CN110151755B - Application of a kind of Brefeldin A in the preparation of inflammatory factor activity inhibitor drugs - Google Patents

Abstract

The invention discloses application of Brefeldin A in preparation of an inflammatory factor activity inhibitor drug, and finds that Brefeldin A can remarkably reduce the release of TNF-alpha in MH-S and the generation of KC in MLE-12 cells, can remarkably improve pathological changes of lung tissues of mice, reduce the contents of white blood cells and TNF-alpha in BALF, can remarkably reduce the MPO activity in the lung tissues of the mice, remarkably increase the level of cAMP, and can remarkably inhibit the phosphorylation of ERK. Brefeldin a can produce a protective effect on acute lung injury, and the mechanism of Brefeldin a may be related to pathways of increasing intracellular cAMP content, inhibiting ERK phosphorylation and the like. The invention provides a new research direction for the treatment target of acute lung injury, and the development of inflammation can be blocked by inhibiting the generation and secretion of inflammatory factors by regulating the protein transport in cells.

Description

A kind of Brefeldin A in the preparation of inflammatory factor activity inhibitor medicine application

(1) Technical field

The invention relates to the application of brefeldin A, in particular to the application of brefeldin A in the preparation of inflammatory factor activity inhibitor medicines.

(2) Background technology

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are acute and progressive hypoxic respiratory failure caused by various pathogenic factors outside of the heart. The specific molecular mechanisms of ALI/ARDS disease progression are poorly understood, and there are no specific therapeutic drugs for ALI/ARDS. Lipopolysaccharide (LPS) is the main component of Gram-negative bacteria and is widely used to induce ALI/ARDS to study the molecular mechanism of acute inflammatory injury in the lungs. Brefeldin A (Brefeldin A, BFA) is a macrolide antibiotic synthesized by fungi, also known as clinoptilocin or ascosporin. It is isolated from Penicillium decumben fermentation broth. It can inhibit the translocation of proteins from the endoplasmic reticulum to the Golgi body by inducing the decomposition of the Golgi body. It has anti-nematode, anti-mitotic, anti-fungal, anti-virus, anti-tumor and other biological academic activity. At present, Brefeldin A, as a protein transport inhibitor, has become an important molecular tool and has been widely used in mammalian signal transduction pathway research, but whether it is effective in inflammation such as acute lung injury has not yet been studied. Therefore, the present invention intends to explore the effect of Brefeldin A on LPS-induced ALI in mice and its possible action mechanism through in vitro cell experiments and in vivo experiments, so as to provide experimental basis for clinical treatment of ALI.

(3) Contents of the invention

The purpose of the present invention is to provide the application of brefeldin A in the preparation of inflammatory factor activity inhibitor drugs, to provide a new drug for treating acute lung injury or acute inflammation, and to suggest a new drug target.

The technical scheme adopted in the present invention is:

The invention provides the application of brefeldin A in the preparation of inflammatory factor activity inhibitor medicine.

Further, the inflammatory factor is tumor necrosis factor-α or chemokine KC.

Further, the inflammatory factor activity inhibitor drug is a drug for the treatment of acute lung injury.

Further, the drug for treating acute lung injury is a drug for preventing or treating lipopolysaccharide-induced acute lung injury.

Further, the Brefeldin A includes a Brefeldin A derivative.

Described Brefeldin A structural formula is:

As a protein transport inhibitor, Brefeldin A can anti-competitively inhibit the transport of proteins from the endoplasmic reticulum to the Golgi by inducing the decomposition of the Golgi apparatus. It has anti-nematode, anti-mitotic, anti-fungal, anti-virus, anti-tumor and other biological properties academic activity.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention finds that Brefeldin A can significantly reduce the release of TNF-α in MH-S and the production of KC in MLE-12 cells (p<0.001), which can significantly It improved the pathological changes of lung tissue in mice and decreased the content of leukocytes (p<0.001) and TNF-α (p<0.05) in BALF, but had no significant effect on albumin, IL-1β and IL-6 in BALF, and could significantly reduce the levels of leukocytes (p<0.001) and TNF-α (p<0.05) in BALF. MPO activity in mouse lung tissue (p<0.05), significantly increased the level of cAMP (p<0.001), and significantly inhibited the phosphorylation of ERK (p<0.05). Brefeldin A has protective effect on acute lung injury, and its mechanism may be related to the increase of intracellular cAMP content and inhibition of ERK phosphorylation.

