The Role And Related Mechanisms Of NRF2 In High-fat Diet-induced Fatty Liver Disease In Mice

Objective: With the improvement of the material living standard,the food achieved by Chinese people is more refined and the material types are more abundant,which results in the increase of the energy intake.The excessive intake of high-fried/sugar food will cause the energy surpluse,which changes the nutriment into poisonous substance.In addition to obesity,it will cause serious burden on other organs and impair homeostasis of the body.One of the main organs involved is the liver.Nonalcoholic fatty liver disease(NAFLD)mainly refers to a kind of fatty liver which is not induced by alcohol,drugs or other viruses,including simple steatosis,nonalcoholic steatohepatitis(NASH).Without effective intervention,the secondary fibrosis can progress to cirrhosis and even increase the incidence of hepatocellular carcinoma.NAFLD is a kind of metabolic disease,which is closely related to a variety of diseases such as insulin resistance,obesity and diabetes.Recently,the international liver disease expert group proposed that NAFLD should be renamed as MAFLD(Metabolic associated fatty life disease).According to the epidemiological survey,the prevalence of NAFLD in China has reached 29.2%,which is equal to that in Europe and the United States.What’s worse,the incidence of NAFLD is gradually getting younger.Due to the interaction between NAFLD and other chronic metabolic diseases,it has brought a great burden to the society.NAFLD has become a serious public health problem in China,so prevention and intervention are urgent.The main pathogenic factors of NAFLD are summarized as "second strike" theory,in which oxidative stress and inflammatory response are most important.Nuclear factor erythroid-2-related factor 2(NRF2)is a classical and efficient antioxidant transcription factor,which can regulate a variety of antioxidant and phase II detoxification enzymes against oxidative stress.With the development of NRF2 research,it has been found that NRF2 also plays important role in anti-inflammatory,glucose and lipid regulation.Previous studies utilizing global Nrf2-KO or constitutive Nrf2-activated mice showed that NRF2 plays a critical role in the development of NAFLD.However,due to the differences of mouse strain,exposure time and feeding condition,the final results are controversial and the mechanism is not clear.In addition,the lack of Nrf2 in the whole body does not accurately indicate the effect of NRF2 on liver and NAFLD,so we established cell-specific Nrf2 knockout mouse model.The main parenchymal cells of the liver include hepatocytes and Kupffer cells(macrophages in liver),closely related to lipid metabolism and inflammation.In the first part of our study,we focused on oxidative stress and lipid metabolism in hepatocyte specific Nrf2 knockout mice.In the second part,we studied the inflammatory response of macrophage specific Nrf2 knockout mice.Finally,we intend to find its specific molecular mechanism in primary hepatocytes.Through the three parts of the study,the aim is to find the role of NRF2 in NAFLD in different cell types,and provide a new theoretical basis for the prevention and treatment of NAFLD.Methods: 1.NAFLD induced by 12 weeks high-fat diet exposure in cell-specific Nrf2 knockout miceThe hepatocyte specific Nrf2 knockout(Nrf2(L)-KO)mice and the control group(Nrf2-loxP)mice born in the same nest were obtained by hybridization of Nrf2-loxP and albuminCre tool mice.Male and female 12 week-old mice were divided into two groups,respectively,one was chow diet group(CD),the other was high-fat diet group(HFD): Nrf2-Lox P,CD group,Nrf2(L)-KO,CD group,Nrf2-Lox P,HFD group,Nrf2(L)-KO,HFD group.Each group was fed with the corresponding diet for 12 weeks.Food intake and water consumption were recorded three times a week;the weight of mice was measured every week;and the body fat composition and blood glucose index were monitored.At the end of observation,the plasma and the key organs of mice were collected.The macrophage specific Nrf2 knockout(Nrf2(Mφ)-KO)mice and nest born control(Nrf2-Lox P)mice were obtained by hybridization of Nrf2-Lox P with Lysozyme2-Cre tool mice.Male 12-16 week-old mice of two genotypes were randomly divided into four groups: Nrf2-Lox P,CD group,Nrf2(Mφ)-KO,CD group,Nrf2-LoxP,HFD