Compounds of RS Extracts by TLC Analysis
1. Introduction
High level fat, obesity and metabolic syndrome may increase oxidative stress, and/or influence the levels of cellular homeostasis (Gao et al., 2011; Furukawa et al., 2004). Thus, oxidative damage and its consequences may result in many chronic health problems. For example, atherosclerosis, cancer, hyperlipidemia, hyperglycemia, and arthritis have been correlated with oxidative damage (Brown and Bicknell., 2001; Alexander, 1995). Diabetes mellitus (DM) can be defined as a group of syndromes due to defects in pancreatic secretion of insulin or insulin action, which characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins along with an increased risk of complications from vascular disease (Taskinen et al., 2002). Hyperglycemia impairs the prooxidant/antioxidant balance, increasing free radical and reducing antioxidant levels (Aragno et al., 2004). Free radicals react with lipids and cause peroxidative changes that result in enhanced lipid peroxidation (Girotti, 1985). The level of lipid peroxidation in cells is controlled by various cellular defense mechanisms consisting of enzymatic and nonenzymatic scavenging systems. The efficiency of the antioxidant defense mechanism is altered in diabetes (Wohaieb and Godin, 1987). Increased free radical production exerts cytotoxic effects on the membrane phospholipid, resulting in formation of toxic products such as MDA. The antioxidant scavenging enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) offer protection to cells and tissues against oxidative injury (Bonnefont-Rousselot et al., 2000).
There are various reports indicating the beneficial effects of antioxidant supplementation in preventing dyslipidemia. Diet modification may be one way to reduce serum lipid level. Numerous studies have reported that lactic acid bacteria fermented food display hypolipidemic effects by inhibiting cholesterol biosynthesis and decreasing low-density lipoproteins (Haberer et al., 2003; Kawase et al., 2000). Momordica charantia fermented milk is effective in preventing and retarding hyperlipidemia and atherosclerosis in hamsters (Tsai et al., 2009), and some kinds of LAB could adjust blood lipid and lower cholesterol, which can prevent and treat some diseases by activating antioxidant enzymes (Jain et al., 2009). Some herbal drugs are a good source of natural antioxidants, and increased utilization of medicinal plants became a World Health Organization policy on 1970.
Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the antioxidant response element (ARE)-dependent gene regulation in response to oxidative stress. Nrf2 regulates the transcriptional activation of more than 200 antioxidant and protective genes that constitute the so-called phase II response. Examples of phase II enzymes (p2Es) include the rate-limiting enzyme in the GSH synthesis pathway, glutathione peroxidase (GPx), as well as superoxide dismutase (SOD), heme oxygenase 1, γ-gluta-mylcysteine ligase, glutathione S-transferase, and reduced nicotinamide adenine dinucleotide phosphate–quinone oxidoreductase (Matafome et al., 2011; Gao et al.,2009 ). Some strains and species of lactic acid bacteria (LAB) have the antioxidative activity (Gao et al., 2011; Sotiroudis et al., 2010). A number of
The present study was performed to investigate the antioxidant therapeutic effects and mechanism of
2. Antidiabetic and antioxidative potentials of Rhodiola sachalinensis polysaccharide
2.1. Preparation and characterization of R. sachalinensis
Dried
Indicator | examined Components | Ratio of Flow(Rf) | Color |
phenol / sulfuric acid | polysaccharide | 0.193-0.553 | brown |
10% NaOH | flavone | 0.248-0.463 | yellow |
bromophenol blue / ethanol | organic acid | 0.278-0.564 | dark yellow |
phosphomolybdic acid / ethanol | saponin | 0.217-0.493 | dark blue |
ferric trichloride / water | hydroxybenzene | - | - |
acetic anhydride / sulfuric acid | terpene and steroid | - | - |
10%KOH | anthraquinone | - | - |
iodine / potassic iodide | alkaloide | - | - |
sulfuric acid / ethanol | lignin | - | - |
2.2. Preparation of STZ-induced diabetic rats
Male wistar rats weighing 180-200 g were obtained from the animal department of Beijing institute of traditional medical and pharmaceutical sciences. The animals were housed under suitable lighting and temperature. Food and drinking water were provided. Following one week of acclimation, eight rats were randomly assigned as normal control group, and the rest rats, treated with STZ, became the diabetic model rats according to the standard method (Gao et al., 2007). The rats with blood glucose levels above 15.0 mM were defined as diabetic model rats. Thirty-two rats (eight normal rats, twenty-four diabetic rats) were chosen and randomly divided into four groups: normal control group (NC), DM control group (DC), DM + RS low dose group (DM+RS LD), and DM+RS high dose group (DM+RS HD).
