Abstract
The present disclosure provides use of malic enzyme 2 (ME2) in preparation of a diagnostic reagent or a medicament for silicosis or pulmonary fibrosis-related diseases, and belongs to the technical fields of medical treatment and medicine. Research results of the present disclosure show that ME2 knockout significantly alleviates inflammatory response and fibrotic lesions in mice with silicosis. Based on the above research results, the present disclosure provides use of ME2 in treatment of pulmonary inflammatory responses and pulmonary fibrotic lesions of silicosis or pulmonary fibrosis-related diseases. Expression of ME2 is inhibited to alleviate the inflammatory response and fibrotic lesions of the silicosis, providing support for exploring a targeted drug for treating pulmonary inflammatory responses and pulmonary fibrosis of silicosis or pulmonary fibrosis-related diseases.
Claims
1. A method for preparing a diagnostic reagent or a medicament for silicosis or pulmonary fibrosis-related diseases using malic enzyme 2 (ME2).
2. The method according to claim 1, wherein the ME2 is used as a biomarker for screening the silicosis or the pulmonary fibrosis-related diseases in preparation of related products for diagnosing or treating the silicosis.
3. The method according to claim 1, wherein the silicosis comprises pulmonary inflammatory response of silicosis and pulmonary fibrosis.
4. The method according to claim 1, wherein the pulmonary inflammatory response and the fibrosis are diagnosed by detection of an expression level of the ME2 in a lung tissue.
5. The method according to claim 4, wherein the detection comprises mRNA and/or protein levels of the ME2.
6. The method according to claim 1, wherein ME2 gene in a macrophage is knocked out to down-regulate levels of inflammatory factors in lungs.
7. The method according to claim 1, wherein the ME2 gene in the macrophage is knocked out to reduce hydroxyproline content in the lung tissue and degree of pulmonary fibrosis.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a schematic diagram of experimental results of levels of ME2 in lung tissues of pneumoconiosis patients. FIG. 1A illustrates a real-time qPCR assay result; FIG. 1B illustrates a Western blot result. ACTIN serves as an internal reference for real-time qPCR assay and Western blot.
[0020] FIG. 2 is a schematic diagram of experimental results of levels of ME2 in lung tissues of pneumoconiosis model mice. FIG. 2A illustrates a real-time qPCR assay result; FIG. 2B illustrates a Western blot result. ACTIN serves as an internal reference for real-time qPCR assay and Western blot.
[0021] FIG. 3 is a schematic diagram of experimental results of cellular localization of ME2 in lung tissues of pneumoconiosis patients and pneumoconiosis model mice; FIG. 3A and FIG. 3B illustrate result analyses of single-cell sequencing; FIG. 3C illustrates immunofluorescence staining results of lung tissue sections of patients with pneumoconiosis; FIG. 3D illustrates immunofluorescence staining results of lung tissue sections of silicosis model mice.
[0022] FIG. 4 is a schematic diagram of experimental results of mRNA expression levels of inflammatory factors in lung tissues of pneumoconiosis model mice. FIG. 4A illustrates expression levels of IL-1 mRNA in mouse lung tissues; FIG. 4B illustrates expression levels of IL-6 mRNA in mouse lung tissues; FIG. 4C illustrates expression levels of TNF- mRNA in mouse lung tissues. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001). Actin serves as an internal reference for real-time qPCR assay.
[0023] FIG. 5 is a schematic diagram of experimental results of levels of inflammatory factors in bronchoalveolar lavage fluids (BALF) of pneumoconiosis model mice. FIG. 5A illustrates expression levels of IL-1 in mouse BALF; FIG. 5B illustrates expression levels of IL-6 in mouse BALF; FIG. 5C illustrates expression levels of TNF- in mouse BALF. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001).
[0024] FIG. 6 illustrates experimental results of inflammatory responses and collagen accumulation in lung tissues of pneumoconiosis model mice. FIG. 6A illustrates hematoxylin-eosin (HE) staining results; FIG. 6B illustrates inflammatory response scoring results; FIG. 6C illustrates Masson staining results; FIG. 6D illustrates fibrosis scoring results. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001).
[0025] FIG. 7 is a schematic diagram of experimental results of expression levels of fibrotic marker Col1a1 in lung tissues of pneumoconiosis model mice. FIG. 7A illustrates expression results of Col1a1 mRNA in mouse lung tissues, and Actin serves as an internal reference for real-time qPCR assay; FIG. 7B illustrates statistics of the positive area of Col1a1 in mouse lung tissues. FIG. 7C illustrates immunohistochemistry (IHC) of Col1a1 mouse lung tissues. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<0.05, **, P<0.01, ***, P<0.001, ****, P <0.0001).
[0026] FIG. 8 is a schematic diagram of expression results of fibronectin in lung tissues of pneumoconiosis model mice. FIG. 8A illustrates experimental results of fibronectin mRNA levels; FIG. 8B illustrates experimental results of fibronectin protein levels; FIG. 8C illustrates statistics of expression of fibronectin protein. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<**, P<0.01, ***, P<0.001, ****, P<0.0001). Actin serves as an internal reference for Western blot.
[0027] FIG. 9 is a schematic diagram of detection results of hydroxyproline in lung tissues of pneumoconiosis model mice. ME2.sup.F/F represents macrophage conditional knockout control mice, and ME2.sup.F/F/Lyz2.sup.Cre represents macrophage conditional ME2 knockout mice (n=12, *, P<0.05, **,P<0.01,***, P<0.001,****,P<0.0001).
[0028] FIG. 10A-E illustrates pathological sections of lung tissues of five pneumoconiosis patients.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0029] The technical solution provided by the present disclosure will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present disclosure.
