Eicosapentaenoic acid loaded silica nanoemulsion attenuates hepatic inflammation through the enhancement of cell membrane components

Encapsulation of EPA into silica nanoemulsion was carried out using water/oil nanoemulsion. Emulsifying agent such as Tween 80 and chemphore were used to further disperse the formed nanoemulsion. Thus, our work is to utilize the pores of silica nanoemulsion as a holder for EPA drug. The porous silica nanoemulsion was prepared via using TEOS as precursor and chemphore as surfactant to kep the particles of the formed silica nanoemulsion a way from each others with the aid of ultrasonic homogenizer that achieve the full dispersition for the formed nanoemulsion with there is no phase separation for the components of the resultant nanoemulsion.

As clearly remarkabe from Fig. 1a that the silica nanoemulsion are prepared with porosity and formed as hollow spherical particles. The DLS of silica nanoemulsion (Fig. 1b) displayed that the particles are phormed with very small size (61.58 nm) with PdI = 0.05. As known from the value of PdI, it can be concluded that the particles are formed with monodisperes particles with no noticable for the agglomeration. In comparison with EPA encapsulated silica nanoemulsion, the particles are filled with EPA. Via eye visullization, silica nanoemulsion (Fig. 1a), the spherical particles are appeared as porous particles, these porosity are appeared as black particles while encapsulation of EPA (Fig. 1c). The average particle size analyzer for EPA loaded silica nanoemulsion (Fig. 1d) is increased to 90 nm with pdI equal to 0.074. The particle size was enlarged due to the encapsulation of EPA. Overall, the hydrodymanimc average size is around 100 nm which is not significantly affected on the efficiency of EPA. Additionally, there is a difference for the diameter size for the produced nanoemuslion when evalauted via TEM and DLS which is mainly attributed to the difference in the utilized technique.

Fig. 1figure 1

TEM, average particle size of (a, b) silica nanoemulsion and (c, d) EPA loaded silica nanoemulsion

DEN is a secondary alkylating agent which generates ROS leading to an oxidation of DNA and/or RNA molecules. It is generally found in smoked and fried food items. It is a confirmed hepatotoxic in rodent models [30].

ALT and AST, the liver biomarker enzymes, are suggestive of the beginning of hepatocellular injury. These enzymes are existed in the cytoplas and liberated into the blood due [31].

It was found that the rats administered DEN significantly increased levels of serum ALT and AST (Fig. 2).

Fig. 2figure 2

Levels of serum ALT and AST in different studied groups. Pa value: significant difference compared to control group. Pb value: significant difference compared to DEN induced hepatic inflammation group. Pc value: significant difference compared to treated I group

Excessive generation of free radicals or depletion of antioxidant enzymes cause oxidative damage by disrupting the cellular redox balance between the production of oxidants and antioxidant defense [32].

The parenchymal cells of the liver are the most vulnerable cells to oxidative stress [33], which is owing to the capacity of certain organelles found inside parenchymal cells (mitochondria, microsomes and peroxisomes) to produce free radicals that can cause fatty acid oxidation, making the liver a key target for ROS damage [34]. Oxidations of biological macromolecules especially DNA, proteins, and lipids are considered the most trademark oxidative stress induced by DEN [35]. Level of  liver GSH,    activity of antioxidant enzyme     SOD, and level of TBARS were measured during this investigation, and the results revealed that administration of DEN significantly elevated the extent of lipid peroxidation, as shown by the elevation of TBARS and decreased level of GSH, and SOD activity (Fig. 3).

Fig. 3figure 3

Liver oxidant / antioxidant markers in different studied groups. Pa value: significant difference compared to control group. Pb value: significant difference compared to DEN induced hepatic inflammation group. Pc value: significant difference compared to treated I group

SOD and GSH are the first line of antioxidant defense system in cells; they protect the cells by scavenging free radicals, altering them to less toxic metabolites [36]. Results from this study showed a reduction in the concentration of GSH and activity of SOD enzyme after administered of DEN in hepatic inflammation group.

These findings are consistent with the observations of Adebayo and his colleagues who reported the increase in the levels of TBARS with a concomitant decrease in antioxidant defense system in the DEN-administered mice [31].

In the present investigation, the supplementations of EPA especially EPA loaded silica nanoemulsion ameliorated activities of serum ALT and AST, enhanced the anti-oxidative status (GSH, SOD), and blunted the oxidative stress level (TBARS) in the treated groups (Figs. 2 and 3). In support, in animal models of liver injury evoked by drugs, alcohol or other, EPA terminated oxidative stress, and mitochondrial dysfunction [37, 38].

