CN106692972B - Formulations containing autophagy inhibitors and their use in the treatment of airway mucus hypersecretion - Google Patents
Formulations containing autophagy inhibitors and their use in the treatment of airway mucus hypersecretion Download PDFInfo
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Abstract
The invention provides a preparation containing an autophagy inhibitor and application thereof. Specifically, the invention provides application of the cell autophagy inhibitor in preparing a preparation for treating airway mucus hypersecretion, and the preparation containing the cell autophagy inhibitor as an effective ingredient. Experiments show that the autophagy inhibitor can remarkably reduce the expression of mucin MUC5AC in an animal model, and further remarkably relieve high secretion of airway mucus.
Description
Technical Field
The invention relates to the field of medicaments, in particular to a preparation for treating high airway mucus secretion of an autophagy inhibitor and application thereof.
Background
Autophagy (autophagy) is an important defense and protection mechanism for the body. The cells can eliminate, degrade and digest damaged, denatured, aged and nonfunctional organelles and biological macromolecules such as denatured proteins, nucleic acids and the like through autophagy and lysosomes, provide necessary raw materials for reconstruction, regeneration and repair of the cells, and realize recycling and reusing of the cells. In this sense, autophagy is an important self-regulatory and protective mechanism for the body to maintain homeostasis and adapt to changes in the microenvironment. However, under certain specific conditions, excessive consumption of organelles and various biological macromolecules ultimately leads to autophagic death of the cells. Thus, whether autophagy ultimately promotes cell survival or death will vary depending on the cell, microenvironment, and induction conditions.
Recent studies have shown that the molecular mechanisms of the autophagy process are associated with at least more than 30 "Atg" genes and proteins. Wherein, Beclin 1(BECN1, Atg6) can regulate the separation and formation of an initial double-layer membrane structure, and in the process of forming the autophagosome by capturing organelles and biological macromolecules of the double-layer membrane structure, LC3B is lipidated (lipidation) and added to the autophagosome double-layer membrane under the action of protease such as Atg5-Atg12-Atg 16. These molecules thus play a key regulatory role in the development of autophagy.
Airway mucus is a dilute solution of lipids, glycocomplexes, and proteins, and the mucin MUC5AC is one of the major components of airway mucus. Airway mucus hypersecretion is a characteristic pathophysiological change of chronic airway diseases such as asthma, and is mainly characterized by goblet cell metaplasia and mucus production increase. Smoking, allergens and other external irritants can cause the recruitment of inflammatory cells and the release of inflammatory mediators in the lung, activate various signal pathways and transcription factors, and promote the high expression of MUC5AC and other mucus proteins.
High airway mucus secretion is an important reason for acute attack and aggravation of chronic airway diseases such as asthma and cystic pulmonary fibrosis, but the molecular mechanism for regulating the airway mucus secretion is not clear. At present, an effective treatment means for airway mucus hypersecretion is still lacking clinically.
In view of the foregoing, there is a pressing need in the art to develop formulations effective in treating or ameliorating airway mucus hypersecretion.
Disclosure of Invention
The invention aims to provide a preparation capable of effectively treating or relieving high secretion of airway mucus.
In a first aspect of the invention, there is provided the use of an inhibitor of autophagy for the preparation of a formulation for the treatment of airway mucus hypersecretion or for inhibiting the expression of mucus proteins.
In another preferred embodiment, the autophagy inhibitor is selected from the group consisting of:
(i) bafilomycin a1(Bafilomycin a1), Chloroquine (Chloroquine), or a combination thereof;
(ii) an antagonist that specifically inhibits the expression and/or activity of an autophagy-related protein;
(iii) any combination of (i) and (ii) above.
In another preferred embodiment, the antagonist comprises iRNA, siRNA, shRNA, or a combination thereof.
In another preferred embodiment, the antagonist comprises an antibody.
In another preferred embodiment, the formulation is a pharmaceutical composition, or a food composition.
In another preferred embodiment, the composition comprises (a) an inhibitor of autophagy; and (b) a pharmaceutically acceptable carrier, or a dietetically acceptable carrier.
In another preferred embodiment, the formulation contains (1) Bafilomycin a1(Bafilomycin a1) and/or Chloroquine (Chloroquine) and (2) interfering RNA for blocking autophagy in a cell.
