The Protective Effect of Garden Cress Lepidium sativum against Lipopolysaccharide (LPS) Induced Hepatotoxicity in Mice Model

Garden cress or Lepidium sativum is a member of a large family Brassicaceae, which till now has about 1500 species (Tackholm, 1974).  It is a herbal plant grows in many different countries and all of its parts (leaves, shoots, seeds and roots) can be eaten (Facciola, 1990). Seeds used as a milk-increasing factor in important animals like camels and horses. The seed component analysis showed that it has about 25% of the weight is protein, around 44% is carbohydrate and around 19% lipids and the rest is crude fibers (Diwakar et al., 2010). The aqueous extract of the plant seed has hypoglycemic effect (Maghrani et al., 2005). Lepidium sativum is able to protect DNA from the dangerous effect of free radicals and other toxins (Sahu et al., 2014). LS showed a modulatory effect on liver enzymes AST, ALT and ALP by using the seeds oil (Ranjani et al., 2019; Sood et al., 2019). The seeds extracts protect the rat liver from the harmful effect of carbon tetrachloride (CCl₄) (Mazin et al., 2019). Seed extract accelerates healing of bone fracture (Al-Yahiya et al., 1994). Seeds of the plant have an anti-inflammatory effect and other effects such as coagulant activities, analgesic and anti-pyretic effect. Lpidium sativum suppress the production of lymphotoxin-β (LTB4), nitrogen oxide (NO) and tumor necrosis factor (TNF) and increase the a-linoleic acid (ALA), Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) levels in the cell membrane lipids of the immuno-competent cells (Diwakar et al., 2010). In addition, Lepidiun sativum showed many medicinal effects such as antidiabetic, antibacterial, anti-diarrheal, anti-hypertensive, anti-oxidant and laxative effects (Chand et al., 2010; Hero and Akrayi, 2012).
         
Many strains of gram-negative bacteria have another outer lipid bilayer in addition to the cell membrane. This called outer membrane (OM) (Ellen et al., 2010). This OM has a complex glycolipid of lipopolysaccharide (LPS) (Bos et al., 2007) those responsible for the resistance of gram-negative bacteria. LPS consists of three main structural parts: lipid A, a core oligosaccharide and variable O-antigen. LPS is a very powerful immunological stimulator and is called bacterial endotoxins. Where it can be recognized by innate immune receptors and then signals and activate many cascades those finally leads to release many proinflammatory cytokines (Sayed, 2019). Purified LPS is used as a potent antigen by so many searcher groups (Brydon et al., 2009) where it has a powerful antigenic characters that may finally leads to failure of many body organs. LPS-induced liver injuries were reported by many studies (Sayed, 2019). One exposure to LPS considered as hepatotoxic xenobiotic and leads finally to direct liver necrosis (Abdel-Razaq et al., 2007; Sayed 2019). Many groups were recorded that plant seeds have an antioxidant and hepatoprotective effects (Najeeb et al., 2011). The present study aims to investigate the modulatory effect of Lepidum sativum on the liver of mice. We made a biochemical analysis for the proinflammatory, anti-inflammatory cytokines and some enzymes those measure the oxidative stress of the liver. In addition, we used gene expression profile analysis to investigate the expression profile of proinflammatory and anti-inflammatory genes as a confirmative data those support the biochemical ones. Finally, we made a histopathological study to declare the effect of LS on LPS treated liver histology.

Animals
 
We used 40 male mice in this study. Animals grouped into five main groups as following.
a)  Group I (8 mice) received phosphate buffer saline as a control group.
b)  Group II (8 mice) received LPS at 300 µg/kg body weight (LPS- group).
c)  Group III (8 mice) received LPS at 300 µg/kg body weight followed by Lepidium sativum extract (50mg/kg) for fifteen days. Animals sacrificed at 6h, 12h, 24h and 48h after receiving LPS.
d) Group IV (8 mice) received Lepidium sativum extract (50mg/kg) for one month and followed by LPS at 300 µg/kg body weight then received lepidium sativum seed extract (50mg/kg) for another fifteen days.
e)  Group V (8 mice) received Lepidium sativum extract (50mg/kg) fifteen days followed by LPS at 300 µg/kg body weight and again Lepidium sativum extract for fifteen days.
 
Biochemical analysis
 
Different liver makers AST, ALT, CAT, Glutathion, Glutation peroxidase, were investigated in serum and liver homogenate. Cytokines (IL-6, TNF-α, IL-4 and IL-10) were investigated using enzyme-linked immunosorbent assay kit (ELIZA) (Biosource, USA) according to manufacturer protocol.
 
