Slake Jingda Experimental Animal Co., Ltd. provided 30 SPF-grade C57BL / 6 male mice, Yunnan Aierfa Biotechnology Co., Ltd. provided Haematococcus pluvialis powder ( Haematococcus Pluvialis , HP) 2. Experimental Methods Thirty male mice were placed in an SPF standard environment for adaptive feeding for 7 days and then randomly divided into a control group, a model group, a low-dose group of Haematococcus pluvialis, a high-dose group of Haematococcus pluvialis, and a positive drug pirfenidone group, with 6 mice in each group. A bleomycin (BLM)-induced pulmonary fibrosis model was established in mice. First, the required surgical instruments (ophthalmic scissors, 1mL syringes, tweezers, absorbable surgical sutures, etc.) were prepared for autoclave sterilization, and Shutai 50, atropine sulfate, and bleomycin solutions were prepared. Then, each mouse was subcutaneously injected with 0.04mg / kg atropine sulfate solution, and 15min later, 60mg / kg Shutai 50 solution was injected intramuscularly to anesthetize the mice. After anesthesia, the mice were weighed, and the mice were placed supinely with their limbs spread out and fixed on the experimental operation board. The neck incision position was preset, and the neck skin at the incision position was wiped and disinfected with iodine cotton balls. The neck skin was clamped with sterile tweezers in the left hand and fixed, and the right hand used ophthalmic scissors to cut a horizontal incision of about 0.5cm, and then cut a longitudinal incision of about 1cm. Then, forceps were used to separate the subcutaneous muscle tissue of the neck to fully expose the trachea. The mice in the model group, Haematococcus pluvialis administration group, and pirfenidone administration group were instilled with BLM solution at a dose of 5 mg / kg into the trachea using a syringe, and the mice in the sham operation group were instilled with the same dose of saline into the trachea. Immediately after the instillation, the operating board was shaken left and right and tilted upward for 5 minutes to make the solution as evenly distributed as possible in the mouse lung tissue, and the wound was sutured with sutures. From the next day, the drugs were administered orally according to the grouping.
[0016] Control group: gavage with the same dose of normal saline daily; Model group: gavage with the same dose of normal saline daily; The group of 3 mg / kg Haematococcus pluvialis was intragastrically administered with the same dose of saline containing 3 mg / kg Haematococcus pluvialis daily; The group of 21 mg / kg Haematococcus pluvialis was intragastrically administered with the same dose of saline containing 21 mg / kg Haematococcus pluvialis daily; Pirfenidone group: daily gavage with the same dose of saline containing 300 mg / kg of Haematococcus pluvialis; The drug was administered continuously for 21 days. On the 22nd day, the experimental mice were tested for lung function, the surgical instruments were sterilized by high pressure, and atropine sulfate and Shutai 50 solutions were prepared to calibrate the PFT system. Atropine sulfate was used to subcutaneously inject 0.04 mg / kg atropine sulfate solution, and 60 mg / kg Shutai 50 solution was injected intramuscularly 15 minutes later. After waiting for the mice to be anesthetized, they were fixed on the experimental operation board in a supine position, the neck incision of the mice was cut, and the trachea was exposed for endotracheal intubation. The mice were placed in the animal fixing chamber of the PFT system, and the intubation was connected to the pulmonary function test system to detect various indicators of the mouse's lung function. Then, the mouse lung tissue was taken, fixed with 4% paraformaldehyde, dehydrated, and embedded in paraffin, and then sliced (5 mm thick), and then HE staining and Masson staining were performed to observe the pathological conditions and fibrosis of the lung tissue, respectively.
[0017] 3. Experimental Results The present invention uses BLM-induced pulmonary fibrosis model mice to detect the anti-pulmonary fibrosis effect of HP in vivo. After continuous intragastric administration of two different doses of HP for 21 days, the respiratory function indicators of each group of mice are detected using a pulmonary respiratory function instrument. The results are as follows Figure 1 As shown: (1) Compared with the normal group mice, the model group mice showed a significant decrease in FEV100 (100 millisecond forced expiratory volume), FVC (forced vital capacity), IC (deep inspiratory volume), VC (vital capacity), and Cchord (lung compliance), while the high-dose HP group mice showed a significant increase in these parameters, which could restore the respiratory function damage caused by BLM; in terms of RI (airway resistance), compared with the normal group mice, the model group mice showed a significant increase, while the high-dose HP mice showed a significant decrease, recovering to the level of the normal group mice; (2) High-dose HP showed a therapeutic effect similar to that of the positive drug pirfenidone; (3) Although low-dose HP did not show a significantly different therapeutic effect, its various parameter indicators were between the model group and the high-dose HP group, which may have a certain alleviating effect.