The present invention provides a new research direction for the therapeutic target of acute lung injury, which may inhibit the production and secretion of inflammatory factors by regulating intracellular protein transport, thereby blocking the development of inflammation.

(4) Description of drawings

Figure 1 The effect of BFA on LPS stimulation-induced release of TNF-α from alveolar macrophages (A) and KC from epithelial cells (B), compared with the model group, ^a p<0.05, ^b p<0.01, ^c p<0.001 .

n＝8-12)，与模型组对比，^ap<0.05,^bp<0.01,^cp<0.001。Figure 2 The effect of BFA on the number of leukocytes in the BALF of mice with acute lung injury (

n=8-12), compared with the model group, ^a p<0.05, ^b p<0.01, ^c p<0.001.

n＝8-12)，与模型组对比，^ap<0.05,^bp<0.01,^cp<0.001。Figure 3 The effect of BFA on albumin content in BALF of mice with acute lung injury (

n=8-12), compared with the model group, ^a p<0.05, ^b p<0.01, ^c p<0.001.

Fig. 4 The effect of BFA on the pathological changes of lung tissue in LPS-induced acute lung injury mice (HE staining, X400).

n＝8-12)，与模型组对比，^ap<0.05,^bp<0.01,^cp<0.001。Figure 5 The effect of BFA on the content of TNF-α, IL-1β and IL-6 in BALF of LPS-induced acute lung injury mice (

n=8-12), compared with the model group, ^a p<0.05, ^b p<0.01, ^c p<0.001.

n＝8-12)，与模型组对比，^ap<0.05,^bp<0.01,^cp<0.001。Figure 6 The effect of BFA on the content of cAMP in lung tissue of ALI mice (

n=8-12), compared with the model group, ^a p<0.05, ^b p<0.01, ^c p<0.001.

n＝8-12)，与模型组对比，^ap<0.05,^cp<0.001。Figure 7 The effect of BFA on MPO activity in the lung tissue of ALI mice (

n=8-12), compared with the model group, ^a p<0.05, ^c p<0.001.

n＝8-12)，与模型组对比，^ap<0.05。Figure 8 The effect of BFA on the phosphorylation level of MAPK signaling pathway in the lung tissue of ALI mice (

n=8-12), compared with the model group, ^a p<0.05.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1

1 Materials and methods

1.1 Experimental animals C57/BL6 mice, male, body weight 19-21 g, clean grade, provided by the Experimental Center of Zhejiang University School of Medicine, certificate number: (SCXK2013-0016). All operations and experimental procedures abide by the Regulations on the Administration of Laboratory Animals. Experimental environment: constant temperature (22±2)°C, humidity 60%-70%, free drinking and eating.

1.2 Reagents and instruments LPS (Escherichia coli LPS O55:B5, L2880, sigma); Brefeldin A (BFA, sigma); Dexamethasone (DXM, 5mg/ml, Zhejiang Xianju Pharmaceutical Co., Ltd.), myeloperoxidase (myeloperoxidase, MPO) kit (Nanjing Jiancheng Biotechnology Co., Ltd., A044); TNF-α (tumornecrosis factor-α), IL-1β (interleukin-1β) and IL-6 (interleukin-6) ELISA kits were purchased from R&D Systems (R&D Systems, Mineapolis, MN, USA, DY410, DY401, DY406); protein assay kit (Bio-Rad Laboratories, Hercules, CA, 500-0120); cyclic adenosine monophosphate (cAMP) ELISA reagent Box (R&D Systems, Mineapolis, MN, 128USA); mouse alveolar macrophage cell line (MH-S) (CRL-2019) and mouse epithelial cell line (MLE12) (CRL-2110) were purchased from the American ATCC cell bank ( Mannassa, VA, USA). Vertical homogenizer: German Fluko company; ELX800UV microplate reader: American Bio-Tek Instrument Company; DHG-9145A electric heating constant temperature blast drying oven: Shanghai Yiheng Technology Co., Ltd.

