SIGNALING PATHWAYS REGULATED BY BRASSICACEAE EXTRACT INHIBIT THE FORMATION OF ADVANCED GLYCATED END PRODUCTS IN RAT BRAIN.

BACKGROUND: The goal of this study was identification signaling molecules mediated the formation of AGEs in brain of rats injected with CdCl2 and the role of camel whey proteins and Brassicaceae extract on formation of AGEs in brain.
METHODS: Ninety male rats were randomly grouped into five groups; Normal control (GpI) and the other rats (groups II-V) were received a single dose of cadmium chloride i.p (5 μg/kg/b.w) for induction of neurodegeneration. Rats in groups III-V were treated daily with whey protein (1g/kg b.w) or Brassicaceae extract (1mg/kg b.w) or combined respectively for 12 weeks.
RESULTS: It was found that whey protein combined with Brassicaceae extract prevented the formation of AGEs and enhance the antioxidant activity compared with untreated group (p <0.001). Serum tumor necrosis factor (TNF-α) and interleukine (IL-6) levels were significantly decreased (p<0.01) in rats treated with whey protein and Brassicaceae extract formation compared with untreated. The combined treatment showed a better impact than individual ones (p<0.001). The level of cAMP but not cGMP were lowered in combined treatment than individual (p<0.01).
CONCLUSION: It can be postulated that Whey protein + Brassicaceae extract formation could have potential benefits in the prevention of the onset and progression of neuropathy in patients.

Introduction

Signal transduction is involved in cell function [Agrawal etal., 2005], development [etal.,2003], differentiation [Alberti etal.,1998], apoptosis and cell death. Signaling molecules including hormones, neurotransmitters and growth factors involve binding to target receptors initiates a process of cascade and finally response [AL-Lawati etal.,2002]. The amplified signal is then propagated to the nucleus, resulting in induction or repression of gene expression [Ashour etal.,1999]. Oxidative damage are often related with path physiology of many diseases [Badr etal.,2011]. Signaling pathways including protein dependent kinases (PK) and secondary messenger as cAMP, cGMP, IP3 and calcium. Identification of these mediators are important rational of identification of pathogenesis and therapeutic protocol [Beg etal.,1986]

Cadmium (Cd) is a toxic heavy metal, considered as a human carcinogen by National Toxicology Agency [Floweler,2009]. Cadmium (Cd) has a long half-life due to its low rate of elimination from the body. Thus, long-term exposure to Cd will lead to toxicity due to its accumulation in different tissues as brains and central nervous system (CNS). Cd can be taken by inhalation and cross the blood brain barrier (BBB) permeability [Satarug etal., 2010]. The previous study indicated the hepatic-toxic effect of Cd that was associated with oxidative stress and tissue damage. The primary neurodegenerative diseases lead to dementia and loss of concentration [Renugadevj and Miltonprabu,2009] In neurodegenerative disease, the early stages, characterized by his inability to acquire new memories and difficulty in recalling When late stage, it is diagnosed with behavior assessments. The symptoms of late stage include confusion, mood swings, language breakdown, long-term memory loss, [Eldemerdash etal.,2004]. loss of body functions, leading to death.

Although the mechanism of Cadmium is not yet been well documented as a result of the pathophysiology of Cadmium, several factors are reported to play a role as activation of NF-κB [Ebaid etal.,2005]. Activation of the renin-angiotensin system, acceleration of oxidative stress, activation of specific kinase and subsequent formation of advanced glycation end products (AGEs) are major risk factors. As a result of the injection with CdCl2, increased overproduction reactive oxygen species (ROS). These ROS results in the glycation of proteins which enhance the formation of AGE which accumulated in different tissues causing its damage. Previous studies reported that camel milk have beneficial role in Cadmium toxicity and many other diseases. Therefore, using of camel milk is a useful addition to the insulin used for the treatment of type I diabetes [Eeg etal.,2008]. Whey protein is believed to be the best available protein, when compared to other proteins. Due to its regulatory effect on the key enzymes that regulate glucose and glycogen metabolism. In addition to the fact that, whey protein contains all amino acids that are necessary for cell growth [Floyed etal.,1970]. Brassicaceae extract was traditionally used in treatment of colic disease, diarrhea, sedative (13).

The potential anti oxidant activity of Brassicaceae extract are due to their content of phenolic compounds (14). It was reported that water extracts of functional foods proved to have a potent antioxidant function. It was proved that, a correlation between serum levels of Brassicaceae extract and metabolic disease as diabetes. The signaling pathway is missing for this mechanism.

The aim of this study was identification of signaling molecules including secondary messenger as cAMP, cGMP. Secondly, evaluate the role of camel whey proteins and Brassicaceae extract on the signals mediated the formation of AGEs in CdCL2 induced neurodegeneration and the mechanism of its action in an experimental rat model.

