Type 2 Diabetes and its Effects on MicroRNA-155 and MicroRNA-133a Expression in Lung Tissue of Male Rats

Background: The presence of an extensive vascular extent and large amounts of collagen and elastin in lung tissue make the lung parenchyma vulnerable to damage in diabetes. However, there are few studies on the pathophysiological effects of diabetes on lung tissue. In this study, we investigated the effects of type 2 diabetes on lung tissue pathology and the expression of miRNA-155 and miRNA-133a in lung tissue of male rats. Methods: In this study, 14 male rats were divided into a control group and a group with diabetes. The group with diabetes received a high-fat diet and a single dose of streptozotocin for one month. After induction of T2D, the rats received a normal diet for the next four weeks. At week 8, the rats were euthanized, and lung tissue was collected to measure microRNAs and examine tissue changes. Results: When lung tissue sections from diabetic rats were examined, the normal structure of the alveoli and alveolar sacs and bronchioles was disturbed. The thickness of interalveolar septa was increased due to the inltration of inammatory cells. The extensive alveolar collapse was the main cause of lung tissue structure disruption, and the accumulation of inammatory cells and exudate secretions resulted in an interstitial pneumonia-like appearance. The expression of miRNA-155 was increased, and the expression of miRNA-133a was decreased in the lungs of diabetic rats compared with control rats. Conclusion: We found signicant changes in the lung tissue of diabetic rats. By studying changes in the expression of microRNAs in diabetes, they can be a diagnostic and therapeutic biomarker in the lungs of patients with diabetes.


Introduction
Diabetes mellitus refers to several metabolic disorders, all characterized by chronic hyperglycemia. In this disease, the ability to produce the hormone insulin is lost (type 1), or the body's cells become resistant to insulin (type 2). When blood sugar levels increase and affect metabolism, complications of the disease become noticeable [1,2]. Cardiovascular, kidney and eye complications, problems of the nervous system, disorders of peripheral organs due to nerve damage and nally, heart and ischemic stroke are complications of diabetes in the body [3]. Diabetes is the seventh most common disease in the world in terms of economic and social burden. According to the World Health Organization, more than 488 million people were diagnosed with diabetes in 2014, and more than 1.6 million deaths were directly attributable to diabetes in 2016 [4].
Lung tissue is highly prone to damage and microvascular complications in diabetes because of its large vascular extent and abundant connective tissue [5]. Decreased lung function in people with diabetes is directly related to blood glucose levels, duration of diabetes, and severity of nonsmoking disease and obesity [6].
High blood glucose levels and the duration of the disease also increase the clinical symptoms of chronic lung disease [7]. Changes in the lung tissue of patients with diabetes include 1) an increase in the thickness of the basement membrane, which is due to an in ammatory process in the lungs of a patient with diabetes. 2) neutrophil in ltration and accumulation, and an increase in alveolar wall thickness, and 3) brosis toward in ammatory cell in ltration, as seen in hematoxylin and eosin stains [8,9]. The effects of diabetes on the lungs and pulmonary disease have been demonstrated in many clinical studies.
However, the pathophysiology and mechanism of the effects of diabetes on pulmonary problems are still largely unknown and unclear. Previous studies have shown pathologic changes in the lung tissue of patients with diabetes, but the exact mechanism of these changes is unknown [6,8,10].
MicroRNAs (miRNA) are small, uncoded, protected RNAs 18-25 nucleotides in length that control gene expression after transcription by inhibiting translation or initiating degradation of mRNAs [11]. It has also been shown that each miRNA has hundreds of target genes. Thus, even if the expression of a miRNA is changed, it has a signi cant impact on regulating target genes [12]. Their expression may be a marker of tissue health or disease. These miRNAs can be used as potential biomarkers for diagnosis, prognosis, and treatment [13].
To date, the involvement of many miRNAs in diabetic complications such as retinopathy, nephropathy, neuropathy, and cardiovascular and pulmonary complications has been demonstrated [14,12]. Also decrease the expression of m miRNA-155 in severe asthma and its inhibition in people with lung in ammation and ductal mucus secretion. It decreases the number and secretion of Th2 lymphocytes. In asthma, the level of miRNA-133a also decreases. Increasing the expression of this miRNA decreases airway remodeling (by affecting airway smooth muscle) [15,16]. MiRNA-133a expression is decreased in the bronchial smooth muscle of asthma patients, which increases RhoA levels and improves bronchial response and sensitivity [17]. MiRNA-133a plays an anti brotic role in idiopathic pulmonary brosis (IPF) disease and inhibits differentiation by inhibiting TGF-B. Myo broblasts and prevention of pulmonary brosis [18].
The level of miRNA-155 in the serum of patients with type 2 diabetes is low, suggesting that miRNA-155 controls blood glucose levels in patients with diabetes. Genetically modi ed mice expressing higher levels of miRNA-155 were more to have hypoglycemia and increased insulin sensitivity. miRNA-155 causes higher glucose uptake in all cell types [19,20]. The level of miRNA-155 in foot ulcers of study participants in the diabetes group is higher than usual, which suppresses FGF7, effective in wound healing and wound epithelialization. By inhibiting miRNA-155, the wound healing rate was signi cant [20]. Studies on the inhibition of miRNA in preclinical models of asthma, cystic brosis (CF), and IPF have shown that therapeutic targets are satisfactory [21][22][23]. In animals, these miRNA inhibitors are administered via sprays and nebulizers, and this is good news for the search for a new way to treat chronic lung diseases [24].
Glycosylated protein in the lung and thoracic tissues after hyperglycemia, the resistance of collagen to proteolysis, and nally, the accumulation of this protein in lung connective tissue may cause lung disease in patients with diabetes. Understanding the mechanism of these disorders will help diagnose and treat the lung damage caused by diabetes. MiRNAs are markers for various diseases. Therefore, in this study, we investigated the effects of type 2 diabetes on lung tissue damage and the expression of miRNA-155 and miRNA-133a in lung tissue of male rats.

