Effects of Coriandrum sativum on distant organ inflammation and apoptosis due to liver ischemia/reperfusion injury

Abstract

Liver ischemia/reperfusion (IR) often affects distant organs, such as small intestine, kidney, and lung. Coriandrum sativum (CS) has an antioxidant and anti-inflammatory effect on liver damage. The aim of this study was to investigate the anti-inflammatory and antiapoptotic effects of CS extract on small intestine, lung, and kidney after the liver IR injury. Small intestine, lung, and kidney tissues were evaluated and scored in terms of cell degeneration, inflammation, and congestion, as well as caspase-3 (Cas-3) and cluster of differentiation 31 (CD31) immunostainings were carried out. Renal enzymes, creatinine and urea levels were measured biochemically in serum. After IR, a decrease in villi size, diffuse degeneration, epithelial cell shedding and extensive congestion in the capillaries were observed. Meanwhile, the number of degenerated villi and congestion decreased in the IR+CS group. Due to IR, increased congestion was detected in the interalveolar septum of the lungs and in the capillaries between the kidney tubules. It was also observed that the positively stained cells with Cas-3 and CD31 were increased in the lung, kidney, and small intestine tissues of the IR group, and decreased in the IR+CS group. Kidney enzymes, urea and creatinine levels were significantly increased in the IR group and decreased in the IR+CS group. In conclusion, it was observed that liver IR caused changes in distant organs, especially in the small intestine, lung, and kidneys. Damaging effects of IR as well as apoptosis and inflammation were found to be decreased in the groups treated with CS.

Introduction

Ischemia/reperfusion (IR) damage in organs is one of the most important causes of morbidity and mortality [1]. IR injury is associated with microvascular dysfunction and inflammatory cytokine expression [2]. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), secreted from leukocytes and oxidative stress released disrupt the glycocalyx and increase the cellular permeability. At the same time, capillary occlusion occurs due to the platelets and leukocytes accumulation, therefore, hypoxia and IR damage increase in the related tissue [3]. Inflammatory mediators are secreted because of hepatic IR, hemorrhagic shocks, transplantation, liver resection, and injuries. Increase in the expression of inflammatory molecules, such as Kupffer cell number, TNF-α and nuclear factor-kappaB (NF-κB), were observed in liver IR injury [4]. Inflammatory cytokines due to IR damage in the liver have been shown to cause damage in distant organs, such as small intestine, kidney, and lung [5, 6]. Villus, epithelial cell degeneration and malabsorption occur in the small intestine [7, 8, 9], related oxidative stress and changes in enzymatic and metabolic pathways from the basis of bilateral liver–kidney injury [10, 11]. Hepatic IR causes pulmonary barrier dysfunction and reduction of the gap junction protein [12], resulting in neutrophil infiltration from the pulmonary capillaries via released endotoxins, and also apoptotic cell death due to oxidative stress [6]. Cluster of differentiation 31 (CD31), also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), is an important transmembrane glycoprotein receptor and is normally located on the surfaces of macrophages, Kupffer cells, granulocytes, lymphocyte cells, and regulates the migration of these cells [13]. There is no clinical answer for primary or secondary IR damage in various organs. Studies on defining the molecular mechanisms that are effective in IR injury and strategies for effective organ protection are continuing [14, 15, 16, 17]. The Coriandrum sativum (CS) plant has antioxidant, anti-inflammatory effects, and the protective effect of CS on liver damage caused by various hepatotoxic substances has been reported [18, 19].

Aim

In the literature reviews, it has been seen that there are very few studies on liver damage related distant organ effects and the protective effect of CS. The aim of this study was to investigate the effect of CS on inflammation and apoptosis in distant organs, such as the small intestine, kidney, and lung, after acute liver IR injury.

Materials and Methods

Plant material and extraction

C. sativum leaves were collected from the Alayköy region in North Cyprus and dried. Dried leaves were extracted with methanol using the maceration method and the extract was evaporated. It was done according to the high-performance liquid chromatography (HPLC) analysis (Agilent Technologies 1200 series HPLC) and separated with Eclipse XDB-C18 column (150 mm×4.6 mm, 5 μm). As mobile phases, (A) water with 0.5% formic acid and (B) methanol were used at a flow rate of 1 mL/min. The gradient was initially 90–70% (A) for 18 minutes, then 55–80% (A) for 5 minutes, and finally, 90% (A) for 5 minutes. The detection wavelength was set at 254 nm. The injection volume was 10 μL for each sample and standard solutions, and according to the results of data validation, the percentage of isoquercitrin was found to contain 139.25 ppm (0.21%).