2.3. Examination of effect of RS in the oral glucose tolerance test
On the first day, after overnight fasting with free access to water, the rats were administered RS solution by oral gavage at the following doses: 200 or 400 mg/kg bw for the DM+RS LD & DM+RS HD groups, and with the same volume of dH2O alone for the NC and DC groups. Tail blood samples were drawn from each rat, then a glucose (2.0 g /kg bw) solution was orally administered by oral gavage after 30 min following RS administration. Blood samples were taken at 30, 60, 90, and 120 min intervals following glucose administration, and blood glucose levels were measured at various time points. The supplement of RS improved the acute blood glucose levels in the rats (Figure 1). There was no significant difference at 0 h between the DC and RS-treated groups, but in the RS LD and HD groups, hypoglycemic effect of RS became significant 1 h following oral administration comparing with DC group (
2.4. Determination of subacute effect of RS treatment on blood glucose level
In the DM+RS LD and HD groups, the rats were given RS solution (200 & 400 mg/kg bw) daily by gavage for 40 days, respectively. The control rats (NC and DC groups) were given the same volume of dH2O. On days 0, 10, 20, 30 and 40, the blood samples were collected from rat’s tail veins and measured, followed by an overnight fast. Changes of plasma glucose level as a result of RS treatment for 40 days are shown in Figure 2. Before treatment of diabetic rats, there was no significant difference for the blood glucose levels among diabetic rats (
The oral administration of RS for 40 days for the STZ-induced diabetic rats showed a significant reduction in blood glucose, which elucidate that
2.5. Determination of effects of RS treatment on blood biochemical parameters
At the end of the experiment, the blood samples of fasted tested rats were collected from the eyes under ether anaesthesia, to determine the levels of TG, TC and insulin, according to commercial advice by the Automatic Biochemical Analyzer. The levels of TG and TC in DC rats were significantly higher compared with those of NC group (Table 2,
Groups | TC (mg/dl) | TG (mg/dl) |
NC | 83.288±3.084* | 72.513±3.169* |
DC | 135.375±4.203 | 170.438±3.376 |
DM+RS LD | 106.913±4.491* | 94.075±3.837* |
DM +RS HD | 96.125±4.591*# | 84.188±4.121*# |
Diabetes is a metabolic disorder affecting carbohydrate, lipid and protein metabolisms, complicated with multiorgans regression in the later period. The levels of serum lipids are usually raised in diabetes mellitus (Sakatani et al., 2005). The increase of blood glucose is accompanied with the rise of TC and TG (Sharma et al., 2003). The significant rise of blood glucose, TC and TG levels has been observed in STZ-induced diabetic rats, whereas those were significantly decreased by RS treatment. Our results suggested that RS not only possess significant hypoglycemic ability but also have remarkable hypolipidemic effect. RS also enhanced serum insulin release in STZ-diabetic rats by 40 days treatment, which was obviously different with the diabetic control rats. We presume that RS appears the hypoglycemic effect in diabetic rats is partly attributed to its stimulation of insulin secretion.
