CN110151755B - Application of brefeldin A in preparation of inflammation factor activity inhibitor medicine - Google Patents
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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
(I) technical field
The invention relates to an application of brefeldin A, in particular to an application of brefeldin A in preparation of an inflammation factor activity inhibitor drug.
(II) background of the invention
Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are acute and progressive hypoxic respiratory failure caused by various internal and external pathogenic factors of the lung except for cardiogenic factors, and currently, a specific molecular mechanism of the ALI/ARDS disease process is not well known, and meanwhile, a treatment medicament specially aiming at the ALI/ARDS is not available. Lipopolysaccharide (LPS) is a major component of gram-negative bacteria and is widely used to induce ALI/ARDS to study the molecular mechanism of acute inflammatory injury of the lung. Brefeldin A (BFA) is a macrolide antibiotic synthesized by fungi, is also called decumbent or ascosporin, is separated from Penicillium decumben fermentation broth in 1958 by Singleton et al, can inhibit protein transport from endoplasmic reticulum to Golgi through induction of decomposition of Golgi, and has various biological activities such as nematode resistance, mitosis resistance, fungus resistance, virus resistance, tumor resistance and the like. At present, Brefeldin A as a protein transport inhibitor becomes an important molecular tool and is widely applied to the research of signal transduction pathways of mammals, but whether Brefeldin A is effective to inflammation such as acute lung injury or not is not researched at present. Therefore, the invention aims to discuss the influence of Brefeldin A in mice ALI induced by LPS and the possible action mechanism thereof through in vitro cell experiments and in vivo experiments, and provides experimental basis for clinical treatment of ALI.
Disclosure of the invention
The invention aims to provide application of brefeldin A in preparation of an inflammatory factor activity inhibitor drug, provides a novel drug for treating acute lung injury or acute inflammation, and suggests a novel drug target.
The technical scheme adopted by the invention is as follows:
the invention provides an application of brefeldin A in preparation of an inflammatory factor activity inhibitor drug.
Furthermore, the inflammatory factor is tumor necrosis factor-alpha or chemokine KC.
Further, the inflammatory factor activity inhibitor drug is a drug for treating acute lung injury.
Further, the acute lung injury treatment drug is a drug for preventing or treating lipopolysaccharide-induced acute lung injury.
Further, the brefeldin A comprises a brefeldin A derivative.
The structural formula of brefeldin A is as follows:
brefeldin A as protein transport inhibitor can inhibit protein transport from endoplasmic reticulum to Golgi apparatus via inducing decomposition of Golgi apparatus and counter-competitively, and has various biological activities of resisting nematode, mitosis, fungus, virus and tumor.
Compared with the prior art, the invention has the following beneficial effects: the Brefeldin A is found to be capable of remarkably reducing the release of TNF-alpha in MH-S and the generation of KC (p <0.001) in MLE-12 cells, remarkably improving the pathological change of lung tissues of mice, reducing the contents of white blood cells (p <0.001) and TNF-alpha (p <0.05) in BALF, but has no remarkable influence on albumin, IL-1 beta and IL-6 in BALF, remarkably reducing the MPO activity (p <0.05) in the lung tissues of mice, remarkably increasing the level of cAMP (p <0.001) and remarkably inhibiting the phosphorylation of ERK (p < 0.05). 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.
(IV) description of the drawings
FIG. 1 Effect of BFA on TNF- α (A) release from mouse alveolar macrophages and KC (B) release from epithelial cells induced by LPS stimulation, in comparison to model groups,ap<0.05,bp<0.01,cp<0.001。
FIG. 2 Effect of BFA on the number of leukocytes in BALF in mice with acute Lung injury: (
n-8-12), compared to the model set,ap<0.05,bp<0.01,cp<0.001。
FIG. 3 Effect of BFA on Albumin content in BALF of mice with acute Lung injury ((S))
n-8-12), compared to the model set,ap<0.05,bp<0.01,cp<0.001。
figure 4 effect of BFA on pathological changes in lung tissue in LPS acute lung injury mice (HE staining, X400).