[0018] To further explore the therapeutic effect of HP on mouse pulmonary fibrosis, HE staining and Masson staining were performed on the mouse lung tissue to evaluate the morphological structure and fibrous structure deposition of the mouse lung tissue. Figure 2 As shown, HE staining results showed that compared with the normal control group mice, the alveolar structure of the model group mice was destroyed, and there were a lot of inflammatory infiltrations in the lung tissue; after receiving different doses of HP treatment, the damage to the lung tissue was alleviated to varying degrees compared with the model group, and the high-dose HP group showed a lung tissue structure similar to that of the normal group; the positive drug PFD group mice also showed a lung tissue structure similar to that of the normal group.
[0019] Masson staining Figure 2 As shown in the figure, the lung tissue of mice in the normal control group showed no fibrosis, while the lung tissue of mice in the model group showed a large amount of collagen deposition, increased fibroblasts, and destroyed alveolar structure; after treatment with different doses of HP, compared with the lung tissue of mice in the model group, the deposition of collagen fibers in the lung tissue was alleviated to varying degrees, and the degree of alveolar structural disorder was reduced, especially the high-dose HP group showed very little collagen deposition, and the alveolar structure was relatively complete, similar to the normal control group; the positive drug PFD group also had no obvious collagen fiber deposition, similar to the normal control group. The above results show that HP can reduce the destruction of mouse lung tissue structure and collagen deposition caused by BLM, and improve pulmonary fibrosis; high-dose HP shows a similar therapeutic effect to the positive drug PFD.
[0020] In summary, Haematococcus pluvialis can reduce the damage of BLM to the lung tissue structure of mice and restore lung function by reducing inflammatory cell infiltration and alleviating collagen deposition, and its effect is comparable to that of pirfenidone.
[0021] Example 2 Haematococcus pluvialis improves the expression of fibrosis-related proteins in human embryonic lung fibroblasts induced by TGF-β1 at the genetic level 1. Experimental Materials Human embryonic lung fibroblast HFL1 (catalog number: GNHu28) was purchased from the Cell Bank of the Chinese Academy of Sciences, and Ham's F-12K complete medium was purchased from Shanghai Meiwan Biotechnology Co., Ltd. 2. Experimental Methods Human embryonic lung fibroblast HFL1 cells were cultured in a 37°C, 5% CO2 incubator. The culture medium used was Ham's F-12K complete culture medium containing 10% fetal bovine serum. Penicillin-streptomycin solution (1%) can reduce culture medium contamination. When the cell confluence reached 80%-90%, 0.25% trypsin was used for digestion for about 3 minutes. HFL1 cells were cultured at 1 × 10 5 Each well was evenly inoculated in a six-well plate.
[0022] After serum-free treatment for 24 hours, the corresponding TGF-β1, Haematococcus pluvialis, and pirfenidone were added to the corresponding groups. The specific groups are as follows: Control group (Control): treated with serum-free medium for 24 hours and then added with F-12K complete medium; Model group: After 24 hours of serum-free treatment, F-12K complete medium containing 5 ng / ml TGF-β1 was added; Haematococcus pluvialis group (HP): after 24 h of serum-free treatment, F-12K complete medium containing 5 ng / ml TGF-β1 and 240 μg / mL HP was added; Pirfenidone group (PFD): after 24 h of serum-free treatment, complete medium containing 5 ng / ml TGF-β1 and 10 μM F-12K was added; After culturing for 48 hours, RNA from HFL1 cells was extracted. RNA was reverse transcribed into cDNA using the PrimeScript™ RT Master Mix kit and used as a template for quantitative PCR to detect the relative expression of the target gene. The kit used for quantitative PCR was TB Green Premix Ex Taq II. b-actin was used as the internal reference gene. -ΔΔCt The relative quantification method was used to calculate the expression level of the target gene.
[0023] 3. Experimental Results The mRNA expression levels of fibrosis markers α-SMA and FN in HFL1 cells were detected by qRT-PCR. The results showed that compared with the normal group, the expression levels of these two genes in the model group were significantly upregulated; after treatment with high doses of HP (240 μg / mL), the mRNA expression levels of α-SMA and FN were significantly decreased ( Figure 3 ). It can be concluded that Haematococcus pluvialis can improve the expression of fibrosis-related factors (α-SMA and FN) mRNA in human embryonic lung fibroblasts induced by TGF-β1.
