Application of hyssopus officinalis volatile oil and composition containing same in preparation of medicine for treating idiopathic pulmonary fibrosis

Abstract

According to the hyssopus officinalis volatile oil and the application of the composition containing the hyssopus officinalis volatile oil in preparation of the medicine for treating idiopathic pulmonary fibrosis, the hyssopus officinalis volatile oil and the composition containing the hyssopus officinalis volatile oil can remarkably reduce lung tissue inflammatory cell infiltration, reduce deposition of fibrotic mouse pulmonary interstitial collagen and fiber, and improve the pulmonary fibrosis effect. By down-regulating a TGF-beta1 / smad2 / 3 fibrosis classical signal channel and a TGF-beta1 / MAPK fibrosis non-classical signal channel, pulmonary interstitial collagen fiber deposition and expression of fibrosis marker proteins alpha-SMA and cologen I are reduced, so that the development process of pulmonary fibrosis is improved and delayed.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of Hyssopus officinalis volatile oil and a composition containing the same in preparing a medicine for treating idiopathic pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary fibrosis disease caused by multiple factors. It is mainly manifested by fibrosis of the extracellular matrix of lung tissue, severe destruction of alveolar structure, decreased pulmonary ventilation, obstructed gas exchange, and clinical manifestations of cough, dyspnea, and even respiratory failure. At present, the understanding of the pathogenesis of IPF generally believes that after lung injury, the inflammatory response is activated, which then stimulates transcription factors such as TGF-β1 to bind to the surface receptors of fibroblasts, resulting in the phosphorylation of downstream smad2 and smad3, and regulating the transformation of fibroblasts into myofibroblasts by regulating the expression of target genes; secondly, after TGF-β1 binds to the receptor, it can also activate downstream MAPK signals, further inducing the transformation of fibroblasts into myofibroblasts, as well as the production of fibrosis markers such as α-smooth muscle actin (α-SMA) and collagen.

[0003] Intratracheal instillation of bleomycin is the most widely used method to induce IPF animal models, which is mainly divided into two stages: 0-7 days after a single dose, activated macrophages and other cells secrete a large number of inflammatory factors, causing lung inflammation and alveolar epithelial cell apoptosis, which is also a sign of early pulmonary fibrosis; while continued damage and inflammation will trigger lung epithelial cell-mesenchymal transformation, and use fibroblasts to repair tissue cell damage caused by inflammatory response, ultimately leading to pulmonary fibrosis, that is, signs of fibrosis are observed on the 7th day, and total collagen deposition increases significantly on the 14th day, which is highly similar to the pathogenesis of human IPF.

[0004] Epidemiology shows that the course of most IPF patients deteriorates rapidly, but treatment options are very limited. Currently, clinical treatment mainly relies on drugs, such as pirfenidone and nintedanib, to slow the progression of pulmonary fibrosis, but these drugs have severe allergic reactions and liver and kidney damage, and the mortality rate is still high after treatment with the drugs. In addition, only a few cases involve lung transplantation in clinical practice, and the use of some anti-inflammatory drugs can only relieve symptoms and cannot organize the progression of fibrosis. With the understanding of the pathogenesis of IPF, therapeutic drugs that block fibrosis signaling pathways may be a more promising treatment for IPF.

[0005] Natural products are the most common source of drugs, and volatile oils are active ingredients extracted from traditional Chinese medicine. Hyssopus officinalis is a common medicinal material used by the Uyghur people to treat respiratory diseases such as colds, bronchitis, asthma, and pneumonia. Hyssopus officinalis volatile oil is extracted from the whole plant of Hyssopus officinalis by steam distillation. In our previous studies, it has been shown to have a good anti-asthmatic effect, but its effects in other aspects have not yet been developed. Summary of the invention

[0006] In order to solve the problems existing in the background technology, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides use of Hyssopus officinalis volatile oil and a composition comprising the same in the preparation of a medicament for treating idiopathic pulmonary fibrosis.

