| Part 1The effects and mechanistic studies of safflower yellow on alleviating obesityObjectivesObesity and its related complications are important diseases threatening the health of human beings.Oxidative stress and adipocytokines are closely linked to the occurrence and development of obesity.Traditional Chinese medicine safflower yellow(SY)can be used in the treatment of cardiovascular and cerebrovascular diseases.It has been found in our previous study that SY possessed the anti-obesity effects,but the specific mechanism is still unclear.Therefore,the purpose of the present study is to investigate the effects of SY and its main active component hydroxysafflor yellow A(HSYA)on weight loss in high-fat diet-induced obesity(DIO)mice,ob/ob obese mice and high-fat diet-fed mice,as well as the roles of antioxidant enzymes and the adipocytokine leptin in the process.MethodsThis study involves animal experiments and cell experiments.The animal experiments include the following four parts:1.Effects of SY and HSYA on DIO mice by intraperitoneal injection.C57BL/6J mice were randomly assigned to standard food(SF)group and high-fat diet(HFD)group to establish DIO mice model.Ten weeks later,mice were divided into five groups(n=10 in each group):SF-Saline group,SF-SY group,HFD-Saline group,HFD-SY group and HFD-HSYA group.Mice in the SY and HSYA intervention groups were intraperitoneally injected with 200 mg/kg/d SY or HSYA for ten weeks.The effects of SY and HSYA on body weight,food intake,fat mass,glucose metabolism,liver function,oxidative stress marker,antioxidant enzymes,leptin,glucose-dependent insulinotropic polypeptide(GIP),and so on,were measured.2.Effects of SY and HSYA on DIO mice by gavage.Similarly,DIO mice model was established and randomly divided into five groups.Mice in the SY and HSYA intervention groups were given 250 mg/kg/d SY and HSYA,respectively,by gavage for nine weeks.The changes of the above obesity and metabolism related indicators were also observed.3.Effects of SY on ob/ob mice by gavage.The ob/ob mice with leptin gene mutation were fed with SF and randomly divided into three groups(n=3 in each group).Mice were administrated with 250 mg/kg/d(low-dose group)or 500 mg/kg/d(high-dose group)SY by gavage for nine weeks.The changes of the above obesity and metabolism related indicators were also observed.4.Effects of SY and HSYA on HFD-fed mice by short time gavage.C57BL/6J mice were fed with HFD and randomly divided into three groups(n=10 in each group).Mice were administrated with 250 mg/kg/d SY(HFD-SY group)or 250 mg/kg/d HSYA(HFD-HSYA group)by gavage.One week later,the respiratory metabolic measurement was performed.Three weeks later,leptin sensitivity experiment was conducted to observe the reactivity of mice to exogenous leptin in appetite suppression and weight loss.Four weeks later,the changes of the above obesity and metabolism related indicators were observed.The cell experiments include the following three parts:1.HepG2 cellsHepG2 cells were treated with 20 ?M H2O2 solution to establish cellular model of oxidative stress damage,and then the effects of SY on the expression of antioxidant factor and antioxidant enzymes were measured.2.3T3-L1 preadipocytes3T3-L1 preadipocytes were induced to differentiate to mature adipocytes.The 3T3-L1 adipocytes were treated with 200 ?M H2O2 solution to establish cellular model of oxidative stress damage,and then the effects of S Y and HSYA on the expression of antioxidant factor and antioxidant enzymes were measured.Besides,3T3-L1 adipocytes without oxidative stress damage were also treated with SY to observe the expression of antioxidant enzymes,leptin and the negative regulators of the leptin signaling pathway.3.Mice stromal