FIG. 5 Effect of BFA on TNF- α, IL-1 β and IL-6 levels in BALF of LPS-induced acute Lung injury mice ((II))
n-8-12), compared to the model set,ap<0.05,bp<0.01,cp<0.001。
FIG. 6 Effect of BFA on cAMP content in lung tissue of ALI mice (C)
n-8-12), compared to the model set,ap<0.05,bp<0.01,cp<0.001。
FIG. 7 Effect of BFA on MPO Activity in lung tissue of ALI mice (II)
n-8-12), compared to the model set,ap<0.05,cp<0.001。
figure 8 effect of BFA on MAPK signaling pathway phosphorylation levels in lung tissue of ALI mice(s) ((s))
n-8-12), compared to the model set,ap<0.05。
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1
1 materials and methods
1.1 Experimental animal C57/BL6 mouse, male, body mass 19-21g, clean grade, available from the Experimental center of Zhejiang university college of medicine, and certification number: (SCXK 2013-0016). All procedures and experimental procedures obeyed the regulations on the management of laboratory animals. The experimental environment is as follows: the temperature is kept at 22 +/-2 ℃ and the humidity is 60-70 percent, and water and food can be freely drunk.
1.2 reagent and Instrument LPS (Escherichia coli LPS O55: B5, L2880, sigma); brefeldin a (BFA, sigma); dexamethasone (DXM, 5mg/ml, zhejiang juanju pharmaceutical company, ltd.), Myeloperoxidase (MPO) kit (tokyo bio-technology ltd., a 044); TNF- α (tumor necrosis factor- α), IL-1 β (interleukin-1 β) and IL-6(interleukin-6) ELISA kits were purchased from R & D Systems (R & D Systems, Mineapolis, MN, USA, DY410, DY401, DY 406); protein assay kit (Bio-Rad Laboratories, Hercules, CA, 500-0120); cyclic Adenosine monophosphate (cAMP) ELISA kit (R & D Systems, minepolis, MN,128 USA); mouse alveolar macrophage cell line (MH-S) (CRL-2019) and mouse epithelial cell line (MLE12) (CRL-2110) were purchased from American ATCC cell bank (Mannassa, VA, USA). Vertical refiner: germany Fluko corporation; ELX800UV type microplate reader: Bio-Tek instruments, USA; DHG-9145A type electric heating constant temperature forced air drying cabinet: shanghai, a constant technology, Inc.
1.3 Experimental methods
1.3.1 measurement of cytokines/chemokines in alveolar macrophages and alveolar epithelial cells alveolar macrophages (MH-S) were cultured in RPMI-1640 medium (corning 10-040-CVR) containing 10% FBS (corning Australian fetal bovine serum, purchased from Shanghai prairie) and a diabody, under the following culture conditions: 37 ℃, 5% CO2, Thermo cell incubator. 200. mu.l of MH-S cells were seeded in 96-well plates at a cell density of 1X 104cell/well, overnight at 37 ℃, and a normal group and a model group (LPS final concentration 500ng & ml) are established-1Stimulation), BFA single administration group (final concentration 1. mu.M, 10. mu.M, 100. mu.M) and BFA combination administration group (final concentration 1. mu.M + LPS final concentration 500 ng. ml)-1The final concentration of 10. mu.M + the final concentration of LPS of 500 ng/ml-1The final concentration was 100. mu.M + the final concentration of LPS was 500 ng/ml-1) After 30 minutes of administration, the culture supernatants were collected at 3 hours, 9 hours, and 24 hours, respectively, and stored at-80 ℃ to measure the TNF-. alpha.value of the supernatants using an ELISA kit. Alveolar epithelial cells (MLE-12) at 5X 10 per well3The cells were cultured at a concentration, dosed as described above, and the supernatant was collected and the KC value was measured.
1.3.2 preparation of animal model and administration of drug mice ALI model was prepared by dropping LPS into airway, and mice were randomly divided into Normal saline control group (Normal group), model group (LPS group), dexamethasone group (Dex5 mg. kg)-1) Brefeldin group A (BFA, 10 mg. kg)-1) And 12 per group. The mouse dose is 280 mg/kg-1Intraperitoneal injection of 100 g.L-1Under anesthesia with chloral hydrate, isolating trachea inactively, and dripping LPS (2 mg. kg) into the airway of mice except for normal saline control group-1Saline, saline formulation), a saline control group was given an equal volume of saline. After 10min, corresponding doses of drugs are respectively injected into the abdominal cavity of the dexamethasone group and the Brefeldin A group at one time, and equal volume of normal saline is injected into the abdominal cavity of the normal saline control group and the model group. Left at 37 ℃ and sacrificed after 6h, the left lung is subjected to bronchoalveolar lavage, and the right lung is collected and stored.
