| Object ive:In this study,db/db diabetic mice were used as the research model to observe the effect of water extract of Cyclocarya paliurus leaf on glucose and lipid metabolism and adipose tissue inflammation in db/db diabetic mice,and to explore its mechanism of improving insulin resistance from the perspective of macrophage polarization,to provide a scientific basis for its clinical application.Method:1.Animal experiment:Select 40 4-week-old db/db mice adaptively for 1 week,and monitor random blood glucose?11.1mmol/L on different days as a successful T2DM model.According to the weight and blood sugar level of the mice,40 db/db mice were randomly divided into 5 groups using a randomized control method,with 8 mice in each group:model group(an equal volume of distilled water per day),metformin group(0.130g/kg.d-1),water extract of Cyclocarya paliurus leaf high-dose group(2.0g/kg.d-1),water extract of Cyclocarya paliurus leaf medium-dose group(1.0g/kg.d-1)and water extract of Cyclocarya paliurus leaf low-dose group(0.5g/kg.d-1).8 animals of the same age C57BL/6J mice were set as the normal group(an equal volume of distilled water per day),and continuous intervention for 6 weeks.Observe the general conditions of the mice every day,measure the weight,water and diet intake,and fasting blood glucose(FBG)of the mice every week;perform the mouse OGTT experiment at the 6th week;after the intervention for 6 weeks,the mice will be sacrificed to collect the serum and tissues to determine the FBG.Lipid metabolism indicators:triglycerides(TG),total cholesterol(TC),free fatty acids(FFA),low-density lipoprotein(LDL),high-density lipoprotein(HDL),liver function indicators:alanine transferase(ALT)),aspartate transferase(AST),renal function indicators:uric acid(UA),urea nitrogen(UREA),blood creatinine(CREA)and fasting insulin levels(INS)and calculate HOMA-IR;epididymal fat and liver pathological changes were observed by HE staining;liver glycogen content was measured by PAS method;epididymal fat inflammatory factor was measured by ELISA method the expression levels of TNF-?,IL-1? and IL-10;the mRNA expression levels of CD86 and cd206 in epididymal fat were determined by real time PCR;the fluorescence expression of M1 and M2 macrophages in epididymal fat was determined by immunofluorescence;the expression of polarization related proteins in macrophages was detected by Western blot.2.Cell experiment:RAW264.7 macrophages were resuscitated and cultured;RAW264.7 macrophages were induced by LPS;CCK8 method was used to determine the effect of water extract of Cyclocarya paliurus leaf on the activity of RAW264.7 macrophages;ELISA method was used to determine the expression levels of TNF-?,IL-1? and MCP-1 of RAW264.7 macrophages;real time PCR was used to determine TNF-?,IL-10,IL-6,CD86 and CD206 of RAW264.7 macrophages.Western blot was used to detect the expression of polarization related proteins.Result:Animal experiment:1.General situation:compared with the normal group,the drinking water,urine,diet and body weight of the model group mice increased significantly,the reaction was slow,and the activity ability decreased.Metformin and water extract of Cyclocarya paliurus leaf could inhibit the weight gain of db/db mice,alleviate the symptoms of polydipsia and polyuria,and improve the activity ability of the mice.2.Serum indexes:1)glucose metabolism indexes:the FBG of db/db mice in model group increased significantly every week(P<0.01),the delay of OGTT blood glucose was serious(P<0.01),the fasting insulin level and HOMA-IR increased significantly(P<0.01);compared with db/db mice in model group,metformin and extract of Cyclocarya paliurus leaf could reduce FBG of db/db diabetic mice in varying degrees(P<0.05 or P<0.01),fasting insulin level and HOMA-IR decreased significantly(P<0.01).2)Lipid metabolism index:compared with normal group,TG,TC,FFA and LDL of db/db mice in model group were significantly increased(P<0.01),while HDL level was significantly decreased(P<0.05);compared with