Experimental Study Of Tea Polyphenols Regulating MiR-155 Against Inorganic Arsenic-induced Liver And Kidney Toxicity

Objective:Arsenic（As）exposure has seriously endangered the health of the population.The NF-E2-related factor 2（Nrf2）signaling pathway is widely involved in various toxic mechanisms of arsenic,including oxidative damage,inflammation,histopathology,carcinogenesis and teratogenesis.Tea polyphenol（TP）is a general term for polyphenols in tea,which has a wide range of biological activities such as antioxidant,anti-inflammatory and anti-tumor.Micro RNA（miRNA）is a kind of non-coding small RNA that is widely involved in regulating various biological processes in the body.More and more studies have shown that TP could further regulate the expression of oxidative stress,inflammation,tumor and other related genes by regulating the expression of miRNA.However,whether TP could regulate the expression of miR-155 to exert an antagonistic effect on the toxicity of inorganic arsenic has not been reported yet.Therefore,this experiment intends to use inorganic arsenic exposure and TP intervention to establish the corresponding animal model.On the one hand,observe whether TP can antagonize the pathological changes and functional damage of liver and kidney tissue induced by inorganic arsenic;on the other hand,observe the effect of TP intervention on the influence of Nrf2 signaling pathway in liver and kidney tissue of mice exposed to arsenic.It is planned to use inorganic arsenic to treat HK-2cells and EGCG intervention to establish a corresponding cell model to observe whether TP can regulate miR-155 to participate in the process of antagonizing the toxicity of inorganic arsenic.Methods:1.At the end of the experiment,recorded the mouse weight,liver and kidney weights,and got the corresponding organ indexes.2.The atomic fluorescence method to determine the total arsenic content（T-As）in tissues and urine 3.Hematoxylin and eosin staining（HE）was used to observe the pathological morphology of liver and kidney tissues.4.The kit detected the alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,blood urea nitrogen（BUN）,serum creatinine（SCRE）and whole blood glutathione（glutathione,GSH）level.5.Calculated the total number of white blood cells（WBC）,mononuclear macrophages（MONO）,neutrophils（Neutrophils,NEUT）,lymphocytes（LYMPH）in the alveolar lavage fluid（ALF）using the cytometer.6.Western Blotting and qPCR experiments to determine the Nrf2 signaling pathway gene transcription and protein expression levels in liver and kidney tissues of each experimental group.7.The database predicted the target miRNAs of genes related to the Nrf2 pathway.8.The qPCR experiment quantitatively detected the expression of miRNAs in each group（miR-155,miR-122,miR-101,miR-21,miR-223,miR-125）.10.Cell counting test（Cell counting Assay,CCK-8）detected the viability of human kidney epithelial cell 2（Human kidney epithelial cell 2,HK-2）treated with NaAsO₂and epigallocatechin gallate（Epigallocatechin gallate,EGCG）11.Used Lipofectamine 3000 to transfect miR-155 mimic into HK-2 cells.12.qPCR and Western Blotting were used to detect the gene transcription and protein content of Nrf2 signaling pathway and the expression level of miR-155 in HK-2 cells in each experimental group.Results:1.The effect of TP intervention on the body weight,organ coefficient,tissue and urine T-As of arsenic-exposed mice.Compared with the control group,the body and organ weight of the mice after NaAsO₂exposure did not change significantly.Compared with the NaAsO₂exposure group,the body and organ weight of the mice decreased slightly after TP intervention（P<0.05）;T-As in liver tissue decreased significantly（P<0.05）;T-As in urine increased significantly（P<0.05）.2.The effect of TP intervention on the morphological structure of liver and kidney tissue in arsenic-exposed mice.Compared with the control group,NaAsO₂exposure caused nucleocytoplasmic separation and aquatic degeneration in liver cells,venous congestion and renal tubular atrophy in kidney cells;and TP intervention all improved the above