Application of poncirin in preparation of medicine for treating and / or preventing hypoxic pulmonary hypertension

Abstract

The invention provides application of poncirin in preparation of a medicine for treating and / or preventing hypoxic pulmonary hypertension, and belongs to the technical field of medicines. According to the application of poncirin in preparation of the medicine for treating and / or preventing hypoxic pulmonary hypertension, the pulmonary hypertension, inflammatory factor release and pulmonary artery smooth muscle proliferation caused by hypoxia can be effectively improved, and the right ventricular pressure and the right ventricular hypertrophy index are remarkably reduced; effective drugs can be provided for treatment of diseases characterized by hypoxic pulmonary hypertension.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of citrus glycosides in preparing medicines for treating and / or preventing hypoxic pulmonary hypertension. Background Art

[0002] Hypoxic pulmonary hypertension is a cardiovascular disease characterized by long-term low pressure and low oxygen. It is one of the types of pulmonary hypertension. It mainly refers to the continuous excessive increase of pulmonary artery pressure under long-term low pressure and low oxygen conditions, which leads to increased pulmonary vascular resistance and pulmonary vascular remodeling, and eventually right heart failure, and even death in severe cases. Its pathophysiological characteristics are that chronic hypoxia causes pulmonary artery endothelial damage and dysfunction, the number of macrophages increases significantly, and the induction of inflammatory response further causes pulmonary vascular contraction and abnormal proliferation of pulmonary artery smooth muscle cells, which promotes the formation of hypoxic pulmonary hypertension. Its pathogenesis is relatively complex, for example, including endothelial dysfunction, pulmonary vasoconstriction, inflammation, extracellular matrix deposition, abnormal proliferation and apoptosis of medial smooth muscle cells. Although calcium ion antagonists, prostacyclin and endothelin receptor antagonists, phosphodiesterase inhibitors, etc. have been used in hypoxic pulmonary hypertension in recent years, there are still problems of poor efficacy and serious adverse drug reactions. Citrus glycosides are a flavonoid glycoside derivative isolated from clover. They have multiple pharmacological effects, including antibacterial, anti-inflammatory, and anti-cancer effects. They are widely used as drugs for the treatment of colitis, gastric cancer, gastritis, and Alzheimer's disease, with minimal toxic side effects. Currently, there are no reports on the effects of citrus glycosides on hypoxic pulmonary hypertension. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide the use of citrus glycosides in the preparation of drugs for the treatment and / or prevention of pulmonary hypertension, which can effectively improve pulmonary hypertension, inflammatory factor release and pulmonary artery smooth muscle proliferation caused by hypoxia, and significantly reduce right ventricular pressure and right ventricular hypertrophy index, and can provide an effective drug for the treatment of diseases characterized by hypoxic pulmonary hypertension.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] Application of citrus glycosides in the preparation of medicines for treating and / or preventing pulmonary hypertension.

[0006] Preferably, the pulmonary hypertension is hypoxic pulmonary hypertension.

[0007] Preferably, the effective dosage of the citrus glycoside is 30-50 mg / kg.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The use of the citrus glycosides provided by the present invention in the preparation of a drug for treating and / or preventing pulmonary hypertension can effectively improve pulmonary hypertension, inflammatory factor release and pulmonary artery smooth muscle proliferation caused by hypoxia, and significantly reduce right ventricular pressure and right ventricular hypertrophy index, and can provide an effective drug for the treatment of diseases characterized by hypoxic pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The treatment with citrus glycosides effectively improved pulmonary hypertension and the release of inflammatory factors caused by hypoxia. AB: The right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (right ventricle / left ventricle + ventricular septum) of HPH rats were detected after citrus glycosides were treated with different doses (10, 30 and 50 mg / kg). CF: Representative Western blot results (E) and quantitative graphs (FH) showed the protein expression levels of NLRP3, Caspase1 and N-GSDMD in the Con group and HPH group after citrus glycosides treatment, with β-actin as the control. G: ELISA was used to detect the expression level of IL-1β in the serum of rats in the Con group and HPH group after citrus glycosides treatment. HJ: Representative HE lung sections of rats in the Con group and HPH group treated with citrus glycosides, medial wall thickness percentage (WT%) and medial wall area percentage (WA%);

[0011] Figure 2 The results show that citrus glycosides act on arterial smooth muscle cells of HPH rats and effectively reduce the proliferation of pulmonary artery smooth muscle. AB: CCK-8 was used to detect the effects of different citrus glycosides concentrations and hypoxia treatment on cell survival rate. CD: EdU method was used to determine the proliferation rate of smooth muscle cells after adding different supernatants and treating them under normoxic or hypoxic conditions for 24 hours.