In another preferred embodiment, the interfering RNA is selected from the group consisting of: beclin-1siRNA, LC3B siRNA, ATG5siRNA, ATG12siRNA, or a combination thereof.
In another preferred embodiment, the mucin comprises MUC5 AC.
In another preferred embodiment, the airway mucus hypersecretion is induced by a factor selected from the group consisting of: smoke, airborne particulates PM, and/or allergens (e.g. house dust mites).
In another preferred embodiment, the airway is that of a human or non-human mammal.
In a second aspect of the invention, there is provided a formulation for the treatment of airway mucus hypersecretion, said formulation comprising (a) an inhibitor of autophagy, said inhibitor of autophagy being bafilomycin a1 and chloroquine; and (b) a pharmaceutically acceptable carrier, or a dietetically acceptable carrier.
In another preferred embodiment, the composition comprises 0.001-99 wt%, preferably 0.1-90 wt%, more preferably 1-80 wt% of the autophagy inhibiting agent, based on the total weight of the composition.
In another preferred embodiment, the formulation comprises a pharmaceutical composition, or a food composition.
In another preferred embodiment, the preparation further comprises interfering RNA for blocking autophagy of cells.
In another preferred embodiment, the interfering RNA is selected from the group consisting of: beclin-1siRNA, LC3B siRNA, ATG5siRNA, ATG12siRNA, or a combination thereof.
In a third aspect of the invention, there is provided a non-therapeutic, in vitro method of inhibiting expression of mucin, comprising the steps of: culturing airway epithelial cells in the presence of an autophagy inhibitor, thereby inhibiting expression of mucus protein by the airway epithelial cells.
In another preferred embodiment, the mucin comprises MUC5 AC.
In another preferred embodiment, the airway epithelial cells are human airway epithelial cells.
In another preferred embodiment, the concentrations of the autophagy inhibitors Bafilomycin a1(Bafilomycin a1) and Chloroquine (Chloroquine) are 10nM and 10 μ M, respectively.
In another preferred embodiment, the autophagy inhibitor is selected from the group consisting of: bafilomycin A1(Bafilomycin A1), Chloroquine (Chloroquine), or a combination thereof
In another preferred embodiment, the autophagy inhibitor is an interfering RNA selected from the group consisting of: beclin-1siRNA, LC3B siRNA, ATG5siRNA, ATG12siRNA, or a combination thereof.
In a fourth aspect of the invention, there is provided a non-therapeutic method of alleviating airway mucus hypersecretion, comprising the steps of: (a) administering an autophagy inhibitor to a subject in need thereof.
In another preferred embodiment, the subject comprises a non-human mammal.
In another preferred embodiment, the subject comprises a rodent, such as a mouse, rat.
In another preferred embodiment, the subject is an animal model induced by smoke exposure, induction by airborne particulates PM, or induction by house dust mite extract.
In another preferred embodiment, the method further comprises the steps of: (b) detecting mRNA expression of MUC5AC, and/or immunofluorescence detecting MUC5AC in the subject.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
Figure 1 shows that blocking autophagy in HBE cells can significantly reduce cigarette smoke extract-induced MUC5AC expression. Wherein figure 1A shows mRNA expression of MUC5 AC. Figure 1B shows immunofluorescence detection MUC5 AC.
Figure 2 shows that the use of an autophagy inhibitor significantly reduced the expression of MUC5AC induced by cigarette smoke extract. Wherein figure 2A shows mRNA expression of MUC5 AC. Figure 2B shows immunofluorescence detection MUC5 AC.
Figure 3 shows that blocking autophagy in HBE cells can significantly reduce the expression of MUC5AC induced by airborne particles PM. Wherein figure 3A shows mRNA expression of MUC5 AC. Figure 3B shows immunofluorescence detection MUC5 AC.
FIG. 4 shows that a defect in the autophagy-related gene is effective in relieving airway mucus hypersecretion induced by cigarette smoke exposure. In which fig. 4A and 4C show representative pictures of airway mucus secretion following smoke exposure in LC3B and Beclin-1 mice, respectively. FIGS. 4B and 4D show the results of semi-quantitative analysis of PAS staining in mice of each group, respectively.