[0008] According to the above scheme, the Hyssopus officinalis volatile oil is prepared by steam distillation.

[0009] According to the above scheme, the Hyssopus officinalis volatile oil and the composition containing the same are used to reduce the infiltration of inflammatory cells in the lung tissue of fibrotic mice.

[0010] According to the above scheme, the Hyssopus officinalis volatile oil and the composition containing the same are used to reduce the deposition of collagen and fiber in the lung interstitium of fibrotic mice.

[0011] According to the above scheme, the Hyssopus officinalis volatile oil and the composition containing the same are used to reduce the expression of pulmonary fibrosis markers α-SMA and collagen I.

[0012] According to the above scheme, the Hyssopus officinalis volatile oil and the composition containing the same are used to reduce the expression of fibrosis signaling pathway related proteins TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK.

[0013] In a second aspect, the present invention provides a medicine for treating idiopathic pulmonary fibrosis, comprising Hyssopus officinalis volatile oil.

[0014] According to the above scheme, the medicine for treating idiopathic pulmonary fibrosis comprises a combination of Hyssopus officinalis volatile oil and other medicinal materials / compounds.

[0015] According to the above scheme, the drug for treating idiopathic pulmonary fibrosis also includes pharmaceutically acceptable additives, carriers and / or excipients.

[0016] The beneficial effects of the present invention are as follows: the present invention provides the use of Hyssopus officinalis volatile oil in the preparation of a drug for treating idiopathic pulmonary fibrosis; Hyssopus officinalis and a composition containing the same can significantly reduce the infiltration of inflammatory cells in lung tissue, reduce the deposition of collagen and fibers in the lung interstitium of fibrotic mice, and can reduce the deposition of lung interstitial collagen fibers and the expression of fibrosis marker proteins α-SMA and collagen I by down-regulating the TGF-β1 / smad2 / 3 classical fibrosis signaling pathway and the TGF-β1 / MAPK non-classical fibrosis signaling pathway, thereby improving and delaying the development of pulmonary fibrosis, and can be further developed as a drug for treating pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of HE staining in the present invention to evaluate the effect of Hyssopus officinalis volatile oil on lung inflammation;

[0018] Figure 2 The results of evaluating the effect of Hyssopus officinalis volatile oil on pulmonary fibrosis using Masson and Sirius red staining in the examples of the present invention;

[0019] Figure 3 The results of Western blot study on the effects of Hyssopus officinalis volatile oil on fibrosis markers α-SMA and collagen I in the examples of the present invention are shown;

[0020] Figure 4 The results of Western blot study of the effect of Hyssopus officinalis volatile oil on proteins related to fibrosis signaling pathways in the embodiments of the present invention. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] In order to solve the problem of lack of effective drugs for treating pulmonary fibrosis in the prior art, the present invention provides the use of Hyssop volatile oil and a composition containing the same in treating idiopathic pulmonary fibrosis. In the present invention, a mouse pulmonary fibrosis model is constructed by intratracheal instillation of bleomycin, and the effect of Hyssop volatile oil in treating pulmonary fibrosis is discussed. It is found that Hyssop volatile oil can significantly reduce the infiltration of inflammatory cells in lung tissue, and can reduce the deposition of pulmonary interstitial collagen fibers and the expression of fibrosis marker proteins α-SMA and collagen I by down-regulating the TGF-β1 / smad2 / 3 fibrosis classical signaling pathway and the TGF-β1 / MAPK fibrosis non-classical signaling pathway, thereby improving and delaying the development of fibrosis, and can be further developed as a drug for treating pulmonary fibrosis.

[0023] The invention provides application of Hyssopus officinalis volatile oil and a composition containing the same in preparing a medicine for treating idiopathic pulmonary fibrosis.

[0024] The hyssop volatile oil is prepared by steam distillation.