[0012] Figure 3 Figure 1 is the expression and distribution of TDP43 in HPH, where Figure A is the expression of TDP43 mRNA in rat lung tissue detected by qPCR. Figure BC is the Western blot result (B) and the quantitative graph (C) showing the expression of TDP43 protein in lung tissue of rats in the Con group and HPH group, with β-actin as the control. Figure D is the expression of TDP43 mRNA in rat lung tissue detected by qPCR after intervention with different drugs. DETAILED DESCRIPTION

[0013] (1) Hypoxic pulmonary hypertension modeling and intervention with citrus glycosides

[0014] Rats were randomly divided into a normoxic control group (Con group), a hypoxic pulmonary hypertension group (HPH group), and a citrus glycoside intervention group (HPH+Con group). The Con group was placed in a normobaric and normoxic environment (about 500 m above sea level) for 4 weeks; the HPH group and the HPH+Con group were placed in a hypobaric and hypoxic high-altitude oxygen chamber simulating an altitude of 5000 m for 4 weeks. During this period, the living environment of the rats was maintained in a 12-hour light-dark cycle, and food and water were freely provided. The Con group and the HPH group were continuously intraperitoneally injected with the same volume of normal saline (due to the different weights of rats, the injection volume was different, and 6 ml / rat was injected in this experiment) from the 15th to the 30th day, and the HPH+Con group was continuously intraperitoneally injected with the same volume of citrus glycoside solution from the 15th to the 30th day.

[0015] The results are as follows Figure 1 As shown in AB, hypoxic environment successfully induced pulmonary hypertension in rats. The right ventricular systolic pressure and right ventricular hypertrophy index of rats in the HPH group were significantly increased. Treatment with citrus glycosides could significantly alleviate the increase of right ventricular systolic pressure and right ventricular hypertrophy index.

[0016] (2) HE staining of lung tissue

[0017] The lung tissue was sliced ​​and stained with hematoxylin and eosin (HE). The distal pulmonary arterioles with a diameter of 50-100 μm were selected for observation under a microscope and the slices were scanned. The extent of pulmonary vascular remodeling was evaluated by calculating the medial wall thickness percentage (WT%) and medial wall area percentage (WA%). The results were shown in Figure 1 As shown in HJ, hypoxic exposure successfully induced pulmonary artery remodeling, which was manifested by the significant thickening of the pulmonary arteriolar walls in rats in the HPH group; the addition of citrus glycosides for intervention could significantly alleviate the hypoxia-induced pulmonary small vessel remodeling.

[0018] (3) Hemodynamic measurement and right ventricular hypertrophy index analysis

[0019] Rats were anesthetized by injecting an appropriate amount of anesthetic according to their body weight, the right external jugular vein was isolated, a microcatheter was inserted, and connected to the Powerlab physiological recorder through a pressure sensor. When the catheter entered the right heart, blood could be seen pumping out regularly from the tube. At this time, the physiological recorder was connected, and the depth and direction of the catheter were adjusted until a regular pressure waveform was presented. The PowerLab physiological recorder recorded the corresponding waveform curve and the right ventricular pressure value. The rat heart was then isolated, the atrium was cut off with microscissors, and the right ventricle (RV), left ventricle (LV) and ventricular septum (S) of the rat were separated and weighed, and the right heart index [RV / (LV+S)] was calculated to reflect the right heart hypertrophy index. The results are shown in the figure. Figure 1 As shown in AB, citrus glycosides can significantly reduce the right ventricular pressure and right ventricular hypertrophy index of HPH rats, thereby effectively slowing down the occurrence of HPH in rats.

[0020] (4) CCK-8

[0021] According to the experimental requirements, different concentrations (5, 15, 25, 50, 100, 200 μM) of citrus glycosides were added for treatment, and then 10 μL of CCK-8 solution was added to each well. The culture plate was incubated in an incubator for 2 hours, and the absorbance at 450 nm was measured using an enzyme reader.

[0022] The results are as follows Figure 2 As shown in AB, citrus glycosides at concentrations below 100 μM did not cause cytotoxicity (P>0.05). Then, the effect of adding citrus glycosides at different concentration gradients on the cell survival rate of hypoxia-induced cells was detected. The results showed that citrus glycosides increased the cell survival rate of cells in a dose-dependent manner. Therefore, the concentration of citrus glycosides selected for subsequent experiments was 50 μM.

