Persistent Hyperglycemia Worsens the Oleic Acid Induced Acute Lung Injury in Rat Model of Type II Diabetes Mellitus

ABSTRACT

Aim:

This research aimed to study the impacts of persistent hyperglycemia on oleic acid (OA)–induced acute lung injury (ALI) in a rat model of type II diabetes mellitus.

Materials and Methods:

Healthy adult male albino rats that weigh 150 to 180 g were divided into four groups (n = 6). Group I-saline (75 μL i.v.) was injected and served as a control; group II-OA (75 μL i.v.) was injected to induce ALI. Group III-pretreated with a high-fat diet and streptozotocin (35 mg/kg), was injected with saline, and served as a control for group IV. Group IV was pretreated with a high-fat diet, and streptozotocin (35 mg/kg) was injected with OA (75 μL i.v). Urethane was used to anesthetize the animal. The jugular venous cannulation was done for drug/saline administration, carotid artery cannulation was done to record blood pressure, and the tracheal cannulation was done to maintain the respiratory tract’s patent. Heart rate, mean arterial pressure, and respiratory frequency were recorded on a computerized chart recorder; an arterial blood sample was collected to measure PaO₂/FiO₂. Additionally, the pulmonary water content and lung histology were examined.

Result:

Hyperglycemic rats showed no significant change in the cardio-respiratory parameter. Histology of the lungs shows fibroblastic proliferation; however, rats survived throughout the observation period. There was an early deterioration of all the cardio-respiratory parameters in hyperglycemic rats when induced ALI (OA- induced), and survival time was significantly less compared to nonhyperglycemic rats.

Conclusion:

Persistent hyperglycemia may cause morphological changes in the lungs, which worsens the outcome of acute lung injury.

INTRODUCTION

Diabetes mellitus (DM) is a set of metabolic illnesses characterized by chronic hyperglycemia that results in long-term hyperglycemia and interferes with the level of lipids proteins and carbohydrate metabolism because of the lack of insulin synthesis or action or both.[1,2] According to the clinical study, the main cause of mortality in people who have diabetes is glucotoxicity-induced complications.[3] Evidence shows that persistent hyperglycemia damages many organs, including the lungs, by many mechanisms.[4,5] Researchers and physicians often ignore lung injury. With continuous chronic hyperglycemia in an aging population, diabetic pulmonary consequences are expected to be responsible for increasing pulmonary dysfunction.[5]

Acute lung injury (ALI) is an acute diffuse inflammatory form of lung injury.[6] It is characterized by increased difficulty in breathing, decreased gas exchange, increased pulmonary vascular permeability, and loss of aerated tissue.[6,7] ALI is linked to noticeably higher rates of morbidity, death, and use of critical care resources.[7] Although many ALI interventions have been suggested, overall mortality is still high because distinct subphenotypes of ALI respond differently to treatment, according to current research.[8] The variability of ALI patients is influenced by the range of ALI predisposing factors and triggering events. This heterogeneity has been proposed as a potential explanation for why successful medicines in exploratory investigations failed to demonstrate success in phase III trials.[9,10] It could be more beneficial to target interventions on patients who are at a greater risk for developing ALI. As a result, it is critical to identify the patient populations that are most at risk for ALI. History of chronic alcohol use, hypoalbuminemia, transfusions, pulmonary source of infection, and presence of comorbidity like DM[9] are major modifiers of ALI expression.[11-15]

Some clinical studies reported that DM might protect against the development of acute respiratory distress syndrome (ARDS),[15-17] whereas some studies demonstrated the deteriorating effect of diabetes in acute lung injury,[18-20] others nevertheless did not show DM’s protective effects.[21,22] The relationship between DM and the risk of death in critically sick patients is likewise not obvious; most, but not all, evidence points to a possible absence of such an association.[23-25] Since the New Berlin definition of ARDS was introduced, no research has examined this connection. Therefore, any connections between DM and this condition must still be defined and clarified. Any such link might substantially influence how a doctor assesses a patient’s likely clinical outcome and could provide information for clinical trials examining medicines for ARDS/ALI prevention and treatment.