[0024] Example 3 Haematococcus pluvialis improves the expression of fibrosis-related proteins in human embryonic lung fibroblasts induced by TGF-β1 at the protein level 1. Experimental Materials Human embryonic lung fibroblast HFL1 (catalog number: GNHu28) was purchased from the Cell Bank of the Chinese Academy of Sciences, and Ham's F-12K complete medium was purchased from Shanghai Meiwan Biotechnology Co., Ltd. 2. Experimental Methods Human embryonic lung fibroblasts HFL1 were cultured in a 37°C, 5% CO2 incubator. The culture medium used was Ham's F-12K complete culture medium containing 10% fetal bovine serum. Penicillin-streptomycin solution (1%) can reduce culture medium contamination. When the cell confluence reached 80%-90%, 0.25% trypsin was used for digestion for about 3 minutes. A cover glass was placed in a six-well plate in advance, and 1×10 5 The cells were placed in a 5% CO2 incubator for 24 h until the cells adhered to the wall.
[0025] Corresponding TGF-β1 and Haematococcus pluvialis were added to the corresponding groups, and the specific groups were as follows: Control group (Control): treated with serum-free medium for 24 hours and then added with F-12K complete medium; Model group: After 24 hours of serum-free treatment, F-12K complete medium containing 5 ng / ml TGF-β1 was added; Haematococcus pluvialis group (HP): after 24 h of serum-free treatment, F-12K complete medium containing 5 ng / ml TGF-β1 and 240 μg / mL HP was added; After 48 hours of continuous culture, the cells were fixed with 4% paraformaldehyde for 15 minutes and treated with 0.5% triton for 15 minutes, then blocked with 10% goat serum for 30 minutes, and incubated with FN antibody (1:200 dilution) and a-SMA antibody (1:200 dilution) at 4°C overnight. Fluorescent secondary antibody (1:100 dilution) was then added and incubated at room temperature in the dark for 1 hour. Finally, DAPI was added and incubated in the dark for 15 minutes, then the sections were sealed and observed under a fluorescence microscope.
[0026] 3. Experimental Results like Figure 4 As shown in the figure, the results of immunofluorescence staining were basically consistent with the results of RT-qPCR and the staining results of mouse lung tissue sections induced by bleomycin. α-SMA, as a marker of myofibroblasts, had a low fluorescence intensity in the control group; after treatment with TGF-β1, the fluorescence intensity was significantly enhanced; after treatment with HP, the fluorescence intensity was significantly reduced and returned to the level of the control group. FN, as a fibronectin, had a low fluorescence intensity in the control group; after treatment with TGF-β1, the fluorescence intensity was significantly enhanced; after treatment with HP, the fluorescence intensity was significantly reduced and returned to the level of the control group. Based on the above results, Haematococcus pluvialis can improve the process of fibroblast-to-myofibroblast transformation in TGF-β1-induced human embryonic lung fibroblasts, and the expression of fibrotic proteins α-SMA and FN were significantly downregulated.
[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the specific implementation modes of the present invention are described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation modes of the present invention can still be modified or replaced by equivalents, and any modifications or equivalent replacements that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. An application of Haematococcus pluvialis or a preparation, characterized in that: The application includes any of the following: A1) Application of Haematococcus pluvialis in the preparation of drugs for the prevention and treatment of pulmonary fibrosis; A2) Use of Haematococcus pluvialis as a medicament for inhibiting the process of fibroblast-to-myofibroblast transformation induced by TGF-β1 in human embryonic lung fibroblasts; A3) Use of Haematococcus pluvialis to reduce Fibronectin protein expression in human embryonic lung fibroblasts; A4) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of Fibronectin protein in human embryonic lung fibroblasts; A5) Use of Haematococcus pluvialis to reduce the expression of a-smooth muscle actin protein in human embryonic lung fibroblasts; A5) Use of Haematococcus pluvialis in the preparation of a drug for reducing the expression of a-smooth muscle actin protein in human embryonic lung fibroblasts; A6) Use of Haematococcus pluvialis in the preparation of a medicament for preventing and / or treating diseases related to pulmonary fibrosis.
2. A drug for treating pulmonary fibrosis, characterized in that: The active ingredient of the medicine is Haematococcus pluvialis.