- Academic Editor
†These authors contributed equally.
Objective: To investigate the effects of Ginkgo biloba extract (GBE) on
autophagy in human macrophages stimulated by cigarette smoke extract (CSE).
Methods: The human monocyte cell line U937 was cultured in
vitro, and phorbol ester (PMA) was added to the cell culture medium to induce
differentiation into human macrophages. CSE was prepared by traditional methods
for experiments. The cells were divided into four groups: the blank group, the
CSE model group, the GBE + CSE group, and the rapamycin + CSE group.
Immunofluorescence was used to identify human macrophages, transmission electron
microscopy was used to observe the ultrastructure of human macrophages in each
group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant
from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were
measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7,
and Rab7 were measured by Western blotting. Results: U937 cells were
successfully differentiated into human macrophages after induction with PMA. The
CSE model group had many more autophagosomes than the blank group. Compared with
the CSE model group, the GBE + CSE group and the rapamycin + CSE group had
significantly more autophagolysosomal. Compared with the other groups, the CSE
model group had a higher level of IL-6 but a lower level of IL-10 in the
Chronic obstructive pulmonary disease (COPD) is a highly disabling chronic respiratory disease characterized by persistent respiratory symptoms and airflow limitation caused by airway and/or alveolar abnormalities. From 1980 to 2017, COPD was the third leading cause of death in the world, and the mortality rate will continue to increase in the next 20 years . Chronic and long-period inhalation of cigarette smoke (CS) is considered a major cause of COPD . CS can alter airway epithelial barrier function, increase oxidative stress, promote airway epithelial aging and activate proinflammatory pathways . CS triggers the recruitment of blood monocytes to the lung, leading to an increase in macrophages in the alveoli and lung parenchyma of COPD patients . Macrophages play an important role in the innate and adaptive immune responses to alien particles and pathogens , and alveolar macrophages (AMs) are responsible for recognizing, phagocytosing and destroying pathogens and processing inhaled particulate matter (including cigarette smoke). The key innate immune effector cells, AMs, play a key role in initiating and maintaining chronic inflammatory responses, and at the same time they play an important role in the pathogenesis of chronic inflammation and abnormal repair in COPD . It was found that the AMs of smokers and COPD patients have functional defects, and CS can impair the phagocytic function of AMs by influencing their autophagy function, reducing their ability to clear airway bacteria .
Autophagy, an automatic cellular component degradation system performed by lysosomes, promotes the recycling of damaged intracellular organelles, denatured proteins and other biological macromolecules, and is essential for maintaining the intracellular environment and cell survival . In addition to regulating inflammatory responses by maintaining protein and organelle homeostasis and cell viability , autophagy also plays a key role in regulating the proliferation, recruitment, polarization and phagocytosis of macrophages . CS exposure leads to protein deposition dysfunction resulting in the accumulation of ubiquitinated proteins and impairs the function of a key player in autophagy, p62. CS-induced autophagy impairment accelerates lung aging and COPD emphysema exacerbation, which is a potential mechanism to initiate the pathogenesis of COPD emphysema . Studies have found that CSE can inhibit the maturation and degradation of autophagosomes in macrophages, inhibit autophagic flux, lead to defective autophagosome maturation and abnormal accumulation of autophagosomes in macrophages, and damage the normal autophagy function of macrophages . GBE is a mixture containing a variety of effective medicinal ingredients extracted from the dried leaves of Ginkgo biloba. Previous studies have found that GBE can reduce the levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) and serum of COPD rats , but the specific mechanism is still unclear. In this study, CSE-stimulated human macrophages were used to establish a COPD cell model in vitro to investigate whether GBE could enhance the autophagy function of human macrophages and to explore the possible mechanism of GBE in the treatment of COPD.
Human histiocytic lymphoma cells (U937 cells) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Item No.: ZQ0087, Shanghai, China); phorbol ester (PMA) was produced by Dalian Meilun Biotechnology Co., Ltd. (Item No.: MB5349, Dalian, China). Ginkgo biloba extract was purchased from Hebei Shenwei Pharmaceutical Group Co., Ltd. (Z13020795, Shijiazhuang, China). Rapamycin was purchased from Dalian Meilun Biotechnology Co., Ltd. (lot No.: MB1197, Dalian, China), and filter cigarettes were produced by Hongyun Honghe Tobacco Co., Ltd. (tar content 10 mg, smoke nicotine content 1.1 mg, smoke carbon monoxide content 12 mg, Kunming, China). IL-6 (lot No.: H201127-007a) and IL-10 (lot No.: H201127-009a) ELISA kits were purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd. (Shenzhen, China), and SYBR FAST qPCR Master Mix (product number: KM4101) was purchased from KAPA Company (Woburn, MA, USA) in the United States. Primary antibodies against p62 (lot No.: bs-55207R), ATG5 (lot No.: bs-4005R), ATG7 (lot No.: bs-2432R), Rab7 (lot No.: bs-6703R), and CD68 (lot No.: bs-0649R) were purchased from Beijing Boaosen Biotechnology Co., Ltd (Beijing, China).
