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Publicly Available Published by De Gruyter May 16, 2019

Enteroprotective effect of Tsukamurella inchonensis on streptozotocin induced type 1 diabetic rats

Tsukamurella inchonensis’in streptozotocin kaynaklı Tip 1 diyabetik sıçanlar üzerindeki bağırsak koruyucu etkisi
  • Mehran Mesgari-Abbasi , Solin Ghaderi , Monire Khordadmehr ORCID logo EMAIL logo , Katayoon Nofouzi , Hossein Tayefi-Nasrabadi and Graham McIntyre

Abstract

Objectives

Enteropathy is one of the most important complications of diabetes mellitus. The present study determined the possible effects of Tsukamurella inchonensis (Ti) on diabetes enteropathy on rat small intestine.

Materials and methods

A total of 40 rats were divided into four groups of 10. Diabetes was induced by streptozotocin. Oral administration of Ti at dose of 105 and 107 CFU/rat was performed in two groups continuously for 14 days. The third and fourth groups received normal saline as the diabetic and negative control groups, respectively. The blood and intestine tissue samples were taken on 21st day post treatment for biochemical and pathological evaluations.

Results

Significant differences were found in serum glucose, cholesterol and triglycerides values together with in CAT and SOD activities, MDA level and IL-6 concentration in both Ti treated groups in comparison with the diabetic rats. Moreover, there were severe pathological changes including degeneration of intestinal mucosa, mononuclear cell infiltration, decreasing number of goblet cells and villous length associated with increasing in villous thickness on the diabetic rats which markedly attenuated in both Ti recipient groups.

Conclusion

In conclusion, it seems that oral administration of Ti may improve intestinal damage in diabetic patients by modulation of intestinal antioxidant defense system.

Öz

Amaç

Enteropati, diabetes mellitusun en önemli komplikasyonlarından biridir. Bu çalışma Tsukamurella inchonensis’in (Ti) sıçan ince bağırsağında diyabet enteropatisi üzerindeki olası etkilerini belirledi.

Gereç ve Yöntem

Toplam 40 sıçan, her birinde 10 sıçan bulunan dört gruba ayrıldı. Diyabet, streptozotosin tarafından uyarıldı. Ti oral yolla 105 ve 107 CFU/sıçan dozunda iki gruba 14 gün boyunca sürekli olarak verildi. Üçüncü ve dördüncü gruplara sırasıyla diyabetik ve negatif kontrol grubu olarak normal salin verildi. Kan ve bağırsak doku örnekleri, biyokimyasal ve patolojik değerlendirmeler için uygulamadan sonraki 21. günde alındı.

Bulgular

Serum glukoz, kolesterol ve trigliserit değerlerinde, CAT ve SOD aktiviteleri, MDA düzeyi ve IL-6 konsantrasyonlarında her iki Ti grubunda da diyabetik sıçanlara kıyasla anlamlı fark bulundu. Ayrıca, diyabetik sıçanlarda intestinal mukoza dejenerasyonu, mononüklear hücre infiltrasyonu, villöz kalınlığının artmasıyla ilişkili villöz uzunluğunun ve goblet hücrelerinin sayısının azalmasını içeren ciddi patolojik değişiklikler vardı. Bu durum her iki Ti alıcı grubunda da belirgin şekilde azalmıştı.

Sonuç

Sonuç olarak, Ti’nin oral uygulamasının, diyabetik hastalarda bağırsak antioksidan savunma sisteminin modülasyonu yoluyla bağırsak hasarını iyileştirebileceği görülmektedir.

Introduction

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia resulting from defects in both insulin secretion and/or insulin action [1]. Generally, the DM can lead various organ dysfunctions such as nephropathy, neuropathy, hepatopathy, retinopathy, sexual failures and gastroenteropathy [2]. Recently, it was expected that the number of diabetic patients in the world will increase from 194 million in 2003 to 330 million in 2030, with 75% who living in developing countries [3]. In this regard, the gastrointestinal (GI) complications of DM, particularly in T1DM, have become increasingly prevalent as the rate of diabetes has increased [4]. The gastroenteropathy due to diabetes include gastroparesis and enteropathy which classically caused by abnormal GI tract motility and may present with diarrhea, constipation or fecal incontinence. It is well known that small intestinal and colorectal dysfunctions are common in longstanding diabetic patients [5]. While enteropathy can impact patients with Type 1 DM (T1DM) and Type 2 DM (T2DM), it appears more commonly in people with T1DM [6]. The suggested mechanisms involved in the pathogenesis of enteropathy in diabetes include high levels of serum glucose, reduction of insulin-growth factor I (IGF-I), dysfunction of GI autonomic nerve, alteration of GI hormone secretion, proinflammatory diathesis associated with genetic predisposition, disrupted synthesis of neuronal nitric oxide, increased oxidative stress, autoimmune diathesis, and imbalance rations of inhibitory and stimulatory enteric neuropeptide [7], [8].

