Introduction Diabetic lung disease is already known as one of the diabetes complications, but report on its therapeutic strategy is rare. The present study aimed to add novel therapeutic strategy for diabetic lung disease, to reveal the protective effect of ghrelin on diabetic lung disease both in vivo and in vitro, and to discuss its probable molecular mechanism.
Research design and methods Diabetic mice and 16HBE cells were our research objects. We surveyed the effect of ghrelin on streptozotocin-induced lung tissue morphology changes by H&E staining. Furthermore, the changes of proinflammatory cytokines (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) were detected by ELISA. To expound the molecular mechanism, we detected critical proteins of TLR4 pathway and observed their changes by immunohistochemistry (IHC), real-time PCR and western blot analysis in vivo and in vitro, respectively.
Results The results of H&E staining showed that pathological alterations of the lung induced by hyperglycemia were ameliorated by ghrelin. The results of ELISA demonstrated that the elevated levels of IL-1β and TNF-α induced by hyperglycemia turned to decrease in the lung after ghrelin treatment. In the results of IHC, real-time PCR and western blot analysis, we found that the TLR4 pathway was elevated by hyperglycemia or high glucose and is remarkably inhibited by the treatment of ghrelin both in vivo and in vitro.
Conclusions Ghrelin could inhibit inflammation of diabetic lung disease by regulating the TLR4 pathway. This study might affect research on diabetic lung disease, and the therapeutic potential of ghrelin for diabetic lung disease is worth considering.
Data availability statement
Data are available upon reasonable request.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Diabetic lung disease was first reported in 1970s.
In our previous study, we indicated that high glucose could induce apoptosis of the lung cells in vitro, whereas ghrelin could eliminate this effect by regulating the Wnt pathway.
WHAT THIS STUDY ADDS
This study showed that hyperglycemia could cause inflammatory effect in the lung, while ghrelin could decrease the release of inflammatory cytokines.
The probable molecular mechanism is that ghrelin could suppress the inflammatory reaction of diabetic lung disease by downregulating the TLR4 pathway both in vivo and in vitro.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
This study finds novel way to treat diabetic lung disease, which affects the research on diabetic lung disease, and the therapeutic potential of ghrelin for diabetic lung disease is worth considering.
Diabetes injures almost all human organs, and the lung is one of the target viscera, which was first reported by Schuyler et al.1 The present study aimed to reveal the anti-inflammatory effect of ghrelin on diabetic lung disease both in vivo and in vitro, and to discuss its probable molecular mechanism. The structural and physiological abnormalities of the lung have been observed, and severe damage to lung function has been found in patients with diabetes.2 Patients with diabetes have lower pulmonary microvascular distensibility in microvascular complications,3 and these features are also related to pulmonary hypertension.4 5 The newest study demonstrated that patients with diabetes tend to be infected with COVID-19 compared with patients without non-diabetes.6 7 Despite the evidence of the diabetic lung disease, the specific molecular mechanism remains unclear. Therefore, more research is urgently needed to clarify the process of diabetic lung disease. In our previous research, we demonstrated that high glucose (HG) could induce epithelial cell death in the lungs in vitro, but the relative phenomenon in vivo still needs to be investigated.8
As a well-known orexigenic peptide, ghrelin is derived predominantly in the gastric area and is also found in the lungs, heart and lymph nodes.9 It was demonstrated previously that ghrelin has an anti-inflammatory effect10 in addition to its growth hormone-releasing properties. Furthermore, it was reported that ghrelin could protect lungs from diseases.11 12 To suppress the activity of ghrelin, D-lys-3-GHRP-6 (an antagonist of ghrelin) is often used in studies in vivo and in vitro.13 14 Our previous studies found that ghrelin could attenuate 16HBE apoptosis induced by HG by regulating the Wnt/β-catenin pathway.8 However, it is still unknown whether other pathways participate in this process.
Increasing evidence indicates that inflammatory mechanisms play a key role in the occurrence and development of diabetes and its complications.15 The Toll-like receptor 4 (TLR4) pathway is closely related to the inflammatory process. The stimulation of TLR4 might activate MyD88 via a series of cascade reactions and contact the promoters of proinflammatory genes. These promoters might activate gene expression and stimulate the inflammatory response, and many cytokines are released to stimulate the occurrence of inflammation.16 Recent studies have investigated the function of the TLR4 pathway in lung diseases.17 Lu et al18 recently found that cyclic peptide extracts relieved the symptoms of chronic obstructive pulmonary disease (COPD) via TLR4 signaling.
