The Diabetic Lung Can Be Ameliorated by Citrullus colocynthis by Reducing Inflammation and Oxidative Stress in Rats with Type 1 Diabetes

Background Diabetes impacts various organs in the body and some reports showed that the lung is also affected by diabetes, and an imbalance of inflammation and oxidative stress may participate to diabetic lung impairments. The present study is conducted to assess the impacts of Citrullus colocynthis (CC) on some aspects of these impairments. Methods Frothy two male Wistar rats (3-4 months old and weighing 200–250 g) were used in the present research. Animals were divided into 3 groups of control, Diabetes, and Diabetes + Drug. CC was administered to diabetic rats orally. The lung tissue and BALF oxidative stress and inflammatory indices including the MDA, TAC, SOD, Gpx, TNFα, IL-6, IL-17, and IL-10 were evaluated by the ELISA method. Results Our observations disclosed the ameliorative impacts of CC administration against oxidative stress and inflammation imbalance. Also, it was found that CC improved body weight and fasting blood sugar in rats with diabetes. Conclusion We can conclude that the administration of CC can be effective in improving diabetic lungs in rats.


Introduction
Type 1 diabetes or Diabetes mellitus (DM) is a systemic malady represented by a chronic hyperglycemic circumstance and infammation and oxidative stress are serious consequences of this disease [1]. Based on the data released by World Health Organization (WHO), about 442 million persons sufered from DM in 2014. Te estimates indicated that the number of diabetic patients in the world will raise to 592 million persons by 2035 [2]. Te main side efects of DM are caused by its microangiopathic and macroangiopathic symptoms, afecting the eyes, kidneys, nerves, heart, major vessels, and lungs [3]. Tese complications mainly result from vascular damage that plays a vital pathophysiological role in diabetes [4]. Te link between lung dysfunction and diabetes is assumed to be the outcome of biochemical alterations developed in the pulmonary and respiratory structures. Tese components involve a set of mechanisms potentially caused by hypoxemia, oxidative stress, systemic infammation, or direct damage due to chronic hyperglycemia [5][6][7]. Impaired lung performance is explored in more than 73% of diabetic patients [8]. Inadequate glucose control can lead to lung dysfunction and elevated systemic levels of infammatory cytokines [9]. TNF-α (Tumor necrosis factor α) can mediate the diabetic process and in association with Oxidative stress may impair the metabolism of glucose [10]. Scientifc data suggest that diabetes involves infammatory changes in the lung [11]. Additionally, serum levels of IL-6 (Interleukine-6) in T2DM patients disrupt endothelial cell function [12]. Experimental data also disclosed that IL-6 plays a vital role in developing infammatory diseases in the respiratory system [13]. Recent studies have found a link between islet amyloid deposition and islet infammation. Accordingly, islet amyloid involves in producing infammatory cytokines mainly interleukin-1β (IL-1 β) by macrophages and dendritic cells [14]. Oxidative stress is one of the factors accounting for pulmonary alterations [15]. An increasing bulk of studies proposes that hyperglycemia and high oxidants accumulation induced by free fatty acid (FFA) can more easily impair the function of β-cells due to antioxidant (SOD (Superoxide dismutase), CAT (Catalase), and GPx (Glutathione peroxidase)) subnormal expression in β-cells [16]. Oxidative stress may also lead to lung endothelial impairment in diabetic patients [17][18][19]. Traditional plants are widely used for treating DM [20]. Citrullus colocynthis (CC) (melon) belonging to the Cucurbitaceae family [21] produces signifcant levels of antioxidant phenolics and favonoids [22]. Te seeds of this plant are used for their medicinal efects, such as soothing, antioxidant, and anti-infammatory properties [21]. Despite the growing number of clinical studies on the diabetic lung, less attention has been paid to the mechanism accounting for diabetes-induced pulmonary disorders. Tus, the current investigation designed to assess the efects of the aqueous extract of CC seeds on glycemic, infammatory cytokines, TNF-α, IL-6, IL-1β, and IL-10 levels, and oxidative status by detecting SOD, GPx activity, TAC (Total antioxidant capacity), NO (Nitric oxide), and MDA (Malonyl dialdehyde) levels in tissue and BALF (Broncho alveolar lavage fuid) in rats with T1DM.