group,Nrf2(Mφ)-KO,HFD group,six mice in each group for 12 weeks.Other treatments are the same as above.2.Mice sample treatmentThe contents of free fatty acids,glycerol and triglycerides in plasma and liver tissues were detected by corresponding kits.Liver tissues fixed with 4% paraformaldehyde were stained with H&E,oil red and immunohistochemistry.The content of malondialdehyde in liver was determined.Total RNA was extracted from liver tissues,and transcriptional levels of classical genes in oxidative stress,glucose metabolism,mitochondrial oxidation,lipid metabolism,inflammatory response and fibrosis response were detected.Total proteins from liver tissues were extracted to detect the content of NRF2 and peroxisome proliferators activated receptors(PPARs).3.To study the molecular mechanism of lipid regulation by NRF2 in primary hepatocytesThe primary hepatocytes were extracted from Nrf2(L)-KO and control mice.Treated with PPARγ agonists,Pparg and its downstream gene expression were detected.To simulate physiological condition,saturated fatty acids were used to primary hepatocytes.PPARγ activity,the TG content in primary cells was measured,and the accumulation of TG was marked by fluorescence staining.The uptake efficiency of fatty acids was tracked by fluorescence.The mitochondrial capacity of two types of hepatocytes was measured by the Seahorse cell stress test.Transient transfer of PPARγ1 and PPARγ2 overexpressed plasmids into primary hepatocytes was used to detect the expression levels of lipid metabolism genes downstream of Pparg to improve its molecular mechanism.Results:1.Nrf2 deficiency in hepatocyte had no effect on the body weight,blood lipid and blood glucose of mice,but showed smaller livers,milder hepatic steatosis and less hepatic TG accumulation.The energy intake of mice in HFD group was higher than that in CD group(p < 0.05),but there was no difference between the two genotypes.The results of body weight and body fat mass were consistent with the trend of energy intake.At the end of 12 weeks exposure,glucose tolerance test was carried out in HFD group,and it was found that the loss of Nrf2 had no significant effect on glucose metabolism.We calculated the organ coefficient of the important organs according to the relatively stable absolute weight of the heart.It was found that the liver,paragonadal fat,and subcutaneous fat were significantly increased in the HFD group(p < 0.05),and there was no difference between the two types of fat in the two genotypes(p > 0.05),but the liver of the Nrf2(L)-KO group was significantly smaller than that of the control group(p < 0.05),which means the liver size was smaller.Triglycerides(TG)in liver decreased significantly in Nrf2(L)-KO,HFD groups(p < 0.05).Due to the individual differences of mice,we classified the results of H&E staining by NAS score analysis,and found that the lipid accumulation in hepatocytes of Nrf2(L)-KO group decreased after HFD.2.Nrf2 deficiency in hepatocyte had no effect on oxidative stress inflammation and fibrosis,but played important role in lipid metabolism.Consistent with the deficiency of Nrf2 in hepatocytes,the mRNA levels of Nrf2 and its downstream genes,such as NAD(P)H:quinone oxidoreductase 1(Nqo1),glutamatecysteine ligase catalytic subunit(Gclc)and hemeoxygenase 1(Ho1),were all decreased in the liver of Nrf2(L)-KO mice compared to Nrf2-Lox P under CD or HFD exposure,while HFD had no significant effect on these mRNA expression.The protein expression of NRF2 was similar to the results of mRNA levels.We measured the MDA level in the liver of four groups,and the results showed that there was no significant difference.In HFD groups,the mRNA levels of Ccl2 and Tnf increased significantly(p < 0.05),but there was no difference between the two genotypes.In the protein level detection of phosphorylation level of NF-κB(p65),it was found that Nrf2-Lox P,HFD group were significantly higher than those in CD group,but not in Nrf2 deletion group.Immunohistochemistry analysis against F4/80,a macrophage marker,showed that HFD exposure induced macrophage infiltration in the livers of Nrf2-Lox P control mice.In conclusion,HFD can induce inflammation,but the absence of Nrf2 will reduce it.There was no significant change in the detection of fibrosis related genes.The expression of the rate-limiting enzyme of β-oxidation,carnitine