Figure 5 shows that a defect in the autophagy-related gene can significantly reduce PM intervention-induced airway mucus hypersecretion and MUC5AC expression. Wherein figure 5A shows representative pictures of airway mucus secretion following PM intervention in various groups of mice. Figure 5B shows the results of semi-quantitative analysis of PAS staining of various groups of mice. Figure 5C shows mRNA expression levels of MUC5AC in mouse lung tissue.
Fig. 6 shows that a defect in the autophagy-related gene can significantly reduce airway mucus hypersecretion induced by HDM intervention. Fig. 6A shows representative pictures of airway mucus secretion following HDM intervention in various groups of mice. Figure 6B shows the results of semi-quantitative analysis of PAS staining of various groups of mice.
In each figure, "CTL" or "control" represents a control.
Detailed Description
The present inventors have made extensive and intensive studies and have unexpectedly found for the first time that an autophagy inhibitor is very effective in treating airway mucus hypersecretion. Experiments show that the expression of mucin MUC5AC in an animal model can be remarkably reduced by inhibiting autophagy of cells, so that high secretion of airway mucus is remarkably relieved. The present invention has been completed based on this finding.
Definition of terms
As used herein, "Airway mucus hypersecretion" or air mucus hyper-production refers to the pathological process in which the Airway produces large amounts of mucus (known as sputum), resulting in a restricted flow of respiratory gases. The symptoms of high mucus secretion of the airway are characterized by sputum production, a large amount of mucus in the airway lumen, goblet cell hyperplasia and the like, and pathological sequelae thereof comprise airway obstruction, airflow limitation, gas exchange reduction and the like.
As used herein, "Mucin protein MUC5 AC" refers to the Mucin protein Mucin 5AC (MUC5AC) encoded by human MUC5 AC. The protein is the most prominent and representative mucus protein of the various components of airway mucus.
As used herein, "Beclin-1" refers to a key regulatory protein in the development of autophagy, which directly regulates the formation of the bilayer membrane structure of autophagosomes. The Beclin1 gene is also known as BECN1 gene.
As used herein, "LC 3B" refers to a key regulatory protein in the development of autophagy, which is one of the key constituent proteins of autophagosomes.
As used herein, "ATG 5" refers to a key regulatory protein in the development of autophagy, which forms a complex with ATG12, in combination with the regulation of involvement of LC3B in the formation of autophagosomes.
As used herein, "ATG 12" refers to a key regulatory protein in the development of autophagy, which forms a complex with ATG5, in combination with the regulation of involvement of LC3B in the formation of autophagosomes.
Autophagy inhibitor
As used herein, the term "autophagy inhibitor" refers to a substance that inhibits autophagy of cells, and the inhibitor can be a small molecule compound or a large molecule compound (e.g., an antibody). Representative autophagy inhibitors include Bafilomycin a1, Chloroquine, interfering RNA, antibodies, and the like.
Bafilomycin a1 includes Bafilomycin a1 or a pharmaceutically acceptable salt thereof. The molecular formula of the bafilomycin A1 is C35H58O9(molecular weight: 622.83), the structural formula is as follows:
chloroquinone refers to Chloroquine or a pharmaceutically acceptable salt thereof. The molecular formula of chloroquine is C18H26ClN3(molecular weight: 319.87) having the following structural formula
RNA interference (RNAi)
In the present invention, one class of potent inhibitors of autophagy is interfering RNA.
As used herein, the term "RNA interference (RNAi)" refers to: some small double-stranded RNAs can efficiently and specifically block the expression of a specific gene in vivo, promote mRNA degradation, and induce cells to exhibit a specific gene-deleted phenotype, which is also referred to as RNA intervention or RNA interference. RNA interference is a highly specific gene silencing mechanism at the mRNA level.
As used herein, the term "small interfering RNA (siRNA)" refers to a short segment of double-stranded RNA molecule that targets the mRNA of a homologous complementary sequence to degrade a specific mRNA, a process known as the RNA interference pathway.
In the present invention, interfering RNA includes siRNA, shRNA and corresponding constructs.
One typical construct is double-stranded, and has either its positive or negative strand comprising a structure of formula I:
Seqforward direction-X-SeqReverse directionFormula I
In the formula (I), the compound is shown in the specification,
Seqforward directionA nucleotide sequence that is an autophagy-related gene or fragment;
Seqreverse directionIs and SeqForward directionA substantially complementary nucleotide sequence;
x is at SeqForward directionAnd SeqReverse directionA spacer sequence therebetween, and the spacer sequence and SeqForward directionAnd SeqReverse directionAre not complementary.
