| Biological transformation of chemical substances normally undergoes two metabolizing phases called phase 1 and phase 2 metabolism in the liver.During the phase 1 metabolism, oxides and toxins are inactivated through direct reaction with small molecules such as GSH, glucuronic acid, vitamin E,etc. to reduce the harm to the body. This reaction generally is fast. During the phase 2 metabolism, the polarity of endogenous small molecules such as glucuronic acid, glutathione (GSH), etc. covalently bound to external harmful substances or metabolic compounds in phase 1 to make them detoxifed or excretion by phase 2 metabolismenzymes. Those phase 2 metabolismenzymes are catalase (CAT), glutathione-S-transferase (GST), epoxide hydrolase, sulfotransferase, aldehyde aminotransferase (scetyltransferase), quinone oxidoreductasel (NQO1). They can eliminate the harmful substances which can functionally modify DNA or critical proteins resulting in diseases in order to maintain a stable internal environment. Therefore, induction of the phase 2 metabolism genes expression can enhance the antioxidation, anti-mutation and anti-tumor capabilities. The promoter region of the phase 2 enzyme has a specific DNA sequence called antioxidant response element (ARE), which can be induced and activated by chemopreventive agents, oxidative stress or electronic materials. Nrf2 (nuclear factor E2-related factor 2), a transcription factor homologous to basic leucine zipper (bZIP) protein family, is of the strongest activity in CNC transcription factor family, which includes p45, Nrf1, Nrf2, Nrf3. It mainly exists in metabolic and detoxifying tissues, such as liver, kidney, and other organizations continuously exposed to the environment, such as skin, lung, digestive tract, etc. Nrf2 is an important transcription factor in response to oxidative stress. Nrf2 plays an important role in oxidative stress through interaction with antioxidant response element. Nrf2 protects cells from oxidative damage and maintains cellular redox balance through inducing the expression of phase 2 metabolismenzymes. In physiological state, Nrf2 is in the cytoplasm binded with Keap1 (Kelch-like ECH-associated protein 1), which plays an active role in regulation, by directing it for proteosomal degradation. Keap1 is a polypeptide, which binds to cytoplasmic actin, contains 624 amino acids and includes five regions (NTR, BTB/POZ, IVR, DGR and CTR). DGR area (double glycine repeat) which contains six pairs of glycine repeats, is the binding site of Keap1 and Neh2, and is also the binding site of Keap1 and actin in Cytoplasm. The function of BTB/POZ region is mediating the dimer conformation of Keap1 and is related with Nrf2-Keap1 binding. The Ser-104 mutations of BTB/POZ district affects Keap1 dimerization and interferes with the function of Keap1 anchoring Nrf2. Keap1 and electrophilic compounds or oxidants react in the area of IVR, which is rich in cysteines. IVR zone takes part in the formation of ubiquitin ligase, so it is related with Nrf2 stability. In the reactive oxygen species (ROS) or other nucleophilic agents signal stimulation, Keap1 or DGR of Keap1 changes, then Nrf2 dissociates out from Keap1. Nrf2 is activated and transfers into the nucleus, combines with other protein Maf, JunD, cJun, ATF4, to form a stable heterodimer. Then it binds to other bZIP proteins to form stable heterodimer. After that, the heterodimer binds to the sites GCTGAGTCA on the ARE(antioxidant responsive element) to regulate the downstream phase 2 gene expression and increase cell resistance to oxidative stress. Thus the Keapl-Nrf2-ARE pathway plays a key role in the gene expression of phase 2 detoxification enzymes. Present studies have confirmed that Keapl-Nrf2-ARE pathway plays a wide range role in cell protection including anti-tumor, anti-stress, regulation of GSH synthesis, anti-apoptosis, anti-inflammatory response, anti-atherosclerosis, regulation of cardiovascular response and neuro-protection. Keapl-Nrf2-ARE pathway is an essential part of the defense mechanism of resistance to environmental stress and endogenous stress. Nrf2 activation barrier or deletion can lead to several common pathological conditions:including the formation of tumors induced by chemical susceptibility, drug-induced acute liver toxicity, as well as increased sensitivity to foreign compounds and UV. Currently Keap1-Nrf2-ARE pathway inducers contain michael reaction acceptor class, biphenyl-fen class, benzoquinones isothiocyanates, peroxidase class, mercaptans, trivalent arsenic species and heavy metals.Ozone (O3) is the allotrope of oxygen, consisting of three oxygen atoms. Under the conditions of normal temperature and pressure, O3 will transform into oxygen.The half-life period is 45 minutes. Medical ozone is mixture gas with major oxygen and minor ozone made by ozone concentration well-controlled ozone generator and considered as an new kind of gas medicine. Medical ozone can be dissolved in water, saturated in 5 minutes, and its solubility increases with higher medical ozone concentration. The half-life of medical ozone in water is 110 hours at 5℃. Medical ozone can improve circulation, modulate immunity, alleviate inflammation and pains, so it is widely used for healing wounds or pressure ulcers, peripheral vascular disease, diabetes, cancer adjuvant therapy, viral hepatitis, rheumatoid arthritis, joint pain and disc disorders. Medical ozone is a strong oxidant, but it can induce the expression of phase 2 metabolism enzymes at appropriate dose or in suitable concentration. We found that pretreatment with medical ozone rectal infusion could reduce liver damage induced by carbon tetrachloride (CCl4)in dogs, but its mechanism is still unclear. Bocci found that saline could