[0025] Specifically, the dried aerial part of the Hyssopus officinalis volatile oil is crushed, and the volatile oil is extracted according to the determination of volatile oil in Appendix 2204 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0026] In some specific embodiments, the Hyssopus officinalis volatile oil and the composition comprising the same are used to reduce the infiltration of inflammatory cells in lung tissue of fibrotic mice.

[0027] In some specific embodiments, the Hyssopus officinalis volatile oil and the composition comprising the same are used to reduce the deposition of collagen and fiber in the lung interstitium of fibrotic mice.

[0028] In some specific embodiments, the Hyssopus officinalis volatile oil and the composition comprising the same are used to reduce the expression of pulmonary fibrosis markers α-SMA and collagen I.

[0029] In some specific embodiments, the Hyssopus officinalis volatile oil and the composition containing the same are used to reduce the expression of fibrosis signaling pathway related proteins TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK.

[0030] The invention also provides a medicine for treating idiopathic pulmonary fibrosis, comprising hyssopus officinalis volatile oil.

[0031] In some specific embodiments, the medicament for treating idiopathic pulmonary fibrosis comprises a combination of Hyssopus officinalis essential oil and other medicinal materials / compounds.

[0032] In some specific embodiments, the drug for treating idiopathic pulmonary fibrosis further comprises a pharmaceutically acceptable additive, carrier and / or excipient.

[0033] The following is a specific example: the effect of Hyssopus officinalis volatile oil in the treatment of idiopathic pulmonary fibrosis (IPF) was determined.

[0034] This experiment used 8-week-old SPF male C57BL / 6J mice weighing 20±2g, which were housed in the barrier system of the Transformation Building of the Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology, and given standard diet and drinking water. The room temperature was controlled at 23±2℃, and the experiment began after 7 days of adaptation. The experimental protocol involving mice in the experiment was approved by the Animal Experiment Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology.

[0035] Mice were randomly divided into a normal group, a model group, a positive drug pirfenidone group, and a Hyssopus volatile oil group. Except for normal mice, mice in other groups were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, fixed on the operating table with endotracheal intubation in a supine position, and a plastic cannula with a flat end and connected to a syringe was inserted into the trachea. After success, a pipette was used to draw the bleomycin hydrochloride working solution and drip it into the cannula port. The dose was 3 mg / kg. After the mice inhaled all the drugs, they were immediately rotated upright to evenly distribute the inhaled drugs in the lungs. Then the cannula was gently pulled out and the mice were returned to their respective cages. Treatment began 3 hours after airway administration. The Hyssopus volatile oil group: the volatile oil dose was 0.739 μL / kg, and the positive drug pirfenidone group: the pirfenidone group dose was 100 mg / kg. The administration method was oral administration, once a day, for 7 and 14 days respectively. The mice were weighed during administration, and the status of the mice was observed and recorded.

[0036] According to the literature, mice are in the acute inflammatory stage when they are given bleomycin for 0-7 days. The left lung tissue of mice that were given bleomycin for 7 consecutive days was taken, immersed in 4% paraformaldehyde, fixed at room temperature for 24 hours, embedded in paraffin, sliced, and stained with HE to observe the inflammation of each group of mice. The results of HE (100×; scale length 50μm) are as follows Figure 1 As shown in the figure, inflammatory response broke out 7 days after modeling. Compared with the normal group, a large number of inflammatory cells infiltrated the interstitial tissue, bronchi and blood vessels of the lung tissue in the model group, and the alveolar structure was completely destroyed. Compared with the model group, the number of inflammatory cell infiltrations in the pirfenidone group was significantly reduced, and the alveolar structure was clearer and more complete. Similarly, in the Hyssopus officinalis volatile oil group, clear alveolar structure was visible, and the number of interstitial inflammatory cell infiltrations was significantly reduced, indicating that Hyssopus officinalis volatile oil has a good anti-inflammatory effect, can significantly inhibit the infiltration of lung tissue verification cells, and the effect is better than pirfenidone.