2.6. Determination of tissue antioxidative enzyme activities
The animals were sacrificed under ether anaesthesia. Their liver and kidney tissues were immediately removed, washed using chilled saline solution, homogenized in 4 volumes of Tris-HCl buffer (pH 7.4). The homogenates were centrifuged at 4,000 ×
Antioxidative level in liver and kidney tissues | NC | DC | DM+RS LD | DM+RS HD |
Liver MDA (nmol/mg.pro) | 8.105± 0.329** | 10.848± 0.455 | 9.773 ±0.375 | 9.132 ±0.374* |
Kidney MDA (nmol/mg.pro) | 9.701±0.269** | 12.315±0.352 | 11.239±0.441 | 10.604±0.236* |
Liver SOD (U/mg.pro) | 46.05±1.129** | 39.913±1.045 | 43.45±1.018 | 44.375±0.958* |
Kidney SOD (U/mg.pro) | 50.725±1.184** | 44.188±0.834 | 47.263±0.927 | 48.638±0.814* |
Liver GSH-px (U/mg.pro) | 24.849±0.944** | 16.959±0.734 | 20.973±0.925* | 21.848±1.136* |
Kidney GSH-px (U/mg.pro) | 32.321±0.795** | 24.438±0.600 | 27.779±0.691* | 30.853±0.898** |
Liver CAT (U/ml) | 20.176±0.551** | 14.786±0.390 | 17.398±0.416* | 18.776±0.597** |
Kidney CAT (U/ml) | 25.544±0.433** | 20.410±0.549 | 22.604±0.539* | 22.665±0.491* |
It was reported that diabetic subjects are highly sensitive to oxidative stress (Pritchard et al., 1986). In STZ-diabetic animals, STZ generates nitric oxide, which is a powerful free radical oxidant (Kwon et al., 1994) resulting in an increase in blood glucose level. Several studies have documented the relationships between the increase of free radicals and blood glucose, lipid peroxidation as well as low-density lipoprotein in the progress of diabetes (Rabinovitch et al., 1996; Tanaka et al., 2002). Free radicals can diffuse intracellularly and result in mitochondrial enzyme damage and DNA break, impair cellular function and contributes to the pathophysiology of diabetes (Bonnefont-Rousselot et al., 2000). Increased free radical production exerts cytotoxic effects on the membrane phospholipid, resulting in formation of toxic products such as MDA. Several reports have shown the alterations in the antioxidant enzymes during diabetic condition (Preet et al., 2005). The antioxidative defense system like SOD and CAT showed lower activities in liver and kidney during diabetes. The decreased activities of SOD and CAT may be a response to increased production of H2O2 and O2– by the auto oxidation of excess glucose and non-enzymatic glycation of proteins (Argano et al 1997). Pigeolet et al (1990) have reported the partial inactivation of these enzyme activities by hydroxyl radicals and hydrogen peroxide. The decreased activity of SOD and CAT could also be due to their decreased protein expression levels in the diabetic condition as reported recently in liver (Sindhu et al 2004). GSH is often regarded as antioxidant agents, since they protect protein –SH groups against oxidation and can scavenge oxygen radicals and some other reactive species (Robertson, 2004). It reduces different oxidants after increasing of its hydrogen atom. In these reactions, two GSH molecules transform into one molecule of oxidized glutathione (GSSG). This reaction catalyzes the enzyme GSH-px in cells (Reiter, 1995). In our research, the level of SOD, GSH-px and CAT was increased and the concentration of MDA was decreased after RS treatment, which suggests that RS has effective antioxidative properties and could scavenge well excess free radicals, which may prevent the oxidative damage of the tissues and can increase a protective effect on improving diabetic complications.
2.7. Single cell gel electrophoresis experiments
SCGE does not require cell division, and under alkaline conditions, enables the assessment of DNA double-and single-strand breaks and alkali-labile sites (Singh et al., 1988; Belpaeme,
dH2O | 281 | 19 | 0 | 0 | 0 | 3.13 |
RS (100 µg/ml) | 275 | 25 | 0 | 0 | 0 | 3.33 |
RS (200 µg/ml) | 271 | 29 | 0 | 0 | 0 | 3.47 |
0 | 8 | 157 | 125 | 10 | 44.03 |
3. Antidiabetic and antioxidant effects of oleanolic acid in diabetic rats
Our study evaluates the antidiabetic and antioxidant effects of oleanolic acid (OA) from
3.1. Sample preparation and characterization
The extraction of
3.2. Preparation of alloxan-induced diabetic rats
Wistar rats weighing 180-200 g were purchased and house as previous condition. Eight rats were randomly picked up as normal control (NC), and the rest were fed on high-fat diet. After exposure to the high-fat diet for 3 weeks, the rats were fasted overnight with free access to water, and injected intraperitoneally with alloxan that was dissolved in sterile normal saline solution, and used dosage of alloxan was 200 mg/kg bw. After injection 72 h, the fasting blood glucose level of the rats was determined according to glucose oxidase method (Trinder, 1969) using a Glucose Analyzer. The blood glucose level above 15 mM was defined as DM rats. Thirty-two rats (8 normal rats, 24 alloxan-induced diabetic rats) were chosen and divided into four groups: normal control group (NC), diabetic control group (DC), diabetes + low-dose OA group (DM + OA LD) and diabetes + high-dose OA (DM + OA HD).
