Preparation Of Hydrogen-rich Solutions And Effects Of Hydrogen On The Ammonium Chloride Damaged Model Of C.elegans

Protein is the material basis that constitutes life.All important components of tissues and cells in the body have the presence of proteins.Protein is hydrolyzed into amino acids which are absorbed and utilized in the body.The amino acids produce a large amount of ammonia by deamination.In addition,the metabolisms of intestinal microbes can also cause the release of ammonia which is toxic to the health of the body.Under normal physiological conditions,there is a dynamic balance between ammonia production and decomposition.Ammonia forms carbamide in the liver through the urea cycle and then excreted through the kidneys.When the liver is dysfunctional,excess ammonia will accumulate in the body and cause high blood ammonia,this in turn causes a series of pathological reactions.Ammonia intoxication hypothesis is considered as the main pathogenesis of hepatic encephalopathy.Hepatic encephalopathy is a neuropsychiatric syndrome based on metabolic disorders with different degrees of severity caused by severe liver dysfunction or portosystemic shunt.The current theory suggests that the occurrence of hepatic encephalopathy is closely related to the oxidative stress caused by ammonia in the body.Studies have shown that hydrogen has good improvements in various disease models and clinical disease treatments.Hydrogen is a new type of exogenous antioxidant that selectively scavenges hydroxyl radicals and nitrite anions in vivo.At the same time,hydrogen also exhibits good biological effects such as anti-inflammatory,anti-apoptotic and anti-metabolic abnormalities.The main methods of using hydrogen include inhaling hydrogen,drinking hydrogen-rich water,and injecting hydrogen-rich physiological saline.This study focused on the preparation of hydrogen-rich solutions and the biological effects of hydrogen in vivo to further confirm the biomedical effects of hydrogen from the perspective of antioxidant stress.The main research contents are as follows:1.Preparation of hydrogen-rich solutions and its influencing factorsObjective:To measure the catalytic effects on hydrogen production of ammonium salt solutions,and analyze the relationships between hydrogen concentration and oxygen concentration,oxidation-reduction potential,pH in solution.Methods:Magnesium and water reacted in different concentrations of NH₄Cl,（NH₄）₂SO₄,NH₄HCO₃,（NH₄）₂CO₃,NaHCO₃ and Na₂SO₃ in 40℃water bath for 0,2,4,6,8 and 10 hours,and measure the hydrogen concentration,oxygen concentration,oxidation-reduction potential and pH of ammonium salt solutions after finishing reaction.Results:The hydrogen concentration in the solutions increased with the increase of the reaction time and the concentration of the ammonium salt solutions.Oxygen concentration and oxidation-reduction potential decreased with the increase of hydrogen concentration,there was a significant negative correlation between hydrogen concentration and oxygen concentration in solution（r=-0.9818）,pH increased with the increase of hydrogen concentration in the ammonium salt solutions.Conclusion:The ammonium salt solutions have a good catalytic effect on magnesium and water reaction.NH₄Cl has the strongest catalytic effect in the same reaction conditions,（NH₄）₂SO₄ is second,NH₄HCO₃ and（NH₄）₂CO₃ are less.Thus,we provide detailed data of hydrogen preparation in different solutions,which lays the foundation for further study on the function of different types of hydrogen-rich solutions.2.Establishment of ammonium chloride damaged model of C.elegansObjective:To establish a stable ammonium chloride damaged model using C.elegans as model organism.Methods:C.elegans at L3 stage were treated with different concentrations of ammonium chloride solution（10 mM,100 mM,1000 mM）for 24 hours based on the test of ammonium chloride LC₅₀,set a control group culturing in normal conditions.Changes in physiological,motor behavior and biochemical levels of nematodes were test.The toxicities caused by ammonium chloride were observed and the toxic mechanisms were analyzed.Results:Compared with the control group,in ammonium chloride treatment groups,the life span of C.elegans were significantly shortened（p<0.05）,the body length were significantly shortened（p<0.01）,the total number of eggs were significantly reduced（p<0.01）in physiological levels.In motor behavior levels of nematodes,the local motor behavior ability of nematodes of each group after ammonium chloride treatment decreased significantly,which showed that the frequency of head swings,body bendings and pharyngeal pump vibrations per unit time were significantly reduced（p<0.05）.In biochemical levels of nematodes,the levels of ROS were significantly increased（p<0.01）,mitochondrial membrane potential were significantly decreased（p<0.01）,SOD and CAT activities were significantly decreased（p<0.01）,content of MDA were significantly increased（p<0.01）.Ammonium chloride produced concentration-dependent damages to nematodes.Conclusion:Ammonium chloride produces significant physiological and motor behavioral toxicities to nematodes.Toxic effects are related to the formation of excess ROS which is associated with oxidative stress and affects the