1.3 Experimental method

1.3.1 Determination of cytokines/chemokines in alveolar macrophages and alveolar epithelial cells Alveolar macrophages (MH-S) were treated with 10% FBS (corning Australian fetal bovine serum, purchased from Shanghai Pufei) and double antibody of RPMI-1640 medium (corning 10-040-CVR, purchased from Shanghai Pufei), culture conditions: 37°C, 5% CO2, Thermo cell incubator. 200 μl of MH-S cells were seeded in 96-well plates, the cell density was 1×10 ⁴ cells/well, overnight at 37°C, and a normal group, a model group (stimulated with a final concentration of LPS of 500 ng·ml ^-1 ), and a BFA-administered group were established. (final concentration 1μM, 10μM, 100μM) and BFA combined administration group (final concentration 1μM + LPS final concentration 500ng·ml ^-1 , final concentration 10μM + LPS final concentration 500ng·ml ^-1 , final concentration 100μM + LPS final concentration 500ng ·ml ^-1 ), 30 minutes after administration, the culture supernatant was collected after 3h, 9h, and 24h respectively, stored at -80°C, and the TNF-α value of the supernatant was measured by ELISA kit. The alveolar epithelial cells (MLE-12) were cultured at a concentration of 5×10 ³ cells per well, administered in the same manner as above, and the supernatant was collected to determine the KC value.

1.3.2 Animal model preparation and administration The mouse ALI model was prepared by instilling LPS into the airway, and the mice were randomly divided into a normal saline control group (Normal group), a model group (LPS group), and a dexamethasone group ( Dex5mg·kg ^-1 ), Brefeldin A group (BFA, 10mg·kg ^-1 ), 12 in each group. Mice were anesthetized by intraperitoneal injection of 100 g·L ^-1 chloral hydrate at 280 mg·kg ^-1 , and the trachea was bluntly separated. Except for the normal saline control group, the other groups were instilled with LPS (2 mg·kg ^-1 , physiological saline preparation), and the normal saline control group was given an equal volume of normal saline. After 10 minutes, the dexamethasone group and Brefeldin A group were injected with corresponding doses of drugs by one-time intraperitoneal injection, respectively, and the normal saline control group and model group were intraperitoneally injected with the same volume of normal saline. The mice were placed at 37°C, sacrificed after 6 hours, the left lung was subjected to bronchoalveolar lavage, and the right lung was collected and preserved.

1.3.3 Counting white blood cells in bronchoalveolar lavage fluid After 6 hours of modeling, the mice were anesthetized, femoral artery was exsanguinated, and after ligation of the right lung, the trachea was exposed for tracheal intubation, and bronchoalveolar lavage was performed three times with 1.5 mL of PBS. , the recovery rate of bronchoalveolar lavage fluid (BALF) was 90%, stored in an ice bath, a part of BALF was taken and mixed, the cells were counted, and total white blood cells (WBC) were counted. The remaining BALF was centrifuged at 4°C and 250 × g. After centrifugation for 10 min, the supernatant was collected and the supernatant was stored at -80°C.

1.3.4 Determination of related cytokines and proteins in bronchoalveolar lavage fluid Double-antibody sandwich ELISA method was used to determine TNF-α, IL-6 and IL-1β related cytokines in supernatant after centrifugation of BALF according to the instructions of the kit , and the total protein content in BALF was determined by Bio-Rad protein assay kit.