Animals studied
This study approved by the ethics committee of the Tabriz University of Medical Sciences, with code 1398/344. In this study, 14 male Wistar rats (8-10 weeks) weighing 220-250 g were transferred after purchase to Animal Care Center from Tabriz University of Medical Sciences. The animals were kept in standard cages on a 12:12 dark-light cycle at temperatures ranging from 18 to 22°C, with free access to water and food. Ten days after transfection (to relieve stress and acclimate to the new environment), rats were randomly divided into two groups: Control (rats with a normal diet) and diabetic (rats with diabetes induction). The study site was the Applied-Drug Research Center of Tabriz University of Medical Sciences.
Induction of diabetes and Approval of diabetic rats T2D was induced by a HFD (22% fat, 20% protein, and 48% carbohydrate) for four weeks, followed by intraperitoneal injection of a single dose of STZ (35 mg/kg body weight) [25]. To dissolve streptozotocin (STZ), 0.1 M cold citrate buffer with a pH = 4.5 was used. The diabetic and control rats received a normal diet for four weeks after the onset of diabetes. Before being killed, the diabetic and control rats were required to fast for 12 h. A glucose tolerance test (GTT) was then performed by administering 1 g/kg glucose to the diabetic and control rats. Blood glucose levels were measured after 0, 30 min, 60 min, 90 min, and 120 min using a rat glucose meter.
Preparation of lung tissue for analysis of tissue sections and expression of miRNAs On the last day of week 8, lung tissue was collected for the measurement of miRNAs after animals were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) to assess tissue changes. Lung tissue was washed with cold, sterile normal saline. Then, one lung was frozen to examine gene expression, and the other was xed in 10% formalin solution to examine tissue changes. The hematoxylin-eosin (H&E) method was used to evaluate the extent of lung injury. The histopathological examination included tissue changes such as leukocyte in ltration, perivascular edema, in ammation, and pulmonary brosis. Histopathological changes were examined using a light microscope (Olympus CH30 made in Japan) and photographed with an Olympus DP12 camera, U-TVO.5XC-2 Japan.
MicroRNAs extraction, cDNA synthesis and real-time polymerase chain reaction Real-time polymerase chain reaction (PCR) was used to determine the transcription of miRNA-155 and miRNA-133a. For this purpose, lung tissue was rapidly sectioned, and RNA content was extracted using TRIzol (Roche, Germany) according to the manufacturer's instructions. A Picodrop 1000 spectrophotometer (Thermo Scienti c, USA) was used to determine RNA concentration and integrity. Table 1 shows the sequences of all primers used to detect miRNA-155, miRNA-133a, and U6-6p. cDNA Synthesis Kit was used to generate cDNA (TaKaRa). Real-time PCR was performed on a Rotor-Gene 6000 instrument using a cocktail of cDNA sample (1L), SYBR Green Master Mix (5L; TAKARA), DEPC water (3.7L), and primers (0.3L) (Corbett, Australia). The PCR products were standardized to U6. The 2ΔCt technique was used to determine the relative quantitative expression of miRNA.

