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Abstract

BACKGROUND:

Liver is a vital organ and the role of Gamma aminobutyric acid (GABA) conjugated chitosan nanoparticles in enhancing the hepatocyte proliferation was reported. To understand the influence of these nanoparticles on various biochemical parameters during enhanced liver regeneration will improve its clinical significance.

OBJECTIVE:

To highlight the influence of GABA chitosan nanoparticles on ascorbic acid, hydroxyproline and lipid peroxidation levels during liver regeneration.

METHODS:

Intraperitoneal administration of nanoparticles was done to partially hepatectomised female Sprague Dawley rats (GCNP) and compared the biochemical parameters with sham operated control (C) and with no treatment (PHNT) cases.

RESULTS:

The hydroxyproline content was increased in the liver of GCNP when compared to PHNT (P < 0.05). The lipid peroxidation level was increased (P < 0.01) in PHNT compared to the control whereas, decreased in GCNP when compared (P < 0.01) with PHNT. There was a significant increase (P < 0.01) in the ascorbic acid content in PHNT when compared with C. It was significantly decreased (P < 0.01) in GCNP when compared with PHNT.

CONCLUSION:

This highlighted the therapeutic implications of lipid peroxidation, hydroxyproline and ascorbic acid in GABA chitosan nanoparticles mediated liver regeneration, which will have immense clinical relevance in maintaining liver health.

Keywords

Gamma amino butyric acid lipid peroxidation ascorbic acid hydroxyproline liver regeneration

1 Introduction

Liver is an important organ responsible for storage, metabolism and synthesis of major compounds in the body. Among all organs, liver has ability to repair itself after suffering loss of tissue mass. Liver is located in the upper right part of belly under the ribs and is responsible for functions vital to life. It also stores vitamins, cholesterol, hormones and minerals [1]. There are many causes of liver injury such as excessive alcohol consumption, viruses, inherited disorders, drug-related injury and environmental toxin exposure, which lead to fibrosis. As the fibrosis increases, it can lead to alterations in the shape and function of the normal liver, that can be examined through abdominal CT scan accurately [2].

The liver is a special organ, that has a remarkable capacity to regenerate after injury and to adjust its size and function to match the previous condition. Within a week after partial hepatectomy of two-third of the liver under experimental settings, the hepatic mass is regained. These observations have prompted considerable research into the mechanisms responsible for hepatic regeneration. Understanding the process assists in treatment of serious liver diseases and may have important implications for certain types of liver-based gene therapy. Most of this type of research has been conducted using small animals like rats employing partial hepatectomy model, but a confirmatory evidence has obtained from human subjects also [3]. The hepatocytes have a practically unlimited capacity for proliferation and full regeneration after as many as 12 sequential partial hepatectomies. Clearly the hepatocyte is not a terminally differentiated cell [4]. Liver regeneration can be enhanced by several mitogens. The present work deals with one such mitogenic agent, gamma amino butyric acid, and conjugated nanoparticles delivery system for active liver regeneration.

Nanoparticles using biopolymers were used as a magnificent generation of delivery platforms for delicate bioactive molecules as a part of targeted drug delivery. Chitosan is extensively used in medical field as a potential drug carrier and a tissue engineering implant material, due to its biocompatible properties. It can be either partially or completely deacetylated chitin. As chitin occurs naturally (for example in fungal cell walls and crustacean shells), chitosan is a fully biodegradable, biocompatible and easily resorbable natural polymer [5] that is soluble in acidic conditions. In solution, the free amino groups on its polymeric chains can protonate, giving chitosan a positive charge. Chitosan nanoparticles can be formed by incorporating a poly anion such as tri polyphosphate (TPP) into the chitosan solution under vigorous stirring.

