The Role Of C/EBPβ-TFAM Mediated Inflammation In Kidney Injury Induced By Arsenic And The Intervention Of Rosa Roxburghii Tratt

Objective:Arsenic is a common toxic metallike element widely found in nature.Long-term exposure to arsenic will lead to the damage of multiple organs and systems in the body.As the main excretory organ of arsenic,kidney,when damaged,can affect the excretion of arsenic and its metabolites in the body and promote the accumulation of arsenic in the body,resulting in the damage of multiple organs including the kidney.However,the mechanism of arsenic induced kidney injury is still unclear.The purpose of this study is to explore the effects of arsenic on kidney injury in population by utilizing the unique coal-burning arsenism disease resources in Guizhou Province.To investigate the role of C/EBPβ-TFAM and NLRP3 mediated inflammation in kidney injury induced by arsenic in rats.A rat model of kidney injury induced by arsenic exposure was established.In addition to the unclear pathogenic mechanism,another major problem facing the prevention and treatment of arsenic poisoning is the lack of specific therapeutic drugs.Based on the findings of the above mechanism studies,the intervention effect of Rosa roxburghii Tratt（RRT）on arsenic induced kidney injury is further discussed.At present,the diagnostic standard of endemic arsenic poisoning is determined according to the degree of skin damage.The previous field epidemiological study of the ward found that patients with arsenic disease were complicated with liver and kidney damage,and arsenic exposed patients may have developed liver and kidney damage before the obvious skin damage.Therefore,two groups of rat intervention models were designed to protect patients without clear diagnosis in the ward.Including antagonistic model and therapeutic model,the prevention and treatment effects of RRT on kidney injury caused by arsenic were discussed,providing reference for the prevention and treatment of health damage in patients exposed to arsenic in the future.Methods:1.Selection of survey points and survey objects:Taking Jiaole Village,Xingren County,Guizhou Province as the investigation site,according to the Diagnostic Criteria for Arsenic Poisoning（WS/T211-2015）,218 residents were identified as arsenic poisoning patients.In addition,158 residents in Shangbatan village with similar living habits and economic conditions and no history of burning high-arsenic coal were selected as the control group.According to the severity of the symptoms of arsenic poisoning,the patients were divided into mild arsenic poisoning group（87 cases）,moderate arsenic poisoning group（80 cases）and severe arsenic poisoning group（51 cases）.The content of urinary arsenic was determined by inductively coupled plasma mass spectrometry（ICP-MS）.Serum creatinine（SCr）was detected by automatic biochemical analyzer.KIM-1 and NGAL were detected by ELISA.2.Establishment of arsenic poisoning rat model:Twenty-four healthy and clean Wistar rats were randomly divided into 4 groups（including normal control group,low arsenic dose group,medium arsenic dose group and high arsenic dose group）with 6 rats in each group and half male and half female after one week of adaptive feeding.The control group was given deionized water intragastric administration,and the poisoning group was given Na As O₂aqueous solution of2.5(1/16LD₅₀),5(1/8LD₅₀)and 10(1/4LD₅₀)mg/kg·bw intragastric administration for four months.The morphological and structural changes of rat kidney were observed by hematoxylin-eosin（HE）staining.Western blot detection of neutrophil gelatinase-associated lipid carrier protein（NGAL）,CCAAT/enhancer binding protein beta（C/EBPβ）in rat kidney tissue,the expression level of mitochondrial transcription factor A（TFAM）,NLRP3 related protein（NLRP3,ASC,caspase-1）and its downstream inflammatory factor（IL-1β,IL-18）.Immunohistochemical staining was used to observe the localization and expression of related proteins in rat kidney tissue.3.The intervention rat model of RRT on arsenic poisoning:3.1 The antagonistic rat model of RRT on arsenic poisoning:Twenty-four healthy and clean Wistar rats were randomly divided into 4 groups(including control group A,high arsenic dose group,RRT group and high arsenic+RRT group with 6 rats in each group and half male and half female after one week of adaptive feeding.Control group A was the same as the normal control group in part 1.The high arsenic dose