Study Of Antioxidants On Oxidative Stress Damage In Central Nervous System In Obstructive Jaundice Rats

Background: When the biliary system is obstructed, the excretion of bile will be restrained. Then bilirubin can reflux into the blood, causing jaundice, known as obstructive jaundice. When the bile duct is blocked, serum bilirubin and total bile acid can increase significantly, meanwhile serum alkaline phosphatase, gamma glutamyl transferase, glutamic pyruvic transaminase can also increase significantly.Because of its lipophilic characteristics, bilirubin tends to bind with tissues which are rich in lipid and can cause the tissue and cell damage. Besides of adipose tissue, the central nervous system is abundant in lipid. Therefore, indirect bilirubin makes the largest injury on the central nervous system.Bilirubin concentration can interfere with the normal metabolism of brain cells which makes brain cells in oxidative stress and produces much more oxygen free radicals; It can downregulate concentration of closed protein in the brain, causing damage of blood brain barrier, at the same time, the expression of caveolin-1(blood brain barrier damage related) and β-catenin in brain tissue can increase to mitigate the damage of bilirubin on BBB(blood brain barrier). In addition, the damage of bilirubin on nervous system can also produce a lot of small molecules such as oxygen free radical and lipid peroxide which can consume amount of antioxidant enzyme and make the highly reactive molecules deposite in vivo, unbalance the oxidation system and antioxidation system, and finally produce oxidative stress injury on the tissue cells. Oxidative stress injury had much more damage to the biological membrane. And when biological membrane was damaged seriously, a lot of calcium ions will flow into the cells which makes the intracellular Ca2+ overload and causes cell dysfunction or loss. On the basis of activated microglia, bilirubin will initiat inflammatory responses, leading to a lack ofneurotrophic factor, which can eventually lead to neuronal degeneration and death. Chroni E’s study found that obstructive jaundice could cause the increasing or decreasing of direct and indirect oxidative stress markers in the rat’s brain and he found a correlation between brain oxidative stress and obstructive jaundice in rats which suggests that similar pathogenetic mechanisms may play a key role in cholestatic liver disease which ultimately lead to hepatic encephalopathy. This had been confirmed by testing oxidative stress in early and later stage of obstructive jaundice. He also pointed out that oxidative stress was associated with jaundice and oxidative stress was an important mechanism of hepatic encephalopathy induced by bilirubin. The studies on oxidative stress in recent years showed that the Keap1-Nrf2-ARE pathway played an important role in anti-oxidative damage, which could maintain the intracellular redox balance and protect cells from oxidative glutamate toxicity and apoptosis induced by hydrogen peroxide.NMDA(N-methyl-D-aspartate) receptor is a kind of glutamate receptor in the central nervous system ionic which mainly concentrates in the postsynaptic membrane and involves in excitatory synaptic transmission with ligand double door control voltage characteristics and high permeability of Ca2+. Excessive activation of NMDAR can induce excitotoxicity which can finally leading to neuronal cell death. Oxygen free radicals can promote the release of glutamate in neurons and neurogliocytes, and at the same time, it can also inhibit the reuptake of glutamate which can activate NMDA receptor by Ca2+ activated phospholipase A and produce oxygen free radicals which finally generate a vicious spiral. Dizocilpinemaleate(MK-801) is a potent non-competitive antagonist of NMDA receptor which can easily pass the blood brain barrier and act on the site of phencylidine in NMDA receptor channel and then make allosteric regulation. It can effectively block the binding of glutamate and postsynaptic membrane receptor and block the activation of Ca2+ channel coupled with NMDA receptor so as to reduce the Ca2+ internal flow, and finally the function of NMDA receptor is weakened to reduce the toxicity of glutamic acid. Numerous studies have confirmed thatMK-801 has a protective effect on neuronal injury caused by various reasons such as poisoning, ischemia and anoxia, hepatic encephalopathy.Resveratrol is a kind a kind of polyphenol flavonoids which can be found in grapes and red wine. Studies both in vivo and in vitro have confirmed its biological and pharmaceutical activity effect such as resisting cell DNA damage due to free radicals in the body and preventing membrane lipid peroxidation and so on which had been confirmed in cardiovascular diseases, diabetes, kidney diseases et al. Experiments have found that its pharmacological effects were produced at least in part by inducing the expression of HO-1, which could protect the cells gainst oxidative stress damage. But there is still no studies about its effect on central nervous system when obstructive jaundice occurs.In this experiment, we first established the animal model of obstructive jaundice and then we further explored whether NMDA receptor antagonists had protective effects on the brain oxidative stress injury caused by obstructive jaundice and what the mechanism was by measuring the indexes below: superoxide dismutase(SOD), catalase(CAT), malondialdehyde(MDA), Nrf2(transcription factor NF-E2-related factor 2, nuclear factor E2 related factor 2), heme oxygenase-1(heineoxygenase-1, HO-1) and Nfr2 m RNA. The third part of this manuscript evaluated the antioxidative effecct of resveratrol though MDA, SOD and HO-1 in the central nervous system in obstructive jaundice rats. 