Tsukamurella inchonensis, an environmental organism, is a Gram-positive species of the genus Tsukamurella belongs to the suborder Corynebacteriaceae and order Actinomycetales which are noticeable because of immunomodulatory activities [9]. Heat-killed preparation of T. inchonensis and some other genera within the order Actinomycetales including Rhodococcus coprophilus (R. coprophilus) and Gordonia bronchialis (G. bronchialis) demonstrate the beneficial effects in the prevention and improvement of some immune-related disease in animal model and veterinary medicine [10], [11], [12], [13]. For instance, suspensions of heat-killed Actinomycetales have been suggested as adjuvant agents for immune intervention strategies in Chagas’ disease (cause by Trypanosoma cruzi) [10]. Moreover, it was suggested that these heat-killed bacteria can modulate inflammatory response of a balloon catheter in the arterial intima in a rat model [11]. Recently, it was reported that both obesity and T2DM can be attenuate by administration of T. inchonensis and G. bronchialis [12]. Also, it was suggested that T. inchonensis is able to present an advantageous impact on IL-6 and TNF-α levels in a mouse model [13]. On basis of the authors knowledge, there is no report on the effects of these immune modulator activators on GI complications of DM. Since administration of many drugs such as metformin and statins in DM are associated with intestinal side effects [14] and regard to rarely isolation of Actinomycetales species in human infections which are as environmental organisms [9], therefore, the present study evaluated the possible beneficial effects of T. inchonensis on an improvement to intestinal disorders in type 1 DM [induction by Streptozotocin (STZ)] through histopathological and biochemical examinations.

Materials and methods

Animals and experimental design

A total of 40 healthy adult male Wistar rats with age between 12 and 16 months, and weight range 250–360 g, were chosen and divided randomly into four groups with 10 rats in each. Type 1 DM were induced in 30 rats by an intraperitoneal (i.p) injection of 55 mg/kg STZ (Sigma Aldrich Co., St. Louis, MO, USA). After 72 h and measurement of blood glucose levels, the experiment was performed. The first and second groups were treated with T. inchonensis with 105 (low dose) and 107 (high dose) CFU/rat which were selected according to previous studies [11], [12] consecutively for 14 days orally by stomach gavage needle. Groups 3 and 4 were appointed the positive-diabetic control and negative control (no diabetic and no bacteria) respectively, and received only normal saline for the same time. All animals were inspected every day for 21 days.

Ethical approval

The experiments on animals were conducted in accordance with the local Ethical Committee laws and regulations as regards care and use of experimental animals. The experiment was approved by the Research Ethics Committee of Tabriz University of Medical Sciences (ethical approval code: 5-4-1171, date: 4 May 2013) and performed according to the Helsinki’s humanity research declaration.

Sampling

Blood samples were collected after mild anesthesia (with i.p injection of 50 mg/kg BW of ketamine and 8 mg/kg BW of xylazine) on the 21st day after treatment and the sera were separated for biochemical tests at 750×g for 15 min. In addition, five rats in each group were sacrificed by cervical dislocation on the mentioned days, and after necropsy, samples from the small intestine were taken for biochemical and histopathological evaluations and stored in −70°C and formalin solution, respectively.

Biochemical assays

Blood glucose, cholesterol and triglycerides levels

These parameters were also measured by colorimetric enzymatic methods and commercial kits (Pars Azmoon, Tehran, Iran) as previously described [15] by using a spectrophotometer (Photometer 5010, Robert Riele GmbH & Co KG, Berlin, Germany) at 546 nm.

Interleukin-6 (IL-6) measurement

The concentrations of IL-6 was measured in obtained serum samples on the 21st day after treatment using Rat IL-6 ELISA kit (Koma Biotech, Korea) according to the instructions of the manufacturer [13].