In a preferred embodiment of the present invention, SeqForward direction、SeqReverse directionIs 19-30bp, preferably 20-25 bp.
In the invention, a typical shRNA is shown as a formula II,
in the formula (I), the compound is shown in the specification,
Seq’forward directionIs SeqForward directionAn RNA sequence or sequence fragment corresponding to the sequence;
Seq’reverse directionIs of Seq'Forward directionA substantially complementary sequence;
x' is nothing; or is located in Seq'Forward directionAnd Seq'Reverse directionAnd the spacer sequence is related to Seq'Forward directionAnd Seq'Reverse directionThe two parts are not complementary to each other,
i is expressed in SeqForward directionAnd SeqReverse directionHydrogen bonds formed between them.
In another preferred embodiment, the length of the spacer sequence X is 3-30bp, preferably 4-20 bp.
Wherein SeqForward directionTarget genes to which the sequences are directed include (but are not limited to): beclin-1, LC3B, ATG5, ATG12, or a group thereofAnd (6) mixing.
Compositions and methods of administration
The present invention also provides a composition for treating or relieving the airway mucus hypersecretion, comprising an autophagy inhibitor as an active ingredient. The composition includes (but is not limited to): pharmaceutical compositions, food compositions, dietary supplements, beverage compositions, and the like.
In the present invention, the autophagy inhibitor can be directly used for disease treatment, for example, for treatment of high airway mucus secretion. When the autophagy inhibitor of the present invention is used, other therapeutic agents such as drugs contributing to sputum excretion and the like may be used together.
The invention also provides a pharmaceutical composition comprising a safe and effective amount of the autophagy inhibiting agent of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention may also be formulated as a powder for inhalation by nebulization. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the autophagy inhibiting agents of the invention may also be used with other therapeutic agents.
For the pharmaceutical compositions of the present invention, administration to a subject in need thereof (e.g., human and non-human mammals) can be by conventional means. Representative modes of administration include (but are not limited to): oral administration, injection, aerosol inhalation, etc.
In the case of pharmaceutical compositions, a safe and effective amount of the inhibitor of autophagy is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The main advantages of the present invention include:
(a) the autophagy inhibitor has obvious effect of relieving high secretion of airway mucus.
(b) The effect of inhibiting autophagy on preventing and treating airway mucus hypersecretion induced by atmospheric pollution and the like is achieved.
(c) The cell autophagy can be used as a brand new target for preventing and treating mucus hypersecretion of airway diseases induced by various reasons.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
General methods and materials
1. Cell culture
Human airway epithelial cell lines (HBE) used in the examples were purchased from Shanghai cell bank of Chinese academy of sciences and cultured in RPMI1640 medium containing 10% fetal bovine serum, 50U/ml penicillin and 50U/ml streptomycin. 3-12 substitutes were used for cell experiments after resuscitation.
2. Preparation of Cigarette Smoke Extract (CSE)
The CSEs used in The examples were prepared using standard research cigarettes (2R1F, available from The Tobacco research institute, University of Kentucky, Lexington, KY) with The filter tips cut off, The smoke was combusted for 6 minutes per cigarette, The smoke was slowly pumped into 10 ml of RPMI1640 medium, The resulting solution was adjusted to pH 7.4 and sterilized with a 0.22 μm filter, i.e., 100% CSE, and stored in a freezer at-80 ℃.
PM particle preparation
The PM particles used in the examples were provided by professor Cormier at louisiana state university, usa, and were artificially synthesized by high-temperature combustion to have a particle size of about 0.2 μm and contain stable radicals.
Transfection with siRNA
The siRNAs used in the examples are commercially available products, all of which are available from Santa cruz company (ATG5-siRNA, SC-41445; ATG12-siRNA, SC-72578; LC3B-siRNA, SC-29390; Beclin-1-siRNA, SC-29797). These siRNAs specifically inhibited ATG5, ATG12, LC3B, Beclin-1, respectively.