Referring to the method of Li Minjing et al. , first, a piece of
unfiltered cigarette (tar content 10 mg, smoke nicotine content 1.1 mg, smoke
carbon monoxide content 12 mg) was ignited. One end of a rubber tube was
connected to a cigarette filter, and the other end of the rubber tube was
connected to a 50 mL syringe. The syringe was pulled slowly 2–3 times first, and
when the smoke in the tube was thick, the syringe was slowly pulled and cigarette
smoke was collected in the syringe (each syringe collects 50 mL, 1 cigarette
produces 8 tubes, or approximately 400 mL), and was injected into 20 mL RPMI1640
medium containing 10% fetal bovine serum. The samples were allowed to sit until
the cigarette smoke was fully dissolved by sufficient shaking, and the above
method was repeated 4 times. The pH value was adjusted to 7.4 with 1 mol/L NaOH
solution and sterilized by filtration through a 0.22
U937 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum
(FBS) at a temperature of 37 °C and a CO
CD68 is a lysosome-associated membrane protein and the most well-known
panmacrophage marker, often used as a specific marker for identifying macrophages
. After the U937 cells were induced to differentiate with PMA for 24 h, the
expression of CD68 was measured by immunofluorescence staining to identify
whether U937 cells differentiated into human macrophages; the original medium was
aspirated, digested with trypsin, resuspended and counted, and the cells were
seeded in a 24-well plate. The plate contained approximately 1
The well-grown human macrophages were randomly divided into 4 groups: the blank
group: cultured with culture medium for 48 h; the CSE model group: after
culturing with culture medium for 24 h, CSE at a final concentration of 15% was
added for another 24 h; the GBE + CSE group: after culturing for 24 h with GBE at
a final concentration of 1.75
After drug intervention, the culture medium was removed by suction, and the cells were washed with PBS and digested with trypsin. After centrifugation, the cells were allowed to sink to the bottom of the centrifuge tube. The supernatant was removed by suction, fixed with 2.5% glutaraldehyde, placed at 4 °C, and then treated with 1% osmic acid. The samples were fixed, dehydrated with ethanol and acetone, impregnated with acetone and pure epoxy resin, embedded, polymerized, ultrathin sectioned, and then stained with uranyl acetate and lead citrate in the dark. The macrophage ultrastructure was observed and photographed under a transmission electron microscope (TEM) (Hitachi HT7700, Tokyo, Japan).
After drug intervention, the cell culture supernatants from each group were collected, and ELISA was used to measure the contents of IL-6 and IL-10 in the cell supernatants from each group. The specific operations were carried out according to the kit instructions.
The cells were collected from each group, the total RNA was extracted according
to the instructions of the TRIzol reagent, the RNA was reverse-transcribed into
cDNA and the product was stored at 4 °C for later use. The cDNA was used
as the template for PCR amplification, and PCR was carried out according to the
following conditions: Predenaturation at 95 °C for 3 min, denaturation
at 95 °C for 5 s, annealing at 56 °C for 10 s, extension at 72
°C for 25 s, and amplification for 40 cycles. The relative expression
levels of p62, ATG5, ATG7, and Rab7 mRNA were
calculated by the 2
|Gene name||Direction||Primer sequence (5′ to 3′)||Product length (bp)|
The cells from each group were collected, and 200
In terms of cell morphology, U937 cells were round or oval under a light microscope and were small. They were suspended cells without protrusions or pseudopodia on the surface. After 24 h of differentiation induced by PMA, the cell morphology changed. The cell shapes were different, the volume was obviously increased, the cell aggregation phenomenon was obvious, and some cells grew pseudopodia. After immunofluorescence (IF) staining for CD68, the cell cytoplasm was obviously stained red, the nucleus was blue, and the cell differentiation rate reached more than 95%, which indicates that the U937 cells had differentiated and become human macrophages. Please refer to Fig. 1.
Almost all U937 cells were differentiated into macrophages after
24 h of induction with PMA. (A) Representative live-cell images demonstrating
the morphological differences between U937 cells (left) and macrophages (right).