Statistical analysis
Analysis was performed using SPSS 19 software. All quantitative data were presented as mean± SEM and analyzed with the student t-test. In this study, a value of P <0.05 was considered signi cant.

Results
GTT con rms type 2 diabetes.
Because we autopsied the rats one month after they developed diabetes, we performed a GTT to con rm that the rats continued to have diabetes. GTT showed that glucose tolerance was impaired in the group with diabetes compared with the control group at week 8 ( Figure 1).

The effect of diabetes on lung tissue
The lungs of both diabetic and control groups were examined by light microscopy with hematoxylin and eosin staining. The lung tissue sections of the rats in the control group showed normal tissue structure.
Normal alveoli with the septum between the thin alveoli and alveolar sacs were visible. The alveoli walls were lined with type 1 and type 2 pneumocytes, each with its characteristics (type 1 pneumocytes are apartments and type II pneumocytes have large round domes) (Figure 2A).
When we studied the lung tissue sections of the diabetic rats, we observed the disruption of the natural structure of the alveoli and sacs and the bronchioles and interstitial vessels. The interstitial septa were thickened, mainly due to in ltration of in ammatory cells, including macrophages. We also saw an increase in the thickness of the epithelial layer covering the surface of the bronchioles. In some tissue sections, homogeneous material accumulated in the arteries, and we also noted exudative secretions between the alveoli. One of the most common ndings on most slides and tissue sections was an extensive alveolar collapse, the leading cause of disruption of normal lung tissue structure. The accumulation of in ammatory cells and exudative secretions resembled interstitial pneumonia ( Figure   2B).
The effect of diabetes on the expression of MiRNA-155 and MiRNA-133a in the lungs of rats According to the results, the expression level of the miRNA-133a gene was lower in diabetic rats than in control rats ( Figure 3A) (p <0.05). However, the expression level of the miRNA-155a gene was higher in diabetic rats than in control rats ( Figure 3B). (p <0.01).

