Biochemical Study and Gene Expression of Glutathione-S-Transferase (GST) in Induced Asthma in Rat

This researchaims to stud the role of Glutathione-S-transferases in asthma under stress conditions. Also, evaluate the ability of Iraqi propolis to inhibit the free radicals production in asthmatic rats too.GST enzyme was partially purified from rats liver after induction asthma. The experimental Sprague-Dawly rats were divided into four groups. (G1) control group, (G2) asthma group, (G3) asthma treated with propolis and (G4) was treated with propolis first and then induce asthma. The results showed reduction GST activity in (G2) when compared with control group (G1), and increase activity of GST in (G3) when compared with (G2).

mitochondrial, and microsomal. cytosolic GSTsare subdivided into seven distinct classes designated ,named as Alpha (α), Mu (µ), Pi (π), Sigma (σ), Theta (δ), Zeta (ξ), Omega (ω) and Kappa (κ) 5 . Three classes Alpha, mu and pi are involved in drug metabolism and detoxification 3,4 . Four additional classes of this super family, called Beta (α), Delta (δ), Phi (ϕ), and Tau (τ) are represented in bacteria, insects and plants (6) . In glutathione redox cycle cycle, most of the enzymes catalyze the conjugation of reduced Glutathione (GSH) with compounds that include an electrophilic center through the formation of a thioether bond between the sulphur atom of GSH and the substrate (7) . Asthma is characterized by chronic airway inflammation and oxidative stress in the lungs has been implicated in its pathogenesis. Most of ROS and other free radicals are generated as by-products of normal cellular metabolic activities. Firstly in the mitochondria or during the enzymatic oxidation.ROShave long been recognized as important mediators of inflammation and cell death. Oxygen free radicals are highly reactive molecules result in oxidative damage of biological molecules such as, lipids, and DNA and proteins and alter their functions (8) . The shift in the balance between oxidants and antioxidants in favor of oxidants is termed oxidative stress. These are countered by enzymatic and non-enzymatic antioxidants, including dietary antioxidants, such as vitamins C, glutathione (GSH), phenolic compounds, alkaloids, non-protein amino acids and atocopherols 9 . A family of enzymes, the glutathione-S-transferases (GSTs), has the general function of conjugating glutathione with electrophilic substances that are capable of generating free radicals, thus leading to detoxification of their effects. Genetic polymorphisms associated with reduced activity of GSTs are therefore of interest in the study of disease susceptibility. Two common deletion polymorphisms of GSTM1 and GSTT1 genes have been associated with asthma 10 .

Experimental design
Sixty malesratsused in this study,the average weight of rats were (140 ± 10) gm and (8)(9) week age were divided into fourgroups. Every group contained 15 rats. First group received distilled water daily for three weeks then injected with (0.5ml) normal saline one dose on 1 st , 3rd, 5thday as a control. Second group received distilled water for three weeks then was injected subcutaneous with three doses of 100µg of egg albumin on 1 st ,3 rd ,5 th day . Third groupinduced asthma by injected of 100µg of egg albumin on 1 st ,3 rd ,5 th dayand then received local propolis for three weeks at doses 200mg /kg.The last groupreceived local propolis at doses 200mg /kg for three weeks, theninjected subcutaneous with three doses of 100µg of egg albumin on 1 st , 3rd, 5thday

Biochemical Tests
Determination of Serum Reduced Glutathione (GSH) level 11 The disulfide of 5, 5-Dithiobis (2nitrobenzoic acid) (DTNB) is readily reduced bysulfahydryl group of GSH to an intensely yellow compound .That reduced Compound measured absorbance at 412 nm and is directly proportional to the GSH concentration, by using UV-VIS Spectrophotometer(APLEPD-303 UV).

Determination of Serum Nitric Oxide Level 12
A reaction between nitrite and sulfanilamide and N-(1-naphthyl) ethylene diamine produced a pink colored complex which is measured by UV-VIS Spectrophotometer(APLEPD-303 UV)at 540nm wave length. 13 The activity was determined by using 1chloro-2, 4-dinitrobenzene (CDNB) as Substrate,The conjugation of (CDNB) with GSHproceeds by nucleophilic aromatic substitution of chlorine by thiol group of GSH, producing a dinitrophenylthioether and chloride ion, measured absorbance at 340nm by using UV-Visible spectrophotometer(SHIMADZU UV-1650PC).

Enzyme partially purified-2 PurificationofGlutathione-s-Transferase (GST) fromRat liver
All steps were performed at 4 o C unless stated otherwise.