Therefore, this study aimed to study the impacts of persistent hyperglycemia on ALI (oleic acid–induced acute lung injury) in the streptozotocin (STZ)–induced diabetic rat.

MATERIALS AND METHODS

The present work was carried out as per the recommendation of the Institutional Animal Ethics Committee, Institute of Medical Sciences, Banaras Hindu University, and the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, with Institutional Review Board number Dean/2021/IAEC/2540, ECR/526/Inst/UP/2016/RR-60. Male albino adult rats of the Charles-Foster strain, weighing 150–180 gm, were used for the experiment. The animal house of the Institute of Medical Sciences, Banaras Hindu University, Varanasi, is where the animals were obtained. The animals were kept in the animal room of the Department of Physiology at an under-regulated room temperature (22 ± 2°C) with light (12 hr: 12 hr light-dark interval). Each cage had two rats. The animals were fed a commercially available normal pellet diet (NPD, Hindustan Lever Ltd.) and water ad libitum. All institutional and governmental regulations for the handling and using experimental animals were followed.

All the rats were erratically separated into two dietary regiments by feeding either a high-fat diet (HFD; 35.10% fat, 18.30% protein, and 20% carbohydrate) or NPD (3% fat, 21% protein, 48.8% carbohydrate, and 27.2% other micronutrients) ad libitum, respectively, for the initial 2 weeks.[26] After dietary modification, a set of HFD-fed rats (6 and 6) with a low dose of STZ (35 mg/kg, i.p) was given.[26] The body weight and biochemical estimations of triglycerides (PTG), plasma glucose (PGL), insulin (PI), and total cholesterol (PTC) were conducted just before and 7 days after the vehicle or STZ injection. Under mild ether anesthesia, blood was drawn from the rat’s retro-orbital plexus into Eppendorf tubes containing heparin (20 microliters, 200 IU mL) for biochemical examination. Centrifugation was used to separate the plasma (5 min, 5000 rpm) and was examined for glucose [GOD-POD, (Glucose Oxidase-Peroxidase)], triglycerides (TG) [GPO-POD, (Glycerol-Phosphoric Acid Oxidase Peroxidase)] and total cholesterol [CHOD-POD, (Cholesterol Oxidase Peroxidase)] levels with commercially available colorimetric diagnostic kits (Auto span, Arkray Healthcare Pvt. Ltd., Surat, Gujarat, India). The rest of the plasma samples were then kept at -20°C until an enzyme-linked immunosorbent assay test using rat insulin as the standard was performed to determine the insulin level. Rats having a nonfasting PGL of more than >300 mg/dL were considered diabetic and chosen for further research.[26] The animals’ feed and water consumption were also measured. The rats’ individual meals were permitted to be consumed until the study’s conclusion. The animals were starved the whole night before being given urethane anesthesia (1.3 to 1.5 gm/kg body weight). The carotid artery was cannulated to measure blood pressure using a pressure transducer. The mean arterial pressure (MAP) was calculated from it. The jugular vein was cannulated to provide saline or drugs, and the trachea was cannulated to maintain the respiratory tract’s patent [Figure 1]. By attaching a thread to the skin above the xiphisternum and connecting it to the force-displacement transducer, it was measured the breathing rate. The needle electrodes in a limb lead II set-up were used to record electrocardiographic potentials, and it was used to calculate HR. All data were recorded using a computerized chart recorder (Lab Chart 7, AD Instruments).

Figure 1.

Experimental setup: (a and b) Jugular vein and carotid artery cannulation

Experimental protocol

Rats were divided into four groups (six in each group). All animals were stabilized for 30 minutes after anesthesia and dissection before beginning the experimental protocol.

Group I (NPD-fed rats) served as a time-matched control group. The jugular vein was injected with 75 μL of saline after the initial recording, and RF, MAP, and HR were subsequently recorded for the first 2 minutes and then every 15 minutes for the next 120 minutes.