、Part I The effects of MK-801 on central nervous antioxidative stresssystem in rats with obstructive jaundiceObjective: the aim of this study was to establish the rat model of obstructive jaundice and investigate the effects of MK-801 on antioxidant system activity in the central nervous system of rats with obstructive jaundice.Methods: Twenty rats were divided into four groups: group I(sham operation group), group II(control group), group III(low dose group) and group IV(high dose group). Group II, III and IV are the obstructive jaundice model group. In order to continiue other parts of our study, from the secondday, group III were processed intraperitoneal injection of MK-801 0.025mg/kg.d and group IV were processed intraperitoneal injection of MK-801, 0.25mg/kg.d. Meanwhile, group I and group II were injected the same volume of normal saline everyday for 10 days. Three days after the operation, rats’ tail vein blood was collected for examining the total bilirubin and direct bilirubin by automatic biochemistry meter in order to determine whether the model was successfully established. And MDA, CAT, total superoxide dismutase(T-SOD) and total antioxidant capacity(T-AOC) were determined on the 10 th day to evaluate the oxdative status of the rats.Results:1 The day after the operation, the rats of sham operation restored normal diet and free activity, and the activity was sensitive while the pee of the rats in this group was light yellow. Two days after the operation, the model of obstructive jaundice began to appearing yellow dye in thin parts of the skin such as the tip of the tail and ears. Gradually yellow dye of the whole body appeared while the urine turned to deep yellow, and the rats showed slow response. Three days after the operation, the concentration of direct bilirubin and total bile acid(μ mol/L) in the rats brain tissues of control group, MK-801 low dose group and high dose group were significantly increased compared with the sham operation group, and there was statistical significance(P<0.01) which meant obstructive jaundice model was established successfully.2 The content of malondialdehyde in control group, MK-801 low dose group and MK-801 high dose group were significantly increased than the sham operation group, and there was statistical difference(P<0.05). The content of MDA decreased in MK-801 groups compared with the control group(P<0.05).3 Compared with the sham operation group, the activity of catalase in control group decreased significantly. The activity of catalase in the MK-801 groups increased compared with the control group with significant difference(P<0.05). There is no statistical difference on the activity of catalase between MK-801 low dose group and high dose group(P>0.05).4 Compared with sham operation group, the activity of total superoxide dismutase were decreased significantly in control group with statistical significance. The activity of total superoxide dismutase were increased in the MK-801 groups compared with control group with significant difference(P<0.05). But there is no statistical significance(P<0.05) on total superoxide dismutase activity between the low dose and high dose group were compared.5 In the Oj groups, the total antioxidant capacity were significantly increased compared with the sham operation group, and there was statistical significance(P<0.05). The total antioxidant capacity in MK-801 groups was increased compared with the control group with statistical significance(P<0.05). But there was no statistical difference between the MK-801 groups.Conclusions:1 By ligation the common bile duct we can successfully establish the animal model of obstructive jaundice. Oxidative stress exists when obstructive jaundice occurs, and obstructive jaundice can aggravate the oxidative stress damage in the rats’ CNS and cause increasing of the catalase expression which enhances antioxidant capacity of the whole body. This is consistent with the current research at home and abroad.2 Low dose of MK-801 can increase activity of SOD but have no effect on the total antioxidant activity of rat central nervous system. High dose of MK-801 could enhance the capacity of anti-oxidative stress in obstructive jaundice rat CNS by increasing some antioxidant except CAT. More experiments are needed to explore the indexes which can reflect the antioxidative effect of MK-801.3 Lipid peroxidation in central nervous system of obstructive jaundice rats failed to be reduced by MK-801, but we still need some other clear indexes to evaluate the effect of MK-801 when oxidative stress injury occurred in central nervous system of rats with obstructive jaundice.Part II investigation of the role of keap-Nrf2-ARE