3.3. Determination hypolipidemic effect of OA
The rats of DC, DM + OA LD and DM + OA HD groups were fasted overnight with free access to water, blood glucose level of each animal was determined as zero-time blood glucose. The animals of DC group were received 0.5% carboxymethylcellulose (CMC) solution by gavage. The rats were orally administered OA 60 mg/kg bw (for the DM + OA LD group) and 100 mg/kg bw (for the DM + OA HD group), OA was dissolved in 0.5% CMC solution. Blood samples of all the rats were taken at 0.5, 1, 2, 4 and 6 hour intervals following the administration and blood plasma glucose levels at various time points were measured. Whereafter, in the DM + OA LD and HD groups, the rats were given OA (60mg/kg bw, 100mg/kg bw) daily by gavage for 40 days, respectively. In contrast, the control rats (NC& DM groups) were given the same volume of 0.5% solution CMC only for 40 days. On day 0, 10, 20, 30 and 40, blood samples were collected from a tail vein, following overnight fasting, and measured.
The supplement of OA improved the acute blood glucose levels in the rats (Figure 6). There was no significant difference at 0 h between the DC and OA-treated groups. But in the DM + OA LD and HD groups, hypoglycemic effect of OA became significant 1 h following oral administration, and reached the peak 2 h (
In the long term test, changes of plasma glucose level as a result of OA treatment are shown in Figure 7. Fasting blood glucose levels were measured on day 0, 10, 20, 30 and day 40. Before treatment of diabetic rats, there was no significant difference for the blood glucose levels among diabetic rats (
3.4. Determination of blood lipid parameters
On the day 41, the rats were fasted overnight, and blood samples were collected from eyepit of all rats under ether anaesthesia. The blood samples were used for the measurement of TG, TC, HDL-c and LDL-c levels, according to commercial advice by the Automatic Biochemical Analyzer (Scientific and Technical Center of Beijing Hospital Clinic Medicine, China). Afterthat, the animals were sacrificed under ether anaesthesia. The liver and kidney were immediately removed, weighed and washed using chilled saline solution. Kidney and liver were homogenized in 4 volumes of Tris–HCl buffer (pH 7.4). The homogenate was centrifuged at 4000 ×
The long term effect of OA treatment on blood lipid levels of tested rat groups is given in Table 5. The results showed that the levels of TG, TC and LDL-c in DM control rats were significantly higher (
Groups | TG (mmol/L ) | TC (mmol/L) | LDL-c (mmol/L) | HDL-c (mmol/L) |
NC | 0.946 ± 0.039** | 1.973± 0.049** | 0.983 ± 0.033* | 0.971 ± 0.028** |
DC | 1.396 ± 0.038 | 2.460 ± 0.033 | 1.145 ± 0.026 | 0.825 ± 0.0278 |
DM+OA LD | 1.078 ± 0.048** | 2.279 ± 0.031* | 1.036 ± 0.032 | 0.895 ± 0.025 |
DM+OA HD | 1.014 ± 0.039** | 1.211 ± 0.031** | 0.991 ± 0.034* | 0.951 ± 0.024* |
Before OA treatment of alloxan-induced diabetic and hyperlipidemic rats, the significant rise in blood glucose was accompanied with increases in TC, TG and LDL-c. After OA treatment, the levels of blood glucose, TC, TG and LDL-c were significantly decreased, and the level of HDL-c in OA-treated rats was higher than those of diabetic rats. These findings indicate that OA might be beneficial to diabetic patients with atherosclerosis, since elevated HDL-c level is associated with the reduced risk of the development of atherosclerosis in diabetes mellitus (Taskinen
3.5. Analysis of antioxidative enzyme activities