Gamma amino butyric acid (GABA) is a prominent inhibitory neurotransmitter seen in the central nervous system. The cell proliferative role of GABA is observed in different regions of the body including the development of outer retina in rabbits [6], leydig cell multiplication in testis [7] and promotes neuronal cell proliferation and migration [8]. The liver cell proliferation during regeneration is initiated by the activation of cyclic adenosine monophosphate (cAMP) regulated transcription factors. The preliminary study identified that GABA chitosan nanoparticles enhance cell proliferation during liver regeneration [9]. Various other cellular parameters needed to be understood to extend the nanoparticle to clinical levels. The ascorbic acid levels, lipid peroxidation and hydroxyproline contents were deeply studied here.

Lipid peroxidation is the oxidative deterioration of cell lipids that contain carbon-carbon double bonds [10]. A large number of toxic byproducts are formed during lipid peroxidation [11]. Lipid peroxidation associated with unsaturated lipids produces a wide variety of oxidation products like lipid hydro peroxides, malondialdehyde (MDA), propanal, hexanal, and 4-hydroxynonenal. Malondialdehyde is a naturally occurring product of lipid peroxidation [11]. It reacts with amino groups on proteins and other biomolecules to form a variety of adducts, including adducts with DNA bases that are mutagenic and possibly carcinogenic. The MDA content during increased levels of lipid peroxidation is linked with various pathological states of diseases. Therefore, the analysis of characteristics and function of MDA, a direct marker for lipid peroxidation, is an effective tool for monitoring the patient condition. Malondialdehyde is a very reactive three carbon dialdehyde formed as a byproduct of polyunsaturated fatty acid peroxidation. It interacts with different functional groups of biomolecules like proteins, RNA, lipoproteins and DNA [12]. Therefore, MDA is extensively considered as a convenient biomarker for many years for lipid peroxidation of omega-3 and omega-6 fatty acids because of its instant reaction with thiobarbituric acid (TBA).

Vitamin C or ascorbic acid is a water-soluble vitamin. It has reducing risk of hepatitis infection [13]. Ascorbic acid regulates enzymatic reactions, transport of neurotransmitters and in hormone biosynthesis [14]. Ascorbic acid neutralizes superoxide, singlet oxygen and hydroxyl radical [15].

Hydroxyproline is synthesized in endoplasmic reticulum during the hydroxylation of proline following the protein synthesis. The tissue regeneration was confirmed by increased cell proliferation with a higher hydroxyproline content and degree of neovascularization. The up-regulation of epidermal growth factor and vascular endothelial growth factor was also associated with regeneration [16]. The design and characterization of GABA chitosan nanoparticles, modulation of cellular mechanisms initiating hepatocyte proliferation and the impact at neurological levels were achieved previously by our team. Apart from this, the functional role of cell managing systems like lipid peroxidation, hydroxyproline content and ascorbic acid regulation are highlighted which will elevate the nanoparticles to next level clinical application.

2 Materials and methods

2.1 Materials

Penta sodium Tripolyphosphate, Phenolphthalein, Copper sulphate, Disodium Hydrogen Phosphate, Hydrogen peroxide, Sulfuric acid, Perchloric acid (Merck Specialities Private Limited, Mumbai, India). Glacial Acetic Acid, Potassium chloride, Gamma-aminobutyric acid, Sodium hydroxide, Sodium carbonate, Sodium Potassium Tartarate, Folin Ciocalteu’s, Isopropyl Alcohol (Nice Chemical Private Limited, Kochi, India). Bovine Serum Albumin, Sodium chloride, Potassium Phosphate, Para di methyl amino benzaldehyde, Hydroxy L-Proline, 2-Thiobarbituric acid (Himedia Laboratories Private Limited, Mumbai). Chitosan flakes (Central Institute Of Fisheries Technology, Cochin, India).

2.2 Methods

2.2.1 Preparation of GABA chitosan nanoparticles

The nanoparticles were prepared by ionic gelation method [17]. 25 mg of chitosan flakes was added into 25 ml, 2% acetic acid solution. 1 mg/ml concentration of GABA was added into the chitosan solution. The solution was stirred for 1 hour at 500 rpm to dissolve the chitosan flakes using magnetic stirrer. Then 1 mg/ml concentration of penta sodium tri polyphosphate (TPP) was added drop by drop into the solution with constant stirring until suspension of the nanoparticles was formed. Nanoparticles were obtained by centrifugation of the solution at 10,000 rpm for 30 minutes. After the centrifugation; the pellets (nanoparticles) were taken and resuspended in saline.