Discussion
The prevalence of diabetes is increasing every day. Therefore, study its adverse effects, including the effects of diabetes on patients' lungs. Previous studies have demonstrated pulmonary changes in patients with diabetes [26-28]. We studied the lung tissue of rats to control the pulmonary changes in diabetes. In this study, we investigated the effects of diabetes on the expression of two genes, miRNA-155 and miRNA-133a, in the lung tissue of diabetic rats.
In a study on the effects of diabetes on lung function, two groups of diabetic and non-diabetic overweight patients were examined. According to studies, all lung capacities (FVC, FEV1, DLCO, TLC) of patients with diabetes were lower than those of non-diabetic patients [29,30]. In a study to evaluate the histopathological changes in the lungs of diabetic rats, changes in lung tissue were investigated in two groups: the group with diabetes and the control group. It was found that a signi cant increase in interalveolar septa was observed after four weeks, indicating cellular in ltration.
Some bronchioles showed patchy hyperplasia of the lower epithelium. Eight weeks after the onset of diabetes, an accumulation of alveolar macrophages and in ammatory cells was observed in the lung tissue, which increased the thickness of the septum between the alveoli and caused many alveoli to collapse, disrupting the normal tissue structure. The normal tissue structure was disturbed. Obstruction and thickening of the vessels occurred with exudate of uid and blood into the lung tissue. Collagen ber accumulation was also increased in the group with diabetes compared with the control group [31].In our study, the lungs of both the control and groups with diabetes were examined. We observed signi cant changes in the lung tissue of diabetic rats compared with the control group. Increased thickness of interalveolar septa, in ltration of in ammatory cells, exudative secretions in the interstitial space, disruption of the typical appearance of normal lung tissue due to the collapse of alveoli and bronchioles, and similar occurrence of interstitial pneumonia are the sum of our ndings from the study of lung tissue sections from diabetic rats.
According to searches in databases and articles, miRNA-133a has not been studied in the lungs of patients with diabetes. Also, the role of miRNA in type 2 diabetes has rarely been discussed. This miRNA is highly expressed in cardiomyocytes. According to a study by Shali Chen et al. [32] the expression of this miRNA was decreased in diabetic rats with cardiac brosis.
Expression of miRNA-133a in bronchial smooth muscle is markedly reduced in asthma patients, which increase RhoA levels and enhances bronchial responsiveness and sensitivity [15]. MiRNA-133a plays an anti brotic role in IPF by inhibiting myo broblast differentiation and preventing lung brosis by inhibiting TGF-β [18]. In our study, miRNA-133a expression was lower in diabetic rats than in controls, consistent with studies in diabetes. Because miRNA-133a in the lungs of patients has been little studied, our study may represent a turning point in the study of miRNA-133a in the lungs of study participants in the diabetes group and other diseases. In our study, the expression of miRNA-155 was increased in the lungs of diabetic rats compared with control rats. A study investigating the effect of miRNA-155 on the pathogenesis of diabetes complications found that the expression of miRNA-155 was reduced in tissues other than the liver, including peripheral blood, heart, liver, sciatica, and aorta of diabetic rats. In this article, they also con rmed changes in the expression of this miRNA in diabetic rats [33]. In another study, the expression of miRNA-155 was also lower in diabetic rats [34]. However, these studies did not examine lung tissue. Thus, their results contradict our results, but they support the importance of this miRNA in diabetes.
A study by Dong Zhang et al. also showed that a de ciency of miRNA-155 plays a protective role in preventing cardiac brosis in diabetic rats [35]. In another study, a group concluded that miRNA-155 reduced the prophylactic effect of TGF-B [36]. In a study by Kuroska et al. [37] miRNA-155 increased collagen deposition and the production of TGF-B, which mediates pulmonary brosis. And Moses mentions that miRNA-155 increased collagen production mediated by TGF-B [38]. MiRNA-155 plays a vital role in the progression of systemic sclerosis. Its expression is markedly increased in the lungs of patients with systemic sclerosis, and miRNA-155 is an essential biomarker for the progression of pulmonary brosis in these patients. This effect could be an important target for treating this disease [39]. And high levels of this miRNA are used in the diagnosis and severity of asthma. It is directly related to IL-13 level and negatively correlates with FEC and FEV1 [40].
In non-SCC lung cancer, we have a high expression of miRNA-155, which increases disease expression [41] and is essential because one of the signi cant lung changes in diabetes is lung brosis and increased collagen production. We also obtained the same result in this study. Additionally, all these articles agree with our study in which an increase in miRNA-155 was found. Although we did not investigate the exact pathogenesis of miRNA-155 expression in diabetic lung lesions, previous studies suggest that miRNA-155 cause brosis and increased collagen deposition by acting on in ammatory and brotic factors. According to recent articles, using miRNAs as diagnostic biomarkers and therapeutic targets is strongly recommended. These miRNAs could be a good target for the treatment and, more importantly, preventing diabetic lung problems.

Conclusion
The prevalence of diabetes is increasing every day. Therefore, research into its side effects, including the effects of diabetes on patients' lungs, is essential. In this study, we found changes in the lung tissue of diabetic rats. We also found that the expression of miRNA-155 increased in the lungs of diabetic rats, and the expression of miRNA-133a decreased in the lungs of diabetic rats. Based on all these ndings, we suggest that miRNA-155 is a signi cant cause of lung injury in diabetes when its expression is increased and that miRNA-133a can cause lung injury when its expression decreases. To further support these ndings, we recommend conducting studies with more extensive samples and investigating changes in lung tissue by manipulating the expression of these genes to evaluate their therapeutic or prophylactic effects. We also recommend that future studies investigate the effects of diabetes on the expression of genes and miRNAs at the proteomics level. Figure 1 Shows the GTT test in the control group and the groups with diabetes (n = 7) for each group. Bars represent means ± SEM.