The Role And Mechanism Of KLF4 In The Improvement Of Hepatic Steatosis By Metformin

Nonalcoholic fatty liver disease(NAFLD)is in addition to alcohol and other specific,liver injury may be caused by excessive fat deposition in liver cell as the main characteristics of a kind of clinical syndrome in the 21 st century,with the progress of science and technology and people’s living standards improve,the incidence of NAFLD rise year by year,has now become the world within the scope of the most common chronic liver disease Epidemiology shows the global prevalence of NAFLD is approximately 25%,by 2018,the incidence of NAFLD in our country is as high as 29.2%,has become China’s first big chronic liver disease The pathogenesis of NAFLD have the characteristics of diversity and complexity,insulin resistance,lipid metabolism disorder,oxidative stress,inflammatory factors may result in the occurrence of NAFLD In addition,will also affect the genetic environment,yet people still know a little research on the pathogenesis of NAFLD For the treatment of NAFLD is relatively limited at present,mainly in the control diet,increasing sports intervention guidance in the way of life in the patients,exclusive first-line drugs for the treatment of NAFLD has not been found Metformin is the first-line drugs of choice for treatment of type 2diabetes,has been in clinical use for nearly 60 years,about 70% of the patients with type 2 diabetes associated with NAFLD,numerous studies have confirmed that metformin can effectively reduce the patients’ serum low density lipoprotein cholesterol,free fatty acids,triglycerides and lipid levels,improve liver fatty degeneration,but the lipid-lowering mechanism is still not clear Kruppel sample factor 4(Kruppel-like factor4,KLF4)is a kind of multi-functional transcription factor of zinc refers to the structure,process,participate in the regulation of a variety of cell biology studies hint KLF4 play an important role in insulin resistance This study aims to KLF4 as the breakthrough point,to explore the lipid-lowering effect of metformin mechanism,to in-depth study of the pathogenesis of NAFLD,choose effective drug targeted therapy,for clinical provides the theoretical foundation to develop effective treatments.Objective: To explore the effect and mechanism of KLF4 on the improvement of triglyceride accumulation in liver by metformin.Methods:1.To C57 BL / 6 j male 8 weeks model group mice were divided into control group and experimental group conventional feed,the control group and experimental mice model group were given a high-fat diet feeding,feeding 16 weeks,the successful establishment of fatty liver in mice model group give metformin(300 mg/kg.Day lavage treatment of 4 weeks,the control group and model group are saline lavage treatment volume,record the change of the mice body weight every week Finally,the serum of mice was extracted and separated by the way of eyeball blood collection,and the liver weight was weighed.The changes of triglyceride in serum and liver were detected,and the frozen sections of liver tissue were stained with oil red O,which proved the lipid-lowering effect of metformin on animal level.2.The hepatocytes were treated with 0.5m M oleic acid for 24 h to establish a fatty liver cell model and the model was treated with 1m M metformin for 24 h.The lipid-lowering effect of metformin on the cell level was demonstrated by oil red O staining and TG content determination.3.The 8-week C57BL/6N male mice were divided into two groups.After 16 weeks of high-fat diet,the mice were treated with AAV-SHKLF4 mediated KLF4 knockdown adeno-associated virus(AAV-SHKLF4)control virus(AAV-GFP)via tail vein for 12 days.The mice in both groups were given metformin gavage(300mg/kg.day)for 4 weeks.4.Used to express KLF4 adenovirus vector(Ad-GFP-KLF4)knock and low KLF4 expression of si RNA transfection fatty liver cell model,by determine the content of intracellular triglyceride and oil red O staining,analysis of KLF4 expression regulation of fatty degeneration of liver cell function Then use the 1 mm metformin treatment,from cellular level that KLF4 mediated metformin lipid-lowering effect.5.The expression of KLF4 CPT-1a after metformin intervention was detected by Western blot real-time fluorescence quantitative PCR and other methods.6.The interaction between miR378a-5p and KLF4 and metformin was detected by Western blot real-time quantitative PCR.Results:1.Metformin can significantly reduce TG accumulation in fatty liver models in vitro and in vivoLiver into general morphology observation and oil red O staining results showed that metformin treatment group of mice liver weight index and hepatic TG content reduce,suggests that metformin can significantly reduce the HFD fatty degeneration of liver cells in mice induced by oil red O staining and TG content shows that metformin can obviously improve the liver steatosis induced by oleic acid.2.KLF4 is an important regulator of TG metabolism in liver.The expression of KLF4 was significantly decreased in fatty liver models in vitro and in vivo.Western blot results showed that KLF4 expression was significantly decreased in the liver of mice in the HFD group compared to the control group,while it was significantly increased after 4 weeks of metformin treatment.In oleacid-induced fatty liver cells,KLF4 expression was significantly decreased while metformin treatment increased KLF4 expression.3.Metformin can increase the expression of KLF4,knock KLF4 expression is low attenuation of metformin lipid-lowering effect.Through KLF4 expression of adenovirus vector(Ad-GFP-KLF4)knock and low KLF4 expression of si RNA respectively too low expression and knock KLF4 expression of oil red O staining and TG content detection can be found that KLF4 expression can significantly improve liver steatosis,knock on low KLF4 expression can decay metformin lipid-lowering effect Through the study of the general morphology observation of the liver with oil red O staining,according to the results found that low levels in the body to knock KLF4 The expression of metformin can also attenuate the lipid-lowering effect of metformin4.By down-regulating miR-378a-5p,metformin promoted the expression of KLF4,resulting in the increased expression of CPT1 A,and promoted the oxidative decomposition of fatty acidsThe qRT-PCR results showed that the expression of miR-378a-5p was increased in the fatty liver cell model,while metformin could down-regulate its expression.Western blot and q RT-PCR results showed that miR-378a-5p could negatively regulate the expression of KLF4.Conclusions:1.Metformin can significantly reduce TG accumulation in fatty liver models in vitro and in vivo.2.KLF4 is an important regulator of TG metabolism in liver.KLF4 expression was significantly decreased in fatty liver models in vitro and in vivo.Overexpression of KLF4 can improve hepatocyte steatosis.3.Metformin can up-regulate the expression of KLF4,and knock down the expression of KLF4 attenuate the lipid-lowering effect of metformin.4.By down-regulating miR-378a-5p,metformin promoted the expression of KLF4,resulting in the increased expression of CPT1 A and promoting the oxidative decomposition of fatty acids.