Regulating HDAC1/TAF9 Pathways By Danshensu Alleviates Non-alcoholic Fatty Liver Disease

Background: Nonalcoholic fatty liver disease is a reversible disease that is not associated with alcohol consumption and is characterized by hepatic steatosis and lipid droplet accumulation.The incidence of nonalcoholic fatty liver disease has increased due to changes in diet.Studies have shown that TBP-associated factor 9 is a component of the complex TFIID of the core promoter element and has the effect of directly regulating the transcription of the target genes.It has been found that after knocking out TBP-associated factor 9,the lipid droplets in the Drosophila body become larger,and acetylated TAF9 may lose activity.Histone deacetylases(HDAC1),which has the ability to deacetylate histones and non-histones,may affect angiotensin II and its receptors.DSS,an active ingredient extracted from the root of the Danshen(Salvia miltiorrhiza),has been used to treat cardiovascular disease.In addition,the renal protective effect of DSS has previously been associated with the suppression of oxidative stress,inflammation,and fibrosis.TAF9 has been shown to be a target protein of HDAC1,its deacetylated form is directly affected by HDAC1.In addition,HDAC1 can be regulated by polyphenolic compounds.However,the role of the HDAC1/TAF9 pathway in NAFLD induced by high fat diet(HFD)and whether DSS protects NAFLD by affecting HDAC1/TAF9 pathway has not been reported yet.Objective: To explore the therapeutic effect of Danshensu on nonalcoholic fatty liver disease and its related molecular mechanisms,as well as to study the effect of HDAC1 on TAF9 in NAFLD.Methods:1.(1)50 male Sprague-Dawley rats weighing 180-220 g were randomly divided into the following five groups:(A)control group;(B)DSS(40 mg/kg)group;(C)high-fat model group;(D)model+ DSS low-dose(20 mg/kg)group;(E)model+ DSS high dose(40mg/kg)group.The rats were fed with HFD for 10 weeks to simulate the NAFLD model,and the rats of B,D and E were gavaged with DSS every day.After 10 weeks,blood was collected from the abdominal aorta,and liver tissue samples were isolated and stored in a negative 80 refrigerator Alanine Aminotransferase(ALT),aspartate Aminotransferase(AST),triglycerides(TG)and total cholesterol(Total Cholesterol,TC)in rat serum were dectected.Liver tissue specimens were weighed,and liver pathological changes were examined by H&E and Oil Red O staining.Electron microscopy was used to observe the accumulation of lipid droplets in liver tissue.Western blotting was used to detect liver TAF9,HDAC1,Acetyl-TAF9,CPT1 A,ACOX1,PPARa,ACSL1,ADRP and β-actin protein levels.The m RNA levels of TAF9,HDAC1,CPT1 A,ACOX1,PPARa,ACSL1,ADRP and β-actin in liver tissues were detected by q RT-PCR.2.(1)AML-12 cells were divided into five groups: control group;PA group(0.4m M);the DSS pretreatment(2.5 μM,5 μM,10 μM)+ PA group.DSS was incubated with the cells for 6 hours,then treated with PA for 24 hours and finally tested for cell viability by CCK-8 method.(2)AML-12 cells were divided into four groups: control group;DSS(10 μM)group;PA(0.4 m M)group;DSS(10μM)+ PA group.Nile Red staining was used to detect lipid accumulation in AML-12 cells.3.(1)A total of 40 C57BL/6 mice were randomly divided into the following four groups: control group;TAF9 lentivirus group(lentivirus,LV);high fat model group;LV-TAF9+ high fat model group.The mice were fed on a high-fat diet for 8 weeks,and TAF9 was overexpressed by injection of lentivirus into the tail vein before modeling.After 8 weeks,blood was taken from the abdominal aorta,and liver tissue samples were isolated and stored in a negative 80 refrigerator.(2)AML-12 cells were divided into four groups: control group;pcDNA-TAF9 group;PA group;PA+pc-TAF9 group.Western blotting was used to detect TAF9,β-actin,CPT1 A,ACOX1,PPARa,ACSL1 and ADRP protein levels.4.(1)AML-12 cells were randomly divided into five groups: control group;PA group;PA+DSS(10 μM)group;si RNA HDAC1+PA group;si RNA HDAC1+PA+DSS(10 μM)group.After transfection,DSS was incubated with the cells for 6 hours,and then induced with PA(0.4 m M)for 24 h to extract the whole protein from the cells.The protein expression levels of TAF9,HDAC1,β-actin,Acetyl-TAF9,CPT1 A,ACOX1,PPARa,ACSL1 and ADRP in AML-12 cells were detected by Western Blotting and TAF9 was used in the control group and PA group by immunoprecipitation.The levels of HDAC1 and β-actin were detected.(2)AML-12 cells were randomly divided into 5 groups: control group;PA group;PA+pc DNA-HDAC1 group;PA+pc DNA-HDAC1+si-Control group;PA+pc DNAHDAC1+si-TAF9 group.After transfection,the cells were induced with PA(0.4 m M)for 24 h,and the whole protein was extracted.The protein expression levels of β-actin,CPT1 A,ACOX1,PPARa,ACSL1 and ADRP were detected by Western Blotting.Results: The high-fat diet caused an increase in the levels of ALT,AST,TG,and TC in the serum of rats,and the volume of liver tissue increased significantly.Danshensu treatment can significantly improve liver color and volume changes caused by high-fat diet.In vivo and in vitro experiments,TAF9-specific overexpression promotes the activation of fatty acid β-oxidation and alleviates lipid droplet accumulation.DSS significantly up-regulates the expression of HDAC1 and TAF9,and promotes β-oxidation of fatty acids and inhibits lipid droplet accumulation.In vitro,after the HDAC1 gene was knocked out,the fatty acid β-oxidation index protein were significantly down-regulated and the lipid droplets index protein were up-regulated,and the effect of DSS on fatty acid β-oxidation and lipid droplets was weakened,indicating that DSS-mediated protective effects involve HDAC1 pathways.Overexpression of HDAC1 and DSS treatment plays the same role in improving NAFLD,but after interfering with TAF9,the effect of overexpressing HDAC1 was partially weakened,further indicating that TAF9 activation by HDAC1 promotes fatty acid β-oxidation and prevents LDs accumulation.These results demonatrates that DSS alleviates nonalcoholic fatty liver disease in rats,mainly by regulating the HDAC1/TAF9 pathway.Conclusions:1.Danshensu has obvious control effect on NAFLD.2.Activation of TAF9 promotes β-oxidation of fatty acids,inhibits lipid droplet accumulation,and thereby alleviates NAFLD.3.Danshensu reduces NAFLD through the HDAC1/TAF9 signaling pathway.