Further, EPA was described to motivate reactions that regulate the expression of detoxifying/ antioxidant genes and obstruct inflammation [39]. EPA reacts directly with the negative regulator of Nrf2, Keap1, and initiates dissociation of Keap1 with Cullin 3, thereby inducing Nrf2-directed antioxidant gene expression [40, 41].

Moreover, Tanaka et al. found that EPA administration to mice significantly reduces hepatic TBARS through enhanced expression of zinc, copper, and manganese-SOD [42].

Oxidative DNA damage is associated with hepatocarcinogenesis development. Over production free radicals such as ROS is considered an important factor in genetic instability during liver inflammation [43]. 8-OHdG is another significant biomarker of ROS-induced oxidative DNA damage, as well recognized a risk factor for hepatocellular carcinoma in patients with chronic liver inflammation [44]. In this study, the 8-OHdG level in urine of DEN-administered rats was significantly elevated as compared with the control group (Fig. 4). Consistent with this finding, recent studies reported that cigarette smoke, which is rich in DEN, causes an accumulation of 8-OHdG in the lungs as a result of increases oxygen free radicals, leading to inflammatory responses, fibrosis, and tumor growth [45, 46].

Fig. 4figure 4

Urinary 8- hydroxyguanozine levels in different studied groups. Pa value: significant difference compared to control group. Pb value: significant difference compared to DEN induced hepatic inflammation group. Pc value: significant difference compared to treated I group

On the other hand, we found that EPA supplementations particularly EPA loaded silica nanoemulsion reduced the level of urinary 8-OHdG urine levels compared to DEN induced hepatic inflammation group (Fig. 4); which confirmed the ability of EPA to counteract oxidative modification of DNA. Previous study demonstrated that fish oil rich with EPA were significantly decreased the levels of oxidative DNA damage (8-OHdG) in male cigarette smokers and attributed that the beneficial effect of EPA on suppressing the generation of reactive oxygen species [47]. Furthermore, dietary fish oil protects against colon cancer in rats by reducing oxidative DNA damage, as measured by a quantitative immunohistochemistry study of 8-OHdG [48].

Chronic inflammation of the liver is a well-recognized risk factor for carcinogenesis, the molecular link between inflammation, hepatic fibrogenesis, and hepatocellular carcinoma [49]. Regarding DEN-induced hepatic inflammation group, concentrations of TNF-α, IL-1b were significantly elevated (Fig. 5). Consistent with our study, Ding et al. found that TNF-α, IL-1b where up-regulated during DEN-exposed, causing hepatic inflammation [50]. These results were due to the de-alkylation of DEN to its active mutagenic metabolites which modulated substances such as 3-methylcholanthrene and phenobarbital (PB) which in turn increase hepatic demethylase activity. Besides, oxidative stress induced by DEN is well documented as a factor to the pathogenesis of hepatic carcinogenesis [51].

Fig. 5figure 5

Serum Inflammatory markers and liver hydroxyproline content in different studied groups. Pa value: significant difference compared to control group. Pb value: significant difference compared to DEN induced hepatic inflammation group. Pc value: significant difference compared to treated I group

DEN activates the myeloid differentiation primary response 88 (MyD88) dependent and MyD88-independent signaling cascades [52]. The MyD88-dependent signal transduction activates NF-kB through activation of its inhibitory protein IkBa, which allows NF-kB nuclear translocation and controls the expression of a multitude of pro-inflammatory cytokines and other immune-related genes, such as TNF-a, IL-1, IL-1β, IL-6, and IL-12 [49].

Liver inflammation is a hallmark of early-stage fibrosis, which can extend to severe fibrosis and cirrhosis [53]. Fibrosis is characterized by the accumulation of collagen and other extracellular matrix components[54]. One of the main approaches for fibrosis quantification and also for the therapeutic assessment of new anti-fibrotic drugs is to determine the hydroxyproline (Hyp) level of the liver [55]. In the current study, increased production of fibrillary collagens was confirmed by a significantly increased hydroxyproline level in the DEN- administered group (Fig. 5). A number of studies have also shown the fast buildup of collagen during nitrosamine-induced liver fibrosis [56, 57].