Group II (NPD + OA) - Following the initial recording of HR, RF, and MAP, OA (75 μL i.v. bolus) were injected via the jugular vein. Recordings were made for the first 2 minutes and then every 15 minutes up to 120 minutes or until the animals died. According to earlier report, the dose of OA was used.

Group III (HFD + STZ + NS) served as a time-matched control for group IV. In these HFD-fed rats, pretreated with STZ (35 mg/kg) with the nonfasting PGL of >300 mg/dL after the initial recording of HR, RF, and MAP; NS (75 μL i.v. bolus) was injected. The first 2 minutes of the recordings were made, followed by 15-minute intervals up to 120 minutes or until the animals died.

Group IV (HFD + STZ + OA) in this HFD-fed rats pretreated with STZ (35 mg/kg) with the nonfasting PGL of >300 mg/dL after the initial recording of HR, RF, and MAP; OA (75 i.v. bolus) was injected. The first 2 minutes of the recordings were made, followed by 15-minute intervals up to 120 minutes, or until the animals died.

To measure the blood sample’s PaO₂ value, the carotid artery blood sample was collected into a heparin-rinsed syringe after 15 minutes of saline/OA injection in the jugular vein in all groups. The PaO₂/FiO₂ ratio was calculated.

Determination of pulmonary water content

The physical method was used to determine the pulmonary water level in each rat.[27,28] The right side of the lung was kept in formal saline for histological examination. At the same time, the other side of the lung was weighed and dried to a consistent weight in an electric oven at 90°C (for 48 hours). Pulmonary water content was determined by the differences between dry and wet lung tissue as a percentage of wet lung tissue.

Histology of lung

The formal saline-preserved lungs underwent routine histological testing and were stained with eosin and hematoxylin for microscopic analysis.

Drugs and solutions

Urethane from Sigma-Aldrich, OA from HiMedia Laboratories, STZ from Sigma-Aldrich, and heparin were the drugs and solutions used.

Statistical analysis

For data analysis and graphical display, the SPSS (Statistical Package for the Social Sciences) software version 16.0 (SPSS Inc., Chicago, USA) and Sigma Plot software version 10.0 (Systat Software, San Jose, CA, USA) were used. The percentage of initial values was used to indicate the changes in HR, MAP, and RF. The data were pooled, and mean ± standard deviation (SD) was determined. One-way analysis of variance was used to compare the groups, and a post hoc test (Tukey’s) was used to see if there were any pairwise differences in the mean values. Paired student t-tests were also used for comparing the means of paired data. Kaplan-Meier’s survival curves and log-rank (Mantel-Haenszel) test were used to compare the survival time. Statistics were considered significant at P < 0.05.

RESULT

Effect of STZ on HFD-fed rats

Table 1 shows that when rats were given an HFD for 2 weeks instead of NPD, their body weight and nonfasting PI, PGL, PTC, and PTG levels significantly increased. After 2 weeks of dietary adjustment, STZ (35 mg/kg body weight) injection substantially raised PGL, baseline PTG, and PTC and showed a significant decrease in PI level in HFD rats. Compared with the NPD rats, these rodents exhibited polydipsia, polyphagia, and polyuria signs (data not shown).

Table 1.

Effect of HFD and streptozotocin on HFD-fed rats on biochemical parameters

Parameters	Mean±SD
	NPD fed rat	HFD-fed rats	STZ (35 mg/kg) + HFD
Body weight (g)	159.15±7.02	226.7±15.03*	218.65±2.72*
Plasma glucose (mg/dL)	124.96±8.96	132.96±15.03	370.26±41.29*,#
Plasma triglyceride (mg/dL)	98.41±12.17	142.93±18.92*	190.48±34.10*,#
Plasma total cholesterol (mg/dL)	116.31±2.07	122.30±11.47	163.59±53.49#
Plasma Insulin (pmol/L)	260.00±22.52	464.50±30.43*	214.65±24.68#

*P<0.05 compared to the NPD-fed rats. ^#P<0.05 compared to the HFD-fed rats

Saline administration produced no significant change in the respiratory and cardiac parameters in NPD and STZ pretreated HFD-fed rats