vascular fraction(SVF)SVF were isolated from the inguinal subcutaneous adipose tissue of C57BL/6J mice,and induced to differentiate to mature adipocytes.The SVF-derived adipocytes were treated with SY to observe the expression of antioxidant enzymes,leptin and the negative regulators of the leptin signaling pathway.ResultsThe results of the animal experiments are as follows:1.Effects of SY and HSYA on DIO mice by intraperitoneal injection.After SY treatment,the body weight gain,white adipose tissue(WAT)mass and WAT percentage of the DIO mice significantly decreased to 56.8%(3.31 ± 1.71g vs.5.83 ±2.27g),57.7%and 62.7%of the HFD-Saline group,fasting blood glucose(FBG)levels significantly decreased(9.49±2.25mmol/L vs.11.62±1.61mmol/L),and homeostasis model assessment of insulin resistance(HOMA-IR)also decreased(all P<0.05).After HSYA treatment,the body weight gain,WAT mass and WAT percentage of the DIO mice also significantly decreased to 28.6%(1.67±2.66g vs.5.83±2.27g),61.7%and 66.3%of the HFD-Saline group,FBG levels significantly decreased(9.80±1.89mmol/L vs.11.62±1.61mmol/L),and HOMA-IR also decreased(all P<0.05).Compared with HFD-Saline group,the blood glucose levels and the area under the curve(AUC)of IPGTT and IPITT in HFD-SY group and HFD-HSYA group were decreased,and liver steatosis and alanine aminotransferase(ALT)levels also decreased(all P<0.05).The superoxide dismutase(SOD)activities in the liver tissue of SY and HSYA treated DIO mice increased by 34.9%and 20.3%,and the mRNA levels of glutamate-cysteine ligase catalytic subunit(GCLC)and NAD(P)H dehydrogenase(quinone 1)(Nqo1)in the liver tissue of SY treated DIO mice increased to 1.8-and 1.7-folds of that in HFD-Saline group(all P<0.05).In the epididymal WAT(eWAT),the mRNA levels of nuclear factor erythroid 2 related factor 2(Nrf2),heme oxygenase-1(HO-1)and GCLC in HFD-SY group increased to 1.3-,3.9-and 2.0-folds,and the mRNA levels of SOD1,HO-1 and GCLC of HFD-HSYA group also increased to 1.2-,1.5-and 1.5-folds of that in HFD-Saline group(all P<0.05).Besides,after SY and HSYA treatment,serum leptin levels of DIO mice significantly decreased by 68.3%(0.82±0.59ng/mL vs.2.59±1.66ng/mL)and 82.6%(0.45±0.79ng/mL vs.2.59±1.66ng/mL),leptin mRNA levels in subcutaneous WAT(sWAT)and eWAT also decreased,while LepRb mRNA levels significantly increased to 2.2-and 1.8-folds of that in HFD-Saline group(all P<0.05).There was no effect of SY and HSYA on the weight of liver tissue,food intake and serum GIP levels.2.Effects of SY and HSYA on DIO mice by gavage.Due to the poor compliance of weight-loss drugs by injection administration,we further explored whether the administration of SY and HSYA by gavage also had the weight-loss effect.The results showed that the body weight of DIO mice started to decrease after SY and HSYA treatment for one week(P<0.05).After SY treatment for nine weeks,the body weight of the DIO mice decreased by 18.1%(36.6±1.5g vs.44.7±4.2g),the body weight gain and WAT mass significantly decreased to 10.9%(0.81 ± 1.68g vs.7.46±2.85g)and 72.2%of the HFD control group(all P<0.05).After HSYA treatment for nine weeks,the body weight of the DIO mice also decreased by 22.1%(34.8±3.2g vs.44.7±4.2g),the body weight gain,WAT mass and WAT percentage significantly decreased to-13.7%(-1.02±2.97g vs.7.46±2.85g),56.3%and 67.5%of the HFD control group(all P<0.05).The weight of liver tissue and ALT levels,and the blood glucose levels and AUC of both IPGTT and IPITT in HFD-SY and HFD-HSYA groups all significantly decreased(all P<0.05).Compared with HFD control group,serum levels of GIP and leptin in HFD-SY group decreased by 30.1%(578.3±170.8pg/mL vs.827.7±259.8pg/mL)and 74.3%(0.64±0.66ng/mL vs.2.49±1.43ng/mL),and leptin mRNA levels in sWAT and