Materials and Methods

Nine week-old (120±20g) male Albino rats (n=90) will be obtained from KFMRC, King Abdulaziz University (Jeddah). The animals will be housed in cages and foods and tap water were given ad libitum. Rats will be randomly grouped into control (Group I) (n=15), received a single dose of 0.1 mol/L citrate buffer. And the other rats (groups 2-5, 75 rats) were received a single dose of injected with CdCl2 i.p, at dose of 5 μg/kg. Rats in group II were considered diabetic untreated. Rats in groups III-V were treated daily with whey protein (1g/ kg b.w) or Brassicaceae extract (1 mg/kg b.w) or combined for 6 weeks. Animals were fasted overnight; blood was collected directly from the heart. Serum was used for the determination of glucose, glycated hemoglobin, advanced glycated end products (AGES), malondialdhyde, superoxide dismutase, total antioxidant activity, urea and creatinine, inflammatory cytokines as tumor necrosis factor (TNF-α) and interleukine (IL-6) levels will be determined by using ELISA kit.

Biochemical analysis in brain tissues

Brain tissue (0.1g) was homogenized in 2 ml phosphate buffer (pH 7.3) contain protease inhibitor. Samples were centrifuged at 12,000 rpm/10 minutes at 5°C. The filtrate was used for the determination of malondialldhyde (MDA), reduced glutathione (GSH), catalase, superoxide dismutase by using commercial kits from BIORAD (England), tumor necrosis factor, interleukine-1, and advanced glycated end products (AGEs) levels by using ELISA kits. Protein levels was determined by Foline reagent using a standard curve [Joseph etal.,1999]. The levels of IL-6 and TNF-α were determined by ELISA using the commercially available kits according to the instructions of the manufacturer. Signaling molecules including secondary messenger as cAMP, cGMP were determined.

Assay of cAMP and cGMP.

Guanosine 3,5-cyclic monophosphate (cGMP) and cAMP in tissues were determined by direct immunoassay Kit with sensitivity, 1~100 fmol /ug protein per assay, than the cGMP Direct Immunoassay Kit, from antibody Biovision company. The level expressed as fmol of cAMP while for cGMP as pmol quantities.

Statistical analysis

Statistical analysis will be done by SPSS using student’s t-test. Value of P < 0.05 was considered as statistically significant. ANOVA test will be performed to correlate between different groups.

Results

Administration of rats with CdCl2 resulted in a significant increase in blood glucose levels in the diabetic group compared with the control group (p<0.001), while treatment with whey protein or Brassicaceae extract or combined resulted in a significance decrease in blood glucose compared with the untreated diabetic animals (p<0.05, table 1). As a result of diabetes, HbA1c was significantly increased (p<0.05) in the diabetic untreated group. Treatment of animals with Whey protein + Brassicaceae extract improves the two parameters (table 1). The Whey protein + Brassicaceae extract exert hypoglycemic action but less potent than insulin (p<0.05). The antioxidant activities GSH reduced, catalase, SOD in brain of diabetic animals were significantly reduced as a result of CdCl2 injection. Supplementation of whey protein or Brassicaceae extract or combination resulted in a significant elevation in GSH (p<0.05 for each) level and the activities of catalase and SOD (p<0.001, <0.01 and <0.05) respectively (table 1). Administration of combination resulted in better decrease of MDA levels than individual ones (table 2).

Table 1

body weight, glucose and HbAlc levels in different groups.

	Group 1	Group II	Group III	Group IV	Group V
Initial body weight (gm)	Group 1 120.13±9.5	Group II 125.44±7	Group III 129.19±6	Group IV 122±7.2	Group V 131.33±6.5
Final body weight (gm)	199.5±8.2	150.8±7.2[a]	167.3±16.5[ab]	165.52±8.9[ab]	210±12.3[a],[b]
Glucose (mg/dl)	92.78±0.45	275.35±1.45[a]	145.92±1.4[ab]	137.71±1.23[b]	102.21±0.57[a],[b]
HbA1c (%)	5.54±0.41	8.42±0.34[a]	6.8±0.54[b]	6.8±0.38[b]	5. 8±0.52[b]

p value significant versus control.

p value significant versus diabetic.