Crude Extract Preparation.
Te method of crude extract preparation of CC was shown in our previous study [23]. Briefy, CC fruits were gathered from the Ilam province in Iran and after removing of their seeds, the seeds dried for 72 h. Tese seeds are a rich source of some fatty acids. In addition, the seeds are rich in essential amino acids and minerals. We grounded 100 gram of seeds using by a mixer and we added the grounded seeds to 1 liter of distilled water. Te mixture was heated for 2 h at 80°C. After that the extract was passed through Whatman No. 1 flter paper. At the end of extraction, the fractions obtained were gathered in a balloon and lyophilized, yielding the lyophilized aqueous extract [23][24][25].

Animals.
All protocols and treatments carried out in this were confrmed (Ethics code: IR.KMU.REC.1398.127) by Kerman University of Medical Sciences. We tried to provide maximum comfort to the animals at all research procedures. Te age and weight of animals used in this experiment were 3-4-month-old and 200-250 gr respectively. Tey were kept under controlled conditions. 42 male wistar rats were assigned to the three groups: Te healthy rats (Control group), Te rats receiving STZ (Diabetes group), and Te diabetic rats receiving CC extraction orally (Diabetes + Drug group).

Induction of Diabetes.
Te type 1 diabetes model was developed after overnight fasting (8 hours). Te induction of severe diabetes was performed in the animals by a single STZ dose (50 mg/kg body weight). Moreover, STZ was prepared in sodium citrate bufer at pH 4.5 and administered intraperitoneally with insulin syringes [26]. Type 1 diabetes symptoms such as severe urination was recorded four days after injection. However, to ensure diabetes, fasting blood sugar (FBS) was measured after 15 days. Te 40-day curation started on the 15th day after the induction of diabetes [23].

Drug
Administration. 15 days after the injection of STZ, the remediation began with CC. Tus, the aqueous extract of CC (200 mg/kg) was administered orally every day for 40 consecutive days [27]. A dose response study was conducted to reaching the dose with maximum efectiveness. Te effective dose was the dose that had the best efect in reducing blood sugar. Te rats received glucose (2 g/kg body weight) and two diferent doses of Citrullus-colocynthis aqueous extract (CCAE) (100 or 200 mg/kg body weight). Te average blood glucose was 80-126 mg/kg. Blood glucose was measured every 30, 60, 90, and 120 minutes in a blood drop taken from the tail by a glucometer. Te CCAE dosage of 200 mg/ kg was more efective than 100 mg/kg in correcting the twohour postprandial blood sugar levels. Consequently, the diabetic rats in all the treated groups received CCAE (200 mg/kg) as the daily oral treatment [23]. Also, other authors such as Kalva et al. used a dose of 200 mg/kg [27].

Biochemical Evaluations.
Te Bradford method utilized for total proteins assessment. Infammatory and oxidative parameters were measured in tissue supernatant and BALF. Te exact methods for evaluation of oxidative parameters were mentioned in our previous study [29]. Briefy, Te level of the SOD enzyme was evaluated using a colorimetric assay. GPx activity was measured by reducing cumene hydroperoxide. Malondialdehyde (MDA), was assessed using the TBARS method. Total antioxidant capacity (TAC) was evaluated by the FRAP assay. In addition, nitrite, total protein and all oxidant and anti-oxidant parameters were evaluated based on the kit's instructions (Navand Salamat Co., Iran). Quantitative investigations of cytokines were accomplished using the double-antibody Sandwich enzyme linked immunosorbent assay (ELISA) kits based on the manufacturers' instructions (Karmania Pars gene Co., Iran) [33][34][35][36].
2.7. Lung Injury Score. Te right lungs and airways were gathered from rats and immersed in 10% formalin. Ten, the hematoxylin/eosin (H&E) was utilized for tissues staining. Te histopathological scoring was performed based on our previous study [34].