Intestinal antioxidant system evaluation

At first, intestinal tissue was homogenized and prepared as previously described [16] and the obtained supernatant was used for all the following assays. Then, measurement of antioxidant enzymes activity (including superoxide dismutase and catalase) and lipid peroxidation (malondialdehyde level) were performed using commercial kits (Pars Azmoon, Tehran, Iran) according to the instructions of the manufacturer by using a spectrophotometer (Photometer 5010, Robert Riele GmbH & Co KG, Berlin, Germany). Activity of superoxide dismutase (SOD) was evaluated by inhibition of pyrogallol antioxidant for 3 min at 430 nm on basis of the previous method described by Marklund and Marklund [17]. Catalase (CAT) activity was examined by the absorption rate of H2O2 as previously described [18] for 1 min at 240 nm. Malondialdehyde (MDA) level was measured by the means of the thiobarbituric acid reactive substance (TBARS) method in the intestinal homogenate at 535 nm as previously described [19].

Histopathological examination

The tissues were fixed in 10% buffered formalin, processed routinely, sectioned, stained with hematoxylin and eosin (H&E), and finally studied microscopically with a light microscope (OLYMPUS-CH30 and using PF10X lens). The observed histopathological lesions were scored generally as previously described [20] with some modification. Additionally, the morphometric intestinal variables including villous height (measured from the villous-crypt junction) and villous thickness (measured at mid-villous height) were measured according to the previous method [21].

Statistical analysis

Statistical analysis of the data was performed using SPSS software version 22 for windows (SPSS, Armonk, NY, USA). The ANOVA and non-parametric tests (Kruskal-Wallis H and Mann-Whitney U-test) were used for comparison of serum biochemical indicators and histopathological changes between the different groups, respectively, and the differences were considered significant with p<0.05.

Results

Biochemical findings

Blood glucose, cholesterol and triglycerides levels

Higher blood glucose, cholesterol and triglycerides values were identified in all the control diabetic rats which were significantly different compared to the other experimental groups (Figures 13). The increase in blood glucose level was observed as early as 72 h after the STZ injection and was maintained during the study. The lower level of hyperglycemia was observed in low dose group in comparison with the diabetic group (Figure 1). By contrast, the lower levels of cholesterol and triglycerides were found in the high dose group (Figures 2 and 3). Although, the decreased levels of cholesterol in the high dose treated group was not significant compared to the diabetic group.

Figure 1: Blood glucose levels (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 1:

Blood glucose levels (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Figure 2: Serum cholesterol levels (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 2:

Serum cholesterol levels (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Figure 3: Serum triglycerides levels (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 3:

Serum triglycerides levels (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

IL-6 evaluation

IL-6 concentration exhibited a markedly difference between the diabetic group in comparison with the control and the treated groups. Likewise, there was notable difference in the negative control when compared with both treated groups. However, no significant difference was found between the low dose and high dose groups (Figure 4).

Figure 4: IL-6 levels (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 4:

IL-6 levels (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Evaluation of intestinal biochemical oxidative stress markers

There were the significant differences in MDA level and also in SOD and CAT activities (Figures 57) in the diabetic group in comparison with the control and treated groups. No significant differences were observed between low dose and high dose groups. However, the low dose group showed near values in all mentioned parameters with the control group.

Figure 5: MDA levels (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 5:

MDA levels (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Figure 6: SOD activity (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 6:

SOD activity (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Figure 7: CAT activity (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 7:

CAT activity (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Histopathological findings

Histological examination of the intestinal tissue sections shown in Table 1 and Figures 8 and 9. In the negative control group was found a normal healthy histoarchitecture in the small intestine tissue. However, in the diabetic rats without treatment, there were observed severe pathological changes including degeneration and desquamation of intestinal mucosa associated with mononuclear cell infiltration (comprising macrophages, lymphocytes and plasma cells), decreasing number of goblet cells, fibrosis, capillary congestion and subepithelial edema. Moreover, decreasing in the villous length together with increasing in villous thickness was occurred which were marked difference in comparison with the control group (Figure 7). Interestingly, all of the pathological lesions were significantly attenuated in the both treated groups, particularly in the low dose group (Figure 8). Indeed, in epithelial degeneration and also lamina propria cell infiltration, most of the evaluated tissue sections (4/5) in low dose group showed normal score and, only one sample (1/5) exhibited mild score. Meanwhile, the high dose group presented various scores from normal to severe in both parameters. Surprisingly, in the low dose group was found normal score in vascular congestion and submucosal edema. Whereas, most of the studies sections in the high dose group displayed mild score in vascular congestion (3/5) and submucosal edema (4/5).

Table 1:

Histopathological findings in various groups on the 21st day of sampling on basis of general scoring system as previously described [20] with some modifications.

