Pathophysiology/complications••

Ghrelin attenuates inflammation in diabetic lung disease by TLR4 pathway in vivo and in vitro

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Abstract

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.

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.

Introduction

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.

H&E staining

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

Immunohistochemistry (IHC)

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

Cell culture

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 analyses

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.

Results

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).

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Figure 1

The pathological changes in the lung tissue by H&E staining (100 µm): (A) the control group; (B) the control+ghrelin group; (C) the STZ group; (D) the STZ+ghrelin group; (E) the STZ+ghrelin/D-lys-3-GHRP-6 group; (F) the total lung injury score was evaluated by the Diffuse Alveolar Damage (DAD) score. Black arrows: alveolar wall thickening; red arrow: inflammatory cells infiltration. Data are presented as means±SDs (n=10). *p<0.05 versus control group, #p<0.05 versus STZ group. STZ, DAD score, Diffuse Alveolar Damage score; STZ, streptozotocin

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).

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Figure 2

Immunohistochemistry detected the expression levels of TLR4, MyD88, TRAF6 in vivo (100 µm): (A) the control group; (B) the control+ghrelin group; (C) the STZ group; (D) the STZ+ghrelin group; (E) the STZ+ghrelin/D-lys-3-GHRP-6 group. Expression levels of TLR4, Myd88, TRAF6 were at a remarkably higher level in (C) (p<0.05 versus (A)). Expressions of TLR4, Myd88, TRAF6 were reduced in (D) (p>0.05 versus (A) and p<0.05 vs (C)). D-lys-3-GHRP-6 reversed the effect of ghrelin (p<0.05 vs (A)). Data are presented as means±SDs (n=10). *p<0.05 versus control group, #p<0.05 versus STZ group. MOD, mean optical density; STZ, streptozotocin.

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Figure 3

The expression levels of TLR4, MyD88 and TRAF6 in vivo were analyzed by real-time PCR and western blot analysis. The expression levels of TLR4, Myd88, and TRAF6 were increased in the STZ group (p<0.05 vs control group), while ghrelin inhibited the expression levels of TLR4, MyD88, and TRAF6 in the STZ+ghrelin group (p<0.05 vs STZ group and p>0.05 vs control group). D-lys-3-GHRP-6 eliminated the effect of ghrelin (p<0.05 vs control group). The data are presented as means±SDs (n=10). *p<0.05 versus control group; #p<0.05 versus STZ group. STZ, streptozotocin.

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Figure 4

The expression levels of TLR4, MyD88 and TRAF6 in vitro were analyzed by real-time PCR and western blot analysis. The expression levels of TLR4, Myd88, and TRAF6 were increased in the HG group (p<0.05 vs control group), while ghrelin inhibited the expression levels of TLR4, MyD88, and TRAF6 in the HG+ghrelin group (p<0.05 vs HG group and p>0.05 vs control group). D-lys-3-GHRP-6 eliminated the effect of ghrelin (p<0.05 vs control group). The data are presented as means±SDs (n=10). *p<0.05 versus control group; #p<0.05 versus HG group. HG, high glucose.

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).

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Figure 5

Effect of ghrelin treatment in STZ-induced diabetic mice on IL-1β and TNF-α. (A) Hyperglycemia increased the concentration of IL-1β (p<0.05 vs the control group), while ghrelin decreased IL-1β in vivo (p>0.05 vs control group and p<0.05 vs STZ group) and (B) hyperglycemia increased the concentrations of TNF-α (p<0.05 vs control group), while ghrelin reduced TNF-α in vivo (p>0.05 vs control group and p<0.05 vs STZ group). The data are shown as means±SDs (n=10). *p<0.05 versus control group; #p<0.05 versus STZ group. IL-1β, interleukin-1β; STZ, streptozotocin; TNF, tumor necrosis factor-α.

Discussion

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.