The effect of OA on MDA, SOD and GSH-px in the rats is given in Table 6. The results showed that the level of MDA was significantly increased in diabetic control rats, while the activities of SOD and GSH-px were decreased comparing with NC group. The MDA level was decreased in DM +OA HD group (
Group | Tissue | MDA(nmol/mg.pro) | SOD(U/mg.pro) | GSH-px(U/mg.pro) |
NC DC DM+OA LD DM+OA HD | Liver kidney liver kidney liver kidney liver kidney | 8.22 ± 0.32** 9.70 ± 0.27** 11.18 ± 0.46 12.32 ± 0.35 9.82 ± 0.35 11.69 ± 0.24 9.19 ± 0.42* 10.6 ± 0.27** | 45.80 ± 1.14** 50.35 ± 1.02** 40.16 ± 0.73 45.06 ± 0.54 42.95 ± 0.99 47.43 ± 0.86 44.13 ± 0.67* 48.52 ± 0.70* | 24.29 ± 1.00** 32.25 ± 0.93** 16.55 ± 0.73 24.49 ± 0.73 19.84 ± 0.73 27.30 ± 0.76 21.35 ± 1.04** 30.68 ± 0.77** |
Alloxan establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Oxygen free radicals exert their cytotoxic effects on membrane phospholipids resulting in the formation of MDA. As the secondary product of lipid peroxidation, MDA would reflect the degree of oxidation in the body. It is a three-carbon dialdehyde, and consists of lipid hydroperoxides. SOD is a scavenger of free radicals, which has important effects on the control of oxidation reactions in the body. The concentration of SOD in diabetes was significantly lower than that of normal (Wohaieb and Godin . 1987).The cause was probably decreased activity of SOD because higher blood glucose could combine with SOD (Fuliang et al., 2005). The level of SOD was increased and the concentration of MDA was decreased after OA treatment. This suggests that OA has effective anti-oxidative properties and could scavenge well excess free radicals and reduce the production of MDA. In our study, the results showed that the GSH-px activity was significantly increased in OA-treated diabetes group compared with diabetes control group. Our research suggests that OA possesses antidiabetic potential in alloxan-induced diabetic rats. Oxidative stress was involved in the early diabetic dysfunction that led to reduced activities of antioxidant enzymes. OA treatment recovered activities of antioxidant enzymes and improved liver and kidney function resultantl, which indicated that oleanolic acid was benefit to early diabetic rats due to its antioxidant property partly at least.
4. Antioxidant therapies of lactic acid bacteria on hypolipidemia
Pickled cabbage is popular Chinese traditional food. Our study was explored to characterize effects of lactic acid bacteria (LAB) isolated from the pickled cabbages on activities of antioxidant enzymes and hypolipidemia in normal and high fat diet mice. 28 LAB strains were isolated from pickled cabbage, and two strains with high acid tolerance and bile salt resistance were screened. The strains were identified to be
4.1. Strains isolation and identification
LAB strains were isolated using MRS broth from Chinese pickled cabbage that was bought from the Shandongpu market, and identified according to Gram stain positive and catalase test negative. The isolated LAB strains were stored in -80°C.
4.2. Determination of acid tolerance
The 28 strains were grown in MRS broth at 37°C overnight, and subcultured in 10 mL of fresh MRS broth adjusted to pH 3 with hydrochloric acid (3.0 M). The initial bacterial concentration was 106 cfu mL-1 and was checked by viable count determination on MRS as described above. Samples were incubated for 4h at 37°C. Cells were serially diluted 10-fold in phosphate buffer (0.1 M, pH 6.2) in order to neutralize the medium acidity. The residual viable count was determined by dilution and plate counting on MRS agar after 24-48 h of incubation. The survival rate was calculated as the percentage of colonies grown on MRS agar compared to the initial bacterial concentration.