2.2.2 Animals

Animal experiments were carried out on adult female Sprague Dawley rats of 200–250 g body weight purchased from Kerala Agricultural University, Mannuthy, India. They were housed in separate cages under 12 hours light and 12 hours dark periods and were maintained on standard food pellets and water ad libitum. All animal care and procedures were taken in accordance with the Institutional, National Institute of Health and CPCSEA guidelines (The approval number—PIMS&RC/IAEC-9/2014). All efforts were made to minimize animal suffering. Each group consisted of four animals. Sham operated control (C), partially hepatectomised group without any treatment (PHNT), partially hepatectomised group with GABA chitosan nanoparticle treatment (GCNP), were the three experimental groups.

2.2.3 Partial hepatectomy and sacrifice

Two-thirds of the liver constituting the median and left lateral lobes were surgically excised under light ether anaesthesia, following a 16 hours fast [18]. Sham operations involved median excision of the body wall, followed by all manipulations except removal of the lobes. All the surgeries were performed between 7 and 9 AM to avoid diurnal variations in responses. After surgery, 1 ml of 30μg/μl GABA chitosan nanoparticles, suspended in saline were injected intraperitoneally into the rats. The rats were sacrificed by decapitation 4th day post hepatectomy and liver was dissected out quickly and kept over ice [19]. The tissues were stored at –80°C until assayed.

2.2.4 Hydroxyproline assay

10 mg Hydroxyproline was dissolved in 1 ml water to make stock solution. Standard concentrations of hydroxyproline were taken as S₁-0.16 mg/0.1 ml, S₂-0.33 mg/0.1 ml, S₃-0.66 mg/0.1 ml, and S4-1 mg/0.1 ml. Liver tissues from each experimental group were weighed (10 mg) and hydrolysed with (1 ml) 6 N HCl for 12 hours at 50°C. These sample tubes mouth was covered with cotton because in order to avoid escape of vapours through it. After 12 hours test tubes were cooled at room temperature. The hydrolysed sample was homogenised and 500μl were added into the test tubes. The hydrolysate was diluted with 2.25 ml deionised water. To the diluted hydrolysate one drop of phenolphthalein indicator was added and the solution was neutralised by using 10 N NaOH. Deionised water was taken as blank. To all the standard and experimental samples,1 ml of 2.5 N NaOH, 3 ml of 0.01M CuSO₄ and 1 ml of hydrogen peroxide (30%) were added. The test tubes were kept in water bath at 80°C for 16 minutes. The tubes were taken out and cooled for 5 minutes. To all the test tubes freshly prepared 2 ml of paradimethylaminobenzaldehyde solution (5%) and 4 ml of 3 N H₂SO₄ were added. The test tubes were kept in water bath at 80°C for 15 minutes. After the incubation, test tubes were taken out and cooled for 5 minutes. 200μl of blank, standards and test solutions were pipetted into micro titre plate and the absorbance was read at 540 nm. A standard graph was plotted between absorbance in the Y-axis and concentration of hydroxyproline in X-axis. Hydroxyproline concentration in the tissue sample was calculated using the standard graph [20].

2.2.5 Determination of protein in the liver of experimental animals

The liver tissue was taken from –80°C deep freezer the liver tissue was taken and kept in ice. 0.1 g liver tissue was weighed from all the samples (C, PHNT, and GCNP). It was homogenised with 1 ml Phosphate Buffer Saline (PBS), pH7.4. Then the homogenate was centrifuged at 3000 rpm for 10 minutes. After centrifugation, 20μl supernatant was taken from each tissue sample. Prepared various concentrations of standard solutions (10, 20, 40, 60, 80, 100 mg/ml) from the stock standard (1.25 mg BSA/250μl PBS). Protein content in the liver of experimental rats, were determined [21]. The absorbance was measured at 660 nm. Standard graph was plotted by taking absorbance on Y-axis and concentration on X-axis. Protein concentration in the tissue sample was calculated using the standard graph.