1.3.5 Determination of MPO activity and cAMP content in lung tissue Accurately weigh the right lower lobe tissue, prepare a tissue homogenate of 100 g·L ^-1 with normal saline according to the kit requirements, and centrifuge at 4°C and 12000 × g for 10 min. The supernatant was collected, and the cAMP ELISA kit was used to measure the cAMP content in the tissue homogenate according to the steps, and the MPO activity was measured by the enzymatic kinetic method.

1.3.6 Lung Histopathological Examination The left lower lobe of the lung was fixed in formalin for 24 hours, then dehydrated, transparent, paraffin-embedded, sectioned, stained with hematoxylin-eosin (HE), and observed by high-power microscope for pulmonary edema and inflammatory cells Infiltration, the pathological changes of lung tissue were observed.

1.3.7 Determination of phosphorylation level of MAPK signaling pathway The phosphorylation level detection kit (ab119674, abcam) was used to determine ERK1/2 (pT202/Y204), p38MAPK (pT180/Y182) and JNK1/2/3 in lung tissue homogenate (pT183/Y185) concentration, follow the instructions.

1.4 Statistical methods SPSS 13.0 software was used for statistical processing. The data of each group were tested for homogeneity of variance first, and the comparison of sample means was performed by variance analysis and Dunnett t test, and P<0.05 was considered as statistically significant.

2 results

2.1 The effect of Brefeldin A on the content of TNF-α in mouse alveolar macrophages and KC in epithelial cells

In mouse alveolar macrophage MH-S cells, compared with the control group, 1 μmol and 10 μmol of BFA had no significant effect on the release of TNF-α, but 100 μmol of BFA could significantly reduce the release of TNF-α (p<0.001). ), and the effect was significant at 3, 6, and 9 h in the early stage, and the effect was significantly weakened at 24 h, but the difference was still statistically significant (p<0.001). In mouse lung epithelial cells MLE-12 cells, BFA reduced the production of KC in a dose-dependent manner, the most obvious at the 9h time point, and 100μmol BFA could significantly reduce the production of KC (p<0.001). At the same time, the duration of action was longer than that of MH-S cells, and the inhibitory effect on KC was still significant at 24h. see picture 1. This result suggests that BFA can inhibit the release of inflammatory cytokines and chemokines and may be used to control the development of inflammation.

2.2 The effect of Brefeldin A on the number of leukocytes and protein exudation in the bronchoalveolar lavage fluid of ALI mice From the results of in vitro cell experiments, we found that Brefeldin A has a strong inhibitory effect on inflammatory cytokines. Therefore, we conducted in vivo studies, In order to clarify whether Brefeldin A is effective in acute lung injury. After instillation of LPS into the airway of mice for 6 hours, compared with the normal saline control group (Normal group), the total amount of leukocytes in the BALF of the LPS group increased by about 6.9 times (p<0.001; Figure 2). Compared with the LPS group, the BFA group The total amount of leukocytes decreased by 62% in the Dex group (p<0.001), and the Dex group was 60%, and the effect of the two groups was equivalent; by measuring the exudation of albumin in the BALF, it can reflect the barrier function of alveolar endothelial cells/epithelial cells, that is, the pulmonary microvessels. of permeability. After LPS stimulation, albumin exudation increased, indicating a significant increase in pulmonary vascular permeability, and Dex could significantly inhibit the increase in vascular permeability (p<0.05, Figure 3), but the effect of BFA was not obvious, suggesting that BFA has a significant effect on microvascular Permeability has little effect.

