The Protective Effect Of Micheliolide Derivative On Liver And Kidney Injury Of Db/dbmice Via Activating PPAR-?-mediated NF-?B And AMPK/mTOR Pathway

Diabetes mellitus is a widespread disease worldwide and diabetic complications seriously threaten human health.Among them,diabetic liver and kidney complications are more extensive.Therefore,this study,under the guidance of TCM theory of "liver and kidney homologous",explores the effectiveness and possible mechanisms of micheliolide derivative(DMAMCL)on liver and kidney protection in type 2 diabetes db/db mice,which will provide theoretical and experimental basis for the development of new drugs for diabetic liver and kidney complications.Chapter 1 Effect of micheliolide derivative on hepatic protection in diabetic db/db mouseObject:To observe the effect of DMAMCL on fatty liver in type 2 diabetes db/db mice,and to explore the possible mechanism of treatment of diabetic liver injury.Methods:Db/db mice were randomly divided into model group,positive control group,and DMAMCL low,medium and high dose group.Additionally,db/m mice were used as the normal control group.The mice were treated with DMAMCL,irbesartan,and normal saline respectively.After 16 weeks,automatic biochemical analyzer was used to detect fasting blood glucose(FBG),triglyceride(TG),total cholesterol(TC),alanine aminotransferase(ALT)and aspartate aminotransferase(AST).ELISA and ultraviolet spectrophotometry were used to detect the contents of TNF-?,IL-1? TG,TC and free fatty acids(FFA)in liver tissue.The expression of related proteins and genes were measured by quantitative real-time PCR and Western Blot.Confocal microscopy and transmission electron microscopy(TEM)were used to observe the expression of LC3B and autophagosomes.Results:In db/db mice,the body and liver weights,FBG,lipid content and liver aminotransferase levels in serum,lipid content and inflammatory cytokine levels in liver tissue,and extent of hepatic steatosis were increased,compared with those in db/m mice.These increases were reversed by DMAMCL treatment.DMAMCL significantly increased the level of PPAR-y mRNA and upregulated the protein expression of PPAR-y,p-AMPK,Atg7 and Beclin-1 and elevated the LC3B-II/I ratio,and reduced the level of p-Ii?B?,p-NF-KB p65,and p-mTOR than those in the model group(P<0.05).Confocal microscopy analysis revealed that LC3B expression in db/db mice was reduced compared to that in db/m mice,whereas these effects were reversed by DMAMCL.TEM analysis suggested a tendency towards fewer autophagosomes in db/db mice compared with that in db/m mice,while the number of autophagosomes was increased by DMAMCL treatment.Conclusion:DMAMCL upregulated the expression of PPAR-y,thereby inhibiting NF-?B signaling pathway and reducing hepatic inflammatory response.Moreover,DMAMCL induced autophagy by activating AMPK/mTOR signaling pathway,which further improved liver steatosis and liver injury in db/m mice.Chapter 2 Effect of micheliolide derivative on hepatic steatosis in lipid mixture-induced AML12 and L02 cellsObject:To further explore the effect and mechanism of DMAMCL on liver protection in type 2 diabetes db/db mice.Methods:AML12 and L02 cells were cultured in high glucose medium and lipid mixture(LM)was used to produce the cell steatosis model.The cells were treated with DMAMCL,PPAR-? inhibitor(T0070907)and AMPK inhibitor(Compound C)respectively.ELISA and ultraviolet spectrophotometry were used to detect the contents of TNF-?,IL-1? TG,TC in cells.Cells were stained with Oil Red O solution to further assess lipid content.Western Blot was used to measure the expression of related proteins.The expression of LC3B was observed with confocal microscopy.Results:Intracellular lipid content and inflammatory cytokine levels in medium in LM-treated AML12 and L02 cells were increased compared with those in the controls,whereas these increases were reversed by DMAMCL.LM significantly increased lipid accumulation in cells,while DMAMCL treatment notably reduced LM-induced cellular lipid accumulation.However,treatment with T0070907 remarkably aggravated LM-induced steatosis in cells,compared with those in model group(P<0.05).DMAMCL increased the expressions of PPAR-? and p-AMPK,and decreased the levels of p-I?B?,p-NF-?B p65,and p-mTOR in LM-induced AML12 and LO2 cells.However,T0070907 administration reduced the expressions of PPAR-? and p-AMPK,and increased the levels of p-I?B?,p-NF-?B/p65,and p-mTOR.In addition,DMAMCL upregulated the expression of Atg7 and Beclin-1 and elevated the LC3B-?/?ratio compared with those of the controls(P<0.05),but compound C reduced the expression levels of p-AMPK,Atg7 and Beclin-1 as well as the LC3B-?/? ratio,which was enhanced by DMAMCL.Confocal microscopy analysis revealed that LC3B was abundantly expressed in LM-induced AML12 and L02 cells treated with DMAMCL,while these effects were reduced by T0070907.Conclusion:DMAMCL improved the hepatic steatosis in LM-induced AML12 and LO2 cells by activating PPAR-?-mediated NF-?B and AMPK/mTOR signaling pathway.Chapter 3 Effect of micheliolide derivative on renal protection in diabetic db/db mouseObject:To explore whether DMAMCL can improve the kidney injury in type 2 diabetes db/db mice by the same mechanism.Methods:Experimental animals,groups and drug interventions were set in the same way as above described.Automatic biochemical analyzer was used to detect the ratio of urine microalbumin to urinary creatinine(UACR),blood urea nitrogen(BUN)and serum creatinine(Scr).ELISA was used to detect the contents of TNF-? and IL-1? in renal tissue.The pathological changes of kidney were detected with optic microscope.Western Blot and immunohistochemistry were used to measure the expression of related proteins.Results:The kidney weight,BUN,Scr,and UACR,and inflammatory cytokine levels in kidney tissue,and extent of renal pathological changes in db/db mice were increased compared with those in db/m mice,whereas these increases were reversed by DMAMCL treatment.Moreover,DMAMCL upregulated the expression of PPAR-?,p-AMPK,Atg7,Beclin-1?LC3B and elevated the LC3B-?/? ratio,and reduced the levels of p-I?Ba,NF-?B p65,p-NF-?B p65,and p-mTOR than those in the model group(P<0.05).Conclusion:DMAMCL can improve the renal function and renal pathological changes in db/db mice,which is also related to the activation of PPAR-?-mediated NF-?B and AMPK/mTOR signaling pathway.