1.3.3 white blood cell count modeling in bronchoalveolar lavage fluid after 6h, anesthetizing the mouse, bleeding the femoral artery to kill, ligating the right lung, exposing the tracheal trachea cannula, performing bronchoalveolar lavage 3 times with 1.5mL of PBS fluid, recovering 90% bronchoalveolar lavage fluid (BALF), storing in ice bath, taking part of BALF, mixing, counting cells, counting total White Blood Cells (WBC), centrifuging the rest BALF at 4 ℃, centrifuging at 250 Xg for 10min, taking the supernatant after centrifuging, and storing at-80 ℃.
1.3.4 determination of the content of related cytokines and proteins in bronchoalveolar lavage fluid by double antibody sandwich ELISA method, according to the instruction of the kit, determining TNF-alpha, IL-6 and IL-1 beta related cytokines in supernatant after BALF centrifugation, and determining the content of total proteins in BALF by using Bio-Rad protein determination kit.
1.3.5 measurement of MPO Activity and cAMP content in Lung tissue Right Lung lower lobe tissue is accurately weighed and prepared into 100 g.L with physiological saline according to the requirements of the kit-1The homogenate of (4 ℃), centrifugation at 12000 Xg for 10min, supernatant collection, and measurement of cAMP content in the homogenate by steps using cAMP ELISA kit, MPO activity measurement using an enzymatic kinetic method.
1.3.6 pathological examination of lung tissue the left lung lobes were fixed in formalin for 24h, dehydrated after fixation, cleared, paraffin embedded and sectioned, hematoxylin-eosin (HE) stained, high power microscope observation of pulmonary edema and inflammatory cell infiltration, and observation of pathological changes in lung tissue.
1.3.7 measurement of phosphorylation levels of MAPK Signal pathways the concentrations of ERK1/2(pT202/Y204), p38MAPK (pT180/Y182) and JNK1/2/3(pT183/Y185) in lung homogenates were determined using the phosphorylation level detection kit (ab119674, abcam) and performed according to the procedures described herein.
1.4 statistical methods statistical processing was performed using SPSS 13.0 software. The homogeneity of variance test is carried out on each group of data, and the average number comparison of samples adopts the analysis of variance and the Dunnett t test, and the data is regarded as having statistical significance when P is less than 0.05.
2 results
2.1 Effect of Brefeldin A on TNF- α in mouse alveolar macrophages and KC content in epithelial cells
In mouse alveolar macrophage MH-S cells, 1 mu mol and 10 mu mol of BFA have no obvious effect on the release of TNF-alpha compared with a control group, but 100 mu mol of BFA can obviously reduce the release of TNF-alpha (p is less than 0.001), the effect is obvious in early 3, 6 and 9 hours, the effect is obviously weakened in 24 hours, and the difference is still statistically significant (p is less than 0.001). In mouse lung epithelial cells MLE-12 cells, the reduction of KC production by BFA is obvious in dose dependence, and most obvious at the time point of 9h, 100 mu mol of BFA can obviously reduce the KC production (p is less than 0.001). Meanwhile, the duration of action is longer than that of MH-S cells, and the inhibition effect on KC is still obvious at 24 h. See fig. 1. This result suggests that BFA can inhibit the release of inflammatory cytokines and chemokines, and may be useful in controlling the development of inflammation.
2.2 influence of Brefeldin A on leukocyte count and protein exudation in the alveolar 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, and therefore, we conducted in vivo studies to determine whether Brefeldin A is effective on acute lung injury. After LPS is dripped into the mouse airway for 6 hours, the total amount of white blood cells in BALF of the LPS group is increased by about 6.9 times (p is less than 0.001; figure 2) compared with a Normal saline control group (Normal group), and the total amount of white blood cells in BFA group is reduced by 62 percent (p is less than 0.001) and 60 percent of Dex group, and the action intensity of the white blood cells and the Normal saline control group is equivalent; the barrier function of alveolar endothelial/epithelial cells, i.e., permeability of pulmonary microvasculature, can be reflected by measuring the exudation of albumin in BALF. After stimulation by LPS, albumin exudation is increased, which shows that the pulmonary vascular permeability is remarkably increased, Dex can remarkably inhibit the increase of the vascular permeability (p is less than 0.05, and figure 3), but the function of BFA is not obvious, which indicates that the BFA has little influence on the microvascular permeability.