(B) Representative IF staining images of CD68 in human macrophages. Nuclei were
visualized using Hoechst-33342 staining. Scale bars, 100
Electron microscopy is still an important tool for observing autophagic activity and the ultrastructure of macrophages . When certain organelles or local cytoplasm degenerate and disintegrate in cells they are surrounded by double-membrane, vesicle-like bodies called autophagosomes. After fusion with primary lysosomes, autophagolysosomes are formed, and hydrolases are activated to decompose aging organelles, proteins, mitochondria, glycogen and other substances, providing energy for cellular metabolism, maintaining cellular homeostasis and avoiding protein abnormalities. From the electron microscope results, it could be seen that there was autophagy in the blank group, with a small amount of autophagolysosomes, but no vacuolization, and the cell state was good [17, 18, 19]; the CSE model group had enhanced autophagy, with an obvious increase in autophagosomes, with relatively few autophagolysosomes, obvious vacuolization, and poor cell state [17, 18, 19]; the GBE + CSE group and the rapamycin + CSE group had significantly enhanced autophagy. Compared with the CSE model group, the number of autophagolysosomes was significantly increased in these two groups, with a few vacuoles in the cells, and the cell state was acceptable [17, 18, 19]. Please refer to Fig. 2, Table 2, and Fig. 3 for details.
Representative images of the ultrastructure inside macrophages
of different groups. (A) Representative images of the ultrastructure of
macrophages in the blank group. (A
GBE or rapamycin increased autophagolysosomes in CSE-treated
macrophages. Bar graphs showing the average number of autophagosome,
autophagolysosome, and the ratio of autophagolysosomes VS. autophagosomes in
macrophages of different groups. One-way Analysis of Variance (ANOVA) and Tukey’s
multiple comparisons test were performed. * p
|CSE model group||4.400
|GBE + CSE group||3.000
|Rapamycin + CSE group||2.600
Compared with the blank group, the content of IL-6 in the supernatant from the
cells in the CSE model group was significantly increased, while the content of
IL-10 in the cell supernatant was significantly decreased (p
Bar graphs showing the differences between IL-6 and IL-10 in the
supernatants from each group. One-way ANOVA and Tukey’s multiple comparisons
test were performed. *** p
|CSE model group||3.330
|GBE + CSE group||3.218
|Rapamycin + CSE group||3.225
Compared with the blank group, the CSE model group had higher expression levels
of ATG5 and ATG7 mRNA and a lower level of p62 mRNA in
human macrophages (p
Bar graphs showing the differences in the mRNA expression levels
of p62, ATG5, ATG7, and Rab7 in human
macrophages among each group. One-way ANOVA and Tukey’s multiple comparisons
test were performed. * p
|CSE model group||0.528
|GBE + CSE group||0.311
|Rapamycin + CSE group||0.392
Compared with the blank group, the protein expression levels of ATG5 and ATG7 in
human macrophages in the CSE model group were significantly increased, while the
protein expression of p62 was significantly decreased (p
|CSE model group||0.406
|GBE + CSE group||0.166
|Rapamycin + CSE group||0.160
The protein level changes in p62, ATG5, ATG7, Rab7, and LC3 in
human macrophages among each group. (A) Representative WB results showing the
protein levels of p62, ATG5, ATG7, and Rab7 in human macrophages of each group.