model group,metformin and extract of Cyclocarya paliurus leaf could reduce TC,FFA and LDL of db/db mice in varying degrees(P<0.05 or P<0.01),and increase HDL level(P<0.05).3)Liver and kidney function indexes:compared with normal group,AST,ALT,UREA,UA and CREA levels of db/db mice in model group were significantly increased(P<0.01);compared with db/db mice in model group,metformin and extract of Cyclocarya paliurus leaf could reduce AST,ALT,UREA,UA and CREA levels of db/db mice in varying degrees(P<0.05 or P<0.01).3.Pathology and PAS detection of liver:compared with db/db mice in model group,metformin and extract of Cyclocarya paliurus leaf could reduce the volume of adipocytes,inhibit the deposition of lipid in liver and increase the synthesis of glycogen in liver.4.Determination of inflammatory factors:compared with the normal group,the levels of TNF-? and IL-6 in adipose tissue of db/db mice in the model group were significantly increased(P<0.01),while the level of IL-10 were significantly decreased(P<0.01);compared with the model group,metformin and extract ofCyclocarya paliurus leaf could inhibit the secretion of TNF-? and IL-6,and increase the secretion of IL-10(P<0.05 orP<0.01).5.Immunofluorescence assay:compared with normal mice,the expression of CD86 in adipose tissue of db/db mice in model group was significantly increased,while the expression of MR was decreased;compared with db/db mice in model group,metformin and extract of Cyclocarya paliurus leaf could reduce the expression of CD86 and increase the expression of MR(P<0.05 or P<0.01).6.Real time PCR and Western blot analysis:compared with the normal group,the expression levels of CD86 mRNA,TLR4 and NF ? Bp65 protein in adipose tissue of db/db mice in the model group were significantly increased(P<0.05),compared with db/db mice in model group,metformin and extract of Cyclocarya paliurus leaf could up regulate the expression of CD206 mRNA and PPAR-y protein in db/db mice(P<0.05 orP<0.01),and down regulate the expression of CD86 mRNA,TLR4 and NF-? Bp65 protein(P<0.05 or P<0.01).Cell experiment:1.Lipopolysaccharide(LPS)modeling:RAW264.7 macrophages were successfully induced by 100 ng/ml LPS,they become inflammatory macrophages and secrete TNF?,IL-1? and MCP-1.2.CCK8 was used to detect cell viability.The water extract of Cyclocarya paliurus leaf at the concentration of 50mg/L or below had no significant effect on the activity of RAW264.7 macrophages.So 10mg/L,20mg/L and 50mg/L water extracts of Cyclocarya paliurus leaf were used for subsequent experiments.3.ELISA assay of inflammatory factors:compared with the model group,the extract of Cyclocarya paliurus leaf could inhibit the secretion of TNF-?,IL-1? and MCP-1(P<0.05 or P<0.01).4.Real time PCR and Western blot analysis:Compared with the normal group,the mRNA expression levels of TNF-?,IL-6,CD86 and the protein expression levels of TRL4 and NF-? Bp65 in the model group were significantly up-regulated(P<0.01),while the mRNA expression levels of IL-10,CD206 and the protein expression level of PPAR-y in the model group were significantly down regulated(P<0.01).Compared with the model group,the mRNA expression levels of TNF-?,IL-6,CD86 and the protein expression levels of TRL4 and NF-? Bp65 in metformin and water extract of Cyclocarya paliurus leaf in different dose groups were down regulated(P<0.05 or P<0.01),while the mRNA expression levels of IL-10,CD206 and the protein expression level of PPAR-? were up-regulated(P<0.01).Conclusion:1.The water extract of Cymbidium paliurus leaf can significantly improve glucose and lipid metabolism in db/db diabetic mice,and improve insulin resistance in db/db diabetic mice.2.The water extract of Cyclocarya paliurus leaf can inhibit the inflammation of adipose tissue in db/db diabetic mice by regulating the polarization of macrophages.3.The water extract of Cyclocarya paliurus leaf may regulate the polarization of macrophages by activating PPAR-? and regulating TLR4/NF-?B signaling pathway. |
PDF Full Text Request