Diabetic lung disease is a diabetes complication that occurs in the respiratory system, but this illness is always neglected, and there are few reports on it. In this study, we speculated that hyperglycemia could induce inflammation in diabetic lung disease, which might explain the increased pulmonary-related morbidity and mortality in diabetes. To verify this conjecture, we used streptozotocin (STZ) mice as the diabetic animal model in vivo and human bronchial epithelial cells (16HBE) as the lung cell model in vitro. We explored whether ghrelin could alleviate the stimulation of TLR4 caused by hyperglycemia (or HG), and changes in inflammatory cytokines in diabetic lungs were also detected.
Materials and methods
Experimental animals and treatment
Sixty male C57BL/6 mice (6–8 weeks old, weighing approximately 22 g) were purchased from Sino-British SIPPR/BK Lab Animal (Shanghai, China) and fed in the animal laboratory center of Shanxi Medical University. The mice were kept according to the Animal Unit’s Standard Operating Procedures (12 hour light-dark cycle, 23±2℃) and were allowed water and food ad libitum. It was reported that STZ injection is the most common method to construct an animal model of diabetes. A single low-dose administration of STZ averts overt toxicity and induces progressive β-cell damage, local inflammation and insulitis,19 which is the scientific justification for the use of this model here. Out of 60 mice, 36 were selected randomly, fasted and deprived of water for 12 hours before administration of STZ (Amresco, Solon, Ohio, USA). STZ was injected intraperitoneally (60 mg/kg/day) according to published methods,20 and animals with random blood glucose values ≥300 mg/dL after STZ stimulation were regarded as STZ-induced diabetic mice. Thirty-six model mice were stochastically and evenly divided into three groups as follows: STZ, STZ+ghrelin and STZ+ghrelin/D-lys3-GHRP-6 groups. The remaining 24 normal mice were stochastically and evenly divided into a control group and a control+ghrelin group. Mice in the five groups were treated as follows: (1) control group received 2 µL of sterile normal saline (NS) by tail vein injection; (2) control+ghrelin group received ghrelin (Sigma, 200 ng in 2 µL of sterile NS) by tail vein injection; (3) STZ group received sterile 2 µL of NS by tail vein injection; (4) STZ+ghrelin group received ghrelin (Sigma, 200 ng in 2 µL of sterile NS) by tail vein injection; and (5) STZ+ghrelin/D-lys3-GHRP-6 group received ghrelin (200 ng in 2 µL of sterile NS) and D-lys3-GHRP-6 (Sigma, 60 µg in 2 µL of sterile NS) by tail vein injection.21 The concentrations of ghrelin and D-lys-3-GHRP-6 were determined according to a previous report.22 Injections were given once per day at 12 a.m. throughout the following 14 weeks. Any mice with blood glucose levels above 500 mg/dL, excessive weight loss or weak body resistance were excluded from the study; any mice that were ill or deceased were also excluded from the study. The mice in the five groups reached the humane endpoint criteria and were immediately euthanized by cervical dislocation under sodium pentobarbital anesthesia according to a previous report,23 and the lungs were extracted. Every effort was made to minimize the number of animals used and their suffering. Finally, isolated lungs were frozen in liquid nitrogen and stored at −80℃ for further use.
Lung tissue samples from the control group, control+ghrelin group, STZ group, STZ+ghrelin group, and STZ+ghrelin/D-lys3-GHRP-6 group were chosen at random, fixed in glutaraldehyde and osmium tetroxide dehydrated, and finally embedded in epoxy. Lung tissue samples were sectioned into 5-µm-thick sections after paraffin embedding. Then, the sections were dyed with H&E and observed under a light microscope (100 µm). The total lung injury score was evaluated by the Diffuse Alveolar Damage (DAD) score.24
IHC was performed by using TLR4 (1:300 dilution, ab13556, Abcam), Myd88 (1:150 dilution, ab133739, Abcam), and TRAF6 (1:500 dilution, ab33915, Abcam). Four C57BL/6 mice were randomly selected from each group. The lungs of mice in each group were extracted and promptly fixed with 4% paraformaldehyde for 24 hours. We prepared six 20-mm-thick sections throughout the lungs. After eliminating endogenous peroxidases, 10% goat serum was used to block non-specific binding for 15 min at 37℃. After incubation with the primary antibodies at 4℃, we used phosphate-buffered saline to wash the sections and biotinylated secondary antibodies were used. Finally, we used chromogen and the nucleus was stained with hematoxylin. The sections were observed at magnification (100 µm) and the optical density was examined. The brownish yellow areas in the lung tissue were considered positive. To analyze the mean optical density (MOD) value, both the percentage of positive cells and the staining intensity were calculated by ImageJ software. The MOD value reflected the positive expression of TLR4, Myd88 and TRAF6.