4.3. Bile salt tolerance test
MRS broth was inoculated with 106 cfu mL-1 from overnight cultures. Growth in control (no bile) and test cultures (0.3% oxgall, Sigma Chemical Co., St. Louis, MO USA) was monitored and incubated for 4 h at 37°C. The strains were serially diluted 10-fold in phosphate buffer (0.1 M, pH 6.2) in order to dilute the medium bile salt. The residual viable count was determined by dilution and plate counting on MRS agar after 24﹣48 h of incubation. The survival rate was calculated as the percentage of colonies grown on MRS agar compared to the initial bacterial concentration. In the acid and bile salt tolerance tests, two LAB strains showed high acid tolerance and bile salt resistance. The strains were identified to be
4.4. Experimental design in vivo
Male ICR mice weighing 18–22 g were purchased from the Experimental Animal Center of China Academy of Military Medical Science (Beijing, China). Fifty-six mice were divided randomly into 7 groups: normal diet control group (NC), high fat diet control group (HFC), lab1 + normal diet group (lab1 + ND), lab1 + high fat diet group (lab1 + HFD), lab2 + normal diet group (lab2 + ND), lab2 + high fat diet group (lab2 + HFD) and mixed bacteria + high fat diet group (MB + HFD). The mice of the HFC, lab1+HFD, lab2+HFD and MB+HFD groups were fed with high fat diet 30 d continually to construct hyperlipidemic models. On the day 31, the living lab1 and lab2 suspensions were fed to the mice by ig. 28 d continually. Experiment groups and feeding treatments are shown in Table 7.
Group | Dosage(MI),Bacteria Number (cfu mL-1) | Experimental animal Quantity | Feeding method |
NC | 0.2, 2×109 | 8 | Normal diet |
HFC | 0.2, 2×109 | 8 | High fat diet |
lab1+DN | 0.2, 2×109 | 8 | Lab1 suspension gavage, normal diet |
lab2+ND | 0.2, 2×109 | 8 | Lab1 suspension gavage, high fat diet |
lab1+HFD | 0.2, 2×109 | 8 | Lab2 suspension gavage, normal diet |
lab2+HFD | 0.2, 2×109 | 8 | Lab2 suspension gavage, high fat diet |
MB+HFD | 0.2, 2×109 | 8 | Lab1+Lab2 suspension gavage, high fat diet |
4.5. Determination of tissue enzyme activities and blood lipid levels
On the day 59, the mice were killed under ether anesthesia, and blood samples were collected from eyepit of all the mice. Livers and kidneys of the mice were removed immediately, weighed and washed with cold physiological saline, and the 10% tissue homogenate was prepared using physiological saline by a cold glass homogenizer. The homogenate was centrifuged at 4000×g for 15 min at 4°C. The protein concentration of the tissues supernatant was determined by the DC Protein Assay Kit (Bio-Rad Labratories; Richmond, CA) based on the lowry colorimetric assay (Lowry
SOD plays a very important role in the balance between oxidation and antioxidation. It could eliminate superoxide free radicals and protect body cells against superoxide damage. Figure8 indicates that the decrease in the level of SOD was significant in the HFC group compared with NC group (
GSH-px is a key antioxidant enzyme catalyzing the reduction of peroxides to protect against oxidative tissue damage. Figure 9 shows effects of lactic acid bacteria on tissue GSH-px activities which demonstrate that there was significant difference between NC and HFC groups (
CAT is a main enzy me in the microbody of cells, which can oxygenolysis toxic components (Holmes and Masters 1978). The activities of CAT of liver and kidney tissues for the HFC group were different significantly compared with NC group (
Group | CAT (mmol/mg·pro) | |
Liver | Kidney | |
NC | 1.08 ± 0.18 | 1.07±0.24 |
HFC | 0.79± 0.15* | 0.85±0.15* |
lab1+DN | 0.92± 0.17 | 1.01±0.21 |
lab2+ND | 0.94 ±0.14 | 0.99±0.26 |
lab1+HFD | 0.97±0.14 | 0.94±0.14 |
lab2+HFD | 0.98± 0.12 | 0.99±0.21 |
MB+HFD | 0.93±0.15 | 1.01±0.27 |