Animals and experimental design

All experimental protocols were performed according to the Guide for Care and Use of Laboratory Animals approved by the Animal Care and Use Local Ethics Committee of Near East University, Nicosia, Cyprus (No. 2019/01-57). Six-month-old female Wistar albino rats (450–500 g weight) were housed at a constant room temperature (22±2°C) under a 12-hour light/dark cycle. They were fed with standard rat chow (210 kcal/100 g/day) and tap water ad libitum.

Animals were randomly divided into four groups:

Sham group (n=10): a midline incision was made, and the hepatic pedicle (contains ductus hepaticus, hepatic portal vein and proper hepatic artery) was mobilized. No blood supply was blocked.

IR group (n=10): ischemia was achieved by clamping the hepatic pedicle with a vascular clamp for one hour; after the ischemic period, the clamp was opened for an hour for reperfusion [20].

IR+CS group (n=10): CS extract was applied 300 mg/kg per day for three days before ischemia and one hour before ischemia by oral gavage [21].

CS group (n=10): CS extract was given by gavage 300 mg/kg per day for three days.

Light microscopy

Tissue samples were fixed in 4% paraformaldehyde for 48 hours, embedded into paraffin, and 5 μm sections were taken. Hematoxylin–Eosin (HE) staining was carried out to assess the general structure of the organs by histological scoring method. In the small intestine: 0 – if the epithelium was normal; 1 – if there was subepithelial openings and capillary congestion in the villus apex area; 2 – if the epithelium was separated from the underlying lamina propria, and there was expansion in the spaces; 3 – if the epithelium was separated in the villi and there was degeneration at the ends; 4 – if the villous degeneration is widespread and the capillaries are very enlarged [22]. In the lungs, leukocyte infiltration, intestinal congestion and alveolar wall thickening were evaluated. Each assessment was scored as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe) [23]. The kidneys were evaluated in terms of tubular cell degeneration, tubular dilatation, peritubular congestion, and glomerular degeneration. Each assessment was scored as 0 (absent), 1 (mild), 2 (moderate), and 3 (severe) [24].

Immunohistochemical staining

Immunohistochemical (IHC) evaluation of the hepatic tissue samples was performed by the following stainings: anti-caspase-3 (Cas-3) (ab2302, Abcam) and anti-CD31 (ab182981, Abcam) antibodies. The cells stained positive with Cas-3 and CD31 were counted under the light microscope, with a magnification of 40× in 10 different areas and averaged.

Biochemical analyses

Biochemical analyses were performed using a clinical chemistry analyzer (Architect Plus c8000, Abbott Laboratories, Illinois, USA). Renal creatinine and urea levels were measured.

Statistical analyses

All the statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) ver. 17.0. Data obtained from the groups were compared using the non-parametric Kruskal–Wallis test within each group, whereas the groups were compared with each other by using the Mann–Whitney U-test. A value of p<0.05 was considered statistically significant.

Results

Small intestine histology

Small intestinal villus structures were normal in the sham group and the CS-only group. There was extensive degeneration of villi, epithelial cell shedding, and extensive congestion in capillaries of the lamina propria after IR. In the IR+CS group, there was less degeneration of the villi, and reduced congestion. The number of positively stained epithelial cells was low in Cas-3 immunohistochemically stained sham and CS groups, and in the IR+CS group, it was observed to be less common than the IR group. CD31 IHC staining results were similar to Cas-3 staining results. CD31 expression was prominent in the IR group, and the decrease was seen in the treatment group (Figure 1), when the results were compared with the sham group. IHC staining results related to the Cas-3 and CD31 were given in the graphs (Figure 2A, 2B).

Figure 1.

Figure 1 – Intestine tissue. HE staining, ×100. Anti-Cas-3 antibody immunomarking, ×400. Anti-CD31 antibody immunomarking, ×200. Cas-3: Caspase-3; CD31: Cluster of differentiation 31; CS: Coriandrum sativum; HE: Hematoxylin–Eosin; IR: Ischemia/reperfusion

Figure 2.