Inflammatory cytokine determination
The levels of interleukin-1β (IL-1β) and TNF-α in the lung tissue samples were analyzed by ELISA kits (Anogen, Mississauga, Ontario, Canada). Five lung tissue samples from the control group, control+ghrelin group, STZ group, STZ+ghrelin group and STZ+ghrelin/D-lys-3-GHRP-6 group were chosen randomly. The lung tissue samples were first chopped into small fragments, homogenized with 1 mL ice-cold RIPA buffer, and then centrifuged at 1000×g for 20 min at 4℃. Finally, the supernatants were collected and subjected to IL-1β and TNF-α level assays using ELISA kits. ELISA was performed as previously reported by Fathi et al.25
16HBE cells (1×106 cells/cm2) were purchased from Millipore Sigma (SCC150, St. Louis, Missouri, USA) and cultured as in our previous report.8 16HBE cells were randomly divided into five groups when 80%–90% confluence was reached: (1) control group (4.5 mg/mL glucose DMEM), (2) control (4.5 mg/mL glucose DMEM)+ghrelin (100 ng/µL), (3) HG group (13.5 mg/mL glucose DMEM), (4) HG (13.5 mg/mL)+ghrelin (100 ng/µL), and (5) HG (13.5 mg/mL)+ghrelin (100 ng/µL)+D-lys-3-GHRP-6 (30 g/L). The concentrations of ghrelin and D-lys-3-GHRP-6 were determined according to a previous report.8
Total RNA extraction and real-time PCR
As described in a previous report,8 total RNA from each group was isolated from the lung tissue samples and 16HBE cells. The mRNA levels of TLR4, Myd88 and TRAF6 were analyzed by real-time PCR using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). The PCR conditions were as follows: denaturation at 94℃ for 5 min, 94℃ for 30 s, 58℃ for 30 s, 72℃ for 20 s, target genes for 26 cycles and GAPDH for 30 cycles, 72℃ for 10 min. The relative gene expression levels were normalized to GAPDH, and those relative to the calibrator were given by the formula 2-ΔΔCt. The following primer pairs were used for real-time PCR: TLR4 forward—5′-AGA CCT GTC CCT GAA CCC TAT-3′, reverse—5′-CGA TGG ACT TCT AAA CCA GCC A-3′; MyD88 forward—5′-GGC TGC TCT CAA CAT GCG A-3′, reverse—5′-CTG TGT CCG CAC GTT CAA GA-3′; TRAF6 forward—5′-ATG CGG CCA TAG GTT CTG C-3′, reverse—5′-TCC TCA AGA TGT CTC AGT TCC AT-3′; GAPDH forward—5′-GGA GCG AGA TCC CTC CAA AAT-3′, reverse—5′-GGC TGT TGT CAT ACT TCT CAT GG-3′.
Western blot analysis
Western blot analysis was carried out as described by Fathi et al.26 27 We extracted protein from samples of the lung tissue or 16HBE cells in lysis buffer. After detecting protein concentrations, we separated and electrotransferred equal amounts of protein (40 µg) from each sample onto polyvinylidene difluoride membranes. After blocking and incubation with the primary antibodies, the membranes were then incubated with secondary antibodies. In the experiment, we used the following antibodies: rabbit polyclonal anti-TLR4 antibody (1:500 dilution, ab13556, Abcam), mouse monoclonal anti-Myd88 antibody (1:1500 dilution, ab119048, Abcam), and rabbit monoclonal anti-TRAF6 antibody (1:1500 dilution, ab33915, Abcam). After electrochemiluminescence (Millipore, USA), the bands were examined and measured by the ImageJ software.
Statistical analysis was performed by GraphPad Prism software (V.8.0). Data from five groups obtained from each experiment in vitro and in vivo are presented as means±SDs. Differences were identified by one-way analysis of variance or χ2 analysis, and p<0.05 was regarded as significant.