Experimental groupsScoreHistopathologicalLesion
Epithelial degeneration and desquamationCell infiltration in lamina propriaVascular congestionSubmucosal edema
Negative controlN5a4a4a4a
+0111
++0000
+++0000
Diabetic controlN0b0b0b0b
+0000
++0101
+++5454
Low doseN4a4a5a5a
+1100
++0000
+++0000
High doseN1ab1ab1ab1ab
+1234
++2110
+++1100
  1. The changes were scored as normal (N), mild (+), moderate (++) and severe (+++). n=6 for each experimental group. (a) Significant difference with diabetic control; (b) significant difference with negative control (p<0.05).

Figure 8: Intestinal villous length and thickness (mean±SEM) in different groups on the 21st day of sampling.(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).
Figure 8:

Intestinal villous length and thickness (mean±SEM) in different groups on the 21st day of sampling.

(a) Significant difference with the diabetic control; (b) significant difference with the negative control (p<0.05).

Figure 9: Small intestine (jejunum), rat.(A) Normal structure of intestine in control group. (B, C, D) Diabetic group with cell infiltration (ci) in lamina propria, decreased number of goblet cells (gc), thickening and shortening of the villous, epithelial degeneration and desquamation (e), submucosal edema and fibrosis (f). (E) Low dose Ti-recipient group and (F) high dose Ti-recipient group with marked attenuation of pathological changes compared to the diabetic rats. H&E.
Figure 9:

Small intestine (jejunum), rat.

(A) Normal structure of intestine in control group. (B, C, D) Diabetic group with cell infiltration (ci) in lamina propria, decreased number of goblet cells (gc), thickening and shortening of the villous, epithelial degeneration and desquamation (e), submucosal edema and fibrosis (f). (E) Low dose Ti-recipient group and (F) high dose Ti-recipient group with marked attenuation of pathological changes compared to the diabetic rats. H&E.

Discussion

The results of present study showed a significant decreased in glucose values in the diabetic rats after treatment with T. inchonensis, especially with low dose. However, the levels of serum cholesterol and triglycerides demonstrated more differences, particularly in the high dose treated group compared to the diabetic group which was significant in triglycerides values. These alterations were related to marked improvement of the intestinal antioxidant enzyme activity and lipid peroxidation together with decreasing significant severity of pathological lesions the intestinal tissue sections. The present findings are consistent with those of some researchers who reported the marked decreased in plasma triglycerides, cholesterol and glucose levels by both treatments of T. inchonensis and G. bronchialis (Actinomycelatles species) on rats with experimental T2DM and obesity especially in T. inchonensis recipient group [12]. Gastrointestinal autonomic dysfunction is suggested the pathogenesis of diabetic enteropathy which is etiologically associated with hyperglycemia. In this regard, it was indicated that chronic or acute hyperglycemia can alter intestinal motility and also induce external anal sphincter dysfunction leading to diarrhea, constipation or fecal incontinence [8]. Okon the other hand, during diabetes, increased endogenous synthesis of cholesterol and triglycerides are proposed as the causes of functional changes in the small intestine mucosa [16]. In this way, control of glycemia accompanied with the serum levels of cholesterol and triglycerides are considered for the management of diabetic enteropathy [22]. Therefore, according to the present findings which support the previous results [12], it seems that oral administration of T. inchonensis act as an immune modulator and can be regulate the blood glucose, cholesterol and triglycerides values in DM and subsequently, improved the others related diabetic-enteric complications.

During diabetes, hyperglycemia is the trigger propel of the excessive production of free radicals, especially reactive oxygen species (ROS) from glucose autoxidation and protein glycosylation in various tissues [23]. It is well known that the intestinal mucosa is susceptible to oxidative stress on exposure to ROS produced by the luminal contents, bacterial metabolites, salivary secretion and bile acids [24]. The current results showed the significant differences in antioxidant defense system on the diabetic rats with alterations of SOD and CAT activities and MDA levels. These observations are consistent with previous study was conducted experimentally on T1DM induced by STZ on rat model with the significant differences on SOD, CAT and glutathione peroxidase (GPx) activities and MDA level [16]. It was stated that the antioxidant defense system alterations during DM in the small intestine are similar with the other tissues [25]. However, it was suggested that the antioxidant enzymes activities may show time dependent changes during diabetes [26]. Another present finding identified that T. inchonensis can response to diabetic intestinal oxidative stress via markedly decrease lipid peroxidation (MDA level) together with significantly increase antioxidant enzymes activities (SOD and CAT) in comparison with control group. Therefore, it is concluded that oral administration T. inchonensis can modulate ROS production in the intestine of diabetic patients and subsequently modulate antioxidant defense system resulting in improvement of diabetes enteropathy complications. Very recently, it has been suggested that T. inchonensis can inhibit generation of lipopolysaccharide-stimulated NO and pro-inflammatory cytokines (IL-6 and TNF-α) in mouse peritoneal macrophages [13]. It is believed that the serum content of IL-6 in patients with T1DM was significantly greater than healthy controls which involved in the inflammation and immune responses and were associated with development of T1DM [27]. In this respect, it was recently found IL-6 can contribute to increase colon contraction in STZ-diabetic rats through IL-6 receptor pathway [28]. Interestingly, in the present study, T. inchonensis exhibited an improvement effect on IL-6 alterations in both high and low doses treated groups. However, this alteration was not significant between high and low doses groups which may be related to the effects of other inflammatory cytokines that contribute in diabetic enteropathy complication. Our results are consistent with previous studies. Meanwhile, further studies on tissue IL-6 alteration and also other involved inflammatory cytokines such as IL-10 are still required to explain these observed alterations.

