| Objective: Nonalcoholic fatty liver disease(NAFLD)is one of the most significant causes of liver disease in the world,but unfortunately,there is no approved drug therapy and no specific drug for NAFLD treatment.When NAFLD progresses to Nonalcoholic steatohepatitis(NASH)accompanied or not accompanied by liver fibrosis without exact treatment measures,NAFLD is more likely to progress to the end-stage of the disease,or even develop into liver cancer.Even simple fatty liver has been proved to be related to cardiovascular and cerebrovascular diseases,affecting people’s quality of life.The prevention and treatment of NAFLD has been a hot spot in the medical field for many years.At present,there is no ideal treatment for NAFLD.Since NAFLD shares similar lipid metabolism disorders with type 2 diabetes,many ways that are effective in treating NAFLD/NASH have been discovered in the course of anti-diabetes therapy.Sodium-glucose co-transporter inhibitor 2 inhibitors(SGLT2i),as drugs that can reduce blood glucose by inducing renal diabetes,have been confirmed by some scholars to increase insulin sensitivity.However,most of the existing literatures have reported laboratory and clinical results based on animal models or NAFLD patients complicated with type 2 diabetes.The specific mechanism of NAFLD should be further explored.Dapagliflozin is one of the SGLT2 inhibitors that have been widely used in the clinical treatment of diabetes.It has been reported that dapagliflozin can improve NAFLD patients with type 2 diabetes,but the specific mechanism remains unclear.In this study,a NAFLD rat model was established by feeding high-fat diet.Transcriptome analysis was performed on liver tissues of normal and NAFLD rats to screen for differentially expressed genes and speculate the mechanism of NAFLD.Subsequently,dapagliflozin was used in vivo and vitro,to clarify the mechanism of dapagliflozin improving NAFLD.Research methods: Part I: Rat NAFLD model was constructed to explore the related mechanism of NAFLD induced by high fat diet based on transcriptome.Male SD rats aged 4-5 weeks were randomly divided into normal group(NC group) and high-fat diet group(HFD group).HFD group was fed high-fat diet for 12 weeks to establish NAFLD rat model,and hematoxylin-Eosin(HE),oil red O staining and NAS scoring were used to identify liver lesions from the pathological point of view.Transcriptome analysis was performed on liver tissues of NC group and HFD group to screen differentially expressed genes and speculate the possible mechanism of NAFLD.Part II: Liver transcriptome,serum metabolomics,16 S r DNA sequencing,immunofluorescence,Western blot and real-time PCR were used to investigate the therapeutic mechanism of dapagliflozin on NAFLD rats.Immunohistochemical method was used to verify the expression of SGLT2 in rat liver tissue.SD rats were divided into normal group(NC group),dapagliflozin gavaged group(ND group),high-fat diet group(HFD group)and high-fat diet dapagliflozin gavaged group(HD group).1mg/kg/d Dapagliflozin was continuously intervened for 5 weeks.Combined with liver transcriptome and serum metabolomics methods,this study investigated whether dapagliflozin improved NAFLD through transcriptome screening and its possible mechanism.Through 16 S r DNA sequencing of feces and combined analysis with serum metabolites,the effect of dapagliflozin on the structure and function of intestinal flora in high-fat fed rats was determined.The effects of dapagliflozin on physiological indexes of SD rats in each group were detected.The secretion of TNF-α,IL-1β and IL-10 in liver tissues and serum of rats in each group were detected by ELISA kit.Immunofluorescence,Western blot and real-time PCR were used to determine the key factors in the pathway to determine the improvement mechanism of NAFLD by dapagliflozin.Part III: In vitro application of dapagliflozin to intervene lipotoxic liver cells,to further clarify the mechanism of dapagliflozin to improve NAFLD.L02 cells were cultured in RPMI 1640 medium containing 10% FBS and treated with 500μM oleic acid to induce lipid toxicity.Cell counting