Statistical
Analyses. Te data are reported as a mean-± SEM. Normality was checked using by Shapiro Wilk test. One-way ANOVA followed by Tukey's post hoc test, used for data analyzing. Signifcant level was considered at p < 0.05 [37,38].

Te Impacts of CC Administration on Body Weight in Rats with Diabetes.
In the Diabetes and Diabetes + Drug (before commencing therapy) groups, diabetic rats' body weight considerably dropped (p < 0.01). Additionally, repeating research experiments into body weight in the Diabetes + Drug group after the commencement of treatment revealed that the body weight considerably raised after 20 and 40 days of CC administration compared to before beginning treatment (p < 0.01). Te treatment and control groups did not vary statistically signifcantly (Figures 1(a) and 1(b)).

Te Impacts of CC on FBS in Rats with Diabetes.
In rats with diabetes in the Diabetes and Diabetes + Drug (before commencing therapy) groups, when compared to 14 days before to STZ administration, the FBS of rats dramatically raised (p < 0.001). Additionally, measurement of FBS after beginning therapy revealed that in the Diabetes + Drug group, the administration of CC for 20 and 40 days resulted in a marked reduction in FBS as compared to baseline (p < 0.01). No signifcant diference between the treatment groups and the control group was found after repetitionbased analysis (Figures 2(a) and 2(b)).

Te Impacts of CC on Oxidative Stress and Protein Leakage
in Lung and BALF. Te results revealed that NO level in BALF and MDA level in tissue and BALF elevated in the diabetic rats in comparison with the healthy rats (p < 0.001), and remediation with CC resulted in a signifcantly decrease the MDA and NO levels (p < 0.001). In the rats with diabetes, meaningful decreased SOD activity was observed in tissue and BALF (p < 0.001, p < 0.01; respectively) as well as GPx activity and TAC level in tissue and BALF was remarkably reduced (p < 0.001) in comparison with the healthy rats but was remarkably elevated after treatment with CC (p < 0.001). Tere was an elevation (p < 0.001) in the total protein content of the BALF of the T1D animals compared with control subjects. Treatment with CC reduced the protein leakage in the diabetic animals (p < 0.001) (Figures 3 and 4).

Te Impacts of CC on Cytokines.
Te BALF and tissue TNF-α, IL-6, and IL-1β levels in diabetic rats were more than Group Control, and those levels were signifcantly lower after CC treatment than those in group diabetes (p < 0.001) in comparison with the healthy rats, the IL-10 level was reduced in group diabetes, and signifcant increase were found in the group receiving the CC (Figures 5 and 6).

