Function And Mechanism Of Thioredoxin Reductase 3 In Liver Fibrosis Process

Selenium（Se）is an indispensable trace element as previous studies have shown that s e-lenium functions in the body to resist oxidative stress,anti-inflammatory,improve body i m-munity and regulate the reproductive system.More than 40 countries in the world are short of selenium,and China is one of the"worst-hit areas".From the point of our territory plate di s-tribution,72%of our territory belongs to the selenium deficiency or low seleniu m area,seri-ous lack of selenium content in soil,lead to selenium deficiency regional plant selenium co n-tent,then by the food chain,livestock meat,eggs and milk and other products,followed by intake of selenium deficiency plants and livestock and pou ltry products and cause selenium deficiency.Selenium exerts its biological function in the form of s elenoprotein,and Txnrd3 is a member of the thioredoxin reductase family,participates in antioxidant defense and plays an important role in the reproductive system,but the function of Txnrd3 in liver disease and liver fibrosis is unclear.Liver fibrosis is a disease that often transforms into cirrhosis and liver cancer,with i n-creasing incidence,and is closely associated with reduced antioxidant capacity,which is closely relative to non-alcoholic steatohepatitis,hepatitis B/C and hepatitis induced by bil i-ary tract disease.All of these factors can develop a chronic inflammation in the liver,leading to an abnormal wound-healing response.Fibrogenesis is initiated by the activation and pro-liferation of myofibroblasts,as activated myofibroblasts are the main source of the extrace l-lular matrix when causing liver damage.Txnrd3,as a member of the th ioredoxin reductase family,is involved in antioxidant defen se and has been shown to play an important role in the reproductive system,but its function in liver disease as well as in liver fibrosis is unknown.Therefore,we constructed Txnrd3 knockout mice for the first time.In this study,80 mice including wild-type and Txnrd3^-/-genotype were randomly divided into two groups,given normal water and 30%TAA water,fed 12 weeks.Then liver tissue and serum were collected.Masson,PAS staining,HE and electron microscopy was used to confirm establishment of li v-er fibrosis model.AML12 and hepa1-6 were cultured in vitro and Txnrd3/Anxa2 overexpre s-sion and knockdown models were constructed to explore the effects of their altered expre s-sion in calcium homeostasis genes,cell pyropt osis,programmed necrosis and iron deat h in AML12 and hepa1-6 cells and liver fibrosis models.Co-IP was applied to screen and verify the interacting proteins of Txnrd3 and study the biological functions of Txnrd3 and its interacting proteins.The study results are as follows:（1）Liver pathological histology and ultrastructural observation showed that TAA in-duced liver cell swelling,cytosolic cavitation,necrosis and nuclear fragmentation in the liver tissue of wild-type mice.Mitochondria in TAA treated mice,with gross ER swelling and ma s-sive hyperplasia of synovial endoplasmic reticulum,and more significant damage in homoz y-gous mice.Msson and Sirius red staining showed a significant increase in collagen f ibers in TAA,and Prussian blue staining in TAA.These results showed that liver damage was exacer-bated by Txnrd3 knockdown.（2）The antioxidant level test results showed that the activity of antioxidant enzyme SOD,CAT,T-AOC and GSH in wild type mouse liver of TAA drinking water group was si g-nificantly reduced compared with the control grou p,while the content of malondialdehyde（MDA）increased,while knockdown Txnrd3 could significantly reduce the content and activ i-ty of antioxidant enzymes,resulting in reducing the antioxidant capacity of liver and aggr a-vating the occurrence of oxidative damage.The results showed that Txnrd3 knockdown could exacerbate TAA-induced liver oxidative stress.