Saline (75 μL)–treated animals were taken as a control group in NPD-fed and HFD-fed + STZ rats. After stabilization, recording of HR, RF, and MAP was done. The HR, RF, and MAP in the saline-treated rats did not vary significantly during the 120-minute recording period [Figures 2-4]. Throughout the whole time of the observation, every animal in this group remained alive. In NPD-fed, the mean ± SD of PaO₂/FiO₂ was 453.96 ± 1.44 and the pulmonary water level was 77.74 ± 2.03% [Figure 5]. Histological studies showed that well-aerated alveoli comprise the lung parenchyma and simple squamous epithelium lined alveoli.

Figure 2.

The figure depicts respiratory frequency (RF) changes during the experiment on the effect of hyperglycemia on OA-induced ALI. RF at “0” min (time of OA injection) was taken as 100%. Mean ± SD values were obtained from six experiments in control, OA, and hyperglycemic (STZ-induced) groups. *P < .05 compared to the control

Figure 4.

The figure depicts heart rate (HR) changes during the experiment on the effect of hyperglycemia on OA-induced ALI. HR at “0” min (time of OA injection) was taken as 100%. Mean ± SD values were obtained from six experiments in control, OA, and hyperglycemic (STZ-induced) groups. *P < .05 compared to the control

Figure 5.

Histogram showing the pulmonary water content (as% of wet lung tissue) in control, OA, and hyperglycemic (STZ-induced) groups. The values are expressed as mean c SD. The values are obtained from six different experiments. *P < .05 compared to the control

In HFD-fed + STZ rats, the mean ± SD of PaO₂/FiO₂ was 410 ± 3.65 and pulmonary water content (73.57±0.38%, P < .05) was significantly less compared to NPD-fed rats. Histological examination of the lung shows well-aerated alveoli, which form the parenchyma of the lung and give it a lace-like appearance with mild fibroblastic proliferation, areas of alveolar septal infiltration, peribronchiolar infiltration, and vascular congestion without any alveolar edema [Figure 6].

Figure 6.

Photomicrograph of rat lung stained with haematoxylin and eosin (H and E) of control (a) OA (b), and hyperglycemic (STZ-induced) (c) groups. The red arrow shows fibroblastic proliferation, the yellow and orange arrow shows infiltration of leucocytes, the black and brown arrow shows alveolar edema, and the blue arrow shows necrosis

No significant changes were observed in these two groups, so HFD-fed + STZ rats were taken as the control group in the figure.

Oleic acid (75 µl) produces features of acute lung injury in NPD-Fed rats

During the OA (75 μL i.v.) administration in NPD-fed rats, there was a 4% (of the initial value) increase in the respiratory frequency (RF) was observed at 2 minutes, which was significantly increased after 15 minutes (62% of the initial value) followed by respiration stopped [Figure 2]. All animals died due to a gradual reduction in RF over the next 95 minutes. The mean survival time in this group was 95 ± 3.16 minutes. MAP was a significant fall of about 19% (P < .05) of initial values by 2 minutes. Then MAP recovered and MAP was maintained by 30 minutes, then decreased gradually [Figure 3]. However, HR showed an initial fall at 2 minutes, then there was a gradual increase in heart rate as recorded from 15 minutes to 60 minutes, and the increase was significant at 30 minutes (P < .05) as compared to the control, followed by a decrease in heart rate at 75 minutes which continued till the rats survived [Figure 4]. The mean ± SD of PaO₂/FiO₂ was 275.86 ± 2.44, and the pulmonary water level was 82.79 ± 0.80%, significantly increased than the control group [Figure 5]. Histological examination showed multifocal and heterogenous injury, with some areas showing minimal changes. On the other hand, others displayed a thickening of the alveolar septal wall caused by leucocyte infiltration and exacerbated alveolar edema. These rats were also presented with peribronchiolar infiltration and vascular congestion [Figure 6].

Figure 3.