Morphological changes in the lung tissue in vivo
H&E staining of the STZ group showed that the lung structure was affected as follows: the alveolar wall was thickened, accompanied by infiltration of inflammatory cells. For the DAD score, there was no significant difference between the control group and the control+ghrelin group. The DAD score was increased in the STZ group (p<0.05 vs control group). Compared with the control group and the control+ghrelin group, changes in inflammatory cell infiltration and alveolar wall thickening could easily be discovered in the STZ group. In the STZ+ghrelin group, the phenomena of alveolar wall thickening and inflammatory cell infiltration were attenuated, with a decline in the DAD score (p<0.05 vs STZ group) (figure 1).
Ghrelin attenuated hyperglycemia-induced stimulation of the TLR4 pathway in vivo and in vitro
Then, we explored whether ghrelin alleviated hyperglycemia-induced lung inflammation via the TLR4 pathway. In figure 2, we detected TLR4, Myd88 and TRAF6 in the lung tissue samples by IHC. The results showed that there was no significant difference between the control group and the control+ghrelin group, whereas the positive rate of the lung cells was higher in the STZ group (p<0.05 vs control group). After adding ghrelin to the STZ group, the positive rate of the lung cells was markedly reduced (p<0.05 vs STZ group), whereas D-lys-3-GHRP-6 eliminated this effect. In figure 3, the levels of TLR4, Myd88 and TRAF6 were not significantly different between the control group and the control+ghrelin group, but were markedly enhanced in the STZ group (p<0.05 vs control group). Furthermore, we found that ghrelin suppressed TLR4, Myd88 and TRAF6 (p<0.05 vs STZ group). This phenomenon was abolished by D-lys-3-GHRP-6 (p<0.05 vs control group). Meanwhile, we found a similar effect in vitro. In figure 4, the levels of TLR4, Myd88 and TRAF6 were not significantly different between the control group and the control+ghrelin group but were markedly increased in the HG group (p<0.05 vs control group). In addition, we found that ghrelin suppressed TLR4, Myd88 and TRAF6 (p<0.05 vs HG group) in vitro. This effect was abolished by D-lys-3-GHRP-6 (p<0.05 vs control group).
Ghrelin decreased the levels of IL-1β and TNF-α in STZ-induced diabetic animals
To investigate the changes in inflammatory cytokines in the lung, the levels of IL-1β and TNF-α were surveyed. The results showed that there was no significant difference between the control group and the control+ghrelin group. However, prominent augmentation of these two cytokines was measured in STZ-induced diabetic animals (p<0.05 vs control group). After administration of ghrelin, IL-1β and TNF-α levels in STZ mice were similar to those from the control group (p>0.05) and significantly lower compared with the STZ group (p<0.05). The suppression of IL-1β and TNF-α levels controlled by ghrelin was cancelled by treatment with D-lys-3-GHRP-6 (p<0.05 vs control group) (figure 5).
Despite the huge advances in understanding the pathophysiology of diabetic lung disease, the precise pathogenesis and therapeutic options are still unclear. In our previous study, we suggested that HG could induce apoptosis in 16HBE cells: changes in blebbing, nuclear shrinkage, polymorphonuclear cells and other signs of apoptosis were observed by transmission electron microscopy.8 It was also pointed out that inflammation is one of the mechanisms of diabetes and its complications.28 Hence, there must be some relationship between apoptosis and inflammation. Recent reports have disclosed this relationship. The release of inflammatory mediators in cardiomyocytes can promote the expression of Bax, which can induce apoptosis.29 Wang et al30 found that inflammation induced by lipopolysaccharide (LPS) could cause cell apoptosis. In addition, there are reports on hyperglycemia-dependent proinflammatory mechanisms. Chang et al31 recently reported that hyperglycemia could cause chronic inflammation. In this article, we used STZ to construct the diabetic animal model, which is often adopted in studies of experimental diabetes.32–34 It could be concluded that hyperglycemia induced an inflammatory response in diabetic mice, since infiltration of inflammatory cells and alveolar wall thickening were found to be increased in the STZ group (figure 1).
