Effect And Mechanism Of DJ-1 In Hydrogen To Improve Hyperhomocysteinemia

Background:Hyperhomocysteinemia(HHcy)has a high incidence rate in China.HHcy is closely related to the occurrence and development of many diseases,such as cardiovascular disease,neurological and mental diseases,chronic kidney disease,fatty liver,etc.,and is an independent risk factor for many diseases.It mainly affects cell function through various mechanisms such as oxidative stress damage,DNA methylation,and abnormal metabolism of sulfur compounds,leading to pathological changes in various tissues and organs.Currently,the management and treatment strategies for HHcy mainly focus on nutritional intervention treatment for key coenzyme deficiencies such as folic acid,vitamin B6,and vitamin B12.Although the efficacy of this intervention in reducing Homocysteine(Hcy)levels is not controversial,its main drawbacks are the long intervention cycle and generally lower than expected improvement in disease prognosis.Therefore,there is an urgent need for an effective intervention strategy that can reduce Hcy levels,significantly improve patient prognosis,and reduce the risk of complications.Hydrogen medicine has developed rapidly in recent years.Hydrogen has been proven to have positive effects on cardiovascular diseases,chronic kidney disease,hypertension,metabolic syndrome,and other diseases.It has attracted widespread attention due to its advantages of safety,small size,rapid action,and non-toxic side effects.Various animal pathological models have confirmed that hydrogen has significant therapeutic effects and no significant adverse reactions.A large number of studies have shown that hydrogen can not only react directly with reactive oxygen species to reduce oxidative stress and exert biological effects,but also exert protective effects by regulating gene expression.Therefore,hydrogen intervention has broad application prospects in the field of chronic metabolic diseases.Our previous research found that hydrogen intervention can achieve the effect of reducing the plasma Hcy level and improving fatty liver in rats with HHcy.Through histological screening,the differentially expressed genes PARK7(DJ-1)and the glycine serine threonine pathway were involved.After hydrogen intervention,the expression of DJ-1 was significantly upregulated,and the glycine serine threonine pathway was significantly activated.However,the specific mechanism remains unclear,and further research is needed.Objective:To observe the effect of hydrogen intervention on HHcy induced by high methionine diet,and explore the role of hydrogen in reducing Hcy and improving liver function;Using DJ-1 KO animal and cell models,to explore the role and mechanism of DJ-1 in hydrogen to improve HHcy,and through PPAR-γ Agonist intervention further validates its regulatory mechanism at the animal and cell levels.Content and Methods:1.Verify the damage effect of HMD on animals and cells(1)Establishment of mouse HHcy model: SPF grade mice were randomly divided into two groups: CHOW group and HMD group.CHOW was fed with AIN-93 G control feed,and HMD group was fed with AIN-93G+2% methionine feed.The HHcy model was established for 12 weeks;(2)Establishment of cell HHcy model: AML 12 cells were divided into four groups:general diet group(CHOW),methionine group(HMD),and Met culture medium(20mmol/L,50 mmol/L,100 mmol/L,mother liquor concentration of 200 mmol/L,using DMEM: F12=1:1 culture medium for corresponding dilution);(3)Detection of DJ-1 and Nrf2 protein expression using Western Blot(WB)technique;(4)Detection of Hcy source and transcriptional expression of key metabolic enzymes using real-time fluorescence quantitative PCR(RT-q PCR).2.Explore the regulatory effect of DJ-1 on Hcy metabolism(1)DJ-1 KO animal model: DJ-1 KO animal model: purchased by Saiye(Suzhou)Biotechnology Co.,Ltd.,and expanded after breeding.After genotype identification,+/+was selected as the control group(WT),and-/-was the knockout group(KO);(2)DJ-1 KO cell model: construct a lentivirus vector from Shanghai Jikai Gene Technology Co.,Ltd.,infect AML 12 cells,and then identify the knockout