| Objectives1.T0 cxplore a new method of hydrogen administration by measuring the safe dose of hydrogen injection into the tail vein and then detect the hydrogen concentration in blood and liver tissue.2.To investigate the therapeutic effect of the new hydrogen delivery method on rats with liver fibrosis,and study its possible mechanism so as to provide theoretical basis for the further promotion of hydrogen medicine.Materials and methodsPart I:firstly,normal wistar rats were taken as the research object,and the acute toxicity indexes(death and survival)of rats were observed after 14 days by injecting different doses of hydrogen through the tail vein with insulin needles in each group by Bliss method.According to the maximum non-lethal dose(LD0)and absolute lethal dose(LD100)were used to determine the appropriate dosage range of hydrogen injected into the tail vein and calculate the half-lethal dose(LD50).Secondly,according to the principle of subacute toxicology experiment,normal wistar rats were randomly divided into blank control group,tail vein injection of H2 group and tail vein injection of N2 group,and 2 mL·kg-115s-1 of hydrogen less than LD0 in the first step(i.e.2ml per kilogram of body weight was injected at a constant speed within 15 seconds,the same below)was taken as the test dose.The subacute toxicity indexes(average pulmonary artery pressure,heart rate,respiratory frequency,arterial oxygen partial pressure,arterial blood carbon dioxide partial pressure,tricuspid systolic regurgitation and pulmonary artery systolic pressure)were measured by biological function information acquisition system,blood gas analyzer and ultra-high resolution multi-mode small animal ultrasonic photoacoustic imaging system to evaluate the safety of 2mL·kg-115s-1 tail vein injection of H2.Finally,the instantaneous hydrogen concentration in blood and the dynamic hydrogen concentration in liver tissue were measured by Clark hydrogen microsensor at a dose of 1 mL·kg-1 15s-1.Part Ⅱ:We established a model of hepatic fibrosis induced by CCl4 in wistar rats.Twenty-seven male Wistar rats were randomly divided into control group,treatment group and model group.Rats in model group and treatment group were injected with 40%CCl4(2mL/kg)intraperitoneally,while rats in control group were injected with olive oil twice a week for 6 weeks.From the 4th week,the treatment group was injected with hydrogen at a uniform speed of 1 mL·kg-115s-1 per day through the tail vein,while the control group and the model group were injected with the corresponding volume of isotonic saline through the tail vein for 6 weeks.Serum liver function indexes(ALT,AST)were detected after treatment.Pathological observation of liver tissue:HE staining was used to semi-quantitatively observe the degree of hepatocyte degeneration,Masson staining was used to semi-quantitatively analyze the collagen of liver fibrosis in each group and Ishak score of liver fibrosis stage,and the expression of COL1αl in liver tissue was observed by immunohistochemistry.GSH/GSSG,SOD and MDA protein activity detection kits were used to detect the level of oxidative stress damage in liver tissue of three groups of rats.Real-time quantitative PCR was used to determine the mRNA expression level of TGFβ1,Smad3 and Smad7 in rat liver tissue.Western blot was used to determine the expression level of TGFβ1 protein in rat liver tissue.Part Ⅲ:Metabolomics analysis based on QE was performed with the iver tissue samples(the blank group,model group and hydrogen treatment group,respectively 4 cases from Part Ⅱ).According to the hierarchical cluster analysis clue of positive ion differential metabolites in metabolomics,the VitaminA lipid droplets of stellate cells in three groups of samples were qualitatively located by transmission electron microscopy.According to the results of metabolomics and transmission electron microscopy,the expression level of autophagy index LC3B in the liver tissues of rats was determined by Western blot.Results1.The LD50 of caudal vein injection of hydrogen was 5.81 mL·kg-115s-1(95%CI:5.28-6.26 mL·kg-1).The caudal venous dose of 2 mL·kg-115s-1 hydrogen was less than LD0.2.The subacute end points of 2 mL·kg-115s-1caudal venous dose monitoring results showed that mPAP level(P<0.01),heart rate and respiratory rate(P<0.05)were significantly increased in N2 group,but there was no statistical significance in H2 group compared with the control group.Blood gas