The results of TC and TG levels of the experimental mice are presented in Table 9. The levels of TC and TG in lab1 + ND and lab2 + ND groups were slightly lower, but which were not significant compared with NC group. TC and TG levels of hyperlipidemic mice were higher than NC group, and difference was extremely significant (
Group | TC(mmol/L) | TG(mmol/L) | HDL-c(mmol/L) | LDL-c(mmol/L) |
NC | 2.76±0.11 | 2.35±0.25 | 1.88±0.18 | 3.81±0.18 |
HFC | 9.03±0.05 | 5.21±0.08 | 4.81±0.02 | 4.99±0.18 |
lab1+DN | 2.58±0.16++ | 2.27±0.15++ | 2.01±0.16 | 3.37±0.16 |
lab2+ND | 2.674±0.15++ | 2.31±0.24++ | 2.16±0.10 | 3.45±0.19 |
lab1+HFD | 7.12±0.14 | 3.86±0.10 | 4.98±0.14 | 4.19±0.26 |
lab2+HFD | 6.59±0.04++ | 3.25±0.04++ | 6.22±0.14+ | 3.93±0.32+ |
MB+HFD | 6.06±0.09++ | 3.02±0.01++ | 7.74±0.03++ | 3.71±0.19+ |
4.6. Analysis of Nrf2 expression in liver tissues
Single hepatocyte suspensions of the mice were prepared in ice-cold 0.1 M PBS with 0.1% sodium azide. Nrf2 antibodies (Santa Cruze biotechnology Inc., USA) were diluted in 0.1 M PBS with 0.1 NaN3. 106 cells were incubated with primary anti-Nrf2 antibodies for 30 min on ice. After two washes in 0.1 M PBS with 0.1% sodium azide, 0.5 µg of secondary FITC-conjugated rabbit anti-mouse antibody were added and incubated on ice for 30 min. Finally, the cells were resuspended in 1 mL of 0.1 M PBS with 0.1% sodium azide. The hepatocytes were scanned using a FACSCalibur (Becton-Dickinson, USA), and fluorescence of Nrf2 positive cells was quantified. Nonspecific binding of secondary antibody was excluded by incubating the cells only with the FITC-labelled secondary antibody, and the experiment was repeated three times. The software used was BD CellQuest Pro (Becton Dickinson Biosciences, USA) and the data were expressed as fluorescence intensity formula (
Group | Nrf2 expression fluorescence intensity |
NC | 219.86±3.42 |
HFC | 144.67±6.82 |
lab1+ND | 211.88±3.15* |
lab2+ND | 285.56±2.49* |
lab1+HFD | 200.27±4.00+ |
lab2+HFD | 275.62±2.16+ |
MB+HFD | 286.25±2.93+ |
Single-colour histograms represent hepatocyte staining with anti-Nrf2 antibodies; x-axis, DTAF flurescence intensity; y-axis, frequency of cells displaying certain fluorescence intensity. A was normal control group; B was lab1 treated normal mice; C was lab2 -treated normal mice; D was lab+lab2
Probiotics are commonly used as viable microbial feed supplements that affect the host animal by improving its intestinal microbial “balance” (Holzapfel, et al., 1998). Several studies demonstrated that some lactobacilli possess antioxidative activity, and could decrease the risk of accumulation of reactive oxygen species during the ingestion of food (Ito, et al., 2003; Kuda et al., 2010). However, probiotic bacteria must be resistant to the acidity of the stomach, lysozyme, bile, pancreatic enzymes. High acidity in the stomach and high concentration of bile components in the proximal intestine are the first host factors, which affect strain selection and adhesion. In our study, two high acid tolerance and bile salt resistance strains were screened, which were
respectively. Levels of serum TC, TG and LDL-c were slightly decreased and HDL-c level was a little higher by the LAB suspension treating on the normal mice. However, compared to the HFC group, levels of TC and TG were decreased extremely in lab2 + HFD and MB + HFD groups. The finding indicates that the two strains might decrease the risks for cardiovascular and arteriosclerosis diseases in various degrees, and it also could fall significantly the cholesterol level in hyperlipidemic mice. Akalin et al. (1997) found that consumption of acidophilus yogurt significantly lowered the values for plasma TC, LDL-c in the mice. After the male SD rats were fed high-fat diet with