The figure depicts mean blood pressure (MAP) changes during the experiment on the effect of hyperglycemia on OA-induced ALI. MAP at “0” min (time of OA injection) was taken as 100%. Mean ± SD values were obtained from six experiments in control, OA, and hyperglycemic (STZ-induced) groups. *P < .05 compared to the control

Effect of STZ pretreatment on OA-induced ALI

In these rats, increase of 25% of initial value in RF was observed at 2 minutes after administration of OA. At 30 minutes of the observation point, the RF decreased by 4% of the initial value, followed by a progressive fall in RF till the death of all animals [Figure 2]. The mean survival time in this group was 65 ± 9.22 minutes, significantly less than the OA group (Kaplan-Meier’s survival curves and log-rank [Mantel-Haenszel] test, P < .05). MAP shows an immediate fall of about 22% of the initial value in MAP as observed at 2 minutes. The MAP continued to decrease till rats survived, with a 55% of initial value of fall was observed at 60 minutes [Figure 3]. HR shows a gradual increase in HR from 10% of initial value at 2 minutes to 24% of initial value at 30 minutes, followed by a decrease in HR at 45 minutes, which continued till the rats survived [Figure 4]. The mean ± SD of PaO₂/FiO₂ was 275.86 ± 2.44, and the pulmonary water level was 81.40 ± 1.2%, similar to the OA group [Figure 5]. The lungs’ histological analysis revealed multifocal and heterogenous damage including regions of alveolar edema and leucocyte infiltration that thickened the wall. The areas of alveolar wall destruction with mild fibroblastic proliferation and necrosis are also present [Figure 6].

DISCUSSION

Dissident of mild fibroblastic proliferation in lung histology and hyperglycemic rats exhibited no significant change in the cardio-respiratory parameter and survived throughout the observation period. The result also shows that persistent hyperglycemia leads to early deterioration of all the cardio-respiratory parameters on OA-induced ALI in a rat model of type II DM.

HFD-fed rats treated with STZ (35 mg/kg) exhibit frank hyperglycemia despite circulating insulin concentrations almost identical to those of normal rats. The histological slide of the pancreas of these rats did not show the destruction or atrophy of beta cells (data not shown). HFD has been exhibited to produce insulin resistance syndrome in these rats, similar to insulin-resistant states in prediabetic humans.[26] These rats already had compensatory hyperinsulinemia to maintain glucose homeostasis, making them insulin-resistant.[26] Therefore, even a minor disruption of low-dosage STZ (35 mg/kg) that might impair beta cell activity could result in a severe hyperglycemic impact.[29] It caused prediabetes to transform into a true hyperglycemia syndrome that is comparable to type II diabetes in people. The disease is accompanied by a decrease in the pancreatic beta cells’ ability to secrete hormones that may counteract insulin resistance. The higher weight of HFD rats may be brought about by their consumption of a diet high in energy from saturated fat, its accumulation in different body fat pads, and their reduced energy expenditure compared to NPD-fed animals.[30] The increased absorption and formation of TG in the form of chylomicrons after exogenous consumption of a diet high in fat or through increased endogenous production of TG-enriched hepatic very low-density lipoprotein may both contribute to the hypertriglyceridemia seen in these HFD-fed or hyperglycemic rats. Moreover, it decreased TG uptake in peripheral tissue.[30] Heightened dietary cholesterol absorption from the small intestine after the consumption of HFD in diabetic conditions may cause numeric hypercholesterolemia.[31]

The STZ (35 mg/kg)–induced hyperglycemic rats showed no significant change in the cardio-respiratory parameter and survived throughout the observation period. The histology of the lung shows mild fibroblastic proliferation, areas of alveolar septal infiltration, and vascular congestion without any alveolar edema, similar to literature reports. However, the pulmonary water content was significantly decreased compared to control rats. Other investigators also reported that STZ-induced diabetic rats presented a significant increase in pulmonary lymphocyte infiltration, patches of organized pneumonia, mild fibrosis, and no pulmonary edema or acute alveolar injury.[4] Clinical studies have reported that there is a correlation between diabetes/hyperglycemia and interstitial fibrosis of the lungs.[5]