Present observation of histopathological changes in the diabetic rats included disruption of intestinal mucosa associated with mononuclear cell infiltration leading to thickening of mucosa and intestinal edema which confirmed the obtained biochemical changes which can lead to decrease digestive capacity of the GI tract. According to the authors knowledge, there is not more information in the intestinal histopathological changes during diabetes. Recently, proliferation of the intestinal epithelial layers accompanied with cellular infiltration in lamina propria and epithelial degeneration were reported in the acute phase of STZ-induced diabetic rats [20] which supported the present microscopical observations. Surprisingly, low dose treated rats showed milder microscopic pathological lesions when compared with high dose treated group, particularly in epithelial degeneration and presence of inflammatory cells. However, both groups exhibited approximately similar lesions in vascular congestion and submucosal edema. The exact cause of more improvement effect of low dose of bacteria is not clear. But, it seems that interactions of inflammatory mediators involved in DM, population of inflammatory cells and defense antioxidant system may play an important role on improvement of pathological lesions. Supporting our results in the previous research, it was presented that the medium does of T. inchonensis exhibited more improvement effect on TNF-α and IL-6 alterations rather than both high and low doses of T. inchonensis [13]. But, little is known about the main mechanism of T. inchonensis on immune system and DM complications and further extensive studies are needed to determine obvious beneficial immunomodulatory impact of this bacteria.

When excessive ROS are produced in the GI tract due to DM, oxidative stress can accelerate cell injuries by alteration in the proteins functions and resulting in lipid peroxidation [29]. Superoxide and NO produce during cellular infiltration (such as neutrophils and macrophages) to the lamina propria of the small intestine. The infiltrated cells generated more ROS and NO which can damage cytoskeleton proteins and resulting in cellular junctions and epithelial permeability alterations lead to barrier disruption and finally presence of pathological changes [30]. Interestingly, oral administration of T. inchonensis showed marked impact on intestinal pathological changes after diabetes induction, particularly in the low dose which showed only mild cellular infiltration and capillary congestion. A recent study had shown the beneficial effect of G. bronchialis on intestinal histology and biochemical parameters of healthy rainbow trout (Oncorhynchus mykiss) [31]. It was understood that increasing the intestinal villous length can enhance the surface area capable of greater absorption of various nutrients [32]. Additionally, it is stated that the intestinal villous height is an indicator of villi activity for normal digestive function [33]. Moreover, it is suggested that shortening of the villi may lead to poor nutrient absorption associated with increased secretion in the gastrointestinal tract, and finally lower performance [34]. On the other hand, it seems that the number of goblet cells can stimulate the villi growth [35], and goblet cells secretion also induces an adherent gel on the mucosal surface of GI tract which may play an important role in epithelial cell protection and repair [36]. Therefore, it is proposed that oral administration of heat-killed Actinomycetales species can affect positively on intestinal morphology and functions through increasing intestinal villous length and goblet cells number which damaged severity during DM.

On the whole, the present data and previous similar results demonstrated a marked beneficial impact of heat-killed T. inchonensis on the biochemical and pathological alterations of diabetic enteropathy in rat model and T. inchonensis may be useful as an immunomodulatory activator for the treatment of diabetic enteropathy complications.

Acknowledgements

The authors express their gratitude to Professor Graham McIntyre, of BioEos Ltd. for preparation and provision of heat-killed Tsukamurella inchonensis. The authors are also grateful to the Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran and also the Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran for the financial support.

  1. Conflict of interest statement: There is no conflict of interests regarding the publication of this article.

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Received: 2018-07-24
Accepted: 2018-11-27
Published Online: 2019-05-16
Published in Print: 2019-10-25

©2019 Walter de Gruyter GmbH, Berlin/Boston

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