efficiency;(3)Detection of homocysteine(t Hcy)concentrations in animal plasma and cells by enzyme circulation method;(4)Cell counting kit 8(CCK-8)method was used to detect the cell viability of AML 12 cells in each group.3.Effect of Rog intervention on DJ-1 and Hcy metabolism(1)Rog Intervention Cell and Animal Model Construction: Using 20mmol/L rosiglitazone to culture AML 12 cells and detect its impact on key enzymes of DJ-1 and Hcy metabolism;(2)Detection of liver function indicators,plasma lipid metabolism,and t Hcy levels in animals;(3)Observation of histopathological changes and liver steatosis in animals with hematoxylin eosin(HE)and oil red staining.Results:Effect of HMD on liver function and DJ-1 expression in mice(1)HMD mice showed a significant increase in t Hcy,a significant decrease in body weight,and a significant increase in liver weight to body weight ratio;(2)The liver ALT and AST of HMD mice significantly increased,and the liver was damaged;(3)In HMD mice,liver TC,TG,and plasma TC,TG significantly increased,and liver lipid metabolism was impaired;(4)Liver damage and fatty liver in HMD mice was observed by pathological sections of the liver;(5)Significant decrease in liver DJ-1 protein and m RNA expression in HMD mice.2.Effect of HMD intervention on Hcy metabolism in DJ-1 KO mice(1)DJ-1 KO mice are more resistant to HMD,and t Hcy significantly increases;(2)Significant decrease in m RNA expression of key enzymes in Hcy metabolic sulfur transfer pathway and GSH synthesis pathway in liver of DJ-1 KO mice;(3)The results of liver transcriptomics showed that after HMD intervention in DJ-1KO mice,the glycine serine threonine metabolic pathway and Vit B6 metabolism were significantly downregulated;Impaired vitamin synthesis,utilization,and fatty acid metabolism in DJ-1 KO mice,resulting in decreased ability to metabolize Hcy and fatty acids.Study on the effect and mechanism of DJ-1 knockout on intracellular Hcy metabolism(1)HMD interferes with a significant decrease in cell DJ-1 protein and m RNA expression,cell activity,and Nrf2 transcription levels;(2)HMD interferes with a significant decrease in the transcriptional expression of key enzymes in the Hcy metabolic synthesis pathway,a significant decrease in the transcriptional expression of key enzymes in the demethylation pathway,and an upward trend in the transcriptional expression of key enzymes in the sulfur transfer pathway and GSH synthesis pathway in cells;(3)HMD interferes with a significant decrease in the activity of DJ-1 KO cells and intolerance to HMD;(4)The m RNA of Nrf2 in DJ-1 KO cells decreased significantly,the transcriptional expression of the key enzyme Mat2 a derived from Hcy decreased significantly,the transcriptional expression of the key enzymes MTR and MTHFR in the demethylation pathway decreased significantly,and the transcriptional expression of the key enzymes in the sulfur transfer pathway and GSH synthesis pathway decreased significantly.4.Construction of animal and cell rosiglitazone intervention models and research on their effects on DJ-1 and Hcy metabolism(1)Rog intervention downregulates DJ-1 protein and transcriptional expression in AML 12 cells;(2)Rog intervention upregulates Nrf2 in AML 12 cells and promotes Hcy metabolism,sulfur transfer pathway,and GSH synthesis;(3)Rog intervention leads to further increase of plasma Hcy in HHcy mice;(4)Rog intervention leads to liver metabolic damage and further aggravation of fatty liver in HMD mice.Conclusion:1.HMD diet can cause liver function damage and fatty liver in mice,and significantly inhibit the expression of liver DJ-1.2.Hydrogen can upregulate the expression of DJ-1,and DJ-1 KO cells and mice are not tolerant to HMD intervention.DJ-1 has a positive regulatory effect on Hcy metabolism.3.The GSH synthesis and Vit B6 metabolism pathways of DJ-1 KO mice are blocked,and the Hcy metabolism sulfur transfer pathway and demethylation pathway are weakened,indicating the key role of DJ-1 in Hcy metabolism.4.PPAR-γ The agonist Rog has the effect of inhibiting the expression of DJ-1,causing metabolic disorders of Hcy,and aggravating the symptoms of Hcy.