（3）Results of Co-IP and LC-MS showed an interaction relationship between Txnrd3and Anxa2.Through bioinformatics analysis,Txnrd3 and its interacting proteins were mainly enriched in cellular signaling pathways including ER protein progression,calcium tr ansport,Alzheimer’s disease,Parkinson’s disease,prion disease,amino acid biosynthesis,carbon m e-tabolism,glycolysis/glycogenesis.Molecular docking results showed that the energy required for the interaction between Txnrd3 and Anxa2 is-530.89k J,which means that the possibility of mutual interaction is great.In vitro,the overexpression or knockdown of Txnrd3 signif i-cantly increased or inhibited the expre ssion of Anxa2 in hepa1-6/AML12 cells.The above results indicate a targeted regulatory relatio nship between Txnrd3 and Anxa2.（4）The expression of IP₃R₂,BIP,PERK,and RyR increased m RNA,and the difference of homozygous mice（P<0.01）;IP₃R₂,RyR and IRE1 proteins were significantly increased compared with TAA treatment,but only IP₃R₂ was most significant after TAA treatment.Txnrd3 overexpression of hepa1-6 cells significantly upregulated the expression of IP₃R₂,PERK and BIP at m RNA level（P<0.01）,but PERK,XBP1 and BIP m RNA in AML12 cells（P<0.05）,while the m RNA expression of IP₃R₂ was reduced（P<0.05）.Meanwhile,the ex-pression of IP₃R₂ also decreased at the protein level.The results showed that Txnrd3 knoc k-down was able to promote TAA water-induced mouse liver by causing ER calcium homeost a-sis imbalance.（5）Expression of pyroptosis channel genes NLRP3,GSDMD,ASC,Caspase1 and IL-1βin wild-type mice of TAA drinking group is differentially increased compared with NC homozygous group.With NLRP3,ASC,Caspase1 increased in protein level.In Txnrd3knockdown hepa1-6 cell model,the expression of Caspaspase1,NLRP3 and GSDMD was significantly decreased,while that in AML12 cells,Caspase1 and GSDMD apoptosis in AML12 cells（P<0.01）.These results showed that Txnrd3 knockdown increased liver damage and liver fibrosis in mice by regulating pyroptosis channels.（6）Genes related to cell programmed necrosis pathway in the liver of wild-type mice in the TAA drinking water group.The expression of cellular programmed necrosis-related genes RIPK1,RIPK3;RIPK3 and MLKL in the liver of TAA water-induced liver fibrosis was significantly increased at the m RNA level versus control.The Txnrd3 knockdown group of hepa1-6 cells showed the opposite trend of RIPK1,RIPK3,MLKL and reduction in PGAM5expression（P<0.01）.Interestingly,the overexpre ssion of Txnrd3 significantly reduced the expression of MLKL,RIPK3 and PGAM5 at the m RNA level in AML12 cells;these results showed that Txnrd3 knockdown further exacerbated liver damage and li ver fibrosis in mice by upregulating genes related to cell programmed necrosis channels.（7）Genes related to the iron death pathway in the liver of wild-type mice in the TAA drinking water group,the m RNA expression abundance of TFRC,PTGS2 and SLC7A11 was significantly increased in TAA water-induced liver fibrosis mice（P<0.01）;a significant in-crease in expression abundance of homozygotes with significantly increased expression at the m RNA level versus the control group.The TFRC and PTGS2 showed a very significant d e-cline;in the AML12 cells,expression of the iron death-related genes TFRC,PTGS2,SLC7A11,FTH1 and FTL were significantly increased at the m RNA level upon Txnrd3 ove r-expression（P<0.01）.However,the GPX4 expression was reduced.The above results indica t-ed that Txnrd3 knockdown exacerbated liver damage and liver fibrosis in mice by activating the iron death pathway.In conclusion,results showed that Txnrd3 overexpression induced an imbalance in calc i-um homeostasis through IP₃R₂ channel,with increased ER stress and oxidative stress,cau sing pyroptosis and necrosis in hepa1-6 cells.The interacting protein Txnrd3-Anxa2 can cause pyroptosis and necrosis in hepa1-6 cells.This study provides insight into the biological fun c-tion of Txnrd3 in liver and provides a rationale for the early clinical diagnosis of liver fibr o-sis and cancer.This study provides insight into the biological function of Txnrd3 in liver di s-ease and provides a rationale for the early clinical diagnosis of liver fibrosis and ca ncer.The results of this study clarify the biological function of Txnrd3 and also pr ovide new targets for the prevention and treatment of liver fibrosis for further exploration of Txnrd3 function.