eWAT also decreased by 58.9%and 49.5%(all P<0.05).Serum levels of GIP and leptin in HFD-HSYA group also decreased by 29.7%(581.5±159.9pg/mL vs.827.7±259.8pg/mL)and 76.7%(0.58±0.39ng/mL vs.2.49±1.43ng/mL),and leptin mRNA levels in sWAT decreased by 65.8%compared with HFD control group(all P<0.05).There was no effect of SY and HSYA on food intake,but the feed efficiency of DIO mice was significantly decreased(P<0.05).3.Effects of SY on ob/ob mice by gavage.The effects of SY on the ob/ob mice,leptin gene mutation induced obese mice,were further explored to investigate whether the weight loss effect of SY was associated with leptin.The results showed that there was no effect of SY on the body weight,food intake,fat mass,glucose metabolism,liver function and serum biochemical parameters,indicating that leptin play an important role in the weight loss effects and improvement of glucose metabolism and liver function by SY.4.Effects of SY and HSYA on HFD-fed mice by short time gavage.In order to investigate the effects of SY and HSYA on energy metabolism and leptin sensitivity before weight loss and fat mass decreased,the effects of SY and HSYA on HFD-fed mice by short time gavage were further explored.There was no change in body weight after SY and HSYA treatment for one weeks.Meanwhile,there was also no change in energy metabolism.However,food intake after leptin injection of the mice in HFD-SY group was decreased compared with saline injection(P<0.05),and the body weight gain after leptin injection of the mice in HFD-SY and HFD-HSYA groups also had a trend to decrease,suggesting that SY can increase the sensitivity to exogenous leptin.Serum triglyceride levels also dereased after HSYA treatment(P<0.05).But there was no effect of SY and HSYA on body weight,body weight gain,fat mass,food intake,feed efficiency,glucose levels,HOMA-IR,liver function and serum levels of GIP and leptin after four weeks' intervention.The results of the cell experiments are as follows:1.HepG2 cellsIn the HepG2 cells with oxidative stress damage,after 10 mg/L SY administration for 24h,Nrf2 mRNA levels increased to 1.3 folds of control group(P<0.05).SY also increased the mRNA levels of GCLC and Nqo1 in a dose-dependent manner.After 10,50 and 100 mg/L SY treatment,mRNA levels of GCLC and Nqo1 increased to 1.4?2.3-folds of control group(all P<0.05).Besides,SOD1 mRNA levels all increased to 1.3-folds,and CAT mRNA levels increased to 1.2-1.4-folds of control group(all P<0.05).2.3T3-L1 preadipocytes3T3-L1 preadipocytes were induced to differentiate to mature adipocytes.In the 3T3-L1 mature adipocytes with oxidative stress damage,after 10,50 and 100 mg/L SY intervention for 24h,mRNA levels of Nrf2 and SOD1 increased to 1.3?1.5-folds of control group,and HO-1 mRNA levels increased to 1.7-3.3-folds of control group in a dose-dependent manner(all P<0.05).HO-1 mRNA levels also increased to 1.4-folds of control group after 100 mg/L HSYA intervention(P<0.05).In 3T3-L1 adipocytes without oxidative stress damage,50 and 100 mg/L SY also directly increased mRNA levels of SOD 1 and HO-1 to 1.1-3.2-folds of control group(all P<0.05).Besides,after 50 and 100 mg/L SY treatment,leptin mRNA levels of 3T3-L1 adipocytes derceased by 51.2%-78.2%,and mRNA levels of the negative regulators of leptin signaling pathway,suppressor of cytokine signaling 3(SOCS3)and protein tyrosine phosphatase 1B(PTPIB),also derceased when compared with the control group(all P<0.05).3.Mice SVFIn the mature adipocytes differentiated from mice SVF,after 100 mg/L SY administration for 48h,the mRNA levels of SOD1 and HO-1 increased to 1.2-and 1.8 folds of control group,while leptin mRNA levels decreased by 35.4%(all P<0.05).But there was no effect of SY on the exression