EXAMPLE 1
[0030] ME2 was significantly expressed in lung tissues of pneumoconiosis patients and mouse models.
[0031] In the present disclosure, expression levels of ME2 were detected by qPCR and Western blot after proteins and RNAs were extracted from lung tissues collected from five normal volunteers and five pneumoconiosis patients. Results found that levels of ME2 mRNA and protein in lung tissues of pneumoconiosis patients were significantly upregulated compared with normal volunteers (FIG. 1). Samples in the present disclosure were from lung transplant samples from pneumoconiosis patients of the China-Japan Friendship Hospital. The present study was approved by the Institutional Review Board of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Project No.: 062-2019). All subjects signed informed consent forms.
TABLE-US-00001 TABLE 1 Pathological sections of lung tissues of five pneumoconiosis patients Pathological Patient Sex Medical history section 1 Male Pneumoconiosis, interstitial lung disease, FIG. 10A and gastroesophageal reflux 2 Male Pneumoconiosis, and history of pulmonary FIG. 10B tuberculosis 3 Male Silicosis FIG. 10C 4 Male Interstitial lung disease Previous history FIG. 10D of contact with coal for 10 years 5 Male Interstitial lung disease, history of FIG. 10E pulmonary tuberculosis, and working in a coal mine for more than 10 years
[0032] To further determine the change in expression of ME2 in pneumoconiosis, the mice were modeled by single-dose intratracheal instillation of 600 mg/kg silica; after six weeks, modeling was completed, mouse lung tissues were collected, and expression levels of ME2 mRNA and protein in mouse lung tissues were detected by qPCR and Western blot. Results showed that compared with the control group (PBS), levels of both ME2 mRNA and protein in lung tissues of silicosis model mice (FIG. 2). This result was consistent with the trend of change in expression of ME2 in lung tissues of the above pneumoconiosis patients.
EXAMPLE 2
[0033] ME2 was mainly expressed in macrophages of lung tissues.
[0034] The lung is a heterogeneous organ orderly composed of a plurality of types of cells. To further explore an effector cell where ME2 serves a function, the present disclosure found from the analysis of single cell transcriptome data of lung tissues of silicosis model mice that the expression of ME2 was significantly upregulated after silica stimulation, and the ME2 was mainly present in macrophages (FIGS. 3A and 3B). Subsequently, verification was conducted by immunofluorescence co-localization. Results found that ME2 was highly expressed in macrophages of lung tissues of both pneumoconiosis patients and model mice (FIGS. 3C and 3D). Macrophage is the most important effector cell known during the pathogenesis and development of silicosis. Biological processes such as its phagocytosis of dust, damage of lysosome, and change of cell metabolism level may activate downstream immune inflammatory response and fibrosis pathway. During the progression of silicosis, upregulation of ME2 can promote disease progression by altering macrophage functions.
EXAMPLE 3
[0035] Macrophage conditional ME2 knockout significantly relieved the secretion of inflammatory factors and the inflammatory cells infiltration in lung tissues of pneumoconiosis model mice.
[0036] To further reveal the function of the ME2 highly expressed in macrophages in pneumoconiosis, the present disclosure used macrophage conditional ME2 knockout mice to construct a model of pneumoconiosis; lung tissues and BALF were collected from normal mice and model mice, mRNA expression levels of inflammatory factors IL-1, IL-6, and TNF- in mouse lung tissues were detected by real-time qPCR (FIG. 4), and expression levels of these three inflammatory factors in BALF were detected by enzyme-linked immunosorbent assay (ELISA) (FIG. 5). In addition, the inflammatory cells infiltration in lung tissues was detected by HE staining (FIGS. 6A and 6B). According to the above three results, it was found that compared with the control group, mRNA and secretion levels of inflammatory factors IL-1, IL-6, and TNF- in lung tissues of macrophage conditional ME2 knockout mice were significantly downregulated, and the degree of inflammatory cells infiltration in lung tissues was significantly improved. Thus, it concludes that knockout of highly expressed ME2 in macrophages can reduce the secretion of inflammatory factors in pneumoconiosis lung tissues and relieve pulmonary inflammatory responses.
EXAMPLE 4
[0037] Macrophage conditional ME2 knockout significantly reduced fibrosis levels of lung tissues of pneumoconiosis model mice.
[0038] To determine the effect of macrophage conditional ME2 knockout on pulmonary fibrosis, the present disclosure conducted Masson staining on paraffin sections of lung tissues of pneumoconiosis model mice. Results showed that the fibrotic degree of lung tissues of macrophage conditional ME2 knockout mice was significantly reduced (FIGS. 6C and 6D). Meanwhile, the present disclosure used real-time qPCR, IHC, and Western blot to detect mRNA and protein expression levels of pulmonary fibrosis marker proteins collagen I (Col1a1) and fibronectin (Fn-1) in lung tissues of the above pneumoconiosis model mice. It was found that compared with the control group, both mRNA and protein expression levels of Col1a1 and Fn-1 were significantly downregulated in lung tissues of macrophage conditional ME2 knockout pneumoconiosis model mice (FIGS. 7 and 8). In addition, the content of hydroxyproline (HYP) in mouse lung tissues was detected, the collagen accumulation in lung tissues was directly detected, and the degree of pulmonary fibrosis was judged. Results showed that, after macrophage conditional ME2 knockout, the content of HYP in lung tissues of pneumoconiosis model mice was reduced by approximately 20% (FIG. 9, P<0.05). The above results demonstrate that macrophage conditional ME2 knockout can significantly reduce fibrosis levels of lung tissues of pneumoconiosis model mice.
[0039] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.