Hyperglycemia harms the lungs due to the pulmonary interstitial injury caused by microangiopathy, which also contributes to autonomic neuropathy.[32] Hyperglycemia is a condition of chronic inflammation with an impaired alveolar-capillary barrier.[3] Additionally, in individuals with diabetes, lung endothelial dysfunction and thickening of the interstitial lung layer may be caused by chronic systemic inflammation linked to oxidative stress and loss of antioxidant capacity.[33,34] According to recent research, NADPH oxidase or the polyol pathway activation may be the mechanism causing diabetic lung damage.[3,34] In hyperglycemia, a rise in the STAT3 (‘Signal Transducer and Activator of Transcription 3’) genes also contributes to cell injury.[4,34] At the same time, the cellular mechanisms for these abnormalities are still unclear.

In this study, we observe the early deterioration of all the cardio-respiratory parameters in HFD-fed STZ (35 mg/kg)–induced hyperglycemic rats when induced OA-induced acute lung injury. The mean survival time in this group was 65 ± 9.22 minutes, significantly less than the nonhyperglycemic/diabetic rats when induced OA-induced ALI (figure; Kaplan-Meier’s survival curves and log-rank (Mantel-Haenszel) test, P ≤ .05). ALI induced by the intravenous lipopolysaccharide model also demonstrated that hyperglycemia exacerbates lung injury.[35] Other studies of intratracheal instillation of lipopolysaccharide and hyperoxia-induced ALI showed less lung injury in diabetic rats than nondiabetic rats.[35] Furthermore, the study shows that acute hyperglycemia during ALI aggravates lung injury in nondiabetic rats by activating the SGK1-NKCC1 pathway.[36] However, clinical studies show diabetic patients develop ALI less often than nondiabetic patients.[15,19] Other observational studies showed no correlation between diabetes and developing ARDS or outcomes from ARDS.[29,31] Although, hyperglycemia is linked with increased mortality in nondiabetic patients.[36] Patients with severe sepsis, trauma, and other systemic inflammatory response syndromes may develop acute hyperglycemia, exacerbating inflammation and promoting lung injury.

In cases of hyperglycemia or diabetes, oxidative stress may come from a variety of causes: the increased production of reactive oxygen species (ROS), nitric oxide synthase, xanthine oxidase, and NADPH oxidase by their enzymatic sources; glucose may undergo antioxidation and produce oxygen-free radicals.[3,20] Another source of nonenzymatic ROS production has also been identified as the mitochondrial respiratory chain.[19,35] Diabetes also inhibits natural antioxidant enzymes, including superoxide dismutases, glutathione, and catalase, causing ROS to accumulate and activating the transcription factor NF-β to activate the production of stress-sensitive genes.[19,35] TNFα and IL6 levels that were elevated acted as indicators of a systemic inflammatory response. Oxygen radicals are linked to lung damage because they lipid peroxide the pulmonary capillary membrane, are rich in polyunsaturated fatty acids, and reduce the permeability of the alveolar-capillary barrier.[37]

Advanced glycated end products (AGE), which are now known to cause endothelial dysfunction and inflammation, are also formed due to the hyperglycemic condition.[38] In the presence of hyperglycemia, the rate of AGE production naturally increases significantly. The ROS production in endothelial cells, elevated procoagulant activity on macrophages and endothelial cells, and the release of cytokines and growth factors, including transforming growth factor beta, which causes the deposition of the extra basement membrane, are all negative effects of the AGE-RAGE signaling axis in the vascular compartment.[38,39]

Numerous studies have shown chronic hyperglycemia causes functional damage in many organs, including the lungs. However, the mechanism of cell injury and its effect on lung function remains largely unexplored. In this study, we observed that persistent hyperglycemia causes morphological changes in the lungs, which worsens the outcome of ALI. Hyperglycemia clinical and experimental data are contradictory. The outcome of ALI in diabetic patients was also confounded by disease severity, and diabetes treatment or management is yet to be fully explored. To establish effective therapeutic strategies for diabetic lung damage, additional research on the dysfunction in the diabetic lung is needed in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.