[0023] (5) EdU staining

[0024] Cells in logarithmic growth phase were inoculated in 96-well plates and treated according to experimental needs. 100 μL of 50 μM EdU culture medium was added to each well and incubated for 2 hours. Then, the cells were fixed and permeabilized. 100 μL of Apollo staining solution was added to each well and incubated in the dark for 30 minutes. An appropriate amount of Hoechst33342 reaction solution was prepared and incubated in the dark for 30 minutes. The cells were immediately observed and photographed using a fluorescence microscope. Figure 2 As shown in CD, hypoxic exposure induced significant proliferation of pulmonary artery smooth muscle cells, and citrus glycoside treatment could significantly reduce the proliferation of smooth muscle cells, thereby effectively alleviating the occurrence of HPH in rats.

[0025] (6)ELISA

[0026] The ELISA kit for rat IL-1β was purchased from Ruixin Biotechnology Co., Ltd. The corresponding rat serum was collected according to the experimental grouping to detect the expression level of IL-1β. The ELISA experiment was performed according to the instructions of the kit. The absorbance value was read at 450nm, and the results were expressed in pg / ml (pg / ml).

[0027] like Figure 1 As shown in G, hypoxia exposure successfully induced the production of an inflammatory storm in rats, which was manifested by a significant increase in serum IL-1β levels. Treatment with citrus glycosides could significantly alleviate the hypoxia-induced inflammatory storm.

[0028] (7) Western blot

[0029] Whole cell proteins were extracted with RIPA lysis buffer. After SDS-PAGE gel electrophoresis to separate proteins, the proteins were transferred from the gel to a PVDF membrane. After blocking with 5% BSA for 1 h at room temperature, the desired protein bands were cut out and incubated with primary antibodies (TDP-43 antibody was purchased from CST, USA; β-actin antibody and Caspase1 antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.; N-GSDMD antibody and NLRP3 antibody were purchased from Beijing Bioson Biotechnology Co., Ltd.) at 4°C for 12 h. On the second day, the corresponding HRP-labeled secondary antibody (diluted at 1:5000) was added, incubated at room temperature for 1 h, and the ECL luminescent solution was added for full reaction and then scanned for color development. The experiment was repeated 3 times. Finally, the corresponding protein expression level was calculated using ImageJ software.

[0030] The results are as follows Figure 1 As shown in CF, the hypoxic environment successfully induced pulmonary hypertension in rats, and the expression level of pulmonary artery tissue cell pyroptosis-related proteins was significantly increased. Intraperitoneal injection of citrus glycosides could significantly reduce hypoxia-induced pulmonary artery cell pyroptosis.

[0031] like Figure 1 As shown, hypoxic exposure successfully induced the occurrence of pulmonary hypertension, and the levels of inflammation, cell pyroptosis and pulmonary artery remodeling in pulmonary artery tissue were significantly increased. Treatment with citrus glycosides can significantly reduce the levels of inflammation and cell pyroptosis, while improving pulmonary artery remodeling, thus playing a protective role against hypoxia-induced pulmonary hypertension.

[0032] like Figure 2 As shown, hypoxic exposure successfully induced the production of pulmonary hypertension, accompanied by pulmonary artery remodeling, which was mainly manifested in the abnormal proliferation of pulmonary artery smooth muscle cells. Treatment with citrus glycosides significantly reduced the proliferation level of pulmonary artery smooth muscle cells, thereby alleviating hypoxia-induced pulmonary artery remodeling.

[0033] (8) Expression and distribution of TDP43 in HPH

[0034] To verify the expression and distribution of TDP43 in HPH, we established a rat HPH model in a plateau simulation cabin and detected the mRNA and protein expression levels of TDP43 in lung tissue using RT-qPCR and Western blot techniques. Subsequently, we used different drugs to intervene in the rats (30 mg / kg of citrus glycosides, 50 mg / kg of melatonin, 20 mg / kg of lycopene, 50 mg / kg of Piper longum. Due to the different weights of rats, the injection volume was different, and 6 ml was injected per rat in this experiment).

[0035] The results are as follows Figure 3As shown in Figures AD, the mRNA and protein levels of TDP43 in the HPH group were significantly higher than those in the Con group. At the same time, the comparison of the effects of multiple drug interventions showed that citrus glycosides had the best intervention effect on TDP43.

Claims

1. Use of citrus glycosides in the preparation of drugs for treating and / or preventing hypoxic pulmonary hypertension.

2. The use according to claim 1, characterized in that: The effective dosage of the citrus glycoside is 30-50 mg / kg.

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