| BackgroundKidney stone is a common disease in the world,which can lead to Chronic Kidney Disease（CKD）and End Stadge Renal Disease（ESRD）,which brings a heavy social and economic and medical burden to many countries.A national epidemiological survey in China on kidney stones shows that the incidence of kidney stones is 6.4%,and the 10-year recurrence rate is 52%.The incidence rate of kidney stones is increasing worldwide.These increases can be seen in gender,race and age.Metabolic dysfunction and dietary habits may be a key driving force.Besides,global warming may affect these trends.Recent epidemiological studies have shown that kidney stones are associated with cardiovascular disease,hypertension,obesity,diabetes and metabolic syndrome,which are recognized risk factors for CKD.Therefore,it is urgent to study the mechanism of occurrence and development of kidney stone disease and take effective prevention and treatment measuresA large number of studies have confirmed that 80-90%of the stones are calcium oxalate（Calcium Oxalate,Ca Ox）.Both animal and clinical experiments have shown that the renal tubular epithelial cells are exposed to high concentrations of oxalic acid or calcium oxalate crystals will produce excessive reactive oxygen species（Reactive Oxygen Species,ROS）,leading to injury and inflammation.The main markers of oxidative stress and inflammation can be detected in the urine of patients with kidney stone and rats with Ca Ox nephrolithiasis.Antioxidant treatment can reduce renal damage in animal model of calcium oxalate crystallization in kidney.It can be seen that oxidative stress and inflammation play an important role in the formation and progression of Ca Ox stones deposition.As the simplest and most abundant element in nature,hydrogen was once considered to have no biological effect on higher organisms.Since Japanese scientists discovered that hydrogen can treat diseases through selective antioxidation,hydrogen has been widely concerned and studied in various fields,and it has been found that hydrogen shows preventive and therapeutic effects in a variety of disease models and human diseases.In recent years,the biological effects of solid hydrogen carriers have become a hot topic in hydrogen medicine.Nanometer Magnesium Hydride（Magnesium Hydride,Mg H2）is a new and efficient magnesium-carrier hydrogen storage material,which has the advantages of high hydrogen storage and light weight.It can hydrolyze slowly and release hydrogen for a long time.Mg H2 was first used in biological research in 2016.The results suggest that Mg H2 can improve serum triglyceride levels and prolong the average life span of wild-type mice fed a high-fat diet.The possible mechanism is that Mg H2 produces hydrogen in the body,and hydrogen stimulates p GC-1αgene expression to promote fatty acid metabolism.Because Mg H2 is easier to carry and use than hydrogen,and releases hydrogen slowly in the body,it has a certain dose-effect relationship,so it is speculated that Mg H2 can improve the model of crystalline kidney injury to a certain extent.However,the protective mechanism of Mg H2 on crystalline kidney injury is still unclear and needs further study.PLIN2 is a kind of fat-related differentiation protein,which is mainly located on the surface of intracellular lipid droplets,promotes the formation of intracellular lipid droplets and protects triglycerides from lipolysis.PLIN2 is not only used as a specific marker to judge the accumulation of atherosclerotic lipids,but also has effects on many metabolic diseases such as pathological obesity,fatty liver and insulin resistance.The purpose of this study was to investigate whether Mg H2 can improve the degree of renal injury in crystalline kidney model and Whether it can play an improvement role through PLIN2.objective1、The first objective of this study was to clarify the role of Magnesium Hydride improving epithelial cells injury in the mice model of Glyoxylate induced Calcium oxalate crystalline,Find a safe and effective new treatment for kidney stones.2、The second objective of this study was to search for potential target screening of Magnesium Hydride to alleviate crystal kidney injury.3、The third objective of this study was to investigate the mechanism of Magnesium Hydride in regulating PLIN2 to alleviate the injury of crystalline kidney.Methods1、Animal models and groupingAnimal experiment 1:thirty six male wild-type C57B/L6 mice（8 weeks old）were randomly divided into six groups:saline group（con,n=6）,Glyoxylate group（Gly,n=6）,Glyoxylate+Magnesium Hydride 50mg/kg group（Gly+MH50,n=6）,Glyoxylate+Magnesium Hydride 100mg/kg group（Gly+MH100,n=6）,Glyoxylate+Magnesium Hydride 200mg/kg group（Gly+MH200,n=6）,Glyoxylate+Magnesiu Hydride400mg/kg group（Gly+MH400,n=6）.Animal experiment 2:another 40 male C57BL/6 mice（8 weeks old）were randomly divided into 5 experimental groups:saline group（Con,n=8）;Glyoxylic Acid induced calcium oxalate crystallization group（Gly,n=8）;Glyoxylic Acid+Magnesium Hydride group（GH,n=8）;Glyoxylic Acid+Magnesium