| Objective:To observe the effect of liraglutide combined with human umbilical cord mesenchymal stem cells on type 2 diabetes mellitus with non-alcoholic fatty liver disease rats liver injury.And then to investigate relation of the effect on the liver injury between the toll-like receptor 4 / nuclear factor-kappa B inflammatory signal pathway and oxidative stress mechanism.Methods:1.Animal model establishment and experimental grouping and intervention: 90 male SD rats were randomly divided into normal control group(NC,n=10)and type 2 diabetes mellitus with non-alcoholic fatty liver disease model group(n=80).The normal control group was fed with conventional feed,and the model group was fed with high glucose and high fat diet.At 8th week,two normal and five model rats were randomly killed.It was observed that compared with the normal group,fasting blood glucose,liver function and blood lipid profile were increased in the model group,and light microscopic examination showed moderate liver diffuse steatosis identified as NAFLD model standard.Then intraperitoneal injection of STZ(30mg/kg),the standard of eligible T2 DM with NAFLD model rats was by measuring three times of fasting blood glucose no less than 11.1 mmol/L,a total of 54 rats and modeled at a rate of 72%.The rats were randomly selected and divided into the following four groups: T2 DM with NAFLD model group(MC),hUC-MSCs treatment group(MC+hUC-MSCs),Liraglutide treatment group(MC+LIRA),Liraglutide combined with hUC-MSCs treatment group(MC+LIRA+hUC-MSCs).The number of rats in each group is ten.Then give the appropriate drμg therapy 8 weeks respectively.HUC-MSCs positive intervention groups(MC+hUC-MSCs,MC+LIRA+hUC-MSCs)were given 100 ul cell nutrient solution with 106 hUC-MSCs by caudal vein injection in the first and fifth week.HUC-MSCs negative intervention groups(NC,MC,MC+LIRA)were given 100 ul cell nutrient solution without hUC-MSCs by caudal vein injection in the first and fifth week.LIRA positive intervention groups(MC+LIRA,MC+LIRA+hUC-MSCs)were given liraglutide 200μg/kg by subcutaneous injection twice a day.LIRA negative intervention groups(NC,MC,MC+hUC-MSCs)were given equal dose saline by subcutaneous injection twice a day.2.Detection Indicator: After 8 weeks treatment,the body weight and liver weight were measured to calculate the liver index;The serum FPG,HbA1 c,TC,TG,HDL,LDL,ALT and AST were measured respectively.The levels of serum TNF-α,IL-6,8-OHdG,SOD and FINS were measured by ELISA and then caculate HOMA-IR.The changes of liver tissue pathology were observed throμgh HE stain under light microscope and transmission electron microscope.The expression of TLR4 and NF-κB protein in liver tissues were detected by immunohistochemistry.The expressions of TLR4 and NF-κB mRNA were detected by RT-PCR.Results:1.Rat general indicators: After 8 weeks treatment,compared with NC group,the levels of LI,FPG,HbA1 c,TC,TG,LDL,ALT,AST and HOMA-IR in MC group were significantly increased(P<0.05),HDL decreased(P<0.05).Compared with MC group,the levels of LI,FPG,HbA1 c,TC,TG,LDL,ALT,AST and HOMA-IR were significantly decreased in the three treatment groups(P<0.05),HDL increased(P<0.05).Compared with MC+hUC-MSCs group,the levels of LI,FPG,HbA1 c,TC,TG,LDL,ALT,AST and HOMA-IR were decreased in MC+LIRA group and MC+LIRA+hUC-MSCs group(P<0.05),HDL was increased(P<0.05).Compared with MC+LIRA group,the levels of LI,FPG,HbA1 c,TC,TG,LDL,ALT,AST and HOMA-IR were decreased in MC+LIRA+hUC-MSCs group(P <0.05),HDL was increased(P<0.05).2.Rat serum levels of TNF-α,IL-6,8-OHdG and SOD: After 8weeks treatment,compared with NC group,the levels of TNF-α,IL-6 and 8-OHdG in MC group were significantly increased(P<0.05),SOD decreased(P<0.05).Compared with MC group,the levels of TNF-α,IL-6 and 8-OHdG were significantly decreased in the three treatment groups(P<0.05),SOD increased(P<0.05).Compared with MC+hUC-MSCs group,the levels of TNF-α,IL-6 and 8-OHdG were decreased in MC+LIRA group and MC+LIRA+hUC-MSCs group(P<0.05),SOD was increased(P<0.05).Compared with MC+LIRA group,the levels of TNF-α,IL-6 and 8-OHdG were decreased in MC+LIRA+hUC-MSCs group(P <0.05),SOD was increased(P<0.05).3.Pathological changes of rats liver tissue: liver tissue was stained with HE and observed under light microscope: the liver tissue of NC rats is normal,the liver tissue of MC rats filled with a large number of lipid droplets,liver cells were severe steatosis,the nucleus was squeezed deformation or disappearance,hepatic cord disorder,hepatic