2.3 The effect of Brefeldin A on the pathological changes of lung tissue in ALI mice After LPS stimulation, spot and sheet hemorrhages were seen in the lungs in the model group, a large number of neutrophils were infiltrated, and there was a mild hyaline membrane at the edge of the alveolar space, showing obvious obvious Inflammation and mild pulmonary edema, marked thickening of alveolar septa, and narrowing of alveolar spaces indicated that the modeling was successful. The above pathological changes were significantly improved in BFA group and Dex group (Fig. 4).

2.4 The effect of Brefeldin A on the contents of TNF-α, IL-1β and IL-6 in the bronchoalveolar lavage fluid of ALI mice The contents of inflammatory cytokines and chemokines in BALF after LPS stimulation for 6 h, and the inflammation in the blank control group were measured. Compared with the model group, the content of TNF-α in BFA was significantly reduced (P<0.05), but IL-6 and IL-1β had no significant changes. Dex could significantly reduce TNF-α, IL-1β and IL-1β. 6 content (P<0.001). These results suggest that the role of BFA differs from that of glucocorticoid hormones in LPS-induced ALI (Figure 5).

2.5 Effects of Brefeldin A on cAMP content and MPO activity in lung tissue of ALI mice Compared with the control group, the cAMP content in the model group was significantly decreased (p<0.001), and the MPO activity was significantly increased (p<0.05). Dex and BFA significantly increased the level of cAMP and decreased the activity of MPO (p<0.05) (Fig. 6, 7). This result suggests that BFA may up-regulate cAMP to inhibit cell inflammation, and BFA has obvious chemotactic effect on neutral.

2.6 Effect of Brefeldin A on the phosphorylation level of MAPK signaling pathway in lung tissue of ALI mice Phosphorylation of ERK 1/2 (pT202/Y204), p38 MAPK (pT180/Y182) and JNK 1/2/3 (pT183/Y185) in lung tissue ) showed that LPS could significantly enhance the phosphorylation of ERK1/2 and SPAK/JNK, but had little effect on p38. Both Dex and BFA significantly inhibited the phosphorylation of ERK (p<0.05), but had no significant effect on the phosphorylation of JNK (Fig. 8). This result suggests that BFA and Dex may have protective effects on AIL through ERK1/2.

3 Discussion

The pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is complex, the disease progresses very rapidly, and the mortality rate is high. It is a common clinical emergency. ALI/ARDS is a continuous pathophysiological process, and ARDS is its the most serious stage. Lipopolysaccharide located in the outer membrane of Gram-negative bacteria is one of the common and important pathogenic factors of acute lung injury. About the mechanism of ALI In recent years, studies have found that oxidative stress, imbalance of pro-inflammatory/anti-inflammatory response, apoptosis disorder, and imbalance of pro-coagulation/anti-coagulation response play important roles in lipopolysaccharide-induced ALI, and the various mechanisms interact with each other. Promote and influence each other, forming a vicious circle and aggravating the damage of the body. Sepsis is still the main cause of ALI. LPS can lead to acute diffuse damage to alveolar capillary endothelial cells, alveolar epithelial cells and lung interstitium. The release of mediators causes lung damage, and at the same time activates more inflammatory cells to release more inflammatory mediators or cytokines, further amplifying and strengthening the lung damage signal, forming an inflammatory cascade effect, leading to alveolar-capillary membrane damage. The alveolar space of normal lung tissue is dominated by macrophages, and when ALI occurs, the alveolar space is dominated by neutrophils. After ALI, under the action of various chemokines, a large number of neutrophils aggregate in the lung tissue, and combine with alveolar epithelial cells, vascular endothelial cells, macrophages, etc. to release a large number of inflammatory mediators through respiratory burst, degranulation and other mechanisms. The pro-inflammatory mediators have a large amount of accumulation in tumor necrosis factor-death factor, among which pro-inflammatory mediators include IL-1β, IL-6, TNF-α, etc., which lead to inflammatory response.