Hydroxide group（GOH,n=8）;Glyoxylic Acid+corn oil group（GV,n=8）.Animal experiment 1:two days in advance,50,100,200 and 400 mg/kg Mg H2were given by intragastrical administration（IG）.On the third day,mice were intraperitoneally injected with Glyoxylate once a day for 5 days.Animal experiment 2:Magnesium Hydride（200mg/kg）,Magnesium Hydroxide（450mg/kg）and corn oil（0.1ml）were given by IG two days in advance.On the third day,mice were intraperitoneally injected with Glyoxylate once a day for 5 days.2、Histopathological stainingAfter the kidney tissues were taken and fixed,they were dehydrated,transparent,waxed,embedded,sliced and baked.According to the experimental procedures,the specimens were stained with HE,Masson,Von Kossa,immunohistochemistry and immunofluorescence,and the photos were taken by light microscope and fluorescence microscope.3、Detection of renal function and oxidative stress in miceAfter anesthesia,blood was collected from eyeball,static,centrifuged and supernatant was taken for standby.Competitive enzyme-linked immunosorbent assay（ELISA）was adopted to purchase kit of corresponding detection indexes,and the test was carried out according to the experimental steps.4、Cell culture,grouping and interventionUsing mouse renal tubular epithelial cells（TCMK-1）as experimental cells in vitro,DMEM medium was prepared.10%fetal bovine serum and 1%double antibody（penicillin streptomycin amphotericin B solution）were added into DMEM medium.Cell experiment groups:Con group:TCMK-1 cells were observed at regular intervals every day,and fresh medium was replaced without any stimulation;Na Ox group:co-incubated with sodium oxalate concentration（0.5 m M）and cultured in incubator for 24 hours;Na Ox+H2 group:after adding sodium oxalate（0.5 m M）,the cells were put into the hydrogen containing cell incubator for 12 hours,and then taken out and put into the incubator for 12 hours;Con+H2 group:TCMK-1 cells were also put into the hydrogen containing cell incubator for 12hours,and then were taken out and put into the incubator for further culture after 12 hours.5、Cell viability testAccording to the experimental steps,sodium oxalate of different concentrations was added into the 96 well plate and incubated in the incubator for different times.The other 96well plate was added with hydrogen culture.Finally,CCK-8 solution was added.The OD value at 450nm was determined by enzyme-linked immunosorbent assay.6、Intracellular ROS detectionDCFH-DA was diluted with serum-free medium at 1:1000,and the final concentration was 10μmol/L.The cells were collected and suspended in diluted DCFH-DA and incubated in cell incubator at 37℃for 20 minutes.Flow cytometry and fluorescence imaging were used to detect DCF.7、Cell mitochondrial membrane potential detectionAccording to the instructions of the kit,the CCCP（10mm）recommended in the kit was added to the cell culture medium at the ratio of 1:1000,diluted to 10μm,treated with JC-1 for 20 minutes,and then loaded with JC-1.The mitochondrial membrane potential was detected by flow cytometry.8、Tissue and cell protein detectionTissue and cell proteins were extracted with total protein extraction reagent（PMSF,protease inhibitor and phosphatase inhibitor were added to the protein lysate according to1:100）.The supernatant was grinded,centrifuged,and the protein was quantified with BCA protein quantitative kit.The sample was added with 1/4 of the supernatant volume of SDS-PAGE protein loading buffer（5x）,heated by metal at 100℃for 5-10 min.The samples were stored in-20℃refrigerator and detected by Western blot.9、Tissue and cell RNA detectionTrizol reagent was used to extract total RNA from tissues or cells,and the concentration of RNA was detected by a microplate reader.The c DNA of the samples was obtained by using m RNA reverse transcription reagent,and the samples were detected by real-time fluorescent quantitative PCR using SYBR green dye method.10、Metabonomics,high throughput transcriptome detection and bioinformatics predictionThe metabonomics of kidney tissue was detected by UPLC/Q-TOF-MS.High throughput transcriptome detection steps:Total RNA extraction,total RNA purity and integrity detection,library construction,computer sequencing,sequencing data quality control,sequence comparison analysis,expression analysis,expression difference analysis,RNA-SEQ screening differential genes,GO analysis and KEGG analysis,differential gene screening and validation.The expression level of plin2 in kidney was verified by NCBI GEO Datasets.Steps to predict transcription factors upstream of PLIN2:first,use NCBI website（https://www.ncbi.nlm.nih.gov/）The upstream promoter sequence of PLIN2 was searched,and then the UCSC database was used（http://genome.ucsc.edu/）The transcription factors binding to PLIN2 promoter were predicted,and then the JASPAR database was used（http://jaspar.genereg.net/）Analysis of predicted transcription factors and PLIN2 upstream promoter sequence Binding sites.11、Statistical methodsThe experimental data were expressed as（mean±standard