Figure 2 – Cas-3-positive (A) and CD31-positive (B) cells in intestine. **p<0.002

Lung histology

Normal histological structure was seen in the sham and CS-only groups. In the IR group, increased congestion in the interalveolar capillaries was observed. Less congestion and macrophage were detected in the IR+CS group. Cas-3 and CD31 expressions were increased in the IR group, according to the sham and CS group results and less expression was detected in the IR+CS group (Figure 3). Cas-3 and CD31 IHC staining results were given in the graphs (Figure 4A, 4B).

Figure 3.

Lung tissue. HE staining, ×400. Anti-Cas-3 antibody immunomarking, ×400. Anti-CD31 antibody immunomarking, ×400.

Figure 4.

Cas-3-positive (A) and CD31-positive (B) cells in lung. *p<0.05, **p<0.002.

Renal histology

Compared with the sham group, it was observed that there were enlargements in the kidney distal tubules and increase in the intertubular capillaries congestion. On the contrary, there was a decrease in the IR+CS group. Compared to the sham and CS groups, Cas-3 and CD31 expression increased in the IR group and decreased in the IR+CS group. CD31 expression was intense especially in the capillaries of the glomeruli (Figure 5). Cas-3 and CD31 IHC staining results were exhibited in the graphs (Figure 6A, 6B).

Figure 5.

Kidney tissue. HE staining, ×100. Anti-Cas-3 antibody immunomarking, ×400. Anti-CD31 antibody immunomarking, ×200

Figure 6.

Cas-3-positive (A) and CD31-positive (B) cells in kidney. *p<0.05, **p<0.002

Histological scoring

Damage in the small intestine, lung and kidney tissues was defined in the IR group (intestine p=0.002, lung p=0.005, kidney p=0.002). While less damage was observed in the small intestine and kidney in the IR+CS group compared to the sham (intestine p=0.002, kidney p=0.019) and CS group (intestine p=0.002). There was no significant decrease in the IR+CS group in the lung (Figure 7A, 7B, 7C).

Figure 7.

Histological scorings of damage in groups of kidney (A), lung (B), and intestinal (C) tissues. *p<0.05, **p<0.002

Renal enzymes

Serum creatinine and urea levels were higher in the IR group than the control (creatinine p=0.004, urea p=0.004). While a significant decrease in creatinine level was observed in the IR+CS group compared to the IR group (creatinine p=0.004), however, the decrease of the urea level was not statistically significant (Figure 8A, 8B).

Figure 8.

Renal creatinine (A) and urea (B) levels in groups. *p<0.05, **p<0.002

Discussions

In this study, the changes caused by liver IR injury in the distant organs of the small intestine, kidney and lung tissues and the effect of CS on these changes were investigated. It has been determined that the small intestine is mostly affected by liver IR damage, and the structural changes are severe. It was observed that the kidneys and lungs were less affected. While the effect of CS was significant in the small intestine and kidneys histological scoring results, it was not statistically significant in the lungs.

Liver and distant organ damage after hepatic IR remains a serious medical problem that requires effective and preventive strategies [25]. Systemic inflammation after liver transplantation or liver IR is an important risk factor for distant organs [26]. The circulatory disorder causes apoptosis and necrosis in both the liver and distant organs, and degeneration is more common in the organs depending on the length of the IR period [27]. Intestinal microbiota is very important for the regulation of liver functions. The bidirectional relationship between the gut and the liver is called the gut–liver axis. As a result of liver IR injury, decrease in intestinal motility and malabsorption, mucosal degeneration, loss of villi, disruption of intestinal barrier, apoptosis, change in microbiota profile, bacterial translocation, and activation of systemic proinflammation occur [7, 8, 9]. In our study, it was observed that the small intestine was the most affected organ by the liver IR damage. Villous degeneration, epithelial cell shedding, capillary congestion, and increased apoptosis are common findings.