Interestingly, our results showed less severe inflammatory response in groups administered EPA essentially EPA loaded silica nanoemulsion reported by the reduction in serum levels of TNF-α, IL-1b compared to DEN induced hepatic inflammation group (Fig. 5). Our findings are following Albracht-Schulte et al. who reported that EPA exhibits protective effects in liver steatosis and inflammation through decreased NF-kB and pro-inflammatory cytokines, such as TNF-α and Mcp-1, as well as increases in anti-inflammatory cytokines, such as IL-10 via up-regulation of miR-let-7 and inhibition of MAPK/ERK/JNK pathways. Furthermore EPA can well incorporate into the liver, and suppress gene expression of the pro-inflammatory cytokines, IL-1b, IL-6 and interferon-γ [37].

We have reported in this study that EPA, considerably EPA loaded silica nanoemulsion represses DEN-induced nodule formation and suppresses sub-sequential fibrosis which is demonstrated through decreased hepatic Hyp content in the treated groups compared to DEN induced hepatic inflammation (Fig. 5). These results supported by findings of Harada et al. who reported the inhibitory effect of EPA on hepatic fibrosis by lowering hepatic Hyp content, and gene expression of collagen, and transforming growth factor -β1 in rats fed a methionine- and choline-deficient diet by immediately reducing the level of ROS [42].

We evaluated the erythrocyte membrane fatty acid fractions including ALA, AA, LA, and OA as shown in Table 1. Our results showed a significant decrease in the erythrocyte membrane ALA content accompanied with a significant increase in the erythrocyte membrane AA, LA, and OA content in DEN- induced hepatic inflammation group compared to the control group (Table 1).

Table 1 Cell membrane fatty acid fractions in different studied groups

In the current investigation, DEN-induced liver inflammation resulted in oxidative stress, as demonstrated by a rise in liver TBARS and a decrease in antioxidant enzyme activity (GSH and SOD), resulting in a substantial reduction in erythrocyte membrane ALA concentration. Rolo et al. proposed a link between low n-3 PUFA levels and oxidative stress, claiming that excessive reactive oxygen species formation owing to mitochondrial malfunction causes lipid peroxidation, which promotes inflammation and stellate cell activation, leading to fibrogenesis. In individuals with liver inflammation, higher levels of oxidative stress and lipid peroxidation have been found [58].

The increase in the erythrocyte membrane AA, LA, and OA content in DEN- induced hepatic inflammation group may be related to the oxidative stress and associated with inflammatory cascades observed in our study. We believe that raised AA levels are the initial stage in the progression of inflammation and eventually, cell death since the AA catabolism pathway, performed by cyclooxygenase and lipoxygenase, produces lipid pro-inflammatory mediators [59]. Consistent with this, we observed increased expression of the pro-inflammatory cytokines including IL-1β and TNF-α along with the increased level of AA (Table 1 and Fig. 5).

The cellular membranes and the capabilities we have to modify its composition and function constitute a strong weapon in the treatment of hepatic inflammation and cancer. The cell membrane and its components must be considered as important aspects in cancer treatment, and novel therapeutic techniques should be developed [4]. From this point of view, we evaluated the potential effect of EPA alone and EPA loaded silica nanoemulsion in improving the cell membrane efficiency upon administration of DEN. Data obtained showed that along with the reduction of the oxidant and inflammatory markers after administration of EPA alone or EPA loaded silica nanoemulsion, a significant increase in the erythrocyte membrane content of ALA and a significant decrease in AA, LA, and OA was observed compared to DEN induced hepatic inflammation group (Table 1and Fig. 5). It is worth to mention that there was a significant increase in the erythrocyte membrane content of ALA accompanied with a significant decrease in AA, LA, and OA in the EPA loaded silica nanoemulsion group compared to EPA only (Table 1) confirming the effectiveness of the prepared EPA in a nanoemulsion form. Besides, EPA is an omega 3 fatty acid which is characterized by its role in replacing AA in the cell membranes and gave the more elasticity and flexibility [4]. Thus in this work, EPA as an animal source of omega 3 effectively increased omega 3 fatty acids and decreased omega 6 and 9. Additionally, in EPA loaded silica nanoemulsion treated (Table 1).

In agreements with our results, Giordano and Visioli reported that moderate/appropriate amount of n-3 PUFA has an antioxidant effect. In addition, a recent study found that supplementing with LC n-3 PUFAs reduced hepatic oxidative stress and triglyceride accumulation in fatty liver caused by a high-fat diet [60]. In the current study, EPA and in particular EPA loaded silica nanoemulsion supplementation improve antioxidant status by restoring antioxidant enzyme activity and GSH levels, preventing DEN-induced hepatic oxidative damage.