error）.statistical chart drawing and statistical data analysis were performed by graphapad 7.0 software;statistical analysis among experimental groups was conducted by two independent sample t-test and one-way ANOVA analysis of variance,and Tukey post-hoc test was used for comparison among groups.P<0.05 indicated that there was statistical difference between the two groups.Results1、Magnesium Hydride ameliorates renal injury induced by Glyoxylate in mice（1）Dose effect relationship of Magnesium Hydride in the treatment of crystal kidney injuryThrough the intervention of different doses of Magnesium Hydride on Glyoxylate induced crystal kidney injury mice,the renal tissues were stained with HE,Masson,Von Kossa.it was found that with the increase of Magnesium Hydride dose,the degree of renal injury was gradually reduced,and the crystal/Stone deposition was reduced.There was no significant difference in the improvement of 200mg/kg and 400mg/kg Magnesium Hydride,which laid the foundation for the follow-up animal experiments.（2）Magnesium Hydride improves renal function and renal tubular injury in crystalline kidney injury modelThe pathological results of renal tissue showed that the acute injury of renal tubule induced by Glyoxylate was obvious,accompanied by a large amount of calcium oxalate crystal deposition,and fibrosis related indicators FN,TGF-βwere significantly high expression,TUNEL staining showed that apoptosis cells were obvious.Magnesium Hydride treatment group not only improved the acute tubular injury,but also significantly reduced calcium oxalate crystal,FN,TGF-βand apoptotic cells Less.At the same time,the renal function of mice treated with Magnesium Hydride was improved.Magnesium Hydroxide group and corn oil group had no effect.2、Molecular hydrogen improves the effect of Sodium Oxalate on the viability and oxidative stress of tubular epithelial cells（1）The results of CCK8 indicated that molecular hydrogen could improve the decrease of TCMK-1 cell viability induced by Sodium Oxalate.（2）The ROS level in TCMK-1 cells co incubated with Sodium Oxalate increased and the mitochondrial membrane potential decreased.Molecular hydrogen could inhibit ROS induced by Sodium Oxalate and improve mitochondrial function by up regulatingmitochondrial membrane potential.3、Metabonomics analysis of mouse kidneyThe renal tissue homogenates（Con,Gly,GH,GOH,GV）of the five groups wereanalyzed by UPLC/Q-TOF-MS.the results showed that the renal injury model of calcium oxalate crystal had metabolic disorder,mainly including the changes of energy metabolism,phospholipid metabolism and amino acid metabolism pathway.Magnesium Hydridetreatment could reverse the above metabolic disorders to a certain extent,especially phospholipid metabolism.Phospholipid,as the main component of biofilm,wassignificantly reversed after Magnesium Hydride treatment,indicating that the treatment of Magnesium Hydride may be achieved through cell membrane protection.4、High throughput transcriptome sequencing of renal tissue and screening and validation of RNA-SEQ differential genesThe total number of differentially expressed genes was 2539,including 1543up-regulated genes and 996 down-regulated genes.GO analysis and KEGG analysis were performed on the differentially expressed genes.The results of q RT-PCR were consistent with the sequencing results.According to the results of metabonomics,PLIN2 gene,which is highly related to lipid metabolism,was selected as the next research object.5、Bioinformatics prediction of related transcription factors and validation in vitro and in vivoFour important upstream transcription factors of PLIN2 were screened bybioinformatics.They are STAT4,C-JUN,PPARγand PPARα.It was found that p-C-JUN was highly expressed in the kidney tissue of crystalline kidney and located in the nucleus of renal tubular epithelial cells.WB detection in vivo and in vitro also confirmed thatp-C-JUN was increased in the model group,and could be reversed after MagnesiumHydride/hydrogen treatment,which was consistent with PLIN2 trend.P-STAT was not highly expressed in the mouse model and was not localized in the epithelial nucleus.6、Magnesium hydride improves oxidative stress and inflammation in mice with crystalline kidney induced by calcium oxalateThe serum inflammatory indexes（TNF-α,IL-1β）,oxidative stress（GSH,NO）,lipid peroxidation（MDA,4HNE）in mice with crystal kidney induced by calcium oxalate were detected by ELISA.The results showed that magnesium hydride could effectively improve the inflammatory reaction,oxidative stress and lipid peroxidation levels of crystallinekidney injury.Conclusions1、Magnesium hydride can effectively alleviate the damage of crystalline renal tubular,improve renal function,improve the level of inflammation,oxidative stress and apoptosis.2、The therapeutic effect of Magnesium Hydride/Hydrogen may be achieved byscavenging ROS,inhibiting C-JUN activation,downregulating PLIN2,and improving lipid peroxidation and iron death. |
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