Schuyler MR, Niewoehner DE, Inkley SR, et al. Abnormal lung elasticity in juvenile diabetes mellitus. Am Rev Respir Dis 1976; 113:37–41.
doi:10.1164/arrd.1976.113.1.37•Google Scholar
Kolahian S, Leiss V, Nürnberg B, et al. Diabetic lung disease: fact or fiction? Rev Endocr Metab Disord 2019; 20:303–19.
doi:10.1007/s11154-019-09516-w•Google Scholar
Roberts TJ, Burns AT, MacIsaac RJ, et al. Diagnosis and significance of pulmonary microvascular disease in diabetes. Diabetes Care 2018; 41:854–61.
doi:10.2337/dc17-1904•Google Scholar
Mauricio D, Alonso N, Gratacòs M, et al. Chronic diabetes complications: the need to move beyond classical concepts. Trends Endocrinol Metab 2020; 31:287–95.
doi:10.1016/j.tem.2020.01.007•Google Scholar
Südy R, Schranc Á, Fodor GH, et al. Lung volume dependence of respiratory function in rodent models of diabetes mellitus. Respir Res 2020; 21.
doi:10.1186/s12931-020-01334-y•Google Scholar
Hussain A, Bhowmik B, do Vale Moreira NC, et al. COVID-19 and diabetes: knowledge in progress. Diabetes Res Clin Pract 2020; 162:108142.
doi:10.1016/j.diabres.2020.108142•Google Scholar
Caruso I, Giorgino F. The diabetic lung: an easy target for SARS-CoV-2? Diabetes Metab Res Rev 2020; 36.
doi:10.1002/dmrr.3346•Google Scholar
Liu X, Chen D, Wu Z, et al. Ghrelin inhibits high glucose-induced 16HBE cells apoptosis by regulating Wnt/β-catenin pathway. Biochem Biophys Res Commun 2016; 477:902–7.
doi:10.1016/j.bbrc.2016.06.156•Google Scholar
Wang Q, Lin P, Li P, et al. Ghrelin protects the heart against ischemia/reperfusion injury via inhibition of TLR4/NLRP3 inflammasome pathway. Life Sciences 2017; 186:50–8.
doi:10.1016/j.lfs.2017.08.004•Google Scholar
Xiao X, Bi M, Jiao Q, et al. A new understanding of GHSR1a--independent of ghrelin activation. Ageing Res Rev 2020; 64:101187.
doi:10.1016/j.arr.2020.101187•Google Scholar
Rocha N de N, de Oliveira MV, Braga CL, et al. Ghrelin therapy improves lung and cardiovascular function in experimental emphysema. Respir Res 2017; 18.
doi:10.1186/s12931-017-0668-9•Google Scholar
Imazu Y, Yanagi S, Miyoshi K, et al. Ghrelin ameliorates bleomycin-induced acute lung injury by protecting alveolar epithelial cells and suppressing lung inflammation. Eur J Pharmacol 2011; 672:153–8.
doi:10.1016/j.ejphar.2011.09.183•Google Scholar
Rasineni K, Kubik JL, Casey CA, et al. Inhibition of ghrelin activity by receptor antagonist [d-Lys-3] GHRP-6 attenuates alcohol-induced hepatic steatosis by regulating hepatic lipid metabolism. Biomolecules 2019; 9.
doi:10.3390/biom9100517•Google Scholar
Li X, Zhao X, Li C, et al. Inhibitor of ghrelin receptor reverses gefitinib resistance in lung cancer. Hum Cell 2019; 32:360–6.
doi:10.1007/s13577-019-00245-5•Google Scholar
Bowers E, Singer K. Obesity-induced inflammation: the impact of the hematopoietic stem cell niche. JCI Insight 2021; 6.
doi:10.1172/jci.insight.145295•Google Scholar
Kim MS, Lee S, Jung N, et al. The vitamin D analogue paricalcitol attenuates hepatic ischemia/reperfusion injury through down-regulation of Toll-like receptor 4 signaling in rats. Arch Med Sci 2017; 13:459–69.
doi:10.5114/aoms.2016.60650•Google Scholar
Song L, Luan B, Xu Q-R, et al. Effect of TLR7 gene expression mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention role of IFN-γ. Eur Rev Med Pharmacol Sci 2021; 25:866–79.
doi:10.26355/eurrev_202101_24655•Google Scholar
Lu F, Yang H, Lin S-D, et al. Cyclic peptide extracts derived from pseudostellaria heterophylla ameliorates COPD via regulation of the TLR4/myd88 pathway proteins. Front Pharmacol 2020; 11.
doi:10.3389/fphar.2020.00850•Google Scholar
Maheandiran M, Mylvaganam S, Wu C, et al. Severe hypoglycemia in a juvenile diabetic rat model: presence and severity of seizures are associated with mortality. PLoS One 2013; 8.
doi:10.1371/journal.pone.0083168•Google Scholar
Al Hroob AM, Abukhalil MH, Alghonmeen RD, et al. Ginger alleviates hyperglycemia-induced oxidative stress, inflammation and apoptosis and protects rats against diabetic nephropathy. Biomed Pharmacother 2018; 106:381–9.
doi:10.1016/j.biopha.2018.06.148•Google Scholar
Chen J, Liu X, Shu Q, et al. Ghrelin attenuates lipopolysaccharide-induced acute lung injury through NO pathway. Med Sci Monit 2008; 14:BR141–6.
Google Scholar
Ma L, Zhang D, Tang Y, et al. Ghrelin-attenuated cognitive dysfunction in streptozotocin-induced diabetic rats. Alzheimer Dis Assoc Disord 2011; 25:352–63.
doi:10.1097/WAD.0b013e31820ce536•Google Scholar
Su Z, Robinson A, Hu L, et al. Acupuncture plus low-frequency electrical stimulation (Acu-LFES) attenuates diabetic myopathy by enhancing muscle regeneration. PLoS ONE 2015; 10.