Histopathological analysis
 
We collected liver samples from each group at the time of sacrifices and fixed in 10% formal saline (name and quantity) for 24 hours. Samples then rinsed in tape water and dehydrated through ethanol alcohol series then infiltrated using xylene and finally embedded in paraffin wax. The paraffin blocks then cut into 5 µm thickness sections. The paraffin removed by xylene then passed in a decreasing series of alcohol for rehydration. Slides then stained by hematoxylin and eosin and subjected to light microscope investigation.
 
Gene expression analysis
 
Total RNA was isolated from the liver of different groups in the experiment. Then mRNA was purified from the total one using oligo-dT, mRNA was transcript into cDNA. cDNA then be used for RT- polymerase chain reaction (PCR). Both forward and reverse primer were used for each gene analyzed in this study which are genes of pro-inflammatory genes (IL-6 and TNF-α) and anti-inflammatory genes (IL-4 and IL-10).
 
Statistical analysis
 
All values presented as mean ± S.E.M. Statistical analysis were performed using one-way analysis of variance (ANOVA). Tukey’s post hoc test used for multiple group comparisons. Significant data were considered at (P value ≤ 0.05).

The serum ALT and AST levels were significantly elevated in mice injected by LPS (Fig 1 a and b). Furthermore, serum HDL and LDL also showed significant increase in LPS treated mice (Fig 1 c and d). The pretreatment with LS (Group IV) markedly decreased the serum AST and ALT levels in LPS-treated mice. From these data, we suggested that LS could prevent LPS-induced acute liver injury and improved liver functions in mice.
 

       
Moreover, LPS-induced hepatic necrosis and injury also provokes oxidative stress to result in an increase of oxidants like GLU, GPX and CAT (Fig 2 a-c), which is included in the pathophysiology of acute and chronic liver injury.
       
Serum liver enzymes ALT and AST considered as indicators for the evaluation of liver necrosis and dysfunction (Kim et al., 2008). In hepatotoxic mice, these enzymes pour into the bloodstream resulting in enhanced serum ALT and AST levels. The serum ALT and AST levels positively correlated with liver histopathology (Liu et al., 2015). LS significantly decreased the elevated serum ALT and AST levels in LPS-treated mice.
 
In addition to hepatic necrosis and loss of hepatocytes around the central vein-as regards to immune reaction- it was found that LPS lead to the secretion of chemokines and cytokine production (IFN γ, IL 4, IL 10, TGF β, or IL 6), resulting in inflammation in the liver. The necrotic hepatocytes secrete ligands, inducing further inflammation and chemokines in the liver. IL-10 induced by LS, which reported to participate in various immune mediated hepatotoxicity in mice.
   
LPS also leading to increase in TNF-α mRNA expression when compared to that noted in (untreated) controls (Fig 2b). The increase in TNF-α expression reflected by increased levels of TNF-α protein in liver homogenates of mice treated with LS. Increasing of IL-10 induced by LPS, which was reported to participate in various immune mediated hepatotoxicity in mice (Fig 2c).
 

       
The marked infiltration of inflammatory cells in the liver tissue in the LPS treated group may be due to the provoke production of diverse proinflammatory cytokines and chemokines in response to LPS mediated inflammation and induce infiltration to inflammatory sites and regulation of immune responses such as cytokine production IFN γ, IL 4, IL 10, TGF β, or IL 6 (Liu et al., 2006; Araújo Júnior et al., 2012). LPS induce cytokines production of TNF-α and IL-6 that are upregulated, leading to the imbalance of pro-/anti-inflammatory response and damage to liver tissues (Su et al., 2014). LS has a protective effect on LPS–treated group, it was found to recovery liver tissue, retained integrity of tissue, also decreased the plasma AST and ALT levels and increase TNF-α mRNA expression when compared to that noted in (untreated) controls. The increase in TNF-α expression was also reflected by increased levels of TNF-α protein in liver homogenates of mice treated with LS (Zou et al., 2011, Yao and Brownlee 2010).
       
The major cellular alterations involve is hepatocytes apoptosis, as well as death of endothelial cells. Furthermore, other studies found that autophagy induced by LPS in the   liver cells or cultured hepatocytes accelerates LPS-induced liver cell death (Su et al., 2014).
       
There are two forms of hepatotoxicity generally a granulomatous hepatitis accompanied with liver dysfunction and an acute hepatitis followed by hepatocellular necrosis with inflammation. As a result, the plasma AST and ALT levels were significantly increased gradually after the LPS administration (Su et al., 2014; Reber et al., 2017). LPS activates cytokines such as TNF-α and IL-6 that are upregulated, leading to the imbalance of pro-/anti-inflammatory response and damage of liver tissues (Su et al., 2014).
 

