| Obesity and diabetes are chronic metabolic diseases,which can cause a variety of complications.They have become a worldwide epidemic,seriously endangering human health,and lack of safe and effective prevention and treatment means.Lactoferrin(Lf)is a multifunctional iron-binding glycoprotein with many biological activities,and it has been found that Lf can regulate lipid metabolism,but its specific mechanism has not been fully clarified,and the research results are inconsistent.Objectives:In this study,male C57BL/6J mice were fed a high fat diet with Lf intervention,and type 2 diabetes model was induced by high fat diet combined with streptozotocin to investigate the prevention and treatment effect of Lf on obesity induced by high fat diet and diabetes in mice.Methods:Experiment 1:40 male C57BL/6J mice aged 8 weeks.After one week of adaptive feeding,they were randomly divided into four groups:① normal diet control(CON)group,②high fat diet(HFD)group,③low Lf(HFD+0.5%Lf)group and④high Lf(HFD+2%Lf)group.The control group was fed with normal diet,while the other three groups were fed with high fat diet(fat energy ratio:60%).The Lf was intervened by drinking water,with low concentration of 5mg/mL Lf aqueous solution and high concentration of 20mg/mL Lf aqueous solution,and the intervention lasted for 12 weeks.During the experiment,the body weight,water and food intake of mice were monitored.After the intervention,oral glucose tolerance test(OGTT)was performed.At the end of the experiment,the following indexes were measured:1.Organ index:measure the weight of subcutaneous fat and visceral fat in mice.2.Serological indexes:Serum biochemical indexes of mice were determined,including four blood lipids:total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-c)and high density lipoprotein cholesterol(HDL-c),and insulin.Insulin resistance was assessed using HOMA-IR model.3.Histopathological indexes:the vacuole size of subcutaneous fat and visceral fat was observed by HE staining of adipose tissue,and the average fat area was calculated.4.Related indexes in adipose tissue:the expression of lipolysis related enzymes in adipose tissue homogenate was determined.RT-qPCR was used to detect the expression of genes related to lipid dilation,lipid synthesis and lipolysis in subcutaneous fat and visceral fat.Experiment 2:48 male C57BL/6J mice aged 8 weeks.After one week of adaptive feeding,10 mice were randomly divided into normal diet control group(CON)and fed with normal diet,while the other 38 mice were fed with high fat diet(fat energy ratio:60%).After one week of injection,the fasting blood glucose of mice was measured,and the mice with fasting blood glucose level≥250mg/dL were successfully used as type 2 diabetic mice.The mice were randomly divided into three groups:① diabetes(DM)group,②low Lf(DM+0.5%Lf)group and③high Lf(DM+2%Lf)group.The Lf was intervened by drinking water,with low concentration of 5mg/mL Lf aqueous solution and high concentration of 20mg/mL Lf aqueous solution,and the intervention lasted for 12 weeks.During the experiment,the body weight,water intake and food intake of mice were monitored.After the intervention,oral glucose tolerance test(OGTT)and intraperitoneal insulin tolerance test(IPITT)were performed.At the end of the experiment,the following indexes were measured:1.Organ index:measure the liver weight of mice.2.Serological indexes:the serum biochemical indexes including blood lipid(TC,TG,LDL-c and HDL-c),alanine aminotransferase(ALT),aspartate aminotransferase(AST),glycosylated serum protein(GSP)and insulin were determined.HOMA-IR model was used to evaluate insulin resistance.Serum pro-inflammatory cytokines including tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-8(IL-8)and interleukin-1β(IL-1β)were measured.3.Histopathological indexes:liver lipid degeneration was observed by HE and oil red O staining.Pancreatic β cell function was observed by HE and insulin immunohistochemical staining.4.Related indexes in liver tissue:the levels of pro-inflammatory cytokines TNF-α,IL-6,IL-8 