doi:10.1371/journal.pone.0134511•Google Scholar
Bai J, Bai Y, Wang XP, et al. Carbon monoxide-releasing molecule-3 ameliorates acute lung injury in a model of hemorrhagic shock and resuscitation: roles of p38MAPK signalling pathway. Shock 2021; 55:816–26.
doi:10.1097/SHK.0000000000001684•Google Scholar
Fathi E, Farahzadi R, Vietor I, et al. Cardiac differentiation of bone-marrow-resident c-kit+ stem cells by L-carnitine increases through secretion of VEGF, IL6, IGF-1, and TGF-β as clinical agents in cardiac regeneration. J Biosci 2020; 45.
Google Scholar
Fathi E, Farahzadi R, Valipour B, et al. Alginate/gelatin encapsulation promotes NK cells differentiation potential of bone marrow resident C-kit+ hematopoietic stem cells. Int J Biol Macromol 2021; 177:317–27.
doi:10.1016/j.ijbiomac.2021.02.131•Google Scholar
Fathi E, Farahzadi R, Javanmardi S, et al. L-carnitine extends the telomere length of the cardiac differentiated CD117+-expressing stem cells. Tissue Cell 2020; 67.
doi:10.1016/j.tice.2020.101429•Google Scholar
Liu X, Xiao Q, Zhao K, et al. Ghrelin inhibits high glucose-induced PC12 cell apoptosis by regulating TLR4/NF-κB pathway. Inflammation 2013; 36:1286–94.
doi:10.1007/s10753-013-9667-2•Google Scholar
Huang J, Peng W, Zheng Y, et al. Upregulation of UCP2 expression protects against LPS-induced oxidative stress and apoptosis in cardiomyocytes. Oxid Med Cell Longev 2019; 2019.
doi:10.1155/2019/2758262•Google Scholar
Wang Y, Wang Y, Yang D, et al. Β1-Adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation. Crit Care 2015; 19:76.
doi:10.1186/s13054-015-0820-1•Google Scholar
Chang S-C, Yang W-CV. Hyperglycemia, tumorigenesis, and chronic inflammation. Crit Rev Oncol Hematol 2016; 108:146–53.
doi:10.1016/j.critrevonc.2016.11.003•Google Scholar
Rossini AA, Like AA, Chick WL, et al. Studies of streptozotocin-induced insulitis and diabetes. Proc Natl Acad Sci U S A 1977; 74:2485–9.
doi:10.1073/pnas.74.6.2485•Google Scholar
Jiang G-J, Han X, Tao Y-L, et al. Metformin ameliorates insulitis in STZ-induced diabetic mice. Peer J 2017; 5.
doi:10.7717/peerj.3155•Google Scholar
Granata R, Settanni F, Julien M, et al. Des-Acyl ghrelin fragments and analogues promote survival of pancreatic β-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats. J Med Chem 2012; 55:2585–96.
doi:10.1021/jm201223m•Google Scholar
Baatar D, Patel K, Taub DD, et al. The effects of ghrelin on inflammation and the immune system. Mol Cell Endocrinol 2011; 340:44–58.
doi:10.1016/j.mce.2011.04.019•Google Scholar
Fu T, Wang L, Zeng Q, et al. Ghrelin ameliorates asthma by inhibiting endoplasmic reticulum stress. Am J Med Sci 2017; 354:617–25.
doi:10.1016/j.amjms.2017.08.022•Google Scholar
Miki K, Maekura R, Nagaya N, et al. Effects of ghrelin treatment on exertional dyspnea in COPD: an exploratory analysis. J Physiol Sci 2015; 65:277–84.
doi:10.1007/s12576-015-0366-7•Google Scholar
Song B, Yan X, Li R, et al. Ghrelin ameliorates chronic obstructive pulmonary disease-associated infllammation and autophagy. Biotechnol Appl Biochem 2021; 68:356–65.
doi:10.1002/bab.1933•Google Scholar
Li B, Zeng M, He W, et al. Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/β-catenin signalling and suppresses lung inflammation. Endocrinology 2015; 156:203–17.
doi:10.1210/en.2014-1539•Google Scholar
Herder C, Dalmas E, Böni-Schnetzler M, et al. The IL-1 pathway in type 2 diabetes and cardiovascular complications. Trends Endocrinol Metab 2015; 26:551–63.
doi:10.1016/j.tem.2015.08.001•Google Scholar
Saltiel AR, Olefsky JM. Inflammatory mechanisms linking obesity and metabolic disease. J Clin Invest 2017; 127.
doi:10.1172/JCI92035•Google Scholar
Cicek M, Unsal V, Doganer A, et al. Investigation of oxidant/antioxidant and anti-inflammatory effects of apigenin on apoptosis in sepsis-induced rat lung. J Biochem Mol Toxicol 2021; 35.
doi:10.1002/jbt.22743•Google Scholar
Imai Y, Kuba K, Neely GG, et al. Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury. Cell 2008; 133:235–49.
doi:10.1016/j.cell.2008.02.043•Google Scholar
Helal MG, Megahed NA, Abd Elhameed AG, et al. Saxagliptin mitigates airway inflammation in a mouse model of acute asthma via modulation of NF-kb and TLR4. Life Sci 2019; 239:117017.
doi:10.1016/j.lfs.2019.117017•Google Scholar
Sidletskaya K, Vitkina T, Denisenko Y, et al. The role of toll-like receptors 2 and 4 in the pathogenesis of chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2020; 15:1481–93.
doi:10.2147/COPD.S249131•Google Scholar

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Abstract
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