Discussion
Mechanisms associated with chronic hyperglycemia can induce oxygen radicals formation and systemic infammation in diabetic patients [9,39]. Te main contribution of this research was that diabetes enhances the lung injury and infammation in rats due to elevated levels of TNF-α, IL-6, and IL-1β and lower levels of IL-10, lower SOD and GPx activity, TAC levels, and increased MDA and NO levels in tissue and BALF. Moreover, the diabetic lung went through marked histological changes leading to high lung injury scores. Traditional medicinal plants have been long used all over the world for a wide range of diabetic symptoms [17]. In this study, the aqueous extract of CC seeds was used to treat diabetes, and the data conformed the improvement in lung status diminished impairments in rats with diabetes.
As demonstrated in the current research, infammatory cytokines remarkably elevated in the lung of the rats with diabetes. Moreover, infammatory cell infltration such as neutrophil, eosinophil, and lymphocyte infltration was signifcantly correlated with high lung pathological change scores. In contrast, the protective mechanisms including IL-10 declined in diabetic rats. Dennis et al. found lower lung function in diabetic subjects. Tis decline can be attributed to elevated levels of infammatory cytokines such as TNF-α, as confrmed in the current research [9]. Xiong et al. demonstrated that the increased levels of TNF-α and IL-6 can act as indices of an infammation [40]. Hu et al. reported elevated TNF-α expression with diabetes [41]. Te present study revealed that lung injury was accompanied by early increase in TNF-α, IL-1β, and IL-6 in tissue and BALF. Our data demonstrated that treatment of rats with CC declined the TNF-α, IL-1β, and IL-6 levels in comparison with the nontreated rats, and increased IL-10, suggesting that increased IL-10 levels may shift the balance in favor of an antiinfammatory condition. A review of the literature did not fnd any studies exploring the immunoinfammatory Histological evidence confrmed the infltration of neutrophils, eosinophils, and lymphocytes in the lung of the diabetic group. Histopathological data in diabetic patients confrmed thickened alveolar, epithelial, and pulmonary capillary basal lamina [4]. Regarding with the fndings of the current research, Zhang et al. confrmed the histological alterations in the diabetic lung after 8 weeks of diabetes in rats [16]. Kolahian et al. also confrmed the infltration of mononuclear cells and edema in the submucosa of the trachea and lung of diabetic animals [2]. Tese results confrmed the infammation in the lungs of diabetics. Furthermore, our fndings indicated CC reduced infammatory cell infltration and pathological change scores and eventually decreased airway damage score. Hofmann et al. found that bitter apple is a useful remediation for treating infammation in a colitis model [44].
Mucins mainly secretes from airway goblet cells and leading to the formation of a mucus layer that protects the    [2]. Similarly, the data in the present study revealed an elevation in lung oxidative stress in diabetic rats compared the controls, and a decline in antioxidant enzyme SOD and GPx activity and TAC level. Tese fndings conformed the results reported by other authors, who showed an increase in oxidative stress in the lungs of diabetic rats compared to the controls. Tey also reported a reduction in the antioxidant enzyme SOD activity [15]. Furthermore, Xiong et al. showed the increased MDA level, a lipid peroxidation marker, associated with the depressed activity of SOD, as the most signifcant endogenous antioxidase in diabetic lung injury [40]. Gumieniczek et al. reported the intensifed levels of the lipid peroxidation process and lower activities of antioxidative compounds in the diabetic lung [45]. Endothelial NO is a remarkable factor in vascular function. Diabetes can lead to an increment of this molecule. Our data demonstrated this increment in diabetic lungs. Te present study confrmed signifcantly lower GPx and SOD scavenger and TAC levels in diabetes, but an increase in CC. Our data also confrmed that CC treatment can signifcantly reduce the production of free radicals as demonstrated by the ameliorated MDA and NO levels. Rizvi et al. also found that CC acts against oxidative stress [46]. Likewise, Ostovan et al. demonstrated the benefcial efects of CC against oxidative stress in the diabetic rats [47]. Tese fndings further confrmed the antioxidative efect of CC as a useful mechanism in diminishing lung damage in diabetics.
Overall, the data in the current research revealed the protective efect of CC on the diabetic lung in rats. CC can reduce infammatory cell infltration into lung tissues. Te data also showed that the administration of CC can prevent oxidative stress and infammation in BALF and lung tissue. Tus, a further exploration of such medicines might ofer a natural key to fnding new anti-diabetic drugs.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
Tis research was approved in Kerman University of Medical Sciences, Kerman, Iran (Ethical Number IR.KMU.REC.1398.127).

Conflicts of Interest
Tere is no conficts of interest to be declared.

Acknowledgments
Tis work was supported by Kerman University of Medical Sciences, Kerman, Iran. Evidence-Based Complementary and Alternative Medicine 9