2.3 after the lung tissue pathological change of the ALI mice is stimulated by LPS (LPS), visible point and sheet bleeding of the lung in a model group, a large amount of neutrophils infiltrate, a slight transparent film exists at the edge of an alveolar gap, obvious inflammation and a slight pulmonary edema phenomenon are displayed, the alveolar space is obviously thickened, and the alveolar cavity is narrowed, thereby prompting the success of molding. The above pathological changes were significantly improved in the BFA and Dex groups (FIG. 4).
2.4 Effect of Brefeldin A on TNF-alpha, IL-1 beta and IL-6 content in alveolar lavage fluid of ALI mice respectively determining inflammatory cytokine and chemokine content in BALF 6h after LPS stimulation, few inflammatory mediators in blank control group, and TNF-alpha content in BFA is significantly reduced (P <0.05), but IL-6 and IL-1 beta are not significantly changed, Dex can significantly reduce TNF-alpha, IL-1 beta and IL-6 content (P <0.001) compared with model group. These results suggest that the effect of BFA is different from that of the glucocorticoid in LPS-induced ALI (figure 5).
2.5 Effect of Brefeldin A on cAMP levels and MPO activity in lung tissue of ALI mice compared to control group, cAMP levels were significantly reduced in the model group (p <0.001) and MPO activity was significantly increased in the model group (p < 0.05). Dex and BFA were able to significantly increase cAMP levels and decrease MPO activity (p <0.05) (FIGS. 6, 7). This result suggests that BFA may up-regulate cAMP to produce an inhibitory effect on cellular inflammation, while BFA has a significant chemotactic effect on neutrality.
2.6 Effect of Brefeldin A on phosphorylation levels of MAPK signaling pathways in lung tissue of ALI mice the results of phosphorylation of ERK1/2(pT202/Y204), p38MAPK (pT180/Y182) and JNK1/2/3(pT183/Y185) in lung tissue showed that LPS significantly enhanced phosphorylation of ERK1/2 and SPAK/JNK, but had no significant effect on p 38. Both Dex and BFA significantly inhibited ERK phosphorylation (p <0.05), but had no significant effect on JNK phosphorylation (fig. 8). This result suggests that BFA and Dex may exert protective effects on AIL through ERK 1/2.
Discussion of 3
Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS) is complex in pathogenesis, very rapid in disease progression, high in fatality rate and clinically common in critical illness, ALI/ARDS is a continuous pathophysiological process, and ARDS is the most severe stage of the ARDS. Lipopolysaccharide located on the outer membrane of gram-negative bacteria is one of the common important pathogenic factors of acute lung injury. Recent studies on the ALI mechanism have found that: oxidative stress, proinflammatory/anti-inflammatory response imbalance, apoptosis disorder, procoagulant/anticoagulation response imbalance and the like play an important role in ALI caused by lipopolysaccharide, and all mechanisms are mutually promoted and influenced to form a vicious circle and aggravate body injury. Sepsis is still the main cause of ALI, and LPS can cause acute diffuse damage to alveolar capillary endothelial cells, alveolar epithelial cells and pulmonary interstitium, and its essence is that various inflammatory cells in the lung activate infiltration and release of a series of inflammatory mediators, causing damage to the lung, and at the same time, more inflammatory cells are activated to release more inflammatory mediators or cytokines, so that lung damage signals are further amplified and strengthened, an inflammatory cascade effect is formed, and alveolar-capillary membrane damage is caused. Macrophages predominate in the alveolar cavity of normal lung tissue, and neutrophils predominate in the alveolar cavity when ALI occurs. After ALI, under the action of various chemokines, neutrophils are greatly accumulated in lung tissues, and release a large amount of inflammatory mediators by combining with mechanisms such as respiratory burst, degranulation and the like of alveolar epithelial cells, vascular endothelial cells, macrophages and the like, wherein the proinflammatory mediators are greatly accumulated in tumor necrosis factor-death factors, and the proinflammatory mediators are IL-1 beta, IL-6, TNF-alpha and the like which cause inflammatory response.