(a) blank group; (b) CSE model group; (c) GBE + CSE group; (d) Rapamycin + CSE
group. (B) Bar graphs showing the quantified protein expression levels results of
p62, ATG5, ATG7, and Rab7 in human macrophages of each group. One-way ANOVA and
Tukey’s multiple comparisons test were performed. (C) Representative WB results
showing the protein levels of LC3-I and LC3-II in human macrophages of each
group. (a) blank group; (b) CSE model group; (c) GBE + CSE group; (d) Rapamycin +
CSE group. (D) Bar graphs showing the ratio of LC3-II VS. LC3-I [optical density
(OD) value] in human macrophages of each group. One-way ANOVA and Tukey’s
multiple comparisons test were performed. * p
COPD is a disease characterized by progressive airflow obstruction primarily caused by chronic inhalation of CS, and CS exposure induces oxidative stress, resulting in transcription factor activation and increased expression of inflammatory mediators and proteases . In addition, smoking can be a major cause of COPD by impairing the mucus transport system of the airway, impairing cough reflex sensitivity, and inducing inflammation . Therefore, in this study, CSE was used to stimulate human macrophages to construct an in vitro COPD cell model. Macrophages are a major group of innate immune cells present in various tissues, where they play an important role as sentinels of the immune system . AMs are the main phagocytic and immunoregulatory innate immune cells in the airway, and their main function is to phagocytose and destroy invading pathogens, while the phagocytic function of macrophages in COPD patients is defective and cannot effectively remove pathogenic bacteria, which in turn leads to persistent bacterial colonization of the lower respiratory tract . In vitro studies have shown that CSE can stimulate mouse AMs to secrete the chemokine ligands CXCL-1, CXCL-2 and IL-6 . IL-6 is a multifunctional proinflammatory cytokine that plays a key role in the development of inflammatory responses . IL-10 is an anti-inflammatory factor that inhibits the expansion of inflammatory responses . In this study, the expression of the proinflammatory factor IL-6 in the cell supernatant from the CSE model group was significantly higher than that from the blank group, while the expression of the anti-inflammatory factor IL-10 was significantly decreased. After the intervention of GBE and rapamycin, compared with the CSE model group, the content of IL-6 in the supernatant was significantly lower while the content of IL-10 was increased, indicating that CSE can stimulate macrophages to secrete inflammatory factors, while GBE and rapamycin can reduce the inflammatory stimulation effect of CSE on macrophages.
Autophagy is an automated cellular component degradation system performed by lysosomes that controls protein and organelle degradation for homeostasis . There are three main types of autophagy: macroautophagy (often referred to as autophagy), microautophagy, and chaperone-mediated autophagy. Macroautophagy is the primary mechanism of autophagy that involves the utilization of transient double membrane vesicles called autophagosomes to recycle damaged proteins and organelles, and then these vesicles fuse with lysosomes, causing lysosomal acid hydrolases to degrade their contents . Autophagy is critical in the regulation of innate and adaptive immune responses; it plays a key role in lung inflammation and the pathogenesis of many chronic lung diseases, and autophagy plays a central role in host defense through multiple mechanisms . Chronic exposure to CS-induced oxidative stress and inflammation leads to impaired autophagy, which in turn accelerates the pathogenesis of COPD emphysema . Macrophages play a key role in the immune response and tissue repair in COPD . AMs constitute the first line of the host innate immune response and play a critical role in early CS exposure. Autophagy mediates CS-initiated activation of macrophage matrix metallopeptidase 12 (MMP12) and subsequent airway epithelium injury, which is an important mechanism to induce the occurrence and development of airway inflammation in COPD . CSE can induce autophagic injury in lung macrophages and cause lung macrophage dysfunction, which on the one hand leads to a decline in their phagocytic function and bacterial clearance capacity and on the other hand promotes the release of inflammatory mediators and proteases .
P62 is a multifunctional adaptor protein that is generally considered to play a key role in autophagy . As one of the markers of autophagy, it is often used to determine the state of autophagy. When autophagy is activated, the level of p62 protein is low, while the p62 level is high when autophagy is inhibited . ATG proteins play a key role in regulating the process and activity of autophagy  and constitute the most important molecular platform that plays a central role in autophagy by regulating the initiation and maturation of autophagosomes . ATG5 and ATG7 are key ATG proteins , and the expression of ATG5 and ATG7 proteins increases significantly when autophagy is activated . ATG5 maintains normal developmental processes by mediating various signals and is a core component of autophagy . It has been reported that ATG5 plays a key role in autophagosome formation and that free ATG5 can facilitate the formation of more autophagosomes . Fluctuations in ATG5 levels largely affect autophagy and are directly involved in the formation of autophagosomes . ATG7 is essential for autophagy, drives the binding of ATG12 to ATG5, and binds LC3-I to lipids of phosphatidylethanolamine to generate LC3-II . Rab7 (or Ypt7 in yeast) is one of the conserved housekeeping Rabs in all eukaryotes and acts as a master regulator of membrane trafficking in eukaryotes. Localized in late endosomes and lysosomes, Rab7 plays a key role in the autophagosome-lysosome fusion step [43, 44]. Its deletion of lysosomes could cause a reduction in the number of lysosomes fused to autophagosomes, which in turn could lead to autophagolysosome maturation impairment .