signaling pathway in obstructive jaundice rats CNS when oxidative strass injury occurredObjective: To investigate the role of Keap1-Nrf2-ARE signalingpathway in CNS of obstructive jaundice rats when oxidative stress injuryoccurred and explore the effect of NMDA receptor blocker on the activationof transcription factor NF-E2 associated factor 2(Nrf2) in the process ofoxidative stress damage in obstructive jaundice rats CNS.Methods: The rats were divided into four groups: group I(shamoperation group), group II(control group), group III(low dose group) andgroup IV(high dose group). Group II, III and IV are the obstructive jaundicemodel group. From the second day, group III were processed intraperitonealinjection of MK-801 0.025mg/kg.d and group IV were processedintraperitoneal injection of MK-801,0.25mg/kg.d which continued for 10 days.Meanwhile, group I and group II were injected the same volume of normalsaline everyday for 10 days. Then the Nrf2, Ho-1 and NQO1 protein weredetermind by Wester blot and Nrf2 m RNA by RT-PCT method.Results:1 Compared with the sham operation group, expression of Nrf2 m RNAwas increased in the OJ groups, and there was statistical significance(P<0.05).Compared with the control group, the expression of Nrf2 m RNA wasincreased in the MK-801 group, and there was statistical significance, P<0.05.Compared with the low dose group, the expression of Nrf2 m RNA wasdecreased in the high dose group, but without statistical significance, P>0.05.2 Compared with the sham operation group, expression of Nrf2 proteinwas increased in the OJ groups, and there was statistical significance(P<0.05).Compared with the control group, the expression of Nrf2 protein wasincreased in the MK-801 group, and there was statistical significance, P<0.05.Compared with the low dose group, the expression of Nrf2 protein wasdecreased in the high dose group with statistical significance, P>0.05.3 Compared with the sham operation group, expression of HO-1 proteinwas increased in the OJ groups, and there was statistical significance(P<0.05). Compared with the control group, the expression of HO-1 protein was Part II investigating of the role of Keap1-Nrf2-ARE signaling pathway in obstructive jaundice rats CNS when oxidative stress injuryincreased in the MK-801 group, and there was statistical significance, P<0.05. Compared with the low dose group, the expression of HO-1 protein was decreased in the high dose group with statistical significance, P<0.05.4 Compared with the sham operation group, expression of NQO1 protein was increased in the OJ groups, and there was statistical significance(P<0.05). Compared with the control group, the expression of NQO1 protein was increased in the MK-801 group, and there was statistical significance, P<0.05. Compared with the low dose group, the expression of NQO1 protein was decreased in the high dose group with statistical significance, P>0.05Conclusions:1 The experiments proved the presence of oxidative stress in obstructive jaundice rats once again.2 The body upregulated the expression of HO-1 and NQO1 and Nrf2 protein through the Keap1-Nrf2-ARE signaling pathway and enhanced the antioxidative ability of the body.3 NMDA receptor antagonist MK-801 upregulated the expression of II-phase detoxification enzymes HO-1 and NQO1 through Keap1-Nrf2-ARE signaling pathway, and thus exert its neuroprotective effect. However, high doses of MK-801 made expression Nrf2 m RNA, Nrf2 protein, HO-1 and NQO1 protein decreased compared with the low dose group, which may due to the toxicity of MK-801. Part III Effects of resveratrol on oxidative damage in central nervoussysterm of rats with obstructive jaundiceObjective: To study the effect of different levels of resveratrol on oxidative damage in central nervous systerm of rats with obstructive jaundice.Method: 32 female SD rats of 8 weeks old were used as experimental object. The animals were randomly divided into four groups and feed seperately, 8 rats in each group. Group A was sham operation group(the common bile duct was seperated without ligation) while B, C, D groups were obstructive jaundice group. Groups C and D were treated with different levels of resveratrol. On the the 14 th day after operation, blood were tested for TBIL, DBIL, ALT and AST. And cerebral cortex specimen were collected, then malondialdehyde, superoxidedismutase activity and HO-1 protein expression in the rats brain of the four groups were measured.Result: The levels of TBIL and DBIL suggested obstructive jaundice model was estabilshed successfully. In the OJ groups, levels of ALT, AST, MDA were increased while levels of SOD and HO-1 were decreased when compared with Group I. In the OJ groups, levels of ALT, AST which reflected the liver function and MDA had a II< III<IV trend while levels of SOD and HO-1 which reflects the oxidative stress had a II >III >IV trend). Different doses of resveratrol had different effects on ALT、AST、MDA、SOD、HO-1 with statisticl significance.(P < 0.05).Conclusion: Resveratrol have little effect on TBIL and DBIL of obstrctive jaundice rats, but it can protect the liver function, and it has antioxidant properties of decreasing MDA and incresing SOD, HO-1 levels in the CNS of obstructive jaundiced rats.