sinus stenosis or disappearance,and hepatic lobular inflammatory cell infiltration.MC+hUC-MSCs group showed hepatocellular steatosis reduced,lipid droplets were significantly reduced and the inflammatory response to reduce.MC+LIRA group is similar to MC+hUC-MSCs group.MC+LIRA+hUC-MSCs group showed no significant lipid droplets and inflammatory cell infiltration,liver tissue tends to normal.Compared with NC group,the NAS score in MC group were significantly increased(P<0.05).Compared with MC group,the NAS score were significantly decreased in the three treatment groups(P<0.05).Compared with MC+hUC-MSCs group,the NAS score were significantly decreased in MC+LIRA group and MC+LIRA+hUC-MSCs group(P<0.05).Compared with MC+LIRA group,the NAS score were decreased in MC+LIRA+hUC-MSCs group(P <0.05).The changes of liver tissue pathology were observed under transmission electron microscope: liver cells of NC group were large and round,a large number of mitochondria with clear structure and endoplasmic reticulum arrangement rules.liver cytoplasm of MC group showed a large number of fat droplets,the nucleus was squeezed deformation,mitochondrial structure blurred and endoplasmic reticulum edema.The liver tissue of MC+hUC-MSCs group and MC+LIRA group showed a small amount of small bubble fat droplets,mitochondrial structure is more clear and endoplasmic reticulum mild edema.MC+LIRA + hUC-MSCs group showed only a single small lipid droplets,the number of mitochondria and the structure is clear,the endoplasmic reticulum arranged neatly,and the liver cell morphology is close to normal.4.Expression of TLR4 and NF-κB protein in rat liver tissue: After 8 weeks treatment,compared with NC group,the expression of TLR4 and NF-κB protein in MC group were significantly increased(P<0.05).Compared with MC group,the expression of TLR4 and NF-κB protein were significantly decreased in the three treatment groups(P<0.05).Compared with MC+hUC-MSCs group,the expression of TLR4 and NF-κB protein were significantly decreased in MC+LIRA group and MC+LIRA+hUC-MSCs group(P<0.05).Compared with MC+LIRA group,the expression of TLR4 and NF-κB protein were decreased in MC+LIRA+hUC-MSCs group(P <0.05).5.Expression of TLR4 and NF-κB mRNA in rat liver tissue: After 8 weeks treatment,compared with NC group,the expression of TLR4 and NF-κB mRNA in MC group were significantly increased(P<0.05).Compared with MC group,the expression of TLR4 and NF-κB mRNA were significantly decreased in the three treatment groups(P<0.05).Compared with MC+hUC-MSCs group,the expression of TLR4 and NF-κB mRNA were significantly decreased in MC+LIRA group and MC+LIRA+hUC-MSCs group(P<0.05).Compared with MC+LIRA group,the expression of TLR4 and NF-κB mRNA were decreased in MC+LIRA+hUC-MSCs group(P <0.05).6.Related analysis: After 8 weeks treatment,(1)Serum ALT and AST were positively correlated with serum TNF-α,IL-6 and 8-OHdG(r=0.936,0.953,0.915;r=0.93,0.959,0.912;P<0.01);and negatively correlated with SOD(r=-0.961;r=-0.953;P<0.01);and positively correlated with TLR4 and NF-κB mRNA in liver tissue(r=0.908,0.948;r=0.907,0.939;P<0.01).(2)NAS score were positively correlated with serum TNF-α,IL-6 and 8-OHdG(r=0.946,0.939,0.9,P﹤0.01);and negatively correlated with SOD(r=-0.949,P<0.01);and positively correlated with TLR4 and NF-κB mRNA in liver tissue(r=0.969,0.954,P<0.01).Conclusion:1.The glycolipid metabolism and liver damage of type 2 diabetes mellitus combined with non-alcoholic fatty liver disease rats are closely related to inflammatory response and oxidative stress.2.Liraglutide or human umbilical cord mesenchymal stem cells single and the combination of the two intervention on the glycolipid metabolism,insulin resistance and liver damage of type 2 diabetes mellitus with non-alcoholic fatty liver disease rats have different degrees of improvement.3.Compared with a single treatment of liraglutide or human umbilical cord mesenchymal stem cells,the effect of improving glycolipid metabolism,insulin resistance and liver damage on type 2 diabetes mellitus with non-alcoholic fatty liver disease rats were so much better in the combined treatment of liraglutide and human umbilical cord mesenchymal stem cells.4.The mechanism of liraglutide combined with human umbilical cord mesenchymal stem cells on type 2 diabetes mellitus with non-alcoholic fatty liver disease is closely related to the down-regulation of TLR4/NF-κB inflammation pathway and oxidative stress. |
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