and IL-1β in liver tissue homogenate were determined;the contents of TC and TG in liver tissue homogenate were determined.The expression of insulin signaling pathway protein in liver tissue was detected by western blot.5.Related indexes in pancreatic tissue:GSH,SOD and MDA related indexes in pancreatic tissue were determined.The pro-inflammatory cytokines TNF-α,IL-6 and IL1β in pancreas were detected by RT-qPCR.Results:Experiment 1:the effects and mechanism of Lf on lipid metabolism in obese mice induced by high-fat diet1.Body weight,water consumption,food intake and energy intake:after the intervention,the body weight of mice fed with high fat diet was significantly higher than that of CON group(P<0.01),and the body weight of HFD+0.5%Lf group was significantly lower than that of HFD group(P<0.05).The amount of drinking water in Lf intervention group was significantly lower than that in CON group and HFD group(P<0.05),and the average daily intake of Lf in low and high concentration Lf was 0.46g/kg and 1.81 g/kg respectively.The food intake of mice fed with high fat was significantly lower than that of CON group(P<0.05),but there was no significant difference in energy intake among each group(P>0.05).2.Serum glucose and lipid metabolism level:compared with HFD group,fasting blood glucose,glucose tolerance,insulin level and HOMA-IR of mice in HFD+0.5%Lf group and HFD+2%Lf group had no significant changes(P>0.05).The four blood lipids in HFD group were significantly higher than those in CON group(P<0.05),but there was no significant change after Lf intervention(P>0.05).3.Fat tissue vacuole size and average area size:compared with HFD group,the vacuoles of subcutaneous fat and visceral fat in HFD+0.5%Lf group decreased significantly,and the number of cells increased in the same visual field.Quantitative analysis showed that the average fat cell area also decreased significantly(P<0.01).4.Expression level of lipid dilatation related genes:compared with HFD group,the expressions of Sfrp5,PTRF/Cavin and Mest in HFD+0.5%Lf group were significantly down-regulated by 72.90%,36.36%and 62.62%respectively(P<0.01,P<0.05,P<0.01).In visceral adipose tissue,the expression of Sfrp5 and Mest genes in HFD+0.5%Lf group were significantly down-regulated by 46.8%and 19.53%respectively(P<0.01),while the expression of Sfrp5 gene in HFD+2%Lf group was significantly down-regulated(P<0.01).5.Expression level of lipid synthesis related genes:compared with HFD group,FAS,SREBP-1c,C/EBPa and FABP4 gene expression in HFD+0.5%Lf group and HFD+2%Lf group were significantly down-regulated(P<0.01).In visceral adipose tissue,the expressions of FAS,C/EBPα and FABP4 in HFD+0.5%Lf group and HFD+2%Lf group were significantly down-regulated by 44.29%,43.67%and 63.97%,respectively(P<0.01),but there was no significant difference in the expression of SREBP-lc gene among the four groups(P>0.05).6.Expression level of lipolysis-related enzymes:compared with HFD group,the expression of HSL in HFD+0.5%Lf group increased significantly(P<0.05),while Lf intervention had no significant change on the expression of ATGL and LPL(P>0.05).In visceral adipose tissue,the expression of LPL and HSL in HFD+0.5%Lf group increased significantly(P<0.01),and the expression of HSL in HFD+2%Lf group also increased significantly(P<0.01).Lf intervention had no significant effect on ATGL expression(P>0.05).7.Expression level of lipolysis-related genes:compared with HFD group,HSL gene expression in HFD+2%Lf group was significantly increased by 1.9 times(P<0.05).while Lf intervention had no significant effect on ATGL and LPL gene expression(P>0.05).In visceral adipose tissue,the expression of LPL and HSL genes in HFD+0.5%Lf group increased significantly by 2.19 times and 2.91 times respectively(P<0.05,P<0.01),and the expression of HSL gene in HFD+2%Lf group increased significantly by 1.55 times(P<0.05).Experiment 2:the effects and mechanism of Lf on diabetic mice induced by high fat diet combined with streptozotocin1.Body weight and liver weight:compared with DM