of SOCS3 and PTP1B.Conclusion1.Intraperitoneal administration of SY and its main active component HSYA could reduce the body weight and fat mass,and improve glucose metabolism and liver function in HFD-induced obese mice.2.Gavage administration of SY and HSYA could also reduce the body weight and fat mass,and improve glucose metabolism and liver function in HFD-induced obese mice,and the anti-obesity effects of SY and HSYA by gavage was more significant than that by intraperitoneal administration.3.The anti-obesity effects of SY and HSYA were probably associated with the directly increased expression of antioxidant enzymes in liver and adipose tissue,and the reduction of leptin resistance in adipose tissue.4.SY could increase the sensitivity of HFD-fed mice to exogenous leptin before weight loss and fat mass decreased.Part 2The effects and mechanistic studies of ileal transposition surgery on alleviating obesityObjectivesIleal transposition(IT)surgery is a novel metabolic surgery that has been reported to induce weight loss,improve glucose and lipid metabolism and increase insulin sensitivity.Fibroblast growth factor 21(FGF21)is an important metabolic regulator that can increase energy expenditure,reduce body weight,increase insulin sensitivity and improve glucose and lipid metabolism.However,whether IT surgery plays the anti-obesity effect by regulating FGF21 has not been reported.Besides,metabolomics is an important tool in the field of metabolic research and can be applied in the study of metabolic surgery.However,the serum metabolite alterations after IT surgery also has not been reported.Therefore,the purpose of the present study is to explore the effects of IT surgery on weight loss and metabolic improvement,as well as the changes in FGF21 signaling pathway after IT surgery.Meanwhile,non-targeted metabolomics is applied to observe the changes of metabolites in serum and adipose tissue of the rats after IT surgery to explore its mechanism of action.MethodsTen-week-old male Goto-kakizaki(GK)rats were fed with HFD and randomly assigned to three groups(n-7 per group):no surgery group,Sham-IT group and IT group.Rats in IT group were subjected to IT surgery with translocation of an ileal segment 5 cm proximal to the ileocecal valve to the proximal jejunum about 5cm from the Treitz ligament.Rats in the Sham-IT group underwent sham surgery,which involved the same incision,transection,and re-anastomosis of the gastrointestinal tract at multiple sites corresponding to the IT except for ileum transposition.The rats in the no surgery group did not receive any surgical intervention.Body weight,food intake and FBG were recorded.Six weeks later,oral glucose tolerance test(OGTT)and IPITT were performed.Then mice were anesthetized to collect blood sample,adipose tissue and liver.Serum levels of biochemical parameters,insulin,FGF21,leptin and gut hormone were measured.The expression of the FGF21,fibroblast growth factor receptor(FGFR1)and ?-klotho(KLB)in liver and adipose tissue,the expression of WAT browning-related genes in adipose tissue,and the expression of glycogen synthase 2(GYS2)and inflammatory cytokines in liver tissue were measured by RT-qPCR and western blot.Besides,metabolites in serum and eWAT were extracted,and non-targeted metabolomics was performed by ultra-high performance liquid chromatography tandem mass spectrometry.The differential metabolites were identified using principal component analysis and orthogonal partial least squares discriminant analysis.Then hierarchical cluster analysis and metabolic pathway analysis were performed.Results1.At the overall levelsCompared with Sham-IT group,the body weight and food intake started to decrease at the second week after IT surgery,and