Alveolar macrophages are critical in inflammatory diseases of the lung and play a role in maintaining the balance of lung tissues and increasing the rapid response to exogenous and endogenous stimuli. They are the first line of defense to eliminate pathogens, act as a key coordinator of the immune response, ultimately promoting apoptotic debris clearance, eliminating inflammation and repairing tissue. Alveolar macrophages are also the major source of various inflammatory cytokines, such as IL-1 β, IL-6, TNF- α, etc., whose role in inflammation is critical. After ALI occurs, the permeability of endothelial cells of pulmonary capillaries is increased, and a large amount of inflammatory transmitters such as free radicals, cell adhesion molecules, tumor necrosis factors, selectins, interleukins, integrins and the like enter alveoli through capillary barriers, so that neutrophils are chemotactic and deformed and then pass through the pulmonary capillaries with increased permeability to enter pulmonary interstitium and alveoli, and a large amount of proteins also enter the pulmonary interstitium and alveoli from the pulmonary capillaries with increased permeability to cause pulmonary edema; meanwhile, related inflammatory media cause the generation of active substances on the surface of alveoli to be reduced by damaging the epithelial cells of the alveoli, so that the water retention in the alveoli is weakened in fluidity, and pulmonary edema is aggravated; regeneration of alveolar epithelial cells is essential for recovery of ARDS, and in a virus-induced ALI mouse model, if the mouse lung loses more than 10% of type i alveolar epithelium, the respiratory function of the mouse is significantly reduced, after which the ALI condition rapidly progresses and is difficult to recover.
Brefeldin a, a potential macrolide antitumor antibiotic, can inhibit the action of proteins in a competitive way, can not only significantly affect the secretory pathway of mammals, but also cause the morphological change of cells, particularly induce the decomposition of golgi bodies, leading to the redistribution of enzymes back to endoplasmic reticulum after the decomposition of the golgi bodies, has been proved to reduce the generation and secretion of chemical mediators of inflammation and immune response, and can reduce the generation of inflammatory mediators stimulated by TNF-alpha by inhibiting Akt, mTOR and NF- κ B pathways in human keratinocytes. Brefeldin A is currently researched more in antitumor treatment, but relatively few researches in the field of anti-inflammation relate to that acute lung injury is mainly inflammatory reaction, a large amount of inflammatory factors and inflammatory mediators are involved in the early stage of acute lung injury, such as IL-1 beta, IL-6, TNF-alpha and the like, and whether brefeldin A can act on ALI through the influence on the release of related inflammatory mediators is needed to be further researched. Therefore, the experiment utilizes the research methods of molecular biology and functional science to research the effect of Brefeldin A on acute lung injury induced by LPS on animal and cell level, so as to further explore the specific action mechanism of Brefeldin A in acute lung injury.
The research result of the experiment shows that the cell level shows that 100 mu mol of Brefeldin A can effectively inhibit the release of inflammatory factors/chemotactic factors such as TNF-alpha in mouse alveolar macrophage and KC content in epithelial cells; brefeldin A on animal level can effectively inhibit increase of BALF leukocyte count caused by LPS, reduce MPO activity in lung tissue and TNF-alpha level in BALF, increase cAMP content in lung tissue, obviously inhibit phosphorylation of ERK protein kinase in MAPK signal path, improve pathological changes of ALI such as various inflammations, and is worthy of attention, Brefeldin A is different from Dex, and has no obvious influence on inflammatory factors such as IL-1 beta and IL-6 on animal level, which suggests that Brefeldin A can generate unique action different from hormone, and a mechanism of Brefeldin A for relieving acute lung injury caused by LPS is probably related to increase of cAMP content and inhibition of TNF-alpha generation, thereby relieving lung sequestration of neutrophils and release of inflammatory factors.
Claims (4)
1. An application of brefeldin A in preparing medicine for treating acute lung injury is disclosed.
2. The use according to claim 1, wherein the medicament is an inhibitor of inflammatory factor activity.
3. The use according to claim 2, wherein the inflammatory factor is TNF- α or the chemokine KC.
4. The use according to claim 1, wherein the medicament is a medicament for the prevention or treatment of lipopolysaccharide-induced acute lung injury.
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