group,there was no significant change in body weight of mice after Lf intervention(P>0.05).Compared with DM group,there was no significant change in liver weight after Lf intervention(P>0.05).2.Serum glucose and lipid metabolism level:there was no significant difference in serum ALT and AST levels after Lf intervention.Compared with DM group,fasting blood glucose level and area under the curve of glucose had no significant change after Lf intervention(P>0.05);Glycosylated serum protein level of DM+0.5%Lf group was significantly lower than DM group(P<0.01);Area under the curve of insulin had no significant difference among the four groups(P>0.05);Compared with DM group,serum insulin level and HOMA-IR value of DM+0.5%Lf group were significantly decreased(P<0.05,P<0.01).In addition,the serum TG level was significantly decreased after Lf intervention,and the serum TC and TG levels in DM+0.5%Lf group and DM+2%Lf group were significantly lower than those in DM group(P<0.05).3.Levels of serum and liver proinflammatory cytokines:compared with DM group,serum proinflammatory cytokines TNF-α and IL-1β in DM+2%Lf group decreased significantly(P<0.01,P<0.05),and TNF-α recovered to the level of CON group.Serum proinflammatory factors IL-6 and IL-8 in DM+0.5%Lf group decreased significantly(P<0.01,P<0.05),and IL-6 recovered to the level of CON group.In liver,only DM+2%Lf group significantly decreased the level of IL-1β(P<0.05).4.Pancreas oxidative stress and inflammation:Compared with DM group,the expression of IL-6 gene in DM+0.5%Lf group was significantly down-regulated(P<0.05),while the expression of TNF-α and IL-1β gene in DM+0.5%Lf group had no statistical significance(P>0.05).GSH in pancreas of mice in DM+0.5%Lf and DM+2%Lf groups.SOD in DM+0.5%Lf group was significantly higher than that in DM group(P<0.05),and there was no statistical difference in MDA content among the four groups.5.Pancreaticβ cell function:immunohistochemical staining of pancreatic HE and insulin showed that pancreatic β cells in DM group were accompanied by obvious hypertrophy,deformity and amyloid deposition,and the morphology of β cells was significantly improved after Lf intervention.Compared with DM group.IOD decreased and AOD increased after Lf intervention,but there was no significant difference(P>0.05).6.Lipid metabolism level and lipid accumulation in liver:compared with DM group,TC level in liver of DM+2%Lf group was significantly decreased by 55.80%(P<0.05),while TG level in liver had no significant change after Lf intervention(P>0.05).The results of liver HE and oil red O staining showed that the liver lipid accumulation of mice fed with high-fat diet decreased after Lf intervention.The liver lipid degeneration and balloon degeneration of mice were evaluated by NASH score method.The liver lipid degeneration and balloon degeneration were improved after Lf intervention,but the difference was not significant.7.Expression of insulin signaling pathway protein in liver:compared with DM group,IRS1 protein expression in liver of DM+2%Lf group was significantly increased by 1.14 times(P<0.01).AKT phosphorylation level was significantly increased by 0.26 times(P<0.05),PI3K phosphorylation level was significantly increased after Lf intervention,but there was no significant difference(P>0.05).Compared with DM group,the expression of GLUT4 protein and IR protein in liver of DM+2%Lf group increased significantly by 0.66 times(P<0.05)and 0.65 times(P<0.01).Conclusions:1.Lf can promote lipid decomposition by inhibiting lipid expansion and lipid synthesis in adipose tissue,thus reducing dyslipidemia in obese mice induced by high-fat diet,and finally reducing the weight of mice.Among them,0.5%Lf has the best intervention effect.2.Lf improves insulin resistance in diabetic mice induced by high fat diet combined with STZ by reducing lipid and lipid accumulation in liver,regulating insulin signaling pathway in liver,enhancing insulin sensitivity,and improving pancreatic β-cell function. |
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