The transfection reagent was GenMutetM transfection reagent. The transfection method is described in the specification for the transfection reagent. 48 hours after transfection, the silencing efficiency of the gene was examined.
RNA extraction and Q-PCR
In all the examples, a conventional Trizol reagent is used for extracting cell RNA, 1g of extracted total RNA is taken, a TaKaRa reverse transcription kit is adopted for synthesizing cDNA, a SYBR Green Ex TagTM system is applied for Q-PCR research, and finally a 2-delta Ct method is adopted for determining the relative expression quantity of mRNA of a target gene, wherein the delta Ct is calculated according to the formula shown in the specification, wherein the delta Ct is delta Ct, Q-delta Ct, c b, the delta Ct and Q represent the difference value between the Ct value of a Q target gene of a sample to be detected and the Ct value of a housekeeping gene (β -actin) of the same sample, and the delta Ct and b represent the difference value between the Ct value of the target gene of a control group and the Ct value of the housekeeping gene.
In the specific embodiment, the primers of the human MUC5AC are as follows:
sense:5′-CAGCACAACCCCTGTTTCAAA-3′(SEQ ID NO.:1)，
antisense:5′-GCGCACAGAGGATGACAGT-3′(SEQ ID NO.:2)；
the mouse MUC5AC primers in the specific examples were:
sense:5′-CTGTGACATTATCCCATAAGCCC-3′(SEQ ID NO.:3)，
antisense:5′-AAGGGGTATAGCTGGCCTGA-3′(SEQ ID NO.:4)。
6. immunofluorescence detection MUC5AC
Cells were seeded on glass slides, after intervention, washed with PBS, fixed with 4% formalin, treated with 0.1% Triton-X100 for membrane rupture, and then stained with antibody. MUC5AC immunofluorescence expression was detected by Zeiss LSM confocal laser microscopy.
7. Mouse cigarette smoke exposure model
Study mice were given smoke exposure (100 cigarettes/day, 5 days/week) and control mice were exposed to air for 12 weeks using a mouse smoke exposure device from the U.S. Teague Enterprises.
8. Mouse PM intervention model
Each mouse was subjected to continuous airway instillation for 4 days at a daily dose of 100 μ g PM (dissolved in 50 μ L PBS), and control mice were instilled with the same dose of PBS alone. Lung tissue from mice was taken 24 hours after the last intervention and analyzed.
9. Mouse house dust mite extract (HDM) intervention model
An asthma model was established by HDM sensitization and challenge. Sensitization: day 0, day 3 and day 5 mice of each asthma model group were individually instilled with 10 μ g HDM (dissolved in 50 μ L PBS) via the airways. Excitation: day 10, day 12 and day 14, each asthma model group mouse was also instilled with 10 μ g of HDM (dissolved in 50 μ L PBS) via the airway of each asthma model group mouse. Control mice were instilled with the same dose of PBS alone. Lung tissue from mice was taken 24 hours after the last intervention and analyzed.
10. Lung tissue treatment
Injecting 4% formalin into left lung via trachea cannula for internal fixation, placing into 5ml test tube filled with 4% formalin for external fixation, and staining PAS on specimen at room temperature.
11. Statistical analysis
The experimental data in all examples were counted using GraphPad prism5.0 software and the results were expressed as mean ± standard error (x ± SEM) and either t-test or one-way ANOVA test was performed on independent samples. P <0.05 indicates significant differences.
Example 1
Blocking the effects of autophagy on reducing MUC5AC expression in CSE-induced HBE cells
In the examples, expression of MUC5AC was detected in HBE cells by blocking autophagy with sirnas from autophagy-related genes such as ATG5, ATG12, or LC3B, CSE induction from cigarette smoke extract.
The method comprises the following steps: HBE cells were first given different siRNA interventions for 24 hours followed by 1% CSE subsequent interventions for 24 hours, cells were harvested and RNA was extracted or immunofluorescence was performed to detect mRNA or protein expression of MUC5AC, respectively.
The results are shown in figure 1, that blocking autophagy in HBE cells can significantly reduce cigarette smoke extract-induced MUC5AC expression. Wherein FIG. 1A shows mRNA expression of MUC5AC and FIG. 1B shows immunofluorescence detection of MUC5 AC. (. about, p <0.001)
Example 2
Effect of administration of an autophagy inhibitor on reducing MUC5AC expression in CSE-induced HBE cells
In the examples, the expression of MUC5AC was tested in HBE cells induced by cigarette smoke extract CSE using the autophagy inhibitors Bafilomycin A1(Baf A1) and Chloroquinone (CQ).