[0037] According to the literature, mice are in the fibrosis formation stage 7-14 days after administration of bleomycin. The left lung tissue of mice that were continuously administered for 14 days was taken, immersed in 4% paraformaldehyde, fixed at room temperature for 24 hours, embedded in paraffin, sliced, and stained with Masson and Sirius red to observe the pulmonary fibrosis of each group of mice. The results of Masson staining are shown in Figure 2 As shown in A, at 14 days after modeling, compared with the normal group, the model group had more blue-stained collagen in the lung tissue, with a large amount of collagen deposition, showing blue areas; compared with the model group, the pirfenidone group and the Hyssop volatile oil group could significantly inhibit the deposition of collagen. Figure 2B) Evaluation of the degree of fibrosis in the lung tissue of each group of mice showed that the model group had a significant increase in the score (average score of 7.7 in the model group and 0.7 in the normal group), indicating that the degree of fibrosis increased significantly. The scores of the pirfenidone group (average score of 3.7) and the hyssop essential oil group (average score of 2.7) decreased significantly, indicating that the degree of fibrosis was significantly improved, and the effect of hyssop essential oil was better than that of the positive drug pirfenidone. The results of Sirius red staining are shown in Figure 2. Figure 2 As shown in C, compared with the normal group, the model group had more red-stained fibers in the lung tissue, with a large amount of collagen fiber deposition, showing red areas; compared with the model group, the pirfenidone group and the Hyssop volatile oil group could significantly inhibit the deposition of collagen fibers. Furthermore, ImageJ software was used to quantify the fibrosis area of ​​the lung tissue of each group of mice. The results are shown in Figure 2 As shown in D, compared with the normal group, the fibrosis area in the model group was significantly increased (model group: 13.7%, normal group: 1%), and the fibrosis area in the pirfenidone group and the hyssop essential oil group was significantly decreased (pirfenidone group: 5.0%, hyssop essential oil group: 3.2%), and the effect of hyssop essential oil was even better than that of the positive drug pirfenidone.

[0038] The right lung tissue of mice that had been given the drug for 14 consecutive days was taken, and the expression of fibrosis marker proteins was detected by Western blot. The lung tissue was ground, and a lysis buffer containing enzyme inhibitors was added to fully lyse the protein. The supernatant was collected for BCA protein concentration determination, and the loading buffer was prepared. SDS-PAGE electrophoresis was performed, and the protein was transferred to a PVDF membrane, blocked with 5% skim milk powder for 2 hours, and incubated with primary antibodies α-SMA (55135-1-AP, Proteintech, 1:1000) and collagenI (14695-1-AP, Proteintech, 1:1000) at 4°C overnight. After incubation with the primary antibody, the membrane was rinsed with TBST, and incubated with HRP-labeled goat anti-rabbit secondary antibody at room temperature for 1 hour. After rinsing with TBST again, the ECL luminescent substrate was developed, and the grayscale value was analyzed with ImageJ software. The protein analysis results are shown in the figure below. Figure 3 As shown in the results, compared with the normal group, the expression of α-SMA and collagen I in the model group increased significantly (α-SMA: 1.7 times; collagen I: 3.2 times), while the Hyssop volatile oil group could effectively inhibit the expression of α-SMA and collagen I in the lung tissue of mice (α-SMA: 0.9 times; collagen I: 1.8 times). The above results suggest that Hyssop volatile oil can significantly reduce the expression of fibrosis marker proteins α-SMA and collagen I and inhibit pulmonary fibrosis.