2.2.6 Lipid peroxidation assay

Lipid peroxidation (LP) can be defined as the oxidative deterioration of lipids containing a number of carbon–carbon double bonds [22]. One of the end products of lipid peroxidation is malondialdehyde (MDA). The level of lipid peroxidase was assayed [23]. The estimation of MDA concentration will directly link with lipid peroxidation. The amount of TBARS was calculated using a molar extinction coefficient of ɛ= 1.56×105 m^–1/cm^–1. Lipid peroxidation was calculated with the formula: $(OD \times Sample volume)$ $C = (ɛ \times Total volume \times Tissue wt . / ml \times Path length \times DF)$

C = Concentration nM/g tissue, OD = Optical Density, ɛ= Extinction coefficient M^–1cm^–1, DF = Dilution Factor,

Liver tissue (0.2 g) was weighed and homogenized with 2 ml 0.15 M KCl solution. 1 ml of homogenate was pipetted out and kept in water bath at 37°C for 2 hours. After the incubation, 1 ml of 10% TCA was added and mixed well. The mixture was centrifuged at 3000 rpm for 10 minutes. Supernatant was taken and mixed with 2 ml, 0.67% Thiobarbituric acid (TBA reagent). Then kept in boiling water (100°C) bath for 10 minutes. After incubation, cooled the solution and diluted with 1 ml distilled water. The coloured solution was read at 535 nm in micro plate reader. TBARS level was expressed as nMoles/g tissue.

2.2.7 Ascorbic acid (vitamin c) assay

Liver tissue (0.5 g) was weighed and homogenized with 5 ml NaCl (0.9%) solution. After the homogenization, 0.5 ml homogenate was taken, 0.5 ml of TCA was added and mixed well. The mixture was centrifuged at 3500×g for 20 minutes. To 0.5 ml of the supernatant, 0.1 ml of the DTC reagent was added and incubated at 60°C for 1 hour. Then 0.75 ml of 65% ice cold sulphuric acid was added, mixed well and the solution was allowed to stand for 30 minutes at room temperature. The colour developed was read at 520 nm. A standard graph was plotted by taking absorbance on Y-axis and concentration on X-axis, where ascorbic acid solution was used as a standard (1.2 mg/1.5 ml). The level of Vitamin C in the liver tissue was found from the standard graph and expressed as mg/g tissue [24].

2.2.8 Statistical analysis

Statistical evaluations used analysis of variance (ANOVA) in GraphPad Instat (version 2.04a; Graph Pad, San Diego, CA). Student Newman-Keuls test was used to compare different groups after ANOVA.

3 Result

3.1 Hydroxyproline content in experimental animals

The hydroxyproline content was increased in the liver of GCNP when compared to PHNT (P < 0.05). There was a significant decrease (P < 0.05) in hydroxyproline content in PHNT when compared to C. There was no significant change in the content when compared between C and GCNP (Table 1).

Table 1
Hydroxyproline content in the experimental rats

Sl. No. Experimental groups Hydroxyproline content in rat liver (mg/g liver tissue)

1 C 264±2.1

2 PHNT 165±1.3^*

3 GCNP 228±2.0^$

Sl. No.	Experimental groups	Hydroxyproline content in rat liver (mg/g liver tissue)
1	C	264±2.1
2	PHNT	165±1.3^*
3	GCNP	228±2.0^$

C-Sham operated control, PHNT-Partially hepatectomised group with no treatment, GCNP-Partially hepatectomised treated rat with gamma-aminobutyric acid chitosan nanoparticles. ^*P < 0.01 when compared to C, ^$P < 0.01 when compared to PHNT.