group was the same as the high arsenic dose group in part 1.RRT group was given 10 m L/kg·bw RRT,High+RRT group was given 10mg/kg·bw Na As O₂aqueous solution and 10m L/kg·bw RRT.The rats were given intragastric administration once a day,six days a week,for four months.3.2 The treatment rat model of RRT on arsenic poisoning:Eighteen healthy and clean Wistar rats were randomly divided into 3 groups（including control group B,recovery group and treatment group）with 6 rats in each group and half male and half female after one week of adaptive feeding.The rats in control group B were treated with deionized water for 5.5 months.The recovery group was administered with 10mg/kg·bw Na As O₂aqueous solution for four months and then given deionized water for 1.5 months.The treatment group was administered 10mg/kg·bw Na As O₂aqueous solution for four months and 10 m L/kg·bw RRT for 1.5 months.The rats were given intragastric administration once a day,six days a week,for 5.5 months.Index detection is the same as part 2.4.The intervention effect of RRT on kidney injury in patients with arsenic poisoning:RRT intervention investigation sites and investigation objects selection:Taking Jiaole Village,Xingren County,Guizhou Province as the investigation site,the sample of arsenic poisoning intervention group was used to select arsenic poisoning patients（46 cases）as intervention objects by using typical sampling method,and the patients were given RRT intervention for 3 months.Index detection is the same as part 1.5.Statistical Analysis:Using SPSS 25.0 statistical software for data analysis,first to normality test,sample normally distributed,data are expressed as（?）±s;One-way analysis of variance was used for comparison between groups.The Bonferroni test was used when the variances were homogeneous,and Tamhane’s T2test was used when the variances were not homogeneous.When the samples were not normally distributed,the median（interquartile spacing）was used to represent the data,and the non-parametric test was used for comparison between groups.Pearson correlation coefficient was used for correlation analysis,and the difference was statistically significant when P<0.05.Results:1.The effect of arsenic on kiendy function and its mechanism:1.1 Effects of arsenic on human kidney function:In order to understand the status of kidney injury in the residents of the arsenic poisoning area,205 patients in the disease area and 150 patients in the control group were included in this study,and the concentration of arsenic in their urine was detected.The results showed that the concentration of arsenic in the arsenic poisoning population was significantly higher than that in the control group.The results of renal function indexes showed that the concentrations of NGAL（6.73μg/m L）,KIM-1（297.32 pg/m L）and SCr（0.80 mg/d L）in urine of arsenic poisoning group were significantly higher than those in control group（6.38μg/m L,280.93 pg/m L,0.72 mg/d L）.The e GFR（94.49 m L/min/1.73m²）was significantly lower than that of the control group（99.31 m L/min/1.73m²）.Correlation analysis results showed that with the increase of urinary arsenic concentration,urinary NGAL and SCr concentrations increased significantly,and e GFR was significantly negatively correlated with UAs.1.2 Preliminary study on the mechanism of kidney injury induced by sodium arsenite exposure in rats:On the basis of the discovery in the first section of the study that arsenic exposure leads to decreased renal function in the population,aiming at the unclear mechanism,a rat model of arsenic exposure is established and the mechanism of kidney injury induced by arsenic is preliminarily discussed.The results show that:（1）Compared with the control group,the expression of NGAL in the kidney tissues of the arsenic exposure group was increased,and the structural integrity of the kidney tissues was damaged.There were different degrees of glomerular vacuolation,tubular dilatation and inflammatory cell infiltration.（2）Inflammation plays an important role in the arsenic induced kidney injury:the expression levels of IL-1βand IL-18 in the medium arsenic dose group were significantly increased compared with the control group（P<0.05）,but there were no significant differences in the expression levels of IL-1βand IL-18 in the high arsenic dose group compared with the control group.Immunohistochemical results showed that,compared with the control group,the expression of IL-1βand IL-18 in renal tubular epithelial cells was significantly increased in the high and medium arsenic dose groups（P<0.05）.