activities of antioxidant enzymes.It provides a stable biological model for subsequent experiments.3.Effects of hydrogen on the ammonium chloride damaged model of C.elegansObjective:Using the ammonium chloride damaged model of C.elegans,explore the biological effects of hydrogen and its mechanisms.Methods:In hydrogen groups,nematodes were cultured in hydrogen environment for 8hours every day for 10 consecutive days after nematodes self-synchronization.Nematodes at L3 stage were treated with ammonium chloride solution of sublethal concentration（10 mM,100 mM）for 24 hours in the same way as before.Set low and medium-concentration ammonium chloride treatment groups and groups cultured in hydrogen environment after low and medium-concentration ammonium chloride treatment,set a normal group and a group of nematodes cultured in hydrogen environment as well.Changes in physiological,motor behavior and biochemical levels of nematodes were test on 10-day-old nematodes.The changes of nematode intestinal microbes were also test in normal group,normal group cultured in hydrogen environment,10mM ammonium chloride treatment group and 10mM ammonium chloride treatment group cultured in hydrogen environment.Analyze the biological effects of hydrogen and its mechanisms.Results:Compared to the ammonium chloride treatment groups,in groups of nematodes cultured in hydrogen environment after low and medium-concentration ammonium chloride treatment,the body length were significantly increased（p<0.05）,the total number of eggs were significantly increased（p<0.05）,the maximum life span and average life span had an increasing trend in physiological levels（p>0.05）.In motor behavior levels of nematodes,the frequency of head swings,body bendings and pharyngeal pump vibrations per unit time were significantly increased（p<0.05）.In biochemical and molecular levels of nematodes,the levels of ROS were significantly decreased（p<0.05）,mitochondrial membrane potential were significantly increased（p<0.05）,SOD and CAT activities were significantly increased（p<0.05）,content of MDA were significantly decreased（p<0.05）,apoptosis were significantly decreased（p<0.05）,expressions of age-1,hsp16.2,skn-1 and ape-1 gene of nematodes were significantly decresed（p<0.05）.The detection of nematode intestinal microbes was divided into 4 groups:normal group,normal group cultured in hydrogen environment,10mM ammonium chloride treatment group and 10mM ammonium chloride treatment group cultured in hydrogen environment,there were no significant changes in Chao1index and Observed species index between the four groups（p>0.05）.In the aspect ofβdiversity index,there were statistically significant biomarkers between the four groups by LEfSe analysis.The specific species of the normal group were Alphaproteobacteria,Rhizobiaceae and Pseudochrobactrum,the specific species of the normal group cultured in hydrogen environment was Lactobacillus,the specific species of the 10mM ammonium chloride treatment group was Gammaproteobacteria,the specific species of10mM ammonium chloride treatment group cultured in hydrogen environment was Pseudomonas.At the phylum classification level,the relative abundance of Proteobacteria and Firmicutes were not significantly changed between the normal group and normal group cultured in hydrogen environment（p>0.05）,the relative abundance of Bacteroidetes was lower in normal group cultured in hydrogen environment than that in normal group（p=0.001）,the relative abundance of Actinobacteria was higher in normal group cultured in hydrogen environment than that in normal group（p=0.006）.At the genus classification level,the relative abundance of Pseudochrobactrum,Pseudomonas,Ochrobactrum and Stenotrophomonas were lower than that in normal group（p=0.001）,the relative abundance of Brevundimonas and Microbacterium were higher than that in normal group（p=0.001）.At the phylum classification level,the relative abundance of Proteobacteria,Actinobacteria and Firmicutes were not significantly changed between 10mM ammonium chloride treatment group and 10mM ammonium chloride treatment group cultured in hydrogen environment（p>0.05）,the relative abundance of Bacteroidetes was lower in 10mM ammonium chloride treatment group cultured in hydrogen environment than that in10mM ammonium chloride treatment group（p=0.001）.At the genus classification level,the relative abundance of Pseudochrobactrum and Microbacterium were higher in10mM ammonium chloride treatment group cultured in hydrogen environment than that in 10mM ammonium chloride treatment group（p=0.001）,the relative abundance of Pseudomonas and Ochrobactrum were not significantly changed between 10mM ammonium chloride treatment group and 10mM ammonium chloride treatment group culturedinhydrogenenvironment（p>0.05）,therelativeabundanceof Stenotrophomonas and Brevundimonas were lower in 10mM ammonium chloride treatment group cultured in hydrogen environment than that in 10mM ammonium chloride treatment group（p=0.001）.Conclusion:Hydrogen inhibits the toxic effects of ammonium chloride on nematodes.The biological effects of hydrogen related to the removal of ROS,promote the antioxidant enzyme activities,protection against mitochondrial damages and down-regulation of expression of age-1,skn-1 and ape-1.