kit-8(CCK8),TG content and oil red O staining were used to detect the effect of different concentrations of dapagliflozin(10 μM,40 μM,80 μM、120 μM)on the survival rate and lipotoxicity of L02 cells under OA.Select the appropriate concentration of dapagliflozin.The cells were separated into BSA group(serum-free BSA medium),OA group(500μM oleic acid medium)and DAPA group(40μM dapagliflozin medium).Real-time PCR,WB and si RNA gene silencing methods were used to further clarify the pathophysiological mechanism of dapagliflozin improvement of NAFLD at the cellular level.Results: Part I: NAFLD rat model was successfully established by feeding high-fat diet for 12 weeks.HE staining,oil red O staining and NAS scoring of liver tissue proved successful modeling.Transcriptome results showed that stearoyl-Co A desaturase(Scd1)was the most significantly changed gene in NC group and HFD group.Scd1 was mainly enriched in PPAR signaling pathway,unsaturated fatty acids biosynthesis pathway and AMPK signaling pathway.Part II: Immunohistochemical showed the expression of SGLT2 in rat liver tissue.Transcriptome results showed that dapagliflozin inhibited the expression of Scd1 by activating the PPAR pathway and regulating CPT1 A.Untargeted metabolomics results: The differential metabolites were mainly enriched in glycerophospholipid metabolic pathway in NAFLD rats treated with dapagliflozin.Pearson correlation results suggested that Ppara,Cpt1 a and Scd1 were closely correlated with the differential metabolites.Dapagliflozin improved the physiological indexes of rats.The results of immunofluorescence,real-time PCR and WB showed that dapagliflozin inhibited the expression of SCD1 while promoted the expression of PPARα and CPT1 A.16S r DNA sequencing and combined analysis with serum metabolites showed that dapagliflozin improved the metabolic phenotype of NAFLD model rats by improving the structure and function of intestinal flora.WB results showed that compared with NC group,the expression of RXR,FGF21,CES1 and ACADSB protein in liver tissue of HFD group decreased significantly.Compared with HFD group,the expression of FGF21,CES1 and ACADSB protein in liver tissue of HD group was significantly increased.In addition,consistent with the transcriptome data,the expression of SREBP1 also increased significantly in HD group compared with HFD group.Dapagliflozin also decreased the expression of fatty acid synthesis proteins FASN,ACC and p-ACC in the liver,and promoted the expression of BCAT and BCKDH,the rate limiting enzymes of branched chain amino acid catabolism,as compared with HFD group.Part III: L02 cells were treated with 500μM oleic acid(OA group)for 24 h to induce lipotoxicity.The cells were incubated with 500μM oleic acid and different concentrations of dapagliflozin for 24 h.CCK8 indicated that when the net concentration of dapagliflozin exceeded 80μM,the cell survival rate decreased(P<0.05).The results of Oil red O staining and blood lipid determination showed Dapagliflozin can improve the lipotoxicity of L02 cells,and the effect of 40μM dapagliflozin was the best.Real-time PCR and WB results showed that the PPARα and CPT1 A gene and protein expressions of L02 cells in OA group were lower than those in BSA group,and SCD1 gene and protein expressions were higher than those in BSA group(P<0.01).The expression levels of PPARα and CPT1 A gene and protein in DAPA group were higher than those in OA group,and SCD1 gene and protein in DAPA group were lower than those in OA group(P<0.01).Pparα gene was silenced by si RNA.Dapagliflozin could inhibit the expression of SCD1 gene and protein and improve the level of TG in hepatocytes.Conclusion: Dapagliflozin activated PPARα pathway,and positively and inversely regulates the expression of CPT1 A and SCD1 m RNA and protein,and activates the glycerol phospholipid metabolic pathway.It regulates the metabolism and synthesis of fatty acids and amino acids in liver tissue,regulates lipid metabolism and increases energy metabolism by improving the structure and function of intestinal flora,and improves NAFLD. |
PDF Full Text Request