Alveolar macrophages are critical in pulmonary inflammatory diseases and play a role in maintaining lung tissue homeostasis and increasing the rapid response to exogenous and endogenous stimuli. They are the first line of defense against pathogens and act as key coordinators of the immune response, ultimately facilitating the clearance of apoptotic debris, eliminating inflammation and repairing tissue. Alveolar macrophages are also the main source of a variety of inflammatory cytokines, such as IL-1β, IL-6, TNF-α, etc., which play a crucial role in inflammation. When ALI occurs, the permeability of pulmonary capillary endothelial cells increases, and a large number of inflammatory mediators such as free radicals, cell adhesion molecules, tumor necrosis factor, selectin, interleukin, integrin, etc. The neutrophils are chemotactic and deformed, and then pass through the pulmonary capillaries with increased permeability into the pulmonary interstitium and alveoli. At the same time, related inflammatory mediators damage the alveolar epithelial cells, causing the production of active substances on the alveolar surface to decrease, thereby weakening the fluidity of water retained in the alveoli and aggravating pulmonary edema; the regeneration of alveolar epithelial cells is very important for the recovery of ARDS. In the virus-induced ALI mouse model, if the mouse lung loses more than 10% of the type I alveolar epithelium, the respiratory function of the mouse will be significantly reduced, after which the ALI disease will progress rapidly and be difficult to recover.

As a potential macrolide antitumor antibiotic, Brefeldin A can anti-competitively inhibit the action of proteins. Brefeldin A can not only significantly affect the secretion pathway of mammals, but also cause morphological changes in cells, especially the induction of Golgi apparatus. Breakdown, leading to the redistribution of enzymes back to the endoplasmic reticulum after Golgi breakdown, Brefeldin A has been shown to reduce the production and secretion of chemical mediators of inflammation and immune responses by inhibiting Akt, mTOR and NF-κB pathway to reduce the production of TNF-α-stimulated inflammatory mediators. Brefeldin A is currently studied more in anti-tumor therapy, but relatively few studies in the field of anti-inflammatory are involved. Acute lung injury is mainly an inflammatory response. In the early stage of acute lung injury, there are a large number of inflammatory factors and The involvement of inflammatory mediators, such as IL-1β, IL-6, TNF-α, etc., whether brefeldin A can affect ALI by affecting the release of related inflammatory mediators remains to be further studied. Therefore, this experiment used molecular biology and functional research methods at the animal and cell levels to study the effect of Brefeldin A on LPS-induced acute lung injury, in order to further explore the specific mechanism of Brefeldin A in acute lung injury.

The results of this experimental study showed that 100 μmol of Brefeldin A at the cellular level can effectively inhibit the release of inflammatory factors/chemokines such as TNF-α in mouse alveolar macrophages and KC content in epithelial cells; Brefeldin A at the animal level can effectively inhibit the release of inflammatory factors/chemokines It inhibits the increase of BALF white blood cell count caused by LPS, reduces the activity of MPO in lung tissue and the level of TNF-α in BALF, increases the content of cAMP in lung tissue, significantly inhibits the phosphorylation of ERK protein kinase in the MAPK signaling pathway, and improves various types of ALI. Pathological changes such as inflammation, and it is worth noting that Brefeldin A is different from Dex, and has no significant effect on inflammatory factors such as IL-1β and IL-6 at the animal level, suggesting that Brefeldin A can produce unique effects different from hormones. Brefeldin A The mechanism of alleviating LPS-induced acute lung injury may be related to increasing cAMP content and inhibiting the production of TNF-α, thereby reducing the pulmonary sequestration of neutrophils and the release of inflammatory factors.

Claims (4)

1. Application of Brefeldin A in the preparation of a medicine for the treatment of acute lung injury. 2. The use according to claim 1, wherein the drug is an inhibitor of inflammatory factor activity. 3. The application according to claim 2, wherein the inflammatory factor is tumor necrosis factor-α or chemokine KC. 4. The application according to claim 1, wherein the medicine is a medicine for preventing or treating acute lung injury induced by lipopolysaccharide.

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