analysis showed obvious hypoxemia in N2 group,but not in H2 group.The results of ultra-high resolution multi-mode ultrasonic photoacoustic imaging system for small animals showed that the systolic period of the tricuspid valve region was not detected and obvious regurgitation was not detected in the blank group and the H2 group after caudal vein injection.and the pulmonary artery systolic pressure measured by CWD was not significantly increased.But in the N2 group.obvious regurgitation signal appeared at the tricLispid valve orifice during systolic period.Meanwhile.CWD measurement showed that the regurgitation pressure difference at the tricuspid valve orifice was significantly increased compared with the resting state,suggesting the presence of pulmonary embolism.3.A dose of 1 mL·kg-115s-1 hydrogen was injected into the caudal vein,and the concentration of hydrogen in the blood and liver was simultaneously and immediately detected.The immediate concentration was 37.6±3.7 μmol/L in the blood and 24.3±2.0 μmol/L in the liver.The hydrogen concentration in liver increased gradually,reaching a peak of 60.6±0.5 μmol/L around 20 minutes.The half-life of hydrogen after caudal intravenous injection of 1 mL·kg-115s-1 was t1/2=0.6801h=40.81min.4.Under light microscope,inflammatory cell deposition was reduced in the caudal vein hydrogen treatment group,and hepatocyte degeneration,edema and necrosis were alleviated.The hyperplasia of fibrous tissue was reduced,and the fibrous septum was reduced in some rats,especially the improvement of collagenmanifold bridging fibrosis and collagen-central bridging fibrosis.The collagen area and Ishak score in hydrogen treatment group were lower than those in model group(P<0.05).The COL1α1 positive staining area in hydrogen treatment group was decreased compared with model group(P<0.05).5.Compared with normal control group,the activity of antioxidant factor GSH and SOD protein in model group was lower than that in normal group(P<0.01),and the level of lipid damage index MDA was higher than that in normal group(P<0.01);The level of GSH and the activity of SOD protein in liver tissue of hydrogen treatment group were increased compared with model group(P<0.05),while the level of MDA was decreased compared with model group(P<0.05).6.mRNA expression levels of TGF-β1 and TGF-β1-related signaling factor Smad3 in liver tissue of rats in model group were significantly increased compared with those in normal group(P<0.0 1),while mRNA expression levels of TGF-β1 and Sinad3 in liver tissue of rats in hydrogen treatment group were significantly lower than those in model group(P<0.05).Smad7 mRNA in model group was lower than that in normal group(P<0.01)while hydrogen treatment group higher than model group(P<0.05).The expression of TGF-β1 protein in liver tissue of model group was significantly higher than that of control group(P<0.01).The expression of TGF-β1 protein in liver tissue of rats in caudal vein treatment group was significantly lower than that in model group(P<0.05).7.The hierarchical cluster analysis of differential metabolites of positive ions showed that VitaminA content decreased in the CCl4-induced hepatic fibrosis model group and recovered after hydrogen injection in caudal vein.8.Compared with blank group,the body of the stellate cells in model group enlarged,the number of the stellate cells increased,and the vitamin A lipid drops in the stellate cells decreased.In the hydrogen caudal vein treatment group,vitamin A lipid drops recovered.9.The relative expression level of LC3BII in liver tissue of model group was significantly higher than that of control group(P<0.01);The relative expression level of LC3BII in liver tissue of rats in caudal vein treatment group was significantly lower than that in model group(P<0.05).ConclusionsIt was a novel,safe,and fast method to intravenously administer H2 at a dose of 1mL/kg over 15s via tail vein and had an anti liver fibrosis effect with the method.The possible mechanism of anti liver fibrosis was that hydrogen maintained the homeostasis of stellate cells by reducing the loss of Vitamin A lipid droplets and regulating expression levels of TGF-β1,Smad3 and Smad7,through the reduction of the oxidative stress induced by CCl4 and liver autophagy.These results showed that tail vein injection of hydrogen can be a safe and effective treatment method,which can provide a new scientific basis for the treatment of liver fibrosis. |