In this study, compared with the blank group, the mRNA and protein expression levels of ATG5 and ATG7 in human macrophages of the CSE model group were significantly increased, and the mRNA and protein expression levels of p62 were significantly decreased, indicating that autophagy was induced after CSE treatment, with increased autophagosomes. Compared with the CSE model group, the mRNA and protein expression levels of ATG5, ATG7, and Rab7 in human macrophages in the GBE + CSE group and the rapamycin + CSE group were significantly increased, while the mRNA and protein expression levels of p62 were significantly decreased, indicating that autophagy was further enhanced by the addition of GBE or the autophagy agonist rapamycin. However, no difference in the expression of Rab7 mRNA or protein levels was found between the blank group and the CSE model group. Moreover, the levels of Rab7 in the CSE model group were significantly lower than those in the GBE + CSE group and the rapamycin + CSE group, indicating that the number of autophagolysosomes was relatively deficient and that there were impaired autophagosome maturation in the CSE model group. In the CSE model group, ATG5, a key protein in autophagosome formation, was significantly increased compared with the blank group, while Rab7, one of the main indicators of lysosome quantity, had no significant difference compared with the blank group. These results indicated that CSE intervention can cause relatively insufficient lysosome formation, a reduced number of autophagolysosomes, and impaired autophagosome maturation in macrophages, which in turn affects normal metabolism in macrophages.
Autophagy is dependent on the lysosomal degradation pathway and plays a crucial role in the development of COPD . Defective autophagy fails to completely clear aged or defective mitochondria, misfolded or damaged protein aggregates, and autophagosomes through lysosomes, thereby further disrupting the cellular clearance process, which drives the initiation and progression of COPD . CS can trigger active autophagy but cause insufficient autophagic clearance in the lung, resulting in a significant accumulation of autophagosomes . Monick found that CS impairs autophagy in AMs in COPD patients, and this autophagy impairment leads to reduced protein aggregation clearance, mitochondrial dysfunction, and defective bacterial delivery to lysosomes . Another study found that CS exposure enhanced the autophagy process, which could cause insufficient autophagic clearance in macrophages, resulting in an increase in autophagic proteins .
In this study, the level of autophagy in the CSE model group was significantly changed compared with the blank group, as suggested by the increased expression of autophagy related proteins. Moreover, under the electron microscope, the number of autophagosomes in macrophages in the CSE model group was significantly increased compared with that in the blank group, but the formation of autophagolysosomes was less, indicating that there is a fusion barrier between autophagosomes and lysosomes in the CSE model group, resulting in defective autophagy, which in turn causes abnormal aggregation of proteins and autophagosomes, hinders the transmission of damaged organelles to lysosomes, further affects the normal metabolism of macrophages, and eventually leads to a poor cell state and obvious vacuolation. Compared with that in the CSE model group, autophagy was further activated in the GBE + CSE group and the rapamycin + CSE group, and the expression of the autophagy proteins ATG5, ATG7, and Rab7 was significantly increased. Under an electron microscope, the number of autophagolysosomes in human macrophages in the GBE + CSE group and rapamycin + CSE group was significantly higher than that in the CSE model group. Hence, we can conclude that GBE and rapamycin can promote the fusion of autophagosomes and lysosome bodies, accelerate the removal of aging mitochondria, damaged protein aggregates and autophagosomes, and thereby maintain the autophagy function of macrophages.
Nonetheless, there are some limitations in this study. First, due to limited resources, the design of this study is relatively simple. Second, the methods used in this study to reflect autophagic activity have their own limitations. For example, although ATG protein levels are increased in other groups with different treatments compared with the blank group, this alone is not sufficient to conclude that autophagy is being induced, but rather suggestive of the changes in autophagy caused by these treatments. Besides, in addition to TEM, other techniques would be necessary to performed to further confirm the hypothesis, such as quantifying the percentage of autophagolysosomes using LysoTracker reagent or indirect immunofluorescence against Lamp1. Third, the CSE exposure time used to establish the COPD model is relatively short compared with the real situation in patients which usually takes years.
In conclusion, we find that GBE can promote the fusion of autophagosomes and lysosomes in macrophages, enhance the autophagy function of macrophages, and reduce the damage to the autophagy function by CSE in macrophages. This study provides a new perspective for the treatment of COPD.
All data needed to evaluate the conclusions are present in the paper.
ZP, SH, and PZ conceptualized the idea. PZ, JZ, and ZP designed the research studies. PZ and JZ conducted the experiments, and LZ, XL, DG, and LL helped with the experiments. PZ provided resource, and DG helped resource. PZ and ZP analyzed the data. PZ, SH, and ZP wrote the manuscript.
We sincerely thank Professor Yuping Tan (Guangxi University of Chinese Medicine, Nanning, China) for her great help in the establishment of research model and guidance in the previous study.
This work is funded by National Natural Science Foundation of China (No. 81904111), and Guangxi Natural Science Foundation (No. 2020GXNSFBA297022).
The authors declare no conflict of interest.
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