The method comprises the following steps: HBE cells intervene with Baf A1(10nM) or CQ (10. mu.M) and 1% CSE for 24 hours, collect cells, extract RNA or do immunofluorescence to detect mRNA or protein expression of MUC5AC, respectively.
The results are shown in figure 2, and the application of the autophagy inhibitor can significantly reduce the expression of MUC5AC induced by cigarette smoke extract. Wherein FIG. 2A shows mRNA expression of MUC5AC and FIG. 2B shows immunofluorescence detection of MUC5 AC. (. p, p < 0.01;. p <0.001)
Example 3
Blocking the effects of autophagy on reducing MUC5AC expression in PM-induced HBE cells
In the examples, in HBE cells, the expression of MUC5AC was detected by blocking autophagy with siRNA of autophagy-associated genes Beclin-1 or ATG5, inducing by air pollution particles PM.
The method comprises the following steps: HBE cells were first given different siRNA interventions for 24 hours followed by PM (100 μ g/ml) subsequent interventions for 24 hours, cells were harvested and RNA was extracted or immunofluorescence was performed to detect mRNA or protein expression of MUC5AC, respectively.
The results are shown in figure 3, blocking autophagy in HBE cells can significantly reduce the expression of MUC5AC induced by airborne particles PM. Wherein FIG. 3A shows mRNA expression of MUC5AC and FIG. 3B shows immunofluorescence detection of MUC5 AC. (. p <0.05)
Example 4
Effect of autophagy-related gene deficiency on reduction of smoke-induced airway mucus hypersecretion in mice
In the examples, cigarette smoke exposure models induced autophagy-associated Beclin-1, or LC3B gene-deficient mice, tested airway mucus secretion in mouse lung tissue, and positive PAS staining in epithelial cells suggested epithelial cell cupping and mucus hypersecretion.
The method comprises the following steps: after 3 months of cigarette smoke exposure (5 days per week, 2 hours per day) using a Teague E10 small animal smoke exposure device, mice were sacrificed and lung tissue PAS staining examined airway epithelial cell cupping and mucus hypersecretion.
The results are shown in fig. 4, and the autophagy-related gene defect can effectively relieve the airway mucus hypersecretion induced by cigarette smoke exposure. Wherein fig. 4A and 4C show representative pictures of airway mucus secretion after smoke exposure in LC3B mice and Beclin-1 mice, respectively, and fig. 4B and 4D show semi-quantitative analysis results of PAS staining in groups of mice, respectively. (. p < 0.001; N.D., not detectable)
Example 5
Effect of autophagy-related gene defect on reduction of airway mucus hypersecretion in mice induced by PM intervention
In the examples, PM intervention induces autophagy-associated Beclin-1 gene deficient mice, and airway mucus secretion in lung tissue of mice is examined.
The method comprises the following steps: each mouse was subjected to continuous airway instillation for 4 days at a daily dose of 100 μ g PM (dissolved in 50 μ L PBS), and control mice were instilled with the same dose of PBS alone. Lung tissues of the mice are taken 24 hours after the last intervention, the mRNA expression level of MUC5AC is detected, and PAS staining of the lung tissues detects goblet formation of airway epithelial cells and high mucus secretion.
The results are shown in fig. 5, and the autophagy-related gene defect can significantly reduce the airway mucus hypersecretion and MUC5AC expression induced by PM intervention. Wherein fig. 5A shows representative pictures of airway mucus secretion following PM intervention in various groups of mice, fig. 5B shows semi-quantitative analysis of PAS staining in various groups of mice, and fig. 5C shows mRNA expression levels of MUC5AC in lung tissue of mice. (. p < 0.01; N.D., not detectable).
Example 6
Effect of autophagy-related gene defect on reduction of high secretion of airway mucus of mice induced by HDM intervention
In the examples, HDM intervention induced autophagy-associated LC3B gene deficient mice, and airway mucus secretion was detected in lung tissue of the mice.
