| Medical Ozone is a new kind gas molecule drugs. The medical biology and pharmacology characteristic of low-dose medical ozone is that it can induce cells to produce antioxidant enzymes and eliminate free radicals. Medical ozone can be dissolved in water, saturated in 5 minutes, and its solubility increases with the medical ozone concentration. Under the same conditions, the solubility of medical ozone is 9 times more than that of oxygen.The half-life of medical ozone in water is 110 hours at 5℃. Bocci found that saline could be ozonized and ozonized saline(OS) could be a very good preservation of the ozone and its derivatives. However, they believe that a high concentration of OS (ozone concentration is 80μg/ml) intravenous infusion may cause chemical phlebitis. Russian scientists found in their clinical practice that low concentrations of OS intravenous drip treatment for age-related macular disease worked well, with no obvious adverse reactions. However, they still need basic research and evidence-based medical data to prove their findings.This study try to observe effect of intravenous injection with OS on acute liver injury of rats induced by carbon tetrachloride (CCl4), and to explore its rational therapeutic concentrations, adverse reactions and effects on antioxidant system. In the first part, the effects of different concentrations of OS on the liver biochemistry, histopathology and antioxidant system in rats treated with CCl4 were studied.METHODS:45 healthy male Sprague Dawley (SD) rats were randomly divided into nine groups. Group 1:normal control group (NC group); Group 2:OS control group (OSC group); Group 3:Oxygen saline group (model group); Group 4～9:OS groups (experimental groups, with ozone concentrations of 5,10,20,30,40,50μg/ml respectively). The rats of OS groups or model group were intravenously administered with 5ml/kg OS of a particular ozone concentration or 5ml/kg oxygen saline once daily for 15 days respectively. On the 16th day, rats were intraperitoneally injected with 2ml/kg 50% CCl4 dissolved in oliver oil. Rats of OSC group also were intravenously administered with 50μg/ml OS 5ml/kg once daily for 15 days, while the rats of NC group didn't receive any pretreatment except for normal feeding for 15 days. On the 16th day, rats of two groups were intraperitoneally injected with 2ml/kg oliver oil but not CCl4. The general condition and adverse effects were recorded during the treatment of OS and CCl4. After 24 hours of CCl4 or olive oil intraperitoneal injection, rats were sacrificed by intraperitoneal injection with 3% sodium pentobarbital in dose of 25mg/kg. The serum levels of ALT and AST, survival rates were measured. The liver tissues were also collected for detection of total antioxygen capability (TAOC), glutathione (GSH), Glutathione peroxidase (GPx), catalase (CAT), malondialdehyde (MDA), and damage area of liver histopathological as well.RESULTS:This study showed that rats of model group and OS groups both had significantly increased in the levels of ALT, AST, MDA and the area of liver histopathological damage (p<0.01 or p<0.05), and significantly reduced in the contents or activities of TAOC, GPx,GSH and CAT (p<0.01 or p<0.05) compared with rats of NC group. While compared with rats of model group, rats of OS groups had decreased in the levels of ALT, AST, and some of which had significant differences (p<0.05).and the area of liver histopathological damage in (5～50)μg/ml OS groups were decreased than model group by 27.1% (p=0.008),37.9%(p=0.000), 39.2%(p=0.000),51.3%(p=0.000),35.4%(p=0.001),24.1%(p=0.018), respectively. The protective effects varied with different ozone concentration, and 30μg/ml OS showed maximal effects. The levels of ALT, AST in rats of 30μg/ml OS group decreased by 56.5%(p=0.007),56.2%(p=0.003) respectively compared with model group. The levels of ALT, AST in rats of 30μg/ml OS group also were less than those of other OS groups, but all of them showed no significantly different (p>0.05). Compared with model group, OS groups increased in the contents or activities of TAOC, GSH, GPx, CAT, while decreased in the contents of MDA, and some of which had significant differences (p<0.05). The contents or activities of TAOC, GSH, GPx, CAT in rats of 30μg/ml OS group significantly increased by 91.6%(p=0.000), 111.1%(p=0.000),51.3%(p=0.001),50.9%(p=0.012) respectively and the activities of MDA reduced by 42.2%(p=0.000) compared with model group Ozone of different concentrations had different effects, of which 30μg/ml OS group was the best. All parameters in rats of 30μg/ml OS group were better than those of other OS groups, and some of them were significantly different (p<0.05). Compared with normal control group, OS control group had no significant difference in ALT, AST, TAOC, GSH, GPx, CAT, MDA levels and the liver pathological damage area. No chemical vasculitis, other adverse reactions and death were occurred in the rats treated solitarily with OS.In the second part of this study, the time-effect relationship of liver injury induced by CCl4 and the effects of OS on the survival rates