be ozonized and ozonized saline(OS) could be in a good state of preservation of the ozone and its derivatives, but dripping high concentrations of ozonized saline (ozone concentration 80μg/ml) was invasive for blood vessels. Injection of ozonized saline may cause chemical phlebitis in human body. Russian scientists found in their clinical practice that low concentrations of OS intravenous drip treatment for age-related macular diseases worked well, without obvious adverse reactions.According to above analysis, This study try to observe the protective effect of intravenous injection with OS on acute liver injury of rats induced by CCl4, and also to explore its rational therapeutic concentrations, adverse reactions and effect on antioxidant system.This study plans to confirm whether OS can activate transcription factor Nrf2 and increase downstream gene of Nrf2 functional activity.Study part 1. The protective effect of ozonized saline against CCl4 induced hepatocyte oxidative damage Methods:Forty,Male,Sprague-Dawley rats were randomly divided into ozonized saline(OS) group,model group and normal control(NC) group.The rats in OS group(in 5,10,20,30,40,50μg/ml six different concentrations) were intravenously administered with OS(5ml/kg),while the rats in model group with oxygen saline every day,for 15 days. On the 16th day, rats of the two groups were intraperitoneally injected with 2ml/kg 50% CCl4 dissolved in oliver oil. The rats in NC group were fed normally for 15 days. On the 16th day, rats of this group were intraperitoneally injected with 2ml/kg oliver oil without CCl4.After 24 hours of CCl4 or olive oil intraperitoneal injection, all of the rats were killed by intraperitoneal injection with 25mg/kg 3% sodium pentobarbital, and then, the serum levels of alanine transaminase (ALT) and aspertate aminotransferase (AST) were measured. The hepatic tissues were also collected for detection of total antioxygen capacity (TAOC), glutathione (GSH),catalase (CAT), Glutathione peroxidase (GPx). Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2.Results:This study showed that the serum ALT and AST levels of the rats in model group and OS groups were both significantly higher (P<0.01 or P<0.05), and the contents or activity of TAOC, GPx,GSH and CAT were significantly lower (P<0.01 or P<0.05) compared with the rats in NC group. While compared with rats in model group, rats in OS groups had significantly decreased the serum levels of ALT(P<0.05) and AST(P<0.05).The protective effects of preconditioning with OS on acute liver injury in rats induced by CCl4 varied in ozone concentration.30μg/ml OS had maximal effects. The serum ALT and AST levels of rats in 30μg/ml OS group decreased by 56.5%(P=0.007),56.2%(P=0.000) respectively compared with the rats in model group. The ALT and AST levels of rats in 30μg/ml OS group also were lower than those in other OS groups, but not all of them were significantly different. Compared with model group, the rats in OS groups increased in the contents or activity of TAOC, GSH, GPx, CAT and some of them had significant differences (P<0.05). The TAOC, GSH, GPx, CAT contents or activity of rats in 30μg/ml OS group significantly increased by 90.3%(P=0.000),114.3%(P=0.000), 51.3%(P=0.001),50.1%(P=0.013) respectively compared with the rats in model group. Ozonized saline in different concentrations had different effects, of which 30μg/mlOS group was the best. All parameters of rats in 30μg/ml OS group were better than those in other OS groups, and some of them were significantly different (P<0.05). Compared with the other two groups, Nrf2 expression of rats in OS groups was strengthened in the liver cells not only by western blot but also by immunofluorescence staining assay. Ozonized saline in different concentrations had different effects, of which 20μg/ml-40μg/ml OS group were better. There were no chemical vasculitis and other adverse reactions in the rats treated with OS, no adverse events and death in all rats.Study part 2. The effect of ozonized saline in 30μg/ml concentration on the antioxidant capacity of normal liver tissue in ratsMethods:Accordingto previous experimental modelparameters,ten,Male,Sprage-Dawley rats were randomly divided into ozonized saline(OS) group and normal control(NC) group. The rats in OS group were pretreated with 30ug/ml OS(5 ml/kg) once a day for 15 days. The rats in NC group were fed normally for 15 days. On the 16th day, rats in the two groups were killed by intraperitoneal injection with 25mg/kg 3% sodium pentobarbital.Nuclear proteins of hepatic tissues were extracted to detect TAOC, GPx and CAT activity. Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2.Results:The TAOC, GSH, GPx, CAT contents or activity of rats in 30μg/ml OS group significantly increased by 31.4%(P=0.005),21.0%(P=0.023),14.2%(P =0.040)respectively compared with rats in model group. Compared to NC group, Nrf2 expression of rats in OS group was strengthened in the liver cells not only by western blot but also by immunofluorescence staining assay. There were no chemical vasculitis and other adverse reactions in the rats treated with OS, no adverse events and death in all rats.Conclusions:Preconditioning intravenous infusions of ozonized saline could reduce the liver injury in rats induced by carbon tetrachloride. The pharmacological effects of ozonized saline had a dose-dependent characteristic, with the concentration in 30μg/ml ozonized saline as the best efficacy. OS can activate the Keapl-Nrf2-ARE signaling pathway and its downstream antioxidant enzymes genes expression,which reduce the oxidative damage of radical oxygen species(ROS) and the deleterious substance through a dose dependent way. |
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