3.2 Protein content in the liver of experimental animals

The protein content was decreased in PHNT when compared to C (P < 0.01) and GCNP (P < 0.01). The protein content was increased in the liver of GCNP when compared to PHNT (P < 0.01) (Table 2).

Table 2
Protein content in the experimental rats

Sl. No. Experimental groups Protein content in rat liver (mg/g liver tissue)

1 C 3.250±0.05

2 PHNT 2.350±0.03^*

3 GCNP 3.950±0.06^# , $

Sl. No.	Experimental groups	Protein content in rat liver (mg/g liver tissue)
1	C	3.250±0.05
2	PHNT	2.350±0.03^*
3	GCNP	3.950±0.06^# , $

C-Sham operated control, PHNT-Partially hepatectomised group with no treatment, GCNP-Partially hepatectomised treated rat with gamma-aminobutyric acid chitosan nanoparticles. *P < 0.01 when compared to C, ^$P < 0.01 when compared to PHNT, ^#P < 0.05 when compared to C.

3.3 Lipid peroxidation in the liver of experimental animals

The lipid peroxidation level was increased (P < 0.01) in PHNT compared to the control. The lipid peroxidation was decreased in GCNP when compared (P < 0.01) with PHNT and it reached near to the control (C) (Table 3).

Table 3
MDA content in the liver of experimental rats

Sl. No. Experimental group MDA content in rat liver (nM/mg tissue)

1 C 4.941±0.21

2 PHNT 6.056±0.34^*

3 GCNP 4.335±0.22^$

Sl. No.	Experimental group	MDA content in rat liver (nM/mg tissue)
1	C	4.941±0.21
2	PHNT	6.056±0.34^*
3	GCNP	4.335±0.22^$

C-Sham operated control, PHNT-partially hepatectomised group without any treatment, GCNP-partially hepatectomised group with GABA Chitosan nanoparticle treatment. ^*P < 0.01 when compared to C and ^$P < 0.01 when compared to PHNT.

3.4 Ascorbic acid content in the liver of experimental animals

There was a significant increase (P < 0.01) in the ascorbic acid content in the liver of PHNT when compared to C. The ascorbic acid content was significantly decreased (P < 0.01) in GCNP when compared to PHNT and increased (P < 0.05) when compared to C (Table 4).

Table 4
Ascorbic acid content in the liver of experimental rats

Sl. No. Experimental group Ascorbic acid content in rat liver (mg/g tissue)

1 C 212.85±1.1

2 PHNT 510.8±3.2^*

3 GCNP 334.36±2.0^# $

Sl. No.	Experimental group	Ascorbic acid content in rat liver (mg/g tissue)
1	C	212.85±1.1
2	PHNT	510.8±3.2^*
3	GCNP	334.36±2.0^# $

C-Sham operated control, PHNT-partially hepatectomised group without any treatment, GCNP-partially hepatectomised group with GABA Chitosan nanoparticle treatment. ^*P < 0.01 and ^#P < 0.05 when compared to C and ^$P < 0.01 when compared to PHNT.

4 Discussion

Chitosan nanoparticles conjugated drugs are utilized for delivering drugs to liver, eye [25], brain [26], cancer tissues, treatment of spinal cord injury and infections [27]. In the last decade, these nanoparticles are widely utilised for clinical application. Chitosan is also a preferred drug carrier or an implant material because of its biocompatible and bioresorbable properties [28]. Gamma amino butyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and the treatment with GABA-chitosan nanoparticles in partially hepatectomised rat improves hepatocyte proliferation [9]. After partial hepatectomy, GABA-chitosan nanoparticles were administered to rats through the peritoneal cavity for a noticeable absorption of nanoparticles to the liver. Partial hepatectomy is a widely accepted model to study liver damage and regeneration. Liver regeneration is an organized process involving the interaction of many cell types like hepatocytes, stellate cells, various inflammatory cells and endothelial cells. The inflammatory cells influence the regeneration by scavenging dead cells, in situ and promoting fibrosis [29]. Various biochemical compounds also influence active liver regeneration. Hydroxyproline is known to be a neutral protein amino acid found in collagen. So, collagen can be quantitatively determined by estimating hydroxyproline concentration [30]. In the present study we observed that, GABA chitosan nanoparticles helped to increase the hydroxyproline and protein during liver regeneration. The collagen synthesis in liver was inversely correlated with cathepsin L activity and also with the intracellular degradation of newly formed collagen. These findings suggest that, a combination of decreased intracellular collagen degradation and increased collagen synthesis contributes to the rapid availability of collagen during the early phase of liver regeneration [31]. Liver regeneration is a process of both hypertrophy and hyperplasia i.e., increases in cell size or protein content during the pre-replicative stage and increase in number of cells [32]. Our study showed an increase in hydroxyproline and protein contents in GABA chitosan nanoparticles treated group. This shows that increase in collagen during liver regeneration induced by GABA chitosan nanoparticles rendered faster hepatocyte proliferation.