Table 2

Serum and brain malondialdhyde (MDA) level and reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) activity in brain of different groups (Mean ±S.D)

Groups	Serum MDA (μ mol /L)	Brain
		MDA μmol/mg protein	GSH μg /mg protein	Catalase U/mg protein	SOD U/mg protein
Group I (n = 15) RangeMean ±S.D.	24 − 15.675 ± 12	98-19666.7 ± 28	104 − 546284 ±52	992-23491249 +140	956 − 26231545 +191
Group II (n= 12) RangeMean ±S.E.p* value	40- 8948 ± 5.9< 0.01	190-261188 ± 37N. S.	68.7-221.5123.6 ±19.3< 0.01	109.5 − 642358 ±65<0.001	453-1342760±78<0.001
Group III (n = 13) RangeMean ± S.E.p* value	33 -5631±12.2< 0.01	149 − 213142 ± 32<0.001	121 − 496259 ±50<0.001	706 − 2002843 ±134<0.001	876 -19871021±163<0.001
Group IV (n= 13) RangeMean ±S.E.p** value	16 − 73.433 ±2.7< 0.001	136-261133± 25<0.001	69.6-213.9128±16.4p< 0.05	456 -1632987 ±56<0.001	962-1316836±53<0.001
Group V (n= 14) RangeMean ±S.E.p** value	22 − 73.438 ±6.7< 0.001	156-261121± 25N.S.	69.6-213.9128 ± 16.4p< 0.05	546 -17561112 ±86<0.001	624-21161233±153<0.001

P value, all groups vs control

P value, treated Vs untreated

Figures (1, 2) showed that, serum aldolase reductase and total antioxidant activities were significantly decreased in diabetic arts compared with control ones (p<0.01 and 0.001) respectively. Whey protein or Brassicaceae extract or combined showed an elevation in these activities with dose dependent. The enhancement in insulin injection is better than combination (p<0.05).

Figure 1

Serum aldose reductase activity in all studied groups (Mean ±SD).

Figure 2

Serum total antioxidant activity in all studied groups (Mean ±SD).

Fig.3 showed that, both TNFa and IL-1 play an important role in the pathogenesis of diabetic neuropathy. As a result of CdCl2 injection the brain levels of TNF-α and IL-1 were significantly elevated (p<0.001) for each indicating a considerable level of inflammation compared with the normal control rats. Administration of whey protein or Brassicaceae extract or combined results in a significant reduction of these elevated levels (p<0.01. Whey protein + Brassicaceae extract supplementation resulted in a significant reduction of AGEs in a dose dependent manner as indicated in Figure 3. Combination effect attenuated AGE production compared with individual treatment (p<0.001).

Figure 3

Brain tumor necrosis factor (TNF-α), interleukine-1, and advanced glycated end products (AGEs) levels

Brain cAMP and cGMP levels (Mean ± SE)

Discussion

Whey protein showed in suppression of nuclear factor kappa B (NF-κB) signaling, which is activated by TNF-α or the receptor activator of NF-κB ligand. The present study demonstrated that the interference with the overproduction of ROS by Whey protein + Brassicaceae extract in the CdCl2 injected rats. Whey protein + Brassicaceae extract reduced normalized downstream effectors of vascular response to injury. In addition, the reduction of free radicals overproduction suggest an indirect AGE-inhibiting effect of Combination treatment. Our data suggested that part of the beneficial effect of Combination includes the disruption of the detrimental AGE-RAGE-NFκB pathways. It was found that whey protein effectively protect brain from CdCl2-mediated protein glycation in vitro. Recent investigations suggested that Brassicaceae extract is an anti-inflammatory agent used frequently in Japanese traditional medicine, are also AGEs inhibitors [Ganong,1997]. This is in accordance of our results which found that, Whey protein + Brassicaceae extract prevent the formation of AGEs and the inhibition neurodegenerations.

In the present study combined treatment with Whey protein + Brassicaceae extract have a good glycemic index control than individual one. This is reflected by increase body weight of treated diabetic rats compared with untreated.

The antioxidant capacity of combined treatment elevated significantly compared with untreated. This indicate the efficacy of these compounds in prevention of ROS production. The antioxidant property of whey protein [Hsu et al., 2006] and Brassicaceae extract [Kar and Mishra,1976] explained their role in improving antioxidant capacity and suppress ROS generation in diabetic rats [Lands etal.,1999 and Hamadeh,2000].

The elevation of cGMP level in CdCl2 rats mediated the formation of AGEs was indicated its role in diabetic neuropathy. Combination lowered cGMP level but not cAMP level that indicate mediator specificity in its action. The present study clearly demonstrates that controlling CdCl2 and catalytic reactive oxygen scavenging are effective approach for the prevention of diabetic retinopathy. Combination whey protein + Brassicaceae extract treatment is a paradigm natural food supplement with a broad spectrum of beneficial biochemical and cell biological effects, based on its ability to reduce the CdCl2 -induced ROS overproduction. Since Whey protein + Brassicaceae extract has beneficial effects on other target tissues of diabetic angiopathy, this concept appears attractive for the prevention or delay of diabetic angiopathy. In conclusion, it can be postulated that Whey protein + Brassicaceae extract could have potential benefits in the prevention of the onset and progression of neuropathy in diabetic patients.