       
Fig 4 showed the gene expression profile of proimflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines. Samples were collected after 2, 4, 8, 16, 24 and 48 hs. of LPS stimulation. Results of proinflammatory cytokines either IL-6 or TNF- α showed a marked differential expression of mRNA of both cytokines in LPS group (Fig 5) and the expression showed a maximum rate after 6hs. stimulation till 24h. regarding IL-6 and till 8hs. in case of TNF-α. The decreasing of TNF- α level after 8hs. may be due to the engagement of this cytokine in many pathways of cytokine network (Weiyi et al., 2019). After that, the expression decreased due to the elimination of the antigen (Qusti et al., 2016). The expression of both IL_6 and TNF in group IV where lepidium sativum was followed LPS recorded an increase in both cytokines in late hours of stimulation. The pretreatment with lepidum sativum reduced the inflammation status of the liver as it shows decreasing of IL-6 and TNF-α (Fig 4). These data are in agree with many reports (Ranjani et al., 2019). The group five those pre and post-treated with Lepidium sativum showed an expression profile nearly similar to that of control group.
 

       
With regard to anti-inflammatory cytokines we investigate IL-4 and IL-10 (Fig 5). The expression profile recorded a high expression rate in LPS challenged group in case of IL-4. Nearly the same resulted recorded in IL-10 but to a lower extent. IL-10 showed fluctuation in expression rate among the other groups. It showed a gradual increasing in expression rate until 8hs. After stimulation decreasing again until 48hs. In group three and four. Group five showed a steady expression rate in all times except in case of 8 and 16hs. After stimulation. In case of IL-4 results recorded more or less as same as IL-10. Results of gene expression analysis support data obtained by biochemical analysis as we recorded that Lepidunm sativum reduced the oxidative status of the liver. These results were in agree with many studies those mention a great protective effect of seeds of Lepidium sativum on liver oxidative stress status (Fig 5 c, d and e).
 

       
From the histopathological liver sections, results revealed that LS pretreatment retained the integrity of hepatocyte architecture and decreased infiltration of the inflammatory cells Fig (6 b, 7). Inflammatory cell infiltration and aggregation in liver tissue is an initial reaction of liver injury reflecting the degree of inflammation (Reber et al., 2017).
 

       
The histopathological examination of liver sections of normal control group showed linear arrangement of hepatocyte cords, with clearly visible nuclei, central vein and sinusoids (Fig 7a).  LPS-treated group established that even a single dose induces obvious liver tissue histopathological changes, including large areas of congestion of sinusoids, central vein congestion, necrosis of hepatic cells was observed in addition to microabscesses in liver parenchyma (green arrow), cellular necrosis (yellow arrow), infiltrated inflammatory cells (white arrow), hemorrhage bleeding leukocytes within hepatic sinusoids (red arrow) (Fig 7b). Group III: LPS+LS Lesser in sinusoidal congestion, moderate necrosis of hepatic cells and hemorrhage hepatocyte cords retained their structures (Fig 7c). Regarding group IV: LS+LPS, there was marked reduction sinusoidal congestion (out of circle) and few infiltrated inflammatory cells (white arrow) and areas of regeneration of hepatocyte cords (Fig 7d, circle). LS+LPS+LS treated-Group V. marked retention of tissue integrity (normal hepatocyte cords central vein swelling exhibit recovery (Fig 7e). Though LPS has been demonstrated to be toxic and these results were coinciding with that reported by (Shaw et al., 2007; Su et al., 2014; Chen et al., 2017; Wang et al., 2019). The histological observations of tissue inflammatory infiltrate, necrosis, in the livers of mice treated for LS and compared the findings with that reported by (Lotze et al., 2007; Yao et al., 2010; Akai et al., 2016). Likewise, LPS -treated mice exhibited an acute liver necrosis similar to what we have noted in TCDD-treated mice (Eghbal et al., 2013; Akai et al., 2016). The absence of LS in LPS alone treated group delays the recovery of liver injury.
 

       
An increase of the hepatic injury resulting in liver necrosis and dysfunction. Our studies with LPS- treated-mice group II established that, there were significant increases in the liver enzymes compared with the normal control.
       
We surmise that group five those pre and post-treated with LS showed a protective effect against LPS and results showed very close to that of control one so, it was found to recover liver tissue, retained integrity of tissue. Our results encourage further research on carcinogenicity and mechanism of action of different Flavonoids to address its potential safety for human use as an anti-toxic therapy.

Our results conclude that Lepidium sativum seed extract ameliorate significantly the LPS-induced hepatotoxicity throughout investigating liver markers such as AST, ALT, GLU. GPX. Proinflammatory, anti-inflammatory cytokines, mRNA of TNF, IL-6, IL-4 and IL-10 expression. In addition, histopathological analysis results recorded a highly protective role for the extract on liver tissue. Apoptotic and inflammatory cell infiltration count showed confirmative results.

Authors acknowledge the deanship of Scientific Research at King Faisal University for the financial support under the annual project number (Project NO: 170100).

The author(s) declare(s) that there is no conflict of interests regarding the publication of this article.