palmitoyl transferase(Cpt),was reduced to nearly 50% by HFD exposure in both Nrf2-Lox P and Nrf2(L)-KO genotypes.Importantly,the levels of Pparg2 mRNA were found to be induced by HFD exposure in both genotypes,whereas the expression of Pparg2 showed a trend to be lower in Nrf2(L)-KO mice compared to control mice.The protein expression of PPARγ was consistent with mRNA levels.3.Nrf2 deficiency in macrophages had no effect on mice exposed to 12 weeks HFDThe energy intake of HFD group was significantly higher than that of CD group(p < 0.05);there was no significant difference in drinking water;the trend of body weight was consistent with energy intake,but there was no significant difference between the two genotypes.The fat mass and blood glucose of mice exposed to HFD were significantly higher than those of CD group(p < 0.05),but there was no significant difference between Nrf2(Mφ)-KO and the control group.There was no significant difference between genotypes in glucose tolerance test at the end of HFD groups.After HFD,the organ index of liver,paragonadal fat and subcutaneous fat increased significantly(p < 0.05),but there was no difference between genotypes.Although the TG contents of livers in HFD groups increased,there was no difference between the two genotypes.No difference was found in the result of H&E stainings.The mRNA expression of Ccl2 and Tnf in the liver was significantly elevated by HFD exposure in both genotypes of mice.However,there were no significant difference in the mRNA expression between the genotypes either under CD or HFD exposure.Other mRNAs measured showed no significance among the four groups.4.Nrf2 deficiency in primary hepatocytes down-regulated PPARγ activation and lipogenesis,but had no effect on mitochondrial energy consumption.The mRNA and protein expression of PPARγ1 and PPARγ2 were significantly induced by ROSI or PIOG in Nrf2-Lox P hepatocytes.Nrf2(L)-KO hepatocytes showed dramatically attenuated mRNA and protein expression of PPARγ1 and PPARγ2 under vehicle or against-challenged conditions.In addition,the mRNA expression of Fabp4 and Cd36,which are known PPARγ target genes,Scd1 and Srebf1 displayed a similar pattern as PPARγ.Compared to Nrf2-LoxP cells,Nrf2(L)-KO hepatocytes were less responsive in their Pparg1 and Pparg2 mRNA induction.In agreement with the mRNA expression,the protein levels of PPARγ1 and PPARγ2 also displayed a trend of increase in both genotypes,while the Nrf2 deletion weakened the tendency.In addition,the mRNA expression of Fabp4,Scd1 and Fasn,the major downstream lipigenic genes of PPARγ,also showed significant increases in response to palmitate treatment in Nrf2-LoxP cells,whereas Nrf2(L)-KO hepatocytes had reduced induction in their expression.Following a 24-hrs palmitate treatment,the TG levels in Nrf2(L)-KO hepatocytes were much lower than those in Nrf2-Lox P cells.The rate of BODIPY uptake in Nrf2(L)-KO hepatocytes was slower than control cells.The mitochondrial function in hepatocytes,as measured by OCR and ECAR,showed no significant difference between the two types of cells.5.Overexpression of PPARγ1 or γ2 distinctively reverses the reduced expression of lipogenic genes caused by Nrf2 deficiency in primary hepatocytes.Pparg1 and Pparg2 were overexpressed in primary hepatocytes from Nrf2(L)-KO and Nrf2-Lox P mice.Accordingly,the direct downstream gene of PPARγ,Fabp4,increased in both Nrf2-deleted and control hepatocytes to the same extent.Consistent with this notion,the mRNA levels of Lpl and Scd1 and Fasn were also reversed by overexpression of Pparg1 and Pparg2,respectively.Conclusion:1.Nrf2(L)-KO mice fed with HFD showed smaller livers,milder hepatic steatosis and less hepatic TG accumulation.There was no significant effect on hepatic oxidative stress,but hepatic steatosis could induce inflammation.2.The loss of Nrf2 in macrophages had no significant effect on lipid metabolism,oxidative stress and inflammatory response of NAFLD induced by 12 weeks HFD exposure.3.Mechanistic investigations in primary hepatocytes indicated that the attenuation from HFD-induced NAFLD in Nrf2(L)-KO mice might be attributable to the decreased expression of PPARγ,PPARγ2 in particular.These findings demonstrate that NRF2-dependent expression of PPARγ plays a critical role in hepatocytes for the initiation of NAFLD induced by HFD exposure.