Ghrelin is one of the endogenous ligands for growth hormone secretagogue receptor (GHSR), which includes two subtypes: GHS-R1a and GHS-R1b. Ghrelin is mainly mediated by GHS-R1a, and the biological activities of GHS-R1b, a C-terminally truncated isoform of the ghrelin receptor, are still unclear since it cannot be bound and activated by ghrelin.35 Ghrelin has been regarded as a vital regulator of numerous physiological effects, including therapeutic effects for diabetes and its complications. It has also anti-inflammatory therapeutic effects on many pulmonary diseases.36–38 Li et al39 found that ghrelin protects alveolar macrophages against apoptosis by regulating growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/β-catenin signaling and suppresses lung inflammation. Here, we discussed whether ghrelin treats diabetic lung disease by exerting its anti-inflammatory effect. H&E staining showed that the administration of ghrelin alleviated the inflammatory response caused by hyperglycemia by reducing inflammatory cell infiltration and alveolar wall thickening in the STZ+ghrelin group, which confirmed the protective effect of ghrelin on diabetic lung disease. Nevertheless, the administration of ghrelin did not affect the control mice (figure 1).
Many surveys have indicated that many inflammatory mediators are disordered in patients suffering from diabetes.40 41 It was also shown that IL-1β and TNF-α cause inflammation in lung tissue and lead to lung cell apoptosis.42 Hence, we detected IL-1β and TNF-α by ELISA and found that hyperglycemia exhibited increased levels of IL-1β and TNF-α (p<0.05 vs control group), while ghrelin could reduce the levels of IL-1β and TNF-α in STZ mice (p<0.05 vs STZ group). The administration of ghrelin in control mice had no significant effect on IL-1β and TNF-α (figure 5). From these results, we discovered that ghrelin ameliorates the pathological injuries of diabetic lung disease by anti-inflammation. We further discussed the relative molecular mechanisms.
In our previous study, we showed that ghrelin could exert its curative effect on lung cells by regulating the Wnt/β-catenin signaling pathway.8 However, whether other signaling pathways participate in this process is still unknown. A previous study discovered that ghrelin could mitigate inflammation in diabetic encephalopathy by inhibiting TLR4/NF-κB signaling in vitro.28 Therefore, the TLR4/NF-κB pathway was probed here to disclose the mechanism of diabetic lung disease. TLRs, such as TLR4, are membrane-associated receptors that are expressed by both innate (macrophages, dendritic cells, endothelial cells, neutrophils) and adaptive immune cells. TLRs are well known to stimulate the innate inflammatory response and lead to pulmonary dysfunction.43 44 Sidletskaya et al45 showed that TLR4 plays critical roles in the immunoregulation of inflammation in COPD. To clarify the changes in this pathway, we selected the most critical target proteins in this pathway as our study objects, which were TLR4, MyD88 and TRAF6. The mRNA and protein expression levels of TLR4, MyD88 and TRAF6 were surveyed by real-time PCR and western blot analysis, and the increased expression of these factors was observed in the STZ or HG group, whereas ghrelin inhibited their expression both in vivo and in vitro. The administration of ghrelin in control mice had no significant effect on these three factors (figures 3 and 4). Similar changes in TLR4, MyD88 and TRAF6 protein induced by hyperglycemia and ghrelin were also verified by IHC (figure 2). Hence, the effect of hyperglycemia could be removed by downregulating the TLR4 pathway, which might be the molecular mechanism by which ghrelin inhibits the inflammation in diabetic lung disease.
In summary, our findings demonstrated that hyperglycemia could cause inflammation of the lung, while ghrelin could decrease the release of inflammatory cytokines, such as IL-1β and TNF-α. The probable molecular mechanism is that ghrelin could suppress the inflammatory reaction of diabetic lung disease by downregulating the TLR4 pathway. However, whether other signaling pathways simultaneously participate in this process still needs to be clarified in future studies. Our discoveries here showed that ghrelin has therapeutic potential for diabetic lung disease as a new drug, which is the clinical impact here. These findings are significant for patients who suffer from diabetic lung disease.
Data availability statement
Data are available upon reasonable request.
Patient consent for publication
All animal protocols were reviewed and approved by the Committee on Animal Research and Ethics of Shanxi Medical University (Permit Number: SCXK (JIN) 2009-0001). All of the animal studies were performed consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The specific process was in accordance with the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals (2020).
Contributors X-YL designed and performed experiments, analyzed data, and wrote the manuscript and acted as guarantor. D-GW participated in the experiments. R-SL conceived and supervised the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.
Funding This work was supported by the basic research project of Shanxi Science and Technology Department (20210302123357).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.