Reports on the role of liver regeneration induced by PH on the liver lipid peroxidative system of well-fed rats have been promising. Several previous studies have reported changes in lipid peroxidation and antioxidant activity during liver regeneration [33]. It was also reported a significant decrease of both blood and liver ascorbic acid concentrations in Dimethyl nitrosamine (DMN)-induced hepatic fibrosis in rats [34]. The study also highlighted an induction in extensive liver necrosis and ultimate fibrosis due to increased lipid peroxides, oxidative stress, and simultaneous formation of free radicals [35]. Vitamin C is a water-soluble antioxidant that involved in decreasing lipid peroxidation through regenerating vitamin E, the major lipid-soluble antioxidant [36]. Vitamin C was also reported to inhibits lipid peroxidation by scavenging aqueous reactive oxygen species through rapid electron transfer [37]. An increase in intracellular reactive oxygen species was contributed by growth factor stimulation by platelet-derived, epidermal and insulin-like growth factors. This can activate kinases, regulate transcription factors and inactivate phosphatases at the cell membrane, leading to cell cycle progression. An increase in reactive oxygen species concentration leads to a decrease in SOD activity and SOD enzyme gene expression during hepatic regeneration by GABA chitosan nanoparticle treatment when compared to the one without treatment [38]. The current study results also showed that Vitamin C can inhibit lipid peroxidation. In this study, the lipid peroxidation level is increased in PHNT when compared to control. The increased level of lipid peroxidation is due to the partial hepatectomy. Our result demonstrated the important role of GABA chitosan nanoparticles treatment and the change in ascorbic acid content that will modulate lipid peroxidation after partial hepatectomy. The ascorbic acid content was increased in PHNT when compared to control and decreased in GCNP when compared to the PHNT. The ascorbic acid content in GCNP reached near to the control. The decrease in lipid peroxidation and decrease in ascorbic acid content during nanoparticle treatment highlights the importance of GABA chitosan nanoparticle assisted enhanced liver regeneration. This has immense therapeutic significance in regenerative medicine research.

5 Conclusion

The liver is involved in many complex metabolic functions essential to life. Liver damage leads to destruction of hepatocytes and thereafter, its healthy regeneration to its original form is difficult. This study identified that lipid peroxidation, hydroxyproline and ascorbic acid content were regulated during liver regeneration that was induced by GABA chitosan nanoparticles. This helps in future research based on the regulation of these parameters in highly controlled and rapid liver regeneration. So, the study concluded that the importance of GABA chitosan nanoparticles in regulating the liver regeneration need to be examined further to implement at clinical levels.

Footnotes

Acknowledgments

The authors sincerely thank late Dr. C.S. Paulose, Cochin University of Science and Technology, Cochin, India for his valuable guidance and Pushpagiri Research Centre, Tiruvalla, India for providing the facilities for the research works.

Author contributions

All authors contributed for writing the manuscript and designing the experiments. All authors involved in performing the experiments and gave approval to the final version of the manuscript.

Conflict of interest

The authors have no conflict of interest in publishing the article.

Funding

The authors report no funding for the work.

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