decreased to 83.4%(259.4±13.0g vs.311.1±18.6g)and 72.1%(15.5 ± 0.7g vs.21.5±2.5g)of that in Sham-IT group,respectively,six weeks after IT surgery(all P<0.05).WAT and WAT percentage of IT surgery group also decreased by 41.6%(4.71±1.66g vs.8.06±1.70g)and 30.8%(all P<0.05).The FBG levels of the rats in IT surgery group decreased from the initial 12.4±1.1mmol/L to 6.9±0.1mmol/L six weeks after surgery,and the FBG levels were all remarkably lower than those of the Sham-IT group at the 2nd,4th and 6th week post-surgery(all P<0.05).Meanwhile,the blood glucose levels and AUC of both OGTT and IPITT,and HOMA-IR in IT surgery group significantly decreased compared with Sham-IT group(all P<0.05).Compared with Sham-IT group,serum levels of FGF21 and leptin in IT surgery group decreased by 26.3%(327.03±54.36pg/mL vs.443.45±38.30pg/mL,P<0.05)and 61.7%(0.57±0.40ng/mL vs.1.49 ± 1.15 ng/mL,P<0.05),respectively.2.At the adipose tissue and liver tissue levelsIn eWAT,the protein levels of FGFR1 and KLB of IT surgery group notably increased to 3.0-and 3.9-folds of the Sham-IT group,and the protein levels of uncoupling protein 1(UCP1),a WAT browning-related gene,also increased to 2.2-folds of the Sham-IT group(all P<0.05).In pWAT,the mRNA levels of FGFR1 and KLB of IT surgery group increased to 1.4-and 2.4-folds of the Sham-IT group,and the protein levels of FGFR1 and UCP1 also significantly increased to 1.7-and 2.3-folds of the Sham-IT group(all P<0.05).In liver tissue,the protein levels of FGF21 and KLB significantly increased to 3.9-and 2.3-folds of the Sham-IT group(P<0.05),while there was no effect on the expression of peroxisome proliferator-activated receptors ?,the upstream regulator of hepatic FGF21 secretion,as well as the downstream molecules of FGF21 signaling pathway,such as GYS2,tumor necrosis factor a and interleukin-6.There was also no change in the phosphorylation of hepatic GYS2 and liver glycogen content.3.Serum and adipose tissue metabolomicsIn the serum metabolomics analysis,ten differential metabolites were identified.Compared with Sham-IT rats,serum LysoPC(O-18:0)and PG(20:4/20:0)of IT rats were decreased,while genistein 4'-O-glucuronide,5,6:8,9-Diepoxyergost-22-ene-3,7beta-diol,PI(16:0/18:2(9Z,12Z)?docosapentaenoic acid,3-Oxo-4,6-choladienoic acid,3-Oxochoic acid,and TG were significantly increased.Pathway analysis highlighted the following pathways,including ether lipid metabolism,alpha linolenic acid and linolenic acid metabolism,incretin synthesis and secretion,free fatty acid receptors,and biosynthesis of unsaturated fatty acids.Besides,in the metabolomics analysis of eWAT,fifty and sixty-eight differential metabolites were identified by positive and negative ion modes,respectively.The hierarchical clustering of these differential metabolites is effective,and pathway analysis highlighted the following pathways,including ubiquinone and other terpenoid-quinone biosynthesis,AMPK signaling pathway,pantothenate and CoA biosynthesis,vitamin digestion and absorption,glycerolipid metabolism,phenylalanine metabolism,steroid hormone biosynthesis and bile secretion.ConclusionIT surgery significantly decreased the body weight and fat mass,and improved glucose metabolism in GK rats.The effects of IT surgery on weight loss and improvement of glucose metabolism were probably associated with the enhancement of FGF21 signaling pathway in visceral adipose tissue to promote browning of white adipose tissue.Besides,the results of metabolomics suggested that the beneficial effects of IT surgery may also be associated with the signaling pathways related to lipid metabolism,incretin synthesis and secretion,and energy metabolism of adipose tissue. |
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