The method comprises the following steps: mice were sensitized 3 times on days 0, 3, 5 and HDM 3 times on days 10, 12, 14, and lung tissue was taken 24 hours after the last challenge, and PAS staining was performed to detect airway epithelial cell goblet formation and mucus hypersecretion.
The results are shown in fig. 6, and the autophagy-related gene defect can significantly reduce airway mucus hypersecretion induced by HDM intervention. Wherein fig. 6A shows representative pictures of airway mucus secretion following HDM intervention in various groups of mice, and fig. 6B shows the results of semi-quantitative analysis of PAS staining in various groups of mice. (. p < 0.001; N.D., not detectable)
Discussion of the related Art
High airway mucus secretion is one of the major clinical features of many chronic airway diseases, such as asthma, and acute exacerbations of these diseases, including death, are often accompanied by large numbers of mucus plugs in the respiratory tract. At present, an effective prevention and treatment method for mucus hypersecretion of patients with chronic airway diseases is still lacking clinically, so that how to inhibit the mucus hypersecretion of airways is a challenge and opportunity for preventing and treating the chronic airway diseases.
The above experiments of the present inventors suggest that autophagy is a key factor in the development of mucus hypersecretion in chronic airway diseases. In the in vitro cultured airway epithelial HBE cells, the inventor utilizes siRNA of different autophagy related molecules such as Beclin-1, ATG5, ATG12 and the like to block autophagy of the cells, and can effectively reduce the expression of MUC5AC induced by CSE or PM. In addition, in HBE cells, the autophagy inhibitors Baf a1 and CQ also significantly inhibited CSE-induced expression of MUC5 AC. Meanwhile, by using mice with autophagy-related Beclin-1 and LC3B gene defects, the invention found that the level of airway mucus high secretion in autophagy-defective mice is significantly reduced in both smoke exposure induction models, PM-induced airway injury models, and HDM-induced asthma models. The research results of different disease models all suggest the same conclusion, namely that inhibition of autophagy can effectively relieve high secretion of airway mucus, and autophagy is expected to become a brand new target for clinically preventing and treating high secretion of airway mucus.
The experimental result of the invention indicates that the autophagy can be used as a key target point for effectively preventing and treating mucus hypersecretion no matter in a cigarette smoke induced model, allergen induced asthma or air duct mucus hypersecretion induced by atmospheric particle pollution.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (8)
1. Use of an autophagy inhibitor for the preparation of a preparation for the treatment of airway mucus hypersecretion or for inhibiting the expression of mucus proteins;
wherein the airway mucus hypersecretion or expression of mucus proteins is induced by a factor selected from the group consisting of: smoke, airborne particulates PM, or allergens;
and, the autophagy inhibitor is selected from the group consisting of:
barfosfomycin A1, Beclin-1siRNA, LC3B siRNA, ATG5siRNA, ATG12siRNA, or a combination thereof.
2. The use of claim 1, wherein the formulation is a pharmaceutical composition.
3. The use according to claim 1, wherein the mucin comprises MUC5 AC.
4. The use according to claim 1, wherein the allergen is a house dust mite extract.
5. A non-therapeutic, in vitro method of inhibiting expression of mucin comprising the steps of: culturing airway epithelial cells in the presence of an autophagy inhibitor, thereby inhibiting expression of mucus protein by the airway epithelial cells;
wherein said mucin expression is induced by a factor selected from the group consisting of: smoke, airborne particulates PM, or allergens;
and, the autophagy inhibitor is selected from the group consisting of:
barfosfomycin A1, Beclin-1siRNA, LC3B siRNA, ATG5siRNA, ATG12siRNA, or a combination thereof.
6. The method of claim 5, wherein said mucin comprises MUC5 AC.
7. The method of claim 5, wherein the airway epithelial cells are human airway epithelial cells.
8. The method of claim 5, wherein the concentration of bafilomycin A1 is 10 nM.
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| 华雯.雷帕霉素与细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究.《浙江大学学位论文》.2013,正文第54页第2段. * |
| 自噬及其抑制剂的研究进展;喻琴琴，等;《实用医学杂志》;20131231;第29卷(第17期);2908-2910 * |
| 雷帕霉素与细胞自噬在哮喘气道炎症和嗜酸粒细胞分化中的作用研究;华雯;《浙江大学学位论文》;20131231;正文第54页第2段 * |
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