pathological changes to a certain extent.3.The effect of TP intervention on the changes of liver and kidney tissue function in arsenic-exposed mice and the number of inflammatory cells in ALF.Compared with the control group,NaAsO₂exposure caused an increase in the levels of ALT,AST,BUN,and SCRE in mice,a decrease in GSH,and an increase in the number of WBC,MONO,and NEUT in ALF（P<0.05）;this kind of increasing or decreasing trend was reversed after TP intervention（P<0.05）.4.The effect of TP intervention on Nrf2 signaling pathway in liver and kidney tissue of arsenic-exposed mice.Compared with the control group,NaAsO₂exposure induced an increase of NRF2,HO-1 protein levels and Nrf2,Ho-1 mRNA levels in liver（P<0.05）.Compared with the NaAsO₂exposure group,TP intervention increased liver NRF2,HO-1 protein expression levels and Ho-1 mRNA level（P<0.05）.Compared with the control group,NaAsO₂exposure induced an increase in the protein levels of NRF2,HO-1 and GCLM in the kidney（P<0.05）.Compared with the NaAsO₂exposure group,TP intervention increased the protein levels of kidney NRF2,HO-1,GCLM and the mRNA levels of Ho-1 and Gclm（P<0.05）.5.The effect of TP intervention on the nucleus NRF2 protein in the liver and kidney tissues of arsenic-exposed mice.Compared with the control group,NaAsO₂exposure induced an increase of NRF2 protein into the nucleus in the liver and kidney tissue of mice;compared with the NaAsO₂exposure group,NRF2 protein into the nucleus increased after TP intervention.6.The effect of TP intervention on the expression of target miRNAs related to the Nrf2 signaling pathway in arsenic-exposed mice.The expression of liver miR-155 and miR-101 did not show corresponding statistical difference between each group.Compared with the control group,NaAsO₂exposure increased the expression levels of kidney miR-155,miR-125,miR-21,miR-223,and miR-101（P<0.05）;compared with NaAsO₂exposure,the miRNAs mentioned above after TP intervention and the level of miR-122 decreased（P<0.05）.7.The effect of simple EGCG treatment and NaAsO₂exposure on HK-2 cell viability.Compared with the control group,when NaAsO₂treated HK-2 cells at 0.5,1,2.5,5,10μM,the cell viability was not affected,while at 25,50,100μM,the cell viability was significantly decreased（P<0.05）.When EGCG was 5,10,25,50,100μM,cell viability was not affected,and cell viability increased at 200,400μM（P<0.05）.8.The effect of EGCG intervention on the transcription and protein expression levels of Nrf2 pathway-related genes in HK-2 cells exposed to NaAsO₂.Compared with the control group,after NaAsO₂exposed HK-2 cells,the protein levels of NRF2,HO-1,GCLM and the mRNA levels of Ho-1 and Gclm increased（P<0.05）;compared with the NaAsO₂exposure group,after EGCG intervention,the protein expression and transcription level of the above genes were significantly improved（P<0.05）.9.The effect of EGCG intervention on the expression of miR-155 in HK-2 cells exposed to NaAsO₂.Compared with the control group,NaAsO₂exposure increased the expression level of miR-155 in HK-2 cells（P<0.05）.Compared with the NaAsO₂exposure group,EGCG intervention reduced the above-mentioned upward trend（P<0.05）.10.The effect of miR-155 over-expression on the Nrf2 pathway related gene transcription and protein expression levels of EGCG intervention in NaAsO₂exposed HK-2 cells.Compared with the EGCG intervention group,the Nrf2,Ho-1,Gclm gene transcription and protein expression levels of the miR-155 negative control group（NC group）did not show corresponding statistical differences,while the transcription and protein expression levels of Ho-1 and Gclm genes decreased correspondingly in the miR-155 over-expression group（Mimics group）（P<0.05）.Conclusion:1.TP could antagonize the pathological,functional and oxidative damage of liver and kidney tissues caused by inorganic arsenic.2.TP could activate the Nrf2 signal pathway of inorganic arsenic exposed liver and kidney tissues and HK-2 cells.3.TP could regulate miR-155 to target at the Nrf2 signal pathway,therefore participating in antagonizing the kidney toxicity of inorganic arsenic.