（3）Sodium arsenite up-regulates the expression of C/EBPβand TFAM,and activates the NLRP3 inflammasome:The NLRP3 inflammatory body related protein expression in kidney tissue of rats in high arsenic dose group was significantly higher than that in control group（P<0.05）,and the protein expressions of C/EBPβand TFAM in kidney tissue of rats in high arsenic dose group were significantly increased compared with the control group（P<0.05）.The immunohistochemical results showed that compared with the control group,the expressions of NLRP3 and caspase-1 in renal tubular epithelial cells were significantly increased in arsenic dose groups,and the protein expressions of c/EBP-βand TFAM were significantly increased,and their expression levels were gradually increased with the increase of arsenic dose（P<0.05）.2.Intervention effect of RRT on kidney injury induced by arsenic:2.1 Intervention of RRT on arsenic induced kidney injury in rats:Based on the preliminary study of 1.2 mechanism,it is found that arsenic may promote the expression of C/EBPβand TFAM,activate NLRP3 inflammasome,trigger inflammatory response,and induce kidney injury.In this part,RRT with anti-inflammatory effect is selected for intervention,and the results show that:（1）RRT can relieve the kidney injury caused by arsenic to a certain extent,reduce the content of NGAL in the kidney tissue of rats,and improve kidney function.（2）Antagonistic effect of RRT on kidney damage of arsenic exposed rats:Compared with the high arsenic does group,the expressions of c/EPBβ,TFAM,NLRP3,ASC,caspase-1,IL-1βand IL-18 in kidney tissue of rats in RRT group were significantly decreased,and the differences were statistically significant.Immunohistochemical results showed that compared with high arsenic group,the expressions of NLRP3 and caspase-1 in the kidney tissues of RRT group and high+RRT group were significantly decreased,and the difference was statistically significant（P<0.05）.（3）The therapeutic effect of RRT on kidney injury in rats exposed to arsenic:compared with the recovery group of arsenic poisoning,the expressions of C/EBPβ,TFAM,NLRP3,ASC,caspase-1,IL-1βand IL-18 in the kidney tissue of rats treated with RRT were significantly decreased,and the differences were statistically significant.Immunohistochemical results showed that the contents of NLRP3 and caspase-1 in kidney tissue of rats in treatment group were significantly lower than those in recovery group,and the difference was statistically significant（P<0.05）.2.2 Observation of intervention effect of RRT on kidney injury in patients with arsenic poisoning:Based on the findings in 2.1,RRT can reduce kidney inflammation in rats with arsenic poisoning,improve kidney pathological changes caused by arsenic,and alleviate kidney injury.In this part,RRT is a natural dietary supplement with rich nutritional components.In order to promote the application of RRT in human population,samples of people with arsenic poisoning intervention in our research group were used to detect related indicators of kidney injury and observe the intervention effect of RRT.The results found that:Compared with before intervention,urine KIM-1（252.27 pg/m L）and NGAL（4.99μg/m L）concentrations of arsenic poisoning patients after intervention were significantly lower than before intervention（404.9 pg/m L,6.52μg/m L）,SCr（0.62 mg/d L）concentrations were significantly lower than before intervention（0.68 mg/d L）.e GFR（123.04m L/min/1.73m²）level was significantly increased（P<0.05）.Conclusions:1.Arsenic exposure can cause an increase in SCr,a decrease in e GFR,and an increase in urinary NGAL and KIM-1,early markers of kidney injury,leading to a decline in kidney function.The risk of renal function decline is associated with an increase in UAs concentration.2.Arsenic exposure may up-regulate the expression of C/EBPβand TFAM,activate the NLRP3 inflammasome,promote the release of inflammatory cytokines IL-18 and IL-1β,trigger inflammatory response,and induce kidney injury.3.RRT can improve the kidney tissue and structure lesions in rats induce by arsenic,plays a role in preventing and treating arsenic-induced kidney injury,the mechanism may be related to C/EBPβ-TFAM mediated inflammatory response.4.RRT can reduce the concentrations of SCr,urinary KIM-1 and NGAL in patients with arsenic poisoning,raise the e GFR,improve kidney function.