Omega-3 fatty acids are promising nominees for treating a wide range of inflammatory responses which accompanies many diseases such as atherosclerosis, diabetes [61], and asthma [62]. EPA is a biologically active polyunsaturated ω-3 FA [38].

Concomitant with the biochemical analysis, histological examination of liver sections of negative control group showed the normal structure of the hepatic lobules. The hepatocytes radiated from the central vein. It exhibited vesicular nuclei, some of it are binucleated and are separated with sinusoids (Fig. 6a). On the other hand, liver sections of negative control group showed normal portal tract with its structures; branches of portal vein, hepatic artery and bile duct (Fig. 6b).

Fig. 6figure 6

Photomicrograph of liver sections of negative control group showing a normal hepatocytes (arrows) radiating from the central vein (asterisk). The hepatocytes showing vesicular nuclei (arrowhead), some of it are binucleated (red arrows) and are separated with sinusoids (blue arrow). b Negative control group showing normal portal tract with its structures; branches of portal vein (arrow), hepatic artery (arrowhead) and bile duct (red arrow). c and d are photomicrograph of liver sections of rat that orally administered with EPA; where c showing normal structure of hepatocytes and sinusoids. d Showing normal structure of portal tract. e and f are photomicrograph of liver sections of rat that administered with EPA-NE; where e showing normal structure of hepatocytes and sinusoids. f Showing normal structure of hepatocytes and sinusoids. Some hepatocytes the surround the portal tract exhibit necrotic feature (arrows). (H&E stain, Scale bar: 5 µm)

Oral administration with EPA in a dose of 500 mg/kg/day for 4 weeks showed normal structure of hepatocytes and sinusoids (Fig. 6c) and portal tracts (Fig. 6d).

On the other hand, administration of EPA loaded silica nanoemulsion in a dose of 500 mg/kg/day for 4 weeks displayed normal structure of hepatocytes and sinusoids (Fig. 6e). Normal structure of the portal tracts was appeared. Some hepatocytes that surround the portal tract exhibited necrotic feature (Fig. 6f).

Examination of liver sections from rat that orally administered with DEN showed loss of the normal hepatic structures, including laminae, sinusoids and dilated portal tract (Fig. 7a). Formations of pseudogland can be observed in high-grade macronodules (Fig. 7b). Complete destruction of the sinusoidal architectural pattern, along with a fibrotic stroma, lytic hepatocellular necrosis, no congestion was observed (Fig. 7c). Congestion in the portal tracts that associated with necrotic feature of the surround hepatocytes were noticed (Fig. 7d).

Fig. 7figure 7

Photomicrograph of liver section from rat that administered with DEN showing: a loss of the normal hepatic structures, including laminae, sinusoids and dilated portal tract. b pseudogland formations (arrows) can be observed in high-grade macronodules. c Architectural pattern, along with a fibrotic stroma, lytic hepatocellular necrosis (arrowheads), no congestion was observed. d congestion in the portal tract that associated with necrotic feature of the surround hepatocytes. (H&E stain, Scale bar: 5 µm)

Histopathological examination of liver sections from rats administered DEN and in the same time treated with EPA showed the structure of hepatic lobules (Fig. 8a). In addition, Fig. 8b showed congestion of portal tracts that associated with mild inflammatory infiltration, and hydropic degeneration.

Fig. 8figure 8

Photomicrograph of liver section showing rat that administered with DEN and in the same time treated with EPA (treated I group) showing: a the structure of hepatic lobule appeared more or less as normal one b congestion of portal tract appeared associated with mild inflammatory infiltration. Hydropic degeneration was seen (H&E stain, Scale bar: 5 µm). c and d showing rat that administered DEN and in the same time treated with EPA loaded silica nanoemulsion (treated II group). Where c showing the hepatic lobule appears nearly like normal control around the portal tract. d showing the portal tract appears nearly like normal control (H&E stain, Scale bar: 5 µm)

Microscopic examination of liver sections from rats that orally administered DEN and in the same time treated with EPA loaded silica nanoemulsion showed the hepatic lobules (Fig. 8c) and the portal tracts (Fig. 8d) appeared nearly like normal control.

This study clearly exhibited that oral administration of EPA alone or EPA loaded silica nanoemulsion ameliorated hepatic inflammation induced by DEN in comparison to DEN induced hepatic inflammation group in both macroscopically and microscopically examinations. Additionally, in EPA nano emulsion treated group; this effect was increased to become more or less near the control group.

We suggesting that this effect is related to the role of nanoemulsion characterized by smaller size that enhance the effect of EPA and facilitate its penetration to the cell membrane.

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