of rats treated with CCl4 were studied.METHODS:91 healthy male SD rats were randomly divided into 3 groups. Group 1:normal control group (NC group) with 15 rats; Group 2:oxygen saline group (model group) with 38 rats; Group 3:OS group (experimental group) with 38 rats. The rats of OS group or model group were intravenously administered with 5ml/kg 30μg/ml OS or 5ml/kg oxygen saline once daily for 15 days respectively. On the 16th day,13 rats that were randomly selected from OS group or model group were intraperitoneally injected with 4ml/kg 60% CCl4 dissolved in oliver oil for making severe liver injury model. Then the survival time and survival rate of those rats were measured. The remaining 25 rats of OS group or model group were intraperitoneally injected with 50% CCl4 dissolved in oliver oil of 2ml/kg for making acute liver injury model. On 1st,2ed,3rd,5th and 7th days post CCl4 injection,5 rats were randomly taken from all group at each time and sacrificed by intraperitoneal injection with 3% sodium pentobarbital in dose of 25mg/kg. The rats of normal control group didn't receive any pretreatment except normal feeding for 15 days. On the 16th day, the rats were intraperitoneally injected with 2ml/kg oliver oil but not CCl4. On 1st,2ed,3rd,5th and 7th days post oliver oil injection,3 rats were randomly taken from NC group every time and killed with 25mg/kg 3% sodium pentobarbital. Then the serum levels of ALT and AST, general condition and adverse effects were measured. The liver tissues were also collected for detection of total antioxygen capability (TAOC), glutathione(GSH), Glutathione peroxidase (GPx), catalase (CAT), malondialdehyde (MDA), and routine liver histopathological damage area assessment as well.RESULTS:This study showed that the livers of rats treated with CCl4 were injured seriously. On 1st,2ed,3rd,5th and 7th days post CCl4 treated, the levels of ALT, AST, MDA and the liver histopathological damage area in rats of OS or model group rose first and then dropped, while the contents or activities of liver TAOC, GSH, GPx and CAT fell first and rose later. The levels of ALT, AST, MDA and the area of liver histopathological damage trended in the same direction, and the contents or activities of liver TAOC, GSH, GPx, CAT trended in the direction on the contrary in the rats of OS or model group. In other words, when the contents or activities of liver TAOC, GSH, GPx and CAT reduced and MDA increased the levels of ALT, AST and the liver histopathological damage area in rats also increased. But when the contents or activities of liver TAOC, GSH, GPx, CAT and MDA returned to normal, the levels of ALT, AST and the liver histopathological damage area in rats came back to normal too. All parameters of normal control group remained sTab.. The levels of ALT, AST, MDA and the liver histopathological damage area in rats of model group rose to the highest value at the first day after injection with CCl4, and the contents or activities of liver TAOC, GSH, GPx, CAT reduced to minimum on the same day. The peak-period of ALT, AST, MDA and the liver histopathological damage area and the valley-period of liver TAOC, GSH, GPx, CAT were delayed (came latter) in rats of OS group. The levels of ALT, AST, MDA and the liver histopathological damage area rose to the peak and the contents or activities of liver TAOC, GSH, GPx, CAT reduced to a minimum value at the second day after injection with CCl4 in rats of OS group. All parameters in rats of OS group or model group restored nearly normal at the 5th day after injection with CCl4. Compared with model group, the peak value of ALT, AST levers in rats of 30μg/ml OS group significantly decreased by 43.0% (p=0.023),42.9%(p=0.011) respectively, the valley value of TAOC significantly increased by 25%(p=0.015), the peak value of liver histopathological damage area significantly decreased (22.7% vs 35.7%, p=0.011), the peak value of MDA decreased by 32.7%, and the valley value of GSH, GPx, CAT increased by 21.5%, 15.6%,25% respectively, though there was no significant difference (p>0.05). No chemical vasculitis, other adverse reactions and death were occurred in the rats treated solitarily with OS.Survival analysis results showed that 7 rats of OS group died and 6 rats were live post CCl4 injection, and 12 rats of model group died and 1 rat was live. The survival rate of OS group significantly increased than that of model group (46.2% vs7.7%, p=0.017).Conclusions:Preconditioning intravenous infusions of ozonied saline could reduce the liver injury of rats induced by carbon tetrachloride, postpone peak-period of liver injury, and improve survival rate in rats. These pharmacological effects of ozonied saline were dose-dependent. The mechanism that ozonied saline can protect rats against acute liver injury induced by carbon tetrachloride might be related to that OS could increase the synthesis of antioxidant enzymes and improve the total antioxidant capacity in rats. Intravenous infusions of ozonied saline showed no significant adverse reactions in rats.
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