Step 1: Preparation of Cytosol Fractions
Rat liver (2 g) was cut into small pieces and homogenized in 10 mM ice-cold Tris-acid pH 8.0 in a blender. The homogenate was filtrated through 2 layers of gauze to remove connective tissue. The homogenate was centrifuged at 5,000 rpm for one hour. The resulting supernatant was then centrifuged for 30 minute. The cytosol fraction was obtained, and stored at -20 CR" (13) .

Step 2: Ammonium Sulfate Fractionation
Protein was precipitated using ammonium sulfate (NH4) 2 SO 4 saturation (50%) or protein salting out 14 . Solid ammonium sulfate (1.45) gm per (5 ml) supernatant was added with stirring to the enzyme extract and the mixture was stirred until all of the ammonium sulfate had dissolved; the suspension was centrifuged for (30) min. The protein in precipitate and supernatant are determined using the modified Lowry method 15 , and the GST is determined in each fraction 13 .
Step 3: Dialysis Dialysis was made using a semipermeable cellophane dialysis membrane with M.wt. cut off (<10000) dalton.The dialysis containing the suspension in (Step 2) was dialyzed against 0.015M phosphate buffer, pH 6.25.The solution was stirred with a magnetic stirrer overnight at (4) oC. The buffer was changed twice during dialysis 16 . The protein of the dialyzed enzyme was estimated by modified Lowry method 15 .

Step 4: Gel Filtration Chromatography
This stepwas done in column dimension of 2 × 100 cm containing gel using Sephadex G-75 to high (80 cm) 16 . 0.5 ml of the protein, which was obtained in (Step 3), was applied on the top of a bed Sephadex G-75, followed by phosphate buffer solution. Elution of protein was carried out at a flowrate (52.3) ml / hour.

Step 5: Freeze-Dryer (Lyophilization) Technique
The enzyme fraction which was obtained from gel filtration was lyophilized to obtain a powder of a concentrated protein. The enzyme was kept in a deep freeze at (-20) °C in a tight sample tube to be used in further investigations.

Gene expression of Glutathione-S-Transferrase (GST) Primers
The primers were used in this study, GAPDH gene primers used as Housekeeping gene, GSTM1 gene and GAPDHgeneprimers used as target genes. These primers were designed by using NCBI-Gene Bank data base and Primer 3 design online, the primers used in quantification of gene expression using qRT-PCR techniques based SYBER Green DNA binding dye, and supplied from (Bioneer, Korea) company.
Quantitative Reveres Transcription Real-Time PCR technique was performed for estimation of relative quantification (gene expression analysis). This technique was done according to method described by (17) .

Biochemical Analysis Glutathione (GSH) level
The asthmatic rats (G2) showed significant decline in the mean of serum GSH level as compared with serum samples in control group(G1)(P <0.05) .The oral administration of EEP and then induced asthma in (G3) caused a significant elevation in GSH as compared with results found in asthmatic group(G2) (P <0.05).Animal'sadministration with EEP before induced asthma in (G4) showed almost the same concentration in GSH level as compared with     GSH concentration plays a central role of antioxidant defense system, which detoxifies ROS 18,19 . The decreased level of serum GSH in asthmatic group (G 2) may be returned to the increase in the oxidative stress ,and so that GSH becomes oxidized to its dimeric form (GSSG)as a result of its function as an antioxidant by acting as a approximate target for reactive oxygen species and other products of lipid peroxidation.This result agrees with (Nouf; 2010) 20 that showed the decrease GSH level in Saudi Asthmatic patients . The enhancement in the GSH level caused by EEP may be returned to its antioxidant components such as flavonoids. Flavonoids are ideal scavengers of peroxyl radicals due to their favorable reduction potentials relative to alkyl peroxyl radicals, and thus they are effective inhibitors of lipid peroxidation 21 . On the other hand, the elevation of GSH level in this group (group 3) may return to increase GSH production,where CAPE ,one of the propolis components ,is known to increase the expression of γ-glutamyl cysteine synthatase,an important enzyme in synthesis of GSH resulted in production of glutathione 22 . The increased level of GSH in (group4) as compared with control group may be backed to that EEP increase the power and capacity of other endogenous antioxidant defense of the body and thus increase the steady state levels of GSH.