[0039] The experiment also used the Western blot method to detect the expression of proteins related to the fibrosis signaling pathway to clarify the mechanism of action of Hyssopus officinalis volatile oil and its combination in inhibiting fibrosis. The primary antibodies were incubated overnight at 4°C, and the dilution ratios were as follows: TGF-β1 (YP-Ab-15967, UpingBio, 1:1000), p-smad2 / 3 (YP-Ab-01305, UpingBio, 1:1000), p-JNK (ab124956, abcam, 1:1000), total-JNK (AF1048, beyotime, 1:1000), p-p38 (4511S, CST, 1:1000), total-p38 (AF7668, beyotime, 1:1000), p-ERK (4370S, CST, 1:1000), total-ERK (AF1051, beyotime, 1:1000). After rinsing with TBST, HRP-labeled goat anti-rabbit secondary antibody was incubated at room temperature for 1 hour, and after rinsing with TBST again, ECL luminescent substrate was developed, and grayscale value was analyzed by ImageJ software. Protein analysis results showed that compared with the normal group, the expression of TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK in the model group increased significantly (TGF-β1: 2.4 times; p-smad2 / 3: 1.8 times; p-JNK: 1.5 times; p-p38: 3.0 times; p-ERK: 6 times), while Hyssopus officinalis volatile oil and its combination can effectively inhibit the expression of TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK in mouse lung tissue (TGF-β1: 1.2 times; p-smad2 / 3: 0.8 times; p-JNK: 1.0 times; p-p38: 0.8 times; p-ERK: 2.6 times). The above results suggest that Hyssopus officinalis volatile oil can significantly reduce the expression of TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK, and inhibit the development of pulmonary fibrosis.

[0040] The above experiments show that Hyssopus officinalis volatile oil has a good therapeutic effect on IPF and can effectively reduce lung inflammatory infiltration. It can reduce lung interstitial collagen fiber deposition and the expression of fibrosis marker proteins α-SMA and collagen I by downregulating the TGF-β1 / smad2 / 3 classical fibrosis signaling pathway and the TGF-β1 / MAPK non-classical fibrosis signaling pathway.

[0041] Hyssop volatile oil can be further developed as a drug for treating pulmonary fibrosis, that is, it can be used to prepare drugs for treating idiopathic pulmonary fibrosis. Furthermore, the composition containing Hyssop volatile oil can also be used to prepare drugs for treating idiopathic pulmonary fibrosis, that is, the drug contains a composition of Hyssop volatile oil and other medicinal materials / compounds; further, the drug for treating idiopathic pulmonary fibrosis can also include pharmaceutically acceptable additives, carriers and / or excipients.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of Hyssopus officinalis volatile oil and a composition containing the same in the preparation of a drug for treating idiopathic pulmonary fibrosis.

2. The use according to claim 1, It is characterized in that The hyssop volatile oil is prepared by steam distillation.

3. The use according to claim 1, It is characterized in that The Hyssopus officinalis volatile oil and the composition containing the same are used for reducing the infiltration of inflammatory cells in the lung tissue of fibrotic mice.

4. The use according to claim 1, It is characterized in that The Hyssopus officinalis volatile oil and the composition containing the same are used for reducing the deposition of collagen and fiber in the lung interstitium of fibrotic mice.

5. The use according to claim 1, It is characterized in that The Hyssopus officinalis volatile oil and the composition containing the same are used for reducing the expression of pulmonary fibrosis markers α-SMA and collagen I.

6. The use according to claim 1, It is characterized in that The hyssop volatile oil and the composition containing the volatile oil are used to reduce the expression of fibrosis signaling pathway related proteins TGF-β1, p-smad2 / 3, p-JNK, p-p38 and p-ERK.

7. A drug for treating idiopathic pulmonary fibrosis, It is characterized in that Contains essential oil of Hyssopus officinalis.

8. The medicament for treating idiopathic pulmonary fibrosis according to claim 7, It is characterized in that A composition comprising essential oil of Hyssopus officinalis and other medicinal materials / compounds.

9. The medicament for treating idiopathic pulmonary fibrosis according to claim 7 or 8, It is characterized in that Pharmaceutically acceptable additives, carriers and / or excipients are also included.

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