In previous studies, thickening in the interalveolar areas of the lungs, edema, congestion, mononuclear cell infiltration [28], inflammation, hemorrhage, atelectasis, hyaline membrane formation, and bronchiole epithelial degeneration have been described [26, 27, 29]. In our study, apoptotic cell death in the lung tissue and capillary congestion in the interalveolar septum were detected. No alveolar wall thickening was observed. In addition, neutrophils number in the interalveolar septum was low.

Acute renal failure due to liver IR injury is a common complication and increases the risk of death. Ischemia causes vasoconstriction in intrarenal vessels, thus activating the renin–angiotensin system. Besides, renal tubular necrosis, decrease in glomerular filtration rate, inflammation, and apoptotic cell death by proinflammatory cytokines occur [6], urea and creatinine levels increase [30]. In addition, dilatation of the renal tubules, platelet accumulation in the glomeruli, increased fibrinogen, disappearance of the striated border, and epithelial cell degeneration have been reported [29]. In our study, significant congestion in inter-tubular capillaries in the renal cortex, shedding of proximal tubule epithelial cells, dilatation of tubules, and apoptosis were observed in the IR group. Again, it was determined that urea and creatinine levels were increased in the IR group compared to the sham group, CS treatment significantly decreased the creatinine level, but the decrease in the urea level was not significant.

Following liver IR, neutrophil and platelet-rich microthrombi are seen in the pulmonary and renal vessels, resulting in a widespread systemic inflammatory effect associated with the accumulation of inflammatory cells [30]. There is a strong relationship between epithelial cell damage, neutrophil activation, and cell death and the CD31 molecule. CD31 is a transmembrane glycoprotein and is also referred to as PECAM-1. This molecule is found on the surfaces of platelets, monocytes, macrophages, neutrophils, and endothelial cells. Its main function is to regulate the migration of inflammatory cells to the intercellular space. CD31 is strongly expressed in fenestrated endothelium of kidney glomeruli, lung alveolar capillaries and intestinal capillaries [31]. It has been reported that hepatic IR injury induced in rats does not induce an inflammatory response and neutrophil accumulation in the kidneys. Inflammatory cell infiltration was not observed in the renal cortex, but tubular injury was detected [32]. In another study, platelet deposition, microvascular thrombi, and fibrinogen deposition were observed in the kidneys and lungs after hepatic IR (1.5 hours ischemia, 24 hours reperfusion). The increase in neutrophils in these organs started six hours after IR and gradually increased, then decreased after 24 hours [30].

Studies show that neutrophil increase or lack of change is associated with IR duration. Since the IR time was one hour in our study, no significant increase in neutrophils was observed in the organs. CD31 expression was defined as increased in IR groups. Expression was strongly detected in the IR group, especially in the kidney glomerular capillaries. No significant difference was found between groups in the small intestine.

Different substances have been used against the effects of experimental liver IR damage on the liver and distant organs [26, 28]. Plants with hepatoprotective potential, such as dill, cumin, anise, fennel, coriander, celery, cypress, angelica, parsley, and carrot, which are from the Apiaceae family, are generally used as vegetables or spices [33]. Coriander (C. sativum) fruits contain an essential oil composed mainly of linalool, which is responsible for its odor and taste. Coriander is “generally recognized as safe” (GRAS) as a food by the U.S. Food and Drug Administration (FDA). Coriander has anti-inflammatory, analgesic and anti-cancer, neuroprotective effects [34, 35, 36]. It has been observed that it has an antioxidant protective effect against the oxidation of heavy metals in the kidneys [37]. Long-term consumption of CS has effects on vascular endothelial function, renal and intestinal sodium absorption, glucolipid homeostasis, and gut microbiota in rats [38].

Conclusions

As a result of this study, it was determined that the degeneration, capillary congestion, inflammation, and apoptosis observed in the small intestine, lung and kidney tissue in the IR group treated with CS were decreased compared to the IR group. Although CS treatment in the lungs caused a decrease in the changes, it was not statistically significant. The decrease in CD31 expression, which was increased in ischemic groups, was not statistically significant in the small intestine. CS treatment significantly reduced apoptotic cell death in all three organs. The antioxidant and anti-inflammatory effect of CS was effective in these reductions. Studies on the use of CS against the changes in distant organs caused by liver IR damage in the literature reviews are very few, and the results obtained will provide a source for studies on this subject.

Conflict of interests

The authors declare that there is no conflict of interests.