B-Nitric Oxide Level
The concentration of NO increased significantly in asthmatic rats (G2) as compared with control group (G1)(P <0.05). This is a marked elevation of NO which was significantly reduced by pre administration ofEEP in (G3)(P <0.05). In addition ,there is a almost the same concentration of NO showed in treated group with EEP and then induced  NO play a critical role in the pathogenesis of airway inflammation in allergic asthma 23 . The increased level of NO in (G2) may be due to OVA albumin which induced asthma .OVA induced airway inflammation, asthmaand its greater expression of NOS in the lung tissue (23 Airway inflammation occur as a result of increased expression and activity of NOS which increase NO realizationand thus the numbers of eosinophils in the airway and airway inflammation 24,25 . The significant decrease in NO values in rats were pretreated with EEP before induce asthmamight be attributed to the ability of EEP to inhibit different pathologic changes associating asthma or may be due to the effect of EEP to inhibited the OVA-induce and decrease level of IgE and NOS in serum of asthmatic rats . This result agrees with (El-Sayedet al 2013) 26 .Where caffeic acid phenethyl ester, a biologically active ingredient of propolis ,showed inhibitory effects on the inflammatory cells 27 and it may be a main regent of

GlutathioneS-Transferase(GST)
The changes in the serum Glutathion -Stransferaseactivity of normal and experimental rats are illustrated in Figure (3) show significant reduction in the activity of GST in serum of rats in (G2) when compared with control group(G1),then simultaneous induced asthma and treatment with EEP (G 3) show increase activity of GST in serum of rats when compared with (G2),while in (G4) treatment with EEP before induced asthma was showed almost same activity of GST enzyme as compared with control group(G1),as shown intable (4).
The body has an effective mechanism to prevent and neutralize the free radicals induced damage.This is accomplished by a set of endogenous oxygen species and oxidant defenses are lost,oxidative stress results (28) . The decrease activity of GST in asthmatic rats (G2) mayreturn to the toxic effects of asthma. They are largely due to its active metabolite, the free radicals and that leads to increased oxidative stress (29,30) ,or may be returned to the decrease availability of GSH in( G 2),the relation between GSH and GST its close, where it was well documented that GST enzyme catalyze the reaction via thiol(-SH) group of glutathione (GSH) by neutralizing and rendering the product more water soluble , this result agrees with (Cervello,et al :1992) (31) . The elevation in serum GST activity of rats received EEP in (G3) might be due to the decline of the production of free radicals and /or by trapping and inhibited the formed free radicals and increasing of toxic metabolites of asthma that lead to increasing of antioxidant system. TheEEP mayinspire theinduction ofGST and other antioxidant enzymes by its chemical ingredients of phenyl propanoids (32).

Enzyme partially purified PurificationofGlutathioneTransferase(GST)fromRat liver
The results predicted that the enzyme activity was found in the (50) % of asaturation ammonium sulfate precipitates (13) .As shown in table (5), the specific activity was increased after dialysis.

Kinetic properties of purified glutathione-Stransferase enzyme
The results showed that the valueV max in (G2)show significant reduction when compared with control group (G1), then simultaneous induced asthma and treatment with EEP (G3) show increase V max when compared with (G2), while in (G4) treatment with EEP before induced asthma was showed almost same V max as compared with control group .As show Figure (1) and table (6).
The Michaelis-Menten constant (Km) of the enzyme was determined from Figure(4) by using a Lineweaver-Burk plot by plotting the reciprocal of the invertedinitial velocity versus the reciprocal of the invertedsubstrate concentration.

Gene expression of Glutathione-S-Transferrase (GST) Quantitative Reverse Transcriptase Real-Time PCR
Quantitative Reveres Transcription Real-Time PCR (RT-qPCR) was performed for measurement of relative quantification (gene expression analysis) for GSTM1 genes expression levels normalized by housekeeping gene expression.RT-qPCR quantification method in Real-Time PCR system was dependent on the values threshold cycle numbers (CT) of amplification plot of target genes and housekeeping gene. Where the result of Real-Time PCR amplification plot of housekeeping gene was appeared no difference in CT value, where the control group (CT=27) while the treatment groups which also appeared (CT=27).

Relative Gene Expression Results
The relative expression of target genes (GSTM1) in rats liver tissue was calculated by using Livak Method (2^-ΔΔCT) that dependent on normalization of RT-qPCR (CT values) of target genes with housekeeping gene (GAPDH) as reference gene in different groups.
The results of relative gene expression in GSTM1 gene which appeared clear difference in fold change of gene expression levels between different groups. Where, G2 was appeared up regulation at (5±1.3), G3 group was appeared up regulation at (2.9±1.6), G4 was appeared down regulation at (0.97±0.4), relative to control groups that is equal to 1 fold change of gene expression levels as in table (7) and table (8).
The statistical analysis of relative gene expression in GSTM1 gene was found significant differences in treatment groups compared with control groups at level (P<0.05) in table (9) Glutathione S-transferases (GST) include a number of subclasses, such as GST-P1 and GST-M1 that are expressed in the lungs and have been implicated in the pathogenesis of asthma. Children with GSTM1 null or GSTP1 val/val genotypes had increased respiratory symptoms upon exposure,As was calculated gene expression and show that result increase gene expression in (G2) when compared with (G1), whilein (G4) was showed almost samegene expression as compared with control group.