Several studies have documented the relationships between increase of free radicals and blood glucose, lipid peroxidation as well as low-density lipoprotein (Tanaka et al., 2002). High fat diet could be used to induce significant oxygen-centered free radicals and ROS generation in the mice. The liver plays a central role in the maintenance of systemic lipid homeostasis and it is especially susceptible to reactive oxygen species (ROSs) damage (Hamelet et al., 2007). Free radicals can diffuse intracellularly and result in mitochondrial enzyme damage and DNA breaks, impair cellular function (Bonnefont-Rousselot et al., 2000). SOD is a scavenger of free radicals, which has important effects on control of oxidation reactions in the body. Some LAB may enhance SOD and GSH-px activities and prevent oxidative damage (Tsai, et al., 2009). GSH is often regarded as an antioxidant agent, since it protects protein –SH groups against oxidation and can scavenge oxygen radicals and some other reactive species. It reduces different oxidants after increasing its hydrogen atom. This reaction is catalysed by enzyme GSH-px in cells (Reiter, 1995). In the research, the concentration of SOD and GSH-px in the high fat diet mice was significantly lower than those of the normal rats. Meanwhile, the activities of SOD and GSH-px were increased in various degrees in Lab1 + ND and lab2 + ND groups compared with the normal control mice after the LAB administering. The levels of SOD and GSH-px in the lab1 + HFD, lab2 + HFD and MB + HFD groups were all increased compared with HFC group, but not achieve the level of the NC group. In this study, the levels of antioxidant enzymes and the function of reducing blood lipid in MB + HFD groups were higher than the other two hyperlipidemic groups. SOD is the most important survival protein and ubiquitously induced antioxidant by various stimulants, so we hypothesized that the strains might play a more crucial role in a severe stressful condition. At the same time, supplementation of Lab also promoted expression of Nrf2 in the liver tissues of the mice. Nrf2 serves as master regular of a cellular defense system against oxidative stress (Motohashi et al., 2004; Nguyen et al., 2004). Under physiological conditions, Nrf2 is sequestered in the cytoplasm by Keap1, which facilitates its ubiquitination and proteasomic degradation (Kang, et al., 2004)Upon exposure to oxidative stress, the sequestration complex breaks down and the dissociated Nrf2 translocates into the nucleus, where it binds to cis-acting antioxidant response elements (AREs) and promotes the transcription of a large number of cytoprotective genes (Kensler et al., 2007; Vries et al., 2008). Nrf2–ARE signaling is also known to be mainly responsible for the upregulation of SOD and GSH-px gene expression and hence constitutes a crucial cellular response to environmental stresses (Surh et al., 2009). L.plantarum is further capable of activation of Nrf2 and preventing HFD-induced inhibition of antioxidant enzymes. Our Flow cytometry data clearly show that high fat diet-induced oxidative stress is associated with activation of Nrf2, as evidenced by a significant elevation of Nrf2 in the nuclear fractions (P<0.05). Supplementation of L.plantarum markedly promoted further translocation of Nrf2 into the nucleus. Thus, L.plantarum could inhibit HFD-induced oxidative stress through the Nrf2-Keap1 signaling pathway. As expected, L. plantarum isolated from fermented cabbage upregulates antioxidative enzymes in high fat diet mice via Nrf2-dependent transcriptional activation of ARE sites. The upregulation of several antioxidative enzymes is associated with the reduced formation of ROS and enhanced survival of liver cells upon the induction of oxidative stress.
Acknowledgement
This work was financially supported by the research grant from the Chinese Ministry of Education Doctor Degree (No. 20101333120011), a grant from Hebei Province Natural Science Fund (No. C2011203137, 11965152D), and a Chinese Postdoctoral grant (480013).
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